Peptides 31 (2010) 184193

Contents lists available at ScienceDirect

Peptides
journal homepage: www.elsevier.com/locate/peptides

Review

Recent developments in liposomes, microparticles and nanoparticles

for protein and peptide drug delivery
Mei Lin Tan a, P.F.M. Choong a,b, C.R. Dass a,*
a
Department of Orthopedics, University of Melbourne, St Vincents Hospital Melbourne, PO Box 2900, Fitzroy 3065, Australia
b
Bone and Soft Tissue Sarcoma Service, Peter MacCallum Cancer Institute, Melbourne, Australia

A R T I C L E I N F O A B S T R A C T

Article history: Proteins and peptides are increasingly recognized as potential leads for the development of new
Received 18 August 2009 therapeutics for a variety of human ailments. Due to their relatively specic mode of action, proteins and
Received in revised form 1 October 2009 peptides can be administered at relatively low doses for therapeutic effects. As natural biological
Accepted 1 October 2009
products, these low doses reduce the risk otherwise caused by other small molecular drugs or larger
Available online 9 October 2009
charged molecules. Unfortunately, their therapeutic potential and clinical application is frequently
hampered by various obstacles to their successful delivery. This review discusses the recent
Keywords:
developments in the elds of liposome, microparticle and nanoparticle pertinent to protein and
Drug delivery systems
Proteins
peptide delivery covering those systems tested and/or validated in vivo.
Peptides 2009 Elsevier Inc. All rights reserved.
Therapy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2. Drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.1. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.2. Microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.3. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

1. Introduction medicines (including proteins and peptides) relating to more than

100 diseases, including cancer, infectious diseases, autoimmune
The pursuit of the human genome revealed many answers, diseases, AIDS/HIV and related conditions. Among these, more
among them, ndings which indicate that more than half of our than 66 of these new medicines were derived from proteins or
30,000 genes encode proteins. The need for an understanding of peptides. These new drugs are in human clinical trials or under
this pool of unknown proteins subsequently led to the establish- review by the Food and Drug Administration [53]. Proteins and
ment of the Human Proteome Organization. As such, many of these peptides are increasingly recognized as potential leads for the
proteins have shown to be valuable reservoirs of drug candidates, development of new therapeutics. Due to their relatively specic
or serve as targets for protein or peptide-based drug research and mode of action, protein and peptide therapy need only in theory to
development (R&D). In 2008, The Pharmaceutical Research and be delivered in low doses. As natural biological products, these low
Manufacturers of Americas (PhRMA) report on Biotechnology doses reduce the risk otherwise caused by other small molecular
Medicines in Development, listed 633 new biotechnology drugs or larger charged molecules (example oligonucleotides).
Unfortunately, their therapeutic potential and clinical application
is frequently hampered by various obstacles to their successful
* Corresponding author at: Orthopedics Department, SVHM, L3, Daly Wing, 35
delivery [20,56]. This review aims to examine the various drug
Victoria Pde., Fitzroy 3065, Australia. Tel.: +61 3 9288 3990/3980; fax: +61 3 9416
3610.
delivery systems (DDSs) designed for the delivery of protein and
E-mail address: [email protected] (C.R. Dass). peptide therapeutics for human disease therapy, with a focus on
URL: http://www.svhm.org.au DDSs that have been investigated in an in vivo setting.

0196-9781/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2009.10.002
 M.L. Tan et al. / Peptides 31 (2010) 184193 185

At a cellular level, the delivery of proteins and large peptides in their dimensions, composition, surface charge and structure, some
vivo can be hindered by their three-dimensional structure, spatial of which designed through a combination of specic types of
occupation and hydrophilic/hydrophobic nature. Thus, diffusion liposomes [15]. For the treatment of cancer, liposomal-DDSs have
transport of these large pharmaceutical proteins is generally been developed for small molecular drugs such as doxorubicin
slower unless specic transporters are available. Protein stability, (Caelyx1/Doxil1) and daunorubicin (DaunoXome1), though few
based on weak non-covalent interactions between secondary, such DDSs have been formulated for protein and peptides.
tertiary and quaternary structures of proteins, is also crucial to A novel attempt to synthesize and deliver pro-apoptotic
prevent any disruptions that will destabilize the proteins [57]. membrane proteins through liposomes were attempted in human
These factors present proteins and peptides as highly vulnerable colorectal carcinoma cells [34]. Natural liposomes derived from
molecules with short in vivo half-lives, due to degradation by spinach thylacoids were prepared through the evaporation/
enzymes and proteases, either at the administration site or en- resuspension technique, together with recombinant VDAC
route to the site of pharmacological action, resulting in poor (voltage-dependent anion channel), Bak and BakDBH3 protein,
bioavailability. In certain cases, frequent administration at high producing LB (Bak liposomes), LBDBH3 (BakDBH3 liposomes), LV
doses might be required to obtain therapeutic effects in vivo, (VDAC liposomes) and LVB (VDAC-Bak liposomes). Upon incuba-
creating a risk for unexpected and unwanted side-effects such as tion of the liposomes with colorectal carcinoma cells, apoptosis
immune responses. In addition, during preparation of the protein/ was induced within 24 h with the release of cytochrome c and
peptide drug, manufacturing processes and environmental activation of caspases-3, -7, -9 and PARP. Thus, these results
factors may damage the proteins, reduce their biological activity, highlight the potential of pro-apoptotic proteins integrated into
induce aggregation, render the proteins immunogenic and lead to natural liposomes, presenting as candidates for cancer protein
their precipitation [20,57]. These processes include sterilization therapy. However, in vivo efcacy still needs to be determined, as
and lyophilisation while the contributing environmental factors the in vitro performance of a DDS does not necessarily predict its
are pH, ionic strength, temperature, high pressure, non-aqueous performance in vivo.
solvents, metal ions, detergents, adsorption, agitation and Studies were also carried out to examine the local delivery of
shearing. therapeutic molecules encapsulated within liposomes as a
For these reasons, a carrier or delivery system for therapeutic potential method to treat ocular inammation [8]. Using the
proteins and peptides would be ideal. Drug delivery systems thin-lm hydration method, the authors formulated rhodamine-
(DDSs) can be advantageous to enhancing disease therapy by: (1) conjugated pegylated (PEG) liposomes composed of phosphaidyl-
enhancing protein/peptide solubility; (2) controlled release of choline (PC), phosphatidylglycerol (PG), cholesterol (Chol), 1,2-
protein/peptide molecules; (3) sustained release instead of burst distearoyl-sn-glycero-3-phosphoethanil-amine-N-[methoxy-
release effect, avoiding undesirable side-effects; (4) improved poly-(ethyleneglycol)-2000] (PEG-DSPE) and phosphatidyle-tha-
biodistribution of protein/peptides; and with improved technology nolamine-N-(lissamine rhodamine B sulfonyl; PE-Rh). Rhodamine-
possibly (5) target the diseased tissue in vivo [2]. In an ideal setting, conjugated liposomes loaded with vasoactive intestinal peptide
protein/peptides administered through DDSs should decrease the (VIP), an immunosuppressive neuropeptide [46], were injected
rate of drug clearance from the system, meaning that lower intravitreally into the eyes of rats for a study of its biodistribution.
volumes or concentrations of the drug can be administered. Twenty-four hours following the intravitreal injection, VIP-Rh-Lip
Lowered volumes or concentrations of proteins/peptides could uorescent VIP-containing liposomes as well as VIP-null uor-
also prove useful for certain proteins/peptides that are inherently escent liposomes, were detected mainly in the posterior segment
difcult to synthesize, extract, purify or become toxic at high of the eye (vitreous, inner layer of the retina) and the conjunctiva
doses. The area under the time versus concentration curve also and episclera. Studies also determined that the liposomes were
increases, thus lengthening the treatment duration and possibly internalized by activated retinal Muller glial cells, ocular tissue
decreasing treatment frequency [21]. In addition, the DDS has to be resident macrophages and rare inltrating activated macrophages.
able to bind the therapeutic protein/peptide in a fashion that does However, it was unclear as to whether both Rh-Lip and VIP-Rh-Lip
not affect its bioactivity but hold it sufciently until the DDS were internalized in a similar manner although some evidence
reaches its site of action and releases the protein/peptide in a pointed to the presence of VIP still contained within the
controlled, sustained manner. In this instance, DDSs formulated conjunctiva 24 h after injection. Both Rh-Lip and VIP-Rh-Lip were
with biocompatible and biodegradable materials would certainly distributed to the cervical lymph nodes and internalized by
be advantageous so as to minimize any adverse host response to resident ED-3 positive macrophages adjacent to CD4 and CD8-
the DDSs. positive T lymphocytes. In addition, it was found that the T
This review aims to present on-going research on drug delivery lymphocytes in close contact with VIP-Rh-Lip-containing macro-
systems for proteins and peptides in an informative manner. phages expressed VIP. The accumulation of liposomes within the
Recent publications containing in vivo experimentation and strictly subcapsular sinus of the cervical lymph nodes following intravi-
belonging to either liposomal, microparticle or nanoparticle treal injection in the conjunctiva was possible through the
delivery systems were chosen to be included in this review. It conjunctival lymphatics. Thus, through this study, results showing
is not intended for the authors to strictly criticize each article the detection of VIP in both macrophages and T cells in cervical
but rather this review serves to update readers regarding new lymph nodes suggested that intravitreal injection of VIP-Rh-Lip
developments in protein and peptide delivery systems while may increase ocular immune privileges by modulating the loco-
providing suggestions for future advancements. regional immune environment. In conclusion, these observations
suggested that these VIP-loaded liposomes could prove a promis-
2. Drug delivery systems ing therapeutic strategy to dampen ocular inammation by
modulating macrophage and T cell activation. As the focus of
2.1. Liposomes the research was tuned towards the therapeutic and benecial
properties of the liposome, there was minimal physical character-
First designed in the late sixties, liposomes are microscopic ization attempted. However, such characterizations should be
vesicles composed of lipid membranes surrounding discrete considered in the future as they can shed light for further
aqueous compartments [59]. Over the decades, various types of improvements on the liposome. In addition, the effects of a
liposome formulations have been made available, each differing in dampened immune response require more thorough analysis so as
186 M.L. Tan et al. / Peptides 31 (2010) 184193

to prevent a lack of immunity when potential pathogens are ism involved upon MOMP vaccination, investigators carried out
encountered. their studies on normal C57BL/6 mice as well as CD4+ T cells
In yet another report utilizing the VIP, a treatment for lung depleted C57BL/6 mice. The results showed that mice immunized
diseases was described [23]. Although the administration of VIP with MOMP/CAF01 had a clear protective effect over mice treated
garnered positive therapeutic potential in human trials for with saline or MOMP/alum, boasting signicantly fewer bacteria
pulmonary artery hypertension, there is yet a VIP agonist in on days seven and 14 after infection. Mice vaccinated with MOMP/
clinical use [22]. This is attributed to the rapid proteolytic digestion alum displayed a strong anti-MOMP humoral response with high
and inactivation of VIP by endopeptidases, thus reducing the IgG1 titers, low levels of IFN-g and tumor necrosis factor (TNF)-a,
bioactivity of the peptide [35]. To enhance the local availability and and a slight reduction in Chlamydia load. On the other hand, mice
therapeutic effect of the peptide, a VIP depot formulation was vaccinated with MOMP/CAF01 displayed high titers of IgG2b, IFN-
designed to provide a retarded release of the peptide, allowing the a and reduced vaginal Chlamydia load. Additionally, MOMP/CAF01
peptide to be protected before its uptake by the appropriate vaccinated mice showed a reduced infection-resolution time
receptor [23]. The authors have previously formulated a uni- compared to saline-treated mice. Also, the results showed that
lamellar liposome and were now developing this liposomal vaccine-mediated protection against Chlamydia was dependent on
formulation as an inhalable peptide carrier. VIP-loaded liposomes, CD4+ T cells. Having demonstrated that the MOMP/CAF01 vaccine
composed of polyethylene glycol-conjugated distearyl phospha- effect was mediated primarily by CD4+ T cells, the authors also
tidyl ethanolamine (DSPE-PEG2000), lysosteryl-phosphatidylgly- showed that similar levels of infection protection were provided by
cerol (lyso-PG) and palmitoyl-oleoyl-phosphatidylcholine (POPC), vaccination with rMOMP/CAF01, verifying that vaccination pro-
were synthesized by a thin-lm rehydration method to incorpo- tection was not dependent on MOMP conformation. In conclusion,
rate an aqueous solution of VIP. PEG also formed a PEG-shell which this study showed that a vaccine based on natively refolded as well
protected surface-attached VIP peptides from enzymatic degrada- as recombinant MOMP administered through the CAF01 delivery
tion in the airway and inhibited particle aggregation. VIP-loaded system induced a CD4+ T cell-dependent immune response that
liposomes were subsequently nebulized at room temperature, efciently protects against vaginal antigen challenge with C.
resulting in liposomes in the spray mist of a mean diameter below muridarum.
14 mm in size. The authors reported that there were no signicant For diabetes mellitus, the major peptide used for treatment is
changes in size before and after nebulization, indicative of the insulin. Despite its importance in diabetes management, conven-
absence of degradation or aggregation of liposomes, and further tional insulin therapies are limited by conditions such as
supporting the suitability of an inhalable VIP liposome. When the retinopathy, nephropathy thickened capillary basement mem-
VIP-loaded liposomes were tested biologically through relaxation branes and cardiovascular complications [16]. In addition, effective
experiments on rat arteries, the liposomes allowed a delay in the insulin delivery could be limited by high elimination rates
peptide release and produced greater vasorelaxation potential, in resulting in a short duration of drug contact with its absorption
comparison to free VIP peptides. In addition, the authors noted that sites and consequently a low bioavailability. Despite the avail-
there was a smooth vasorelaxation response towards effect ability of commercial pre-lled insulin, these treatment options
saturation, indicative of a continuous and smooth peptide release are cumbersome to the patients and might not effectively mimic
from the liposomes. Release of the VIP peptides were also physiological patterns of insulin secretion and action. In an
attributed to triggering by target cells, as the VIP-loaded liposomes attempt to address these issues, the authors investigated the
were stable without loss of peptide during storage. However, applicability of insulin-containing multivesicular liposomes
concerning the applicability of stored VIP-loaded liposomes, the (MVLs), with the addition of novel chitosan and carbopol coating
current technology employed by the authors only allowed storage (CS/P-MVLs) as sustained release protein delivery systems via the
of the liposomes for a maximum of 1 month before activity nasal and ocular routes [26]. As a result, the authors obtained
declined. This VIP-loaded liposomal formulation has shown insulin-loaded MVLs of 2634 mm with a high protein loading
potential towards the adaptation for the encapsulation of other between 58% and 62%. The in vivo applicability of these liposomes
peptide drugs. In summary, the authors have formulated a VIP- were tested through drop-style nasal and ocular administrations in
loaded liposome that increases peptide availability by (i) providing STZ-induced diabetic rats and compared to non-coated MVLs,
protection of peptides from peptidases and other forms of conventional liposomes and free insulin solution. In STZ-induced
degradation and, (ii) prolonging the specic activity of the peptide diabetic rats, CS-MVLs effectively reduced the blood glucose levels
and indicative of a sustained substance release in the lungs [23]. by 35% up to 48 h after nasal administration, compared to a
The authors should proceed to examine the in vivo safety, marginal reduction of 22% by non-coated MVLs up to 12 h after
biocompatibility and biodegradability of these liposomes, given administration. A comparison between chitosan-(CS-MVLs) and
the variety of chemicals and materials used in the liposome carbopol-(CP-MVLs) coated MVLs showed a maximum of 65%
formulation. reduction in blood glucose levels and 55% reduction, respectively,
Liposome delivery of therapeutic proteins was also attempted at 8 h. Upon ocular administration, the CS-MVL carrier showed
as a DDS for vaccination against genital Chlamydia infection [24]. better blood glucose reduction levels of up to 30% reduction at 24 h
The major outer membrane protein (MOMP) is a target of both compared to CP formulation and other tested formulations. This
humoral and cellular immune responses during infections in superior effect observed in both nasal and ocular administrations
humans and is a leading vaccine candidate. Despite the success of has been mainly attributed to the prolonged residence of the
native MOMP vaccines [41,42], recombinant MOMP, MOMP mucoadhesive MVLs in the mucosa as well as the protective effect
peptides or MOMP DNA have not yielded expected protection as of the MVLs against enzymatic attacks of the peptide drug. In
delivery systems could not retain MOMPs strong cell-mediated conclusion, nasal administration of CS-MVLs has shown a slightly
immunity and thus results in a dramatic inuence on the vaccines better hypoglycaemic prole as compared to the ocular route.
efcacy [40,50]. Using a recently developed cationic liposome Nonetheless, these experiments have proved to be very promising
formulation 1 (CAF01) [14], the authors compared the efcacy of to show effective delivery of insulin to the systemic circulation via
MOMP/CAF01 or MOMP adjuvanted with alum (T helper cells type non-invasive routes, and will be further investigated and devel-
2-promoting aluminium hydroxide) by mouse immunizations oped for improved transmucosal insulin formulations.
followed by a Chlamydia muridarum antigen challenge six weeks Investigating another peptide drug, calcitonin, Werle and
later. To further investigate the cell-mediated immunity mechan- Takeuchi aimed to study the efcacy of liposomes coated with
 M.L. Tan et al. / Peptides 31 (2010) 184193 187

the polymer-protease inhibitor conjugate chitosan-aprotinin for microparticle drug delivery formulations approved for clinical use
oral peptide delivery in rats [60]. The calcitonin-containing [64,65] although more clinical trials are currently underway
chitosan-aprotinin multilamellar vesicles (MVL) liposome con- [12,39]. The following articles present various microparticles
jugate prepared ranged from 3 to 4.5 mm in diameter and was designed for peptides and proteins delivery.
loaded with at least 75% of the drug. Formulations of chitosan- Using a synthesized peptide with the major T cell epitope of Ole
coated MLVs, chitosan-aprotinin coated MLVs and free calcitonin e 1, the main allergen of olive pollen, Marazuela et al. evaluated
were administered intragastrically into fasting rats which were whether intranasal administration of peptide containing-poly(-
tested for their blood calcium levels at pre-determined time- lactide-co-glycolide) (PLGA) microparticles (MP) were able to
points. The results showed a signicant decrease of blood calcium prevent mice from allergic sensitivity to the whole protein [37].
levels in chitosan-aprotinin-coated MLVs after 30 min of admin- Peptide-PLGA microparticles prepared by the solvent evaporation
istration as compared to chitosan-coated MLVs. The authors double emulsion method were mostly spherical and measured
speculated that this effect was most likely mediated by the 0.8 mm in diameter. This was encouraging to the authors as other
protease-inhibitory activity of chitosan-aprotinin, as the two studies have shown that microparticles smaller than 10 mm had a
formulations did not differ markedly in terms of drug-loading and higher uptake by M cells of the nasal associated lymphoid tissues
mucoadhesive properties. Additionally, in rats treated with (NALT), implying that the PLGA MP used in the study was suitable
chitosan-coated MLVs, calcium levels returned to the initial for M cells uptake and translocation to lymphoid organs to initiate
readings after 24 h, whereas calcium levels remained decreased antigen-specic immune responses. Also, with a peptide-loading
after 24 h in rats treated with chitosan-aprotinin coated MLVs. capacity of around 3 mg/mg, the quantity was considered high
Comparing the blood calcium concentrationtime curve (AAC) of enough for in vivo administration of the peptide without the need
both liposomes and calcitonin solution, chitosan-coated MLVs for large concentrations of MP or increased MP doses. Although the
increased the AAC about 11-fold above calcitonin solution, while authors were initially concerned that the peptides integrity might
chitosan-aprotinin MLVs yielded a 15-fold increase. This result be compromised by the use of harsh organic solvents in the solvent
again suggested that the efcacy of chitosan-coated MLVs can be evaporation method, results showed that the MP preparation
improved through the covalent attachment of specic protease process did not signicantly affect the peptides potency. It was
inhibitors such as aprotinin to the polymer. Thus, it was concluded reported that the PLGA MP had an initial burst release of 74%
that in order for the liposome to achieve a more pronounced effect peptide followed by a slow and sustained release over several
in vivo, an optimized liposomal chitosan-aprotinin delivery system weeks up to 28 days. This property of the MP was an advantage as it
which displays a maximal amount of covalently-bound aprotinin had previously been reported that an initial burst release of antigen
without exhibiting decreased mucoadhesive properties must be could stimulate the immune system to mucosal vaccines, and then
designed. allow the MPs slow continuous release of peptide to provide a long
Looking at the various liposomal delivery systems presented lasting immune response [1].
above for different proteins and peptides, there were almost no In this study, the authors found that three consecutive doses of
two similar formulation methods or materials. This could be 3 mg peptide in PLGA MP were enough to prevent subsequent
attributed to the difference in protein/peptide structure, charge, sensitization to the whole Ole e 1 allergen. Peptide-PLGA MP pre-
stability, solubility and other properties. When more becomes treatment signicantly reduced IgE Ab levels and specic IgG1 Ab,
understood about the biochemistry of the protein or peptide, other while increasing IgG2a Ab levels, as opposed to less signicant
factors such as the intended administration route for a desired results observed in sham-treated mice. Intranasal pre-treatment
treatment and also the action site of the drug should be taken into with peptide-PLGA MP also resulted in a decrease in IL-5 and IL-10
consideration. However, these varied formulations would not be levels although there were no signicant changes in IFN-g levels.
helpful to researchers seeking a liposome suitable for their Importantly, prophylactic treatment with peptide-PLGA MPs
therapeutic of interest. A more suitable approach would be for prevented lung inammation. It was inferred that a strong Th1-
researchers to rst characterize their therapeutic of interest for type response was present with both the increase of IgG2a Ab and
properties such as structure, surface charge and solubility before the high levels of IFN-g. Thus, the use of PLGA MP containing
identifying a formulation method which is most appropriate. To appropriate antigen can be considered when required for
prevent unwarranted side-effects and negative interactions with establishing a specic Th1-type response in humans. A signicant
the host, researchers should also opt for safer materials that are nding from this report was that through the intranasal
natural, biocompatible, biodegradable and less likely to incite an administration of the major T cell epitope of Ole e 1 peptide,
adverse reaction in the host. In addition, most liposomal-DDSs PLGA MP formed a protective effect against sensitization to the
were formulated using complex techniques requiring specialized whole allergen. Although the observed ndings might not be
equipment or chemicals that might be unreachable for others. directly translated to humans, this model addressed the immu-
Thus, researchers should consider using simpler, less complex nological effects most commonly experienced in clinical features of
chemicals that might additionally prove to be less toxic to the host. allergy, posing the PLGA MP carrier system with a good potential
Another concern might be due to the size of liposomes, which are for peptide-based vaccines.
typically around or above the micrometer range as the larger the In another study, the authors sought to evaluate gelatine
DDS, the higher the possibility of triggering an opsonization effect microparticles for the controlled release of bone morphogenetic
by the macrophages or other components of the immune system protein-2 (BMP-2) [44]. To begin, the authors hypothesized that
[38]. Largely-sized liposomes might also encounter various decreasing gelatine cross-linking and using collagenase-contain-
barriers within the hosts endothelial surfaces and various other ing PBS would result in faster degradation of gelatine and
barriers such as the bloodbrain barrier. consequently higher BMP-2 release. Also, it was hypothesized
that higher loadings of BMP-2 in the gelatine microparticles would
2.2. Microparticles result in higher burst release after 24 h. PLGA microparticles,
commonly studied for BMP-2 controlled release, were used as a
Generally, microparticles can be classied as particles between side-by-side comparison to gelatine microparticles. Both micro-
1 and 1000 mm. Initial experiments with microparticles started in particles were loaded with BMP-2 by the diffusion/adsorption
the early seventies and have since studied extensively with many method and tested for various parameters that affected BMP-2
different materials and polymers. As yet, there are very few release from microparticles. The optimized parameters were then
188 M.L. Tan et al. / Peptides 31 (2010) 184193

used for in vivo testing to evaluate the release of BMP-2 from formulation temperatures stabilized the microparticles by the
composite scaffolds of gelatine microparticles within a porous formation of crystallites through hydrogen bonds among amine,
poly(propylene fumarate) (PPF) scaffold. PLGA microparticles had amide, carboxylic and hydroxyl groups in the mucin and sodium
an average diameter of 2542 mm, depending on the molecular alginate polymer networks [61]. Additionally, the low temperature
weight of PLGA, while gelatine microparticles were sieved for a helped minimize the incompatibilities of peptides with the organic
diameter of 50100 mm. In general, gelatine microparticles solvents so that mucin was not denatured by acetone. No
displayed minimal burst release effect (<10%) at 24 h followed signicant trend was observed in insulin-loaded microparticle
by a moderate or slow release over a period up to 28 days, with sizes with sodium alginate, mucin, sodium alginatemucin (3:1)
gelatine microparticles incubated in collagenase-containing PBS and sodium alginatemucin (1:3) microparticles, which were 310,
exhibiting higher release rates and BMP-2 release. For both high 230, 260 and 680 mm, respectively. Subsequently, the insulin-
and low molecular weight PLGA microparticles, a moderate burst loaded microparticles prepared with different sodium alginate
release was observed followed by sustained release over 28 days. mucin ratios were lled into hard gelatine capsules with each
To examine the in vivo release of BMP-2 from gelatine micro- capsule containing insulin equivalent to 100 IU of soluble insulin.
particles, composite scaffolds with 10 and 40 mm gelatine Diabetic rabbits were orally administered with capsules containing
microparticles were tested in a subcutaneous mouse model. At insulin-loaded microparticles, while distilled water, insulin solu-
each time point where scaffolds were retrieved, a brous capsule tion and subcutaneous administered insulin acted as controls.
with extensive neovascularisation was observed surrounding the Blood glucose levels were taken hourly and the percentage
composites, although no ectopic bone formation was observed. reduction of initial glycaemia was used as evidence of insulin
Both 10 and 40 mm composites showed signicantly lower burst absorption. As expected, distilled water did not cause a reduction
and cumulative release of BMP in comparison to PPF controls. In in blood glucose levels while orally administered insulin solution
addition, observations on higher cross-linking resulting in lower resulted in a slight drop in blood glucose levels (30%) during the
BMP-2 release were also noted in vivo. In conclusion, the authors rst 2 h after administration and returning to normal by the third
correctly hypothesised that decreasing gelatine cross-linking and hour. In comparison, insulin-loaded microparticles produced
addition of collagenase would result in degradation of gelatine and better blood glucose lowering effect. Among the three different
consequently higher BMP-2 release. Interestingly, the hypothesis insulin-loaded microparticles (mucin, sodium alginate and mucin
of higher loaded doses of BMP-2 resulting in higher burst release sodium alginate), microparticles prepared with 1:3 mucin:sodium
was not observed, instead an opposite effect of decreased release alginate produced maximum blood glucose reduction (>70%) 5 h
with higher BMP-2 doses was seen. Finally, both the 10 and 40 mm after oral administration that was comparable to subcutaneously
composite scaffolds of BMP-2 gelatine microparticles showed administered insulin. The authors postulated that this could be due
minimal burst release followed by sustained release over 28 days, to synergism between sodium alginate and mucin that confers
indicating that this system can be used for the controlled delivery insulin protection or enhance absorption in the GIT. In conclusion,
of BMP-2, and thus provide a controlled sustained delivery system the results obtained from this study demonstrated the effective-
which more closely mimics the growth factor prole during ness of mucinated sodium alginate microparticles as a drug
natural bone healing. delivery system for oral insulin delivery, and thus indicating the
In a subsequent study by the same author, vascular endothelial possibility of designing an optimal drug delivery system using the
growth factor (VEGF) was investigated as the polypeptide of right combination of chemicals and formulation technique for the
interest to be delivered by gelatine microparticles [43]. Similarly, oral delivery of insulin towards effective control of blood glucose.
parameters such as the effects of gelatine cross-linking, growth Although positive and encouraging results have been shown in this
factor dose and release medium on VEGF release kinetics were study, more work closely examining the formulation materials,
examined in vitro and in vivo, before forming composite scaffolds of conditions and the efcacy of this potential DDS is required. A
gelatine microparticles within a porous PPF scaffold. Focusing on concern for oral insulin capsules would be the palatability of the
the microparticles in vivo characteristics, it was interesting to note drug and its effect on patient compliance, in addition, biodegrad-
that there was no cross-linking effect on release rates for burst and ability and excretion of these mucinsodium alginate capsules and
continued release which was unlike in vitro observations. its metabolites within a reasonable time is imperative, as insulin
However, the authors were still able to determine that increased treatment is mostly long-term and highly repetitive.
gelatine cross-linking resulted in lower VEGF release, indicating Having biodegradable and biocompatible characteristics,
that by varying cross-linking extent, this protein delivery system hydroethlystarch (HES) was traditionally used in humans as
can be used for the controlled delivery of VEGF in vivo. Although articial colloids for intravascular volume replacement [28,33].
the authors examined the bioactivity of VEGF after in vitro release, Based on these characteristics, the authors developed and
an in vivo study was not performed. Thus the DDS was not characterized HES microparticles suitable for antigen delivery
thoroughly tested for its efcacy and would required further systems, through the examination of its in vivo biocompatibility
investigations before recommendations are made. and biodegradability, shelf-life stability, loading capacity, con-
In another study for diabetes treatment, insulin-loaded trolled release and localization of loaded proteins [17]. In this
microparticles were formulated for oral insulin delivery and its subsequent study, HES microparticles were accessed for their
effectiveness was tested through reduction of initial blood glucose ability to induce an immune response in mice against the bovine
levels in diabetic rabbits [7]. Insulin-loaded microparticles were serum albumin (BSA) protein in comparison with an aluminium
prepared with mucin and sodium alginate combined at different hydroxide (alum) adjuvant [3]. Firstly, HES microparticles were
ratios based on a method of polymer coacervation and diffusion prepared using the interfacial cross-linking method designed
lling at low temperatures of 30 and 5 8C, respectively. The previously [36]. Briey, HES was emulsied in cyclohexane, stirred
authors speculated that this formulation concept of mucin and with terephtaloyl chloride, diluted with chloroform/cyclohexane
sodium alginate cross-linking would enhance the adsorption of before washing in a series of ethanol and water and nally
insulin from the gastro-intestinal tract (GIT) through the provision lyophilized. HES microparticles without BSA loaded measured
of a protective environment for insulin and also improved between 4 to 15 mm with an average diameter of 8 mm. BSA was
mucoadhesion of insulin microparticles. Although the low loaded into HES microparticles through incubation and immedi-
temperatures required for this method could possibly be a ately used for inoculation into mice on days 1 and 21. Each group of
deterrent to this formulation method, it was argued that low mice was immunized with saline, BSA solution, BSA in alum or
 M.L. Tan et al. / Peptides 31 (2010) 184193 189

BSA-HES microparticles, either through the intraperitoneal (ip) or angiogenesis assays while the time-course and sustainability of
subcutaneous (sc) route. Blood samples were then collected peptide release from RGD-HGC nanoparticles were examined
approximately every four days until 77 days after the rst using a non-invasive optical imaging system. HGC nanoparticles
immunization. No primary immune response was induced by all were rst prepared through the conjugation of glycol chitosan with
immunization doses delivered ip, although BSA-HES microparti- hydrophobic 5b-cholanic acid, followed by the encapsulation of
cles elicited secondary response through induction of anti-BSA IgG RGD by solvent evaporation. RGD peptides were efciently loaded
after the second immunization, with a maximal titer at day 38. into HGC nanoparticles (85%) and displayed a spherical nanopar-
However, ip immunization with BSA-HES microparticles appeared ticle structure measuring around 335 nm in diameter. There was a
less efcient than BSA/alum. Upon sc immunization, antibody burst release of RGD peptide (78%) from HGC nanoparticles within
response was detected in all three groups following the rst one day but residual peptide (22%) showed a reasonable sustained
injection. At this stage, BSA-HES microparticles and BSA/alum were release over the next seven days. RGD peptides conjugated to
more efcient than free BSA. After the booster dose, high antibody uorophore Cy5.5 were also encapsulated into HGC nanoparticles
titers were observed in BSA/alum mice until day 77. However, for optical imaging. To examine the in vivo time-dependent drug
there was no signicant difference in antibody titers between mice release prole of RGD-HGC nanoparticles in tumors, RGD-Cy5.5-
immunized with BSA-HES microparticles and free BSA. Therefore, HGC nanoparticles and RGD-Cy5.5 were intravenously (iv) or
the sc route of administration for vaccination against BSA resulted intratumorally (it) injected into B16F10 melanoma-bearing mice.
in a stronger IgG response as compared to ip administration. To Results showed that RGD peptides injected it produced a stronger
determine whether a Th1- or Th2-mediated immune response was uorescence signal within the tumor interstitial as compared to
induced following BSA-HES microparticle immunization, titers of mice injected with RGD peptides iv, although mice injected with
IgG1 and IgG2a were quantied through an ELISA assay. The ip RGD-Cy5.5-HGC nanoparticles it showed the highest uorescence
immunization with BSA-HES microparticles elicited a high IgG1 intensity within the tumor. In addition, RGD-Cy5.5 released from
than IgG2a titer; however SA/alum elicited a higher IgG1/IgG2a HGC nanoparticles co-located with CD31-positive microvessels,
ratio and was more efcient in triggering a Th2-type immune indicating that RGD peptide bound strongly to the angiogenic
response. Unfortunately, sc administered BSA-HES microparticles microvessels in tumor tissues. Comparisons of uorescence
only resulted in IgG1 and IgG2a responses comparable to free BSA, intensities between tumor and normal tissues also showed that
while BSA/alum induced both a sustained IgG1 response and RGD-HGC nanoparticles allowed the maintenance of a high
increasing IgG2a response from days 30 to 77. Interestingly, mice concentration of RGD peptide in the tumor, as compared to free
immunized ip with BSA-HES microparticles had increased levels of RGD. The anti-tumor efcacy of free RGD and RGD-HGC
IFN-g and IL-4. Similarly, mice injected sc with BSA-HES nanoparticles were studied in the melanoma-bearing mice, where
microparticles induced higher IFN-g levels. In conclusion, this free RGD peptides were injected iv and it while RGD-HGC
study demonstrated the following: importance of the choice of the nanoparticles were injected it every two days. After twelve days
route of administration and BSA-HES microparticles can induce of RGD treatment, tumor growth in all mice were retarded, with
both a Th1 and Th2 immune response which is critical for the the RGD-HGC treated mice boasting signicantly smaller tumor
control of malignant cells as well as infectious pathogens. Thus, sizes than other treated groups. H&E staining further conrmed the
HES microparticles may be considered a suitable drug delivery signicant reduction in tumor tissues and CD31 immunostaining
system for antigens to generate vaccines. showed a large decrease (> 70%) in CD31-positive microvessels. In
Similarly to liposome formulation, microparticle DDSs are conclusion, the anti-angiogenic effects of RGD-HGC nanoparticles
formulated with a wide variety of formulation materials and were superior to free RGD peptides as observed by the marked
methods. It is crucial to design DDSs that are both compatible with tumor growth inhibition through suppression of tumor angiogen-
the protein/peptide drugs as well as the host. Once again, the size esis. This could be attributed to the protective effect of HGC
of these DDSs would also contribute to the overall efcacy of the towards RGD against enzymatic degradation and also towards the
drug. As with the concerns about opsonization of liposomes above, accumulation of RGD within the tumor. Thus, the RGD-HGC
microparticles might also encounter similar problems. Thus, nanoparticle model has shown great potential and could be further
nanoparticles being on a much smaller scale, might appear to be investigated for anti-angiotherapy for local and regional tumor
a better DDS. therapy.
The bloodbrain barrier (BBB), made up of tight continuous
2.3. Nanoparticles circumferential junctions between the brain micro-vessel
endothelial cells, forms a barrier against water-soluble molecules
Moving onto a nano-scale, another promising therapeutic and possibly affecting those with molecular weight above 500 Da,
carrier, glycol chitosan modied with hydrophobic analogs, has posing many challenges for the delivery of drugs to the central
been studied extensively due to its ability to absorb drugs with nervous system (CNS). This also constitutes a major impediment to
high avidity within its hydrophobic inner cores and allow for their therapy as drugs are unable to reach the target organs at
prolonged, sustained release. Glycol chitosan nanoparticles have therapeutic concentrations. Recently, nanoparticles were exam-
also been reported to display fast cellular and tissue internalization ined as a possibility for delivering drugs to the brain. In this report,
into tumors [13]. Additionally, chitosan and its derivatives are the authors sought to evaluate the brain transport of large
biodegradable, biocompatible and poorly immunogenic [25]. Thus, molecular weight drug-loaded nanoparticles by a microdialysis
in this study, hydrophobically modied glycol chitosan (HGC) sampling technique [11]. The peptide of interest was neurotoxin-I
nanoparticles were used as a drug delivery system for the (NT-I), an analgesic peptide with limited permeability across the
enhancement of anti-angiogenic and anti-tumor effects of an BBB while the peptide carrier consisted of the polymer PLA, a
RGD peptide in a solid tumor model [30]. RGD peptides were biodegradable polyester approved for clinical use by the Food and
recently developed to target the avb3 integrin on angiogenic Drug Administration agency. NT-I nanoparticles prepared using
endothelial cells and have since shown to be valuable in tumor the double emulsication solvent evaporation method yielded
therapy and imaging although their anti-tumor effects are less particles about 65 nm in diameter, and an encapsulation efciency
promising. This was attributed to the relatively small RGD peptide of about 35%. Rats were subsequently administered either NT-I
analogs which were rapidly cleared from the circulation. RGD- nanopartices or free NT-I solution intranasally (in) or intravenously
containing HGC nanoparticles (RGD-HGC) were tested in in vivo (iv). Prior to treatment administration, a brain microdialysis probe
190 M.L. Tan et al. / Peptides 31 (2010) 184193

was inserted into the pre-prepared right olfactory bulb of the brain dopaminergic neurons and nondopaminergic neurons, including
and sampling began 30 min after treatment. Brain microdialysate spinal cord motorneurons and peripheral ganglia [36]. Previous
samples were collected and analyzed for NT-I levels. Results studies for GDNF treatment have been administered through factor
showed that brain delivery of NT-I was enhanced with PLA infusion, cell-based transplantation or gene therapy, with results
nanoparticles administered via both in and iv routes. In addition, in showing its potential as an agent for CNS treatment [9,10,19,52].
administration of NT-I PLA nanoparticles most signicantly To tap into the therapeutic potential of GDNF, the authors
increased NT-I concentrations in the brain. Thus, with nanopar- hypothesized that a continuous supply of GDNF would give
ticles as peptide carriers and delivery systems, in administration of greater efcacy to neural restoration of the injured spinal cord, and
CNS drugs (proteins and peptides) can be improved for brain thus designed a delivery system for the efcient delivery and
transport. sustained release of GDNF in the injured animals [58]. Firstly, the
In another work further investigating pathologies involving the authors determined whether nano-scaled PLGA nanoparticles
brain and the BBB, for the treatment of seizures, an endogenous could serve as carriers for GDNF by using uorescently-labeled
neuropeptide, thyrotropin-releasing hormone (TRH) is known to latex nanoparticles to investigate on the effects of particle size on
have anticonvulsant effects in animal seizure models and clinical transport within the spinal cord. Intraspinal injection of the
trials involving intractable epilepsies [32,55]. TRHs bioactivity and nanoparticles was chosen so as to avoid a lack of efcacy that might
bioavailability however is hindered by rapid tissue metabolism result during systemic administration and the resistance of the
and the BBB. In this study, the authors designed a direct nose-to- bloodbrain barrier. Subsequently, PLGA-GDNF nanoparticles
brain delivery of neuropeptides in sustained release biodegradable were formulated and their therapeutic effect on neuronal bers
nanoparticles (NPs) as a mode of therapy which enhances CNS was evaluated in SCI rats. In the rst part of the study, latex
neuropeptide bioavailability [31]. In the interests of space, only nanoparticles measuring 20 and 100 nm in diameter were injected
one aspect of this work directly related to nanoparticulate delivery into the lumbar section L1 and imaged by a uorescence reader.
systems of proteins will be presented here. Using the kindling 20 nm nanoparticles were largely located in the neuronal bers of
model of temporal lobe epilepsy, the authors investigated whether the thoracic and cervical spinal sections of rats, while 100 nm
the intranasal administration of polylactide nanoparticles (PLA- nanoparticles were accumulated mainly in the central canal or at
NPs) containing TRH (TRH-NPs) can impede kindling development the injection site. These results suggested that 20 nm nanopar-
in terms of behavioral stage, after-discharge duration (ADD) and ticles could be easily uptaken by neurons at the injection site and
clonus duration. PLA NPs were formulated together with or transported through the healthy spinal cord by retrograde axonal
without TRH by the solvent evaporation method using a double transport. A similar test conducted on SCI rats showed that the
emulsion process. TRH NPs and empty PLA NPs measured 108 and transportation of nanoparticles was interrupted. In the next part
102 nm, respectively, demonstrating a uniformity of size. Two of the study, PLGA nanoparticles encapsulating GDNF were
groups of rats were pre-treated with either TRH-NPs or empty NPs synthesized by the double emulsion solvent evaporation method.
seven days before kindling. Kindling stimulations were initiated on The resultant nanoparticles measured approximately 200 nm and
the eighth day, along with intranasal administration of NPs and had a sustained release of GDNF over seven days. PLGA-GDNF
continued daily until the rats were fully kindled (permanently nanoparticles were injected into the injured spinal cords of SCI
epileptic) or up to 20 stimulations. As compared to free rats and their effect was evaluated through the assessment of the
administration of TRH peptides or empty NPs, positive results rats hindlimb locomotor function every 23 days up to 31 days.
were observed in the TRH-NP group due to: (1) the ADD was Based on the Basso Beattie Bresnahan (BBB) locomotor rating
signicantly attenuated from the 10th to 13th stimulations; (2) the scale, PLGA-GDNF rats were given a score of 3.7 at week one as
number of stimulations required to reach the rst stage V seizure compared to the score of PLGA rats at 1.3, and only showed slight
and to become fully kindled was signicantly prolonged; (3) the movement of one or two joints. Over subsequent weeks, BBB
onset to clonus was signicantly delayed and suppressed overall scores in PLGA-GDNF rats increased signicantly over PLGA rats
clonus duration during stage V seizures. Initial safety proles were or bolus GDNF rats. These ndings strongly suggested for the
also positive given that there were no observable behavioral continuous release of GDNF from PLGA nanoparticles to the
changes or signicant weight change over the course of the study, contused spinal cord leading to better locomotor recovery as
an indirect indication of a non-effect on the pituitary-thyroid compared to other treatment groups. Physically, PLGA-GDNF
hormone axis. The levels of TRH in the brain after intranasal treated rats also afforded weight-supported plantar steps while
delivery were not determined due to the technical difculty rats in other treatment groups only showed slight movements in
involved, though they remain a subject for further research. In the joints. Immunouorescence further supported the nding by
conclusion, the authors believed that the data presented are the showing elongated intact neuronal ber bundles with neurola-
rst direct proof of concept for efcacious use of intranasal ment-positive staining in the lesion center of PLGA-GDNF rat
administration of sustained release anticonvulsant neuropeptide spinal cord, while only ne fragmented neuronal bers were
NPs as a viable new means for suppressing temporal lobe seizures observed in PLGA-treated mice. This was a highly positive sign for
and other intractable seizures. In future, TRH-NPs may provide an spinal recovery treatment as effective neuroprotection was an
alternative treatment for seizures that could signicantly impede important factor in enhancing hindlimb locomotion recovery in
or even prevent epileptogenesis, however, prior to that, more SCI rats. Hence, although the size of PLGA-GDNF nanoparticles
pharmacokinetic and pharmacotherapeutic research is necessary were larger than that suitable for retrograde axonal transport,
to optimize this intranasal neuropeptide carrier system. results demonstrated that GDNF loaded in PLGA was released
In yet another condition that is difcult in its treatment, spinal over time and remained bioactive in the injured spinal cord,
cord injury (SCI) has the potential of destroying the axonal nerve which results in an increased neuronal survival and better
bers and triggering inammation that might lead to neuronal cell hindlimb locomotion in SCI rats. Through further studies on the
death. A potential therapeutic treatment for SCI could occur safety, biocompatibility, shelf-life and efcacy of these nano-
through neuroprotection by the administration of exogenous particles, the potential of this treatment could be extended to
neurotrophic growth factors to improve axonal outgrowth [54]. humans who are suffering the devastating effects of spinal cord
One such polypeptide growth factor, the glial cell line derived injury.
neurotrophic factor (GDNF) is a transforming growth factor (TGF)- Developments of anti-cancer treatments have been especially
b super family member which functions as a trophic factor for difcult for researchers as chemotherapeutic drugs are often toxic
 M.L. Tan et al. / Peptides 31 (2010) 184193 191

to both cancer cells as well as normal cells. This was also true of the release of BMP-2, often suffering from high initial burst release and
naturally occurring tetrapeptide tubulysin [47]. Isolated from inability to provide sustained release of BMP-2. In a previous work
strains of myxobacteria, tubulysin has demonstrated potent by the authors, BMP-2 containing bovine serum albumin (BSA)
antiproliferative activity against multiple cancer cell lines even at nanoparticles (NPs) with a polyethylenimine (PEI) coating were
nmol/L and pmol/L concentrations [48]. Tubulysin act as prepared and analyzed accordingly in an in vitro system [63]. In
antimitotic agents, causing depolymerization of cell microtubules this study, PEI-coated BMP-2 containing BSA NPs were evaluated in
and triggering apoptosis. Tubulysin also worked in tumors a subcutaneous implant model to examine the pharmacokinetics
exhibiting multidrug resistance phenotypes [29]. However, of BMP-2 release and also to assess their osteoinductive activity
tubulysin is also highly toxic to healthy cells and has low [62]. Firstly, a comparison was made between PEI-coated and non-
solubility in solvents. Thus, in this report, the authors synthesized coated BMP-2 NPs to examine the effect of PEI. Non-coated BMP-2
a thiol derivative of tubulysin A (TubA) and conjugated TubA to a NPs experienced signicant burst release (>70%) as compared to
b-cyclodextrin-based polymer (CDP), forming CDP-TubA nano- PEI-coated NPs (2030%) after one day. At the end of seven days,
particles [49]. The CDP-TubA nanoparticles were synthesized in a about 4050% of BMP-2 remained in PEI-coated NPs while less
four step synthesis process resulting in particles of 100 to 130 nm than 15% of BMP-2 remained in non-coated NPs. This was expected
in diameter with a minimal release of TubA (<5%) over 72 h. The as the PEI coating was expected to delay BSA degradation from the
anti-tumor activity of TubA was subsequently evaluated in NPs and thus allow the retarded release of BMP-2 from the BSA
subcutaneous HT29 human colon carcinoma xenografts estab- matrix. The osteoinductive activity of BMP-2 NPs was then
lished in female athymic nude mice. As a comparison, free TubA assessed by subcutaneous implantation in rats. Higher ALP activity,
was administered through three weekly doses of 0.1 mg/kg, which osteocalcin levels and calcium deposits were all observed in non-
unfortunately was higher than the maximum tolerated dose and coated BMP-2 NPs as compared to PEI-coated BMP-2 NPs,
led to 50% mortality. In contrast, three weekly doses of CDP-TubA suggesting of an undesired effect of PEI on the osteoinductive
nanoparticles at 3 mg/kg were well tolerated with minimal body activity of BMP-2, even when free BMP-2 was administered along
weight loss and signicant tumor growth delay. At the conclusion with PEI-coated BMP-2 NPs. To determine if altering the
of the experiment, there were three 90-day survivors, six partial concentration of BMP-2 could reduce the negative effects of PEI,
tumor regressions, three complete tumor regressions and one NPs were loaded with different concentrations of BMP-2. As before,
tumor-free survivor. Even at 50% of the maximum tolerated dose both ALP levels and calcication results showed that non-coated
of CDP-TubA, the treatment was signicantly more effective in NPs had the highest activity as compared to PEI-coated NPs and
terms of tumor size reduction and survival, as compared to free BMP-2. It appeared that the NP formulation process did not
chemotherapeutic drugs vinblastin and paclitaxel at equitoxic adversely affect the osteoinductive activity of BMP-2, however, the
and maximum tolerated dose, respectively. The efcacy of CDP- inclusion of PEI during NP formulation produced cytotoxic effects
TubA nanoparticles were further evaluated in human H460 non- that were detrimental to BMP-2 bioactivity. This observation was
small cell lung carcinoma xenografts. Treatment with CDP-TubA further conrmed when little signs of bone formation were
at 3 mg/kg and 6 mg/kg (50% and 100% maximum tolerated dose, observed with the delivery of PEI-coated NPs together with the
respectively) resulted in a signicant tumor growth delay and addition of free BMP-2. In conclusion, PEI-coated NPs had reduced
reduction in tumor sizes. Tumor growth curves also showed that burst release and sustained delivery of BMP-2, however, PEI
there was tumor stasis for approximately two weeks after the nal adversely interacted with BMP-2 causing BMP-2 to lose its
dose of 6 mg/kg CDP-TubA indicative of its prolonged tumour osteoinductive activity. The results also showed a dose-dependent
growth suppression ability and prolonged release of active drug increase in bone formation with increased BMP-2 loading into NPs.
within the tumors. As expected, treatment of mice with free TubA Hence, the authors believed that the toxicity of PEI can be reduced
did not result in a signicant tumour growth delay or any by optimizing the BMP-2 loading and retaining the osteoinductive
reduction in tumour burden. In conclusion, the thiol derivative of activity of BMP-2.
tubulysin A, TubA conjugated to CDP forming CDP-TubA, has At rst glance, nanoparticle DDSs were used to explore
shown equal or superior efcacy at tumor regression compared difcult diseases (spinal cord injury, seizures and brain tumors)
with conventional chemotherapy drugs vinblastine and paclitaxel involving the penetration of the bloodbrain barrier, the positive
but with reduced toxicity. This study also rst tested this novel results itself attesting to the potential of this carrier system
analog of tubulysin A, TubA, for anti-tumor activity against over liposomes or microparticles. However, much more can be
established tumors and provides the path for further studies with done when formulating DDSs to produce carriers that are
CDP-TubA nanoparticles. In addition, the authors could have compatible with protein and peptide therapeutics. Not forgetting
further probed for the side-effects of CDP-TubA on blood and carriers that are compatible with the host to minimize host
accessory organs for a thorough analysis of its improved rejection.
therapeutic index.
Bone morphogenetic proteins (BMPs) are a group of secreted 3. Future directions
proteins which exerts an osteoinductive effect, of which BMP-2 has
been shown to induce osteogenesis [6]. Due to its potency at With the advancement in protein and peptide technology,
stimulating bone growth at implantation sites, BMPs are an more is being understood about various proteins and their roles
alternative for autogenous bone grafting in bone healing [27,51]. in human health and disease. For proteins with therapeutic
Clinically, BMP is utilized for the treatment of bone fractures and value, improving technologies also mean that they could be more
spinal fusion procedures [5,45]. Bone formation is induced when easily extracted, puried or synthesized for therapeutic purposes.
BMP-2 is present locally at the site of administration and also Unfortunately, a downside to protein and peptides are their
limited to the time when BMP is present. However, its efcacy is susceptibility to proteases and other factors. Thus, DDSs were
reduced due to the fast clearance rate and diffusion rate of BMP-2 envisioned to provide protection to proteins/peptides until they
from the site of administration [17]. Hence, a carrier which have reached their site of action. However, after a decade of
maintains BMP-2 at the treatment site was required. One of the protein/peptide DDS research; there is still a lack of good systems
most effective carriers used recently is the type 1 bovine available. Thus, for more efcient protein and peptide DDSs,
absorbable collagen sponge (ACS), which although has excellent researchers must consider the following: (1) safety and biocom-
properties as a carrier, has inherent problems with the control patibility of the DDS; (2) material and host toxicity; (3)
192 M.L. Tan et al. / Peptides 31 (2010) 184193

encapsulation efciency; (4) retention or enhancement of the [28] Jungheinrich C, Scharpf R, Wargenau M, et al. The pharmacokinetics and
tolerability of an intravenous infusion of the new hydroxyethylstarch 130/
natural therapeutic property of the protein/peptide; (5) tissue- 0.4 (6%, 500 ml) in mild-to-severe renal impairment. Anesth Analg 2002;95:
specic action; (6) uncomplicated preparation procedure and 54451.
administration; (7) cost-effective and not cost-prohibitive; (8) [29] Kaur G, Hollingshead M, Holbeck S, et al. biological evaluation of tubulysin A: a
potential anticancer and antiangiogenic natural product. Biochem J 2006;
biodegradability; (9) controlled, sustained release of therapeu- 396(2):23542.
tics. [30] Kim JH, Kim YS, Park K, et al. Self-assembled glycol chitosan nanoparticles for
the sustained and prolonged delivery of antiangiogenic small peptide drugs in
cancer therapy. Biomaterials 2008;29(12):192030.
References [31] Kubek MJ, Domb AJ, Veronesi MC. Attenuation of kindled seizures by intra-
nasal delivery of neuropeptide-loaded nanoparticles 2009;6(2):35971.
[1] Aguado MT, Lambert PH. Controlled-release vaccines-biodegradable polylac- [32] Kubek MJ, Garg BP. Thyrotropin-releasing hormone (TRH) in the treatment of
tide/polyglycolide (PL/PG) microspheres as antigen vehicles. Immunobiology intractable epilepsy. Pediatr Neurol 2002;26(1):917.
1992;184:11325. [33] Langeron O, Doelberg M, Ang ET, et al. Voluven1, a lower substituted novel
[2] Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science hydroxyethyl starch (HES 130/0.4), causes fewer effects on coagulation
2004;303(5665):181822. in major orthopaedic surgery than HES 200/0.5. Anesth Analg 2001;92:
[3] Balasse E, Odot J, Gatouillat G, et al. Enhanced immune response induced by 85562.
BSA loaded in hydrozyethylstarch microparticles. Int J Pharm 2008;353(1 [34] Liguori L, Marques B, Villegas-Mendez A, et al. Liposomes-mediated delivery
2):1318. of pro-apoptotic therapeutic membrane proteins. J Control Rel 2008;126:21
[5] Boden SD, Zdeblick TA, Sandhu HS, et al. The use of rhBMP-2 in interbody 7.
fusion cages. Spine 2000;25(3):37681. [35] Lilly CM, Draxen JM, Shore SA. Peptidase modulation of airway effects of
[6] Boyne PJ. Application of bone morphogenetic proteins in the treatment of neuropeptides. Proc Soc Exp Biol Med 1993;203:388404.
clinical oral and maxillofacial osseous defects. J Bone Joint Surg Am [36] Lin LF, Doherty DH, Lile JD, et al. GDNF: a glial cell line-derived neurotrophic
2001;83:s14650. factor for midbrain dopaminergic neurons. Science 1993;260(5111):1130
[7] Builders PF, Kunle OO, Okpaku LC, et al. Preparation and evaluation of 2.
mucinated sodium alginate microparticles for oral delivery of insulin. Eur J [37] Marazuela EG, Prado N, Moro E, et al. Intranasal vaccination with poly(lactide-
Pharm Biopharm 2008;70(3):77783. co-glycolide) microparticles containing a peptide T of Ole e 1 prevents mice
[8] Camelo S, Lajavardi L, Bochot A, et al. Ocular and systemic bio-distribution of against sensitisation. Clin Exp Allergy 2008;38(3):5208.
rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of [38] Moghimi SM, Szebeni J. Stealth liposomes and long circulating nanoparticles:
Lewis rats. Mol Vis 2007;13:226374. critical issues in pharmacokinetics, opsonization and protein-binding proper-
[9] Cao L, Liu L, Chen ZY, et al. Olfactory ensheathing cells genetically modied ties. Prog Lipid Res 2003;42(6):46378.
to secrete GDNF to promote spinal cord repair. Brain 2004;127(3): [39] Nomura S, Inami N, Shouzu A, et al. The effects of pitavastatin, eicosapen-
53549. taenoic acid and combined therapy on platelet-derived microparticles and
[10] Cheng H, Wu JP, Tzeng SF. Neuroprotection of glial cell line-derived neuro- adiponectin in hyperlipidemic, diabetic patients. Platelets 2009;20(1):16
trophic factor in damaged spinal cords following contusive injury. J Neurosci 22.
Res 2002;69(3):397405. [40] Pal S, Barnhart KM, Wei Q, et al. Vaccination of mice with DNA plasmids coding
[11] Cheng QY, Feng J, Chen J, et al. Brain transport of neurotoxin-I with PLA for the Chlamydia trachomatis major outer membrane protein elicits an
nanoparticles through intranasal administration in rats: a microdialysis study. immune response but fails to protect against a genital challenge. Vaccine
Biopharm Drug Dispos 2008;29(8):4319. 1999;17:45965.
[12] Chiappetta DA, Carcaboso AM, Bregni C, et al. Indinavir-loaded pH-sensitive [41] Pal S, Peterson EM, de la Maza LM. Vaccination with the Chlamydia trachomatis
microparticles for taste masking: toward extemporaneous pediatric anti-HIV/ major outer membrane protein can elicit an immune response as protective as
AIDS liquid formulations with improved patient compliance. AAPS PharmSci- that resulting from inoculation with live bacteria. Infect Immun 2005;
Tech 2009;10(1):16. 73:815360.
[13] Cho YW, Park SA, Han TH, et al. In vivo tumour targeting and radionuclide [42] Pal S, Theodor I, Peterson EM, et al. Immunisation with the Chlamydia
imaging with self-assembled nanoparticles: mechanisms, key factors, and muridarum mouse pneumonitis major outer membrane protein can elicit a
their implications. Biomaterials 2007;28(6):123647. protective immune response against a genital challenge. Infect Immun 2001;
[14] Christensen D, Korsholm KS, Rosenkrands KS, et al. Cationic liposomes as 69:62407.
vaccine adjuvants. Expert Rev Vaccines 2007;6:78596. [43] Patel ZS, Ueda H, Yamamoto M, et al. In vitro and in vivo release of vascular
[15] Dass CR, Walker TL, Burton MA, DeCruz EE. Enhanced anticancer therapy endothelial growth factor from gelatine microparticles and biodegradable
mediated by specialised liposomes. J Pharm Pharmacol 1997;49:9725. composite scaffolds. Pharm Res 2008;25(10):23708.
[16] Davis SN, Granner DK. Insulin, oral hypoglycaemic agents, and the pharma- [44] Patel ZS, Yamamoto M, Ueda H, et al. Biodegradable gelatine microparticles as
cology of endocrine pancreas. In: Hardman JG, Limbird LE, Molinoff PB, delivery systems for the controlled release of bone morphogenetic protein-2.
Ruddeon RW, Gilman AG, editors. Goodman and Gilmans the pharmaco- Acta Biomater 2008;4(5):112638.
logical basis of therapeutics. 9th ed., New York: Mc-Graw Hill; 1996 . p. [45] Riedel GE, Valentin-Opran A. Clinical evaluation of rhBMP-2/ACS in orthopae-
1487517. dic trauma: a progress report. Orthopaedics 1999;22(7):6635.
[17] Devy J, Balasse E, Kaplan H, et al. Hydroxyethylstarch microcapsules: a [46] Said SI, Mutt V. Polypeptide with broad biological activity: isolation from small
preliminary study for tumour immunotherapy application. Int J Pharm intestine. Science 1970;169:12178.
2006;307(2):194200. [47] Sandmann A, Sasse F, Muller R. Identication and analysis of the core biosyn-
[19] Foust KD, Flotte TR, Reier PJ, et al. Recombinant adeno-associated virus- thetic machinery of tubulysin, a potent cytotoxin with potential anticancer
mediated global anterograde delivery of glial cell line-derived neurotrophic activity. Chem Biol 2004;11(8):10719.
factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in [48] Sasse F, Steinmetz H, Heil J, et al. Tubulysins, new cytostatic peptides from
the rat. Hum Gene Ther 2008;19(1):7182. myxobacteria acting on microtubule. Production, isolation, physico-chemical
[20] Frokjaer S, Otzen DE. Protein drug stability: a formulation challenge. Nat Rev and biological properties. J Antibiot (Tokyo) 2000;53(9):87985.
2005;4:298306. [49] Schluep T, Gunawan P, Ma L, et al. Polymeric tubulysin-peptide nanoparticles
[21] Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal with potent antitumour activity. Clin Cancer Res 2009;15(1):1819.
doxorubicin: review of animal and human studies. Clin Pharmacokinet [50] Shaw J, Grund V, Durling L, et al. Dendritic cells pulsed with a recombinant
2003;42(5):41936. Chlamydia major outer membrane protein antigen elicit a CD4+ type 2 rather
[22] Groneberg DA, Rabe KF, Fischer A. Novel concepts of neuropeptide-based drug than type 1 immune response that is not protective. Infect Immun 2002;70:
therapy: vasoactive intestinal polypeptide and its receptors. Eur J Pharmcol 1097105.
2006;533:18294. [51] Szpalski M, Gunzburg R. Recombinant human bone morphogenetic protein-2:
[23] Hajos F, Stark B, Hensler S, et al. Inhalable liposomal formulation for vasoactive a novel osteoinductive alternative to autogenous bone graft? Acta Orthop Belg
intestinal peptide. Int J Pharm 2008;357:28694. 2005;71(2):13348.
[24] Hansen J, Jensen KT, Follmann F, et al. Liposome delivery of Chlamydia [52] Tai MH, Cheng H, Wu JP, et al. Gene transfer of glial cell line-derived
muridarum major outer membrane protein primes a Th1 response that pro- neurotrophic factor promotes functional recovery following spinal cord con-
tects against genital chlamydial infection in a mouse model. JID 2008;198 tusion. Exp Neurol 2003;183(2):50815.
(1):75867. [53] Tauzin B. Report: biotechnology medicines in development. Washington, DC:
[25] Hirano S. Chitin and chitosan as novel biotechnological materials. Poly Int Pharmaceutical Research and Manufacturers Association; 2008.
1999;48:7324. [54] Thuret S, Moon LD, Gage FH. Therapeutic interventions after spinal cord injury.
[26] Jain AK, Chalasani KB, Khar RK, et al. Muco-adhesive multivesicular liposomes Nat Rev Neurosci 2006;7(8):62843.
as an effective carrier for transmucosal insulin delivery. J Drug Target [55] Wan RQ, Noguera EC, Weiss SR. Anticonvulsant effects of intra-hippocampal
2007;15(6):41727. injection of TRH in amygdala kindled rats. Neuro-report 1998;9(4):67782.
[27] Jones AL, Bucholz RW, Bosse MJ, et al. Recombinant human BMP-2 and [56] Wang W. Instability, stabilisation and formulation of liquid protein pharma-
allograft compared with autogenous bone graft for reconstruction of diaphy- ceuticals. Int J Pharm 1999;185:12988.
seal tibial fractures with cortical defects: a randomised, controlled trial. J Bone [57] Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J
Joint Surg Am 2006;88(7):143141. Pharm 2005;289:130.
 M.L. Tan et al. / Peptides 31 (2010) 184193 193

[58] Wang YC, Wu YT, Huang HY, et al. Sustained intraspinal delivery of neuro- [62] Zhang S, Doschak MR, Uludag H. Pharmacokinetics and bone formation by
trophic factor encapsulated in biodegradable nanoparticles following contu- BMP-2 entrapped in polyethylenimine-coated albumin nanoparticles.
sive spinal cord injury. Biomaterials 2008;29(34):454653. Biomaterials 2009;30(28):514355.
[59] Weissmann G, Sessa G. Phospholipid spherules (liposomes) as a model for [63] Zhang S, Wang G, Lin Z, et al. Polyethylenimine-coated albumin nanoparticles
biological membranes. J Lipid Res 1968;9(3):3108. for BMP-2 delivery. Biotechnol Prog 2008;24(4):94556.
[60] Werle M, Takeuchi H. Chitosan-aprotinin coated liposomes for oral peptide [64] Ipsen Pharmaceuticals Limited, Decapeptyl S. R.information for the patient.
delivery: development, characterisation and in vivo evaluation. Int J Pharm URL: http://www.ipsen.ie/dkp_patient.pdf.[Accessed 12/9/09].
2009;370(12):2632. [65] Takeda UK Limited, Prostap 3 Leuprorelin acetate depot injectionsummary
[61] Wu X, Huang S, Zhang J, et al. Preparation and characterisation of novel of product characteristics. URL: http://emc.medicines.org.uk/document.aspx?
physically cross-linked hydrogels composed of poly(vinyl alcohol) and documentId = 2237.[Accessed 12/9/09].
amine-terminated polyamidoamine dendrimer. Macromol Biosci 2004;4:715.