In Vitro Cytotoxicity and in Vivo Antitumor Activity of Lipid Nanocapsules Loaded With Novel Pyridine Derivatives
In Vitro Cytotoxicity and in Vivo Antitumor Activity of Lipid Nanocapsules Loaded With Novel Pyridine Derivatives
In Vitro Cytotoxicity and in Vivo Antitumor Activity of Lipid Nanocapsules Loaded With Novel Pyridine Derivatives
Article
In Vitro Cytotoxicity and In Vivo Antitumor Activity of Lipid
Nanocapsules Loaded with Novel Pyridine Derivatives
Amr Selim Abu Lila 1,2,† , Mohammed Amran 3,4,† , Mohamed A. Tantawy 5,6 , Ehssan H. Moglad 7,8 ,
Shadeed Gad 9 , Hadil Faris Alotaibi 10 , Ahmad J. Obaidullah 11 and El-Sayed Khafagy 7,9, *
1 Department of Pharmaceutics, College of Pharmacy, University of Hail, Hail 81442, Saudi Arabia;
a.abulila@uoh.edu.sa
2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University,
Zagazig 44519, Egypt
3 Department of Pharmacy, Faculty of Health Sciences, Thamar University, Thamar 87246, Yemen;
mohamran2013@gmail.com
4 Department of Pharmacy, Al-Manara College for Medical Sciences, Maysan 62001, Iraq
5 Hormones Department, Medical Research and Clinical Studies Institute, National Research Centre, Dokki,
Giza 12622, Egypt; mohamed_tantawy@daad-alumni.de
6 Stem Cells Lab, Center of Excellence for Advanced Sciences, National Research Centre, Dokki,
Giza 12622, Egypt
7 Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia; e.moglad@psau.edu.sa
8 Department of Microbiology and Parasitology, Medicinal and Aromatic Plants Research Institute,
National Center for Research, Khartoum 2404, Sudan
9 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University,
Ismailia 41522, Egypt; shaded_abdelrahman@pharm.suez.edu.eg
10 Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourah Bint Abdul Rahman
University, Riyadh 11671, Saudi Arabia; hfalotaibi@pnu.edu.sa
11 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia; aobaidullah@ksu.edu.sa
Citation: Abu Lila, A.S.; Amran, M.;
* Correspondence: e.khafagy@psau.edu.sa
† These authors contributed equally to this work.
Tantawy, M.A.; Moglad, E.H.; Gad, S.;
Alotaibi, H.F.; Obaidullah, A.J.;
Khafagy, E.-S. In Vitro Cytotoxicity
Abstract: This study demonstrates high drug-loading of novel pyridine derivatives (S1–S4) in lipid-
and In Vivo Antitumor Activity of and polymer-based core–shell nanocapsules (LPNCs) for boosting the anticancer efficiency and
Lipid Nanocapsules Loaded with alleviating toxicity of these novel pyridine derivatives. The nanocapsules were fabricated using a
Novel Pyridine Derivatives. nanoprecipitation technique and characterized for particle size, surface morphology, and entrap-
Pharmaceutics 2023, 15, 1755. https:// ment efficiency. The prepared nanocapsules exhibited a particle size ranging from 185.0 ± 17.4
doi.org/10.3390/pharmaceutics15061755 to 223.0 ± 15.3 nm and a drug entrapment of >90%. The microscopic evaluation demonstrated
Academic Editors: Vanessa Carla
spherical-shaped nanocapsules with distinct core–shell structures. The in vitro release study depicted
Furtado Mosqueira and Raquel a biphasic and sustained release pattern of test compounds from the nanocapsules. In addition, it was
Silva Araújo obvious from the cytotoxicity studies that the nanocapsules showed superior cytotoxicity against both
MCF-7 and A549 cancer cell lines, as manifested by a significant decrease in the IC50 value compared
Received: 22 May 2023
to free test compounds. The in vivo antitumor efficacy of the optimized nanocapsule formulation
Revised: 10 June 2023
(S4-loaded LPNCs) was investigated in an Ehrlich ascites carcinoma (EAC) solid tumor-bearing mice
Accepted: 15 June 2023
Published: 16 June 2023
model. Interestingly, the entrapment of the test compound (S4) within LPNCs remarkably triggered
superior tumor growth inhibition when compared with either free S4 or the standard anticancer drug
5-fluorouracil. Such enhanced in vivo antitumor activity was accompanied by a remarkable increase
in animal life span. Furthermore, the S4-loaded LPNC formulation was tolerated well by treated
Copyright: © 2023 by the authors. animals, as evidenced by the absence of any signs of acute toxicity or alterations in biochemical
Licensee MDPI, Basel, Switzerland. markers of liver and kidney functions. Collectively, our findings clearly underscore the therapeutic
This article is an open access article
potential of S4-loaded LPNCs over free S4 in conquering EAC solid tumors, presumably via granting
distributed under the terms and
efficient delivery of adequate concentrations of the entrapped drug to the target site.
conditions of the Creative Commons
Attribution (CC BY) license (https://
Keywords: antitumor activity; Ehrlich ascites carcinoma; MTT assay; nanocapsule; pyridine derivatives
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Cancer is a worldwide health problem characterized by the abnormal proliferation of
malignant cells with the ability to infiltrate or spread to other regions of the body [1]. It is
regarded as the second leading cause of mortality globally, after cardiovascular disease, and
it overtakes cardiovascular disease as the leading cause of mortality in high-income coun-
tries [2]. More than 10 million new cases of cancer are diagnosed annually, and the World
Health Organization forecasts that cancer-related deaths will grow to over 13.1 million
by 2030, with the greatest rise in low- and middle-income countries [3]. Despite the fact
that cancer-related mortality has declined in recent years due to a better understanding
of tumor biology, which has led to remarkable progress in cancer detection, prevention,
and treatment, the development of new anticancer agents that show higher therapeutic
efficacies along with minimized adverse effects remains the ultimate goal in cancer research.
Pyridines are a class of synthetic and naturally occurring heterocyclic compounds with
a six-membered heterocyclic moiety that has a wide variety of biological and therapeutic
applications, including anticancer activities [4–7]. Recently, we succeeded in synthesizing
novel 2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile derivatives using a facile
one-pot multicomponent condensation reaction. All the synthesized pyridine derivatives
exhibited promising in vitro cytotoxic activities against various cancer cell lines, which
grants their subsequent use in cancer therapy [7]. Nevertheless, these compounds showed
limited aqueous solubility that might limit their clinical use. Furthermore, the lack of target
selectivity of these synthesized pyridine derivatives might compromise their therapeutic
effectiveness in vivo.
Recently, the application of nanotechnology has enabled a tremendous push to en-
hance the therapeutic efficacies of many anticancer agents. Nanoparticulate delivery
systems have offered many advantages over conventional delivery systems, including
protecting the drug from being degraded/eliminated during its transit to the target site,
modifying the biodistribution of the entrapped therapeutic agents, promoting targeted
delivery of high drug payloads, controlling drug release, and evading multidrug resistance
mechanisms [8–10]. Accordingly, a number of nanoparticulate drug delivery systems
with an average size below 200 nm, such as micelles [11], liposomes [12], lipid/polymeric
nanoparticles [13], dendrimers [14], etc., has been screened for their possible application
in cancer therapy. Among these carriers, nanocapsules (NC) have emerged as poten-
tial nanocarriers in cancer therapy. Nanocapsules are vesicular systems with a typical
core–shell structure that confines active molecules as a reservoir in a cavity surrounded by
a polymer coating [15]. The cavity can hold an active material in solid, liquid, or molecular
dispersion form [16,17].
Lipid nanocapsules (LNCs) are considered a hybrid structure between liposomes and
polymeric nanocapsules owing to their oily core, which is surrounded by a tension-active
rigid membrane [18]. Nevertheless, compared to liposomes, LNCs offer the advantages
of being of higher physical stability, higher gastrointestinal stability, and having a higher
encapsulation capacity of lipophilic drugs in their oily core [18,19]. Most importantly, LNCs
show adjuvant effects, such as P-glycoprotein inhibition properties, which could promote
preferential intracellular drug accumulation within cancer cells, resulting in enhanced
anticancer effects [20]. Recently, many reports have established the in vivo effectiveness
of methotrexate-loaded lipid core nanocapsules [21] and indomethacin-loaded lipid-core
nanocapsules [22] in treating glioblastoma, since lipid-core nanocapsules act as a drug
shuttle that cross the blood brain barrier to deliver the drug to brain tissue.
The aim of this study was to investigate the efficiency of LPNCs in enhancing the
anticancer activity of novel pyridine derivatives. LPNCs, composed of polylactic-co-glycolic
acid (PLGA) with one of different oil phases (caprylic/capric triglyceride, olive oil, or oleic
acid), were prepared by a nanoprecipitation method and were characterized in terms of
morphology and physicochemical properties. The anti-proliferative activities of different
pyridine derivatives entrapped within LPNCs were investigated in vitro against A549 and
MCF-7 cell lines, and in an in vivo tumor model.
The aim of this study was to investigate the efficiency of LPNCs in enhancing the
anticanceranticancer
activity activity
of novelofpyridine
novel pyridine derivatives.
derivatives. LPNCs,LPNCs,
composed composed of polylactic-co-gly-
of polylactic-co-gly-
anticancer activity of novel pyridine derivatives. LPNCs, composed of polylactic-co-gly-
colic acidcolic acid (PLGA)
(PLGA) with onewith one of different
of different oil (caprylic/capric
oil phases phases (caprylic/capric triglyceride,
triglyceride, olive oil,olive
or oil, or
colic acid (PLGA) with one of different oil phases (caprylic/capric triglyceride, olive oil, or
oleic
oleic acid), acid),
were were prepared
prepared by a nanoprecipitation
by a nanoprecipitation methodmethod
and were and were characterized
characterized in termsin terms
oleic acid), were prepared by a nanoprecipitation method and were characterized in terms
of morphology and physicochemical
of morphology properties.
and physicochemical The anti-proliferative
properties. activities
The anti-proliferative of differ-
activities of differ-
of morphology and physicochemical properties. The anti-proliferative activities of differ-
ent pyridine
ent pyridine derivatives
derivatives entrapped entrapped within LPNCs
within LPNCs were investigated
were investigated in vitro in vitro A549
against against A549
Pharmaceutics 2023, 15, 1755 ent pyridine derivatives entrapped within LPNCs were investigated in vitro against A5493 of 19
and MCF-7 and cell
MCF-7 cell
lines, lines,
and in anand
in in an tumor
vivo in vivomodel.
tumor model.
and MCF-7 cell lines, and in an in vivo tumor model.
2. Materials
2. Materials and Methods
and Methods
2. Materials and Methods
2.1. Materials
2.1. Materials 2. Materials and Methods
2.1. Materials
Caprylic/capric
Caprylic/capric 2.1.triglyceride
triglyceride Materials was obtained
was obtained from the from the Gattefosse
Gattefosse company company (St. Priest,
(St. Priest,
Caprylic/capric triglyceride was obtained from the Gattefosse company (St. Priest,
France).France). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
Caprylic/capric triglyceride tetrazolium
tetrazolium wasbromide bromide
obtained(MTT) (MTT)
from reagent,
the reagent,
5-
Gattefosse 5-
company (St. Priest, France).
France). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent, 5-
fluorouracil,
fluorouracil, poly(D,L-lactide-co-glycolide)
poly(D,L-lactide-co-glycolide) lactide:glycolide
lactide:glycolide
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (PLGA
(PLGAtetrazolium
50:50, Mw 50:50, Mw (MTT)
45,000
bromide 45,000 reagent, 5-fluorouracil,
fluorouracil, poly(D,L-lactide-co-glycolide) lactide:glycolide (PLGA 50:50, Mw 45,000
g/mol), g/mol), oleic
oleic acid, acid,
olive olive
oil, andoil, and dialysis
dialysis tubing cellulose
tubing cellulose
poly(D,L-lactide-co-glycolide) membrane membrane (M.Wt.
(M.Wt.(PLGA cutoffMw
cutoff 50:50,
12–14 12–14
g/mol), oleic acid, olive oil, andlactide:glycolide
dialysis tubing cellulose membrane 45,000
(M.Wt.g/mol), oleic acid,
cutoff 12–14
kDa)obtained
kDa) were were obtained
fromolive from Sigma-Aldrich
Sigma-Aldrich
oil, and (St. CA,
(St. Louis,
dialysis tubing Louis, CA,
USA).
cellulose USA).
All All(M.Wt.
other
membrane other cutoff
reagents reagents
and 12–14andkDa) were obtained from
kDa) were obtained from Sigma-Aldrich (St. Louis, CA, USA). All other reagents and
chemicals
chemicals were of were of analytical
analytical grade. grade. Louis, CA, USA). All other reagents and chemicals were of analytical grade.
Sigma-Aldrich
chemicals were(St. of analytical grade.
2.2. Synthesis
2.2. Synthesis Scheme
Scheme for forSynthesis
2‐Amino‐4‐aryl‐6‐substituted
2‐Amino‐4‐aryl‐6‐substituted
2.2. Schemefor Pyridine‐3,5‐dicarbonitrile
Pyridine‐3,5‐dicarbonitrile
for2‐Amino‐4‐aryl‐6‐substituted
2-Amino-4-aryl-6-substituted Derivatives
Derivatives
Pyridine-3,5-dicarbonitrile Derivatives
2.2. Synthesis Scheme Pyridine‐3,5‐dicarbonitrile Derivatives
2-amino-4-aryl-6-substituted
2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile
pyridine-3,5-dicarbonitrile derivatives
derivatives were
2-amino-4-aryl-6-substitutedpyridine-3,5-dicarbonitrile were
synthe- synthe-
pyridine-3,5-dicarbonitrilederivatives
derivativeswere
were synthe-
synthesized
2-amino-4-aryl-6-substituted
sized bysized by a one-pot
a one-pot multicomponent
multicomponent
by a one-potreaction reaction as previously
as previously
multicomponent reported
reaction reported
as [7] [7] (Scheme
(Scheme
previously 1 and 1 [7]
reported and(Scheme 1 and Table 1).
sized by a one-pot multicomponent reaction as previously reported [7] (Scheme 1 and
Table 1).Table 1). The structure
The structure of the
The of thecompounds
target target
structure ofcompounds
the was was completely
completely
target compounds identified
identified
was by IRidentified
by IR spectra,
completely spectra, by IR spectra, mass
Table 1). The structure of the target compounds was completely identified by IR spectra,
mass spectra,
mass spectra, and 1spectra,
and 1H-NMR H-NMR spectra
spectra and as previously
as previously
1 H-NMR shown
spectra shown
[7]. [7].
as previously shown [7].[7].
mass spectra, and 1H-NMR spectra as previously shown
2.3. Solubility
2.3. Solubility Measurement
Measurement 2.3. SolubilityMeasurement
2.3. Solubility Measurement
The solubility
The solubility of 2-amino-4-aryl-6-substituted
of 2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile
pyridine-3,5-dicarbonitrile derivatives
derivatives
The solubility
The solubilityofof2-amino-4-aryl-6-substituted
2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile
pyridine-3,5-dicarbonitrile derivatives
derivatives
(S1–S4) (S1–S4)
in variousin various
solventssolvents
(methanol,(methanol,
ethanol,ethanol,
phosphatephosphate buffer,
buffer, and and was
water) water) was deter-
deter-
(S1–S4)
(S1–S4) in in various
varioussolvents
solvents(methanol,
(methanol, ethanol,
ethanol, phosphate
phosphate buffer,
buffer, and and
water)water) was deter-
was deter-
mined by mined
addingby an
adding anamount
excess excess amount of each compound
of each compound to 10 mLtoof10each
mL solvent
of each solvent in a sealed
in a sealed
mined addingan
mined by adding anexcess
excessamount
amount ofof each
each compound
compound to mL
to 10 10 mL of each
of each solvent
solvent in a sealed
in a sealed
conical
conical flask. flask.
The Thewas
slurry slurrythenwas then agitated
agitated at 100
at 100 rpm in arpm in a temperature-controlled
temperature-controlled incuba- incuba-
conical flask. The
conical Theslurry
slurrywas
wasthen
thenagitated
agitatedatat100
100rpm
rpm inin
a temperature-controlled
a temperature-controlled incuba-
incubator
tor for
tor shaker shaker
72 hfor 72 h toequilibrium.
to reach reach equilibrium. The undissolved
The undissolved particlesparticles were allowed
were allowed to settleto settle
tor shaker
shaker forfor
72 72 h toreach
h to reachequilibrium.
equilibrium. TheTheundissolved
undissolved particles were
particles allowed
were to settle
allowed to settle
by keeping
by keeping the saturated
the saturated solutionsolution
withoutwithout
agitationagitation for another
for another 2 h. The2solution
h. The solution
was then was then
by keeping
by keepingthe thesaturated
saturated solution without
solution agitation
without for another
agitation 2 h. The2 solution
for another h. The was then was
solution
then centrifuged at 10,000 rpm for 10 min. One mL samples of the clear supernatant was
withdrawn, diluted with the suitable solvent, and finally analyzed spectrophotometrically
at pre-determined wavelengths (Table S1).
solution containing 0.25% w/v poloxamer 188 or Tween80 under magnetic stirring (Table 2).
The mixture was maintained under moderate magnetic stirring overnight to allow the
evaporation of the organic solvents and subsequent formation of LPNC. Blank LPNCs were
prepared similarly, but without the addition of pyridine derivatives to oil.
Table 2. Composition of various LPNC formulations.
∑ ni Di4
D [4, 3] =
∑ ni Di3
Dv0.9 − Dv0.1
Span =
Dv0.5
where I is an index of the population, Di is the particle diameter of the population i, and
Dv0.1 , Dv0.5 , and Dv0.9 are the diameters at the percentiles 10, 50, and 90 under the cumula-
tive size-distribution curve based on the volume of particles. For particle size distribution,
test samples were diluted 1:500 v/v with deionized water before the measurement.
The zeta potential of different LPNC dispersions was measured by electrophoretic mo-
bility and analyzed with the Malvern Nano ZS Zetasizer. All experiments were performed
in triplicates.
1
Tumor size (mm3 ) = (Length × Width2 )
2
Pharmaceutics 2023, 15, 1755 7 of 19
Mean tumor volume o f treated group
% TGI = 1 − × 100
Mean tumor volume in control group
" #
MST treated group
%ILS = − 1 × 100
MST control group
3.2.1. Effect of Polymer Content and Oil Content on the Size Distribution of LPNCs
Polymer and oil contents are considered the most important factors influencing the
structure of LPNC formulations [35]. Accordingly, in this study, the impact of both polymer
and oil content on the size distribution of the formulated LPNCs was investigated. LPNCs
were prepared using variable amounts of PLGA (10, 20, and 30 mg) and variable amounts
of CCT (100, 150, and 200 mg) in the presence of poloxamer 188 as a surfactant. The
particle size distribution of the prepared LPNCs, in terms of volume mean diameter (D[4,3])
and polydispersity index (PDI), is summarized in Table 3 (F1–F5). The laser diffraction
analysis confirmed the presence of all formulated LPNCs in the nano-size range with a
mean diameter (D[4,3]) ranging from of 135.33 ± 0.58 to 225.00 ± 10.54 nm and with a span
value fluctuating from 0.99 ± 0.01 to 1.82 ± 0.23. Generally, the span is used to quantify
distribution width; span values below 2.0 indicate narrow particle size distribution for these
formulations. Of note, at fixed PLGA contents the mean diameter (D[4,3]) of different LPNC
formylations was found to increase gradually from 135.33 ± 0.58 to 225.00 ± 10.54 nm
with increasing oil content from 100 to 200 mg. Such an increase in particle size with rising
CCT content might be ascribed to the high viscosity of the organic phase, since the higher
the oil content, the more viscous the organic phase [36]. Furthermore, it was evident that at
fixed oil content, increasing polymer content from 10 to 30 mg triggered a mutual rise in the
mean diameter (D[4,3]) of LPNCs. The mean diameter (D[4,3]) of F1 prepared with 10 mg
PLGA showed a mean diameter (D[4,3]) of 135.33 ± 0.58 nm, which is remarkably smaller
than that of LPNCs prepared with 30 mg PLGA (F5 184.67 ± 6.43 nm). Nevertheless,
increasing the polymer content from 10 to 20 mg did not result in a remarkable change
in the mean diameter of formulated LPNCs. Apart from its effect on particle size, it is
well-recognized that the polymeric shell plays a key role in protecting encapsulated drugs
and maintaining formulation stability. Accordingly, the stability of LPNCs prepared with 10
and 20 mg PLGA was investigated. Two weeks after preparation, a remarkable increase in
particle size of LPNCs prepared with 10 mg PLGA from 135.33 ± 0.58 to 146.33 ± 3.21 nm
was observed, while no remarkable increase in particle size was detected in LPNCs prepared
with 20 mg PLGA. Based on these findings, a polymer content of 20 mg and an oil content
of 100 mg was selected for the formulation of LPNCs for further experiments.
Table 3. Effect of various formulation variables on particle size distribution of blank LPNCs.
3.2.2. Effect of Oil Type and Surfactant Type on the Size Distribution of LPNCs
Next, we investigated the effect of oil type on the size distribution of LPNCs. In this
set of experiments, the size distribution of LPNCs prepared with 100 mg oleic acid or olive
oil was estimated and compared with that of LNCPs prepared with CCT. As depicted in
Table 3, the type of oil significantly affected the mean diameter (D[4,3]) of different LPNC
formulations. The mean diameter (D[4,3]) of LPNCs prepared with different oils was in
the following order: CTT (136.00 ± 1.73 nm) < oleic acid (255.00 ± 40.63 nm) < olive oil
(346.67 ± 26.03 nm). Viscosity regulates the mobility of the organic phase into the aqueous
phase, and thus directly affects the emulsification and nanoparticles size [36]. This might
explain the smaller size of LPNCs prepared with the low viscous oil (CCT) compared to
the higher viscous oils (oleic acid and olive oils).
Many reports have emphasized the contribution of the hydrophilic lipophilic balance
(HLB) of the used surfactants on particle size and formulation stability. In this study,
however, it was evident that surfactant types exerted no distinct effect on the particle size
of the formulated LPNCs (Table 2). LPNCs prepared with 20 mg of PLGA, 100 mg of CCT,
and 0.25% w/v Poloxamer 188 (F4) showed the smallest particle size amongst all tested
formulations; consequently, this formulation was selected as an optimized formula for
encapsulating different pyridine derivatives (S1–S4).
gation [41]. As summarized in Table 4 and Figure S1, the zeta potentials of all LPNC
formulations were in the negative range, fluctuating from −7.55 ± 1.7 (S2-LPNCs) to
−13.4 ± 2.1 mV (S4-LPNCs). The negative zeta potential of all formulations might be
ascribed to the presence of the terminal carboxylic acid group in PLGA used as a shell-
forming polymer [42]. This negative zeta potential might contribute to the colloidal stability
of formulated LPNCs via hindering particle aggregation and/or precipitation by virtue of
electrostatic repulsion between negatively charged surfaces.
Figure 1. TEM micrograph depicting the shape and morphology of (A) S1-loaded LPNCs; (B) S2-
Figure 1. TEM micrograph depicting the shape and morphology of (A) S1-loaded LPNCs;
loaded LPNCs; (C) S3-loaded LPNCs; and (D) S4-loaded LNCPs. The scale bar equals 100 nm.
(B) S2-loaded LPNCs; (C) S3-loaded LPNCs; and (D) S4-loaded LNCPs. The scale bar equals 100 nm.
3.4.4.Drug
3.4.4. Drug Loading
Loading and
and Entrapment
EntrapmentEfficiency ofof
Efficiency Test Compound-Loaded
Test LPNCs
Compound-Loaded LPNCs
Efficient drug loading is a key determinant of the therapeutic efficacy of drug-loaded
Efficient drug loading is a key determinant of the therapeutic efficacy of drug-loaded
nanocarriers. As summarized in Table 4, the drug-loading percentage of different test
nanocarriers. As summarized in Table 4, the drug-loading percentage of different test
compounds within LPNCs varied from 1.75 ± 0.03 (for S3-loaded LPNCs) to 2.39 ± 0.01%
compounds within LPNCs varied from 1.75 ± 0.03 (for S3-loaded LPNCs) to 2.39 ± 0.01%
(for S2-loaded LPNCs), respectively. Regarding encapsulation efficiency, it was evident
(for S2-loaded LPNCs), respectively. Regarding encapsulation efficiency, it was evident
that the entrapment efficiency of different test compounds within LPNCs fluctuated from
that the entrapment efficiency of different test compounds within LPNCs fluctuated from
90.62 ± 0.52% (for S2-loaded LPNCs) to 93.72 ± 0.66% (for S4-loaded LPNCs). The signifi-
90.62
cantly±high
0.52% (for S2-loaded
encapsulation LPNCs)
efficiency of different ± 0.66%
to 93.72pyridine (for S4-loaded
derivatives withinLPNCs). The sig-
LPNCs might
nificantly high encapsulation efficiency of different pyridine derivatives within
be explained by the greater solubility of compounds in the oily component of these for- LPNCs
might be explained
mulations. by thewere
Similar results greater solubility
reported ofMelo
by de compounds in who
et al. [44], the oily component
emphasized the of
im-these
formulations. Similar results
pact of the composition were
of the oily reported
nucleus by encapsulation
on the de Melo et al.of[44], who emphasized
the hydrophobic drug the
impact of thewithin
benzocaine composition of the oily nucleus on the encapsulation of the hydrophobic drug
PLGA nanocapsules.
benzocaine within PLGA nanocapsules.
3.4.5. Differential Scanning Calorimetry Analysis
3.4.5. Differential Scanning Calorimetry Analysis
DSC is an important technique for analyzing the crystallinity and interaction of drugs
withDSC is an components
different important technique for analyzing
of nanocapsules the crystallinity
by determining and interaction
the alteration of drugs
of temperature
with different
and energy components
at phase of nanocapsules
transition. by determining
In order to investigate the effect the alteration
of the of temperature
formulation process
and energy
on the at phase
thermal transition.
behavior of the In order to investigate
formulations, the effect
a DSC analysis of thetest
of each formulation
compoundprocess
in
on theform,
pure thermal
PLGA,behavior of the188,
poloxamer formulations,
and differenta DSC
LPNC analysis of eachwas
formulations testconducted
compound(Fig-
in pure
form,
ure 2).PLGA, poloxamer 188,
DSC thermograms of theand different
pure LPNC
substances (S1,formulations
S2, S3, and S4)was conducted
showed (Figure 2).
characteristic
DSC
sharpthermograms
peaks at 167.13 of the pure substances
°C, 224.98 (S1,and
°C, 186.18 °C, S2, 194.38
S3, and S4)
°C, showed characteristic
respectively, indicating thesharp
crystalline
peaks nature
at 167.13 ◦ C, the test◦ C,
of224.98 186.18 ◦ C,The
compounds. andPLGA ◦ C, respectively,
194.38thermogram showed a sharp endo-
indicating the crys-
thermic melting peak at 47.33 °C, which might correspond to the glass transition temper-
ature of PLGA [45]. Similarly, the Poloxamer 188 thermogram showed the sharp endo-
thermic melting peak at 54 °C, which corresponds to the melting peak of Poloxamer 188.
Of interest, DSC thermograms of test compound-loaded LPNCs did not show the melting
peaks of pure compounds, suggesting that each compound was either converted into an
Pharmaceutics 2023, 15, 1755 11 of 19
talline nature of the test compounds. The PLGA thermogram showed a sharp endothermic
melting peak at 47.33 ◦ C, which might correspond to the glass transition temperature
of PLGA [45]. Similarly, the Poloxamer 188 thermogram showed the sharp endothermic
melting peak at 54 ◦ C, which corresponds to the melting peak of Poloxamer 188. Of interest,
DSC thermograms of test compound-loaded LPNCs did not show the melting peaks of
pure compounds, suggesting that each compound was either converted into an amorphous
form or was molecularly dispersed in the formulated LPNCs. Furthermore, the DSC
study indicated the absence of any drug-excipient incompatibility between the12compounds
Pharmaceutics 2023, 15, x FOR PEER REVIEW of 20
and excipients.
Figure 2. DSC thermograms of free test compounds (S1–S4); PLGA; Poloxamer 188; and various test
Figure 2. DSC thermograms of free test compounds (S1–S4); PLGA; Poloxamer 188; and various test
compounds
compounds (S1–S4)-loaded
(S1–S4)-loaded LPNC
LPNC formulations.
formulations.
would grant the efficient delivery of an adequate concentration of the entrapped drug to
Pharmaceutics 2023, 15, x FOR PEER REVIEW 13 of 20
the target tissue for a minimum of 4–7 days post-formulation-administration, which in turn
would enhance patient compliance.
Invitro
Figure 3. In
Figure vitrorelease
releaseprofile
profileofoftest
test compounds
compounds (S1–S4)
(S1–S4) from
from test test compound-loaded
compound-loaded LPNCs.
LPNCs.
Values are expressed as mean ± SD (n =
Values are expressed as mean ± SD (n = 3).3).
3.6. In Vitro
3.6. In VitroCytotoxicity
CytotoxicityAssay
Assayofof Test
Test Compound-Loaded
Compound‐Loaded LPNCs
LPNCs
order to
In order toaddress
addressthe thepossible
possible potentiating
potentiating effect
effect of test
of test compound
compound encapsulation
encapsulation
within LPNCs
within LPNCs on onthethetherapeutic
therapeuticefficacyefficacyof of
different
differentpyridine
pyridinederivatives, the inthe
derivatives, vitro
in vitro
cytotoxicities of different
cytotoxicities different test compounds
compounds (S1–S4)
(S1–S4) andand test
test compound-loaded
compound-loaded nanocap- nanocapsules
were were investigated
sules investigated againstagainst
bothboth the breast
the breast cancer
cancer MCF MCF 7 cell
7 cell lineline andthe
and thelung
lungcancer
cancerA549
A549
cell linecellatline at concentrations
concentrations (0.1,(0.1, 1, and
1, 10, 10, and
100100µM) µM) using
using MTTMTT assay.Both
assay. Bothcancer
cancercell
cell lines
lines selected
were were selected
basedbased
on our onprevious
our previous results
results [7] depicting
[7] depicting the weak
the weak cytotoxic
cytotoxic poten-
potential of free
tial of
test free test compounds
compounds against both against
cellboth cellThe
lines. lines.
ICThe IC50 values
values of of different
different formulations
formulations against
50
against MCF-7
MCF-7 and A549 and A549 cell lines
cell lines are summarized
are summarized in in Table5.5. The
Table The results
resultsreported
reported in in
Table
Table 5
5 clearly indicate that the test compound-loaded LPNCs were more
clearly indicate that the test compound-loaded LPNCs were more cytotoxic against both cytotoxic against both
cell lines
cell lines compared
comparedtotothe thefree
freecounterparts.
counterparts. AllAll
tested freefree
tested compounds
compounds exerted IC50 values
exerted IC50 values
of >100 µM against either MCF-7 or A549 cells. On the other hand, test compound-loaded
of >100 µM against either MCF-7 or A549 cells. On the other hand, test compound-loaded
LPNCs significantly augmented the cytotoxic potential of entrapped drugs as manifested
LPNCs significantly augmented the cytotoxic potential of entrapped drugs as manifested
by a significant decrease in the IC50 values. The IC50 values against MCF-7 cells were 21.9
by a significant decrease in the IC50 values. The IC50 values against MCF-7 cells were
± 1.3, 28.3 ± 1.7, 28.2 ± 1.4, and 9.33 ± 0.9 µM for S1-, S2-, S3-, and S4-loaded LPNCs, re-
21.9 ± 1.3, 28.3 ± 1.7, 28.2 ± 1.4, and 9.33 ± 0.9 µM for S1-, S2-, S3-, and S4-loaded LPNCs,
spectively. Similarly, the IC50 values against A549 cells were 63.1 ± 3.2, 51.3 ± 2.9, 58.9 ± 2.6,
respectively. Similarly, the IC50 values against A549 cells were 63.1 ± 3.2, 51.3 ± 2.9,
and 28.8 ± 1.1 µM for S1-, S2-, S3-, and S4-loaded LPNCs, respectively. Of note, blank
58.9
LPNCs ± 2.6,
did and 28.8 any
not exert ± 1.1 µM for cytotoxic
significant S1-, S2-, effect
S3-, and S4-loaded
against either testedLPNCs,
cancerrespectively.
cell line at Of
note, blank LPNCs did not exert any significant cytotoxic effect
any of the tested concentration ranges. Consequently, the superior cytotoxic activity against either testedof cancer
cell line at any of the tested concentration ranges. Consequently,
different test compound-loaded LPNCs, compared to free compounds, might be ac- the superior cytotoxic
activity
countedof fordifferent test compound-loaded
by the enhanced LPNCs, compared
cellular uptake/internalization of to free compounds,
nanocapsules might be
by cancer
accounted for by the enhanced cellular uptake/internalization
cells [46]. Of note, among different drug-loaded nanocapsule formulations, S4-loaded of nanocapsules by cancer
cells [46]. Of note, among different drug-loaded nanocapsule
LPNCs showed the highest cytotoxic potential against both tested cancer cell lines. The formulations, S4-loaded
LPNCs
relatively showed the highest
slower release cytotoxic
of S4 from LPNCs potential
compared against
to otherboth tested cancer
formulations might cell lines. The
account
relatively
for the superiorslower release activities
cytotoxic of S4 from of LPNCs
S4-loaded compared to other
LPNCs against bothformulations mightSim-
cancer cell lines. account
for
ilar the superior
results cytotoxic
were reported by activities
Sethi et al.of S4-loaded
[47], LPNCsthe
who attributed against
enhanced bothin cancer
vitro andcellin lines.
Similar results potential
vivo cytotoxic were reported by Sethi et al. nanoparticles
of docetaxel-loaded [47], who attributed the enhanced
to the sustained drug in vitro and
release
in vivo cytotoxic
pattern potential of
from nanoparticles. docetaxel-loaded
Based on our in vitronanoparticles to the sustained
cytotoxicity results, S4-loaded drugLPNCs release
were selected
pattern from for further in vivo
nanoparticles. investigations.
Based on our in vitro cytotoxicity results, S4-loaded LPNCs
wereFinally,
selectedtofor rule out theinpossible
further cytotoxic potential of S4-loaded LPNCs against nor-
vivo investigations.
mal healthy cells, the cytotoxicity of S4-loaded LPNCs was screened against noncancer-
ous, normal MCF-10A human breast cancer cells, and the selectivity index was calculated.
As shown in Figure S2, no remarkable decrease in the viability of MCF-10A cells was
Pharmaceutics 2023, 15, 1755 13 of 19
Table 5. IC50 values of the tested free compounds and test compound-loaded LPNCs against MCF 7
and A549 cell lines.
Finally, to rule out the possible cytotoxic potential of S4-loaded LPNCs against normal
healthy cells, the cytotoxicity of S4-loaded LPNCs was screened against noncancerous,
normal MCF-10A human breast cancer cells, and the selectivity index was calculated.
As shown in Figure S2, no remarkable decrease in the viability of MCF-10A cells was
detected at test compound concentrations up to 100 µM. However, at relatively higher
concentrations (200 and 400 µM), S4-loaded LPNCs could induce an obvious cytotoxic
effect against MCF-10A cells. The IC50 value of S4-loaded LPNCs cells was 198.5 ± 13.2 µM.
Most importantly, the calculated selectivity index, defined as the ratio of the average IC50
value against the noncancerous cell line (MCF-10A) to that in the cancer cell line (MCF-7),
was found to be 21.3, indicating the great selectivity of S4-loaded LPNCs against cancerous
cell lines rather than normal cells.
Figure
Figure 4. 4.InInvivo
vivoantitumor
antitumorefficacy
efficacy ofof various
variousS4
S4formulations
formulationsononEAC
EACtumor-bearing
tumor-bearingmice. At days
mice. At days
8 after tumor inoculation, either blank LPNCs, free 5-FU (10 mg/kg), free S4 (10 mg/kg), or S4-loaded
8 after tumor inoculation, either blank LPNCs, free 5-FU (10 mg/kg), free S4 (10 mg/kg), or S4-loaded
LPNCs (10 mg/kg) were i.p. administered. (A) Tumor size; (B) Survival of EAC-bearing mice; and
LPNCs (10 mg/kg) were i.p. administered. (A) Tumor size; (B) Survival of EAC-bearing mice; and
(C) Body weight changes during the treatments. ** p < 0.01 and *** p < 0.001 vs. positive control, # p
(C)< Body
0.05 andweight
## p <changes during
0.01 vs. free the treatments.
S4-treated mice. ** p < 0.01 and *** p < 0.001 vs. positive control,
# p < 0.05 and ## p < 0.01 vs. free S4-treated mice.
Furthermore, S4-loaded LPNCs exhibited a pronounced survival advantage, where
50%Furthermore,
of the treatedS4-loaded
mice (fourLPNCs exhibited
out of eight) a pronounced
became survivalfor
long-term survivors advantage, where
up to 63 days
50% of the treated mice (four out of eight) became long-term survivors for up
post-treatment compared to other treated groups (p < 0.01) (Figure 4B). In addition, S4-to 63 days
post-treatment compared to other treated groups (p < 0.01) (Figure 4B).
loaded LPNCs triggered a remarkable increase in the life spans of tumor-bearing mice. In addition,
S4-loaded LPNCs
The percent triggered
increase a remarkable
in life span (% ILS) in increase
S4-loadedinLPNCS-treated
the life spans mice
of tumor-bearing mice.
was 1.54, which
The percent increase in life span (% ILS) in S4-loaded LPNCS-treated mice was 1.54, which
Pharmaceutics 2023, 15, x FOR PEER REVIEW 16 of 20
Pharmaceutics 2023, 15, 1755 15 of 19
was significantly higher than that in mice treated with either standard 5-FU (% ILS 1.33)
was significantly higher than that in mice treated with either standard 5-FU (% ILS 1.33) or
or free S4-treated mice (% ILS 1.37). On the other hand, blank LPNCs did not show a sur-
free S4-treated mice (% ILS 1.37). On the other hand, blank LPNCs did not show a survival
vival advantage compared with the control group.
advantage compared with the control group.
Finally, no body weight loss was detected in any of the treated groups under our
Finally, no body weight
experimental conditions (Figure loss
4C). was
Thesedetected in anythat
results suggest of the treatedprotocols
treatment groups under
were our
well-tolerated by mice and that S4-loaded LPNCs could exert their potent antitumor ac- were
experimental conditions (Figure 4C). These results suggest that treatment protocols
well-tolerated
tivity by mice and that
in EAC tumor-bearing miceS4-loaded LPNCsremarkable
without causing could exerttoxicity.
their potent antitumor activity
in EAC tumor-bearing mice without causing remarkable toxicity.
3.7.4. Histopathological Examination of Solid Tumors
3.7.4. Histopathological Examination of Solid Tumors
Microscopic examination of H&E-stained tumor sections from the EAC solid tumor
of theMicroscopic examination
control and blank of H&E-stained
LPNC-treated tumor
groups (Figure 5) sections from the
showed typical EAC solidfea-
pathological tumor of
the control and blank LPNC-treated groups (Figure 5) showed typical pathological
tures of closely arranged tumor cells with clear nuclear structures, numerous bizarre mi- features
of closely arranged tumor cells with clear nuclear structures, numerous
totic figures, and giant tumor cells with slight necrosis. On the other hand, tumor sectionsbizarre mitotic
from animals treated with either 5-FU, free S4, or S4-loaded LPNCs revealed a loss of cel- from
figures, and giant tumor cells with slight necrosis. On the other hand, tumor sections
animals
lular treated with
architecture either tissue
and tumor 5-FU, compactness
free S4, or S4-loaded
as well asLPNCs revealed of
the appearance a loss of cellular
vacuolar
architecture
structures and tumor
within tissueregion
the necrotic compactness
(Figure as
5).well as the appearance
Of interest, there was aofremarkable
vacuolar structures
de-
withininthe
crease thenecrotic
numberregion (Figure
of mitotic 5). and
figures Of interest, there
giant cells was a remarkable
in S4-loaded decrease
LPNC-treated micein the
number of
compared tomitotic figures andorgiant
either 5-FU-treated cells in S4-loaded
free S4-treated LPNC-treated
mice (Figure mice confirm
5). These findings compared to
the superior
either antitumor
5-FU-treated orefficacy of S4-loaded
free S4-treated miceLPNCs
(Figurecompared
5). Theseto either free
findings 5-FU the
confirm or free
superior
S4 compounds.
antitumor efficacy of S4-loaded LPNCs compared to either free 5-FU or free S4 compounds.
Figure
Figure5.5.HH&&
EE staining of tumor
staining tissue
of tumor sections
tissue collected
sections fromfrom
collected (A) control group;
(A) control mice treated
group; with with
mice treated
(B) blank LPNCs; (C) 5-FU (10 mg/kg); (D) Free S4 (10 mg/kg); or (E) S4-loaded LPNCs (10 mg/kg).
(B) blank LPNCs; (C) 5-FU (10 mg/kg); (D) Free S4 (10 mg/kg); or (E) S4-loaded LPNCs (10 mg/kg).
Black arrows refer to mitotic figures and red arrows refer to tumor giant cell, while the necrosis area
isBlack arrows
outlined refer
by the to mitotic figures and red arrows refer to tumor giant cell, while the necrosis area
square.
is outlined by the square.
3.7.5. Biochemical Analysis of Liver and Kidney Functions
3.7.5. Biochemical Analysis of Liver and Kidney Functions
The liver and kidney are the two most significant organs in the body for drug metab-
The liver and kidney are the two most significant organs in the body for drug
olism and elimination of drugs in the body, and they are the most susceptible organs to
metabolism and elimination of drugs in the body, and they are the most susceptible organs
harm by drug-induced effects. Consequently, biochemical assays for serum AST and ALT,
to harm by drug-induced effects. Consequently, biochemical assays for serum AST and
ALT, as indicators for liver function, and serum creatinine levels, as a marker for kidney
function, were performed to ensure the safety of different treatments. Serum levels of
Pharmaceutics 2023, 15, 1755 16 of 19
different liver and kidney markers post different treatments were recorded in Table 6.
Generally, liver injury is associated with elevated levels of ALT and AST [48]. As summa-
rized in Table 5, with the exception of mice treated with 5-FU, no significant alterations
in serum levels of either ALT and AST were observed upon treatment with blank LPNCs,
free S4, or S4-loaded LPNCs. Similarly, kidney function, monitored via estimating serum
creatinine levels, demonstrated a non-significant increase in serum creatinine levels in all
treated animals with the exception of 5-FU, which triggered a significant elevation of serum
creatinine levels in 5-FU-treated mice when compared with control animals. Consequently,
these results signify the safety of S4-loaded LPNCs following systemic administration and
nullify the possibility of the induction of liver injury or renal dysfunction.
Table 6. Effects of different S4 formulations on serum levels of blood biochemical parameters.
4. Conclusions
In this study, we succeeded in developing LPNCs, composed of a CCT oily core
and PLGA shell, for the efficient entrapment of novel pyridine derivatives (S1–S4). The
fabricated nanocapsules could efficiently enhance the aqueous solubility of test compounds
(S1–S4), and thereby could contribute to the enhancement of the anticancer potential of
entrapped compounds compared to their free counterparts. In vitro cytotoxicity studies
clearly demonstrated the superior cytotoxic potential of S4-loaded LPNCs against both
MCF-7 and A549 cancer cells compared to free S4. Most importantly, in vivo antitumor
experiments emphasized the efficacy of S4-loaded LPNCs in defeating tumor growth and
prolonging the survival time of tumor-bearing mice compared to the free test compound
(S4). This potent antitumor activity might be ascribed to the sustained release of the test
compound from LPNCs, which could grant effective delivery of adequate concentrations
of the entrapped drug to tumor tissues following systemic administration. Furthermore,
treatment with S4-loaded LPNCs could exert their efficient antitumor effect without eliciting
any remarkable systemic toxicities. To sum up, test compound-loaded LPNCs might be
a promising candidate for cancer therapy due to their high therapeutic efficacy and low
adverse off-target effects.
University researchers supporting project number (PNURSP2023R205), Princess Nourah bint Ab-
dulrahman University, Riyadh, Saudi Arabia. This work was also supported by the Researchers
Supporting Project number (RSPD2023R620), King Saud University, Riyadh, Saudi Arabia.
Institutional Review Board Statement: The animal study protocol was approved by the Faculty of
Pharmacy Ethical Committee Acts (201611PHDA2) of Suez Canal University, Ismailia, Egypt.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article and
Supplementary Materials.
Acknowledgments: The authors acknowledge the support from Prince Sattam Bin Abdulaziz Univer-
sity project number (PSAU/2023/R/1444). The authors extend their appreciation to the Researchers
Supporting Project number (RSPD2023R620), King Saud University, Riyadh, Saudi Arabia. Addition-
ally, the authors extend their appreciation to Princess Nourah bint Abdulrahman University, Riyadh,
Saudi Arabia for funding this work under researcher supporting project number (PNURSP2023R205).
Conflicts of Interest: The authors declare no conflict of interest.
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