1

UNIVERSIDADE FEDERAL DE SANTA CATARINA

CENTRO TECNOLÓGICO
PROGRAMA DE PÓS-GRADUAÇÃO EM ENGENHARIA
QUÍMICA

ENZYMATIC RING OPENING POLYMERIZATION OF

POLY(GLOBALIDE-CO-ε-CAPROLACTONE) BY MEANS OF
SUPERCRITICAL TECHNOLOGY AND POST
FUNCTIONALIZATION BY THIOL-ENE REACTIONS

CAMILA GUINDANI

FLORIANÓPOLIS - SC
2018
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UNIVERSIDADE FEDERAL DE SANTA CATARINA

CENTRO TECNOLÓGICO
PROGRAMA DE PÓS-GRADUAÇÃO EM ENGENHARIA
QUÍMICA

Camila Guindani

ENZYMATIC RING OPENING POLYMERIZATION OF

POLY(GLOBALIDE-CO-ε-CAPROLACTONE) BY MEANS OF
SUPERCRITICAL TECHNOLOGY AND POST
FUNCTIONALIZATION BY THIOL-ENE REACTIONS

Tese de Doutorado submetida ao

Programa de Pós-Graduação em
Engenharia Química da Universidade
Federal de Santa Catarina para a
obtenção do grau de Doutor em
Engenharia Química.
Orientadora: Profa. Dra. Débora de
Oliveira
Coorientadora: Profa. Dra. Sandra
Regina Salvador Ferreira
Coorientador: Prof. Dr. Pedro Henrique
Hermes de Araújo

Florianópolis - SC
2018
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Ficha de identificação da obra elaborada pelo autor através do Programa

de Geração Automática da Biblioteca Universitária da UFSC.

Guindani, Camila
ENZYMATIC RING OPENING POLYMERIZATION OF
POLY(GLOBALIDE-CO-ε-CAPROLACTONE) BY MEANS OF
SUPERCRITICAL TECHNOLOGY AND POST FUNCTIONALIZATION BY
THIOL-ENE REACTIONS / Camila Guindani ; Orientadora Débora de Oliveira ;
coorientadora, Sandra Regina Salvador Ferreira ; coorientador, Pedro Henrique
Hermes de Araújo.
163 p.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro

Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis,
2018.
Inclui referências.
1. Engenharia Química. 2. ε-caprolactona, globalide. 3. dióxido de carbono
supercrítico. 4. funcionalização. 5. reações tiol-eno. I. Oliveira, Débora. II.
Ferreira, Sandra Regina Salvador. III. Araújo, Pedro Henrique Hermes de. IV.
Universidade Federal de Santa Catarina. Programa de Pós-Graduação em
Engenharia Química. V. Titulo.
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An expert is a person
who has made all the
mistakes that can be
made in a very narrow
field.

Neils Bohr
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AGRADECIMENTOS

Agradeço a Deus, por ter me dado força e sabedoria durante todo

este caminho.
Aos meus pais, Katia e Ivo pelo apoio, incentivo aos estudos e
pelo amor incondicional, e ao meu irmão Igor, pela amizade e por estar
sempre comigo, mesmo quando estamos longe.
Ao meu namorado Graziâni, por todo o amor e companheirismo,
e por sempre me incentivar a alcançar meus objetivos.
Aos meus orientadores, Prof. Débora de Oliveira, Prof. Sandra
R. S. Ferreira e Prof. Pedro H. H. Araújo pela orientação, pelas
oportunidades e pela parceria, fundamental para o desenvolvimento deste
trabalho.
À Prof. Katharina Landfester, do Max-Planck-Institut für
Polymerforschung (MPIP - Mainz – Alemanha) pela oportunidade,
aprendizado e tempo dedicado em orientar parte deste trabalho.
A todos aos colegas do LATESC pela amizade e pelas discussões
sempre produtivas, em especial aos meus queridos amigos Kátia, Eto,
Sara, Simone e Gláucia.
A todos os colegas do MPIP pela acolhedora recepção, em
especial à Johanna Simon pelas discussões e apoio experimental, e Marie-
Luise Frey pelo apoio, conhecimentos compartilhados e pela sincera
amizade.
A todos os colegas do LCP, em especial ao Thiago Ouriques
Machado, pela parceria e amizade que estabelecemos trabalhando juntos
no MPIP.
Aos amigos Josamaique e Jeovandro por todas as discussões em
torno do trabalho e conhecimentos transmitidos.
Ao bolsista de iniciação científica e amigo Pablo Dozoretz pela
parceria e apoio dedicado na realização dos experimentos.
Aos membros da banca examinadora pelas contribuições.
A todos os professores do departamento de Engenharia Química
e Engenharia de Alimentos da UFSC, que contribuíram para a minha
formação profissional.
À UFSC, onde tive a oportunidade de iniciar a construção da
minha carreira acadêmica e conhecer pessoas incríveis ao longo destes
anos.
À CAPES, pelo suporte financeiro que me proporcionou a
oportunidade de realizar parte deste trabalho no exterior.
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A todos que de alguma forma contribuíram e torceram por mim

e pelo sucesso deste trabalho.
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RESUMO

Durante as últimas décadas, muitos esforços têm sido realizados no

desenvolvimento de novos dispositivos biocompatíveis e
biorreabsorvíveis para aplicação biomédica, visando melhorar a
qualidade de vida de pacientes. Poliésteres são uma das classes de
polímeros mais estudadas para estas aplicações, devido a sua capacidade
de serem bioreabsorvidos e/ou biodegradados, além de serem
biocompatíveis. A polimerização de poliésteres por abertura de anel pode
ser realizada por enzimas, consideradas catalisadores green, visto que o
seu uso não gera resíduos tóxicos e a reação pode ser conduzida em
condições brandas de forma eficiente. O uso de fluidos supercríticos
como substituintes de solventes orgânicos tóxicos também vem provando
ser uma alternativa limpa para a produção de polímeros para aplicação
em dispositivos biomédicos. Neste contexto este trabalho relata a síntese
de poli(globalide-co-ε-caprolactona) (PGlCL) por polimerização por
abertura de anel via enzimática (e-ROP) utilizando tecnologia supercrítica
e sua posterior funcionalização via reações tiol-eno. PGlCL foi sintetizada
por e-ROP utilizando como solventes o dióxido de carbono supercrítico
(scCO2) e a mistura scCO2+diclorometano (DCM). Foram utilizadas
diferentes razões de comonômeros Gl/CL. Com relação aos valores de
massa molar, maiores teores de Gl (relativo a quantidade total de
monômeros) levaram a maiores os valores de massa molar. O uso de
scCO2 + DCM causou redução nos valores de massa molar, em
comparação com os valores obtidos para scCO2 puro. O uso de DCM
como cossolvente também causou maior produção de oligômeros
cíclicos, causando um comportamento de fusão duplo. Amostras de
PGlCL com diferentes razões de Gl/CL sintetizadas utilizando scCO2
puro foram então funcionalizadas com N-acetilcisteína através de reações
tiol-eno. A insaturação presente nas unidades de Gl permitiu esta
conjugação, e o PGlCL funcionalizado apresentou menor cristalinidade
(sendo totalmente amorfo para teores de Gl maiores que 50%), e maior
hidrofilicidade, além de potencial antioxidante. Estas características
devem melhorar a degradação do material e são interessantes para
aplicações biomédicas, especialmente quando a biorreabsorção é
desejada. Visando futuras aplicações na nanomedicina, nanopartículas
(NPs) de PGlCL foram também produzidas. A superfície das NPs foi
funcionalizada via reações tiol-eno com a proteína BSA de forma bem-
sucedida, reduzindo a internalização das NPs por células imunológicas.
O PGlCL mostrou ser um copolímero muito funcional com forte potencial
para futuras aplicações como um biomaterial.
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Palavras-chave: ε-caprolactona, globalide, dióxido de carbono

supercrítico, funcionalização, reações tiol-eno.
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ABSTRACT

During the last decades, many efforts have been made in the development
of new biocompatible and bioresorbable polymeric devices for
biomedical application, aiming to improve the quality of life of the
patients. Polyesters are one of the most studied polymers to these
applications, due to its capacity of being bioresorbed/biodegraded,
besides being biocompatible. The ring opening polymerization reaction
of polyesters may be catalyzed by enzymes, considered as green catalysts,
since it does not generate toxic residues and the reaction can be carried
on under mild conditions in an efficient way. The use of supercritical
fluids, in substitution to toxic organic solvents is also proving to be a clean
alternative to the production of polymers for application in biomedical
devices. In this context, this work reported the synthesis of
poly(globalide-ε-caprolactone) (PGlCL) by enzymatic ring opening
polymerization (e-ROP) using supercritical technology and its subsequent
functionalization via thiol-ene reactions. PGlCL was synthesized by e-
ROP using supercritical carbon dioxide (scCO2) and the mixture scCO2 +
dichloromethane (DCM) as solvents. Different Gl/CL comonomer ratios
were used. Regarding the molecular weight values, higher Gl contents
(relative to the total monomers amount), led to higher PGlCL molecular
weight values. The use of scCO2 + DCM caused a decrease on molecular
weight values, in comparison to the use of only scCO2. The use of DCM
as cosolvent also increased the production of cyclic oligomers, causing a
double melting point behavior. Samples of PGlCL with different Gl/CL
ratio synthesized using only scCO2 were then successfully functionalized
with N-acetylcysteine by thiol-ene reaction. The unsaturation present in
Gl units enabled this conjugation, and the functionalized copolymer
PGlCL-NAC presented lower crystallinity (being totally amorphous for
Gl contents higher than 50%) and higher hydrophilicity, besides
presenting antioxidant potential. These characteristics should improve the
material degradation and are interesting for biomedical applications,
especially when bioresorption is desired. Aiming future applications in
nanomedicine, nanoparticles (NPs) made of PGlCL were also produced.
The surface of the NPs was successfully functionalized by thiol-ene
reactions with the protein BSA, reducing the internalization of the NPs
by immune cells. PGlCL showed to be a very functional copolymer with
strong potential for future applications as a biomaterial.

Keywords: ε-caprolactone, globalide, supercritical carbon dioxide,

functionalization, thiol-ene reactions.
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LIST OF FIGURES

Figure 2.1 - Stages of cell adhesion in a surface. .................................. 41

Figure 2.2 - Interactions between cells and protein-nanoparticle
complexes. ............................................................................................. 43
Figure 2.3 - (A) Structure of the monomer e-caprolactone; (B) Structure
of PCL repeating unit. ........................................................................... 47
Figure 2.4 - (A) Structure of the monomer globalide; (B) Structure of the
repeating unit of PGl. ............................................................................ 47
Figure 2.5 - Enzymatic transesterification mechanism proposed by Geus
(2007). ................................................................................................... 52
Figure 2.6 - Reaction pathways for PCL synthesis by e-ROP............... 54
Figure 2.7 - Carbon dioxide phase diagram in the pressure-temperature
plane. ..................................................................................................... 56
Figure 2.8 - Two-step mechanism for free-radical thiol-ene reaction,
alternating propagation (i) and chain transfer (ii).................................. 61
Figure 2.9 - Copolymer poly(globalide-co-4-methyl caprolactone), before
(left) and after (right) crosslinking with dicumyl peroxide. .................. 63
Figure 2.10 - Synthetic routes to functional crosslinked films from
macrolactone using thiol–ene chemistry. .............................................. 64
Figure 2.11 - Crosslinked functionalized PGl films prepared by Ates and
Heise (2014) through routes B and C. ................................................... 65
Figure 2.12 - Synthesis of poly (acrylic acid) grafted from crosslinked and
ATRP initiator modified PGl surfaces, followed by bioconjugation. ... 66
Figure 3.1 - Structure of the cyclic monomers (A) ε-caprolactone and (B)
globalide. Globalide a mixture of two constitutional monomers with the
double bond located either at the 11 or 12 position (dashed line). ........ 74
Figure 3.2 - (A) 13C NMR spectrum of PGlCL 75/25 (Gl/CL feed mass
ratio). (B) Expanded CH2(C=O)O region of 13C NMR spectra, with the
peaks of each respective diad. ............................................................... 80
Figure 3.3 - Number average molecular weight (Mn) as function of feed
globalide content (relative to total monomer amount) using scCO2 and
scCO2+DCM as solvent. ....................................................................... 81
Figure 3.4 - Presence of double melting point behavior: DSC second
heating run for PGlCL 25/75 (Gl/CL feed mass ratio) obtained with
scCO2+DCM, using CO2:MIX = 2:1..................................................... 86
Figure 3.5 - Expanded view of MALDI-TOF spectrum of PGlCL 10/90
(Gl/CL feed mass ratio), obtained using scCO2 as solvent, with DHB
matrix. ................................................................................................... 88
Figure 3.6 - PGlCL molecular weight distribution of PGlCL 10/90 (Gl/CL
feed mass ratio) by the use of different solvents ................................... 89
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Figure 4.1- Poly(globalide-co-ε-caprolactone) side-chain thiol-ene

functionalization with N-acetylcysteine. ................................................98
Figure 4.2 - 1H NMR spectra of (A) PGlCL and (B) PGlCL-NAC (75/25
Gl/CL ratio) and its respective peak assignments to the chemical structure
of the polymers. .....................................................................................98
Figure 4.3 - Schematic representation of the (A) PGlCL chains packaged
arrangement, forming crystalline regions, and (B) PGlCL-NAC chains
disordered arrangement, forming amorphous regions due to the presence
of NAC side-chains. (C) DSC second heating run curves ................... 102
Figure 4.4 - Contact angle values of PGlCL and PGlCL-NAC samples as
function of Gl/CL ratio. ....................................................................... 103
Figure 5.1 - (A) Structure and properties of the copolymer PGlCL in a
mass ratio Gl/CL = 50/50 (B) Scheme representing the conjugation of
PGlCL NPs to modified BSA through thiol-ene reaction. ................... 110
Figure 5.2 - (A) TEM image of PGlCL nanoparticles and information
about particle size and zeta potential; (B) Scheme representing the surface
functionalization of PGlCL nanoparticles with NAC through thiol-ene
reaction; (C) Global double bond consumption and double ................ 118
Figure 5.3 - (A) FACS histograms for unconjugated NPs, non-covalent
and covalent conjugates (B) FCS autocorrelation curves (symbols) and
corresponding fit (straight lines): BSA-FITC (black diamonds ◊), BSA-
FITC non-covalent conjugates (blue diamonds ◊) and BSA-FITC
covalent conjugates (green diamonds ◊) (C) TEM image of uncoated NPs
(1 and 3) and BSA covalent conjugates (2 and 4) and information about
particle size and zeta potential of BSA covalent conjugates ................ 122
Figure 5.4 - Cell uptake: Amount of fluorescent positive cells (%) for
unconjugated NPs and BSA-NPs covalent conjugates during (A) 2h and
(B) 24h of incubation. .......................................................................... 123
Figure A.1 - Yield kinetic behavior for PGlCL 50/50 (Gl/CL feed mass
ratio) synthesis on DCM, under magnetic and mechanical stirring. .... 150
Figure A.2 - Number average molecular weight (Mn) kinetic behavior for
PGlCL 50/50 (Gl/CL feed mass ratio) synthesis on DCM, under magnetic
and mechanical stirring. ....................................................................... 151
Figure B.1 - (A) Degradation curves of PGlCL (Gl/CL = 10/90) in buffer
(circles) and in lipase solution (triangles); (B) Degradation curves of
PGlCL-NAC (Gl/CL = 10/90) in buffer (circles) and in lipase solution
(triangles). ............................................................................................ 158
Figure B.2 - (A) Degradation curves of PGlCL (Gl/CL = 25/75) in buffer
(circles) and in lipase solution (triangles); (B) Degradation curves of
PGlCL-NAC (Gl/CL = 25/75) in buffer (circles) and in lipase solution
(triangles). ............................................................................................ 159
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Figure B.3 - Scanning electronic microscopy images of PGlCL in a Gl/CL

ratio of 25/75 before and after degradation in lipase and buffer ......... 160
Figure B.4 - Scanning electronic microscopy images of PGlCL-NAC in a
Gl/CL ratio of 25/75 before and after degradation in lipase and
buffer ................................................................................................... 160
Figure B.5 - Degradation curves obtained by the OECD biodegradability
assays in activated sludge for PGlCL-NAC (triangles), PGlCL-NAC +
Starch (squares) and Starch (circles). .................................................. 162
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LIST OF TABLES

Table 3.1 - 1H NMR data of PGlCL copolymer composition obtained

using different solvents (scCO2 and scCO2+DCM) and different
globalide/ε-caprolactone feed ratios. ..................................................... 79
Table 3.2 - Thermal properties determined by DSC for PGlCL obtained
using different solvents (scCO2 and scCO2+DCM) for different feed
globalide/ε-caprolactone ratios.............................................................. 85
Table 3.3 - Relation between cyclic and linear chains obtained on e-ROP
under the use of different solvents. ....................................................... 88
Table 4.1 - Thiol-ene reaction conversion and number of functionalized
Gl units per PGlCL-NAC molecule, calculated through 1H NMR
spectroscopy data. ................................................................................. 99
Table 4.2 - Properties of synthesized PGlCL functionalized with NAC for
different globalide/ε-caprolactone repeating units ratio. ..................... 100
Table 4.3 - EC50 values of DPPH and ABTS assays for NAC, PGlCL, and
PGlCL-NAC in a 50/50 Gl/CL ratio. .................................................. 104
Table A.1 - Thermal properties of samples determined by DSC for
samples obtained in different reaction times under mechanical
stirring. ................................................................................................ 153
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LIST OF ABBREVIATIONS AND SYMBOLS

ABTS – 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

AIBN –Azobisisobutyronitrile
BSA – Bovine serum albumin
BSA-FITC – Bovine serum albumin labeled with fluorescein
isothiocyanate
BSA-NP – Bovine serum albumin covalently conjugated with
nanoparticle
CALB – Candida antartica Lipase B
CDCl3 – Deuterated chloroform
CL – Caprolactone
CO2 – Carbon dioxide
DCM – Dichloromethane
DHB – 2,5 - Dihydroxybenzoic Acid
DLS – Dynamic light scattering
Dp – Particle diameter
DPPH – 1,1-diphenyl-2-picrylhydrazil
DSC – Differential scanning calorimetry
EAM – Enzyme-activated monomer
EAPC – Enzyme-activated polymer chain
e-ROP – Enzymatic ring opening polymerization
EtOH - Ethanol
Gl – Globalide
FACS – Fluorescence-activated cell sorting
FBS – Fetal bovine serum
FCS – Fluorescence correlation spectroscopy
FDA – Food and drug administration
FITC – Fluorescein isothiocyanate
GPC – Gel permeation chromatography
KPS – Potassium persulfate
MALDI-TOF – Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry
MIX – Mixture of dichloromethane and monomers
Mn – Number average molecular weight
Mw – Weight average molecular weight
NAC – N-acetylcysteine
NMR – Nuclear magnetic resonance
NP – Nanoparticle
PCL – Poly(ε-caprolactone)
PDI – Polydispersity index
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PEG – Polyethilene glycol

PGl – Polyglobalide
PGlCL – Poly(globalide-co-ε-caprolactone)
PGlCL-NAC – Poly(globalide-co-ε-caprolactone) functionalized with N-
acetylcysteine
R – Degree of randomness
ROP – Ring opening polymerization
scCO2 – Supercritical carbon dioxide
SDS – Sodium dodecyl sulfate
SEM – Scanning electron microscopy
Tc – Crystallization temperature
TEM – Transmission electron microscopy
Tg – Glass transition temperature
THF – Tetrahydrofuran
Tm – Melting temperature
TMS – Tetramethylsilane
UV – Ultraviolet
Xc – Degree of crystallinity
Đ – Dispersity
ΔHm – Fusion Heat
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SUMMARY

CONCEPTUAL DIAGRAM .............................................................. 27

THESIS METHODOLOGICAL SEQUENCE FLOWCHART ..... 31
CHAPTER 1 ........................................................................................ 33
1. INTRODUCTION ...................................................................... 33
1.1. OBJECTIVES....................................................................... 36
1.1.1. General objective ........................................................ 37
1.1.2. Specific objectives....................................................... 37
CHAPTER 2 ........................................................................................ 39
2. LITERATURE REVIEW .......................................................... 39
2.1. BIOCOMPATIBLE, BIODEGRADABLE AND
BIORESORBABLE POLYMER…………………………………...39
2.2. POLYMERS FOR BIOMEDICAL APPLICATIONS ......... 39
2.2.1 Interaction between polymers and biomolecules in a
biological environment............................................................... 41
2.3. POLYESTERS ..................................................................... 44
2.3.1. Poly(ε-caprolactone)................................................... 45
2.3.2. Polyglobalide............................................................... 47
2.3.3. Copolymers of lactones and unsaturated
macrolactones ............................................................................. 49
2.4. ENZYMES ON POLYESTER SYNTHESIS ...................... 50
2.5. ENZYMATIC RING OPENING POLYMERIZATION (e-
ROP)..................................................................................................51
2.5.1. The role of water on e-ROP ....................................... 55
2.6. E-ROP REACTIONS USING SUPERCRITICAL CARBON
DIOXIDE AS SOLVENT ................................................................. 56
2.7. POST-POLYMERIZATION FUNCTIONALIZATION OF
POLYESTERS ................................................................................. 58
2.7.1. Thiol-ene “click” reactions ........................................ 59
2.7.2. Thiol-ene reactions in unsaturated polyesters ......... 61
 2.7.3. Conjugation of nanoparticles and proteins...............66
2.8. THE STATE OF ART...........................................................68
CHAPTER 3 .........................................................................................71
3. ENZYMATIC RING OPENING COPOLYMERIZATION OF
GLOBALIDE AND Ε-CAPROLACTONE UNDER
SUPERCRITICAL CONDITIONS ....................................................71
3.1. INTRODUCTION .................................................................72
3.2. MATERIAL AND METHODS ............................................74
3.2.1. Material preparation ..................................................74
3.2.2. Enzymatic ring-opening polymerization in
pressurized solvents ....................................................................75
3.2.3. Characterization of the copolymer ............................76
3.3. RESULTS AND DISCUSSION ...........................................78
3.3.1. Synthesis and composition of PGlCL ........................78
3.3.2. Molecular weight and dispersity ................................81
3.3.3. Thermal analysis data.................................................83
3.3.4. Effect of dichloromethane on cyclic oligomers
synthesis ………………………………………………………...87
3.4. CONCLUSIONS ........................................................................90
CHAPTER 4 .........................................................................................91
4. N-ACETYLCYSTEINE SIDE-CHAIN
FUNCTIONALIZATION OF POLY(GLOBALIDE-CO-Ε-
CAPROLACTONE) THROUGH THIOL-ENE REACTION.........91
4.1. INTRODUCTION ......................................................................92
4.2. MATERIAL AND METHODS ............................................94
4.2.1. Materials ......................................................................94
4.2.2. Poly(globalide-co-ε-caprolactone) synthesis using
supercritical carbon dioxide.. ………………………………….95
4.2.3. Thiol-ene functionalization of poly(globalide-co-ε-
caprolactone) with N-acetylcysteine ..........................................95
4.2.4. Characterization of the copolymer ............................96
 4.3. RESULTS AND DISCUSSION ........................................... 97
4.3.1. Synthesis of PGlCL-NAC by thiol-ene reaction ............ 97
4.3.2. Effect of functionalization on crystallinity and melting
temperature .............................................................................. 100
4.3.3. Effect of functionalization on the surface
characteristics of the polymer ................................................. 102
4.3.4. Antioxidant potential of the material ..................... 104
4.4. CONCLUSIONS ................................................................ 105
CHAPTER 5 ...................................................................................... 107
5. COVALENTLY BINDING OF PROTEINS TO POLYMERIC
NANOPARTICLES ...........................................................................107
5.1. INTRODUCTION ................................................................... 108
5.2. MATERIAL AND METHODS.......................................... 110
5.2.1. Materials ......................................................................... 111
5.2.2. Poly(globalide-co-ε-caprolactone) synthesis using
supercritical carbon dioxide as solvent .................................. 111
5.2.3. Poly(globalide-co-ε-caprolactone) nanoparticles
preparation ............................................................................... 112
5.2.4. Surface modification of poly(globalide-co-ε-caprolactone)
nanoparticles............................................................................. 112
5.2.5. Proton nuclear magnetic resonance (1H NMR) ........... 114
5.2.6. Ellman’s assay ................................................................ 114
5.2.7. Pierce assay ..................................................................... 114
5.2.8. Dynamic light scattering (DLS) .................................... 114
5.2.9. Zeta potential .................................................................. 114
5.2.10. Transmission electron microscopy (TEM) ................. 115
5.2.11. Flow cytometry - Fluorescence-activated cell sorting
(FACS) measurements ………………………………………..115
5.2.12. Fluorescence correlation spectroscopy (FCS) ............ 115
5.2.13. Cell culture.................................................................... 116
 5.2.14. Cell uptake: Flow cytometry and confocal laser scanning
microscopy ................................................................................. 117
5.3. RESULTS AND DISCUSSION ......................................... 117
5.3.1. PGlCL nanoparticle formation and functionalization with
NAC……………………………………………………………117
5.3.2. Covalent conjugation of PGlCL NPs with BSA ........... 119
5.3.3. Cell uptake ....................................................................... 122
5.4. CONCLUSIONS ................................................................. 124
CHAPTER 6 ....................................................................................... 125
6. CONCLUDING REMARKS ................................................... 125
6.1. SUGGESTIONS FOR FUTURE STUDIES ............................ 127
REFERENCES................................................................................... 129
APPENDIX A - KINETIC STUDY OF PGlCL ENZYMATIC
SYNTHESIS UNDER DIFFERENT KINDS OF STIRRING ....... 149
APPENDIX B – PGlCL DEGRADATION STUDY ....................... 157
 27

CONCEPTUAL DIAGRAM

“ENZYMATIC RING OPENING POLYMERIZATION OF

POLY(GLOBALIDE-CO-ε-CAPROLACTONE) BY MEANS OF
SUPERCRITICAL TECHNOLOGY AND POST
FUNCTIONALIZATION BY THIOL-ENE REACTIONS”

What?
Enzymatic synthesis of poly(globalide-co-ε-caprolactone) (PGlCL)
using supercritical carbon dioxide (scCO2) as solvent, and production
of PGlCL nanoparticles (NPs). Subsequent thiol-ene functionalization
of PGlCL and PGlCL NPs with N-acetylcysteine (NAC) and bovine
serum albumin (BSA), respectively.

Why?
 There is a growing interest in the use of biocompatible and
bioresorbable polymers in biomedical devices, aiming to
improve medical treatments efficiency and give more comfort
and practicality to the life of patients;
 Both globalide (Gl) and ε-caprolactone (CL) can be used to
produce polymers with very interesting properties for
biomedical and pharmaceutical applications;
 Through functionalization reactions it is possible to produce
tailored biomaterials for different purposes;
 The use of enzymes and scCO2 is considered green, and ideal
for biomedical and pharmaceutical applications, since it does
not leave toxic residues in the final product.

State of the art

 The use of scCO2 as solvent in reaction media has been
employed for the enzymatic ring opening polymerization (e-
ROP) of several monomers, including ε-caprolactone,
providing good results.
 The literature has few reports on the polymerization and
functionalization of globalide, but none using N-
28

acetylcysteine (NAC) or the protein bovine serum albumin

(BSA);
 The conjugation of proteins to NPs is usually done through
simple non-covalent methods. However, protein-NPs
conjugates produced by covalent methods are more uniform
and stable in a biological environment.
 This is the first study to propose a platform of biomaterials
based on PGlCL synthesized using green technologies.

Hypotheses
 Different Gl/CL feed ratios lead to different PGlCL molecular
weights and thermal properties;
 The use of dichloromethane (DCM) as cosolvent affects the
phase equilibria and the mass transfer in the system, causing
changes in PGlCL molecular weights;
 The formation of oligomeric cycles is affected by the use of
DCM;
 Thiol-ene functionalization of PGlCL with N-acetylcysteine
(NAC) reduces the crystallinity and hydrophobicity of the
material, besides conferring it an antioxidant character;
 It is possible to produce BSA-NPs covalent conjugates
functionalizing the surface of PGlCL NPs with BSA by thiol-
ene reaction;
 After covalent conjugation with BSA, NPs present reduced
uptake by immune cells (stealth properties).

Which steps?
 e-ROP of PGlCL with different Gl/CL feed ratios using pure
scCO2 and scCO2 + DCM;
 Characterization of the obtained PGlCL;
 Functionalization of PGlCL with NAC by thiol-ene reaction;
 Characterization of the functionalized copolymer PGlCL-
NAC;
 Preparation of PGlCL NPs by the solvent evaporation method;
 Functionalization of PGlCL NPs with BSA by thiol-ene
reaction;
 Characterization of BSA-NPs covalent conjugates and
conduction of cell uptake assays.
 29

Expected results
 To comprehend the effect of Gl/CL ratio and kind of solvent
on the phenomenology involved on PGlCL e-ROP and its
relation with the product final properties;
 To produce amorphous and hydrophilic functionalized
PGlCL-NAC copolymers, with antioxidant characteristic;
 To produce stable and irreversible BSA-NPs covalent
conjugates with stealth properties;
 To produce PGlCL copolymers with tunable properties, for
different biomedical applications.
 31

THESIS METHODOLOGICAL SEQUENCE FLOWCHART

 33

CHAPTER 1

1. INTRODUCTION

The use of biomaterials dates back to the ancient civilization, where

artificial eyes, teeth, and noses were found on Egyptian mummies, and
waxes, glue and tissues were used to reconstruct missing or defective
body parts by Indians and Chinese (BUCHANAN, 2008). The advances
in medicine, engineering and materials science have allowed the progress
on the development of new biomaterials. During the last decades, many
efforts were concentrated on the development of biocompatible and
bioresorbable/biodegradable polymers, used on medical applications, in
substitution to traditional materials, as metal and ceramics, as well as on
food and pharmaceutics area, on packaging and controlled drug delivery
devices.
The application of polymers in the human body requires a high purity
degree of the material, being free of any toxic residue. In this context, the
use of non-toxic biocatalysts as immobilized enzymes is gaining more
and more space in academia and industry. The synthesis of polymers by
enzymatic ring opening polymerization (e-ROP) is a promising
technique, since enzymes are considered green catalysts, being obtained
from animal, plant or microbiological sources (KOBAYASHI, 2010;
KOBAYASHI et al., 2001). In addition, enzymes have high chemo-
regio- and enantioselectivity, which makes them highly specific for
different reaction media (ZHANG et al., 2014).
In spite of the frequent use of enzymes, known as green catalysts,
most e-ROP studies use organic solvents such as toluene (ATES et al.,
2014; CLAUDINO et al., 2012; GEUS et al., 2005; KUNDU et al., 2011;
VAN DER MEULEN et al., 2008, 2011), which is a solvent of high
toxicity, that may leave residues in the final product (NIH, 2009; SMYTH
et al., 1969). scCO2 has been shown to be relatively inexpensive, non-
toxic, non-flammable (KUMAR; MADRAS; MODAK, 2004) and exhibit
transport properties that can accelerate mass transfer in enzymatic
reactions (OLIVEIRA; OLIVEIRA, 2000). In addition, scCO2 can be
easily separated from the final product by simply depressurizing the
system, generating free-solvent products, and being able to be reused in
the process. Supercritical carbon dioxide (scCO2) has been recently used
in several studies (COMIM ROSSO et al., 2013; LOEKER et al., 2004;
POLLONI et al., 2017; THURECHT et al., 2006; VENERAL, 2014) as
solvent in e-ROP of lactones and macrolactones, showing to be a
promising alternative to the use of organic solvents.
34

Poly(ε-caprolactone) (PCL) is a well-studied polymer due to its ease

of molding and manufacturing, and capacity of application in a wide
variety of biomedical devices. In addition, PCL exhibits mechanical and
kinetic degradation properties suitable for driving the growth of living
tissues and for controlled release of drugs contained within their matrix
(WOODRUFF; HUTMACHER, 2010). PCL has a high crystallinity,
which makes its bioresorption by the organism slower (2-3 years).
Another important fact is that PCL-manufactured drug release devices
have Food and Drug Administration (FDA) approval and mark
registration (CE Mark), which allows a faster entry route to the market
(WOODRUFF; HUTMACHER, 2010).
Polyglobalide (PGl) is a biocompatible and non-toxic polyester
(VAN DER MEULEN et al., 2008), obtained by polymerization of the
monomer globalide (Gl). Globalide is typically used in the fragrances and
cosmetics industries, since it possesses musky aroma and a quality of
slowly losing its perfume (VAN DER MEULEN et al., 2008). Globalide,
also known as 15-pentadecenolide or oxacyclohexadecen-2-one, is
derivative from hydroxy fatty acids, being a 16-membered macrolactone,
with a double bond in its structure. In comparison to the smaller lactones,
such as ε-caprolactone (CL) for example, polymerization via chemical
catalysis of macrolactones proceeds slowly and gives low molecular
weight polymers (FOCARETE et al., 2001; NOMURA; UENO; ENDO,
1994). However, enzymatic ring-opening polymerization has been shown
to be very efficient (FOCARETE et al., 2001; GEUS et al., 2010;
POLLONI et al., 2017). The interest in polyesters derivative from
macrolactones comes from the excellent mechanical and chemical
properties of their homopolymers, which are interesting features for
application in biomedical devices. However, in general, the
homopolymers of macrolactones are poorly biodegradable/bioresorbable
due to their semi-crystalline structure and high hydrophobicity (VAN
DER MEULEN et al., 2011). This fact makes it difficult to apply PGl in
cases where bioresorption is desired. However, biocompatible and non-
bioresorbable materials can also be applied in permanent applications
such as bone implants (VAN DER MEULEN et al., 2008). In addition,
the double bond present in globalide structure enables the attachment of
various interest molecules to the polymer chain by thiol-ene reaction. The
functionalization of the polymer has the ability of giving the material
completely new features, depending on the type of desired application
(ATES et al., 2014; ATES; THORNTON; HEISE, 2011).
The development of a copolymer derived from a lactone (ε-
caprolactone) and an unsaturated macrolactone (globalide) is a very
 35

functional alternative for biomedical applications. By the synthesis of this

copolymer, it becomes possible to add new properties to the well-known
PCL through the introduction of globalide units, which are able to be
functionalized. Variables of the copolymer synthesis, such as the ratio
between the monomers (unsaturation density) and the type of molecule to
be attached to the main chain can be modulated. It means that, by these
simple changes, it can be produced a wide range of engineered materials
with different characteristics: tailored bioresorption behavior, enhanced
mechanical properties, compatibility with specific types of tissue and
optimized cell uptake (in the case of nanomaterials) are some examples.
This work proposed the synthesis and functionalization of the
copolymer poly(globalide-co-ε-caprolactone) (PGlCL), produced using
clean technologies. PGlCL and PGlCL nanoparticles (NPs) were
functionalized respectively with N-acetylcysteine (NAC) and bovine
serum albumin (BSA), aiming to obtain biomaterials with enhanced
properties for application in a biological environment. Besides, this work
also approached the study of the process phenomenology involved in the
e-ROP of PGlCL in scCO2. This is an original research that started the
design of a PGlCL based biomaterial platform. This study should
contribute to the development of novel engineered biomedical devices
that improve healthcare and promote more effective medical treatments.
36
 37

1.1. OBJECTIVES

1.1.1. General objective

To develop, using clean technologies, a copolymer composed of ε-

caprolactone and globalide units and functionalize it with different
molecules, obtaining materials with enhanced properties for biomedical
applications.

1.1.2. Specific objectives

a) To obtain PGlCL copolymers by the use of scCO2 and scCO2 +

DCM as solvents in different Gl/CL feed ratios, and evaluate the
effect of these process variables in PGlCL molecular weight
(Mn), dispersity (Đ), melting temperature (Tm), degree of
crystallinity (Xc), final Gl/CL copolymer composition and
formation of cyclic oligomers;
b) To select process conditions that yield PGlCL samples with the
most interesting characteristics, in terms of biomedical
applications, for subsequent functionalization by thiol-ene
reactions;
c) To functionalize PGlCL copolymers with N-acetylcysteine
(NAC) by thiol-ene reaction and characterize the functionalized
copolymers regarding the number of functionalizations, Tm, Xc,
contact angle with water and antioxidant potential;
d) To produce PGlCL nanoparticles (NPs), functionalize its surface
with bovine serum albumin (BSA) by thiol-ene reaction, and
characterize the uncoated NPs and BSA-NPs covalent conjugates
regarding the particle diameter, stability, occurrence of the
covalent attachment, morphology and evaluate cell uptake
behavior.
 39

CHAPTER 2

2. LITERATURE REVIEW

2.1. BIOCOMPATIBLE, BIODEGRADABLE AND

BIORESORBABLE POLYMERS

The term biocompatibility, according to Williams (2008) refers to

the ability of a biomaterial to perform its desired function with respect to
a medical therapy, without eliciting
any undesirable local or systemic effects in the recipient or beneficiary of
that therapy but generating the most appropriate beneficial cellular or
tissue response in that specific situation and optimizing the clinically
relevant performance of that therapy.
The American Standard for Testing and Materials states that
biodegradable polymers are those in which degradation results primarily
from the action of microorganisms such as naturally occurring bacteria,
fungi and algae (ASTM STANDARD D883-11, 2011).
On the other hand, bioresorbable polymers are those that show
degradation through size reduction and are resorbed in vivo, for example:
Materials that are eliminated by the metabolic routes of the organism.
Bioresorption is a concept that reflects the total elimination of the
material, and its by-products degradation (low molecular weight
compounds), without residual side effects. The word "bioresorption" is
used when the elimination is complete (VERT et al., 1992).
Biodegradable and bioresorbable polymers currently have two main
applications. The production of biomedical polymers that contribute to
patient care; and their use as green polymers that keep the environment
clean. Most of the biodegradable and bioresorbable polymers available
are used for one or other of the two purposes, but some of them are
applicable to both (IKADA; TSUJI, 2000).

2.2. POLYMERS FOR BIOMEDICAL APPLICATIONS

The key to the success of polymer-based biomaterials is in the fact

that they can be synthesized with a wide range of properties and
functionality in a relative easy and inexpensive way. Because most of the
structural living tissues (like bone, ligament, connective tissue, etc.) are
macromolecular composites (combinations of different macromolecules
and possible other components), synthetic polymeric composites are an
40

attractive group of materials for development of new, tailor-made

biomaterials for replacement, support, augmentation or fixation of living
tissues (TÖRMÄLÄ; POHJONEN; ROKKANEN, 1998). In many cases
the tissues need only the temporary presence of a biomaterial to support,
augment or replace tissues or to guide their regrowth. In such cases
bioresorbable polymeric materials are very interesting alternatives,
capable of being bioresorbed by the human body when, after healing, the
biomaterial presence is no longer necessary (TÖRMÄLÄ; POHJONEN;
ROKKANEN, 1998).
For such applications, the use of devices such as scaffolds are very
interesting. Scaffolds are porous or semiporous materials that have
suitable properties for the growth and interaction with cells of living
organisms. Scaffolds have the function of mimetizing the extracellular
matrix of the native tissue and providing a suitable environment to
promote cellular migration and adhesion through the pores, repairing the
native tissue (COSTA-PINTO; REIS; NEVES, 2011).
Nanoparticles are another important type of biomedical device.
From the biomedical point of view, the main interest in nanoparticles
comes from the fact that they are small enough to interact with cellular
machinery and reach difficult access targets in the human body
(MAHMOUDI et al., 2011). There are many applications of nanoparticles
in medical treatments, such as nanocarriers for drug delivery systems,
magnetic hyperthermia for tumor treatment and nanoparticle uses with
diagnostic purposes.
For drug delivery applications, the main idea is to avoid systemic
drug effects and guide the medicament to the desired location in the body.
The nanocarrier also have the function of protect the drug from
degradation before it reaches the target location, so the circulation time in
the blood stream can be prolonged (WINZEN, 2015). On the other hand,
magnetic hyperthermia treatment treats tumors by heating them to above
42 °C to destroy the cancerous cells. The benefit of this technique over
chemotherapy is that it specifically targets the tumor and does not damage
the surrounding healthy tissue (MCNAMARA; TOFAIL, 2017). The
application of nanoparticles in for diagnosis aims to visualize pathologies
and to improve the understanding of important (patho-) physiological
principles of various diseases and disease treatments (BAETKE;
LAMMERS; KIESSLING, 2015). For this application, diagnostic
nanoparticles should possess a good and efficient delivery to the target
site and should exhibit highly specific binding and internalization
capabilities (BAETKE; LAMMERS; KIESSLING, 2015). Nanoparticles
can be coupled with radiolabels and detected by positron emission
 41

tomography (PET) or single photon emission computed tomography

(SPECT) and contrast agents can be encapsulated and used to increase the
sensitivity of imaging methods, such as magnetic resonance imaging
(MRI) (WINZEN, 2015).

2.2.1 Interaction between polymers and biomolecules in a biological

environment

2.2.1.1. Interaction between cells and polymer surfaces for tissue

repairing

In a biological environment, when a cell first contacts a polymer

surface, cells receptors (specially integrins) primarily interact with the
polymeric devices via chemical groups (ligands) on the material surface.
In case of materials that mimic extracellular matrix (such as scaffolds or
guided tissue regeneration membranes for tissue repairing), integrin-
ligand binding is followed by the extension of pseudopodia from the cell
body, starting cell adhesion process. The adhesive bond is defined as the
sum of non-covalent interactions, e.g., hydrogen bonds, electrostatic
interactions, van der Waals forces, dipole-dipole interactions between two
macro molecules (MCEVER; ZHU, 2010). As the cell begins to flatten
against the substrate, it forms additional bonds with the extracellular
matrix, rearranging its cytoskeleton to form focal adhesions
(BURRIDGE, 1988). To spread, the cell must exert force against the
surface to allow pseudopodial extension and the stabilization of new
bonds as its contact area expands (REINHART-KING; DEMBO;
HAMMER, 2005). Figure 2.1 illustrates the cell adhesion stages in a
surface.
Figure 2.1 - Stages of cell adhesion in a surface.

Source: Adapted from Khalili and Ahmad (2015).

42

2.2.1.2. Interaction between cells and polymeric nanoparticles

Nanomaterials are usually administered intravenously to be

distributed over the body though the bloodstream (DUNCAN, 2006;
WINZEN, 2015). Blood plasma is the non-cellular component of blood
and consists of 3,700 identified proteins that are capable of interacting
with the nanomaterial surface. The adsorption of this proteins to the
nanoparticle surface gives rise to the so called “protein corona” (LYNCH;
SALVATI; DAWSON, 2009; MONOPOLI et al., 2011). The formed
corona creates a new interface and gives a “new identity” to the
nanoparticle, which influences the recognition of the nanomaterials by
the cells and determine its biological fate (LYNCH; SALVATI;
DAWSON, 2009; WINZEN, 2015).
Typically, attractive forces (some generic and some highly specific,
such as receptor–ligand interactions) will lead to the binding of the
complex particle-protein to the cell surface. The proteins present in the
protein corona that remain adsorbed on the particle surface for longer
times will affect, indeed mediate, the nanoparticle–cell interactions. The
scheme in Figure 2.2 illustrate this interactions.
 43

Figure 2.2 - Interactions between cells and protein-nanoparticle complexes.

Source: Adapted from Caracciolo (2013).

The composition of the protein corona strongly influences the

cellular uptake and biodistribution of the nanoparticles (EHRENBERG et
al., 2009; SCHÖTTLER et al., 2016). For example, a protein corona rich
in opsonins like fibrinogen and Immunoglobulin G (IgG), etc. is believed
to promote phagocytosis and removal of the NPs
from the bloodstream (ISHIDA; HARASHIMA; KIWADA, 2001;
OWENS; PEPPAS, 2006), while binding of dysopsonins like
apolipoproteins, promotes prolonged circulation time in blood
(CARACCIOLO et al., 2015; MONOPOLI et al., 2011). Proteins that are
not recognized by any receptors on the cell will make the particle less
“attractive” to the cell. After all, cells associate and take up only the
proteins they need (LYNCH; SALVATI; DAWSON, 2009).
44

2.3. POLYESTERS

In recent years, environmental concerns have led to a renewed

interest in biodegradable polyesters as an alternative to commodity
plastics. Since ester linkages are frequently encountered in nature it is
reasonable to assume that at least a subset of the polyester family will be
environmentally degradable (MILETIĆ; LOOS; GROSS, 2010).
Polyesters are versatile materials, being the lead candidate in the
biomedical and pharmaceutical applications. Its good mechanical
properties, hydrolysability, biodegradability and biocompatibility make
polyesters suitable for a variety of medical applications (e.g. as
prosthetics, artificial skin, etc.) and in pharmaceutical industries for drug
delivery applications.
The biodegradation and the bioresorption of polyesters occur
initially through the hydrolysis of the ester bonds, resulting in the
decrease of the polymer molecular weight, but without the loss of mass.
This initial degradation occurs until molecular weight less than 5,000 Da,
at which point cellular degradation takes over. The final degradation and
resorption of the material proceed through cells of the organism such as
macrophages, lymphocytes and neutrophils (PACHENCE; BOHRER;
KOHN, 2007). This second step of degradation is characterized by mass
loss, molecular weight reduction, structural changing and loss of
mechanical properties as tensile strength, compressive strength and
hardness (ATHANASIOU et al., 1998; BARBANTI; ZAVAGLIA;
DUEK, 2005).
Different factors affect the degradation kinetics of polyesters, such
as: chemical composition and configurational structure; processing
history; molecular weights (Mn and Mw); dispersity (Đ); environmental
conditions; mechanical stress; crystallinity; chain orientation; distribution
of chemically reactive compounds within the matrix; additives; overall
hydrophilicity and morphology (porosity, for example); and size of the
polymeric device (DUNN; CAMPBELL; MARRA, 2001; HEIDEMANN
et al., 2001).
The physical and biodegradable properties of aliphatic polyesters
can be adjusted by changing the structure and composition of the main
polymer chain, chain flexibility, presence of polar groups, molecular
weight, crystallinity and orientation, in order to match the material to a
given application (ALBERTSSON; VARMA, 2003; COULEMBIER et
al., 2006).
 45

2.3.1. Poly(ε-caprolactone)

Poly(ε-caprolactone) (PCL) is a well-studied polymer, derivative

from the monomer ε-caprolactone (also known as 2-oxepanone), being
one of the first polymers synthesized by the Carothers group in 1930
(VAN NATTA; HILL; CAROTHERS, 1934).
During the resorbable-polymer-boom of the 1970s and 1980s, PCL
and its copolymers were used in a number of drug-delivery devices.
Attention was drawn to these polymers owing to their numerous
advantages over other polymers in use at that time (WOODRUFF;
HUTMACHER, 2010). PCL has mechanical properties, permeability and
degradation kinetics suitable to enable controlled release of drugs
contained within its matrix, besides having the ability of being fully
excreted from the body once bioresorbed. (WOODRUFF;
HUTMACHER, 2010). PCL biodegrades within several months to
several years, depending on the molecular weight, the degree of
crystallinity of the polymer, and the conditions of degradation (LABET;
THIELEMANS, 2009).
In the 1990s and the 2000s, there was the birth of a new area, called
“tissue engineering”. The arising of this area generated many studies on
PCL, due to its superior rheological and viscoelastic properties over many
other resorbable polymers. These properties make PCL easy to
manufacture and manipulate into scaffolds (three-dimensional structures
designed to support cell growth, allowing the regeneration of tissues) with
controlled pore size, enabling appropriate tissue growth (WOODRUFF;
HUTMACHER, 2010). PCL can be used in a wide range of scaffold
fabrication technologies, and its relatively inexpensive production routes
(compared with other aliphatic polyesters) is hugely advantageous
(WOODRUFF; HUTMACHER, 2010).
PCL and its copolymers have demonstrated excellent properties such
as high flexibility, biodegradability and biocompatibility, as well as non-
toxicity. These favorable attributes give PCL the characteristic of being
the most attractive and useful class of biodegradable polyesters (PITT;
MARKS; SCHINDLER, 1980). PCL is a very interesting polymer to be
applied on drug delivery devices/long-term vaccines and suture materials,
since it degrades at a slower rate (2-3 years) than polyglycolides and
polylactides for example, besides having high permeability to many drugs
(NAIR; LAURENCIN, 2006; WOODRUFF; HUTMACHER, 2010).
Some drug delivery devices made from PCL have FDA (Food and Drug
Administration) approval and trademark registration (CE Mark), which
46

allows a faster way out to the market (WOODRUFF; HUTMACHER,

2010).
There are several examples of commercial products based on PCL:
the long-term contraceptive device, CapronorTM, that could yield a zero-
order release of levonorgestrel for over one year (NAIR; LAURENCIN,
2006); the ε-caprolactone/glycolide copolymer suture fibers called
MonocrylTM; a multiblock copolymer composed of bioresorbable ε-
caprolactone, glycolide, lactide and polyethylene glycol, used as a vehicle
for drug delivery, called SynBiosysTM (NAIR; LAURENCIN, 2006); a
bioresorbable implant that is used for covering trephination burr holes in
neurosurgery, OsteoplugTM (LOW et al., 2009); and a thermoplastic
synthetic polymer based root canal filling material made of 25-40% of
poly(ε-caprolactone), called ResilonTM (HIRAISHI et al., 2007; JIA,
2005; JIA; ALPERT, 2003; JIA; TROPE; ALPERT, 2005).
PCL is a semi-crystalline polymer, which has glass transition
temperature of -60 °C and melting point ranging between 59 and 64 °C,
depending upon its crystalline nature of PCL (SINHA et al., 2004). At
room temperature, PCL is highly soluble in chloroform, dichloromethane,
carbon tetrachloride, benzene, toluene, cyclohexanone and 2-
nitropropane; slightly soluble in acetone, 2-butanone, ethyl acetate,
dimethylformamide and acetonitrile; and insoluble in alcohols, petroleum
ether, diethyl ether and water (SINHA et al., 2004). Bordes et al. (2010)
determined the solubility of PCL of different molecular weights in 99
analytical grade solvents, and determined in which solvents PCL is
soluble, partially soluble or insoluble, at room temperature.
Industrially, high molecular weight PCLs are synthesized by ring
opening polymerization of ε-caprolactone (CL) monomer, which can be
obtained by the traditional Baeyer-Villiger reaction using cyclohexanone.
In the last decades, another route has been developed to produce CL
monomer. This route is a ‘‘green synthesis’’, which requires hydrogen
peroxide as oxidizer and zeolite/tin catalysis (WILSON, 2001 apud
COULEMBIER et al., 2006). This process looks very promising since the
waste product is water and the tin-impregnated zeolite is an ecologically
friendly catalyst. Figure 2.3A presents the structure of the monomer ε-
caprolactone, while Figure 2.3B presents the repeating unit structure of
PCL.
 47

Figure 2.3 - (A) Structure of the monomer e-caprolactone; (B) Structure of PCL
repeating unit.

Source: Adapted from Labet and Thielemans (2009).

2.3.2. Polyglobalide

Polyglobalide (PGl) has been recently studied as aliphatic polyester

on biomedical application (ATES et al., 2014; ATES; HEISE, 2014;
CLAUDINO et al., 2012; VAN DER MEULEN et al., 2008, 2011). Until
then, its monomer, globalide (Gl) (Figure 2.4), also known as 15-
pentadecenolide or oxacyclohexadecen-2-one (IUPAC name), was most
often used as a fragrance in the perfumery industry because of its musky
odor and its slow loss of aroma. This way, very few studies on synthesis
of polyglobalide are found in literature.

Figure 2.4 - (A) Structure of the monomer globalide; (B) Structure of the
repeating unit of PGl.

Source: Van der Meulen et al. (2008).

In recent works on synthesis and functionalization of PGl, it was

possible to verify that it is a biocompatible and non-toxic polymer (ATES
et al., 2014; ATES; HEISE, 2014; ATES; THORNTON; HEISE, 2011;
VAN DER MEULEN et al., 2008,VAN DER MEULEN et al., 2011). As
well as other polymers derived from macrolactones, PGl has the
48

characteristic of being a very hydrophobic and semi-crystalline material.

These characteristics are very similar to the characteristics of
polyethylene, one of the most versatile polymers used in the industry.
Despite of being very interesting for a large number of applications, these
characteristics generate limitations for biomedical applications, where
polymer bioresorption is desired (ATES; HEISE, 2014). In their work,
van der Meulen et al. (2008) observed that there was no hydrolytic or
enzymatic degradation of PGl over 100 days, and concluded that the high
crystallinity and hydrophobicity are responsible for this behavior.
These limitations can, however, be overcome by the
functionalization and/or crosslinking of the double bonds present in the
PGl by thiol-ene “click” reaction. Functionalization enables the
introduction of a wide range of chemical groups, changing the
characteristics of the polymer as desired (ATES et al., 2014; ATES;
HEISE, 2014; ATES; THORNTON; HEISE, 2011; CLAUDINO et al.,
2012; VAN DER MEULEN et al., 2011).
PGl has a glass transition temperature lower than -60 °C and a
melting temperature ranging from 46 to 50 °C (VAN DER MEULEN et
al., 2008). According to the literature, PGl showed to be soluble in
dichloromethane, toluene, tetrahydrofuran (THF) and chloroform (ATES;
THORNTON; HEISE, 2011; VAN DER MEULEN et al., 2008). PGl, as
well as other lactones and macrolactones, can be synthesized by ring
opening polymerization of its monomer, globalide. Studies of the ROP of
cyclic esters have primarily focused on the small-rings (glycolide, lactide,
butyrolactone, valerolactone, etc.) and medium-rings (ε-caprolactone
(CL), 1,5-dioxepan-2-one (DXO), etc.) rings, which have angular or
transannular strains. Large-rings, on the other hand, are defined as a ring
containing 12 or more atoms. There is no upper limit to this number,
although very few reports are available on lactones with more than 16
atoms. This way, globalide can be considered a large-ring lactone. Unlike
the small and medium-rings, which are considerably strained, these large
rings are relatively strain-free, which ease ROP reactions
(ALBERTSSON; VARMA; SRIVASTAVA, 2009).
Most studies involving PGl synthesis used enzymes as catalyst on
ROP reactions, since the use of conventional chemical catalysts on
macrolactones ROP generates only oligomeric, low molecular weight and
low yield materials (ALBERTSSON; VARMA; SRIVASTAVA, 2009;
BISHT et al., 1997). Besides, the reaction proceeds much faster, under
mild conditions, and yields materials free of any metal contamination,
which can be considered a great advantage for biomedical applications in
 49

view of the toxicity of many metal containing catalysts (ALBERTSSON;

SRIVASTAVA, 2008).

2.3.3. Copolymers of lactones and unsaturated macrolactones

Many different copolymers based on all kinds of lactones were made

during the last decade. However, the study of copolyesters based on
lactones and unsaturated macrolactones is little reported until now. The
interest in polyesters derived from macrolactones stems from the
excellent mechanical properties of the polymers and the fact that
degradation products should be harmless, as they resemble fatty acid
derivatives (ATES; HEISE, 2014; VAN DER MEULEN, 2010; VAN
DER MEULEN et al., 2008). However, homopolymers of macrolactones
are non-bioresorbable under physiological conditions due to their semi-
crystalline morphology and high hydrophobicity (VAN DER MEULEN
et al., 2008, 2011).
These problems can be overcome by the introduction hydrophilic or
a bulky comonomer to reduce the crystallinity. At the same time, the
transition from a semi-crystalline to an amorphous or low-melting
temperature material may also requires a post-synthesis crosslinking step,
in order to produce copolymers able to be processed into stable shapes for
biomedical applications (VAN DER MEULEN, 2010; VAN DER
MEULEN et al., 2011). The main chain unsaturation provides a
straightforward functionality for crosslinking, since simple free radical
reaction can be employed.
Van der Meulen et al. (2011) investigated the synthesis of
copolymers from unsaturated macrolactones, aiming to increase the
biodegradability of the materials. It was investigated the
copolymerization with smaller biocompatible comonomers like 1,5-
dioxepan-2-one (DXO) and 4-methyl caprolactone (4MeCL), and their
influence on the copolymer properties. DXO and 4MeCL typically
generates amorphous homopolymers. Thermal investigation showed that
the melting point was lowered upon incorporating the comonomers, and
the decrease was dependent on the comonomer ratio. The authors also
showed that crosslinking of the polymer can be performed using dicumyl
peroxide, generating completely degradable crosslinked polyesters.
Preliminary degradation tests confirmed that the crosslinked copolymers
are enzymatically degradable and that the incorporation of hydrophilic
comonomers like DXO enhances degradation through crystallinity and
hydrophobicity reduction.
50

In their work, Claudino et al. (2012) synthesized by e-ROP with

different monomer ratios of poly(globalide-co-ε-caprolactone)
copolyesters. The aim of the work was to investigate if thiol-ene
crosslinking by UV curing could be used with unsaturated copolymers
derived from macrolactones to provide films with efficient curing and
high conversions. The authors were interested in determining the
relationship between functional density (related to the amount of
globalide) and the resulting thiol-ene conversion. It were obtained
copolymers of molecular weight up to 23,000 Da (Mn). All compositions
tested (globalide content ranging from 10% to 100%) were successfully
crosslinked by equimolar reaction of “ene” groups and thiol groups from
trimethylolpropane tris(3-mercaptopropionate), affording fully
transparent amorphous elastomeric materials with different thermal and
viscoelastic properties. The authors concluded that the addition of ε-
caprolactone to globalide is a good way to increase the mobility of the
chains while providing high thiol-ene conversions (>80%, based in the
residual unsaturation after curing) and maintaining the cure behavior
irrespective of the functional density.

2.4. ENZYMES ON POLYESTER SYNTHESIS

In nature, enzymes can be divided into six classes: oxide-reductases,

transferases, hydrolases, lyases, isomerases and ligases. Hydrolases are
the most-investigated enzymes for in vitro synthesis. This class includes
lipase, which is used for the hydrolysis of fatty esters in nature, being
particularly interesting for polymer synthesis. Lipases are known to
catalyze reactions in organic media, since they are active on the water-fat
interface in cells. Moreover, lipases do not require any co-catalyst, and
proved to be the most efficient enzyme for in vitro polyester synthesis.
Lipase can be used for polycondensation and poly-transesterification
reactions, ring-opening polymerizations, and polymer modifications
reactions (GEUS, 2007; MILETIĆ; LOOS; GROSS, 2010).
The most common lipase-catalyst used for polyester synthesis is
Candida antarctica lipase B (CALB). The immobilized CALB catalyst
that has been primarily used is Novozym® 435, manufactured by
Novozymes (Bagsvaerd, Denmark). Novozym 435 consists of CALB
physically adsorbed within the macroporous resin Lewatit VPOC 1600
(poly[methyl methacrylate-co-butyl methacrylate], supplied by Bayer)
(ZHANG et al., 2014). CALB is composed of 317 amino acids and has a
molecular weight of 33,000 Da. This lipase belongs to the alpha/beta
hydrolases family, and was initially obtained from the yeast Candida
 51

antarctica (UPPENBERG et al., 1994). Novozym 435 is a highly

versatile catalyst with thermosetting immobilization and activity on a
wide variety of substrates in various organic solvents (GEUS, 2007).
The high cost of the enzymes for a long time was considered as a
barrier to their use in commercial processes. However, technological
advances that include the study of the use of solvent-tolerant lipases and
immobilized lipases, allowing reuse of the catalyst, have made possible
the development of low cost systems (FUKUDA; KONDO; NODA,
2001)
Enzymatic polymerizations are a promising strategy under study by
many groups throughout the world to develop environmental friendly
processes for polyester synthesis (MILETIĆ; LOOS; GROSS, 2010).

2.5. ENZYMATIC RING OPENING POLYMERIZATION (e-ROP)

The most commonly used route for the production of PCL, PGl, and
its copolymers, is the ring-opening polymerization (ROP), since it is able
to give polymers with high molecular weight and low dispersity
(CLAUDINO et al., 2012; GEUS, 2007; LABET; THIELEMANS, 2009;
PENCZEK et al., 2007; THURECHT et al., 2006; VAN DER MEULEN
et al., 2008, 2011). This reaction can be performed by a wide variety of
catalysts, usually based on tin, zinc and aluminum. However, residues of
organometallic catalysts are not tolerated in biomedical applications due
to their toxicity (LI et al., 2011). In this context, the use of biological
catalysts, such as enzymes, has been gaining more space, being an
efficient alternative to avoid the toxicity problem (VENERAL, 2014).
The first e-ROP of lactones was presented in 1993 when Knani,
Gutman and Kohn (1993) and Uyama and Kobayashi (1993)
independently published works on e-ROP of ε-caprolactone using a lipase
enzyme. Since then, many different lipases have been studied in e-ROP,
transesterification and polycondensation reactions, such as lipases from
Candida antarctica, Candida cylindracea, Candida rugosa,
Pseudomonas fluorescens, Pseudomonas cepacia and from porcine
pancreas (ALBERTSSON; SRIVASTAVA, 2008). Among these, lipase
from Candida antarctica B is the most applied enzyme on e-ROP of
lactones and macrolactones (ZHANG et al., 2014).
Lipase catalyzes ester bond hydrolysis by means of a catalytic triad,
composed of a nucleophilic serine residue activated by a hydrogen bond
in relay with histidine and aspartate or glutamate. This catalytic triad is
responsible for the ROP of lactones (ALBERTSSON; SRIVASTAVA,
2008).
52

Figure 2.5 presents e-ROP mechanism proposed in details by Geus

(2007), based on the works of Henderson et al. (1996) and Uyama et al.
(1995). The catalytic triad in the active site of a lipase is electronically
stabilized. In the e-ROP mechanism, a cyclic ester function as substrate
molecule and undergoes a nucleophilic attack from the primary alcohol
group of serine in the active site of the enzyme (I), leading to the
formation of an intermediate specie (II). In the transesterification, the
original alkoxy group (R2-OH) is then released (III), forming the enzyme-
activated monomer species (EAM) (III). However, in the case of an e-
ROP the alkoxy group will not be released, since the lactones are cyclic.
Subsequently, a nucleophile (a primary alcohol, water, amine or thiol
group) (R3-OH) can attack this EAM-species (III) forming a new
intermediate specie (IV). Then the final product, a short chain polymer is
released and the enzyme is regenerated (GEUS, 2007; VENERAL, 2014).

Figure 2.5 - Enzymatic transesterification mechanism proposed by Geus (2007).

Source: Geus (2007).

The nucleophile R3-OH can be considered as the initiator of the

polymerization, and it is necessary to regenerate the enzyme and create
the (ring-opened) product. This initiator can be water or any other
nucleophile (such as alcohols amines and thiols). The ring-opened
 53

product (oligomer) formed after a catalytic cycle has in its structure a

hydroxyl molecule on one side and the functional initiator on the other.
In this way it is possible to identify the initiator that gave origin to the
molecule (GEUS et al., 2005).
Characteristics such as molecular weight control and
functionalization of the terminal chain can be obtained by the addition of
different nucleophiles in the polymerization, replacing the water in the e-
ROP (KNANI; GUTMAN; KOHN, 1993). However, since the enzyme
needs water to maintain its structure, the use of other nucleophiles results
in products where fractions of the chains produced were initiated by
water, resulting in a mixture of chains with different terminal groups
(HENDERSON et al., 1996).
The reaction propagation stage occurs by a nucleophilic attack of the
hydroxyl molecule present in the oligomer chain (formed in the previous
cycle), to the EAM complexes (ALBERTSSON; SRIVASTAVA, 2008).
New complexes, called enzyme-activated polymer chain (EAPC) are
generated, resulting in the addition of one more monomeric unit in the
polymer chain (as final product of one more cycle). In this way, the
process occurs successively.
During the propagation step via e-ROP, different products can be
obtained. Johnson, Kundu and Beers (2011) described the kinetic
pathway of the polymerization of lactone ε-caprolactone using the
commercial lipase Novozym 435 as a biocatalyst. The authors concluded
that the proposed model is applicable for lactones, macrolactones, lactams
and cyclic carbonates. The following scheme (Figure 2.6) was made by
Johnson, Kundu and Beers (2011), based on the reaction pathways
proposed by Mei, Kumar and Gross, (2002) and shows the kinetic
modeled reactions in PCL synthesis by e-ROP in four steps.
All reaction steps require enzyme to be present, and include the
reaction for ε-caprolactone ring-opening, enzyme and polymer chain
interactions with water, chain propagation or degradation, and cyclic
formation.
The first chain conformation possible to be obtained is the polymer
chain activated by the enzyme, which is a PCL chain bounded to the
active site of the enzyme (EAPC). The second consists of a PCL linear
chain in solution. The third possibility is the formation of cyclic chains,
which are polymer chains in solution that form a cyclic ring from an
enzyme-activated polymer chain.
54

Figure 2.6 - Reaction pathways for PCL synthesis by e-ROP.

Source: Adapted from Johnson et al. (2011).

The first reaction (step 1) involved in the mechanism comprises the

formation of the enzyme-activated polymer chain (EAPC) complex. This
step is considered irreversible, as ring strain should prevent the ring from
reforming. The linear chain formed is more thermodynamically stable
when compared to the cyclic ring. The product generated is an EAPC with
an ester bond at the active site (JOHNSON; KUNDU; BEERS, 2011).
In the second reaction (step 2), a water molecule is consumed in the
EAPC complex, breaking the ester bond between the polymer chain and
the active site of the enzyme, which leads to the formation of a polymer
chain in solution and the regeneration of the active site. In the reverse
pathway, occurs the formation of the active site-polymer chain ester bond
(EAPC), generating a water molecule (JOHNSON; KUNDU; BEERS,
2011).
Enzymatic polycondensation reactions (step 3) may occur when an
EAPC reacts with the hydroxyl group from a linear chain in solution. In
this case, occurs the regeneration of the active site and formation of chains
with high molecular weights. In the reverse reaction, the enzymatic
degradation occurs when the active enzyme site cleaves an ester bond at
a random position of a PCL chain, reforming the ester bond with the
enzyme active site. This results in a lower molecular weight linear chain
and an EAPC (JOHNSON; KUNDU; BEERS, 2011).
In the last reaction (step 4), the formation of cyclic compounds
occurs when the polymer chain backbites on itself at the active site,
forming an ester bond with itself and generating a cyclic chain and
regenerating the enzyme active site. The reverse reaction consists in the
opening of the cyclic ring, similar to step 1, but with a larger ring. This
 55

reaction results in the formation of an EAPC (JOHNSON; KUNDU;

BEERS, 2011).

2.5.1. The role of water on e-ROP

In e-ROP reaction, the control over the amount of water plays a

crucial role, having very strong influence in the size of the polymer chains
obtained (MEI; KUMAR; GROSS, 2002). When in excess, the water
content can affect the polymerization, causing the degradation of polymer
chains and reducing its molecular weight (GEUS, 2007; SIVALINGAM;
CHATTOPADHYAY; MADRAS, 2003).
In their work, Thurecht et al. (2006) and Mei et al. (2003) studied
the effect of different amounts of water as an initiator of poly(ε-
caprolactone) e-ROP using the enzyme Candida antarctica B (CALB).
The authors observed an inverse relationship between the initiator content
and the molecular weight of the polymers. At higher water concentrations,
it were obtained polymers of lower molecular weight, due to the
hydrolysis of the aliphatic ester bonds of the poly(ε-caprolactone) chains
(VENERAL, 2014).
Geus (2007) performed poly(ε-caprolactone) e-ROP reactions,
initiated by HEBI, using dried and non-dried Novozym 435 as catalyst.
The author noted that after 120 minutes, monomer conversion was almost
identical using both dried and non-dried enzymes. However, the evolution
of monomer consumption during the reaction was different. When more
water was present, the initial rate of monomer consumption was
significantly higher. At the same time, the observed Mn was lower, which
can be expected, since water will also act as initiator, together with HEBI.
Besides, the excess of water probably was favoring the degradation of the
polymeric chains, reducing its molecular weight.
Besides acting as a nucleophile, creating carboxylic acid end-
functionalized polymer chains, water has great influence in the
conformation and flexibility of the enzymes, ensuring its stability
(ADLERCREUTZ, 1991; SIVALINGAM; CHATTOPADHYAY;
MADRAS, 2003), which means it cannot be completely removed from
the enzyme.
Enzymes require a small amount of water to retain their active three-
dimensional conformation even when they are covalently attached to a
support. Water also contributes to the structural integrity, active site
polarity and protein stability, and may also limit the solubility of
hydrophobic substrates around the enzyme (DALLA-VECCHIA;
NASCIMENTO; SOLDI, 2004).
56

2.6. E-ROP REACTIONS USING SUPERCRITICAL CARBON

DIOXIDE AS SOLVENT

A supercritical fluid (Figure 2.7) can be defined as a substance that

is under conditions of temperature and pressure above its critical values
(above the critical point) and has intermediate properties between a gas
and a liquid (BRUNNER, 2004).

Figure 2.7 - Carbon dioxide phase diagram in the pressure-temperature plane.

Source: Brunner (1994).

The most important supercritical fluid properties related to its use as

a solvent are density, viscosity and diffusion coefficient. The density
values are close to the values of liquids, which strengthens its solvent
power. The viscosity and the diffusion coefficient, on the other hand, are
close to the values of gases, which makes the transport properties of the
fluid very favorable to the process. In the vicinity of the critical point,
small changes in pressure and temperature lead to large variations in the
density of the supercritical fluid and consequently of its solvent power
(BRUNNER, 1994). All these unique properties make supercritical fluids
a very interesting medium for chemical reactions. These transport
 57

properties, similar to gases, facilitate the mass transfer rates between

reactants and catalysts. In reactions limited by diffusion, kinetics will
generally be more favored in supercritical fluids than in conventional
liquid solvents (MESIANO; BECKMAN; RUSSELL, 1999).
The most widely used supercritical fluid is carbon dioxide (CO2),
due to its low toxicity, low cost, non-flammability and critical parameters
of easy access (critical properties: 31.4 ° C and 78.8 bar). In addition, CO2
is widely available in high purity from commercial and industrial sources
and can also be reused. At ambient temperature and pressure, CO2 is
gaseous, which makes the recovery of the reaction products very simple
and free of solvents (BRUNNER, 1994; JÉRÔME; LECOMTE, 2008).
Because there is no residual CO2 remaining in the material after
processing, CO2 is not considered exactly a solvent, so it does not require
reassessment by the United States FDA. Only water also enjoys this
special situation. Most commercial operations using CO2 as solvent have
been initiated to take advantage of such particular advantages of CO2 in
products designed for intimate contact with the human organism, like
food, biomedical and pharmaceutical devices (BECKMAN, 2004).
Some studies report the use of scCO2 in lactones e-ROP. Loeker et
al. (2004) reported for the first time the synthesis of poly (ε-caprolactone)
via e-ROP in scCO2, using the enzyme Novozym 435 as catalyst. The
authors obtained poly(ε-caprolactone) with molecular weights (Mn) of
12,000 to 37,000 Da, dispersity of 1.4 to 1.6 and yields of up to 98% in
24 hours of polymerization at 65 °C. Thurecht et al. (2006) performed
kinetic studies of ε-caprolactone e-ROP in a high-pressure reactor using
scCO2 as solvent. The authors obtained materials with high molecular
weight (up to 50,000 Da, Mn), yields around 80% and could conclude that
the amount of water in the reaction medium has a direct influence on
molecular weight. Additionally, the authors concluded that e-ROP
kinetics of ε-caprolactone in scCO2 exhibited a similar behavior to e-ROP
under analogous conditions, but using toluene as solvent.
More recently, poly(ε-caprolactone) was synthesized using scCO2 as
solvent in different types of reactors: fixed volume reactor (SANTOS et
al., 2012); variable-volume reactor (COMIM ROSSO et al., 2013); and in
a continuous packed bed reactor (PBR) (COMIM ROSSO et al., 2015).
Comim Rosso et al. (2013) obtained poly (ε-caprolactone) with a Mn of
up to 13,700 Da and dispersity of 1.2 to 1.7, while Santos et al. (2012)
obtained poly (ε-caprolactone) with 7,400 Da (Mn) and dispersity of 1.96.
Comim Rosso et al. (2015), using a PBR, obtained poly (ε-caprolactone)
with molecular weights up to 35,800 Da and dispersity ranging from 1.5
to 2.1.
58

In spite of the good results of yield, molecular weight and dispersity,

shown by the works that performed e-ROP of ε-caprolactone in scCO2
(COMIM ROSSO et al., 2013, 2015; LOEKER et al., 2004; SANTOS et
al., 2012; THURECHT et al., 2006), up to the present moment it was not
possible to find in the open literature works covering e-ROP of globalide,
using scCO2 or any other supercritical fluid as solvent.

2.7. POST-POLYMERIZATION FUNCTIONALIZATION OF

POLYESTERS

Material modification allows specific interactions within biological

systems, which significantly enhances biomaterial performance, offering
the material a dynamic role in its application. However, as highlighted by
Pounder and Dove (2010), the introduction of functional groups
throughout the polymer chain via ROP remains highly labored. Despite
this hindrance, there are numerous examples of side chain functionalized
linear polyesters produced from ROP which underlines the demand for
these materials. Most examples reported are produced via rigorous, often
multi-step, synthetic procedures to derivatize lactone or lactide monomers
(ATES; THORNTON; HEISE, 2011).
Chain modification by post-polymerization allows the production of
functional polymers that cannot be prepared by direct polymerization of
the respective monomers. After the functionalization process, the physical
and chemical properties of the polymers can be modified, such as
viscosity, solubility, adhesion, reduction of crystallinity, increase of
hydrophilicity, and the possibility of synthesis of more complex structures
to obtain new materials.
In polymers from unsaturated macrolactones, such as globalide, the
unsaturation provides a chemical handle for functionalization. A
chemically straightforward modification of aliphatic polyesters is
possible, by using highly efficient and robust thiol-ene “click” reactions
(section 2.7.1) directly on unsaturated polyesters, obtained from ROP of
the unsaturated macrolactone. This reaction may be employed for the
post-polymerization modification of unsaturated polyesters to allow the
introduction of numerous different chemical groups to polymeric
backbones, thereby potentially enabling the formation of biofunctional
materials (ATES; THORNTON; HEISE, 2011; DONDONI, 2008;
LOWE, 2009).
Some authors reported the synthesis of polyglobalide (PGl) via e-
ROP using CALB lipase, where the double bond in the formed polyester
chain was functionalized via thiol-ene reaction, using different thiol
 59

groups. These works aimed to change characteristics of polyglobalide and

its copolymers, such as thermal and mechanical properties, crystallinity,
hydrophilicity and ability for bioconjugation (ATES et al., 2014; ATES;
HEISE, 2014; ATES; THORNTON; HEISE, 2011; CLAUDINO et al.,
2012).

2.7.1. Thiol-ene “click” reactions

Kolb, Finn and Sharpless (2001) described a new concept for the
conduction of organic reactions, which follows examples of nature,
generating substances by joining small units together with heteroatom
links. This concept is called “click” chemistry. Click reactions must have
the following characteristics: being modular (capable of connecting two
molecules), being wide in scope, obtaining high reaction yields, forming
harmless byproducts (which can be removed by non-chromatographic
methods), and being stereospecific. The required process characteristics
include simple reaction conditions, readily available starting materials,
solvent exclusion of the reaction medium or use of non-toxic solvents and
ease of purification of the product.
Thiol-ene reactions consists of a simple and adaptable methodology
to prepare functionalized polymers and polymer networks using
combinations of multifunctional alkenes and thiols (HOYLE; LEE;
ROPER, 2004). Thiol-ene addition reactions are considered click
reactions, since they meet several attributes of this reactions class. Thiol-
ene reactions have high yields and fast reaction rates, need small
concentrations of catalysts, can be conducted without the use of solvent
or using non-toxic solvents, can be carried out under mild conditions, are
insensitive to air/oxygen, and generate pure products (HOYLE;
BOWMAN, 2010; HOYLE; LEE; ROPER, 2004).
The first applications of products obtained through thiol-ene
reactions were implemented in the construction of materials for simple
applications, such as films, protective coatings, coatings for electronic
materials and printed circuit boards (HOYLE; BOWMAN, 2010). The
limited application of these materials was related to problems with the
color of the coatings, caused by initiator residues in the product, allied to
weathering. Benzophenone (photoinitiator) was used in large quantities
in these systems, giving rise to significant light stability problems of the
cured networks associated with colored byproducts formed upon
exposure to interior and exterior light. The use of thiol-ene chemistry was
severely reduced when new low-cost polymers of high purity became
60

available at prices that allowed for widespread applications (HOYLE;

LEE; ROPER, 2004).
However, in recent years, thiol-ene addition reactions have been
applied in the synthesis of new materials with high added value, being the
surface modification, functionalization and crosslinking of polymers of
great importance for applications in biomedical area, since a controlled
polymer structure can be achieved by this method (HOYLE; BOWMAN,
2010; HOYLE; LEE; ROPER, 2004).
Thiol-ene reaction takes advantage of the easily abstractable
hydrogen atom of the thiol group due to the relatively weak sulphur-
hydrogen bond (FOSSEY; LEFFORT; SORBA, 1995). The cleavage of
SH bonds can be promoted either by direct photolysis (or simply by
thermolysis) or indirectly from heat (or light) generated nucleophilic alkyl
radicals obtained from the cleavage of initiators. The resulting
electrophilic thiyl radicals (RS•) are extremely reactive and can add to a
wide variety of unsaturated compounds to form new carbon-carbon
linkages. The RS• addition reaction to the double bond is exothermic and
forms a strong σ C-C bond, (370 kJ.mol-1), at the expense of the weaker
π bond (235 kJ.mol-1), which means this reaction is energetically favored
(CLAUDINO, 2011).
Generally, thiol-ene reactions have been conducted under radical
conditions which proceed through a typical chain process with initiation,
propagation and termination steps (ATES, 2014; CLAUDINO, 2011).
The characteristic two-step mechanism for the addition reaction of an
isolated unsaturation is represented in Figure 2.8.
The thiol-ene reaction starts via initiation (UV or thermally
induced), promoting the hydrogen transfer from the thiol (R-SH) to one
of the initiating free-radicals generated (via photoinitiator or initiator
cleavage), generating the thiyl radical (RS•). During the first propagation
step, the generated thiyl radical adds across the C=C double-bond,
yielding an intermediate β-thioether carbon centered radical. On the
second propagation step occurs the chain transfer to a second thiol group,
giving the final thiol–ene addition product, and regenerating RS• radical.
The mechanism continues to generate thiyl radicals in this cyclic process,
allowing the propagation of the reaction until the depletion of the thiol-
ene species. Termination reactions are frequently considered unimportant
if compared to the rates of propagation and usually involve bimolecular
combination of the intervening radical species (β-carbon or thiyl radicals)
(CLAUDINO, 2011).
 61

Figure 2.8 - Two-step mechanism for free-radical thiol-ene reaction, alternating

propagation (i) and chain transfer (ii).

Source: Claudino (2011).

2.7.2. Thiol-ene reactions in unsaturated polyesters

So far there are very few studies related to e-ROP from unsaturated
macrolactones available in the open literature. The first study reported
was developed by van der Meulen et al. (2008), which performed the
synthesis of two unsaturated polymacrolactones, using globalide (15
carbons) and ambretolide (16 carbons) as monomers. The polymers
obtained showed to be biocompatible and non-toxic (monomer derivative
from hydroxy fatty acids), which is the primary requirement that all
materials have to meet for applications in the body (VAN DER MEULEN
et al., 2008). However, polyesters derived from macrolactones are very
hydrophobic and semi-crystalline, which are undesired characteristics for
applications were bioresorption is necessary, such as drug delivery
devices or scaffolds. In their work van der Meulen et al. (2008) obtained
polymacrolactones of molecular weight (Mn) around 24,000 Da. The
degree of crystallinity obtained for polyambretolide e polyglobalide was
62

62 and 60%. It was not observed hydrolytic and enzymatic degradation

after long monitoring periods.
Since then, this research group has studied the functionalization and
crosslinking of polyesters obtained from the monomer globalide, in order
to obtain materials susceptible to degradation for biomedical purposes.
The globalide monomer is composed of 15 carbons and has an
unsaturation in its chain (position 11 or 12 - mixture of two isomers). This
chain unsaturation allows crosslinking or functionalization through thiol-
ene reaction.
Ates, Thorton and Heise (2011) carried out the functionalization of
polyglobalide via thiol-ene addition reactions using 6-mercapto-1-
hexanol (MH) and N-acetylcysteamine (n-ACA). The authors obtained
coupling efficiency of 95% for both MH and n-ACA, when using high
concentrations of initiator (AIBN) and thiol in excess. The polymers
formed showed branched chains and there was reduction in the
crystallinity of the final material, resulting in totally amorphous materials.
According to the authors, the introduction of these groups also potentially
enabled further polymeric modification, to introduce functional amino
acid groups (among others) that possess a wide range of chemical and
physical properties.
Meulen et al. (2011) carried out the copolymerization of globalide
and ambretolide with ε-caprolactone, 4-methyl caprolactone and 1,5-
dioxepan-2-one, in order to increase the biodegradability of the material.
The authors crosslinked the obtained copolymers with dicumyl peroxide.
In the enzymatic degradation studies with Pseudomonas cepacia lipase,
the authors observed a loss of mass of 30% and greater than 90% after
approximately 100 days for the copolymers poly(Gl-co-4MeCl) - in the
ratio 53/47 (Gl/4MeCL) - and poly(Am-co-DXO) - in ratio 25:75 (Am:
DXO) -, respectively. Figure 2.9 shows the copolymer poly (Gl-co-
4MeCl) before and after crosslinking with dicumyl peroxide. It is possible
to observe the change in the color of the materials as a function of the
transition from the initial crystalline state to the amorphous final state.
 63

Figure 2.9 - Copolymer poly(globalide-co-4-methyl caprolactone), before (left)

and after (right) crosslinking with dicumyl peroxide.

Source: van der Meulen et al. (2011).

Claudino et al. (2012) also synthesized copolymers of globalide and

ε-caprolactone. The formed copolymer was crosslinked through the
addition of a trithiol (trimercapto propionate - TMP) via photoinitiation
(photoinitiator Irgacure 184) in the double bonds derived from the
globalide units. The authors obtained high thiol–ene conversions (>80%)
in all cases. For high globalide contents (high unsaturation density),
crosslinked polymers lost all the crystallinity, becoming amorphous,
since crosslinking inhibits chain mobility to crystallize. To lower contents
of globalide, some crystallinity still remains in the sample.
After these studies, Ates and Heise (2014) evaluated different
strategies to produce stable crosslinked surface functional films from
polyglobalide, employing a dithiol crosslinker and N-acetylcysteamine
(N-ACA) mono-thiol as a model compound. Three routes were tested, as
shown in Figure 2.10. In route A, the authors tried to functionalize the
monomer first, and then polymerize by e-ROP the functionalized
monomer. In route B, first, the polymer was produced by e-ROP. After
the polymerization, a fraction of the double bonds was functionalized
with N-ACA. Lastly, the free double bonds of the functionalized polymer
was crosslinked with the use of the dithiol. Route C was similar to route
B, but performing the crosslinking step before the functionalization step.
64

Figure 2.10 - Synthetic routes to functional crosslinked films from macrolactone

using thiol–ene chemistry.

NZ435: Novozym 435; AIBN: azobisisobutyronitrile; N-ACA: N-

acetylcysteamine; BAET: 2-(Boc-amino) ethanethiol; EGMP: ethylene glycol
bis(3-mercapto propionate).
Source: (ATES; HEISE, 2014).

Routes A and B did not result in products with desirable qualities,

leading to the formation of low yield polymers and molecular weights.
The crosslinked polymers obtained by route B were tacky and of low
mechanical integrity, irrespective of the crosslinker to polymer ratio,
rendering this route unfeasible. Route C formed films with high molecular
weights, 100% crosslinked, totally amorphous and transparent and
presenting a 20% reduction in their mass when submitted to enzymatic
degradation during 50 days. Figure 2.11 shows the difference between the
films obtained by routes B and C. The authors explained that this
difference might happen due to an incomplete network formation, which
might be due to a too large distance between the double bonds preventing
optimal formation of a three-dimensional network. This problem could be
overcome by carrying out the geometrically more demanding
 65

crosslinking reaction first, followed by N-ACA post-functionalization on

the crosslinked film.

Figure 2.11 - Crosslinked functionalized PGl films prepared by Ates and Heise
(2014) through routes B and C.

Source: Ates and Heise (2014).

After these series of studies, Ates et al. (2014) published a work

putting together all the knowledge developed previously about the
synthesis of functionalized crosslinked PGl films. Their idea was to
synthesize a PGl film functionalized with chemical chains capable of
bioconjugation, improving cell adhesion and enhancing cell growth. The
sequence of reactions performed to obtain these films are represented in
Figure 2.12.
The authors synthesized PGl films by e-ROP, the films were
crosslinked and then functionalized with 6-mercapto-1-hexanol. This
functionalized films, also called “hydroxyl decorated films” (PGl-OH),
were converted into atom transfer radical polymerization (ATRP)
initiators, through reaction of the hydroxy decorated film with α-
bromoisobutyryl bromide, generating “initiator decorated films”. The
initiator decorated films reacted to tert-butyl acrylate, forming a poly
(tert-butyl acrylate) grafted PGl film. After deprotection of the tert-butyl
ester groups, reactive carboxylic acid groups are generated, which makes
the decorated PGl films able to bioconjugation. The authors successfully
immobilized fluorescent protein and chitobase enzyme in the obtained
grafted PGl film. This was the first work that reported the production of
biofunctional films derived from natural macrolactones.
66

Figure 2.12 - Synthesis of poly (acrylic acid) grafted from crosslinked and ATRP
initiator modified PGl surfaces, followed by bioconjugation.

Source: Ates et al. (2014).

2.7.3. Conjugation of nanoparticles and proteins

It is known that the composition of the protein corona, formed when

the nanoparticles are in contact to a biological fluid, gives a biological
identity to the nanoparticles and defines its interaction with cells
(LYNCH; DAWSON, 2008; TREUEL et al., 2014). Engineering the
surface of the nanoparticles is an excellent strategy to tune interfacial
properties and produce tailored materials for specific cell targeting,
enabling treatments to occur efficiently (DE et al., 2007; RANA; YEH;
ROTELLO, 2010).
 67

Polyethilene glycol (PEG) is a molecule widely used in order to

functionalize nanoparticles, aiming to suppress cell uptake by defense
cells through the composition control of the protein corona that surrounds
nanoparticles. However, PEG is a non-biodegradable polyether, and its
accumulation in the human body should be avoided, especially when
NPs-PEG conjugates are administered in long-term treatments. In such
cases, the accumulation of PEG in the human body can cause undesired
side effects (SCHÖTTLER et al., 2016). In this context, the
functionalization of nanoparticles directly with proteins rises as an
alternative to the use of PEG, as a way to control the composition of the
protein corona. Apolipoproteins, for example, when conjugated with
nanoparticles, have the ability of inhibit its uptake by defense cells
(CARACCIOLO et al., 2015; MONOPOLI et al., 2011; SCHÖTTLER;
LANDFESTER; MAILÄNDER, 2016). Apolipoprotein J (Apo J), also
known as clusterin, and apolipoproteins A4 and C3 (ApoA4 and ApoC3)
presente high capacity to inhibit phagocytosis (RITZ et al., 2015;
SCHÖTTLER et al., 2016).
When used in the nanoparticle pre coating (non-covalent), ApoA4
and ApoC3 significantly reduced non-specific cell uptake (RITZ et al.,
2015). The non-covalent adsorption method is one of the simplest and
most frequently employed procedures for the attachment of proteins onto
particles(ARVIZO; DE; ROTELLO, 2007). The attachment occurs by
hydrophobic or electrostatic interactions between the nanoparticle and the
protein (ARVIZO; DE; ROTELLO, 2007; CARUSO, 2001). However,
this method have a serious disadvantage, since conjugates produced by
non-covalent methods are reversible. This means that proteins can adsorb
and desorb to the surface of NPs, and it becomes very difficult to maintain
the stability, uniformity and reproducibility of the conjugates, which are
desired characteristics for biomedical applications. Under physiological
conditions, proteins adsorbed to the NPs can desorb and be replaced by
other proteins and biomolecules present in the local environment, making
it difficult to predict the behavior and the composition of this conjugates
for long times. (BOYER et al., 2011; CHEN; LIU LORETO MEGIDO;
DÍEZ, 2018; PINO et al., 2014).
Covalent functionalization of proteins and nanoparticles is an
interesting strategy to produce conjugates that are stable toward
dissociation. The stability and irreversibility of covalent protein-NP
conjugates are decisive factors that determine the biological response of
cells and organisms. The stability of the conjugates is especially
important for applications in complex biological media, where many
interfering species can alter the composition of protein-NPs conjugates
68

and consequently change completely the targeting of nanoparticles over

time (LESZCZYNSKI, 2010; RANA; YEH; ROTELLO, 2010).

2.7.3.1. Thiol-ene functionalization of proteins and nanoparticles

Thiol-ene click reactions are highly efficient and robust reactions

and can be employed in the preparation of biologically active protein–
nanoparticles conjugates, in spite of being still little reported in the
literature to this purpose (DONDONI, 2008; KELEŞ; HAZER;
CÖMERT, 2013; LOWE, 2009; TUNCA, 2014). Site-specific covalent
conjugation strategies, using highly efficient orthogonal chemistries, such
as thiol–ene click reactions, provide good control over the orientation of
the conjugated proteins (BOYER et al., 2011), forming also very uniform
and reproducible conjugates.
Thiol-ene reactions are especially interesting in the presence of free
thiol groups in cysteine-containing proteins. The cysteine thiol group can
be attached to double bonds on the surface of the nanoparticles, leading
to site-selective and irreversible conjugations (BOYER et al., 2011).
In the work of Liu et al. (2017) a cysteine-selective ligand containing
a double bond was designed and anchored to the surface of a hydrophobic
nanoparticle surface. This way, any thiol-containing biomolecule can be
conjugated on the surfaces of nanoparticles. In their work, a cysteine-
containing horseradish peroxidase were selected for thiol-ene
bioconjugation of colloidal nanoparticles. The success of the thiol-ene
linkage occurrence was determined by SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis).
It is important to highlight that is still a challenge to verify the
covalent conjugation efficiency in systems involving nanoparticles and
complex molecules such as proteins and peptides (ABBAS et al., 2017).
In this cases, traditional analytical methods (spectroscopy techniques,
nuclear magnetic resonance and chromatography, for example) are not
always able to detect the formation of this covalent linkages, especially
due to the complexity of proteins structure and difficulties in solubilizing
the conjugates.

2.8. THE STATE OF ART

The arising of polymeric materials in the beginning of the 20th

century has revolutionized the industry and the life of people. Due to its
versatility and practicality, polymers have rapidly replaced traditional
 69

materials such as metals, glass and wood in a wide range of applications,

including the automotive, food, electronics and civil construction
industries.
With the emergence of tissue engineering and nanomedicine,
polymers also started to be intensely used in biomedical and
pharmaceutical applications. The increasing demand for biocompatible
materials for specific applications in biological environment is
encouraging current and future scientists to work on researches towards
designing bioresorbable polymers with enhanced properties and develop
clean processes.
One of the most studied biocompatible/bioresorbable polymers is
poly(ε-caprolactone). PCL is very attractive due to its excellent properties
such as high flexibility, biodegradability and biocompatibility, as well as
non-toxicity (PITT; MARKS; SCHINDLER, 1980). However, PCL has
a high crystallinity, which makes its bioresorption in the organism very
slow, which is not desired for some applications.
Polyglobalide, a polymer derivative from an unsaturated
macrolactone, is a very little studied polymer, which is non-toxic and
biocompatible and has very interesting mechanical properties (VAN DER
MEULEN et al., 2008). Besides, PGl chain can easily be functionalized
and/or crosslinked, due to the presence of unsaturation in its chain,
through thiol-ene reactions. The functionalization is capable to reduce
monomer crystallinity and hydrophobicity, as well as enhance the affinity
of the polymer for different human cells/tissues.
The synthesis of copolymers based on ε-caprolactone and globalide
come in order to unite the flexibility and the biodegradability features of
PCL, to the good mechanical properties and the double bonds versatility
from PGl, enabling the development of a material with tailored properties
by tuning monomer ratios and/or using different functionalizing
molecules.
The most interesting route for obtaining biocompatible and
bioresorbable polymers with potential use in the biomedical and
pharmaceutical areas, is the ring-opening polymerization catalyzed by
enzymes (e-ROP). The use of enzymes avoids the use of toxic
organometallic catalysts and is environmentally friendly. Supercritical
carbon dioxide used as solvent also proved to be interesting from the
environmental and health point of view, being easily separated from the
polymer, and possible to be reused. The use of scCO2, together with e-
ROP have contributed to render the (co)polyesters production a clean
process (COMIM ROSSO et al., 2013; SANTOS et al., 2012;
THURECHT et al., 2006; VENERAL, 2014).
70

Post-polymerizations modifications rise as a strategy to enhance

biomaterials performance. Thiol-ene click reactions are an efficient way
to produce functionalized polymers for tissue engineering purposes, due
to its high yields and formation of harmless byproducts. Also, it is one of
the most interesting methods to produce nanoparticle-proteins covalent
conjugates, yielding uniform and stable nanomaterials for treatments in
specific targeted cells.
In the presented literature review, it was shown important results
published in the scientific literature, which are directly related to the
theme of this project. However, there are still a great number of
challenges to be overcome, especially regarding the functionalization of
polymers to proteins and its characterization, given the complexity of
protein structures and its direct influence on protein-cell interactions.
Considering the actual scenario, there is a great demand for
biocompatible/bioresorbable polymers, and there is wide accessibility for
clean technologies. It is common to find in literature works that have been
carried out individually on the topics here proposed: polymerization in
supercritical fluids, post-polymerization functionalization by thiol-ene
reactions and conjugation of proteins to nanoparticles. However, the
innovation of this work consists in the connection among all these topics,
leading to the construction of an engineered biomaterials platform based
on PGlCL, free of toxic residues, and with enhanced properties for
different biomedical applications. Researches in this area are fundamental
for the development of clean processes and new biomedical devices,
improving the quality of life of the population.
 71

CHAPTER 3

The use of scCO2 and scCO2 in mixture with DCM as solvents was
studied by synthesizing PGlCL on different fed ratios between globalide
and ε-caprolactone. It was evaluated the influence of the different solvents
and monomers ratio on the molecular weight, thermal properties,
formation of oligomeric cycles and ratio between globalide and ε-
caprolactone repeating units present in the obtained copolymers. Through
the understanding of the mass transfer behavior on e-ROP, it was possible
to stablish relation between the variables evaluated and its effects on the
polymer properties. The results presented in this chapter were published
in The Journal of Supercritical Fluids
(doi.org/10.1016/j.supflu.2017.06.008).

3. ENZYMATIC RING OPENING COPOLYMERIZATION OF

GLOBALIDE AND Ε-CAPROLACTONE UNDER
SUPERCRITICAL CONDITIONS

Abstract
The enzymatic ring opening copolymerization of globalide and ε-
caprolactone under supercritical conditions was investigated. Samples
were evaluated in terms of molecular weight, thermal properties, ratio
between repeating units, and production of cyclic oligomers. For the first
time, poly(globalide-co-ε-caprolactone) (PGlCL) was synthesized by e-
ROP using supercritical carbon dioxide (scCO2) and the mixture
scCO2+dichloromethane (DCM) as solvents. The feed ratio between
globalide and ε-caprolactone was evaluated. PGlCL synthesized in scCO2
showed the highest molecular weights, while the use of cosolvent caused
a decrease on molecular weight values. For both scCO2 and scCO2+DCM,
the increase on globalide content (relative to total monomers amount)
increased molecular weight values. Thermal analysis indicated a non-
isomorphic behavior, characteristic of random semi-crystalline
copolymers, which is the case of the PGlCL produced. PGlCL
synthesized on scCO2+DCM presented double melting point, which is
related to a greater presence of cyclic oligomers, in comparison to PGlCL
synthesized using only scCO2, being confirmed by MALDI-TOF
analysis.
72

Graphical Abstract

3.1. INTRODUCTION

During the last decades, the development of

biodegradable/bioresorbable polymeric materials for biomedical
applications has advanced considerably, due to the interest in replacing
traditional materials used as medical devices. In this context, the
enzymatic synthesis of polymers for biomedical applications is very
attractive, since enzymes do not leave toxic residues in the final product,
are active under mild conditions of temperature, and provide very specific
polymers (COMIM ROSSO et al., 2013).
The use of supercritical carbon dioxide (scCO2) in substitution to
organic solvents is an alternative on enzymatic ring-opening
polymerization (e-ROP) reactions. scCO2 is inexpensive, non-toxic, non-
flammable (KUMAR; MADRAS; MODAK, 2004), and exhibits
transport properties that can accelerate mass transfer in enzymatic
reactions (OLIVEIRA; OLIVEIRA, 2000). Also, it can be easily
separated from the final product by system depressurization and can
therefore be reused in the process. In order to improve the system
solubilization, it is also possible to use cosolvents, together with scCO2.
Dichloromethane (DCM) has been successfully used as solvent on e-ROP
reactions, as well as on scaffolds without affect the cultivation and growth
of different kinds of cell (CHEW et al., 2008; ELOMAA et al., 2011;
POLLONI et al., 2017; VENERAL, 2014; XIE et al., 2009). Its low
 73

boiling point (40 °C) (BROWN; STEIN, 2016) allows its easy separation
from the final products, avoiding toxicity problems (JÉRÔME;
LECOMTE, 2008).
The synthesis of poly(ε-caprolactone) (PCL) by e-ROP using
organic solvents has been extensively studied (CÓRDOVA et al., 1998;
KUMAR; GROSS, 2000; KUNDU et al., 2011; MEI; KUMAR; GROSS,
2003; ZHANG et al., 2012a). PCL is one of the most attractive polymers
for medical application, since it is biodegradable and bioresorbable, and
presents mechanical properties proper for this application
(ALBERTSSON; SRIVASTAVA, 2008). Some drug delivery devices
made from PCL already have FDA approval and trademark
(WOODRUFF; HUTMACHER, 2010).
Polyglobalide (PGl) is a biocompatible and non-toxic polymer,
produced from the monomer globalide (VAN DER MEULEN et al.,
2008). Globalide is an unsaturated 16-membered macrolactone, which
contains a double bond. Typically, globalide is used in the fragrance
industry due to its musky odor and its quality of losing its scent slowly
(MICHROWSKA; WAWRZYNIAK; GRELA, 2004; WILLIAMS,
1999). In comparison to small lactones, the chemical polymerization of
macrolactones is a challenge, since in general it proceeds slowly and gives
low molecular weight polymers (FOCARETE et al., 2001; NOMURA;
UENO; ENDO, 1994). However, the enzymatic ring-opening
polymerization of macrolactones has shown to be very effective
(FOCARETE et al., 2001; GEUS et al., 2010; POLLONI et al., 2017).
Polymers obtained from macrolactones also have excellent mechanical
properties (CAI et al., 2010; FOCARETE et al., 2001; GEUS et al., 2010).
Besides, the double bonds in globalide structure lead to many possibilities
of functionalizing the polymer, giving it the most diverse characteristics,
making PGl very interesting for its application in medical devices, as it
could increase biocompatibility or biodegradation.
The synthesis of copolymers of globalide (Gl) and ε-caprolactone
(CL) is a versatile alternative for tissue engineering, since the ratio
between the monomers may be tuned, producing copolymers with
different molecular weights and melting temperatures, depending on the
desired application. Besides, the presence of the double bond enables the
change of other characteristics that cannot be changed by the simple
variation on monomer ratio (increase on hydrophilicity, affinity for
different human tissues, decrease on crystallinity index and increase on
mechanical resistance for example) (ATES et al., 2014; ATES; HEISE,
2014; ATES; THORNTON; HEISE, 2011; CLAUDINO et al., 2012), by
74

functionalizing the copolymer. Figure 3.1 shows the structures of the

monomers globalide (Gl) and ε-caprolactone (CL).

Figure 3.1 - Structure of the cyclic monomers (A) ε-caprolactone and (B)
globalide. Globalide a mixture of two constitutional monomers with the double
bond located either at the 11 or 12 position (dashed line).

The present work aimed to study the synthesis of poly(globalide-co-

ε-caprolactone) (PGlCL) copolymers by e-ROP, using scCO2 and the
mixture scCO2+DCM as solvents, being a subject which was not reported
in literature until now. e-ROP of PGlCL using scCO2 and scCO2+DCM
were performed under different feed ratios between globalide and ε-
caprolactone. It were studied the effects of the different solvents and the
globalide/ε-caprolactone feed ratio on copolymer properties, such as
molecular weight, dispersity, melting temperature, crystallinity index and
formation of cyclic oligomers.
In this way, the present work brings a series of unprecedented
important results, which contributes to the understanding of the process
phenomenology behind PGlCL e-ROP on scCO2, being vital for future
studies on PGlCL functionalization and its use for biomedical devices
development.

3.2. MATERIAL AND METHODS

3.2.1. Material preparation

Dichloromethane P.A. 99.8% (DCM) and ethanol P.A. 99.8%

(EtOH) was purchased from Vetec Química and used as received.
Novozym 435 was kindly donated by Novozymes, Brazil, A/S
(commercial lipase B from Candida antarctica immobilized on cross-
linked polyacrylate beads, esterification activity 42 U/g, measured
 75

according to a procedure adapted from literature (OLIVEIRA et al.,

2006)). Enzymes were dried under vacuum (0.4 bar) and 70 °C, during 16
hours (VENERAL, 2014) and stored in a desiccator over silica and 4 Å
molecular sieves. Globalide was a kind gift of Symrise. ε-caprolactone
were purchased from Sigma-Aldrich. Both globalide and ε-caprolactone
were dried under vacuum (0.1 bar) and 100 °C, during 24 hours
(VENERAL, 2014) and also stored in a desiccator over silica and 4 Å
molecular sieves. Carbon dioxide (99.9% purity) used as solvent was
purchased from White Martins S/A, Brazil.

3.2.2. Enzymatic ring-opening polymerization in pressurized

solvents

Polymerization experiments were carried out in a high-pressure

variable-volume view cell, with a maximum internal volume of 27 mL,
with two sapphire windows for visual observation, an absolute pressure
transducer (Model LD 301, Smar, USA) and a syringe pump (260HP,
Teledyne Isco, Lincoln, NE, USA). The cell contains a movable piston
that allows the control of pressure inside the reactor. The variable-volume
reactor allows an accurate control of pressure and temperature during
reaction.
For polymerization experiments, the enzyme (Novozym-435), the
monomer (globalide and ε-caprolactone) and DCM (when used) were
weighed on a precision scale balance (Shimadzu AUY220, Philippines
with 0.0001 g accuracy) and placed inside the reactor, which was
immediately closed. A known amount of solvent, CO2, was loaded into
the reactor using the syringe pump until the desired composition was
achieved, meeting the mass ratio specified conditions. The system
pressure was increased until the desired pressure was achieved. A metallic
jacket surrounds the reactor, and water from a thermostatic bath was used
as heating/cooling fluid, keeping the cell at the desired temperature. Once
the desired temperature was reached, the reaction time started. The cell
content was kept under constant stirring with the use of a magnetic stirrer
and a TeflonTM-coated stirring bar.
To understand the influence of the different globalide to ε-
caprolactone proportions on enzymatic synthesis of poly(globalide-co-ε-
caprolactone) in scCO2 and scCO2 with the use of cosolvent, a series of
assays were performed, varying the feed mass ratio of globalide/ε-
caprolactone (10/90, 25/75, 50/50, 75/25 and 90/10). These ratios may
also be expressed in percentage of globalide relative to the total monomer
amount. With the aim of improving the mass transfer (increasing the
76

solubility of the system), DCM was used as cosolvent. DCM has an easy
separation from final products due to its low boiling point (JÉRÔME;
LECOMTE, 2008), and promotes good solubilization of globalide and ε-
caprolactone (BORDES et al., 2010; VAN DER MEULEN et al., 2008).
The pressure and temperature conditions of the system were
maintained constant at 120 bar and 65 °C, respecting the temperature
range of higher enzyme activity (OLIVEIRA et al., 2004; RICHETTI et
al., 2010; ROSA et al., 2008). Enzyme content was fixed at 5 wt%
(relative to the total monomer amount). For assays which used only scCO2
as solvent, the CO2:monomers mass ratio was fixed at 1:2. For assays
using scCO2, and DCM as cosolvent (scCO2+DCM), DCM:monomers
mass ratio was maintained constant at 1:2 for all assays, while the mass
ratio CO2:MIX were 1:2 and 2:1 (where MIX = DCM+monomers).
Also, a reaction using only DCM as solvent was performed, using a
feed globalide/ε-caprolactone mass ratio of 10/90, at exactly the same
conditions used in reactions on scCO2, fixing a DCM:monomers mass
ratio of 1:2. The reaction was performed at this condition in order to
compare the formation of cyclic oligomers by the use of different solvents
through MALDI-TOF analysis.
The reaction conditions were chosen based on previous works
(COMIM ROSSO et al., 2013, 2015; POLLONI et al., 2017) of e-ROP
homopolymerization of ε-caprolactone and ω-pentadecalactone, using
scCO2 as solvent, that led to high values of yield and homopolymer
molecular weight. The reaction time was set at 2 h. After polymerization,
the material was purified through solubilization in DCM, followed by the
separation of the enzymes and precipitation in cold EtOH. DCM and
EtOH were used at the volumetric proportion of 1:6. The polymeric
suspension was filtered and dried at room temperature in vacuum, up to
constant mass.

3.2.3. Characterization of the copolymer

Gel Permeation Chromatography - GPC: Number average molecular

weight (Mn), weight average molecular weight (Mw) and dispersity (Đ)
were determined by Gel Permeation Chromatography (GPC). For the
analysis, 0.02 g of the copolymer was dissolved in 4 mL of
tetrahydrofuran (THF). The obtained solution was filtered through a
nylon syringe filter, pore: 0.45 µm, diameter: 33 mm. The molecular
weight distributions were obtained using a high-performance liquid
chromatography equipment (HPLC, model LC 20-A, Shimadzu) and
Shim Pack GPC800 Series columns (GPC 801, GPC 804 e GPC 807),
 77

also from Shimadzu. THF was used as eluent with volumetric flow rate
of 1 mL∙min-1 at 40 °C. The calibration was performed using polystyrene
standards with molecular weight ranging from 580 to 9.225 x 106 g.mol-
1.

Differential Scanning Calorimetry - DSC: Samples of approximately 5

mg of dried copolymer were analyzed using a Perkin-Elmer JadeDSC,
under inert atmosphere (20 mL∙min-1), from 0 to 120 °C at a heating rate
of 10 °C/min. The thermal history was removed prior to the analyses at a
heating rate of 20 °C/min and cooling rate of 10 °C/min. Melting and
crystallization temperatures were determined from the second heating run
and the first cooling from the melt, respectively. The material crystallinity
was determined using the fusion enthalpy (ΔHm) from the second heating
run.
Nuclear Magnetic Resonance - NMR: 1H NMR and 13C NMR
spectroscopy were performed on a Bruker AC-200F NMR, operating at
200 MHz for 1H NMR and 50 MHz for 13C NMR. Chemical shifts are
reported in ppm relative to tetramethylsilane (TMS) 0.01% (v/v)
(δ=0.00). All samples were solubilized in CDCl3 (δ = 7.27 for 1H NMR,
and δ = 77.0 for 13C NMR).

Poly(globalide-co-ε-caprolactone) 1H NMR (CDCl3 200 MHz): δ (ppm)

5.49-5.32 (m, CH=CH), 4.10-4.04 (m, CH2O(C=O)); 2.35-2.26 (m,
CH2(C=O)O); 2.07-1.97 (m, CH2(CH=CH)); 1.71-1.62, 1.29 (m, CH2).

Poly(globalide-co-ε-caprolactone) 13C NMR (CDCl3 50 MHz): δ (ppm)

173.9 (C=O), 133.5-125.0 (CH=CH), 64.1-63.7 (CH2O(C=O)), 34.4-32.0
(CH2(C=O)O), 29.5-23.6 (CH2).
Degree of randomness - R: The degree of randomness of the copolymer
was calculated from 13C NMR data through the following equations
(JANSEN, 2005; VAN DER MEULEN et al., 2011; YAMADERA;
MURANO, 1967):
𝑃𝐺/𝐶 = 𝑃𝐺𝐶 + 𝑃𝐶𝐺 (3.1)
𝑃𝐺/𝐶
𝑃𝐺 𝑡𝑜𝑡𝑎𝑙 = + 𝑃𝐺𝐺 (3.2)
2
𝑃𝐺/𝐶
𝑃𝐶 𝑡𝑜𝑡𝑎𝑙 = + 𝑃𝐶𝐶 (3.3)
2
𝑃𝐺/𝐶
𝑅= (3.4)
2𝑃𝐺 𝑡𝑜𝑡𝑎𝑙 𝑃𝐶 𝑡𝑜𝑡𝑎𝑙
where PGG, PCC, PCG and PGC represents the integral value of the GG, CC,
CG and GC diads, respectively, denoting the mol fraction of each diad.
78

PG/C denotes the total integral of the mixed diads (GC and CG) and PG total
and PC total denotes the total mol fraction of G and C units respectively.

MALDI - TOF mass spectrometry: Matrix-assisted laser desorption

ionization time-of-flight mass spectrometry (MALDI-TOF) was carried
out in a Bruker Autoflex III smartbeam spectrometer. 2,5-
dihydroxybenzoic acid (DHB) was used as matrix, being dissolved in a
methanol/water solution (70% v/v methanol) in a concentration of 20
mg/mL. Samples were solubilized in chloroform and then mixed to the
matrix solution in a 1:1 volume ratio. To semi quantify the cyclic chains
present in the copolymer, the area under the cyclic peaks was compared
to the area under the linear peaks. Analysis were performed in both
reflector positive mode and linear positive mode.
3.3. RESULTS AND DISCUSSION

3.3.1. Synthesis and composition of PGlCL

The e-ROP reaction of the monomers globalide (Gl) and ε-

caprolactone (CL) were carried out in the following systems: with scCO2
in a mass ratio of 1:2 (CO2:monomers); with scCO2 + DCM in a mass
ratio of 1:2 (CO2:MIX); and with scCO2 + DCM in a mass ratio of 2:1
(CO2:MIX). As described in section 3.2.2, MIX = DCM+monomers, and
the mass ratio of DCM:monomers is fixed in 1:2. The feed mass ratio of
Gl/CL was varied (10/90, 25/75, 50/50, 75/25 and 90/10), while other
parameters like pressure, temperature and enzyme content were
maintained constant (section 3.2.2.). Reaction yield values varied from 45
to 60%.
Table 3.1 shows 1H NMR spectroscopy data for samples obtained
with the use of scCO2 and scCO2 + DCM. Differences between Gl/CL
feed ratio and Gl/CL copolymer composition were not relevant for both
solvents used (scCO2 and scCO2+DCM), which means that none of the
monomers reacted preferentially.
Table 3.1 - 1H NMR data of PGlCL copolymer composition obtained using different solvents (scCO2 and scCO2+DCM)
and different globalide/ε-caprolactone feed ratios.
CO2:monomers or Feed Gl/CL Feed Gl/CL NMR Gl/CL copolymer composition
Solvent
CO2:MIX (mass ratio) (mol ratio) (mol ratio)
10/90 5/95 2/98
50/50 33/67 27/73
scCO2 1:2
75/25 59/41 62/38
90/10 81/19 77/23
10/90 5/95 2/98
50/50 33/67 37/63
scCO2 + DCM 1:2
75/25 59/41 63/37
90/10 81/19 84/16
79
80

All spectra obtained are in good agreement to the spectra of

polyglobalide and poly(ε-caprolactone) reported in literature (ATES;
THORNTON; HEISE, 2011; BRANDOLINI; HILLS, 2000; VAN DER
MEULEN et al., 2008).
The degree of randomness (R) was calculated from Equations (3.1)
to (3.4) presented on item 3.2.3., in order to know the distribution of the
repeating units in the copolymer chains. For this calculation it were used
values determined by 13C NMR diads peak integration of the signal from
the carbon next to the carbonyl (CH2(C=O)O), between 32 and 35 ppm.
PGlCL synthesized using pure scCO2, with 75/25 Gl/CL feed ratio
(mass), was selected for the randomness determination. As shown in
Figure 3.2, signals from Gl-Gl repeating units (GG, 32.6 ppm) can be
found next to the signals from CL-CL repeating units (CC, 34.1 ppm) in
the spectrum. Gl-CL (GC, 32.0 ppm) and CL-Gl (CG, 34.4 ppm) diads
can be found to be adjacent to the Gl-Gl and CL-CL peaks.

Figure 3.2 - (A) 13C NMR spectrum of PGlCL 75/25 (Gl/CL feed mass ratio). (B)
Expanded CH2(C=O)O region of 13C NMR spectra, with the peaks of each
respective diad.

The diads values calculated by integration of the peaks were:

GG=0.31, CC=0.13, GC=0.13; CG=0.43, resulting in R=1.1, indicating
that PGlCL has a random distribution, which is in accordance to the
results obtained in other copolymerization studies between lactones and
macrolactones (CECCORULLI et al., 2005; CLAUDINO et al., 2012;
KUMAR et al., 2000; VAN DER MEULEN et al., 2011). For R=1, units
 81

take a random distribution, and the probability of finding a copolymer

unit belongs to Bernoulli statistics. If R < 1 the units tend to cluster in
blocks of each units, R = 0 for homopolymers, whereas for R > 1 the
sequence length becomes shorter, and finally, R = 2 for alternating
copolymers (VAN DER MEULEN et al., 2011; YAMADERA;
MURANO, 1967). The spectra obtained are in good agreement to spectra
obtained in literature (JACOBS et al., 1991; KUMAR et al., 2000; VAN
DER MEULEN et al., 2011).

3.3.2. Molecular weight and dispersity

Figure 3.3 presents Mn as function of the feed globalide content

(relative to the total monomer amount) for reactions carried out in systems
with: scCO2 in a mass ratio of 1:2 (CO2:monomers); scCO2 + DCM in a
mass ratio of 1:2 (CO2:MIX); and scCO2 + DCM in a mass ratio of 2:1
(CO2:MIX).

Figure 3.3 - Number average molecular weight (Mn) as function of feed globalide
content (relative to total monomer amount) using scCO 2 and scCO2+DCM as
solvent.
CO2
CO2:MIX (1:2)
CO2:MIX (2:1)

25000

20000
Mn (Da)

15000

10000

5000
0 10 20 30 40 50 60 70 80 90 100
Globalide content (%)

As reported in literature (KUNDU et al., 2011; POLLONI et al.,

2017; THURECHT et al., 2006; VENERAL, 2014), polyesters
82

synthesized by e-ROP does not generate symmetrical and monomodal

peaks, as typically obtained when chemical catalysts are used. In this
work, similarly to the literature, e-ROP synthesized polymers which
chromatogram presents a broad polymer peak, followed by a long tail
(bimodal distribution). Kundu et al. (KUNDU et al., 2011) also observed
this behavior for e-ROP of PCL, and analyzed by mass spectrometry the
fraction correspondent to the tail in the chromatograms, founding that
they are relative mainly to cyclic oligomers. To obtain self-consistent
results for all experimental conditions studied in this work, it was adopted
as integration criterion the use of the inflection point of the broad polymer
peak as the endpoint for integration procedures. This way, the peaks of
higher molecular weights were used for integration, since these peaks are
more representative (always above 90 % of the chromatogram area), and
the oligomeric tail was not considered. The same integration criterion was
used in literature (KUNDU et al., 2011) and enables valid data
comparisons, maintaining the general trend in this data consistent with
other studies (COMIM ROSSO et al., 2013; VAN DER MEULEN et al.,
2008, 2011).
For all reaction systems evaluated, Mn increased with the increase of
globalide content. Globalide is a large molecule, of molecular weight
twice higher than ε-caprolactone. This way, knowing that the ratio of Gl
and CL repeating units in the copolymer is in accordance to the monomer
feed ratio (section 3.3.1), it is natural that the higher is the content of
repeating units derived from globalide, higher is Mn values.
The systems with scCO2, in comparison to scCO2+DCM systems,
provided higher Mn values. Since the experiments were carried on in a
reactor with a sapphire window, it was possible to visualize that when e-
ROP takes place using scCO2 as solvent, the reaction media stirring was
impaired and practically stops during the reaction, due to the formation
of large copolymer chains, and consequent increase of the media
viscosity. With a poor convection mass transfer, the monomer molecules
which were in contact to the enzyme pellets can react subsequently with
the active sites of the enzymes, generating polymer chains of high
molecular weights. The high concentration of monomers surrounding the
enzymes, lead to a greater growth of the copolymer chains. The use of
DCM as cosolvent diluted the reaction media and improved convection
mass transfer, in comparison to the use of only scCO2. This way, there is
a decrease in the monomer concentration in the surrounding of the
enzyme pellets, which means that the copolymer chains grew less.
Córdova et al. (1998), Thurecht et al. (2006), Comim Rosso et al. (2013)
and Loeker et al. (2004a) also observed that the monomer concentration
 83

has a strong influence on the polymer molecular weight. Many low

molecular weight oligomeric chains may be formed in diluted systems,
due to the greater difficulty of the monomer molecules to find the enzyme
active sites. The formation of low molecular weight cyclic oligomers also
occurs more frequently, since intermolecular reactions are not favored by
the dilution of the reaction medium.
The use of scCO2 + DCM in the two proportions of CO2 studied (1:2
and 2:1 CO2:MIX) provided Mn values very similar. In the beginning of
the reaction, the system with composition 2:1 (CO2:MIX) is formed by
two phases (liquid and vapor), while the 1:2 (CO2:MIX) system is fully
miscible (one liquid phase). This behavior also could be observed during
our assays by visualization through the sapphire window, and also by
other authors (COMIM ROSSO et al., 2013; XU; WAGNER; DAHMEN,
2003). It was observed that few minutes after the reaction starts, a vapor
phase (basically composed of CO2 (XU; WAGNER; DAHMEN, 2003))
appears in the system 1:2, due to exceeding the solubility limit, which
were also observed by Comim Rosso et al. (2013). It suggests that the
amount of CO2 fed in 1:2 system in the beginning of the reaction was
already very close to the solubility limit. After this limit is reached, the
concentration of the liquid phase is the same for both 1:2 and 2:1 systems
(CO2 saturation concentration). Since the reaction occurs in the liquid
phase (composed by monomers, enzyme pellets, DCM and CO2), it
explains the similarity between molecular weight data of 1:2 and 2:1
studied systems.
Dispersity (Đ) values varied from 1.3 to 1.6 for samples obtained in
scCO2, while for samples obtained by the use of scCO2 + DCM (1:2 and
2:1 CO2:MIX ratio), values varied from 1.3 to 1.7 (oligomeric tail not
considered). Đ values are low, and keep practically constant for all
systems evaluated, indicating an uniform polymer chain size distribution.

3.3.3. Thermal analysis data

PGlCL samples obtained on different globalide/ε-caprolactone feed

ratios, using scCO2 and scCO2 + DCM as solvents, were evaluated by
DSC. Results of crystallization temperature (Tc), melting temperature
(Tm), melting enthalpy (ΔHm) and degree of crystallinity are presented in
Table 3.2. The degree of crystallinity was calculated through the relation
between ΔHm of each sample, and the theoretical value of a 100%
crystalline PCL sample, obtained from literature (CRESCENZI et al.,
1972).
84

For the samples obtained by using only scCO2 as solvent, the relation
between the copolymer composition and Tm values presented an eutectic
trend. At first, Tm values decrease as the globalide content increase,
reaching a minimum value around 50% globalide content. After this
point, Tm values begin to increase. For copolymers, this behavior known
as “melting point depression”, and is characteristic of random semi-
crystalline copolymers constituted of very different monomers, in terms
of size and structure (non-isomorphic) (CANEVAROLO JR., 2006),
which is the case of globalide and ε-caprolactone. Each globalide
repeating units is 16-membered an has an unsaturation, while each ε-
caprolactone repeating unit is 7-membered and do not present any
unsaturation. In this case, monomers do not substitute each other within
the unit cell, and it is not possible to follow an additive rule for the
determination of the Tm of the copolymers (CANEVAROLO JR., 2006).
According to Wendling and Suter (1998), considering a copolymer of two
monomers A and B crystallizing in the crystal lattice of A, the monomers
B may either be excluded from the crystals or act as defects in the crystal.
In both cases the Gibbs energy of the crystal will increase and the melting
temperature decrease. Well-known equations (BAUR, 1966; FLORY,
1953; WUNDERLICH, 1980) can be applied to describe the observed
melting temperature depression. Claudino et al. (2012) evaluated thermal
properties of poly(globalide-co-ε-caprolactone) and observed the same
behavior for melting temperature.
Table 3.2 - Thermal properties determined by DSC for PGlCL obtained using different solvents (scCO2 and
scCO2+DCM) for different feed globalide/ε-caprolactone ratios.
Feed Gl/CL CO2:monomers or
Solvent Tc (°C) Tm 1 (°C) Tm 2 (°C) ΔHm (J/g) Xc (%)
(mass ratio) CO2:MIX
PCL 38 56 - 97.0 72
10/90 31 51 - 80.9 60
25/75 35 54 - 85.7 63
50/50 scCO2 1:2 20 39 - 92.1 68
75/25 30 45 - 85.8 63
90/10 33 48 - 74.0 55
PGl 36 51 - 95.3 70
50/50 22 36 - 96.6 71
scCO2 + DCM 1:2
75/25 26 - 44 85.7 63
50/50 20 34 - 79.0 58
scCO2 + DCM 2:1
75/25 31 36 45 91.4 68
Tc: Crystallization temperature; Tm 1: First peak melting temperature; Tm 2: Second peak melting temperature; ΔHm: Heat of fusion;
Xc: Degree of crystallinity, calculated from the fusion enthalpy value of a PCL 100% crystalline sample (CRESCENZI et al., 1972).
85
86

An interesting point to note is that, for samples obtained with the

use of cosolvent, broad melting peaks were obtained, or even double
peaks (double melting point) (Figure 3.4), while samples obtained by
using only scCO2 as solvent presented a single well-defined melting peak.
Multiple melting behavior (which includes double melting behavior) has
been observed to semi-crystalline polymers, including biodegradable
polymers such as poly(ethylene succinate) (AL-RAHEIL; QUDAH,
1995), poly(butylene succinate) (PBSu) (MIYATA; MASUKO, 1998;
YASUNIWA; SATOU, 2002) and poly (L-acid lactic) (YASUNIWA et
al., 2004). This double melting behavior has been explained with a melt-
recrystallization model (WUNDERLICH, 1980). The model suggests that
small and imperfect crystals change successively into more stable crystals
through the melt-recrystallization mechanism, generating a competition
between melting and recrystallization during the heating run. Since
recrystallization is a slow process, and the heat is continuously provided
by the system, the recrystallized chains and original chains which
remained in the crystal lattice finally melt. Then, the second melting peak
appears.
Figure 3.4 - Presence of double melting point behavior: DSC second heating run
for PGlCL 25/75 (Gl/CL feed mass ratio) obtained with scCO2+DCM, using
CO2:MIX = 2:1.

0
Second heating run
Unsubtracted Heat Flow (mW)

10
0 20 40 60 80 100
Temperature (°C)

According to the results presented by Córdova et al. (1998) and

Thurecht et al. (2006), when the reaction system is diluted by a solvent,
 87

the formation of cyclic compounds is favored (in comparison to bulk

reactions). The use of DCM as cosolvent in comparison to the use of only
scCO2, diluted the reaction media. Besides, the fact that DCM solvates
better the monomers/oligomers/polymers molecules in all reaction media
tested in this work, makes molecules more flexible and favors
intramolecular reactions (cyclic oligomers formation). These oligomeric
cycles may be responsible for the formation of less stable regions in the
crystalline arrangement, which causes the melting - recrystallization
behavior during the second heating run. Tm for samples obtained by using
cosolvent were lower than Tm of samples obtained by using only scCO2,
probably due to a higher formation of cyclic oligomers, which may
decrease the stability of the crystalline arrangement.
For all conditions evaluated, the melting enthalpy, which is related
to the amount of crystalline regions, showed little variation, which reflects
on the calculated degree of crystallinity values. All conditions evaluated
formed semi-crystalline copolymers, being consistent to literature data for
PGlCL, PCL and PGl (CLAUDINO et al., 2012; SINHA et al., 2004;
VAN DER MEULEN et al., 2008).

3.3.4. Effect of dichloromethane on cyclic oligomers synthesis

The formation of cyclic oligomeric chains was evaluated through

MALDI-TOF mass spectrometry. It were analyzed three samples of
copolymer, synthesized in the same conditions, but using different
solvents, with the objective of observing the formation of cyclic
oligomeric chains, under the use of different solvents/cosolvent.
The ratio between cyclic and linear compounds were obtained by
relating the area of the MALDI-TOF spectrum peaks which correspond
to these compounds. The calculations were based on cyclic species with
molecular weights greater than 1000 Da (background signal difficult the
peaks identification bellow 1000 Da). Figure 3.5 presents a MALDI-TOF
spectra in the region of 1000 to 2000 Da, were: peak (a) represent cyclic
PGlCL chains (PGlCL without end groups); peak (b) represent linear
PGlCL chains; and peaks (c) and (d) are probably related to PGlCL linear
species (doubly-lithiated species and sodiated species, respectively).
88

Figure 3.5 - Expanded view of MALDI-TOF spectrum of PGlCL 10/90 (Gl/CL

feed mass ratio), obtained using scCO2 as solvent, with DHB matrix.

(a) cyclic PGlCL chains; (b) linear PGlCL chains; (c) and (d) probably related to
PGlCL linear species (doubly-lithiated species and sodiated species,
respectively).

In MALDI-TOF analysis, cationization can occur through the

attachment of a cation to the sample. Due to their natural abundance, Li+,
Na+ and K+ ions are frequently attached to the sample molecules
(PASCH; WOLFGANG, 2003). These peaks series are present in spectra
obtained for all samples and are similar to those obtained by Córdova et
al. (1998) and Thurecht et al. (2006) for PCL.
Samples evaluated are listed on Table 3.3, as well as the ratio
between cyclic and linear chains, obtained by MALDI-TOF.

Table 3.3 - Relation between cyclic and linear chains obtained on e-ROP under
the use of different solvents.
CO2: CO2: DCM: Feed Gl/CL Cyclic:Linea
Solvent
monomers MIX monomers (mass ratio) r ratio
scCO2 1:2 - - 10/90 1:6
scCO2
- 1:2 - 10/90 1:4
+ DCM
DCM - - 1:2 10/90 1:3
 89

As expected, the sample synthesized using only scCO2 as solvent,

showed the lowest content of cyclic compounds, while the sample
synthesized using only DCM, presented the highest content (twice higher
than scCO2). The use of DCM as cosolvent, caused an increase in the
content of cycles, in comparison to the use of only scCO2 as solvent, but
it is still lower than the cycles content obtained by pure DCM. These
results strongly suggest that the use of DCM favors the formation of
cyclic oligomers, helping to explain the double melting point behavior
(item 3.3.3), observed in samples synthesized in the presence of DCM as
cosolvent. These samples presented higher cyclic oligomers content, in
comparison to those obtained in scCO2. The presence of cyclic oligomers
among linear chains generate less stable crystalline arrangements,
conferring double melting points to the copolymer samples, as observed
in the thermal analysis.
Besides, the higher formation of cyclic oligomers also provides a
wider molecular weight distribution. The cyclic oligomers detected by
MALDI-TOF compose the oligomeric tail detected in the chromatograms
of item 3.3.2. Figure 3.6 shows the molecular weight distribution of the
same samples analyzed by MALDI-TOF.

Figure 3.6 - PGlCL molecular weight distribution of PGlCL 10/90 (Gl/CL feed
mass ratio) by the use of different solvents

CO2
CO2+DCM
DCM
1.0

0.8
dw/d(LogMW)

0.6

0.4

0.2

0.0
2 3 4 5 6
Log (MW)
90

The samples synthesized using only scCO2 showed a narrower

molecular weight distribution, while the use of DCM generated samples
with wider molecular weight distribution. As explained in item 3.3.2,
when DCM is added as cosolvent, the concentration of monomers in the
media decreases. This way, when only DCM is used, monomer
concentration decreases even more, since all solvent and reactants are
present in the liquid phase (no vapor phase formation). As explained in
literature (CÓRDOVA et al., 1998; THURECHT et al., 2006) a diluted
medium does not favor the incidence of intermolecular reactions. Besides,
DCM has a greater ability to solubilize the
monomers/oligomers/polymers, giving more flexibility to the chains,
facilitating intramolecular backbiting.

3.4. CONCLUSIONS

This work brought important results about the enzymatic synthesis

of poly(globalide-co-ε-caprolactone) which was until now poorly
reported. Reactions were performed in scCO2 and scCO2+DCM, under
different globalide/ε-caprolactone feed ratios, consisting of an
unpublished study. The synthesis of PGlCL by e-ROP was successful,
especially when conducted under the use of only scCO2 as solvent.
Copolymers of Mn up to 25,000 Da were obtained, with a well-defined
melting point, random repeating units distribution, allied to low formation
of oligomeric products. Thermal analysis of PGlCL showed a non-
isomorphic behavior, which is typical for random semi-crystalline
copolymers, composed by comonomers with very different structures.
The use of DCM as cosolvent generated higher formation of cyclic
oligomers (in comparison to only scCO2), conferring to PGlCL double
melting point and wider molecular weight distribution.
 91

CHAPTER 4

After the study performed in Chapter 3, it was possible to understand

the phenomena that occur during poly(globalide-co-ε-caprolactone)
enzymatic synthesis in scCO2. Besides, it was also possible to find the
process conditions that yield copolymers with the most interesting
characteristics in terms of biomedical applications, such as high
molecular weight, well-defined melting point and small content of cyclic
oligomers. In the present chapter, PGlCL samples synthesized using pure
scCO2 in different Gl/CL ratios were selected to be functionalized with N-
acetylcysteine by thiol-ene coupling reaction. The functionalization of
PGlCL with NAC was performed in order to make the polymer more
amorphous and less hydrophobic, aiming to favor future applications
where a faster bioresorption is required. The resulting materials were
evaluated regarding its final composition, thermal properties (melting
temperature, degree of crystallinity and heat of fusion), hydrophobicity
and antioxidant potential. By varying the ratio between the copolymer
repeating units it was possible to tune the properties of the functionalized
polymer, increasing its range of applications. The results presented in this
chapter were published in the journal Materials Science and Engineering:
C (doi.org/10.1016/j.msec.2018.09.060).

4. N-ACETYLCYSTEINE SIDE-CHAIN
FUNCTIONALIZATION OF POLY(GLOBALIDE-CO-Ε-
CAPROLACTONE) THROUGH THIOL-ENE REACTION

Abstract

N-Acetylcysteine (NAC) is a drug well known for its antimucolytic

action, antioxidant activity and ability to protect cells from oxidative
stress. Conjugation of NAC with double bonds in the main polymer chain
of poly(globalide-co-ε-caprolactone) (PGlCL) through thiol-ene reaction
is reported. Different globalide (Gl) (an unsaturated macrolactone) to ε-
caprolactone (CL) ratios were employed for PGlCL synthesis. The
polymeric materials (PGlCL-NAC) were evaluated in terms of the
number of functionalized double bonds, thermal properties, affinity for
water and antioxidant potential. PGlCL-NAC containing more globalide
repeating units presented higher degree of functionalization, due to the
higher number of double bonds available to react through thiol-ene
coupling. For high globalide contents (Gl/CL ratios above 50/50), NAC
coupling in PGlCL chains resulted in completely amorphous copolymers
92

with a more hydrophilic character, which should enhance bioresorption

and cell adhesion characteristics. Functionalization also gave rise to a
thioether linkage, conferring to PGlCL-NAC an antioxidant character,
important for biomedical applications, where the material could combat
cellular oxidative-stress.

Graphical abstract

4.1. INTRODUCTION

Over the last decades, biodegradable/bioresorbable polymeric

materials has received significant attention, and its development has
advanced considerably, focusing specially in their use as degradable drug
delivery vehicles and in tissue engineering (ALBERTSSON; VARMA,
2003). In this context, many efforts have been concentrated in material
modification studies, which allows specific interactions within biological
systems. These modifications should significantly improve biomaterial
performance, making it very dynamic for a series of applications.
Thiol-ene reactions consist of a simple and adaptable methodology
to prepare functionalized polymers and polymer networks using
combinations of multi-functional alkenes and thiols (HOYLE; LEE;
ROPER, 2004; KELEŞ; HAZER; CÖMERT, 2013). Thiol-ene coupling
have high yields, fast reaction rates and form harmless byproducts, being
considered “click” reactions (HOYLE; BOWMAN, 2010; KOLB; FINN;
SHARPLESS, 2001). These reactions may be employed as a post-
polymerization modification in unsaturated polymers, even as multiple
click reactions, allowing the side-chain functionalization of simple and
 93

complex structures with different chemical groups, enabling the

formation of biofunctional materials (DONDONI, 2008; KELEŞ;
HAZER; CÖMERT, 2013; LOWE, 2009; TUNCA, 2014).
Poly(ε-caprolactone) (PCL) is one of the most attractive polymers
for medical application, since it is biodegradable and bioresorbable, and
presents mechanical properties proper for this application
(ALBERTSSON; SRIVASTAVA, 2008). Some drug delivery devices
made from PCL have already FDA approval and trademark
(WOODRUFF; HUTMACHER, 2010). However, the high crystallinity
of PCL reduces water permeability within the polymer matrix and make
the bioresorption process very slow (2-4 years) (WOODRUFF;
HUTMACHER, 2010). Besides, PCL is very hydrophobic, which in
addition to hinder its bioresorption, also difficult the cell adhesion and
proliferation limiting its potential applications (JEON; LEE; KIM, 2014).
Polyglobalide (PGl) is a biocompatible and non-toxic polyester with
mechanical properties suitable for biomedical applications (CAI et al.,
2010; FOCARETE et al., 2001; GEUS et al., 2010) obtained from ring-
opening polymerization (ROP) of globalide, an unsaturated 16-membered
macrolactone (VAN DER MEULEN et al., 2008). After ROP, the double
bonds remain in the main polymer chain of PGl enabling the
functionalization of the polymer chain by thiol-ene coupling reaction.
The development of copolymers composed by ε-caprolactone and
globalide tends to be a very versatile alternative for biomedical
applications, contributing to add new properties to the well-known PCL
through the addition of globalide units, which can be further
functionalized. The ratio between the monomers and the type of
functionalizing group may be tuned depending on the desired application,
producing copolymers with different characteristics such as different
melting temperatures, degrees of crystallinity, hydrophilicity, affinity for
different human tissues, crosslinking and mechanical properties, for
example.
N-acetylcysteine (NAC) is a drug well known for its antimucolytic
action, besides being used in a wide range of neuropsychiatric disorders
(BERK et al., 2013; DEAN; GIORLANDO; BERK, 2011). NAC presents
antioxidant activity (ANDRADE; MOURA; MARQUES, 2015;
CAZZOLA et al., 2015; DHOUIB et al., 2016) and has the ability to
protect cells from oxidative stress. NAC is a hydrophilic molecule
containing a thiol group (OSOL, 1970) that enables its conjugation by
thiol-ene reaction. The incorporation of NAC molecules covalently
bonded to PGlCL copolymer (PGlCL-NAC) by thio-ether linkages could
increase the affinity of the polymeric material for water and reduce its
94

degree of crystallinity. Besides, it is expected that the final material

presents an antioxidant potential, that is an important tool in the cellular
oxidative stress combat (VAN LITH et al., 2014; WANG et al., 2006).
In this sense, the present study discusses the side-chain
functionalization of the copolymer poly(globalide-co-ε-caprolactone)
(PGlCL) with N-acetylcysteine, through thiol-ene reactions, aiming to
produce modified PGlCL copolymers with enhanced characteristics for
future biomedical applications. Here, PGlCL was synthetized by
enzymatic ring-opening polymerization (e-ROP) using supercritical
carbon dioxide (scCO2) as solvent. After polymerization, PGlCL with
different globalide/ε-caprolactone repeating unit ratios were
functionalized with NAC, and the materials were evaluated in terms of
the number of functionalized double bonds, melting temperature, degree
of crystallinity, contact angle with water and antioxidant potential. To our
knowledge these are the first studies on poly(globalide-co-ε-
caprolactone) (PGlCL) side-chain functionalization with N-
acetylcysteine where the functionalized polymer reduced crystallinity,
increased hydrophilicity and improved antioxidant activity.

4.2. MATERIAL AND METHODS

4.2.1. Materials

Dichloromethane P.A. 99.8% (DCM), ethanol P.A. 99.8% (EtOH),

chloroform P.A. 99.8%, glacial acetic acid P.A. 99.8%, and the free
radical initiator azobisisobutyronitrile 98% (AIBN) were purchased from
Vetec Química (Brazil). Carbon dioxide (99.9% purity) used as solvent
was purchased from White Martins A/S, Brazil. N-acetylcysteine 99.8%
(NAC) was purchased from Gemini (Brazil). Novozym 435 was kindly
donated by Novozymes, Brazil, A/S (commercial lipase B from Candida
antarctica immobilized on cross-linked polyacrylate beads, esterification
activity 42 U g-1, measured according to a procedure adapted from
literature) (OLIVEIRA et al., 2006). Enzymes were dried under vacuum
(0.4 bar) and 70 °C, during 16 hours (COMIM ROSSO et al., 2015) and
stored in a desiccator over silica and 4 Å molecular sieves. Globalide (Gl)
was a kind gift of Symrise. ε-caprolactone were purchased from Sigma-
Aldrich. Both globalide and ε-caprolactone (CL) were dried under
vacuum (0.1 bar) and 100 °C, during 24 hours (COMIM ROSSO et al.,
2015) and also stored in a desiccator over silica and 4 Å molecular sieves.
 95

4.2.2. Poly(globalide-co-ε-caprolactone) synthesis using supercritical

carbon dioxide

Polymerization experiments were carried out as previously

described by Guindani et al. (2017) using supercritical carbon dioxide
(scCO2) as solvent. The pressure and temperature of the system were
maintained constant at 120 bar and 65 °C, and the monomers were
allowed to react for 2 hours. Enzyme content was fixed at 5 wt% (relative
to the total monomer amount), and the CO2 to monomers mass ratio was
fixed at 1:2. Experiments were performed varying the globalide/ε-
caprolactone (Gl/CL) mass ratio (10/90, 25/75, 50/50, 75/25 and 90/10).
After polymerization, the material was purified through solubilization in
DCM, followed by the separation of the enzymes and precipitation of the
polymer in cold EtOH. The polymeric suspension was filtered and dried
at room temperature in vacuum, up to constant weight.

4.2.3. Thiol-ene functionalization of poly(globalide-co-ε-

caprolactone) with N-acetylcysteine

PGlCL copolymers produced in scCO2, with different Gl/CL ratios

(10/90, 25/75, 50/50, 75/25 and 90/10) were functionalized through thiol-
ene reactions, aiming to understand the influence of the different
globalide/ε-caprolactone repeating unit ratio in the final properties of the
functionalized copolymer. NAC was chosen as a thiol compound suitable
for PGlCL functionalization. PGlCL and NAC were placed in a flask
together with the free radical initiator AIBN and a mixture of solvents,
under nitrogen atmosphere. During the reactions, the flask was kept
immersed in an oil bath at 80 °C, for 24 hours, under continuous magnetic
stirring. The amount of NAC used was established as being twice the
minimum amount of NAC required to functionalize all double bonds.
AIBN content was fixed in 5% (mol), relative to NAC amount. A mixture
of acetic acid and chloroform in a volumetric proportion of 3:1 (acetic
acid:chloroform) was used as solvent. In a typical reaction, 300 mg of
PGlCL is dissolved in 4 mL of solvent.
After polymerization, the solvents were removed in air circulation
oven. The dried material was washed in cold water, for the free NAC
removal, followed by a second wash in methanol for AIBN removal.
PGlCL functionalized with NAC (PGlCL-NAC) was then dried under
vacuum at room temperature, up to constant weight. Thiol-ene
conversions were determined through proton nuclear magnetic resonance
96

(1H NMR), by comparing the integral values of the peaks related to the
double bonds before and after thiol-ene reaction.

4.2.4. Characterization of the copolymer

Differential Scanning Calorimetry - DSC: Samples of approximately 5

mg of dried copolymer were analyzed using a Perkin-Elmer Jade DSC,
under inert atmosphere (20 mL min-1), from 0 to 150 °C at a heating rate
of 10 °C min-1. The thermal history was removed prior to the analyses at
a heating rate of 20 °C min-1 and cooling rate of 10 °C min-1. Melting
temperatures were determined from the second heating run. The material
crystallinity was determined using the fusion enthalpy (ΔHm) from the
second heating run.
Nuclear Magnetic Resonance - NMR: 1H NMR spectroscopy were
performed on a Bruker AC-200F NMR, operating at 300 MHz. Chemical
shifts are reported in ppm relative to tetramethylsilane (TMS) 0.01%
(vol%) (δ=0.00). All samples were solubilized in CDCl3 (δ = 7.27 for 1H
NMR).

Poly(globalide-co-ε-caprolactone) 1H NMR (CDCl3 300 MHz): δ (ppm)

5.49-5.32 (m, CH=CH); 4.10-4.04 (m, CH2O(C=O)); 2.35-2.26 (m,
CH2(C=O)O); 2.07-1.97 (m, CH2(CH=CH)); 1.71-1.62, 1.29 (m, CH2).

Poly(globalide-co-ε-caprolactone)- NAC 1H NMR (CDCl3 300 MHz): δ

(ppm) 6.70-6.40 (d, NH); 5.49-5.32 (m, CH=CH); 4.91-4.79 (m, CH-N);
4.12-4.05 (m, CH2O(C=O)); 3.81-3.70 (t, CH-S); 3.20-2.99 (m, S-CH2);
2.41-2.28 (m, CH2(C=O)O); 2.11-1.94 (m, CH2(CH=CH); 1.78-1.88 (t,
CH3(C=O)); 1.73-1.56, 1.27 (m, CH2).

Free radical scavenging activity - DPPH: NAC, PGlCL and PGlCL-NAC

free radical scavenging were evaluated using 1,1-diphenyl-2-
picrylhydrazil (DPPH) as described by Mensor et al. (MENSOR et al.,
2001) Different sample concentrations were evaluated by mixing in
Eppendorf 710 μL of DPPH diluted solution and 290 μL of sample
solution, and the reaction was kept away from the light, at room
temperature. After 30 min, the absorbance values were measured at 517
nm in spectrophotometer and converted into percentage of antioxidant
activity (AA%), and in EC50, i.e., concentration of test solution required
to decrease 50% absorbance compared to a blank solution and expressed
in μg of sample mL-1. EC50 values were calculated from the linear
 97

regression of the AA% curves obtained for all sample concentrations and
considering as the mean value of triplicate assays.

Free radical scavenging activity - ABTS: NAC, PGlCL and PGlCL-NAC

free radical scavenging were evaluated using 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS), based on the procedure
described by Re et al. (RE et al., 1999) ABTS•+ radical was produced by
reacting 7 mM ABTS and 2.45 mM potassium persulfate in the dark at
room temperature during 16 h before use. The aqueous ABTS•+ solution
was diluted with 5 mM phosphate buffer (pH 7.4) to an absorbance of 0.7
(±0.02) at 734 nm. Later on, 20 μL of sample solution (5 different
concentrations) and 980 μL of ABTS•+ solution was mixed in an
Eppendorf vial and the reaction was kept away from the light, at room
temperature. After 45 min of reaction, the absorbance was measured at
734 nm in spectrophotometer. These values were obtained from the
different concentrations of each sample tested in the assay and converted
into percentage of antioxidant activity (AA%), and in EC50, i.e.,
concentration of test solution required to decrease 50% absorbance
compared to a blank solution, and expressed in μg of sample mL-1. EC50
values were calculated from the linear regression of the AA% curves
obtained for all sample concentrations and considering as the mean value
of triplicate assays.

Contact angle assay: PGlCL and PGlCL-NAC films were obtained by

solvent casting in microscope slides, and the contact angle between the
polymeric films and water droplets were measured in a goniometer
(Ramé-Hart Instrument Co. - Ramé-Hart 250). All measurements were
performed in triplicate at room temperature with a drop volume of 10 µL.

4.3. RESULTS AND DISCUSSION

4.3.1. Synthesis of PGlCL-NAC by thiol-ene reaction

The synthesis of PGlCL-NAC was carried out through post-

polymerization thiol-ene reaction of PGlCL with NAC. PGlCl on the
other hand, was synthesized by e-ROP using scCO2 as solvent in a fixed
mass ratio of 1:2 (CO2:monomers) as described previously. After its
synthesis, PGlCL with different Gl/CL ratios were submitted to thiol-ene
reaction with NAC, generating the functionalized copolymer PGlCL-
NAC, as can be seen in Figure 4.1.
98

Figure 4.1- Poly(globalide-co-ε-caprolactone) side-chain thiol-ene

functionalization with N-acetylcysteine.

1H NMR analysis was performed to quantify the degree of NAC

coupling to PGlCL, related to the number of unsaturations at the polymer

chain. All spectra obtained for PGlCL and PGlCL-NAC are in good
agreement to the spectra obtained in the literature for polyglobalide,
poly(ε-caprolactone), N-acetylcysteine and other thiol-ene functionalized
polymers (ATES; THORNTON; HEISE, 2011; BRANDOLINI; HILLS,
2000; HMDB, 2017; VAN DER MEULEN et al., 2008). Figure 4.2
presents PGlCL and PGlCL-NAC 1H NMR spectra for Gl/CL 75/25.

Figure 4.2 - 1H NMR spectra of (A) PGlCL and (B) PGlCL-NAC (75/25 Gl/CL
ratio) and its respective peak assignments to the chemical structure of the
polymers.

Conversion values of the functionalization reactions were

determined through 1H NMR spectroscopy, by comparing the integral
values of the double bond peaks of PGlCL and PGlCL-NAC. For all
Gl/CL ratios evaluated, it was possible to observe a reduction in the area
of double bond peaks, besides the appearance of other new peaks related
 99

to NAC, indicating the occurrence of thiol-ene coupling. Comparing

some peaks that appear after functionalization with NAC and peaks that
remain from the original PGlCL chain, it is possible to notice the areas
are not proportional to the occurrence to the protons relative to each
group. Usually, NMR spectrum of polymers present broader peaks. In
such cases, the differences in chemical shifts of various 1H nuclei, which
are in different stereochemical and compositional environment, are
usually small, causing broad and overlapping signals (BRAR; GOYAL;
HOODA, 2009).
Also, in spite the processes employed to remove the solvents used
during functionalization of purification, it is possible that some residues
still remain in the material, causing peaks overlap. This way, more than
one peak may be appearing together as single peak in the spectra,
presenting then an area larger than the expected.
The number of functionalized globalide units are presented in Table
4.1. Analyzing these data, it is possible to see that the increase in the
amount of globalide units in PGlCL causes an increase in the number of
functionalized globalide units per polymer chain. When a PGlCL sample
containing a smaller amount of globalide units undergo thiol-ene reaction
with NAC, there are less double bonds available for functionalization, in
comparison to samples containing higher amounts of globalide units.

Table 4.1 - Thiol-ene reaction conversion and number of functionalized

Gl units per PGlCL-NAC molecule, calculated through 1H NMR
spectroscopy data.
Gl/CL Number of Gl Number of functionalized
Conversion
(mass) units per Gl units per PGlCL-NAC
a (%) b
PGlCL chain chain
10/90 4 80 3
25/75 17 71 12
50/50 35 53 19
75/25 67 51 34
90/10 96 42 40
a
Gl/CL: feed mass ratio of the monomers globalide (Gl) and ε-caprolactone (CL).
b
Conversion values calculated based on the consumption of the double bonds
present in PGlCL chains.
100

As a result, the PGlCL-NAC containing higher amounts of globalide

are much more prone to functionalization implying in more intense
changes in the material characteristic.

4.3.2. Effect of functionalization on crystallinity and melting

temperature

Samples of PGlCL with different Gl/CL repeating unit ratios

functionalized with NAC were evaluated by DSC. Results of melting
temperature (Tm), melting enthalpy (ΔHm) and degree of crystallinity (Xc)
are presented in Table 4.2.
The degree of crystallinity was calculated through the relation
between ΔHm of each sample, and the theoretical value of a 100%
crystalline PCL sample, obtained from literature (CRESCENZI et al.,
1972). It was not possible to obtain molecular weight data for PGlCL-
NAC, due to the low solubility of the functionalized polymer in solvents
traditionally used in GPC. After functionalization, PGlCL is soluble in
mixtures of solvents with different polarities. The data obtained for
PGlCL-NAC were compared to the data obtained by Guindani et al.
(2017) for non-functionalized PGlCL samples.
Table 4.2 - Properties of synthesized PGlCL functionalized with NAC for
different globalide/ε-caprolactone repeating units ratio.
PGlCLa PGlCL-NAC
Gl/CL Tm ΔHm Xc Mn Tm ΔHm Xc Mn
(mass) (°C) (J g-1) (%) (Da) (°C) (J g-1) (%) (Da)
10/90 51 80.9 60 8,623 43 84.5 62 ND
25/75 54 85.7 63 9,552 39 63.1 47 ND
50/50 39 92.1 68 16,568 - - - ND
75/25 45 85.8 63 21,206 - - - ND
90/10 48 74.0 55 25,360 - - - ND
a
PGlCL properties previously reported by Guindani et al. (2017). Tm, ΔHm and
Xc determined by DSC, Mn determined by gel permeation chromatography
(GPC). Tc: Crystallization temperature; Tm: Melting temperature; ΔHm: Heat of
fusion, Xc: Degree of crystallinity, calculated from the fusion enthalpy value of a
PCL 100% crystalline sample (CRESCENZI et al., 1972), Mn: Number average
molecular weight, ND: not determined.

Typically, for polylactones, when the molecular weight increases,

the degree of crystallinity presents a tendency of decreasing (TUBA;
OLÁH; NAGY, 2014). On the other hand, Tm is reported to not suffer
 101

significant changes with molecular weight increase (TIPTIPAKORN et

al., 2015; TUBA; OLÁH; NAGY, 2014). However, for copolymers, the
molecular weight is a direct consequence of the composition of repeating
units in the copolymer and the level of energy required for the copolymer
chains to acquire some mobility will be the result of the contribution of
each constituent (CANEVAROLO JR., 2006). This way, the molecular
weight itself do not exert a strong influence in Tm and ΔHm, but this values
are influenced by the composition of the copolymer.
The DSC second heating run of each sample is presented in Figure
4.3C. Guindani et al. (2017) obtained semi-crystalline polymers, with a
degree of crystallinity ranging between 55-68%. Generally, semi-
crystalline polymers exhibit a lower degradation rate than amorphous
polymers (CASALINI, 2017), limiting applications where a faster
degradation is necessary.
The functionalization of PGlCL containing a 10/90 Gl/CL ratio
resulted in PGlCL-NAC samples with lower Tm, in comparison to non-
functionalized PGlCL, while the degree of crystallinity (Xc) remained
practically the same after functionalization with NAC. The decrease of
the melting temperature after functionalization is an indicative that the
intermolecular forces in the crystalline arrangement of the material
became weaker, and its structure became easier to be undone. The
presence of branches implies in a free volume increase, which leads to an
easier chain movement of the chains, reducing the energetic level
necessary to overcome the secondary intermolecular forces between the
chains of the crystalline phase, destroying the regular structure of
packaging (CANEVAROLO JR., 2006).
102

Figure 4.3 - Schematic representation of the (A) PGlCL chains packaged

arrangement, forming crystalline regions, and (B) PGlCL-NAC chains disordered
arrangement, forming amorphous regions due to the presence of NAC side-
chains. (C) DSC second heating run curves

This way, the addition of NAC side-chains in the copolymer

structure in a small amount (10/90 Gl/CL, Table 4.1), despite not
changing the amount of crystalline arrangements (practically unchanged
ΔHm and Xc), was enough to reduce the amount of energy necessary to
undo these arrangements (Tm decreases).
PGlCL-NAC samples containing 25/75 Gl/CL presented a decrease
in Xc and Tm values, in comparison to non-functionalized PGlCL (25/75
Gl/CL) and to PGlCL-NAC (10/90 Gl/CL).
Finally, for PGlCL-NAC containing 50/50, 75/25, and 90/10
(Gl/CL), the higher density of NAC side-chains prevented the formation
of crystalline arrangements, resulting in completely amorphous polymers.
As shown in 1H NMR results (Table 4.1), the higher is the globalide
content in the copolymers (and consequently the number of double bonds
available to be functionalized), the higher is the presence of NAC side-
chains, which leads to materials with more amorphous characteristics.
4.3.3. Effect of functionalization on the surface characteristics of
the polymer

Samples that presented a completely amorphous behavior (DSC

assay), and are consequently interesting candidates for biomedical
applications (CASALINI, 2017; FERRARI et al., 2013), were evaluated
in terms of its affinity for water, through contact angle assay. Figure 4.4
 103

compares the contact angle of PGlCL and PGlCL-NAC films with 50/50,
75/25 and 90/10 (Gl/CL) ratios.
PGlCL with different Gl/CL ratios presented contact angle values
around 88°, and may be considered hydrophobic materials (ZHENG et
al., 2005). The surface hydrophilicity/hydrophobicity influences the
adsorption of protein onto the polymer surface. Hydrophobic polymers
are usually not desirable for biomedical applications, since they are
unsuitable for cell attachment (DOWLING et al., 2011; SYROMOTINA
et al., 2016).The surface property most frequently correlated with
adhesion is surface-free energy, a measure of the capacity of a surface to
interact spontaneously with other materials by forming new bonds
(BECKA; LOEB, 1984; CALLOW et al., 2005). At lower surface energy
surfaces (hydrophobic, high contact angle) on rigid surfaces, cell
attachment is generally poor, in comparison to high surface energy (more
hydrophilic, low contact angle) surfaces (GUELCHER; HOLLINGER,
2006; SYROMOTINA et al., 2016).
Bioresorption is another property affected by polymer
hydrophobicity/hydrophilicity, since it occurs initially through the
hydrolysis of the ester bonds. The high hydrophobicity of the polymer
limits water uptake, which decrease the polymer degradation rate
(CASALINI, 2017; GUALANDI et al., 2010; SAHA; TSUJI, 2006).

Figure 4.4 - Contact angle values of PGlCL and PGlCL-NAC samples as function
of Gl/CL ratio.

The results obtained for PGlCL-NAC, on the other hand, presented

lower contact angle values in comparison to PGlCL, for each respective
Gl/CL ratio evaluated. This result, together with 1H NMR and DSC
results, strengthen the idea that the NAC thiol-ene functionalization was
successful. As expected, the higher is the globalide content of repeating
units (higher number of NAC functionalization - Table 4.1), the lower is
104

contact angle values. NAC molecules were capable of giving a

hydrophilic character to the material, yielding PGlCL-NAC with contact
angle values varying from ~60° to ~47°. These values are within the range
of optimal contact angle for good cell attachment, reported in previous
studies as being between 50° and 70° for various polymers and cells
(BERNHARDT et al., 2009; KIM; PARK; PH, 2006; SELVAKUMAR
et al., 2015; TORRES et al., 2017; XU; SIEDLECKI, 2007). Increasing
the surface hydrophilicity induces the attachment of proteins like
fibronectin and cells like osteoblast and fibroblasts (SELVAKUMAR et
al., 2015; TORRES et al., 2017; YOSHINARI et al., 2010). Besides, a
more hydrophilic character is also interesting for applications in drug
delivery systems, were nanocarriers coated with a hydrophilic surface
(stealth nanocarriers) has a prolonged permanence in the bloodstream and
allows specific targeting (GUNASEELAN et al., 2010; MOGHIMI et al.,
1993; SALMASO; CALICETI, 2013; SCHÖTTLER et al., 2016).

4.3.4. Antioxidant potential of the material

N-acetylcysteine is known for its antioxidant activity. After

functionalization, NAC is covalently bonded to the polymer through a
thioether linkage, which also presents antioxidant activity, and is able to
induce the decomposition of hydroperoxides in inert products, being
considered a secondary antioxidant (AGNELLI; CHINELATTO, 1992;
CHANDA; ROY, 2010). PGlCL-NAC with a Gl/CL ratio of 50/50 was
evaluated in relation to its antioxidant potential through DPPH and ABTS
assays. The results are presented in terms of EC50 and are shown in Table
4.3.
The antioxidant activity of non-functionalized PGlCL was evaluated
and did not present antioxidant activity against DPPH• nor ABTS•+
radicals.
Table 4.3 - EC50 values of DPPH and ABTS assays for NAC, PGlCL, and
PGlCL-NAC in a 50/50 Gl/CL ratio.
DPPH assay ABTS assay
EC50 EC50
Sample
(μg mL-1) (μg mL-1)
PGlCL ND ND
NAC 4.31 ± 0.03 137 ± 3
PGlCL-NAC 4065 ± 157 1553 ± 22
 105

NAC in its free form, as expected, presented a strong antioxidant

activity in both DPPH (EC50 = 4.31 ± 0.03 μg mL-1) and ABTS (EC50 =
137 ± 3 μg mL-1) assays, being comparable to other for traditional
antioxidant compounds such as BHT, eugenol and ascorbic acid, for
example (BADANAI et al., 2015; PÉREZ-ROSÉS et al., 2016; TAYLOR
et al., 2014; ZHANG et al., 2012b). The antioxidant potential of the
functionalized polymer PGlCL-NAC, conferred by the thioether linkage,
was evaluated through DPPH and ABTS assays. For DPPH assay,
PGlCL-NAC presented EC50 = 4065 ± 157 μg mL-1.
This value is similar to the values obtained by Chen et al. (2009) for
eugenol-grafted chitosan nanoparticles (EC50 = 2600 μg mL-1) and
carvacrol-grafted chitosan nanoparticles (EC50 > 4000 μg mL-1), with
application focus in wound healing. Also, similar EC50 values were
obtained by Salarbashi et al. (2013) for soybean polysaccharide
incorporated with Mentha pulegium (EC50 = 5225.24 μg mL-1) and
Zataria multiflora Boiss (EC50 = 4188.60 μg mL-1) essential oils, for food
packaging applications.
In other work, Bishai et al. (2014) successfully performed a
modification of polylactic acid with humic acid (a natural antioxidant), in
order to obtain a material with enhanced functional properties ideal for
tissue engineering applications. Bishai et al. (2014) evaluated polylactic
acid modified with humic acid (PLA-HA) antioxidant activity through
DPPH (EC50 = 22450 μg mL-1) and ABTS assay (EC50 = 57500 μg mL-
50 = 1553 ± 22 μg mL . PGlCL-
1). For ABTS assay, PGlCL-NAC EC -1

NAC presented lower EC50 values than PLA-HA against both DPPH• nor
ABTS•+, indicating that PGlCL-NAC has a stronger antioxidant potential.
The importance of developing a functionalized copolymer for
biomedical applications able to scavenge free radicals is something that
should be highlighted. This kind of material that can locally and
continuously (while the polymer is present) inhibit excessive reactive
oxygen species generation may be a useful tool for treatments in many
biomedical areas, allowing a normal function of the cells (VAN LITH et
al., 2014).

4.4. CONCLUSIONS

Poly(globalide-co-ε-caprolactone) (PGlCL) was successfully

functionalized with N-acetylcysteine (NAC) through thiol-ene reaction.
Higher globalide contents in PGlCL led to higher incorporation of NAC
to the polymer chain due to the higher number of double bonds available
106

to react through thiol-ene coupling. Increasing the amount of NAC to

PGlCL reduced the melting temperature and amount of crystalline
domains. For copolymers with high contents of globalide (Gl/CL ratios
above 50/50), functionalization resulted in completely amorphous
copolymers with a more hydrophilic character, which should favor
biomedical applications where bioresorption and cell adhesion are
required. Besides, the pendant thioether linkage formed through thiol-ene
coupling conferred to the polymer an antioxidant character, very
important for tissue engineering applications, where the material could
locally combat oxidative-stress related to inflammatory responses.
 107

CHAPTER 5

The study presented in this chapter was developed at the Max-

Planck-Institut für Polymerforschung, in Mainz, Germany, under the
supervision of Professor Katharina Landfester. This period abroad was
part of a “sandwich-PhD” program, funded by CAPES/DAAD/CNPq. As
mentioned before, through the study performed on chapter 3 it was
possible to understand the mechanisms involved in the e-ROP of
poly(globalide-co-ε-caprolactone) in scCO2, and select conditions that
yield PGlCL samples with the most interesting characteristics in terms of
biomedical applications. On chapter 4, it was possible to functionalize
PGlCL with NAC and confer it completely new properties after this
chemical modification. In the present chapter, the work was expanded to
the nanoparticles field, which have a huge range of applications in the
biomedicine, such as in drug delivery, diagnostic imaging and
hyperthermia treatments for cancer. PGlCL nanoparticles (NPs) were
covalently conjugated with bovine serum albumin (BSA) by thiol-ene
coupling reaction, aiming to obtain engineered nanoparticles that present
enhanced internalization by cells. Uncoated particles and BSA conjugated
NPs were characterized regarding size, stability and morphology. The
success of the covalent conjugation is evaluated by fluorescence-activated
cell sorting and fluorescence correlation spectroscopy. Transmission
electron microscopy imaging was applied to the visualization of the
protein layer surrounding the nanoparticles. Cell uptake assays with
macrophage cells were also performed, comparing BSA-NPs conjugates
and uncoated NP.

5. COVALENTLY BINDING OF PROTEINS TO POLYMERIC

NANOPARTICLES

Abstract

When applied in biological fluids, proteins immediately adsorb onto

nanoparticles surface, forming a protein corona, which gives the
nanoparticle a new “identity” and determine its biological fate. Living
systems usually interact better with protein-nanoparticle conjugates rather
than with uncoated nanoparticles. Non-covalent conjugates are less stable
and more susceptible to desorption in biological media, which makes the
development of engineered nanoparticle surfaces by covalent attachment
an interesting topic. In this work, the surface of poly(globalide-co-ε-
caprolactone) (PGlCL) nanoparticles containing clickable groups is
108

covalently functionalized with bovine serum albumin (BSA) by thiol-ene

chemistry, producing conjugates which are irreversibly attached. The
successful formation of the covalent conjugates is confirmed by
fluorescence-activated cell sorting (FACS) and fluorescence correlation
spectroscopy (FCS). By help of transmission electron microscopy (TEM)
it is possible to visualize the formation of the conjugates and observe the
presence of a protein layer surrounding the NPs. After conjugation with
BSA, nanoparticles present reduced cell uptake by immune cells, in
comparison to uncoated nanoparticles. These are original results that
demonstrate that it is possible to produce stable and irreversible
conjugates by covalently binding BSA to PGlCL nanoparticles through
thiol-ene reaction.

Graphical abstract

5.1. INTRODUCTION

The application of nanomaterials has attracted the attention of many

scientific fields in the last decades. In the biomedical field, there is a
special interest regarding the use of nanomaterials due to their capability
to interact with cells and reach difficult access targets (MAHMOUDI et
al., 2011). The scientific and medical community has now recognized that
when nanoparticles (NPs) are exposed to a biological environment, its
surface suffers modifications by the adsorption of proteins, forming the
so-called protein corona. Nowadays, it is well-accepted that the presence
 109

of the protein corona affects cellular responses to the NPs, determining

its biological fate (LYNCH; DAWSON, 2008; TREUEL et al., 2014). In
this context, engineering the surface of the NPs is an excellent way to tune
its interfacial properties and create a wide material platform for specific
biological and biomedical applications (DE et al., 2007; RANA; YEH;
ROTELLO, 2010).
Nanoparticles with tailored surface properties are potentially useful
in a broad range of applications. The non-covalent adsorption method is
one of the most frequently employed procedures for the attachment of
proteins onto particles (ARVIZO; DE; ROTELLO, 2007). This is the
simplest way to produce NP-protein conjugates, and the attachment
occurs by hydrophobic or electrostatic interactions between the NP and
the protein (ARVIZO; DE; ROTELLO, 2007; CARUSO, 2001).
However, conjugates produced by non-covalent methods are reversible,
which means that proteins can adsorb and desorb to the surface of NPs,
making it difficult to maintain its stability, uniformity and reproducibility.
Under physiological conditions, proteins adsorbed to the NPs can desorb
and be replaced by other proteins present in the local environment, and a
long-term behavior of these conjugates may be difficult to predict
(BOYER et al., 2011; CHEN; LIU LORETO MEGIDO; DÍEZ, 2018;
PINO et al., 2014). In order to avoid these problems, covalent attachment
of proteins to NPs becomes an alternative to produce conjugates that are
stable toward dissociation. The stability and irreversibility of covalent
protein-NP conjugates can be decisive factors governing the biological
response of cells and organisms, making this strategy useful for
applications in complex biological media with other interfering species
(LESZCZYNSKI, 2010; RANA; YEH; ROTELLO, 2010).
Click reaction methods are an excellent approach to the preparation
of biologically active protein–polymer NPs. Thiol-ene reactions are an
example of a type of click reaction, consisting of a simple and adaptable
methodology to prepare functionalized polymers using combinations of
multi-functional alkenes and thiols (HOYLE; LEE; ROPER, 2004;
KELEŞ; HAZER; CÖMERT, 2013). Thiol-ene coupling present high
yields, fast reaction rates and form harmless byproducts (HOYLE;
BOWMAN, 2010; KOLB; FINN; SHARPLESS, 2001). These reactions
are frequently employed as a post-polymerization modification in
unsaturated polymers, enabling the formation of biofunctional materials
(DONDONI, 2008; KELEŞ; HAZER; CÖMERT, 2013; LOWE, 2009;
TUNCA, 2014). Thiol-ene reactions are especially interesting in the
presence of free thiol groups in cysteine-containing proteins, since this
110

allows a click reaction with accessible groups on NPs surface, leading to

site-selective and irreversible conjugations (BOYER et al., 2011).
In the present study, we aimed to produce poly(globalide-co-ε-
caprolactone) (PGlCL) nanoparticles and covalently conjugate them with
bovine serum albumin (BSA) protein, in order to obtain irreversible and
stable conjugates, suitable for biomedical applications. The unsaturated
polyester PGlCL was synthetized by enzymatic ring-opening
polymerization (e-ROP) using supercritical carbon dioxide (scCO2) as
solvent, and then PGlCL NPs were produced by the solvent evaporation
method. The double bonds present on the surface of PGlCL NPs were
then directly functionalized by thiol-ene reactions. At first, the
nanoparticles were functionalized with a small molecule, N-
acetylcysteine (NAC), in order to give us information about the feasibility
of the surface functionalization. Then, BSA was modified to increase the
amount of thiol units in its structure, and it was conjugated to PGlCL NPs
(Figure 5.1).

Figure 5.1 - (A) Structure and properties of the copolymer PGlCL in a mass ratio
Gl/CL = 50/50 (B) Scheme representing the conjugation of PGlCL NPs to
modified BSA through thiol-ene reaction.

(a) Mn, Xc and Tm reported previously by Guindani et al., (2017) Mn: Number
average molecular weight; Xc: Degree of crystallinity; Tm: Melting
temperature; I2959: photoinitiator Irgacure 2959 ®

The success of the covalent conjugation of BSA to PGlCL NPs was

evaluated through fluorescence-activated cell sorting (FACS) and
fluorescence correlation spectroscopy (FCS) measurements. The
conjugate visualization was possible through transmission electron
 111

microscopy (TEM). Finally, cell uptake assays with macrophage cells

were performed, comparing BSA-NPs conjugates and uncoated NPs. To
the best of our knowledge these are the first studies on covalent
conjugation of PGlCL NPs with BSA and we see our experiments as an
important contribution for the development of engineered nanomaterial
surfaces and for safer and more effective medical treatments.

5.2. MATERIAL AND METHODS

5.2.1. Materials

Solvents were purchased from Merck and Sigma Aldrich and used
as received, unless otherwise stated. The thermal initiator potassium
persulfate (KPS) was purchased from Thermo Fisher Scientific and the
surfactant sodium dodecyl sulfate (SDS) was from Alpha Aesar. The
photoinitiator Irgacure 2959® was purchased from BASF.
Carbon dioxide used as solvent was purchased from White Martins
S/A. ε-caprolactone (CL), N-acetylcysteine (NAC), bovine serum
albumin (BSA) and bovine serum albumin-fluorescein isothiocyanate
conjugate (BSA-FITC) were purchased from Sigma Aldrich. Novozym
435 was kindly donated by Novozymes A/S (commercial lipase B from
Candida antarctica immobilized on cross-linked polyacrylate beads,
esterification activity 42 U g-1, measured according to a procedure
adapted from literature (OLIVEIRA et al., 2006)). Globalide (Gl) was a
kind gift from Symrise. The enzymes and the monomers globalide and ε-
caprolactone dried under vacuum (GUINDANI et al., 2017) and stored in
a desiccator over silica and 4 Å molecular sieves. The proteins were used
without further purification.
5.2.2. Poly(globalide-co-ε-caprolactone) synthesis using supercritical
carbon dioxide as solvent

Polymerization experiments were carried out as described by

Guindani et al. (2017), using supercritical carbon dioxide (scCO2) as
solvent. The pressure and temperature conditions of the system were
maintained constant at 120 bar and 65 °C, in a fixed reaction time of 2
hours. Enzyme content was fixed at 5 wt% (relative to the total monomer
amount), and the CO2:monomers mass ratio was fixed at 1:2. The feed
mass ratio of globalide/ε-caprolactone (Gl/CL) was fixed in 50/50. After
polymerization, the material was purified through solubilization in
dichloromethane (DCM), separation of the enzymes by filtration and
112

precipitation of the polymer in cold ethanol (EtOH). The polymeric

suspension was then filtered and dried at room temperature in vacuum,
up to constant mass.
5.2.3. Poly(globalide-co-ε-caprolactone) nanoparticles preparation

The NPs were prepared according to the solvent evaporation

method. The aqueous phase was prepared mixing water (14 g) and the
surfactant SDS (0.2 % w/w). The organic phase was prepared mixing the
pre-synthesized PGlCL (100 mg), and DCM as solvent (3.5 g). The
aqueous phase was added to the organic phase and the mixture was
sonicated. Sonication occurred for 3 minutes (10 seconds pulse on, 10
seconds pulse off) with an amplitude of 70%. An ice bath was used to
reduce the temperature increase during the sonication. The miniemulsion
was left in a thermostatic bath with a temperature of 50 °C until complete
solvent evaporation.
The purification of miniemulsions was carried out by centrifugation
(12,000 rpm, 4 °C for 50 min). The supernatant containing the excess of
surfactant was removed and the NPs re-solubilized in distilled water. This
process was performed twice. Purified miniemulsions were stored in
refrigerator (4 °C) until functionalization steps were performed.

5.2.4. Surface modification of poly(globalide-co-ε-caprolactone)

nanoparticles
5.2.4.1. Functionalization with N-acetylcysteine (NAC)

The surface of PGlCL NPs was functionalized with N-acetylcysteine

(NAC) through thiol-ene reactions. The miniemulsion containing PGlCL
NPs was placed in a flask together with NAC and the water-soluble
initiator KPS, under nitrogen atmosphere. During the reaction, the flask
was kept immersed in an oil bath at 70 °C, during 4 hours, under
continuous magnetic stirring. NAC was used in sufficient amount to
functionalize 5% of the double bonds. Different KPS amounts were
tested, varying from 0.625 to 5% (mol), relative to NAC amount.
After the reaction, the miniemulsion containing NAC functionalized
NPs was purified by centrifugation (described in 5.2.3.) for NAC excess
removal. The functionalized NPs were re-solubilized in distilled water.
Purified miniemulsions were stored in refrigerator (4 °C).
 113

5.2.4.2. Functionalization with Bovine Serum Albumin (BSA)

BSA modification - Before conjugation with nanoparticles, BSA was

modified by reaction with Traut’s reagent (2-Iminothiolane), aiming to
introduce more thiol groups to the BSA structure, which should enhance
its capacity of conjugation with PGlCL NPs through thiol-ene reaction.
A BSA solution was prepared (10 mg BSA mL-1) in sodium phosphate
buffer 0.1 M, pH 8.0 containing 1mM EDTA. The 2-Iminothiolane
solution was prepared in a 2 mg mL-1 concentration and was mixed to the
BSA solution in a proportion of 1:50 (BSA:2-Iminothiolane) (v/v). The
reaction was carried out for 1 h in a shaker, at room temperature. After
reaction, the modified BSA was purified by dialysis for 48 hours and then
stored in a freezer (-18 °C).

Covalent functionalization of the nanoparticles with modified BSA - The

covalent conjugation of PGlCL nanoparticles with the modified BSA was
carried out through thiol-ene reactions, directly in the polymeric chain
double bonds present on the surface of the particle. The miniemulsion
containing PGlCL NPs was placed in a flask together with modified BSA
and the water-soluble initiator Irgacure 2959®, under nitrogen
atmosphere. During the reactions, the flask was exposed to UV light (365
nm) for 4 hours, under continuous magnetic stirring. The amount of BSA
used was established as 10-8 mol of BSA per mL of miniemulsion. This
amount is theoretically enough to cover the surface of all nanoparticles.
Irgacure 2959® content was fixed in 1% (mol), relative to the total amount
of thiol groups from BSA present in the reaction media. After the reaction,
the miniemulsion containing covalent BSA-NPs conjugates was purified
by centrifugation (described in 5.2.3.) for BSA excess removal. The
conjugates were re-solubilized in distilled water. Purified miniemulsions
containing the conjugates were stored in refrigerator (4 °C).

Non-covalent functionalization of the nanoparticles with modified BSA -

The non-covalent conjugation of PGlCL nanoparticles with the modified
BSA was carried out by mixing in a flask the miniemulsion containing
PGlCL NPs and the modified BSA, without any initiator. The mixture
was incubated overnight in refrigerator. The amount of BSA used was 10-
8 mol of BSA per mL of miniemulsion. After the reaction, the

miniemulsion containing non-covalent BSA-NPs conjugates was purified

by centrifugation (described in 5.2.3.) for BSA excess removal. The NPs
were re-solubilized in distilled water. Purified miniemulsions containing
the conjugates were stored in refrigerator (4 °C).
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5.2.5. Proton nuclear magnetic resonance (1H NMR)

1HNMR spectroscopy was performed on a Bruker Avance 300,
operating at 300 MHz. All spectra were referenced internally to residual
proton signals of the deuterated solvent. Samples were solubilized in
CDCl3 (δ = 7.27 ppm for 1H NMR).

5.2.6. Ellman’s assay

The Ellman’s assay was used to determine the final concentration of

thiol groups present on modified BSA, as well as the amount of NAC non-
reacted to the NPs. Ellman’s reagent (5,5'-dithio-bis-[2-nitrobenzoic
acid], DTNB) was solubilized in sodium phosphate buffer (0.1 M, pH 8.0,
containing EDTA 1 mM), in a concentration of 4 mg mL-1. The assay
was carried out in triplicate, in a 96 well plate, mixing in each well 4 µL
of Ellman’s reagent, 200 µL of buffer and 20 µL of sample. The reaction
occurs under constant stirring and protected from light, during 15
minutes. N-acetylcysteine (NAC) was used as standard. Absorbance was
measured at 412 nm with a Tecan infinite plate reader.

5.2.7. Pierce assay

The protein concentration was determined by Pierce 660 nm Protein

Assay according instructions of the manufacturers. Bovine serum
albumin (BSA) was used as standard. Absorbance was measured with a
Tecan infinite plate reader.

5.2.8. Dynamic light scattering (DLS)

Intensity particle average diameters of the uncoated nanoparticles

and the conjugates (Dp) and the polydispersity indexes (PDI) were
measured by dynamic light scattering (DLS —Malvern Instruments,
Zetasizer Nano S). The latex samples were diluted approximately 1:15
with distilled water prior to DLS measurements.

5.2.9. Zeta potential

The zeta (ζ) potential of the uncoated nanoparticles and the

conjugates (10 µL, 10 mg mL-1) was measured in a 1 mM potassium
 115

chloride solution (1 mL) with a Zeta Sizer Nano Series (Malvern

Instruments).

5.2.10. Transmission electron microscopy (TEM)

Transmission electron microscopy was carried out with a FEI Tecnai

F20 transmission electron microscope operated at an acceleration voltage
of 200 kV. The NPs were first diluted with 1 mL water and then one
droplet was placed on a carbon-coated copper grid and dried overnight.
In order to visualize the presence of the protein coating on the
conjugates, the negative staining technique was used. NPs (uncoated and
conjugates) were first diluted with water and then one was placed onto a
lacey grid and let to dry. The samples were stained with 4% uranyl acetate
+ 1% trehalose solution (1:1) (v/v) according to the method from
Kokkinopoulos et al. (2016). Images were taken with an Ultrascan 1000
(Gatan) charge-coupled device (CCD) camera.

5.2.11. Flow cytometry - Fluorescence-activated cell sorting (FACS)

measurements

Flow cytometry measurements were performed for NPs covalently

and non-covalently conjugated with BSA, and also for uncoated NPs. For
these measurements, the conjugates were produced using fluorescein
isothiocyanate (FITC) labeled BSA. BSA-FITC was modified before the
conjugation reactions, in the same way as described in item 5.2.4.2.
Measurements were performed using an Attune NxT Flow Cytometer
(laser: 488 nm laser for FITC excitation; emission: 530 nm band pass
filter). The fluorescent signal was expressed in a histogram and the
median intensity was determined. Attune NxT software was used for data
analysis.

5.2.12. Fluorescence correlation spectroscopy (FCS)

FCS measurements were performed for NPs covalently and non-

covalently conjugated with BSA. For these measurements, the conjugates
were produced using fluorescein isothiocyanate (FITC) labeled BSA.
BSA-FITC was modified before the conjugation reactions, in the same
way as described in item 5.2.4.2. The measurements were also performed
for pure modified BSA-FITC.
116

FCS was carried out on a commercial setup (Carl Zeiss, Germany)

consisting of the modules LSM510, ConfoCor 2 and an inverted
microscope model Axiovert 200 with a C-Apochromat 40×, NA 1.2 water
immersion objective. An argon ion laser (488 nm) was used for excitation
and the emission was collected after filtering with a BP505-550 band pass
filter. 8-well, polystyrene chambered cover glasses (Laboratory-Tek,
Nalge Nunc International) were used as sample cells. For each sample
series 10 measurements with a total duration of 10 s were performed. The
time-dependent fluctuations of the fluorescence intensity δI(t) caused by
the diffusion of the fluorescent species through the confocal observation
volume were recorded and analyzed by an autocorrelation function
G(𝑡) = 1 + 〈𝛿𝐼(𝑡) ∙ 𝛿𝐼(𝑡 + 𝜏)〉⁄〈𝛿𝐼(𝑡)〉2. As has been shown
theoretically, for an ensemble of identical freely diffusing fluorescence
species, G(𝑡) has the following analytical form:
𝑓𝑇 1 1
G(𝑡) = 1 + [1 + 𝑒 −𝑡⁄𝜏𝑇 ] (5.1)
1 − 𝑓𝑇 𝑁 𝜏 𝜏
[1 + 𝜏 ] √1 + 2
𝐷 𝑆 𝜏𝐷

Where N is the average number of diffusing fluorescence species in

the observation volume, 𝑓𝑇 and 𝜏𝑇 are the fraction and the decay time of
the triplet state, 𝜏𝐷 is the diffusion time of the species, and 𝑆 is the so-
called structure parameter, 𝑆 = 𝑧0 /𝑟0, where 𝑧0 and 𝑟0 represent the
axial and radial dimensions of the confocal volume, respectively. The
diffusion time 𝜏𝐷 is related to the respective diffusion coefficient 𝐷,
through 𝐷 = 𝑟0 2 /4𝜏𝐷 . The experimentally obtained G(𝑡) can be fitted
with Equation (5.1), giving as a result the diffusion times and the diffusion
coefficients of the fluorescent species. The hydrodynamic radii 𝑅h can be
calculated (assuming spherical particles) using the Stokes-Einstein
relation: 𝑅h = 𝑘𝐵 𝑇/6𝜋𝜂𝐷, where 𝑘𝐵 is the Boltzmann constant, 𝑇 is the
temperature, and 𝜂 is the viscosity of the solution. As the radial dimension
𝑟0 of the confocal probing volume is not known a priory it was determined
by performing calibration experiments using a fluorophore with known
diffusion coefficient in water, i.e. AlexaFluor488.

5.2.13. Cell culture

RAW264.7 were bought from DSMZ (Deutsche Sammlung für

Mikroorganismen und Zellen, Braunschweig, Germany). The cells were
maintained in Dulbecco´s modified eagle medium (DMEM)
 117

supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/ml

streptomycin and 2 mM glutamine (Thermo Fisher).

5.2.14. Cell uptake: Flow cytometry and confocal laser scanning

microscopy

RAW 264.7 (100 000 cells/well) were seeded out in 24 well plates
(200 µL) for flow cytometry analysis. After overnight incubation at 37
°C, cells were washed and serum-free culture medium was added. It were
also tested the addition of cell culture medium containing 10% human
serum or 10% fetal bovine serum (FBS). For nanoparticle uptake analysis,
the fluorescent dye Coumarin-6 was first encapsulated in the NPs, and
then the BSA-NPs covalent conjugates were produced. Uncoated
nanoparticles and conjugates were applied to cells at a concentration of
37.5 µg mL-1 for 2 h and 24 h.
Afterwards, cells were collected and detached with 2.5 % trypsin
from cell culture wells. Flow cytometry measurements were performed
with Attune™ NxT Flow Cytometer (Invitrogen, USA). The fluorescent
dye Coumarin-6 was excited with a 488 nm laser. Data analysis was
performed using Attune™ NxT software (Invitrogen, USA). Values are
expressed as percentage (%) of fluorescent positive cells as an average of
at least two independent experiments.

5.3. RESULTS AND DISCUSSION

5.3.1. PGlCL nanoparticle formation and functionalization with

NAC

The formation of PGlCL NPs was carried out through the solvent
evaporation method. At first, PGlCl was synthesized by e-ROP using
scCO2 as solvent, and then pre-synthesized PGlCL was used to prepare
the NPs. In order to evaluate size, stability and morphology of the NPs,
DLS and zeta potential measurements were performed, as well as TEM
imaging (Figure 5.2A). The mean particle diameter was determined to be
around 145 nm, with a low PDI value, indicating an uniform particle size
distribution. On the other hand, zeta potential value was determined to be
around -54.0 mV, revealing a good miniemulsion stability. The negative
charge is associated to the presence of SDS (an anionic surfactant)
adsorbed on the surface of the particles (LOOSLI; STOLL, 2017). TEM
imaging corroborate with results obtained by DLS and confirm the
spherical morphology of the NPs.
118

Figure 5.2 - (A) TEM image of PGlCL nanoparticles and information about
particle size and zeta potential; (B) Scheme representing the surface
functionalization of PGlCL nanoparticles with NAC through thiol-ene reaction;
(C) Global double bond consumption and double

Dp: Particle diameter; PDI: polydispersity index; ξ potential: zeta potential

After its preparation, the double bonds present on the surface of

PGlCL NPs were functionalized through thiol-ene reaction with NAC, as
can be seen in Figure 5.2B. The global consumption of the particle double
 119

bonds was determined through 1H-NMR measurements, by comparing

the integral values of the double bond peaks before and after
functionalization with NAC. Besides, the double bond consumption
caused specifically by thiol (SH) groups attachment could be determined.
After the functionalization reaction, the samples were purified and the
supernatant containing excess of NAC was collected. The concentration
of non-reacted NAC was determined by the Ellman’s assay. Figure 5.2C
compares the global double bond consumption and the double bond
consumption caused by SH attachment.
It is possible to observe that the higher the amount of initiator used,
the higher is the global double bond consumption, reaching around 9%
for the highest initiator concentration tested. As described in 5.2.4.1,
NAC was used in sufficient amount to consume 5% of the double bonds,
which indicate that the double bond consumption is not happening only
due to the NAC coupling. On the other hand, the double bond
consumption caused by SH attachment remained almost constant at a
value around 4%, which is consistent to the amount of NAC used. The
consumption of the double bonds may happen not only due to the SH
attachment by thiol-ene reaction, but also because of the occurrence of
other side reactions, such as crosslinking reactions, addition of initiator
derived radicals to the double bonds, head-to-head coupling of carbon-
centered radicals, and cross-termination between carbon-centered and
thiyl radicals (DERBOVEN et al., 2013). The incidence of these side
reactions crucially depend on the applied initiator concentrations. The
higher the initiator concentration applied is, the higher is the incidence of
side reactions (DERBOVEN et al., 2013), and the data obtained in the
present work endorse this behavior. It is possible to notice that small
amounts of initiator are enough to promote effective SH attachment with
a lower incidence of side reactions. This way, in the next steps of this
work, the amount of initiator was fixed in 1 % (mol) relative to the amount
of SH groups.

5.3.2. Covalent conjugation of PGlCL NPs with BSA

After confirming the feasibility of functionalizing the double bonds

on the surface of PGlCL NPs through thiol-ene reaction (item 5.3.1.), the
next step of this work was to use the same type of reaction to covalently
attach cysteine derivative SH groups from BSA to the surface of the NPs.
First, BSA was modified through reaction with the Traut’s reagent, in
order to introduce more SH groups in its structure. This modification is
120

necessary, since BSA has originally only one cysteine with a free thiol
group (SUGIO et al., 1999). After modification, the final protein
concentration was determined by the Pierce assay, and the final
concentration of thiol groups present on modified BSA was determined
by the Ellman’s assay. This way, it was possible to calculate the final
amount of SH groups per BSA molecule, which was on average 5 thiol
groups for each protein molecule.
As shown in Figure 5.3, after purification, DLS, ξ potential and TEM
imaging were performed in order to characterize the conjugates regarding
its size, stability and morphology, respectively. FACS and FCS were
performed to evaluate the success of the covalent conjugation. For FACS
and FCS measurements, the conjugates were produced using the
fluorescently labeled protein, BSA-FITC, which was also modified by
Traut’s reagent before conjugation reactions.
Figure 5.3A shows FACS results for uncoated NPs, non-covalent
and covalent conjugates. FACS measurements give information about the
amount of particles that emitted fluorescent light after excitation by a 488
nm laser in each evaluated sample. For non-covalent conjugates, around
26% of the nanoparticles emitted fluorescence, while for covalent
conjugates, this value was around 36%. For uncoated NPs, a very small
amount of particles presented fluorescence (around 2%), as expected. As
FACS results shows, after purification, covalent conjugates remained
with a higher amount of fluorescent particles, in comparison to the non-
covalent conjugates. Since the fluorescence of the particle is conferred by
the presence of fluorescent protein on its surface, this is a strong
indicative that BSA-FITC was covalently bonded to the nanoparticles,
forming more resistant and stable conjugates.
FCS measurements were also performed, in order to investigate the
covalent attachment of BSA-FITC to the NPs. Figure 5.3B shows the FCS
autocorrelation curves for pure modified BSA-FITC, BSA-FITC non-
covalent conjugates and BSA-FITC covalent conjugates, measured at
excitation wave length of 488 nm. All correlation curves were fitted with
Equation 5.1 (item 5.2.12), and the hydrodynamic radii of the
corresponding fluorescent species were obtained with an experimental
error of ±10%, typical for FCS studies.(WINZEN et al., 2016) For the
pure BSA-FITC sample, a hydrodynamic radius of Rh = 3.3 ± 0.3 nm was
obtained, which matches the values reported for BSA in the literature
(BOHIDAR, 1989). If BSA-FITC molecules get attached to the NPs,
forming conjugates, this attachment should result in a shift of the
autocorrelation curve, since conjugates are large species that diffuses
slower in comparison to small molecules such as BSA, leading to longer
 121

diffusion times. Comparing pure BSA-FITC and BSA-FITC conjugates

autocorrelation curves, it is possible to clearly see these shifts, indicating
that BSA-FITC is binding to the NPs and forming conjugates. BSA-FITC
covalent conjugates curve presented a strong shift in relation to the pure
BSA-FITC curve. For BSA-FITC non-covalent conjugates curve, this
shift also occurred, but in a less intense way. This behavior suggests that
in fact, the interactions between the protein and the particles are stronger
for BSA-FITC covalent conjugates, since they showed to be larger and
slower, which means that more proteins were kept attached to the NPs
surface.
The fitting of the conjugates autocorrelation curves also revealed
that for both covalent and non-covalent conjugates samples, there are two
species responsible for emitting fluorescence: free BSA-FITC, and the
BSA-FITC nanoparticle conjugates, which are the larger and slower
species. In the case of the covalent conjugates, free BSA-FITC is
responsible for the emission of 10% of the detected fluorescence, while
90% of the fluorescence is emitted by the conjugates. On the other hand,
for the non-covalent conjugates, free BSA-FITC is responsible for the
emission of 40% of the detected fluorescence, while 60% of the
fluorescence is emitted by the conjugates. Here we can clearly see that, in
fact, proteins and NPs are interacting much more strongly when BSA-
FITC is covalently bonded to the NPs, forming conjugates that are more
resistant to dissociation.
As shown in Figure 5.3C, after covalent functionalization, the
conjugates presented a very subtle increase in the particle diameter, as
expected, from 145 nm (uncoated NPs - Figure 5.2A), to 152 nm,
probably caused by the formation of the protein layer around the NPs. ξ
potential values also suffered changes after NPs conjugation with BSA,
slightly decreasing from -54 mV (uncoated NPs - Figure 5.2A) to -61.0
mV. This behavior was also observed by other authors (SIMON et al.,
2018) and it is attributed to the BSA charge contribution, since it has an
overall negative charge at pH > 5.5 (PETERS, 1985). Figure 5.3C also
present TEM images of uncoated NPs (images 1 and 3) and covalent
conjugates (images 2 and 4), obtained by the negative staining technique.
These images confirm the formation of a protein layer that surrounds the
nanoparticles. This layer contains proteins that even after purification
remained tightly bonded to the surface of the NPs by covalent bonds or
other strong interactions. Very similar behavior was found in previous
reports (KOKKINOPOULOU et al., 2017; SIMON et al., 2018).
122

Figure 5.3 - (A) FACS histograms for unconjugated NPs, non-covalent and
covalent conjugates (B) FCS autocorrelation curves (symbols) and corresponding
fit (straight lines): BSA-FITC (black diamonds ◊), BSA-FITC non-covalent
conjugates (blue diamonds ◊) and BSA-FITC covalent conjugates (green
diamonds ◊) (C) TEM image of uncoated NPs (1 and 3) and BSA covalent
conjugates (2 and 4) and information about particle size and zeta potential of BSA
covalent conjugates

5.3.3. Cell uptake

The uptake of uncoated NPs and BSA-NPs covalent conjugates into

macrophages (RAW264.7) was analyzed by flow cytometry (Figure 5.4).
NPs and BSA-NPs conjugates were applied to cell culture medium
without additional proteins or supplemented with fetal bovine serum
(FBS) or human serum. This way, it was possible to observe how
 123

conjugation with BSA affects NPs uptake by macrophages in different

environments.
Flow cytometry analysis clearly indicate that, in all cases, the
conjugation with BSA has reduced cell uptake by macrophages. Serum
albumin is often reported as a dysopsonin protein (OGAWARA et al.,
2004; THIELE et al., 2003), which means it inhibits cellular
internalization by phagocytosis. This way, when conjugated to a NP, BSA
hampers the recognition of BSA-NPs conjugates by macrophages,
promoting the so-called “stealth effect” (CARACCIOLO et al., 2015;
SCHÖTTLER et al., 2016). This effect avoids the clearance of the
nanoparticles by the immune system and promotes a prolonged blood
circulation time, enabling the nanoparticle to reach the desired target
(KANG et al., 2015; SIMON et al., 2018).

Figure 5.4 - Cell uptake: Amount of fluorescent positive cells (%) for
unconjugated NPs and BSA-NPs covalent conjugates during (A) 2h and (B) 24h
of incubation.

Another important characteristic that can be observed through these

results is that even when BSA-NPs conjugates are introduced in FBS or
human serum, cellular uptake is still reduced. This means that the stealth
properties obtained by conjugating NPs with BSA were preserved,
reinforcing the idea that stable conjugates were formed.
The covalent modification of the NPs surface with BSA is an
interesting strategy to produce stable nanocarriers, which are able to keep
its stealth properties and to remain in the bloodstream for extended
periods, improving the efficiency of treatments.
124

5.4. CONCLUSIONS

In the present work, PGlCL NPs were successfully conjugated with

bovine serum albumin by thiol-ene reaction, producing BSA-NPs
conjugates which are resistant to dissociation. FACS and FCS
measurements revealed that the conjugates produced by BSA covalent
attachment remained with a larger amount of proteins on its surface even
after the purification processes, indicating a higher stability in comparison
to the conjugates produced by non-covalent method. The conjugates
visualization was possible through TEM imaging, and a protein layer
attached to the surface of the NPs was clearly observed. Cell uptake
assays revealed a reduced internalization of the NPs by macrophages after
conjugation with BSA. These results confirm that covalent BSA-NPs
conjugation is a feasible strategy to produce stable conjugates with stealth
properties.
 125

CHAPTER 6

6. CONCLUDING REMARKS

In this work, the synthesis and functionalization of the copolymer

poly(globalide-co-ε-caprolactone) was performed by using clean high
pressure technologies for future applications with biomedical purposes.
PGlCL e-ROP was performed using scCO2 and scCO2+DCM as
solvents, under different globalide/ε-caprolactone feed ratios. The
reactions were especially successful when conducted under the use of
only scCO2 as solvent. The copolymers synthesized with pure scCO2
reached Mn values up to 25,000 Da, presented a well-defined melting
point and a low formation of oligomeric products, and by this reason they
were chosen to be further functionalized. The use of DCM as cosolvent
increased the formation of cyclic oligomers, since it diluted the reaction
media, favoring intramolecular backbiting. The presence of cyclic
oligomers confers to PGlCL double melting point and wider molecular
weight distribution.
PGlCL produced using pure scCO2 in different Gl/CL ratios was
successfully functionalized with N-acetylcysteine (NAC) through thiol-
ene reaction. Higher globalide contents in PGlCL led to higher
incorporation of NAC to the polymer chain, and the presence of NAC
pendant groups disturbed the formation of crystalline domains, thus
reducing the melting temperature of the copolymers. For copolymers with
high contents of globalide, functionalization resulted in completely
amorphous copolymers and a more hydrophilic character, desirable for
biomedical applications where bioresorption is required. After
functionalization with NAC, the polymer also presented antioxidant
character, probably due to the presence of the the pendant thioether
linkage formed after thiol-ene coupling. This property have an important
role in applications where the material can combat cellular oxidative-
stress.
After PGlCL functionalization with NAC, the work was expanded
to the nanomaterials field, and then PGlCL nanoparticles were prepared
and successfully conjugated with bovine serum albumin by thiol-ene
reaction. As a result, it was produced stable and irreversible BSA-NPs
conjugates. The occurrence of the covalent attachment was observed by
FACS and FCS measurements, revealing that the BSA conjugates
produced by covalent attachment remained with a larger amount of
proteins in its surface in comparison to conjugates produced by non-
126

covalent method. The visualization of the BSA layer attached to the

particles was possible through TEM imaging. Finally, cell uptake assays
revealed that NPs presented stealth properties after conjugation with
BSA, avoiding clearance by immune cells.
This work consisted of an unprecedent study, where it started to be
built a platform of biomaterials based on PGlCL copolymers, synthesized
using clean technologies. Here, it was possible to explore the process
variables of PGlCL synthesis and understand their effect in the final
properties of the material. Besides, it was possible to show different
PGlCL modifications, which enabled the tuning of its properties for future
applications in tissue engineering and in nanomedicine. There are still
many challenges to be overcome in this area and also many modification
strategies to be explored. This work should contribute to the progress of
biomedical engineering and be a basis for future studies leading to
improved biomaterials.
 127

6.1. SUGGESTIONS FOR FUTURE STUDIES

 To produce fibers/scaffolds using PGlCL-NAC and test its

degradation and cell adhesion;
 To functionalize PGlCL with other molecules/biomolecules
containing thiol groups that can improve polymer features for
biomedical applications, and produce fibers/scaffolds with the
modified material;
 To encapsulate medicaments in PGlCL NPs and functionalize the
surface of the the NPs with BSA or other proteins, aiming
specific targeting;
 To produce magnetic PGlCL-NPs for diagnostic imaging or for
tumor treatment, and functionalize it with BSA or other proteins
for specific targeting.
 129

REFERENCES

ABBAS, I. et al. Predictable Peptide Conjugation Ratios by Activation

of Proteins with Succinimidyl Iodoacetate (SIA). Methods and
Protocols, v. 1, n. 1, p. 2, 2017.
ADLERCREUTZ, P. On the importance of the support material for
enzymatic synthesis in organic media: Support effects at controlled
water activity. European Journal of Biochemistry, v. 199, n. 3, p.
609–614, 1991.
AGNELLI, J. A. M.; CHINELATTO, M. A. Degradação de
Polipropileno : Aspectos Teóricos e Recentes Avanços Em Sua
Estabilização. Polímeros: Ciência e Tecnologia, v. 2, n. 3, p. 27–31,
1992.
AL-RAHEIL, A.; QUDAH, A. M. A. On the Triple Melting Behaviour
of Poly ( ethylene succinate ). Polymer International, v. 37, p. 249–
254, 1995.
ALBERTSSON, A.-C.; VARMA, I. K. Recent Developments in Ring
Opening Polymerization of Lactones for Biomedical Applications.
Biomacromolecules, v. 4, n. 6, p. 1466–1486, 2003.
ALBERTSSON, A. C.; SRIVASTAVA, R. K. Recent developments in
enzyme-catalyzed ring-opening polymerization. Advanced Drug
Delivery Reviews, v. 60, n. 9, p. 1077–1093, 2008.
ALBERTSSON, A. C.; VARMA, I. K.; SRIVASTAVA, R. K.
Polyesters from Large Lactones. Handbook of Ring-Opening
Polymerization, p. 287–306, 2009.
ANDRADE, K. Q. DE; MOURA, F. A.; MARQUES, J. Oxidative
Stress and Inflammation in Hepatic Diseases : Therapeutic Possibilities
of N -Acetylcysteine. International Journal of Molecular Sciences, p.
30269–30308, 2015.
ARVIZO, R. R.; DE, M.; ROTELLO, V. M. Proteins and Nanoparticles:
Covalent and Noncovalent Conjugates. Nanobiotechnology II: More
Concepts and Applications, p. 65–78, 2007.
ASTM STANDARD D883-11. Standard Terminology Relating to
Plastics. West Conshohocken, PA.
130

ATES, Z. et al. Functional brush-decorated poly(globalide) films by

ARGET-ATRP for bioconjugation. Macromolecular Bioscience, v. 14,
n. 11, p. 1600–1608, 2014.
ATES, Z. Functional Polymers from Macrolactones. Dublin City
University, 2014.
ATES, Z.; HEISE, A. Functional films from unsaturated
poly(macrolactones) by thiol–ene cross-linking and functionalisation.
Polymer Chemistry, v. 5, n. 8, p. 2936, 2014.
ATES, Z.; THORNTON, P. D.; HEISE, A. Side-chain functionalisation
of unsaturated polyesters from ring-opening polymerisation of
macrolactones by thiol-ene click chemistry. Polymer Chemistry, v. 2,
n. 2, p. 309–312, 2011.
ATHANASIOU, K. A. et al. Orthopaedic applications for PLA-PGA
biodegradable polymers. Arthroscopy, v. 14, n. 7, p. 726–737, 1998.
BADANAI, J. et al. Ability of scavenging free radicals and preventing
lipid peroxidation of some phenols and ascorbic acid. Journal of
Applied Pharmaceutical Science, v. 5, n. 8, p. 34–41, 2015.
BAETKE, S. C.; LAMMERS, T.; KIESSLING, F. Applications of
nanoparticles for diagnosis and therapy of cancer. British Journal of
Radiology, v. 88, n. 1054, 2015.
BARBANTI, S. H.; ZAVAGLIA, C. A. C.; DUEK, E. A. R. Polímeros
Bioreabsorvíveis na Engenharia de Tecidos. Polímeros, v. 15, p. 13–21,
2005.
BAUR, V. H. Einfluß der Sequenzlangenverteilung auf das Schmelz-
Ende von Copolymeren. Die Makromolekulare Chemie, v. 98, n.
2364, p. 297–301, 1966.
BECKA, A.; LOEB, G. Ease of removal of barnacles from various
polymeric materials. Biotechnology and Bioengineering, v. 26, n. 10,
p. 1245–1251, 1984.
BECKMAN, E. J. Supercritical and near-critical CO2 in green chemical
synthesis and processing. Journal of Supercritical Fluids, v. 28, n. 2–
3, p. 121–191, 2004.
BERK, M. et al. The promise of N-acetylcysteine in neuropsychiatry.
 131

Trends in Pharmacological Sciences, p. 1–11, 2013.

BERNHARDT, A. et al. Proliferation and osteogenic differentiation of
human bone marrow stromal cells on alginate – gelatine –
hydroxyapatite scaffolds with anisotropic pore structure. Journal of
Tissue Engineering and Regenerative Medicine, n. November 2008,
p. 54–62, 2009.
BISHAI, M. et al. A comprehensive study on enhanced characteristics
of modified polylactic acid based versatile biopolymer. European
Polymer Journal, v. 54, p. 52–61, 2014.
BISHT, K. S. et al. Enzyme-Catalyzed Ring-Opening Polymerization of
ω-Pentadecalactone. Macromolecules, v. 30, n. 9, p. 2705–2711, 1997.
BOHIDAR, H. B. Light scattering study of solution properties of bovine
serum albumin, insulin, and polystyrene under moderate pressure.
Colloid and Polymer Science, v. 267, n. 4, p. 292–300, 1989.
BORDES, C. et al. Determination of poly(ε-caprolactone) solubility
parameters: Application to solvent substitution in a microencapsulation
process. International Journal of Pharmaceutics, v. 383, n. 1–2, p.
236–243, 2010.
BOYER, C. et al. An overview of protein-polymer particles. Soft
Matter, v. 7, n. 5, p. 1599–1614, 2011.
BRANDOLINI, A. J.; HILLS, D. D. NMR Spectra of Polymers and
Polymer Additives. New York: Marcel Dekker, 2000.
BRAR, A. S.; GOYAL, A. K.; HOODA, S. Two-dimensional NMR
studies of acrylate copolymers. Pure and Applied Chemistry, v. 81, n.
3, p. 389–415, 2009.
BROWN, R. L.; STEIN, S. E. No Title. In: LINSTROM, P. J.;
MALLARD, W. G. (Eds.). NIST Chemistry WebBook, NIST
Standard Reference Database Number 69. Gaithersburg, MD:
National Institute of Standards and Technology, 2016.
BRUNNER, G. Gas Extraction: an introduction to fundamentals of
supercritical fluids and the application to separation processes.
Heidelberg: Steinkopff, 1994.
BRUNNER, G. Supercritical fluids as solvents and reaction media.
132

Elsevier, 2004.
BUCHANAN, F. J. Degradation Rate of Bioresorbable Materials:
Prediction and Evaluation. Cambridge: Woodhead Publishing
Limited, 2008.
BURRIDGE, K. Focal Adhesions: Transmembrane Junctions Between
The Extracellular Matrix And The Cytoskeleton. Annual Review of
Cell and Developmental Biology, v. 4, n. 1, p. 487–525, 1988.
CAI, J. et al. Effects of molecular weight on poly(ω-pentadecalactone)
mechanical and thermal properties. Polymer, v. 51, n. 5, p. 1088–1099,
2010.
CALLOW, J. A. et al. The influence of surface energy on the wetting
behaviour of the spore adhesive of the marine alga Ulva linza (synonym
Enteromorpha linza). Journal of The Royal Society Interface, v. 2, n.
4, p. 319–325, 2005.
CANEVAROLO JR., S. V. Ciência dos Polímeros - Um texto básico
para tecnólogos e engenheiros. 2a Edição ed. São Paulo: Artliber,
2006.
CARACCIOLO, G. The protein corona effect for targeted drug delivery.
Bioinspired, Biomimetic and Nanobiomaterials, v. 2, n. 1, p. 54–57,
2013.
CARACCIOLO, G. et al. Stealth Effect of Biomolecular Corona on
Nanoparticle Uptake by Immune Cells. Langmuir, v. 31, n. 39, p.
10764–10773, 2015.
CARUSO, F. Nanoengineering of particle surfaces. Advanced
Materials, v. 13, n. 1, p. 11–22, 2001.
CASALINI, T. Bioresorbability of polymers: chemistry, mechanisms,
and modeling. In: Bioresorbable Polymers for Biomedical
Applications. Cambridge: Woodhead Publishing-Elsevier, 2017. p. 65–
83.
CAZZOLA, M. et al. Influence of N -acetylcysteine on chronic
bronchitis or COPD exacerbations : a meta-analysis. European
Respiratory Review, p. 451–461, 2015.
CECCORULLI, G. et al. Cocrystallization of random copolymers of
 133

omega-pentadecalactone and epsilon-caprolactone synthesized by lipase

catalysis. Biomacromolecules, v. 6, n. 2, p. 902–907, 2005.
CHANDA, M.; ROY, S. K. Plastics Fundamentals, Properties, and
Testing. Boca Ratón: CRC-press, 2010.
CHEN, E. Y.; LIU LORETO MEGIDO, W. F.; DÍEZ, P. Understanding
and utilizing the biomolecule/nanosystems interface. In:
Nanotechnologies in Preventive and Regenerative Medicine.
Elsevier, 2018. p. 207–297.
CHEN, F. et al. Antioxidant and antibacterial activities of eugenol and
carvacrol-grafted chitosan nanoparticles. Biotechnology and
Bioengineering, v. 104, n. 1, p. 30–39, 2009.
CHEW, S. Y. et al. The effect of the alignment of electrospun fibrous
scaffolds on Schwann cell maturation. Biomaterials, v. 29, n. 6, p. 653–
661, 2008.
CLAUDINO, M. Thiol–ene Coupling of Renewable Monomers: at
the forefront of bio-based polymeric materials. Kungliga Tekniska
Högskolan, 2011.
CLAUDINO, M. et al. Photoinduced thiol-ene crosslinking of
globalide/ε-caprolactone copolymers: Curing performance and resulting
thermoset properties. Journal of Polymer Science, Part A: Polymer
Chemistry, v. 50, n. 1, p. 16–24, 2012.
COMIM ROSSO, S. R. et al. Enzymatic synthesis of poly(ε-
caprolactone) in supercritical carbon dioxide medium by means of a
variable-volume view reactor. Journal of Supercritical Fluids, v. 79,
p. 133–141, 2013.
COMIM ROSSO, S. R. et al. Enzymatic synthesis of poly(ɛ-
caprolactone) in liquified petroleum gas and carbon dioxide. The
Journal of Supercritical Fluids, v. 96, p. 334–348, 2015.
CÓRDOVA, A. et al. Lipase-catalysed formation of macrocycles by
ring-opening polymerisation of ε-caprolactone. Polymer, v. 39, n. 25, p.
6519–6524, 1998.
COSTA-PINTO, A. R.; REIS, R. L.; NEVES, N. M. Scaffolds Based
Bone Tissue Engineering: The Role of Chitosan. Tissue Engineering
Part B: Reviews, v. 17, n. 5, p. 331–347, 2011.
134

COULEMBIER, O. et al. From controlled ring-opening polymerization

to biodegradable aliphatic polyester: Especially poly(β -malic acid)
derivatives. Progress in Polymer Science (Oxford), v. 31, n. 8, p. 723–
747, 2006.
CRESCENZI, V. et al. Thermodynamics of fusion of poly-β-
propiolactone and poly-ε-caprolactone. comparative analysis of the
melting of aliphatic polylactone and polyester chains. European
Polymer Journal, v. 8, n. 3, p. 449–463, 1972.
DALLA-VECCHIA, R.; NASCIMENTO, M. D. G.; SOLDI, V.
Aplicações sintéticas de lipases imobilizadas em polímeros. Quimica
Nova, v. 27, n. 4, p. 623–630, 2004.
DE, M. et al. Biomimetic interactions of proteins with functionalized
nanoparticles: A thermodynamic study. Journal of the American
Chemical Society, v. 129, n. 35, p. 10747–10753, 2007.
DEAN, O.; GIORLANDO, F.; BERK, M. N -acetylcysteine in
psychiatry : current therapeutic evidence and potential mechanisms of
action. Journal of Psychiatry and Neuroscience, v. 36, n. 2, p. 78–86,
2011.
DERBOVEN, P. et al. Kinetic Modeling of Radical Thiol − Ene
Chemistry for Macromolecular Design: Importance of Side Reactions
and Di ff usional Limitations. Macromolecules, v. 46, p. 1732–1742,
2013.
DHOUIB, I. E. et al. A minireview on N-acetylcysteine: An old drug
with new approaches. Life Sciences, 2016.
DONDONI, A. The emergence of thiol-ene coupling as a click process
for materials and bioorganic chemistry. Angewandte Chemie -
International Edition, v. 47, n. 47, p. 8995–8997, 2008.
DOWLING, D. P. et al. Effect of Surface Wettability. Journal of
Biomaterials Applications, v. 26, p. 327, 2011.
DUNCAN, R. Polymer conjugates as anticancer nanomedicines. Nature
Reviews Cancer, v. 6, n. 9, p. 688–701, 2006.
DUNN, A. S.; CAMPBELL, P. G.; MARRA, K. G. The influence of
polymer blend composition on the degradation of polymer /
hydroxyapatite biomaterials. Journal of Materials Science: Materials
 135

in Medicine, v. 2, p. 673–677, 2001.

EHRENBERG, M. S. et al. The influence of protein adsorption on
nanoparticle association with cultured endothelial cells. Biomaterials,
v. 30, n. 4, p. 603–610, 2009.
ELOMAA, L. et al. Preparation of poly(ε-caprolactone)-based tissue
engineering scaffolds by stereolithography. Acta Biomaterialia, v. 7, n.
11, p. 3850–3856, 2011.
FERRARI, R. et al. Tunable PLGA-based nanoparticles synthesized
through free-radical polymerization. Macromolecular Materials and
Engineering, v. 298, n. 7, p. 730–739, 2013.
FLORY, P. J. Principles of Polymer Chemistry. Ithaca: Cornell
University Press, 1953.
FOCARETE, M. L. et al. Physical Characterization of Poly (ω-
pentadecalactone ) Synthesized by Lipase-Catalyzed Ring-Opening
Polymerization. J. Polym. Sci. Part B: Polym. Phys., v. 39, p. 1721–
1729, 2001.
FOSSEY, J.; LEFFORT, D.; SORBA, J. Free Radicals in Organic
Chemistry. New York: John Wiley & Sons, 1995.
FUKUDA, H.; KONDO, A.; NODA, H. Biodiesel fuel production by
transesterification of oils. Journal of Bioscience and Bioengineering,
v. 92, n. 5, p. 405–416, 2001.
GEUS, M. et al. Performance polymers from renewable monomers: high
molecular weight poly(pentadecalactone) for fiber applications.
Polymer Chemistry, v. 1, n. 4, p. 525–533, 2010.
GEUS, M. DE et al. Investigation of Factors Influencing the
Chemoenzymatic Synthesis of Block Copolymers. Macromolecules, v.
38, p. 4220–4225, 2005.
GEUS, M. DE. Enzymatic Catalysis in the Synthesis of New Polymer
Architectures and Materials. Technische Universiteit Eindhoven,
2007.
GUALANDI, C. et al. Scaffold for tissue engineering fabricated by non-
isothermal supercritical carbon dioxide foaming of a highly crystalline
polyester. Acta Biomaterialia, v. 6, n. 1, p. 130–136, 2010.
136

GUELCHER, S. A.; HOLLINGER, J. O. An Introduction to

Biomaterials. In: MICHAEL R. NEUMAN (Ed.). The Biomedical
Engineering Series. Boca Raton: CRC Press, 2006.
GUINDANI, C. et al. Enzymatic ring opening copolymerization of
globalide and ε-caprolactone under supercritical conditions. The
Journal of Supercritical Fluids, v. 128, p. 404–411, 2017.
GUNASEELAN, S. et al. Surface modi fi cations of nanocarriers for
effective intracellular delivery of anti-HIV drugs ☆. Advanced Drug
Delivery Reviews, v. 62, n. 4–5, p. 518–531, 2010.
HEIDEMANN, W. et al. Degradation of poly(D,L)lactide implants with
or without addition of calciumphosphates in vivo. Biomaterials, v. 22,
n. 17, p. 2371–2381, 2001.
HENDERSON, L. A. et al. Enzyme-Catalyzed Polymerizations of ε-
Caprolactone: Effects of Initiator on Product Structure, Propagation
Kinetics, and Mechanism. Macromolecules, v. 29, n. 24, p. 7759–7766,
1996.
HIRAISHI, N. et al. Susceptibility of a Polycaprolactone-based Root
Canal-filling Material to Degradation. III. Turbidimetric Evaluation of
Enzymatic Hydrolysis. Journal of Endodontics, v. 33, n. 8, p. 952–
956, 2007.
HMDB. Human Metabolome Database. Disponível em:
<http://www.hmdb.ca/spectra/nmr_one_d/1783>. Acesso em: 13 nov.
2017.
HOYLE, C. E.; BOWMAN, C. N. Thiol-Ene Click Chemistry.
Angewandte Chemie International Edition, v. 49, n. 9, p. 1540–1573,
2010.
HOYLE, C. E.; LEE, T. Y.; ROPER, T. Thiol-enes: Chemistry of the
past with promise for the future. Journal of Polymer Science Part A:
Polymer Chemistry, v. 42, n. 21, p. 5301–5338, 2004.
IKADA, Y.; TSUJI, H. Biodegradable polyesters for medical and
ecological applications. Macromolecular Rapid Communications, v.
21, n. 3, p. 117–132, 2000.
ISHIDA, T.; HARASHIMA, H.; KIWADA, H. Interactions of
Liposomes with Cells In Vitro and In Vivo: Opsonins and Receptors.
 137

Current Drug Metabolism, v. 2, n. 4, p. 397–409, 2001.

JACOBS, C. et al. Macromolecular engineering of polylactones and
polylactides. 5. Synthesis and characterization of diblock copolymers
based on poly-ε-caprolactone and poly(L,L or D,L)lactide by aluminum
alkoxides. Macromolecules, v. 24, n. 11, p. 3027–3034, 1991.
JANSEN, M. A. G. Modification of poly ( butylene terephthalate ) by
incorporation of comonomers in the solid state. Technische
Universiteit Eindhoven, 2005.
JEON, H.; LEE, H.; KIM, G. A Surface-Modified Poly(ε-caprolactone)
Scaffold Comprising Variable Nanosized Surface-Roughness Using a
Plasma Treatment. Tissue Engineering Part C: Methods, v. 20, n. 12,
p. 951–963, 2014.
JÉRÔME, C.; LECOMTE, P. Recent advances in the synthesis of
aliphatic polyesters by ring-opening polymerization. Advanced Drug
Delivery Reviews, v. 60, n. 9, p. 1056–1076, 2008.
JIA, W. T. Dental filling material, United States Patent Application
20050066854, 2005.
JIA, W. T.; ALPERT, B. Root canal filling material, United States
Patent Application 20030113686, 2003.
JIA, W. T.; TROPE, M.; ALPERT, B. Dental filling material, United
States Patent Application 20050069836, 2005.
JOHNSON, P. M.; KUNDU, S.; BEERS, K. L. Modeling enzymatic
kinetic pathways for ring-opening lactone polymerization.
Biomacromolecules, v. 12, n. 9, p. 3337–3343, 2011.
KANG, B. et al. Carbohydrate-Based Nanocarriers Exhibiting Specific
Cell Targeting with Minimum Influence from the Protein Corona.
Angewandte Chemie - International Edition, v. 54, n. 25, p. 7436–
7440, 2015.
KELEŞ, E.; HAZER, B.; CÖMERT, F. B. Synthesis of antibacterial
amphiphilic elastomer based on polystyrene-block-polyisoprene-block-
polystyrene via thiol-ene addition. Materials Science and Engineering
C, v. 33, n. 3, p. 1061–1066, 2013.
KHALILI, A. A.; AHMAD, M. R. A Review of cell adhesion studies
138

for biomedical and biological applications. International Journal of

Molecular Sciences, v. 16, n. 8, p. 18149–18184, 2015.
KIM, T. G.; PARK, T. A. E. G.; PH, D. Biomimicking Extracellular
Matrix : Tissue Engineering, v. 12, n. 2, p. 221–233, 2006.
KNANI, D.; GUTMAN, A. L.; KOHN, D. H. Enzymatic
polyesterification in organic media. Enzyme-catalyzed synthesis of
linear polyesters. I. Condensation polymerization of linear
hydroxyesters. II. Ring-opening polymerization of ε-caprolactone.
Journal of Polymer Science Part A: Polymer Chemistry, v. 31, n. 5,
p. 1221–1232, 1993.
KOBAYASHI, S. et al. Enzymatic Polymerization. Chemical Reviews,
v. 101, n. 12, p. 3793–3818, 2001.
KOBAYASHI, S. Lipase-catalyzed polyester synthesis - A green
polymer chemistry. Proceedings of the Japan Academy, Series B:
Physical and Biological Sciences, v. 86, p. 338–365, 2010.
KOBAYASHI, S.; UYAMA, H.; TAKAMOTO, T. Lipase-catalyzed
degradation of polyesters in organic solvents. A new methodology of
polymer recycling using enzyme as catalyst. Biomacromolecules, v. 1,
n. 1, p. 3–5, 2000.
KOKKINOPOULOU, M. et al. Visualization of the protein corona:
Towards a biomolecular understanding of nanoparticle-cell-interactions.
Nanoscale, v. 9, n. 25, p. 8858–8870, 2017.
KOLB, H. C.; FINN, M. G.; SHARPLESS, K. B. Click Chemistry:
Diverse Chemical Function from a Few Good Reactions. Angewandte
Chemie - International Edition, v. 40, n. 11, p. 2004–2021, 2001.
KUMAR, A. et al. Efficient Ring-Opening Polymerization and
Copolymerization of -Caprolactone and ω-Pentadecalactone Catalyzed
by Candida antartica Lipase B. Macromolecules, v. 33, p. 6303–6309,
2000.
KUMAR, A.; GROSS, R. A. Candida antartica lipase B catalyzed
polycaprolactone synthesis: effects of organic media and temperature.
Biomacromolecules, v. 1, n. 1, p. 133–8, 2000.
KUMAR, R.; MADRAS, G.; MODAK, J. Enzymatic Synthesis of Ethyl
Palmitate in Supercritical Carbon Dioxide. Industrial and Engineering
 139

Chemistry Research, v. 43, n. 7, p. 1568–1573, 2004.

KUNDU, S. et al. Continuous flow enzyme-catalyzed polymerization in
a microreactor. Journal of the American Chemical Society, v. 133, n.
15, p. 6006–6011, 2011.
LABET, M.; THIELEMANS, W. Synthesis of polycaprolactone: a
review. Chemical Society Reviews, v. 38, n. 12, p. 3484–3504, 2009.
LESZCZYNSKI, J. Bionanoscience: Nano meets bio at the interface.
Nature Nanotechnology, v. 5, n. 9, p. 633–634, 2010.
LI, Q. et al. Ring-opening polymerization of ε-caprolactone catalyzed by
a novel thermophilic lipase from Fervidobacterium nodosum. Process
Biochemistry, v. 46, n. 1, p. 253–257, 2011.
LIU, Y. et al. Thiol-ene click chemistry: A biocompatible way for
orthogonal bioconjugation of colloidal nanoparticles. Chemical
Science, v. 8, n. 9, p. 6182–6187, 2017.
LOEKER, F. C. et al. Enzyme-Catalyzed Ring-Opening Polymerization
of ε-caprolactone in Supercritical Carbon Dioxide. Macromolecules, v.
37, p. 2450–2453, 2004.
LOOSLI, F.; STOLL, S. Effect of surfactants, pH and water hardness on
the surface properties and agglomeration behavior of engineered
TiO2nanoparticles. Environmental Science: Nano, v. 4, n. 1, p. 203–
211, 2017.
LOW, S. W. et al. Use of OsteoplugTM polycaprolactone implants as
novel burr-hole covers. Singapore Medical Journal, v. 50, n. 8, p.
777–780, 2009.
LOWE, A. B. Thiol-ene “click” reactions and recent applications in
polymer and materials synthesis. Polymer Chemistry, v. 1, n. 1, p. 17–
36, 2009.
LYNCH, I.; DAWSON, K. A. Protein-nanoparticle interactions. Nano
Today, v. 3, n. 1–2, p. 40–47, 2008.
LYNCH, I.; SALVATI, A.; DAWSON, K. A. Protein-nanoparticle
interactions: What does the cell see? Nature Nanotechnology, v. 4, n.
9, p. 546–547, 2009.
MAHMOUDI, M. et al. Protein-nanoparticle interactions: Opportunities
140

and challenges. Chemical Reviews, v. 111, n. 9, p. 5610–5637, 2011.

MCEVER, R. P.; ZHU, C. Rolling Cell Adhesion. Annual Review of
Cell and Developmental Biology, v. 26, n. 1, p. 363–396, 2010.
MCNAMARA, K.; TOFAIL, S. A. M. Nanoparticles in biomedical
applications. Advances in Physics: X, v. 2, n. 1, p. 54–88, 2017.
MEI, Y.; KUMAR, A.; GROSS, R. Kinetics and mechanism of Candida
antarctica lipase B catalyzed solution polymerization of ε-caprolactone.
Macromolecules, v. 36, n. 15, p. 5530–5536, 2003.
MEI, Y.; KUMAR, A.; GROSS, R. A. Probing water-temperature
relationships for Lipase-catalyzed lactone ring-opening polymerizations.
Macromolecules, v. 35, n. 14, p. 5444–5448, 2002.
MENSOR, L. L. et al. Screening of Brazilian plant extracts for
antioxidant activity by the use of DPPH free radical method.
Phytotherapy Research, v. 15, n. 2, p. 127–130, 2001.
MESIANO, A. J.; BECKMAN, E. J.; RUSSELL, A. J. Supercritical
Biocatalysis. Chemical Reviews, v. 99, n. 2, p. 623–633, 1999.
MICHROWSKA, A.; WAWRZYNIAK, P.; GRELA, K. Synthesis of
macrocyclic carbonates with musk odor by ring-closing olefin
metathesis. European Journal of Organic Chemistry, n. 9, p. 2053–
2056, 2004.
MILETIĆ, N.; LOOS, K.; GROSS, R. A. Enzymatic Polymerization of
Polyester. In: LOSS, K. (Ed.). Biocatalysis in Polymer Chemistry.
Groningen: Wiley e Sons, 2010. p. 83–129.
MIYATA, T.; MASUKO, T. Crystallization behaviour of
poly(tetramethylene succinate). Polymer, v. 39, n. 7, p. 1399–1404,
1998.
MOGHIMI, S. M. et al. Coating particles with a block co-polymer (
poloxamine-908 ) suppresses opsonization but permits the activity of
dysopsonins in the serum. Biochimica et Biophysica Acta, v. 1179, p.
157–165, 1993.
MONOPOLI, M. P. et al. Physical-Chemical aspects of protein corona:
Relevance to in vitro and in vivo biological impacts of nanoparticles.
Journal of the American Chemical Society, v. 133, n. 8, p. 2525–
 141

2534, 2011.
NAIR, L. S.; LAURENCIN, C. T. Polymers as biomaterials for tissue
engineering and controlled drug delivery. Advances in Biochemical
Engineering/Biotechnology, v. 102, n. October 2005, p. 47–90, 2006.
NIH. Toxicological Data, compiled by the national institute of health
(NIH), USA, selected and distributed by technical database services
(TDS), 2009.
NOMURA, R.; UENO, A.; ENDO, T. Anionic Ring-Opening
Polymerization. Macromolecules, v. 27, p. 620–621, 1994.
OECD. OECD 301 - Ready Biodegradability. OECD Guidelines for
the Testing of Chemicals, v. 301, n. July, p. 1–62, 1992.
OGAWARA, K. I. et al. Pre-coating with serum albumin reduces
receptor-mediated hepatic disposition of polystyrene nanosphere:
Implications for rational design of nanoparticles. Journal of Controlled
Release, v. 100, n. 3, p. 451–455, 2004.
OLIVEIRA, D. et al. Optimization of enzymatic production of biodiesel
from castor oil in organic solvent medium. Applied biochemistry and
biotechnology, v. 113–116, p. 771–780, 2004.
OLIVEIRA, D. et al. Assessment of two immobilized lipases activity
treated in compressed fluids. Journal of Supercritical Fluids, v. 38, n.
3, p. 373–382, 2006.
OLIVEIRA, J. V.; OLIVEIRA, D. Kinetics of the Enzymatic
Alcoholysis of Palm Kernel Oil in Supercritical CO2. Industrial &
Engineering Chemistry Research, v. 39, n. 12, p. 4450–4454, 2000.
OSOL, A. Remington’s Pharmaceutical Sciences. 14th ed. ed. Easton,
PA: Mack Publishing Co., 1970.
OWENS, D. E.; PEPPAS, N. A. Opsonization, biodistribution, and
pharmacokinetics of polymeric nanoparticles. International Journal of
Pharmaceutics, v. 307, n. 1, p. 93–102, 2006.
PACHENCE, J. M.; BOHRER, M. P.; KOHN, J. Biodegradable
polymers. In: LANZA, R.; LANGER, R.; VACANTI, J. (Eds.).
Principles of Tissue Engineering. 3rd Editio ed. Elsevier, 2007. p.
323–339.
142

PASCH, H.; WOLFGANG, S. MALDI-TOF Mass Spectrometry of

Synthetic Polymers. 1st Editio ed. Berlin Heidelberg: Springer-Verlag,
2003.
PENCZEK, S. et al. Living ring-opening polymerizations of
heterocyclic monomers. Progress in Polymer Science (Oxford), v. 32,
n. 2, p. 247–282, 2007.
PÉREZ-ROSÉS, R. et al. Biological and non-biological antioxidant
activity of some essential oils . Author. Journal of Agricultural and
Food Chemistry, 2016.
PETERS, T. Serum Albumin. Advances in Protein Chemistry, v. 37,
n. C, p. 161–245, 1985.
PINO, P. DEL et al. Protein corona formation around nanoparticles -
From the past to the future. Materials Horizons, v. 1, n. 3, p. 301–313,
2014.
PITT, C. G.; MARKS, T. A.; SCHINDLER, A. Controlled Drug
Release of Bioactive Materials. New York: Academic Press, 1980.
POLLONI, A. E. et al. Enzymatic ring opening polymerization of ω-
pentadecalactone using supercritical carbon dioxide. The Journal of
Supercritical Fluids, v. 119, p. 221–228, 2017.
POUNDER, R. J.; DOVE, A. P. Towards poly(ester) nanoparticles:
recent advances in the synthesis of functional poly(ester)s by ring-
opening polymerization. Polymer Chemistry, p. 260–271, 2010.
QIN, J. X. et al. New insight into the difference of PC lipase-catalyzed
degradation on poly(butylene succinate)-based copolymers from
molecular levels. RSC Advances, v. 6, n. 22, p. 17896–17905, 2016.
RANA, S.; YEH, Y.-C.; ROTELLO, V. M. Engineering the
nanoparticle–protein interface: applications and possibilities. Current
Opinion in Chemical Biology, v. 14, n. 6, p. 828–834, 2010.
RE, R. et al. Antioxidant activity applying an improved ABTS radical
cation decolorization assay. Free Radical Biology and Medicine, v. 26,
n. 9–10, p. 1231–1237, 1999.
REINHART-KING, C. A.; DEMBO, M.; HAMMER, D. A. The
dynamics and mechanics of endothelial cell spreading. Biophysical
 143

Journal, v. 89, n. 1, p. 676–689, 2005.

RENZ, P. et al. Imaging of Polymeric Nanoparticles : Hard Challenge
for Soft Objects. p. 1879–1885, 2016.
RICHETTI, A. et al. Assessment of process variables on 2-ethylhexyl
palmitate production using Novozym 435 as catalyst in a solvent-free
system. Bioprocess and Biosystems Engineering, v. 33, n. 3, p. 331–
337, 2010.
RITZ, S. et al. Protein Corona of Nanoparticles: Distinct Proteins
Regulate the Cellular Uptake. Biomacromolecules, v. 16, n. 4, p. 1311–
1321, 2015.
ROSA, C. D. et al. Lipase-catalyzed production of fatty acid ethyl esters
from soybean oil in compressed propane. Journal of Supercritical
Fluids, v. 47, n. 1, p. 49–53, 2008.
SAHA, S. K.; TSUJI, H. Hydrolytic degradation of amorphous films of
L-lactide copolymers with glycolide and D-lactide. Macromolecular
Materials and Engineering, v. 291, n. 4, p. 357–368, 2006.
SALARBASHI, D. et al. Development of new active packaging film
made from a soluble soybean pol‐ ysaccharide incorporated Zataria
multiflora Boiss and Mentha pulegium essen‐ tial oils Davoud. Food
Chemistry, 2013.
SALMASO, S.; CALICETI, P. Stealth Properties to Improve
Therapeutic Efficacy of Drug Nanocarriers. Journal of Drug Delivery,
v. 2013, 2013.
SANTOS, R. D. et al. Lipase-catalyzed synthesis of poly(ε-
caprolactone) in supercritical carbon dioxide. Biocatalysis and
Agricultural Biotechnology, v. 1, n. 4, p. 280–283, 2012.
SCHÖTTLER, S. et al. Protein adsorption is required for stealth effect
of poly(ethylene glycol)- and poly(phosphoester)- coated nanocarriers.
Nature Nanotechnology, v. 11, n. February, p. 1–6, 2016.
SCHÖTTLER, S.; LANDFESTER, K.; MAILÄNDER, V. Controlling
the Stealth Effect of Nanocarriers through Understanding the Protein
Corona. Angewandte Chemie - International Edition, v. 55, n. 31, p.
8806–8815, 2016.
144

SELVAKUMAR, R. et al. Functionalization od scaffolds with

biomolecules for various types of tissue engineering applications. In:
AUGUSTINE, R. et al. (Eds.). Nanomedicine and Tissue
Engineering: State of the Art and Recent Trends. 1. ed. Apple
academic press/CRC press, 2015.
SIMON, J. et al. Exploiting the biomolecular corona: pre-coating of
nanoparticles enables controlled cellular interactions. Nanoscale, v. 10,
n. 22, p. 10731–10739, 2018.
SINHA, V. R. et al. Poly-ε-caprolactone microspheres and nanospheres:
An overview. International Journal of Pharmaceutics, v. 278, n. 1, p.
1–23, 2004.
SIVALINGAM, G.; CHATTOPADHYAY, S.; MADRAS, G. Solvent
effects on the lipase catalyzed biodegradation of poly (ε-caprolactone)
in solution. Polymer Degradation and Stability, v. 79, n. 3, p. 413–
418, 2003.
SIVALINGAM, G.; MADRAS, G. Modeling of Lipase Catalyzed Ring-
Opening Polymerization of ɛ-caprolactone. Biomacromolecules, v. 5, p.
603–609, 2004.
SMYTH, H. F. et al. Range-Finding Toxicity Data: List VII. American
Industrial Hygiene Association Journal, v. 30, n. 5, p. 470–476, 1969.
SUGIO, S. et al. Crystal structure of human serum albumin at 2.5 A
resolution. Protein engineering, v. 12, n. 6, p. 439–46, 1999.
SYROMOTINA, D. S. et al. Surface wettability and energy effects on
the biological performance of poly-3-hydroxybutyrate films treated with
RF plasma. Materials Science and Engineering C, v. 62, p. 450–457,
2016.
TAYLOR, P. et al. Antioxidant and radical scavenging activities of
chamazulene. Natural Product Research, v. 28, p. 2321–2323, 2014.
THIELE, L. et al. Competitive adsorption of serum proteins at
microparticles affects phagocytosis by dendritic cells. Biomaterials, v.
24, n. 8, p. 1409–1418, 2003.
THURECHT, K. J. et al. Kinetics of enzymatic ring-opening
polymerization of ε-caprolactone in supercritical carbon dioxide.
Macromolecules, v. 39, n. 23, p. 7967–7972, 2006.
 145

TIPTIPAKORN, S. et al. Effects of polycaprolactone molecular weights

on thermal and mechanical properties of polybenzoxazine. Journal of
Applied Polymer Science, v. 132, n. 18, p. 1–11, 2015.
TÖRMÄLÄ, P.; POHJONEN, T.; ROKKANEN, P. Bioabsorbable
polymers: materials technology and surgical applications. Proceedings
of the Institution of Mechanical Engineers. Part H, Journal of
engineering in medicine, v. 212, n. 2, p. 101–111, 1998.
TORRES, E. et al. Influence of the Hydrophobic-Hydrophilic Nature of
Biomedical Polymers and Nanocomposites on In Vitro Biological
Development. Macromolecular Materials and Engineering, v.
1700259, p. 1–10, 2017.
TREUEL, L. et al. Impact of protein modification on the protein corona
on nanoparticles and nanoparticle-cell interactions. ACS Nano, v. 8, n.
1, p. 503–513, 2014.
TUBA, F.; OLÁH, L.; NAGY, P. Towards the understanding of the
molecular weight dependence of essential work of fracture in semi-
crystalline polymers: A study on poly(ε-caprolactone). Express
Polymer Letters, v. 8, n. 11, p. 869–879, 2014.
TUNCA, U. Orthogonal multiple click reactions in synthetic polymer
chemistry. Journal of Polymer Science, Part A: Polymer Chemistry,
v. 52, n. 22, p. 3147–3165, 2014.
UPPENBERG, J. et al. The sequence, crystal structure determination
and refinement of two crystal forms of lipase B from Candida antarctica.
Structure, v. 2, p. 293–308, 1994.
UYAMA, H. et al. Lipase-Catalyzed Ring-Opening Polymerization of
12-Dodecanolide. Macromolecules, v. 28, p. 7046–7050, 1995.
UYAMA, H.; KOBAYASHI, S. Enzymatic Ring-Opening
Polymerization of Lactones Catalyzed by Lipase. Chemistry Letters, v.
22, n. 7, p. 1149–1150, 1993.
VAN DER MEULEN, I. et al. Polymers from functional macrolactones
as potential biomaterials: Enzymatic ring opening polymerization,
biodegradation, and biocompatibility. Biomacromolecules, v. 9, n. 12,
p. 3404–3410, 2008.
VAN DER MEULEN, I. Polyesters from natural macrolactones for
146

biomedical applications. Technische Universiteit Eindhoven, 2010.

VAN DER MEULEN, I. et al. Copolymers from unsaturated
macrolactones: Toward the design of cross-linked biodegradable
polyesters. Biomacromolecules, v. 12, n. 3, p. 837–843, 2011.
VAN LITH, R. et al. Engineering biodegradable polyester elastomers
with antioxidant properties to attenuate oxidative stress in tissues.
Biomaterials, v. 35, n. 28, p. 8113–8122, 2014.
VAN NATTA, F. J.; HILL, J. W.; CAROTHERS, W. H. Studies of
polymerization and ring formation. XXIII. ε-Caprolactone and its
polymers. Journal of the American Chemical Society, v. 56, n. 2, p.
455–457, 1934.
VENERAL, J. G. Produção enzimática de poli(ɛ-caprolactona) em
reator de leito empacotado utilizando fluido pressurizado.
Universidade Federal de Santa Catarina, 2014.
VERT, M. et al. Bioresorbability and biocompatibility of aliphatic
polyesters. Journal of Materials Science: Materials in Medicine, v. 3,
n. 6, p. 432–446, 1992.
WANG, Y. et al. Expansion and osteogenic differentiation of bone
marrow-derived mesenchymal stem cells on a vitamin C functionalized
polymer. Biomaterials, v. 27, n. 17, p. 3265–3273, 2006.
WENDLING, J.; SUTER, U. W. A New Model Describing the
Cocrystallization Behavior of Random Copolymers. Macromolecules,
v. 31, n. 8, p. 2516–2520, 1998.
WILLIAMS, A. S. The Synthesis of Macrocyclic Musks. Synthesis, v.
10, p. 1707–1723, 1999.
WILLIAMS, D. F. On the mechanisms of biocompatibility.
Biomaterials, v. 29, n. 20, p. 2941–2953, 2008.
WINZEN, S. Influence of different factors on the interaction
between polymeric nanomaterials and blood plasma proteins.
Johannes Gutemberg Universität, 2015.
WINZEN, S. et al. Colloids and Surfaces B : Biointerfaces Fluorescence
labels may significantly affect the protein adsorption on hydrophilic
nanomaterials. Colloids and Surfaces B: Biointerfaces, v. 147, p. 124–
 147

128, 2016.
WOODRUFF, M. A.; HUTMACHER, D. W. The return of a forgotten
polymer - Polycaprolactone in the 21st century. Progress in Polymer
Science (Oxford), v. 35, n. 10, p. 1217–1256, 2010.
WUNDERLICH, B. Macromolecular Physics. New York: Academic
Press, 1980.
XIE, J. et al. The differentiation of embryonic stem cells seeded on
electrospun nanofibers into neural lineages. Biomaterials, v. 30, n. 3, p.
354–362, 2009.
XU, L.; SIEDLECKI, C. A. Effects of surface wettability and contact
time on protein adhesion to biomaterial surfaces. Biomaterials, v. 28, p.
3273–3283, 2007.
XU, Q.; WAGNER, K. D.; DAHMEN, N. Vapor-liquid equilibria of
different lactones in supercritical carbon dioxide. Journal of
Supercritical Fluids, v. 26, n. 2, p. 83–93, 2003.
YAMADERA, R.; MURANO, M. The determination of randomness in
copolyesters by high resolution nuclear magnetic resonance. Journal of
Polymer Science Part A-1: Polymer Chemistry, v. 5, n. 9, p. 2259–
2268, 1967.
YASUNIWA, M. et al. Thermal analysis of the double-melting behavior
of poly(L-lactic acid). Journal of Polymer Science, Part B: Polymer
Physics, v. 42, n. 1, p. 25–32, 2004.
YASUNIWA, M.; SATOU, T. Multiple melting behavior of
poly(butylene succinate). I. Thermal analysis of melt-crystallized
samples. Journal of Polymer Science, Part B: Polymer Physics, v. 40,
n. 21, p. 2411–2420, 2002.
YOSHINARI, M. et al. Effect of Cold Plasma-Surface Modification on
Surface Wettability and Initial Cell Attachment. International Journal
of Biomedical and Biological Engineering, v. 3, n. 10, p. 2006–2010,
2010.
ZHANG, J. et al. Recent developments in lipase-catalyzed synthesis of
polymeric materials. Process Biochemistry, v. 49, n. 5, p. 797–806,
2014.
148

ZHANG, M. et al. Lipase-catalyzed Continuous Ring-opening

Polymerization of ε-Caprolactone in a Packed-bed Reactor. Chemical
and Biochemical Engineering, v. 26, n. 1, p. 1–6, 2012a.
ZHANG, Y. et al. Antioxidant and a -glucosidase inhibitory activity of
Adina rubella Hance in vitro. African Journal of Pharmacy and
Pharmacology, v. 6, n. 41, p. 2888–2894, 2012b.
ZHENG, C. J. et al. Fatty acid synthesis is a target for antibacterial
activity of unsaturated fatty acids. FEBS Letters, v. 579, n. 23, p. 5157–
5162, 2005.
 149

APPENDIX A - KINETIC STUDY OF PGlCL ENZYMATIC

SYNTHESIS UNDER DIFFERENT KINDS OF STIRRING

This section presents a preliminary kinetic study of poly(globalide-

co-ε-caprolactone) (PGlCL) using dichloromethane (DCM) as solvent,
under magnetic and mechanical stirring. The samples obtained in the
kinetic study were evaluated in terms of reaction yield, molecular weight
and thermal properties to evaluate the effect of time and stirring on PGlCL
e-ROP.
E-ROP reactions in dichloromethane (DCM) were carried out in two
different reactors, with two different kinds of stirring, in order to observe
the effect of non-homogeneities on the kinetics of globalide (Gl) and ε-
caprolactone (CL) copolymerization. The first reactor employed a
magnetic stirrer with a TeflonTM-coated stirring bar. The other reactor
(Parr Instrument Company, model 4592, Moline, IL, USA) has a
mechanical agitation using a straight blade impeller. Both reactors consist
in jacketed stainless steel vessels with temperature and pressure control.
Globalide and ε-caprolactone, both previously dried, were used in a mass
ratio of 50/50 (globalide/ε-caprolactone). Dichloromethane (DCM) was
used as solvent, in a mass ratio of 2:1 (solvent:monomers).
Novozym 435, also previously dried, was used in an amount of 5%
in relation to the mass of monomers (total mass of globalide and ε-
caprolactone). The enzyme, monomers, and the solvent were weighed on
a precision scale balance (Shimadzu AUY220, Philippines with 0.0001 g
accuracy) and fed to the reactor, which was immediately closed. The
reaction temperature was kept in 65 °C, respecting the optimal enzyme
activity temperature range (OLIVEIRA et al., 2004; RICHETTI et al.,
2010; ROSA et al., 2008). The reactor pressure was maintained around
2.5 bar, which is the equilibrium pressure that the system reached at 65 °
C. At the end of the reaction time, more DCM was added to the reaction
mixture to reduce the viscosity, helping to dissolve the product. After
filtering off the enzyme, the filtrate was precipitated in cold ethanol.
DCM and EtOH were used at the volumetric proportion of 1:6. The
polymeric suspension was filtered and dried at room temperature in
vacuum, up to constant mass.
Reactions were performed in different time intervals, ranging from
30 to 720 min, where each kinetic data point represented a different assay
(destructive experiments). Yields were determined through the mass ratio
between the mass of copolymer obtained and the mass of monomer fed.
PGlCL samples obtained on kinetic study were characterized in
terms of molecular weight (Gel Permeation Chromatography – GPC),
150

thermal properties and crystallinity (Differential Scanning Calorimetry –

DSC). The assays were performed according item 3.2.3., on Chapter 3.
A1. YIELD AND MOLECULAR WEIGHT KINETIC CURVES

Figure A.1 presents the yield kinetic data for e-ROP of PGlCL 50/50
(Gl/CL feed mass ratio) synthesized on DCM, submitted to magnetic and
mechanical stirring. For the reaction system stirred magnetically, it is
possible to note that the yield reaction increases in a higher rate up to 120
minutes. After 120 min the yield increases in lower intensity, stabilizing
its value in around 50%. During the polymerization reaction, the size of
the copolymer chains increase, increasing the system viscosity. After 120
min this high viscosity affects the magnetic stirring, making it less intense
and possibly even stopping the stirring completely. This way, after 120
min, mass transfer in the system happens basically by diffusion, which
makes the polymerization reaction practically stop.

Figure A.1 - Yield kinetic behavior for PGlCL 50/50 (Gl/CL feed mass ratio)
synthesis on DCM, under magnetic and mechanical stirring.
Magnetic stirring
Mechanical stirring

70

60

50
Yield (%)

40

30

20

10

0
0 100 200 300 400 500 600 700 800
Time (min)

For the mechanically stirred system, the reaction yield also increases
in a higher rate up to 120 min. However, after 120 min, the system
maintained an increase on reaction yield, in a less intense way, reaching
 151

almost 70% of conversion. The viscosity of the reaction media affects less
the mechanical stirring than the magnetic stirring. Maintaining the stirring
allows the monomer molecules to keep finding the active sites of the
enzymes, leading to higher reaction yields.
Figure A.2 presents number average molecular weight (Mn) kinetic
data of PGlCL 50/50 (Gl/CL feed mass ratio) synthesized on DCM,
determined by GPC. For all kinetic data points, the inflection point of the
broad polymer peak was used as the endpoint for integration procedures,
as described in section 3.3.2, in Chapter 3. The same integration criteria
was used in literature (KUNDU et al., 2011) and enables valid data
comparisons maintaining the general trend in this data consistent with
other studies (COMIM ROSSO et al., 2013; VAN DER MEULEN et al.,
2008, 2011).

Figure A.2 - Number average molecular weight (Mn) kinetic behavior for PGlCL
50/50 (Gl/CL feed mass ratio) synthesis on DCM, under magnetic and
mechanical stirring.
Magnetic stirring
Mechanical stirring
20000

15000
Mn (Da)

10000

5000

0
0 100 200 300 400 500 600 700
Time (min)

For magnetic stirring, Mn grows up to 120 min of reaction. Initially,

after the monomer rings are opened, enzyme-activated polymer chains
152

(EAPC, polymer chains attached to the enzyme active site) reacts in a

very intense way to oligomer/polymer chains in solution. This reaction
regenerate the enzyme active site and generate growing polymer chains,
increasing Mn values. The increase in Mn leads to lower mobility of the
polymer chains, which causes a decrease in the polymerization rate. This
way, the effect of polymer degradation reactions by the presence of water
molecules becomes more prominent (COMIM ROSSO et al., 2013;
GEUS, 2007; THURECHT et al., 2006). After 120 min, Mn keeps
basically constant, which probably happens due to the establishment of
an equilibrium between polymerization and degradation reactions which
occurs simultaneously (KOBAYASHI; UYAMA; TAKAMOTO, 2000;
KUNDU et al., 2011; SIVALINGAM; MADRAS, 2004).
For mechanical stirring, the same kinetic behavior of Mn can be
observed, consisting of a growing chain step, and a step where an
equilibrium of polymerization and degradation reactions is established,
reaching after 90 min, values of Mn similar to those obtained under
magnetic stirring. However, in comparison to the magnetic stirred system,
the chain growth rate for mechanical stirred system is much higher. In 60
min of reaction, Mn reached values around 6,000 Da for magnetic stirred
reaction, and around 13,000 Da for mechanical stirred reaction. The
oligomeric chains formed during this interval of time are probably kept in
contact to the enzyme pellets in a more efficient way with the use of a
mechanical stirring, which allows the chains to grow faster.

A2. THERMAL PROPERTIES

Samples obtained in different reaction times, under mechanical

stirring, were evaluated by DSC. These samples were chosen due to their
more interesting kinetic results (yield and molecular weight). Results of
crystallization temperature (Tc), melting temperature (Tm), melting
enthalpy (ΔHm) and degree of crystallinity are presented in Table A.1.
The degree of crystallinity was calculated through the relation between
ΔHm of each sample, and the theoretical value of a 100% crystalline PCL
sample, obtained from literature (CRESCENZI et al., 1972).
 153

Table A.1 - Thermal properties of samples determined by DSC for

samples obtained in different reaction times under mechanical stirring.
Tm 1 Tm 2 Xc
Time (min) Stirring Tc (°C) ΔHm (J/g)
(°C) (°C) (%)
60 28 37 45 76.3 56
120 26 34 44 76.7 57
Mechanical
240 24 34 42 82.8 61
480 20 35 39 84.3 62
Tc: Crystallization temperature; Tm 1: First peak melting temperature; Tm 2:
Second peak melting temperature; ΔHm: Heat of fusion; Xc: Degree of
crystallinity, calculated from the fusion enthalpy value of a PCL 100% crystalline
sample (CRESCENZI et al., 1972).

All samples presented a double melting point, in the same way that
the samples synthesized on scCO2+DCM (section 3.3.3., in Chapter 3).
This fact led us to further investigate the relation between the use of DCM
and the presence of double melting points. As demonstrated by MALDI-
TOF analysis, the use of DCM in PGlCL favors the formation of
oligomeric cycles (intramolecular backbiting), probably generating small
and imperfect crystals, responsible for the double melting peaks presence
(melting-recrystallization mechanism).
Melting temperatures remained practically constant throughout the
reaction time. ΔHm, and consequently Xc experienced a subtle variation
on their values as the reaction times increased, however, this variation is
too small to be considered. Copolymers formed in all evaluated reaction
times showed to be semi-crystalline.
154
 155

APPENDIX A - REFERENCES

COMIM ROSSO, S. R. et al. Enzymatic synthesis of poly(ε-

caprolactone) in supercritical carbon dioxide medium by means of a
variable-volume view reactor. Journal of Supercritical Fluids, v. 79,
p. 133–141, 2013.
CRESCENZI, V. et al. Thermodynamics of fusion of poly-β-
propiolactone and poly-ε-caprolactone. comparative analysis of the
melting of aliphatic polylactone and polyester chains. European Polymer
Journal, v. 8, n. 3, p. 449–463, 1972.
GEUS, M. DE. Enzymatic Catalysis in the Synthesis of New Polymer
Architectures and Materials. Technische Universiteit Eindhoven,
2007.
KOBAYASHI, S.; UYAMA, H.; TAKAMOTO, T. Lipase-catalyzed
degradation of polyesters in organic solvents. A new methodology of
polymer recycling using enzyme as catalyst. Biomacromolecules, v. 1,
n. 1, p. 3–5, 2000.
KUNDU, S. et al. Continuous flow enzyme-catalyzed polymerization in
a microreactor. Journal of the American Chemical Society, v. 133, n.
15, p. 6006–6011, 2011.
OLIVEIRA, D. et al. Optimization of enzymatic production of biodiesel
from castor oil in organic solvent medium. Applied biochemistry and
biotechnology, v. 113–116, p. 771–780, 2004.
RICHETTI, A. et al. Assessment of process variables on 2-ethylhexyl
palmitate production using Novozym 435 as catalyst in a solvent-free
system. Bioprocess and Biosystems Engineering, v. 33, n. 3, p. 331–
337, 2010.
ROSA, C. D. et al. Lipase-catalyzed production of fatty acid ethyl esters
from soybean oil in compressed propane. Journal of Supercritical
Fluids, v. 47, n. 1, p. 49–53, 2008.
SIVALINGAM, G.; MADRAS, G. Modeling of Lipase Catalyzed Ring-
Opening Polymerization of ɛ-caprolactone. Macromolecules, v. 5, p.
603–609, 2004.
156

THURECHT, K. J. et al. Kinetics of enzymatic ring-opening

polymerization of ε-caprolactone in supercritical carbon dioxide.
Macromolecules, v. 39, n. 23, p. 7967–7972, 2006.
VAN DER MEULEN, I. et al. Polymers from functional macrolactones
as potential biomaterials: Enzymatic ring opening polymerization,
biodegradation, and biocompatibility. Biomacromolecules, v. 9, n. 12,
p. 3404–3410, 2008.
VAN DER MEULEN, I. et al. Copolymers from unsaturated
macrolactones: Toward the design of cross-linked biodegradable
polyesters. Biomacromolecules, v. 12, n. 3, p. 837–843, 2011.
 157

APPENDIX B – PGlCL DEGRADATION STUDY

This section presents a preliminary degradation study of

poly(globalide-co-ε-caprolactone) (PGlCL). PGlCL degradation was
evaluated in buffer and in the presence of a lipase from Pseudomona
cepacia. Besides, a degradation assay was also performed in the presence
of active sludge.

B1. Degradation of PGlCL and PGlCL-NAC films in buffer and lipase

solutions

PGlCL and PGlCL-NAC films were prepared by casting 120 μL of

a polymeric solution (120 mg/mL) onto circle Microscope Cover Slips
with 25 mm diameter. PGlCL samples were diluted in chloroform, while
PGlCL-NAC were diluted in a chloroform:ethanol mixture 9:1 (v/v). The
samples were dried under vacuum at room temperature until a constant
weight was achieved. The weights of the films were about 11-15 mg.
The degradation experiments were conducted at 37 °C by immersing
each polymer film with the Cover Slips in 4 mL phosphate buffer solution
(0.1 M, pH 7.4). In case of degradation by lipase, the films were immersed
in phosphate buffer solution containing 0.05 mg Pseudomonas
cepacia/mL phosphate buffer solution. The buffer-enzymatic solution
was changed every 24 h to maintain the enzymatic activity. The Cover
Slips were picked up over predetermined time intervals, washed with
distilled water and dried under vacuum. The weights of the Microscope
Cover Slips were measured before film casting and the weight of the
Cover Slips with polymer was measured before and after degradation
studies. The morphology of the films before and after degradation were
characterized with a Gemini 1530 (Carl Zeiss AG, Oberkochem,
Germany) scanning electron microscope operating at an accelerating
voltage of 0.35 kV.
Figure B.1. Presents the degradation curves of PGlCL (A) and
PGlCL-NAC (B) in a Gl/CL ratio of 10/90. As expected, degradation in
the presence of a lipase have a significant effect on polymers degradation,
in contrast to a slower hydrolytic degradation. For all cases, the highest
weight loss were obtained during enzymatic degradation, since
Pseudomonas cepacia lipase is able to catalyze the cleavage of ester
bonds (QIN et al., 2016), resulting in the degradation and weight loss of
the polymer. Comparing PGlCL and PGlCL-NAC, the functionalization
with NAC was not enough to cause important effects in the degradation
in buffer, both PGlCL and PGlCL-NAC reaching around 20% of weight
158

loss after 960 h. However, for degradation in enzymes, the

functionalization with NAC resulted in a increase in degradation rate.
After 480h of degradation polymer reached 55% of weight loss. For
PGlCL non-functionalized, after 480h of degradation in the presence of
enzymes, the weight loss was 28%, which means there was an increase of
around 97% in the weight loss caused by enzymatic degradation, after
functionalization with NAC.

Figure B.1 - (A) Degradation curves of PGlCL (Gl/CL = 10/90) in buffer (circles)
and in lipase solution (triangles); (B) Degradation curves of PGlCL-NAC (Gl/CL
= 10/90) in buffer (circles) and in lipase solution (triangles).

On Figure B.2., for PGlCL with a Gl/CL ratio of 25/75, the effect of
the functionalization with NAC in the weight loss caused by degradation
is even more evident. This result is expected, since in this case, the degree
of functionalization with NAC was higher (item 4.3.1.). In this case, it is
possible even to observe an increase in the weight loss for degradation in
buffer, that reached 50% after 240 h, while for non-functionalized PGlCL,
only 24% was reached after 960 h.
As it was discussed in Chapter 4, the incorporation of NAC
molecules covalently bonded to PGlCL increased the affinity of the
polymeric material for water and reduced its degree of crystallinity,
making it easier for the molecules of water to access the polymer chains
and cleavage ester bonds, promoting degradation.
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Figure B.2 - (A) Degradation curves of PGlCL (Gl/CL = 25/75) in buffer (circles)
and in lipase solution (triangles); (B) Degradation curves of PGlCL-NAC (Gl/CL
= 25/75) in buffer (circles) and in lipase solution (triangles).

Scanning electronic microscopy (SEM) images are shown in Figure

B.3 and Figure B.4 and corroborate with the weight loss results. For both
PGlCL and PGlCL-NAC it is possible to see clearly the difference in the
film morphology after degradation in lipase, compared with time zero and
with degradation in buffer, where the presence of the enzymes damaged
more intensively the surface of the films. For PGlCL-NAC films, it is
possible to observe that after 240 h in the presence of lipase, the film was
almost completely degraded, being consistent with weight loss values.
It is also possible to notice that morphology of PGlCL film becomes
completely different after functionalization with NAC (time zero images).
PGlCL-NAC films present much more irregularities in the surface, and it
is possible that this irregularities are caused by the folding of the polymer
chains for the establishment of separate interactions between polar groups
(coupled NAC molecules) and hydrophobic chains (PGlCL chains). The
presence of this irregularities probably also facilitate the degradation of
the functionalized material, since it caused an increase of the surface area
accessible to the molecules of water and enzymes.
160

Figure B.3 - Scanning electronic microscopy images of PGlCL in a Gl/CL ratio

of 25/75 before and after degradation in lipase and buffer

Figure B.4 - Scanning electronic microscopy images of PGlCL-NAC in a Gl/CL

ratio of 25/75 before and after degradation in lipase and buffer

B2. BIODEGRADABILITY IN ACTIVATED SLUDGE

In this assay, it was tested PGlCL-NAC (Gl/CL = 25/75)
biodegradability in activated sludge, using the method based on
Organisation for Economic Cooperation and Development (OECD)
guideline 301F (OECD, 1992). In summary, the biological oxygen
demand (BOD) for each chemical was measured using the OxiTop control
manometric closed system (WTW, Germany) over 28 days. The
percentage of biodegradability was determined comparing the measured
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BOD and the calculated theoretical oxygen demand (ThOD) values. In a

typical run, flasks containing the following compositions were used: (1)
Two flasks for the inoculum “blanks”, containing activated sludge and
mineral medium with nutrients; (2) Two flasks for procedure control,
containing the activated sludge, a reference compound readily
biodegradable (in this case it was used starch) and mineral medium; (3)
Two flasks for toxicity control, containing the inoculum, PGlCL-NAC,
starch and mineral medium; (4) Two flasks for evaluating the
biodegradability of the tested material, containing activated sludge,
PGlCL-NAC and mineral medium.
The activated sludge sample used in all studies was from the same
batch of collected at the waste treatment unit in the city of Mainz,
Germany and it was aerated in the dark at 20 °C for 7 days before the start
of the experiments. Activated sludge (2.8 mL) with a solid content of 3.9
g/L was mixed to 365 mL of mineral medium in amber bottles, giving a
final concentration of approximately 30 mg/L, as described by the OECD
procedure. It was used 24.16 mg of starch and 13 mg of PGlCL-NAC per
bottle. Magnetic stirrer bars were also added. The screw-top measuring
heads, containing sodium hydroxide pellets to adsorb produced carbon
dioxide, were replaced. The flasks were stirred in an incubator cabinet in
the dark at 20 °C. Oxygen consumption data via measurement of pressure
loss were recorded over a 28 days period. At this time, the ThOD of each
polymer was calculated as described in the OECD 301 guidelines.
Blank oxygen consumption values (BOD values representing
background respiration in activated sludge) were deducted from the BOD
of the test compound prior to determining the percentage
biodegradability.
Figure B.5. present the degradation curves of PGlCL-NAC, PGlCL
in the presence of starch as a toxicity control, and pure starch as a positive
control. Starch is a readily biodegradable molecule, and the results
obtained also showed this behavior, since in 28 days, it reached almost
75% of degradation. The mixture PGlCL-NAC + starch was used as a
toxicity control, and the results clearly show that PGlCL-NAC did not
present toxicity to the bacteria present in the activated sludge, since the
mixture presented high degradation by the bacteria. PGlCL-NAC
presented a degradation curve very similar to starch, also reaching almost
75% of degradation. This result shows that PGlCL-NAC is highly
biodegradable and have a strong potential of application not only in the
biomedical area, but also for the manufacturing of green biodegradable
packages, for example.
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Figure B.5 - Degradation curves obtained by the OECD biodegradability assays

in activated sludge for PGlCL-NAC (triangles), PGlCL-NAC + Starch (squares)
and Starch (circles).
PGlCL-NAC
PGlCL-NAC + Starch
120 Starch

100
Degradation (%)

80

60

40

20

0
0 100 200 300 400 500 600
Time (Hours)
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APPENDIX B – REFERENCES

OECD. OECD 301 - Ready Biodegradability. OECD Guidelines for

the Testing of Chemicals, v. 301, n. July, p. 1–62, 1992.
QIN, J. X. et al. New insight into the difference of PC lipase-catalyzed
degradation on poly(butylene succinate)-based copolymers from
molecular levels. RSC Advances, v. 6, n. 22, p. 17896–17905, 2016.