In Vitro Cytotoxicity and in Vivo Antitumor Activity of Lipid Nanocapsules Loaded With Novel Pyridine Derivatives

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pharmaceutics

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/).

Pharmaceutics 2023, 15, 1755. https://doi.org/10.3390/pharmaceutics15061755 https://www.mdpi.com/journal/pharmaceutics


Pharmaceutics 2023, 15, 1755 2 of 19

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

Scheme Scheme 1. Synthesis


1. Synthesis scheme
scheme for for 2-amino-4-aryl-6-substituted
2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile
pyridine-3,5-dicarbonitrile deriva- deriva-
Scheme 1.
Scheme 1. Synthesis
Synthesis scheme
scheme for
for 2-amino-4-aryl-6-substituted
2-amino-4-aryl-6-substituted pyridine-3,5-dicarbonitrile
pyridine-3,5-dicarbonitrilederiva-
derivatives.
tives. tives.
tives.
Table 1. Chemical structure of various pyridine derivatives (S1–S4).
Table 1. Table 1. Chemical
Chemical structurestructure ofpyridine
of various various pyridine derivatives
derivatives (S1–S4). (S1–S4).
Table 1. Chemical structure of various pyridine derivatives (S1–S4).
Compound Code
Compound
Compound Amine Amine
Code Code Amine R R Chemical
R ChemicalChemical
Formula M∙Formula
Formulawt M∙wt M·wt
Compound Code Amine R Chemical Formula M∙wt
S1 S1 S1 CH33CH22CH
CH NH3CH
2 2NHCH
2 2CH33CH2
3CHCH 2 C15H13NC
5 15H13N 13 N5 263
C5 H263 263
S12 CH3CH 2NH2 CH3CH15 2 C15H13N5 263
S2 S2 S2 5 20H15N
C20H15NC C20
5 H 15 N5 325
325 325
S2 C20H15N5 325
S3 S3 S3 NH44OAc NH4OAc H H C15H9N5C15H9NC515 H
235
9 N5 235 235
S3 NH4OAc H C15H9N5 235
S4 S4 S4 C19
5 19H15N
C19H15NC 5 H 15 N5 311
311 311
S4 C19H15N5 311

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).

2.4. Preparation of Lipid-Polymer Nanocapsules (LPNCs)


Lipid-polymer nanocapsules were fabricated by a nanoprecipitation technique [23].
Initially, LPNCs were prepared by an interfacial deposition of polymer. Briefly, a definite
weight of PLGA polymer was dissolved in 5 mL of acetone containing 0.25% w/v Span 60.
Then, 1 mM of different pyridine derivatives (S1–S4) previously solubilized in a definite
amount of caprylic/capric triglyceride (CCT), oleic acid (OA), or olive oil (VO) was added
to the acetonic solution. The resulting organic solution was injected into 10 mL of aqueous
Pharmaceutics 2023, 15, 1755 4 of 19

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.

PLGA CCT Oleic Acid Olive Oil Poloxamer 188 Tween 80


Formula
(mg) (mg) (mg) (mg) (%w/v) (%w/v)
F1 10 100 --- --- 0.25 ---
F2 10 150 --- --- 0.25 ---
F3 10 200 --- --- 0.25 ---
F4 20 100 --- --- 0.25 ---
F5 30 100 --- --- 0.25 ---
F6 20 --- 100 --- 0.25 ---
F7 20 --- --- 100 0.25 ---
F8 20 100 --- --- --- 0.25
F9 20 --- 100 --- --- 0.25
F10 20 --- --- 100 --- 0.25
PLGA, polylactic-co-glycolic acid (PLGA; 50:50); and CCT, caprylic/capric triglyceride.

2.5. Physicochemical Characterization of LPNCs


2.5.1. Particle Size, Polydispersity Index, and Zeta Potential
The particle size and polydispersity index (PdI) of different LPNC dispersions were
measured by dynamic light scattering (Nano ZS Zetasizer, Malvern Instruments Ltd.,
Worcestershire, UK) at 25 ◦ C. Volume-weighted mean diameters (D[4,3]) and polydispersity
(expressed via measuring the distribution width, span) were estimated by the Malvern
software based on the following equations:

∑ 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.

2.5.2. Entrapment Efficiency Percentage


Drug loading (DL%) and entrapment efficiency (EE%) of different pyridine derivative-
loaded LPNCs were determined indirectly by estimating the concentration of the free
compound. Briefly, 1 mL of test compound-loaded LPNCs was centrifuged at 15,000 rpm
for 30 min. After centrifugation, the concentration of free compounds in the supernatant
was estimated spectrophotometrically and the DL% and EE% were calculated using the
following equations:
Total drug − Free drug
DL% = × 100
Total weight o f LPNCs

Total drug − Free drug


EE% =
Total drug
Pharmaceutics 2023, 15, 1755 5 of 19

2.5.3. Surface Morphology


The surface morphology of the prepared LPNCs was examined using a transmission
electron microscopy (JEOL JEM 1010, JEOL USA, Inc., Peabody, MA, USA). Briefly, a drop
of LPNC dispersion was applied to a copper grid coating, and a filter paper was used to
remove the excess droplets. After 5 min, a drop of 2% w/v uranyl acetate solution was
then put onto the grids. After the samples were negatively stained and air-dried at room
temperature, they were imaged by TEM at an acceleration voltage of 74 kV.

2.5.4. Differential Scanning Calorimetry (DSC)


Differential Scanning Calorimetry (DSC) was used to study the physical state, crys-
tallinity of each compound, and possible physical interactions of it with the solid excipient
to be used in LPNC formulations. The measurement was performed with a differential
scanning calorimeter (Shimadzu DSC-60, Tokyo, Japan). Samples weighing 3–7 mg were
sealed in aluminum pans and a heating rate of 10 ◦ C/min was employed in a temperature
range of 25 to 300 ◦ C under nitrogen gas flow (30 mL·min−1 ). An empty aluminum pan
was used as a reference in the study.

2.6. In Vitro Release Studies


The in vitro release of each compound was assessed by the dialysis bag method as
described previously [24]. Briefly, 1 mL of the LPNC formulation of each compound was
tightly sealed in a dialysis tubing (MW cutoff 12−14 kDa) and immersed in 30 mL of
release medium (phosphate buffer saline, pH 7.4) kept at 37 ◦ C and stirred at a constant
stirring speed (100 rpm). At scheduled time intervals (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120,
144, and 168 h), 1 mL samples were collected and replaced by an equal volume of fresh
release medium. The concentration of various pyridine derivatives in each sample was
then quantified spectrophotometrically at specified wavelengths.

2.7. In Vitro Cytotoxicity Assay of Pyridine Derivatives-Loaded LPNCs


A human lung cancer (A549) cell line, human breast adenocarcinoma (MCF-7), and
normal MCF-10A human breast cancer cells were obtained from VASCERA Co. (Vaccines
Sera and Drugs; Cairo, Egypt). Both cell lines were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum,
100 U/mL penicillin/100 µg/mL streptomycin, and incubated under standard conditions
(37 ◦ C, 5% CO2 ).
The in vitro cytotoxicities of various pyridine derivatives loaded into LPNCs against
A549 cells and MCF-7 cells were evaluated by the MTT assay [25]. Briefly, 100 µL cell
suspension equivalent to 1 × 104 cells/well was planted in a 96-well plate and incubated
at 37 ◦ C. At 24 h post-incubation, the cells were treated with 100 µL of serial dilutions of
either free pyridine derivatives (S1–S4) or pyridine derivatives (S1–S4)-loaded LPNCs in
a concentration range of 0.1 to 100 µM. The cells were further incubated at 37 ◦ C for 48 h.
The spent culture medium was then decanted, the cells were rinsed, 50 µL of MTT reagent
(0.5 mg/mL) was added to each well, and the cells were incubated for 4 h at 37 ◦ C. Finally,
to dissolve the formazan crystals, DMSO solution (150 µL) was added to each well. The
absorbance of each well was measured at 570 nm using a microplate reader (TEKAN Japan,
Kawasaki, Japan). The percent cell viability was plotted against the tested compound
concentration to determine the IC50 values.

2.8. In Vivo Studies


2.8.1. Animals
Female Swiss Albino male mice (20–25 g, 6–8 weeks) were provided by the animal
house Faculty of Pharmacy in Suez Canal University and kept under specific pathogen-free
conditions with free access to standard water and food. All animal studies were reviewed
and approved by the Faculty of Pharmacy Ethical Committee Acts (201611PHDA2) of Suez
Canal University, Ismailia, Egypt.
Pharmaceutics 2023, 15, 1755 6 of 19

2.8.2. Median Lethal Dose (LD50 ) of Test Selective Compound


The median lethal dose (LD50 ) test was performed to determine the dose of a test
substance that elicits 50% death in mice [26]. Briefly, a total of 30 mice were categorized
into 6 groups (n = 5). Each group was dosed with a single intraperitoneal injection of the
test compound (S4) dissolved in 3% dimethyl sulfoxide. The screened doses ranged from
100 to 180 mg/kg. The animals were then monitored for 2 h and then at 4, 6, and 24 h for
signs of toxicity. At the end of 24 h, the total number of deaths was recorded. The LD50 was
then calculated using the following equation [27]:

{Σ[Dose difference × Mean dead]}


LD50 = LD100 −
Number of animals per group

where LD100 is the lowest dose capable of killing 100% of animals.

2.8.3. Acute Toxicity Study


Acute toxicity studies are carried out in order to evaluate the short-term negative
effects of a test compound when administered in a single dose during a period of 24 h.
Briefly, the maximum tolerated dose (100 mg/kg) of the test compound (S4), dissolved in
3% dimethyl sulfoxide, was injected intraperitoneally in a group of mice (n = 5). A control
group (n = 5) was i.p. injected with an equivalent volume of 3% dimethyl sulfoxide. The
mice were then observed for general behavior for 1 h post-treatment and then intermittently
for 4 h and finally at 24 h. The mice were then monitored for up to 14 days after treatment
for symptoms of toxicity or mortalities.

2.8.4. Induction of Solid Tumor in Mice


Ehrlich ascites carcinoma (EAC) cells (1 × 106 cells) obtained from the National Cancer
Institute (Cairo, Egypt) were injected intraperitoneally into the peritoneal cavity of Swiss
Albino mice. On day 7 post tumor cell inoculation, ascites fluid was collected. EAC was
diluted with saline to obtain a cell density of 25 × 106 cells/mL. Then, 200 µL of the cells
were injected subcutaneously in the mice’s right flanks. Tumor growth was monitored
post-inoculation, and the size of the tumor was measured with a Vernier caliper. Treatment
protocols were initiated on day 8 post tumor inoculation, when the tumor was palpable.

2.8.5. Treatment Protocol


One week after tumor implantation, when the tumor was palpable, the mice were
randomly categorized into five groups (n = 8). All treatments were initiated on day 8 and
extended to day 29 post implantation (a three-week treatment period).
Group 1 (EAC control group): EAC-bearing mice were i.p. injected with 0.4 mL saline once
every other day for a period of 21 days.
Group 2: EAC-bearing mice were i.p. injected with blank LPNCs once every other day for
a period of 21 days.
Group 3: EAC-bearing mice were i.p. injected the standard anticancer drug (5-fluorouracil;
5-FU) at a dose of 10 mg/kg [28] every other day for a period of 21 days.
Group 4: EAC-bearing mice were i.p. injected with free test compound(S4) at a dose of
10 mg/kg) once every other day for a period of 21 days.
Group 5: EAC-bearing mice were i.p. injected with test compound (S4)-loaded LPNCs at a
dose of 10 mg/kg) once every other day for a period of 21 days.
The dose and route of administration of the test compound and the treatment regimen
were established based on previously published reports [29,30]. The antitumor efficacy
of different treatment protocols was evaluated in terms of tumor size, percentage tumor
growth inhibition (% TGI), median survival time (MST), and percentage increase in life
span (% ILS) using the following equations [31,32]:

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 = (day of first death + day of last death)/2

" #
MST treated group
%ILS = − 1 × 100
MST control group

In addition, body weight was assessed concurrently to determine any remarkable


systemic toxicity.

2.8.6. Histopathological Examination of Solid Tumors


On the last day of the experiment, solid tumors were dissected from all mice and
were fixed overnight in formalin-alcohol for 48 h, dehydrated in an alcohol series, cleared
in xylene, and embedded in paraffin. Sections (4–5 µm in thickness) were stained with
hematoxylin and eosin (H&E). Slides were then imaged using a Universal Infinity System
optical microscope (Olympus® , Tokyo, Japan) equipped with a computer-controlled digital
camera [33]. A histopathological analysis of the tumor sections was focused on finding
a typical mitotic picture, neoplastic giant cells, and the extension of the necrotic area.
Histopathological examinations were evaluated in a blinded way by a pathologist.

2.8.7. Biochemical Analysis for Liver and Kidney Functions


On the last day of the experiment, 4 out 8 mice were euthanized, and blood samples
were collected by cardiac puncture in dry Eppendorf tubes. Blood samples were set aside
for 30 min at room temperature and the tubes were then centrifuged for 15 min at 2000× g to
obtain serum. To assess possible hepatic damage, aspartate transaminase (AST) and alanine
transaminase (ALT) activities in serum samples were assayed by using a commercial kit
(Biodiagnostic, Cairo, Egypt) according to the method of Reitman and Frankel [34]. In
addition, serum creatinine level was quantified as an indicator for possible renal damage.
The level of serum creatinine was analyzed by using a commercial kit that was purchased
from Biodiagnostic (Cairo, Egypt) according to manufacturer instructions.

2.9. Statistical Analysis


A statistical analysis was performed using Graph Pad Prism version 9 by one-way
ANOVA followed by a post-hoc Bonferroni’s comparison test. A value of p < 0.05 was
considered significant. All data were expressed as mean ± standard deviation.

3. Results and Discussion


3.1. Solubility Measurement of Various Pyridine Derivatives (S1–S4) in Different Solvents
The aqueous solubility of a drug is a fundamental property that plays a crucial role in
drug effectiveness. Accordingly, solubility studies of various pyridine derivatives (S1–S4)
were conducted in different solvents, namely, water, ethanol, methanol, and phosphate
buffer (pH 7.4). As shown in Table S2, the maximum solubility of all the tested compounds
(S1–S4) was observed in organic solvents, ethanol, and methanol. On the other hand, all the
tested compounds showed poor solubility in both water and the phosphate buffer. These
results imply that the solubility of test compounds decreases with the increasing polarity of
the solvents. Most importantly, the poor aqueous solubility of test compounds might limit
their therapeutic efficacy since poor aqueous solubility would be associated with poor drug
bioavailability. Consequently, in order to overcome such limitations, the test compounds
were loaded into lipid polymer nanocapsule (LPNC) formulations and investigated for
their anticancer potential both in vitro and in vivo.
Pharmaceutics 2023, 15, 1755 8 of 19

3.2. Preparation of Lipid Polymer Nanocapsules (LPNCs)


During the development process, a variety of materials and their combinations were
adopted for the preparation of LPNCs (Table 2). First, PLGA, at variable amounts, was
employed as the shell-forming polymer, while caprylic/capric triglyceride (CCT), oleic acid,
or olive oil, at variable amounts, were tested as the main oily component of the developing
LPNC formulations. The shell-forming polymer (PLGA) was dissolved in acetone in the
presence of two different surfactants (poloxamer 188 or Tween 80). All the developed
formulations were screened for particle size. The formulation that showed the smallest
particle size was selected as the optimized formula.

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.

Formula Dv0.1 Dv0.5 Dv0.9 D[4,3] Span


F1 73.67 ± 0.58 126.67 ± 0.58 210.00 ± 1 135.33 ± 0.58 1.07 ± 0.01
F2 91.00 ± 6.08 172.00 ± 3 326.67 ± 16.07 193.33 ± 2.08 1.37 ± 0.15
F3 80.67 ± 4.62 187.00 ± 9.17 420.67 ± 35.1 225.00 ± 10.54 1.82 ± 0.23
F4 78.67 ± 1.53 128.00 ± 2.65 205.33 ± 2.31 136.00 ± 1.73 0.99 ± 0.01
F5 76.00 ± 3.61 158.00 ± 4.36 334.00 ± 21.63 184.67 ± 6.43 1.63 ± 0.12
F6 86.00 ± 4.36 203.33 ± 10.26 461.67 ± 80.39 255.00 ± 40.63 1.85 ± 0.37
F7 102.33 ± 6.11 253.00 ± 11.53 693.33 ± 73.38 346.67 ± 26.03 2.33 ± 0.17
F8 79.33 ± 4.73 138.67 ± 3.51 238.33 ± 28.31 153.67 ± 11.55 1.14 ± 0.22
F9 80.67 ± 5.03 172.00 ± 9.54 362.67 ± 56.89 201.67 ± 19.73 1.63 ± 0.30
F10 84.33 ± 2.89 201.67 ± 5.77 472.00 ± 22.52 256.00 ± 15.59 1.92 ± 0.19
Data represent mean ± SD of three independent experiments.
Pharmaceutics 2023, 15, 1755 9 of 19

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).

3.3. Preparation of Each Compound-Loaded LPNC


Based on formulation development data, PLGA (20 mg) as a shell-forming polymer,
CCT (100 mg) as the oily core, and poloxamer 188 (0.25% w/v) as the stabilizer were selected
as the components for the fabrication of pyridine derivatives (S1–S4)-loaded LPNCs. Briefly,
1 mM of the test compounds were initially solubilized in CCT, and then the oily phase was
added to polymer acetonic solution. Finally, the resulting organic solution was injected
into 10 mL of water containing 0.25% w/v poloxamer 188 under magnetic stirring. The
formulated test compound-loaded LPNCs were then characterized for mean particle size
and size distribution, surface charge, and morphology.

3.4. Characterization of Test Compound-Loaded LPNCs


3.4.1. Particle Size and Size Distribution
Particle size plays a crucial role in dictating the therapeutic efficacy of drug-loaded
nanocarriers. Generally, most nanocarrier systems used for cancer therapy are designed
to be between 50 to 200 nm to favor the enhanced permeation and retention (EPR) ef-
fect [37]. As listed in Table 4 and Figure S1, various test compound-loaded LPNCs had a
particle size ranging from 185.0 ± 17.4 (S1-loaded LPNCs) to 223.0 ± 15.3 nm (S4-loaded
LPNCs). This size range of all formulated LPNCs is large enough (>50 nm) to evade hepatic
sequestration [38], while it is small enough to escape phagocytosis (<250 nm) [39]. Of
note, the polydispersity index (PDI) of all formulations is <0.3, indicating narrow size
distribution [40].
Table 4. Physicochemical characteristics of various test compound-loaded LPNCs.

Size Distribution Zeta Potential Entrapment Drug Loading


Formula PDI
(nm) (mV) Efficiency (%) (%)
S1-LPNCs 185.0 ± 17.4 0.098 ± 0.01 −7.55 ± 1.7 92.67 ± 0.84 1.99 ± 0.02
S2-LPNCs 187.5 ± 15.2 0.141 ± 0.02 −11.60 ± 1.8 90.62 ± 0.52 2.39 ± 0.01
S3-LPNCs 218.9 ± 10.9 0.200 ± 0.02 −11.90 ± 1.5 91.20 ± 1.70 1.75 ± 0.03
S4-LPNCs 223.0 ± 15.3 0.218 ± 0.03 −13.4 ± 2.1 93.72 ± 0.66 2.37 ± 0.02
Data represent mean ± SD of three independent experiments.

3.4.2. Zeta Potential of Test Compound-Loaded LPNCs


The zeta potential in another important parameter that contributes to the stability of
colloidal dispersions. Generally, colloidal dispersions having a high zeta potential value
of ±20 mV are believed to have good dispersion stability and a low chance of aggre-
Pharmaceutics 2023, 15, 1755 10 of 19

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.

3.4.3. Morphology of Test Compound-Loaded LPNCs


In order to investigate the morphology of LPNCs, transmission electron microscopy
(TEM) analysis was conducted (Figure 1). As shown in Figure 1, all the formulated LPNCs
were spherical in shape, having smooth surfaces. Of note, TEM photographs showed that
the particle size of the nanoparticles was around 100–200 nm. Small variations in particle
size estimated by TEM, compared to the DLS technique, might be ascribed to differences
in measurement conditions. In TEM imaging, the size of nanocapsules is determined in a
dry state, while in DLS, the nanoparticles are in a hydrated state, where each particle is
Pharmaceutics 2023, 15, x FOR PEERsurrounded
REVIEW 11 of 20 by
by a solvent sheath. This could explain the larger size of LPNCs estimated
DLS compared to that determined by TEM analysis [43].

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.

3.5. In Vitro Release of Test Compounds from LPNCs


3.5. In Vitro Release of Test Compounds from LPNCs
The release profile of various test compounds from LPNC formulations was assessed
The release profile of various test compounds from LPNC formulations was assessed
for 7 days and the cumulative drug release was plotted as a function of time (Figure 3).
for 7 days and the cumulative drug release was plotted as a function of time (Figure 3). As
As shown
shown in Figurein Figure 3, allcompound-loaded
3, all test test compound-loaded LPNC formulations
LPNC formulations showed ashowed
biphasica re-biphasic
lease pattern with an initial burst release of compounds in the first 12 h, followed by a by a
release pattern with an initial burst release of compounds in the first 12 h, followed
sustained
sustained drug
drug release
release for for
up toup168
to h.
168Theh. percent
The percent cumulative
cumulative drug in
drug release release in12
the first the first
h12 h 66.4
was was±66.4
3.2%,±54.0
3.2%,
± 4.4%, ± 4.4%,
54.0 61.9 ± 3.7%, and±26.4
61.9 3.7%, andfor
± 2.9% S1-,±S2-,
26.4 2.9%
S3-,for
andS1-, S2-, S3-, and
S4-loaded
S4-loaded
LPNCs, LPNCs, respectively.
respectively. This biphasic Thisrelease biphasic
pattern release
might bepattern might
attributed to be
theattributed
core–shell to the
core–shell
structure structure of lipid-polymer
of lipid-polymer nanocapsules, nanocapsules, in which the
in which the compounds couldcompounds
be associated could be
associated
with withofthe
the surface thesurface of the nanocapsules
nanocapsules and/or dissolvedand/or
intodissolved intoOf
the core [44]. thenote,
coreamong
[44]. Of note,
among different
different LPNC formulations,
LPNC formulations, S4-loaded S4-loaded
LPNCs showedLPNCs theshowed the lowest
lowest burst releaseburst release of
of com-
compound
pound S4, suggesting
S4, suggesting the efficient
the efficient entrapment entrapment of compound
of compound S4 in
S4 in the oily therather
core oily core
than rather
than adsorbed
being being adsorbed at the
at the outer outer of
surface surface of the nanocapsule
the nanocapsule polymericpolymeric shell. Furthermore,
shell. Furthermore, like
other formulations, LPNCs could sustain the release of compound S4 for
like other formulations, LPNCs could sustain the release of compound S4 for up to 7 days. up to 7 days.
Such
Sucha arelease pattern
release patternwould
would be favored
be favored in cancer therapy,
in cancer where
therapy, a sustained
where drug release
a sustained drug release
would grant the efficient delivery of an adequate concentration of the entrapped drug to
the target tissue for a minimum of 4–7 days post-formulation-administration, which in
turn would enhance patient compliance.
Pharmaceutics 2023, 15, 1755 12 of 19

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.

Formula IC50 (µM) against MCF-7 IC50 (µM) against A549


Pure S1 >100 >100
Pure S2 >100 >100
Pure S3 >100 >100
Pure S4 >100 >100
S1-loaded LPNCs 21.9 ± 1.3 63.1 ± 3.2
S2-loaded LPNCs 28.3 ± 1.7 51.3 ± 2.9
S3-loaded LPNCs 28.2 ± 1.4 58.9 ± 2.6
S4-loaded LPNCs 9.33 ± 0.9 28.8 ± 1.1
Data represent mean ± SD of three independent experiments.

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.

3.7. In Vivo Studies


3.7.1. Median Lethal Dose (LD50 ) of Compound (S4) in Female Albino Mice
Initially, to investigate the appropriate dose of test compound (S4) for in vivo use, mice
were intraperitoneally injected with escalating doses of S4 ranging from 100 to 180 mg/kg.
The animals were then monitored periodically for 24 h, and the LD50 was computed
at 24 h post-injection. As summarized in Table S3, high doses of test compound S4
(120 mg/kg and higher) were lethal to animals, while the lower dose (100 mg/kg) was
not lethal but only induced drowsiness in test animals, which recovered after 24 h. The
calculated LD50 of compound (S4) in adult female mice was found to be 50 mg/kg body
weight after intraperitoneal injection.

3.7.2. Acute Toxicity


An acute toxicity study was carried out to outline the short-term adverse effects of the
maximum tolerated dose (100 mg/kg) of test compound S4. According to the acute toxicity
study, no behavioral abnormalities, toxicity symptoms, or mortalities were detected in mice
at test compound S4 levels up to 100 mg/kg, suggesting the safety of the test compound at
this dose.

3.7.3. Antitumor Activity of S4-Loaded LPNCs


In order to address whether the in vitro cytotoxicity results would be reflected by the
in vivo therapeutic activity, the in vivo antitumor activity of different test compound (S4)
formulations in a subcutaneous EAC tumor-bearing mouse model was assessed. Mice were
treated with distilled water, blank nanocapsules, standard chemotherapeutic agent (5-FU),
free S4 compound (10 mg/kg), or S4-loaded PLNCs (10 mg S4 per kg) three times a week
for three weeks via intraperitoneal injection. The administered dose of S4 was determined
based on preliminary studies, which showed the safety and tolerability of this test dose
following subsequent administrations. As depicted in Figure 4A, the treatment of mice with
standard chemotherapeutic agent (5-FU), free test compound (S4), or S4-loaded LPNCs
resulted in a considerable tumor growth inhibitory effect compared with either control
(non-treated) mice or blank LPNCs-treated mice. Most importantly, consistent with our
Pharmaceutics 2023, 15, 1755 14 of 19

Pharmaceutics 2023, 15, x FOR PEER REVIEW 15 of 20

in vitro cytotoxicity experiments, S4-loaded LPNCs exerted a superior antitumor efficacy


compared
antitumor to efficacy
free S4. In addition,towhen
compared compared
free S4. with all
In addition, treated
when groups,with
compared S4-loaded LPNCs
all treated
strongly suppressed tumor growth in the EAC tumor-bearing mouse
groups, S4-loaded LPNCs strongly suppressed tumor growth in the EAC tumor-bearing model; the tumor
growth
mouse model; the tumor growth inhibition rate (TGI (%)) was 92.83%, which was remark- of
inhibition rate (TGI (%)) was 92.83%, which was remarkably higher than that
either
ably the standard
higher chemotherapeutic
than that agentchemotherapeutic
of either the standard (5-FU) or the freeagent
S4-treated-group
(5-FU) or the free(TGIs
S4-(%)
were 86.81% and 73.42%, respectively). This enhanced antitumor efficacy
treated-group (TGIs (%) were 86.81% and 73.42%, respectively). This enhanced antitumor of test compound-
loaded
efficacy LPNCs
of testmight be ascribed to
compound-loaded the efficient
LPNCs might beentrapment
ascribed to of
thethe test compound
efficient entrapmentwithin
of
thethe
inner oily core ofwithin
test compound nanocapsules, which
the inner oily could
core efficiently protect
of nanocapsules, the entrapped
which could efficientlypayload
pro-
from
tectbeing prematurely
the entrapped payloadreleased into prematurely
from being systemic circulation during
released into its transit
systemic to thedur-
circulation target
tumor tissue,
ing its transitand thereby
to the targetgrant
tumorthe delivery
tissue, and of an adequate
thereby grant theconcentration
delivery of anofadequate
entrapped
drug to the target
concentration site.
of entrapped drug to the target site.

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.

Groups AST (IU/L) ALT (IU/L) Creatinine (mg/dL)


EAC control 36.06 ± 1.72 17.45 ± 1.31 0.49 ± 0.09
EAC + blank LPNCs 34.61 ± 1.33 16.55 ± 0.71 0.54 ± 0.03
EAC + 5-FU 74.23 ± 3.84 * 51.87 ± 1.29 * 1.05 ± 0.15 *
EAC + Free S4 (10 mg/kg) 36.66 ± 1.87 20.50 ± 1.28 0.54 ± 0.02
EAC + S4-loaded LPNCs (10 mg/kg) 36.13 ± 2.15 18.96 ± 1.56 0.51 ± 0.03
AST = Aspartate aminotransferase; ALT = alanine aminotransferase; EAC = Ehrlich’s ascites carcinoma. Data are
exhibited as mean ± SD. * p < 0.05 vs. EAC control.

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.

Supplementary Materials: The following supporting information can be downloaded at:


https://www.mdpi.com/article/10.3390/pharmaceutics15061755/s1, Figure S1: (A) Particle size
and (B) zeta potential of various test compound-loaded LPNCs; Figure S2: In vitro cytotoxicity of
S4-loaded LPNCs against normal noncancerous MCF-10A breast cancer cells; Table S1: Maximum
wavelength (λmax ) of various pyridine derivatives (S1–S4) in acetonitrile, methanol, ethanol, phos-
phate buffer (pH 7.4), and ethanol:phosphate buffer (1:1); Table S2: Solubility of different pyridine
derivatives (S1–S4) in different solvents; Table S3: Results of the lethal doses of compound (S4) for
the determination of the LD50 after i.p. injection in female Albino Swiss mice (n = 5).
Author Contributions: Conceptualization, E.-S.K., S.G. and A.S.A.L.; methodology, M.A., M.A.T. and
S.G.; software, E.H.M., A.J.O. and H.F.A.; formal analysis, E.-S.K. and E.H.M.; investigation, M.A.,
M.A.T. and S.G.; resources, E.-S.K., H.F.A. and A.J.O.; data curation, A.S.A.L.; writing—original draft
preparation, M.A., A.J.O., H.F.A. and A.S.A.L.; writing—review and editing, A.S.A.L. and E.-S.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This study is supported via funding from Prince Sattam Bin Abdulaziz University project
number (PSAU/2023/R/1444). This work was supported by Princess Nourah bint Abdulrahman
Pharmaceutics 2023, 15, 1755 17 of 19

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.

References
1. Hassanpour, S.H.; Dehghani, M. Review of cancer from perspective of molecular. J. Cancer Res. Pract. 2017, 4, 127–129. [CrossRef]
2. Mahase, E. Cancer overtakes CVD to become leading cause of death in high income countries. BMJ 2019, 366, l5368. [CrossRef]
[PubMed]
3. McCormack, V.A.; Boffetta, P. Today’s lifestyles, tomorrow’s cancers: Trends in lifestyle risk factors for cancer in low- and
middle-income countries. Ann. Oncol. 2011, 22, 2349–2357. [CrossRef] [PubMed]
4. Guimarães, D.S.M.; de Sousa Luz, L.S.; do Nascimento, S.B.; Silva, L.R.; de Miranda Martins, N.R.; de Almeida, H.G.; de Souza
Reis, V.; Maluf, S.E.C.; Budu, A.; Marinho, J.A.; et al. Improvement of antimalarial activity of a 3-alkylpiridine alkaloid analog by
replacing the pyridine ring to a thiazole-containing heterocycle: Mode of action, mutagenicity profile, and Caco-2 cell-based
permeability. Eur. J. Pharm. Sci. 2019, 138, 105015. [CrossRef] [PubMed]
5. De, S.; Kumar, S.K.A.; Shah, S.K.; Kazi, S.; Sarkar, N.; Banerjee, S.; Dey, S. Pyridine: The scaffolds with significant clinical diversity.
RSC Adv. 2022, 12, 15385–15406. [CrossRef]
6. Albratty, M.; Alhazmi, H.A. Novel pyridine and pyrimidine derivatives as promising anticancer agents: A review. Arab. J. Chem.
2022, 15, 103846. [CrossRef]
7. Lila, A.S.A.; Abdallah, M.H.; Khafagy, E.-S.; Shehata, T.M.; Soliman, M.S.; Younes, K.M.; Omran, M.M.; Gad, S. Design, synthesis
and cytotoxic evaluation of 2-amino-4- aryl-6-substituted pyridine-3,5-dicarbonitrile derivatives. Trop. J. Pharm. Res. 2021, 20,
2127–2133. [CrossRef]
8. Rizvi, S.A.A.; Saleh, A.M. Applications of nanoparticle systems in drug delivery technology. Saudi Pharm. J. 2018, 26, 64–70.
[CrossRef]
9. Patra, J.K.; Das, G.; Fraceto, L.F.; Campos, E.V.R.; Rodriguez-Torres, M.D.P.; Acosta-Torres, L.S.; Diaz-Torres, L.A.; Grillo, R.;
Swamy, M.K.; Sharma, S.; et al. Nano based drug delivery systems: Recent developments and future prospects. J. Nanobiotechnol.
2018, 16, 71. [CrossRef]
10. Mitchell, M.J.; Billingsley, M.M.; Haley, R.M.; Wechsler, M.E.; Peppas, N.A.; Langer, R. Engineering precision nanoparticles for
drug delivery. Nat. Rev. Drug Discov. 2021, 20, 101–124. [CrossRef]
11. Ghosh, B.; Biswas, S. Polymeric micelles in cancer therapy: State of the art. J. Control. Release 2021, 332, 127–147. [CrossRef]
12. Abu Lila, A.S.; Ishida, T.; Kiwada, H. Recent advances in tumor vasculature targeting using liposomal drug delivery systems.
Expert Opin. Drug Deliv. 2009, 6, 1297–1309. [CrossRef]
13. Mundekkad, D.; Cho, W.C. Nanoparticles in Clinical Translation for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 1685. [CrossRef]
14. Bober, Z.; Bartusik-Aebisher, D.; Aebisher, D. Application of Dendrimers in Anticancer Diagnostics and Therapy. Molecules 2022,
27, 3237. [CrossRef]
15. Montané, X.; Bajek, A.; Roszkowski, K.; Montornés, J.M.; Giamberini, M.; Roszkowski, S.; Kowalczyk, O.; Garcia-Valls, R.;
Tylkowski, B. Encapsulation for Cancer Therapy. Molecules 2020, 25, 1605. [CrossRef]
16. Kothamasu, P.; Kanumur, H.; Ravur, N.; Maddu, C.; Parasuramrajam, R.; Thangavel, S. Nanocapsules: The weapons for novel
drug delivery systems. Bioimpacts 2012, 2, 71–81. [CrossRef]
17. Yurgel, V.; Collares, T.; Seixas, F. Developments in the use of nanocapsules in oncology. Braz. J. Med. Biol. Res. 2013, 46, 486–501.
[CrossRef]
18. Huynh, N.T.; Passirani, C.; Saulnier, P.; Benoit, J.P. Lipid nanocapsules: A new platform for nanomedicine. Int. J. Pharm. 2009, 379,
201–209. [CrossRef]
19. Moura, R.P.; Pacheco, C.; Pêgo, A.P.; des Rieux, A.; Sarmento, B. Lipid nanocapsules to enhance drug bioavailability to the central
nervous system. J. Control. Release 2020, 322, 390–400. [CrossRef]
Pharmaceutics 2023, 15, 1755 18 of 19

20. Béduneau, A.; Saulnier, P.; Hindré, F.; Clavreul, A.; Leroux, J.-C.; Benoit, J.-P. Design of targeted lipid nanocapsules by conjugation
of whole antibodies and antibody Fab’ fragments. Biomaterials 2007, 28, 4978–4990. [CrossRef]
21. Figueiró, F.; de Oliveira, C.P.; Rockenbach, L.; Mendes, F.B.; Bergamin, L.S.; Jandrey, E.H.; Edelweiss, M.I.; Guterres, S.S.;
Pohlmann, A.R.; Battastini, A.M. Pharmacological Improvement and Preclinical Evaluation of Methotrexate-Loaded Lipid-Core
Nanocapsules in a Glioblastoma Model. J. Biomed. Nanotechnol. 2015, 11, 1808–1818. [CrossRef] [PubMed]
22. Bernardi, A.; Frozza, R.L.; Hoppe, J.B.; Salbego, C.; Pohlmann, A.R.; Battastini, A.M.; Guterres, S.S. The antiproliferative effect
of indomethacin-loaded lipid-core nanocapsules in glioma cells is mediated by cell cycle regulation, differentiation, and the
inhibition of survival pathways. Int. J. Nanomed. 2013, 8, 711–728. [CrossRef] [PubMed]
23. Fessi, H.; Puisieux, F.; Devissaguet, J.P.; Ammoury, N.; Benita, S. Nanocapsule formation by interfacial polymer deposition
following solvent displacement. Int. J. Pharm. 1989, 55, R1–R4. [CrossRef]
24. Molaahmadi, M.R.; Varshosaz, J.; Taymouri, S.; Akbari, V. Lipid Nanocapsules for Imatinib Delivery: Design, Optimization and
Evaluation of Anticancer Activity against Melanoma Cell Line. Iran. J. Pharm. Res. 2019, 18, 1676–1693. [CrossRef]
25. Moin, A.; Wani, S.U.D.; Osmani, R.A.; Abu Lila, A.S.; Khafagy, E.S.; Arab, H.H.; Gangadharappa, H.V.; Allam, A.N. Formulation,
characterization, and cellular toxicity assessment of tamoxifen-loaded silk fibroin nanoparticles in breast cancer. Drug Deliv. 2021,
28, 1626–1636. [CrossRef]
26. Akhila, J.S.; Shyamjith, D.; Alwar, M.C. Acute toxicity studies and determination of median lethal dose. Curr. Sci. 2007, 93,
917–920.
27. Chinedu, E.; Arome, D.; Ameh, F.S. A new method for determining acute toxicity in animal models. Toxicol. Int. 2013, 20, 224–226.
[CrossRef]
28. He, Y.C.; Chen, J.W.; Cao, J.; Pan, D.Y.; Qiao, J.G. Toxicities and therapeutic effect of 5-fluorouracil controlled release implant on
tumor-bearing rats. World J. Gastroenterol. 2003, 9, 1795–1798. [CrossRef]
29. Amr, A.E.E.; Ibrahimd, A.A.; El-Shehry, M.F.; Hosni, H.M.; Fayed, A.A.; Elsayed, E.A. In Vitro and In Vivo Anti-Breast Cancer
Activities of Some Newly Synthesized 5-(thiophen-2-yl)thieno-[2,3-d]pyrimidin-4-one Candidates. Molecules 2019, 24, 2255.
[CrossRef]
30. Mohamed, S.F.; Hosni, H.M.; Amr, A.E.-G.E.; Abdalla, M.M. Synthesis of novel substituted pyridines from 1-(3-aminophenyl)-3-
(1H-indol-3-yl)prop-2-en-1-one and their anticancer activity. Russ. J. Gen. Chem. 2016, 86, 672–680. [CrossRef]
31. Hather, G.; Liu, R.; Bandi, S.; Mettetal, J.; Manfredi, M.; Shyu, W.C.; Donelan, J.; Chakravarty, A. Growth rate analysis and efficient
experimental design for tumor xenograft studies. Cancer Inf. 2014, 13, 65–72. [CrossRef]
32. Abu Lila, A.S.; Kizuki, S.; Doi, Y.; Suzuki, T.; Ishida, T.; Kiwada, H. Oxaliplatin encapsulated in PEG-coated cationic liposomes
induces significant tumor growth suppression via a dual-targeting approach in a murine solid tumor model. J. Control. Release
2009, 137, 8–14. [CrossRef]
33. Alotaibi, B.; Tousson, E.; El-Masry, T.A.; Altwaijry, N.; Saleh, A. Ehrlich ascites carcinoma as model for studying the cardiac
protective effects of curcumin nanoparticles against cardiac damage in female mice. Environ. Toxicol. 2021, 36, 105–113. [CrossRef]
34. Reitman, S.; Frankel, S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic
transaminases. Am. J. Clin. Pathol. 1957, 28, 56–63. [CrossRef]
35. Nicolas, S.; Bolzinger, M.A.; Jordheim, L.P.; Chevalier, Y.; Fessi, H.; Almouazen, E. Polymeric nanocapsules as drug carriers for
sustained anticancer activity of calcitriol in breast cancer cells. Int. J. Pharm. 2018, 550, 170–179. [CrossRef]
36. Moinard-Chécot, D.; Chevalier, Y.; Briançon, S.; Beney, L.; Fessi, H. Mechanism of nanocapsules formation by the emulsion–
diffusion process. J. Colloid Interface Sci. 2008, 317, 458–468. [CrossRef]
37. Kim, M.W.; Kwon, S.-H.; Choi, J.H.; Lee, A. A Promising Biocompatible Platform: Lipid-Based and Bio-Inspired Smart Drug
Delivery Systems for Cancer Therapy. Int. J. Mol. Sci. 2018, 19, 3859. [CrossRef]
38. Blanco, E.; Shen, H.; Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat.
Biotechnol. 2015, 33, 941–951. [CrossRef]
39. Nicolete, R.; dos Santos, D.F.; Faccioli, L.H. The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro
inflammatory response. Int. Immunopharmacol. 2011, 11, 1557–1563. [CrossRef]
40. Singh, E.; Osmani, R.A.M.; Banerjee, R.; Abu Lila, A.S.; Moin, A.; Almansour, K.; Arab, H.H.; Alotaibi, H.F.; Khafagy, E.S. Poly
ε-Caprolactone Nanoparticles for Sustained Intra-Articular Immune Modulation in Adjuvant-Induced Arthritis Rodent Model.
Pharmaceutics 2022, 14, 519. [CrossRef]
41. Soliman, W.E.; Khan, S.; Rizvi, S.M.D.; Moin, A.; Elsewedy, H.S.; Abulila, A.S.; Shehata, T.M. Therapeutic Applications of Biostable
Silver Nanoparticles Synthesized Using Peel Extract of Benincasa hispida: Antibacterial and Anticancer Activities. Nanomaterials
2020, 10, 1954. [CrossRef] [PubMed]
42. Tavares, E.J.M.; Araújo, D.R.d.; Fraceto, L.F. Ivermectin-loaded polymeric nanoparticles: Screening the effects of polymers,
methods, and the usefulness of mathematical models. J. Nanosci. Nanotechnol. 2017, 17, 4218–4234. [CrossRef]
43. Al Saqr, A.; Khafagy, E.S.; Alalaiwe, A.; Aldawsari, M.F.; Alshahrani, S.M.; Anwer, M.K.; Khan, S.; Lila, A.S.A.; Arab, H.H.;
Hegazy, W.A.H. Synthesis of Gold Nanoparticles by Using Green Machinery: Characterization and In Vitro Toxicity. Nanomaterials
2021, 11, 808. [CrossRef] [PubMed]
44. De Melo, N.F.; Grillo, R.; Guilherme, V.A.; de Araujo, D.R.; de Paula, E.; Rosa, A.H.; Fraceto, L.F. Poly(lactide-co-glycolide)
nanocapsules containing benzocaine: Influence of the composition of the oily nucleus on physico-chemical properties and
anesthetic activity. Pharm. Res. 2011, 28, 1984–1994. [CrossRef]
Pharmaceutics 2023, 15, 1755 19 of 19

45. Liu, G.; McEnnis, K. Glass Transition Temperature of PLGA Particles and the Influence on Drug Delivery Applications. Polymers
2022, 14, 993. [CrossRef]
46. Chen, C.K.; Law, W.C.; Aalinkeel, R.; Yu, Y.; Nair, B.; Wu, J.; Mahajan, S.; Reynolds, J.L.; Li, Y.; Lai, C.K.; et al. Biodegradable
cationic polymeric nanocapsules for overcoming multidrug resistance and enabling drug-gene co-delivery to cancer cells.
Nanoscale 2014, 6, 1567–1572. [CrossRef]
47. Sethi, M.; Sukumar, R.; Karve, S.; Werner, M.E.; Wang, E.C.; Moore, D.T.; Kowalczyk, S.R.; Zhang, L.; Wang, A.Z. Effect of drug
release kinetics on nanoparticle therapeutic efficacy and toxicity. Nanoscale 2014, 6, 2321–2327. [CrossRef]
48. Giannini, E.G.; Testa, R.; Savarino, V. Liver enzyme alteration: A guide for clinicians. CMAJ 2005, 172, 367–379. [CrossRef]

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