Efficacious Doxorubicin Delivery Using Glutathione-Responsive Hollow Non-phospholipid Vesicles Bearing Lipoyl Cholesterols

Article abstract of DOI:10.1002/cmdc.201900335

In this study, we developed redox-sensitive vesicles using synthesised lipoyl cholesterol derivatives, a non-ionic surfactant and an optimum level of free cholesterol. Interestingly, concentration-dependent self-assembly was observed by scanning electron microscopy, wherein vesicles manifested as hollow spherical (at 0.15 mm) and triangular (0.50 mm). The redoxresponsive characteristics of the vesicles was probed in the presence of dithiothreitol; they underwent a clear increase in size as observed by dynamic light scattering measurements. These vesicles could easily encapsulate an anticancer drug, doxorubicin, and were observed to be stable in the presence of serum. They showed substantial release of the drug in response to biologically relevant stimulus, that is, glutathione. A toxicity assessment on HeLa and HepG2 cancer cells demonstrated activities of the drug-loaded vesicles comparable to that of free drug, whereas significantly enhanced toxicity and apoptotic induction were observed against drug-resistant HeLa cells, which was determined by studying the cellular internalisation of doxorubicin.

Full text of DOI:10.1002/cmdc.201900335

Accepted Article
Title: Efficacious doxorubicin delivery using glutathione responsive
hollow non-phospholipid vesicles bearing lipoyl cholesterols
Authors: Krishan Kumar, Lalit Yadav, Paturu Kondaiah, and Sandeep
Chaudhary
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Efficacious doxorubicin delivery using glutathione responsive
hollow non-phospholipid vesicles bearing lipoyl cholesterols
Krishan Kumar,[a] Lalit Yadav, Paturu Kondaiah,* and Sandeep Chaudhary*[a]
[a]
[b]
Dedication ((optional))
Abstract: Herein, we have developed redox sensitive vesicles using
synthesized lipoyl cholesterols and non-ionic surfactant along with
optimum level of free cholesterol. Interestingly, a concentration
dependent self-assembling behavior was noticed wherein vesicles
manifested as hollow spherical (0.15 mM) and triangular (0.50 mM)
aggregates as observed under scanning electron microscopic (SEM)
analysis. The redox responsive characteristic was probed in the
presence of dithiothreitol (DTT) which demonstrated a clear increase
in size as observed under dynamic light scattering (DLS)
measurements. These vesicles could easily encapsulate an
anticancer drug, doxorubicin and observed to be adequately stable
in the presence of serum. It showed a substantial release of the drug
in response to biologically relevant stimulus, i.e., glutathione (GSH).
Their toxicity assessment on HeLa and HepG2 cancer cells
demonstrated a comparable activity to that of free drug whereas
noticeable toxicity enhancement and apoptotic induction were
observed against drug resistant HeLa cells which were interpreted
through drug cellular internalization.
notable drug delivery systems that have been part of extensive
drug delivery research in the hope of achieving desirable
outcomes in chemotherapeutic treatments. Significant efforts
have been made in order to make these drug carriers bio-
responsive for effective release of the drug.[3,17] In particular, the
relatively low pH of intracellular compartments (endosomes; pH
~5-6/lysosomes; pH ~4-5) to that of blood plasma (pH ~7.4) has
been successfully exploited for the development of pH sensitive
nanocarriers.[3,4,18,19] These nanocarriers remain stable at
physiological pH, but degrade rapidly under mildly acidic
environment of endosomal/lysosomal compartments, thereby
releasing the encapsulated drug inside the cells.[ Likewise, the
equally important and substantially explored disulfide bridge
based redox sensitive nanocarriers have been designed to
disintegrate in response to the relatively high cytosolic
concentration of reducing agent, glutathione (GSH) tripeptide
(~2-10 mM) to that of extracellular fluids (~2-10 µM).[3,17a,21] In
particular, this biological stimulus is crucial owing to the fact that
tumor tissues represent a relative high reducing intracellular
environment in comparison to normal tissues which renders an
opportunity for tumor specific drug transport.[17b] Therefore, we
herein have attempted to develop reduction sensitive vesicles
which are constructed using non-ionic surfactants and
cholesteryl-lipoic acid conjugates (lipoyl cholesterols). The non-
ionic surfactants {e.g., sorbitan esters (spans) and
polyethoxylated sorbitan esters (tweens)} self-assemble in to
liposome-like vesicles in an aqueous media which are suitable
for the encapsulation of lipophilic and hydrophilic drug molecules
20]
Introduction
The design and development of biocompatible drug delivery
systems still remains the promising and leading approach to
bring about desirable outcomes in anticancer therapeutics.[1,2]
Towards achieving the therapeutic goals, the development of
bio-responsive nanocarriers that respond to either exogenous
(
e.g., temperature and magnetic field) or endogenous (e.g., pH
in vesicular membrane and within the aqueous core
respectively.[12b,22] The lower cost and greater stability of self-
assemblies (vesicles) of non-ionic surfactants have been in
marked contrast to those of phospholipids and offer them as
promising alternatives to liposome based systems.[22b,c] In
and redox potential) stimulus has been rigorously exercised in
full capacity over the last ten years.[3-5] The bio-responsive
systems have paved the way for their possible exploitation to
combat marked issues in anticancer therapy such as inefficient
delivery, tumor cell-specific targeting and the development of
resistance to chemotherapeutic drugs.[4,6-8] The existing plethora
of drug transport vehicles is composed of several nano and
micro-structured self assemblies of different amphiphilic
structures.[6,9,10] The liposomes,[11] niosomes,[12] micelles,[13]
nano/micro particles,[14] polymersomes[15] and nanogels[16] are
addition, these are considered to be biodegradable and non-
immunogenic which indicate their safe interaction with biological
system and are reckoned to be potential candidate for drug
delivery application.[22c]
The inclusion of biological small
molecule, i.e., cholesterol during such vesicle preparation is
routinely practiced as it provides stability to vesicles and
improves encapsulation efficiency of the drug.[
22b,c,23]
Additionally,
ethoxylate derivative of cholesterol and other conjugates have
also been reported to result in the formation of vesicular
structures.[24] Therefore, bioconjugates of cholesterol with lipoic
acid linked through oxyethylene spacer groups have been
synthesized in the current study to blend in with non-ionic
surfactant (Figure 1). Lipoic acid is a naturally occurring powerful
antioxidant and has been assessed to be reliably safe for human
use.[ Thus, it has been approved as an ingredient in many
dietary and pharmaceutical supplements. The disulfide
linkage of the dithiolane ring in lipoic acid structure is favourably
prone to rapid cleavage by cytosolic glutathione of cells and
[
[
a]
b]
Dr. K. Kumar, L. Yadav, Dr. S. Chaudhary
Department of Chemistry
Malaviya National Institute of Technology
Jawaharlal Nehru Marg, Jaipur-302017, India
E-mail: schaudhary.chy@mnit.ac.in
Prof. P. Kondaiah
Department of Molecular Reproduction, Development and Genetics
Indian Institute of Science
Bangalore- 560012, India.
25]
E-mail: paturu@iisc.ac.in
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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ChemMedChem
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therefore can be exploited to impart redox sensitivity to drug
delivery systems.[26] Herein, the non-phospholipid vesicles of
polysorbate 20 (Tween 20) containing lipoyl cholesterols (with
varying length of oxyethylene spacer) and optimum level of free
cholesterol were thoroughly characterized for size and
morphological analysis by means of field emission scanning
electron microscopy (FE-SEM), high-resolution transmission
electron microscopy (HR-TEM) and atomic force microscopy
synthesis procedures and characterizations have been placed in
Supporting Information.
Scheme 1a
(
AFM) studies. The hydrodynamic diameters and surface charge
of vesicles were assessed using dynamic light scattering (DLS)
and zeta potential measurements respectively. DLS studies
further assisted in redox sensitive assessment of these vesicles
in terms of change in size which was induced using non-
biological and biological reducing agents, i.e., DTT and GSH
respectively. The vesicles could efficiently encapsulate an
anticancer drug, doxorubicin (DOX) and demonstrated
substantial stability against serum. Triggered drug release was
ensured in reducing environment. Flow cytometry and confocal
microscopic analysis revealed significant drug release inside
DOX sensitive cervical (HeLa) and hepatic (HepG2) cancer cells
alike free drug. Interestingly, the vesicle mediated drug transport
revealed a significantly increased drug cellular internalization in
comparison to that of free drug in DOX resistant HeLa cells (DR-
HeLa) which resulted in marked induction of apoptosis (annexin
V binding) and reduction of cell viability counts (MTT assay).
Summarily, the exploitation of lipoyl-cholesterol bearing non-
phospholipid vesicles may offer exciting strategy for safe and
bio-responsive drug delivery to cancer cells, especially in the
condition of multi-drug resistance.
aReagents and conditions: i) p-TsCl/Py/DCM, rt, 20 h, 80%; ii) di-/tri-ethylene
glycol, 1,4-dioxane, 110 ˚C, 6 h, 65%/62%); iii) Lipoic acid, DCC, DMAP (cat),
DCM, 6 h, rt, 70%/80%.
Preparation and characterization of vesicles. Non-ionic
surfactants are the preferred surface active agents for the
preparation of low cost non-phospholipid based vesicular
structures. The stability and benign hemolytic behaviour of these
Results and Discussion
vesicles remark them to be superordinate to other vesicle
forming amphiphiles.[
22,28]
Similar to liposome organization, non-
ionic surfactants vesicles are produced from amphiphiles with
hydrophilic head and hydrophobic tail. The popularly known
series of surfactants, i.e., Tweens which bear a long alkyl chain
as well as
a
large hydrophilic moiety are approved
pharmaceutical excipients and known to give rise to stable
vesicular structures when co-formulated with cholesterol in an
aqueous phase.[22c,28] These structures are termed as niosomes
and have been reported to enable the entrapment of both
hydrophilic and lipophilic drugs. Having realized the role of
cholesterol in such vesicular structure preparation, we co-
formulated Tween 20 (polysorbate type non-ionic surfactant)
with cholesteryl-lipoic acid conjugates (CL1 and CL2)
synthesized in the present study (Figure 1) to give rise to redox
sensitive vesicles. The disulfide bond of lipoic acid is rapidly
cleaved in the presence of adequate reducing environment and
triggers the disorganization of self-assembled aggregates which
is considered advantageous for the release of entrapped
cargoes.[ Therefore the proportions of CL1/CL2 bioconjugates
and free cholesterol in vesicle composition were first optimized
Figure 1. Molecular structure of (a) cholesteryl-lipoic acid (CL) conjugates and
(b) Tween 20.
Synthesis of lipoyl-cholesterols. The lipoylated conjugates of
cholesterol were prepared as summarized in Scheme 1.[27]
Briefly, cholesteryl tosylate (1) was reacted with diethylene
glycol and triethylene glycol to give rise to diethoxy (2a) and
triethoxy cholesterol (2b) derivatives respectively which were
then coupled to lipoic acid using DCC based esterification
procedure to obtain CL1 and CL2 conjugates. The details of
26]
(
Table S1) based on the response to non biological reducing
agent, dithiothreitol (DTT; 10 mM) to mimic the intracellular
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ChemMedChem
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reductive environment and size of the vesicles using dynamic
light scattering (DLS) measurements. The thin-film hydration
method was employed for the preparation of vesicles. The
resulting vesicle dispersions were appropriately diluted and their
hydrodynamic diameters were determined with and without DTT
The CL1V and CL2V were further subjected to Transmission
electron microscopic (TEM) analysis for their morphological
investigation. It revealed the vesicles to be hollow in nature with
spherical morphology (Figure 2a1 and 2a2). Further imaging of
vesicles through scanning electron microscope (SEM)
demonstrated the presence of hollow spherical structures which
corroborated the observations under TEM analysis (Figure 2b1
and 2b2). Interestingly, different morphological feature was
revealed by the vesicles at relatively low dilution wherein at
concentration ≥0.5 mM, it manifested as triangular structures
with relatively deep and prominent cavities like that of a bowl as
shown in Figure 2b3 and 2b4.[32] The triangular vesicles
appeared to be relatively bigger in size than the spherical
vesicles. The surface morphological characteristic of the
vesicles was further assessed by means of atomic force
microscopy (AFM). The AFM imaging (Figure 2c1-2c3) further
substantiated the presence of hollow structures which appeared
like multimicellar vesicles.[33] Such hollow structured self-
assemblies have been reported to be of great interest because
of their high drug encapsulation efficiency (EE) and an efficient
or complete drug release.[34]
treatment (10 mM;
4 h). The vesicle composition, i.e.,
T20/cholesterol/CL; 15/5.0/10 for both CL1 and CL2 containing
vesicles, termed herein as CL1V and CL2V respectively, was
sorted by its optimal response to DTT treatment and appropriate
size (Table S1). The average hydrodynamic diameters of both
CL1V and CL2V ranged between 120 nm and 130 nm.
Additionally, the DLS measurements exhibited low polydispersity
index (PDI) values (˂0.25) for both vesicle preparations (Table
S1) which suggested nearly monodisperse population of
vesicles.[ Notably, the variation in proportions of cholesterol
and CL conjugates in different vesicle compositions did not
evidence any significant change in the size of vesicles. The zeta
potential measurements revealed that the surface charges of the
CL1V and CL2V were ~-37 and ~-40 mV which indicated the
adequate stability of these formulations due to electrostatic
stabilization of the particles (Figure S1).[30] The DTT treatment of
these vesicles led to an evident increase in their hydrodynamic
diameters (˃350 nm) and PDI values (˃0.60) which suggested
the triggered disorganization of vesicular self assembly owing to
redox sensitivity of disulphides of lipoyl units.[31] The
representative DLS graph depicting the change in size of CL1V
after treatment with DTT has also been shown in comparison to
that of without DTT treatment (Figure S2a and S2b).
29]
Drug encapsulation and release. The optimized formulations
were further investigated for their drug encapsulation efficacy to
profile their possible exploitation as redox sensitive drug delivery
vesicles for the rapid release of drug upon exposure to cell
reducing environments. The routinely prescribed chemotherapy
medication, doxorubicin was encapsulated in CL1V and CL2V
and the encapsulation efficiencies (EE) were observed to be
55.89% and 56.76% respectively as determined by the
calibration plot of doxorubicin. The drug delivery systems should
be adequately stable in the blood circulation and should not
exhibit premature drug release.[17a] Therefore, we subsequently
assessed the stability of doxorubicin loaded vesicles (CL1V-
DOX and CL2V-DOX) in the presence of 10% serum condition
(
fetal bovine serum; FBS).
Figure 2. The morphological investigation of CL1V (a1, b1 and b3) and CL2V
(
0
a2, b2 and b4) using TEM (a1 and a2) and SEM (b1-b4) at concentrations
.15 mM (a1, a2, b1 and b2) and 0.50 mM (b3 and b4). The AFM visualization
Figure 3. Evaluation of the stability of CL1V-DOX and CL2V-DOX against
serum (FBS; 10%) treatment and redox sensitivity against glutathione (GSH;
of CL2V structure (c1) along with 2D (c2) and 3D (c3) representation of a
single CL2V vesicle. Scale bar = 100 nm (TEM) and 200 nm (SEM and AFM).
10 mM) treatment (a). Dithiothreitol (DTT; 10 mM) mediated drug release from
vesicles at different time points (b). Cytotoxicity analysis of CL1V-DOX and
CL2V-DOX against HeLa (c) and HepG2 (d) cells in comparison with free drug
48 h post treatment.
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It was noticed that none of the CL1V-DOX and CL2V-DOX
showed any substantial loss of encapsulated doxorubicin. The
presence of serum could induce only ~16% and ~19% release of
the drug after 10 h incubation period (Figure 3a) which deduced
the stability of these vesicles. On the other hand, rapid and
significant drug release was observed from the vesicles (0.15
mM) in the presence of DTT (10 mM) when ascertained at two
different time points (2 h and 8 h). The DTT induced release of
based assay (Figure 3c and 3d). It exhibited that both the
formulations significantly reduced the cell viability counts in both
the cell lines and the observed IC50 values for CL1V-DOX (2.07
µg/ml; HeLa and 2.86 µg/ml; HepG2) and CL2V-DOX (1.67
µg/ml; HeLa and 2.30 µg/ml; HepG2) were comparable to that of
free doxorubicin treatment (0.68 µg/ml; HeLa and 1.24 µg/ml;
HepG2).
Notably, the respective concentrations of blank
vesicles (CL1V and CL2V) did not show any cytotoxic response
to either of the cell lines studied (Figure S4). This in turn
substantiated the non-toxic nature of non-ionic surfactant based
formulations blended with CL conjugates which make them
preferred tool for various delivery applications.[28b] In addition, we
examined the cytotoxicity of CL1V/CL2V-DOX vesicles against
˃50% of the encapsulated drug within 2 h (Figure 3b). We also
probed the reduction induced drug release behaviour in the
presence of biological reducing agent, glutathione (GSH; 10
mM) which evidenced ~70% release of DOX after 10 h
incubation period (Figure 3a). In order to look into drug release
behaviour from different morphologies (as observed under SEM), non-cancerous fibroblast cells, NIH3T3. Interestingly, it revealed
we also measured the amount of released drug at relatively low
dilution (0.50 mM) of vesicles wherein it appeared as triangular
hollow structures. The DTT treatment (2 h) resulted in a
relatively more prominent drug release (~80%) which suggested
the practical utility of structurally distinct morphologies (Figure
S3). The observations herein concluded that these vesicles are
significantly stable in physiological condition and can cause to
rapid release of encapsulated drug in the presence of
intracellular stimulus (redox potential).[21a]
significantly high cell viability counts for vesicle mediated DOX
treatments when compared to that of free DOX treatment
(Figure S5). This in turn signified that these formulations could
be promising candidates for receptor-targeted drug delivery
application.
The emergence of drug resistance during chemotherapy
treatments has been reported as a vital issue against successful
therapeutics.[35a] The chemotherapeutics under such condition
go with additional and/or frequent drug doses which develop
serious side effects and consequently leads to possible
treatment failure.[35b] The stimuli responsive drug delivery
systems have been ensured to raise the intracellular
accumulation of drugs in vitro for reaching the therapeutic levels
in prospect towards overcoming the drug resistance.[8,11a,20a,35]
Figure 4. Cytotoxicity (a) and annexin V binding analysis (b) of CL1V-DOX
and CL2V-DOX treated DR-HeLa cells in comparison with free DOX at 96 h
and 72 h post treatment respectively. Representative overlaid flow cytometry
histograms for the (c) comparative cellular internalization of drug (7.5 µg/ml;
4h) and (d) annexin V-FITC binding between free DOX and CL2V-DOX
mediated treatments (15 µg/ml; 72h).
Cytotoxicity and cellular internalization of CL1V-DOX and
CL2V-DOX. Reduction triggered intracellular drug release from
nanocarriers has been realized as potential strategy to achieve
improved anticancer therapeutics.[21] Hence, there is a growing
need to develop such responsive and safe nanocarriers for
efficacious drug delivery to reach therapeutic level of the drugs.
We therefore determined the cytotoxicity of CL1V-DOX and
CL2V-DOX formulations against cervical cancer (HeLa) cells
and human liver carcinoma (HepG2) cells in comparison with
free drug for a 48 h treatment following the conventional MTT
Figure 5. The confocal microscopic investigation of DOX cellular
internalization in HeLa (a1 and a2) and HepG2 (b1 and b2) cells after
treatment with free DOX (a1 and b1) and CL2V-DOX (a2 and b2) at the
concentration of
5 µg/ml for 4 h. Each panel, respectively, left to right
annotates DOX fluorescence, DAPI (nuclear stain) fluorescence, bright field
image and overlay of previous three images. Scale bar = 20 µm.
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We therefore examined the cytotoxic effect of CL1V-DOX and
reveal any notable apoptotic induction (~4%) whereas significant
apoptosis (~35% annexin-V positive cells) was detected for
CL2V-DOX mediated treatment (Figure 4b and 4d). The
observation clearly evidenced the efficacious cellular transport of
DOX through vesicles across the membrane of DOX resistant
HeLa cells which resulted in induction of apoptosis and thereby
cell death.
CL2V-DOX against doxorubicin resistant HeLa cells (DR-
HeLa).[
20a]
Owing to significant DOX resistance (IC50; 61.45
µg/ml) of DR-HeLa cells (Figure S6), we refrained from
determination of IC50 values for CL1V-DOX and CL2V-DOX
because it would require relatively high concentration of vesicles
for equivalent DOX concentration that exert potential cytotoxic
effects in vitro pertaining to the concentration of Tween type
surfactant (Figure S4).[36] Therefore, we checked out the cell
viability only at 7.5 and 15 µg/ml. Interestingly, in DR-HeLa cells
a marked difference in cell viabilities was observed between
vesicle mediated DOX treatments and that of free drug. As
shown in Figure 4a, the CL1V-DOX mediated treatments
resulted in ~17 and ~28 % cell death at 7.5 and 15.0 µg/ml,
respectively, 96 h post treatment. At these concentrations the
observed cell death for CL2V-DOX treatment was ~20 and 34%.
However, no significant decrease in cell viability counts was
detected for free doxorubicin treatment of DR-HeLa cells.
The study was then further advanced while looking into
comparative cellular internalization of drug in HeLa and DR-
HeLa cells for the treatments mediated by CL1V-DOX/CL2V-
DOX and free DOX by means of flow cytometry and confocal
microscopy experimentation. The quantitative analysis of flow
cytometry results revealed that CL1V-DOX and CL2V-DOX
mediated treatment showed nearly similar DOX fluorescence
intensity to that of free drug mediated treatment in HeLa cells
(Figure S7). The confocal microscopic analysis revealed
substantial DOX fluorescence in the cell nuclei (of both HeLa
and HepG2 cells) for CL1V/CL2V mediated DOX treatments
which indicated rapid and efficient drug release from the vesicles.
This was evident from the observed magenta colour in merged
images as a result of co-localization of DOX (red) and DAPI
(
nuclear stain; blue) as shown in Figure 5.[12b] Such efficacious
nuclear distribution of drugs has always been desirable in the
case of nanocarrier/nanoparticle mediated drug delivery.[20a] In
addition, the quantitative analysis of cellular DOX fluorescence
in confocal micrographs further substantiated the results of flow
cytometry (Figure S8). In case of DR-HeLa cells, significantly
enhanced cellular DOX internalization manifested for CL1V-DOX
and CL2V-DOX treatments in comparison to that of free DOX as
evidenced from the stark difference in quantitative analysis of
DOX fluorescence intensity using flow cytometry (Figure 4c and
S9). The confocal microscopic analysis corroborated the
observation as CL1V-DOX and CL2V-DOX treated (24 h; 15
µg/ml) DR-HeLa cells exhibited much brighter DOX fluorescence
than those treated with free drug (Figure 6b1 and 6b2). This
enhancement in cellular DOX fluorescence was also quantified
from the micrographs of three independent experiments which
demonstrated almost doubled mean fluorescence intensity of
DOX in DR-HeLa Cells treated with vesicles (Figure S10). This
distinct activity of drug loaded vesicles in DR-HeLa cells was
also evaluated 4 h post treatment and it was observed that
CL1V/CL2V-DOX treatment led to a clearly visible intracellular
DOX fluorescence whereas free drug treatment did not reveal
any noticeable DOX fluorescence inside the cells (Figure 6a1
and 6a2).[38] This can be plausibly ascribed to the ability of
nanocarriers to bypass the drug efflux pumps on the membrane
of drug resistant cells which results in the increased intracellular
concentration of drug.[7,38] The drug internalization study also
helped in understanding the observed cytotoxicity profile against
both drug sensitive and resistant cancer cells which apparently
relied upon the delivered intracellular concentration of drug.[
Based on these observations, it can be suggested that the lipoyl
cholesterols could be exploited to give rise to redox sensitive
vesicles which possess excellent potential of being examined as
drug delivery vehicle, especially in the case of drug resistance.
In addition, the idea of functionalizing these vesicles further for
site-specific drug delivery would be highly advantageous as it
Figure 6. The confocal microscopic investigation of DOX cellular
internalization in DR-HeLa cells after treatment with free DOX (a1 and b1) and
CL2V-DOX (a2 and b2) at the concentration of 15 µg/ml for the periods of 4 h
(a1 and a2) and 24 h (b1 and b2). Each panel, respectively, left to right
annotates DOX fluorescence, DAPI (nuclear stain) fluorescence, bright field
image and overlay of previous three images. Scale bar = 20 µm.
This distinct cytotoxic response of vesicle mediated DOX
treatment was also evaluated in terms of monitoring apoptotic
DR-HeLa cell population following annexin-V binding assay
using flow cytometry. In apoptotic cells, the phosphatidylserine
appears on the cell membrane surface and can easily be
detected using fluorophore bound annexin-V which is in turn
used for quantitative determination of apoptosis.[37] The annexin-
V FITC labelling of free DOX treated DR-HeLa cells did not
20a,38]
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FULL PAPER
may derive pharmacologically effective formulations. The
development of such bioconjugates promises an outright
application to be blended in different liposomal and/or niosomal
formulation with various other amphiphilic components to obtain
biocompatible and bioresponsive delivery vehicles.
(DPBS), Gibco fetal bovine serum (FBS) and annexin V- FITC conjugate
were received from Thermo Fisher Scientific.
Synthesis of lipoyl cholesterols. The details of the synthetic
procedures for lipoylated cholesterol derivatives have been given in
Supporting Information. All compounds were fully characterized by 1H
NMR, 13C NMR and HR-MS. H NMR spectra of compounds were
recorded on an ECS 400 MHz (JEOL) NMR spectrometer and 13C NMR
were recorded at 100 MHz. Mass spectra were recorded on a Xevo G2-S
Q Tof (Waters, USA).
1
Conclusions
Summarily, two bioconjugates (lipoyl-cholesterols) which differ in
terms
−OCH
of
(−CH
the
−O−CH
length
of
oxyethylene
spacer
Preparation and characterization of vesicles. A lipid layer hydration
method was employed for the preparation of vesicles wherein Tween 20
and different proportions of CL1 and CL2 along with cholesterol were
dissolved in chloroform:methanol and evaporated under reduced
pressure to produce a thin lipid film on the wall of a round-bottomed flask.
The film was then hydrated with sterile milli-Q water followed by vortex
mixing for 20 min and bath sonication at 60 ºC for 15 min. The
suspensions were diluted hundred times and subjected to size and
morphological analysis using Tecnai G2 20 (FEI) S-Twin transmission
electron microscope (TEM) and Nova Nano FE-SEM 450 (FEI) scanning
electron microscope (SEM). For TEM; samples were placed on carbon
coated copper grid and negatively stained using 2% phosphotungstic
acid (PTA) solution before imaging. For SEM; samples were loaded on
carbon conductive adhesive tape placed on stubs and sputter coated
using gold before imaging. The AFM imaging was performed using a
multimode scanning probe microscope (Bruker). The size distributions
{dynamic lighting scattering (DLS) measurements} of different vesicle
suspension before and after DTT treatment and zeta potentials were
analyzed on Zetasizer Nano from Malvern Instruments.
[
2
2
2
−) CH O−; n = 1 and 2] have been
n
2
successfully synthesized and exploited as the biocompatible
component in the fabrication of reduction responsive non-
phospholipid vesicles. The optimized formulations (Tween
20/cholesterol/CL; 15/5.0/10) were thoroughly characterized by
means of HR-TEM, FE-SEM, and AFM. Self-assembled hollow
structures (vesicles) of triangular shape were observed at ≥0.5
mM concentration, however, below which (0.1 mM) it appeared
to be spherical in morphology. The polydispersity distribution
index (PDI) values (˂ 0.25) in DLS measurements suggested a
nearly monodisperse vesicle population. Zeta potential
measurements revealed a strongly negative surface charge (˃
3
0) in favour of the stability of the vesicles. The vesicles could
encapsulate chemotherapeutic drug, doxorubicin efficiently
%EE; >55%) and showed adequate response to reducing
(
environment (DTT and GSH) to release the encapsulated drug.
The MTT based cytotoxicity studies revealed that vesicle
mediated drug treatment had comparable IC50 values to that of
free drug against HeLa and HepG2 cells. On the other hand, it
demonstrated a significant decrease in cell viability counts
against doxorubicin resistant HeLa (DR-HeLa) cells when
compared to free drug treatments. Flow cytometric and confocal
microscopic analysis further evidenced in favour of cytotoxicity
profile wherein it demonstrated alike cellular fluorescence of
doxorubicin in doxorubicin sensitive HeLa and HepG2 cells for
both the treatments mediated by either free drug or via vesicles.
However, significantly higher doxorubicin fluorescence was
noticed in DR-HeLa cells for vesicle mediated treatment in
comparison to free drug which resulted in notable induction of
apoptosis and cell death as observed in annexin V binding and
MTT assay respectively. Concisely, these lipoyl cholesterol
based reduction responsive bioconjugates offer a viable means
of developing different surfactant or liposome based vesicles
which can be exploited as promising candidates to improve the
efficacy of chemotherapeutic treatments especially in drug-
resistant carcinomas.
Drug encapsulation and release studies. The drug, doxorubicin
hydrochloride (4.0 mg/ml) was added to optimized formulations of CL1V
and CL2V during hydration step and thereafter followed the same
procedure as discussed above. The unentrapped drug was removed by
centrifugation and the % entrapment was calculated by means of
recording UV/Vis spectrum of the supernatant drug molecules (λmax; 485
nm) on a LAMBDA 750 (Perkin Elmer) UV/Vis NIR Spectrophotometer as
v t v
follows. %EE = (A /A )*100; A = absorbance of vesicle entrapped drug =
A
t
-A ; A and A = absorbance of total drug and that of supernatant.
s
t
s
Thereafter, the amount of vesicle entrapped drug was calculated using a
standard calibration plot of drug. To examine drug release, CL1V and
CL2V suspension were added to buffer solution (10 mM; pH = 7.4) and
treated with serum (FBS; 10%), GSH (10 mM) and DTT (10 mM).
Thereafter extent of drug release (R) was evaluated at desirable time
points by means of UV/Vis spectrophotometer using following
t 0 0 0 t
formula. %R = (A -A /A100-A )*100 where A is the initial absorbance, A is
the absorbance at set time points post treatment and A100 represents
absorbance of samples with complete drug leakage in the presence of
isopropanol.
Cytotoxicity analysis. The cytotoxicity for blank and DOX encapsulated
CL1V and CL2V in comparison with free DOX was determined using
MTT based colorimetric assay against human liver carcinoma cell line
(
HepG2 cells), human cervical cancer cell line (HeLa cells) and DOX
Experimental Section
resistant culture of HeLa cells (DR-HeLa cella). The cells were plated in a
9
4
6-well cell culture plate (HepG2/HeLa; 1×10 cells/well and DR-HeLa;
3
Materials. Analytical grade reagents and solvents were procured from
best-known sources and were used as received. Cholesterol, Lipoic acid
and DAPI, MTT reagent, DMSO, Tween 20 and silica gel (mesh size; 60−
5×10 cells/well) in cell culture medium, Dulbecco’s Modified Eagle
medium (DMEM) supplemented with 10% serum (FBS). After 24 h, cells
were treated with CL1V/CL2V and their DOX containing formulations
along with free DOX at different drug concentrations in 200 μL of cell
culture medium and placed in an incubator set at 37 °C and supplied with
5% CO2, for a period of 48 h (HepG2 and HeLa) and 96 h (DR-HeLa).
Then, 20 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
120) were purchased from Merck. Di-/tri-ethylene glycol, DCC reagent
and DMAP were purchased from Spectrochem Pvt. Ltd. Dulbecco’s
modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline
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ChemMedChem
10.1002/cmdc.201900335
FULL PAPER
tetrazoliumbromide) reagent {stock; 5.0 mg/mL in DPBS (Dulbecco's
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Acknowledgements
S. C. acknowledges DST, New Delhi for DST-NRF Indo-South
Africa Joint Research Project (DST/INT/South Africa/P-19/2016).
K.K. acknowledges CSIR, New Delhi for providing financial
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The cholesteryl lipoic acid
conjugates have been
exploited for the
Krishan Kumar, Lalit Yadav,
Paturu Kondaiah,* and
Sandeep Chaudhary*
development of reduction
responsive hollow non-
phospholipid vesicles which
showed concentration
dependent spherical and
triangular structures. In
comparison to free
Page No. – Page No.
Efficacious doxorubicin
delivery using glutathione
responsive hollow non-
phospholipid vesicles
bearing lipoyl cholesterols
doxorubicin, the vesicle
encapsulated drug
exhibited comparable and
substantially high
intracellular accumulation
against drug sensitive and
resistant cancer cells
respectively, which offer an
exciting strategy for the
development of reduction
responsive nanocarriers.
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Author(s), Corresponding Author(s)*
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Title
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