Molecular Self-Assembly of Bile Acid-Phospholipids Controls the Delivery of Doxorubicin and Mice Survivability

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00105

Lipid composition in general determines the drug encapsulation efficacy and release kinetics from liposomes that impact the clinical outcomes of cancer therapy. We synthesized three bile acid phospholipids by conjugating the phosphocholine headgroup to th

Full text of DOI:10.1021/acs.molpharmaceut.7b00105

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Article
Molecular Self-Assembly of Bile Acid-Phospholipids
Controls the Delivery of Doxorubicin and Mice Survivability
Vedagopuram Sreekanth, Nihal Medatwal, Sanjay Pal, Sandeep Kumar, Sagar Sengupta, and Avinash Bajaj
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00105 • Publication Date (Web): 30 Jun 2017
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Molecular Self-Assembly of Bile Acid-Phospholipids Controls
the Delivery of Doxorubicin and Mice Survivability
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Vedagopuram Sreekanth,1,2 Nihal Medatwal,1,2 Sanjay Pal,1,3 Sandeep
Kumar,1,2 Sagar Sengupta,4 and Avinash Bajaj1,*
1: Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology,
NCR Biotech Science Cluster, 3rd Milestone FaridabadꢀGurgaon Expressway, Faridabadꢀ
121001, Haryana, India.
2: Manipal University, Manipalꢀ576104, Karnataka, India.
3: KIIT University, Bhubaneswarꢀ751024, Odisha, India.
4: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhiꢀ110067, India.
*Corresponding Author:
Email: bajaj@rcb.res.in
Ph: +91ꢀ124ꢀ2848831, Fax: +91ꢀ124ꢀ2848803
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Abstract: Lipid composition in general determines the drug encapsulation efficacy and
release kinetics from liposomes that impact the clinical outcomes of cancer therapy. We
synthesized three bile acid phospholipids by conjugating the phosphocholine head group to
the 3'ꢀhydroxyl group of benzylated lithocholic acid (LCA), deoxycholic acid (DCA), and
cholic acid (CA); and investigated the impact of membrane rigidity on drug encapsulation
efficacy, drug release kinetics, anticancer effects, and mice survival. Liposomes with
hydrodynamic diameter of 100ꢀ110 nm were subsequently developed using these
phospholipids. Fluorescenceꢀprobe based quantification revealed more fluidic nature of
DCAꢀPCꢀ and CAꢀPCꢀderived liposomes, whereas the LCAꢀPCꢀderived are rigid in nature.
Doxorubicin encapsulation studies showed ~75% encapsulation and ~38% entrapment
efficacy of Doxorubicin using more fluidic DCAꢀPC, CAꢀPC derived liposomes as compared
to ~58% encapsulation and ~18% entrapment efficacy in case of LCAꢀPC derived
liposomes. In vivo anticancer studies in the murine model confirmed that Doxorubicin
entrapped CAꢀPC liposomes compromise mice survival, whereas rigid drug entrapped LCAꢀ
PCꢀderivedꢀliposomes increased mice survival with ~2ꢀfold decrease in tumor volume.
Pharmacokinetic and bioꢀdistribution studies revealed ~1.5ꢀfold increase in plasma drug
concentration and ~4.0ꢀfold rise in tumor accumulation of Doxorubicin on treatment with
drug entrapped LCAꢀPC liposomes as compared to Doxorubicin alone. In summary, this
study presents the impact of bile acid derived liposomes with different rigidity on drug
delivery and mice survivability.
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Keywords: Bile Acids, Phospholipids, Doxorubicin delivery, Molecular selfꢀassembly
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Introduction:
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Efficient liposomal based drug delivery systems using different kinds of phospholipids has
been commercialized for treatment of cancer and fungal infections.1 The encapsulation of
active drug ingredients either in the aqueous or lipid bilayer compartments form the premise
of the strategy for liposomal formulations where drug entrapment efficacy and their release
kinetics is contingent on the chemical nature and composition of lipid ingrediants.2 Low drug
encapsulation, poor retention efficiencies, instability of the liposomes and their uptake by
the reticuloꢀendothelial system, nonꢀspecific leakage of the encapsulated drug, and poor
accumulation at tumor sites are major limitations of the encapsulation strategy used for
anticancer drug delivery.3 Therefore, designing of phospholipids for drug delivery is an
active area of research; and recent studies have stressed on fineꢀtuning of the
physicochemical, mechanical, and structural properties of the lipids to control the release
rates of encapsulated drugs.4ꢀ7
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Numerous approaches using smart head group design,8 interfacial chemical linkage in
hydrophobic region,9 or dimeric lipids10 have been used to fineꢀtune the membrane
properties of liposomes. Szoka et al. recently investigated the interactions of cholesterolꢀ
derived phospholipids with membranes and developed effective formulations for drug
delivery.11,12 Impermeable vesicles have been developed using confirmationally restricted
phosphatidylethanolamine derivatives that provide rigidity and mechanical strength to
vesicles.13 Wang and coꢀworkers showed superior stability and enhanced doxorubicin
encapsulation using phospholipid vesicles reinforced with hydrogen bonding.14
Phospholipids produced using thiolꢀyne chemistry were resistant to the action of
phospholipases, thereby generating more stable vesicles.15
Chemical nature and composition of lipids affect the rigidity of liposomal membranes
because the presence of double bonds render the membranes fluid, whereas steroidal lipids
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rigidify the membranes.16 Recent studies have shown that the composition of lipid
membranes affects its fluidity, thereby regulating the release of the encapsulated drugs.17
It has been shown that in vivo anticancer activity of the folate targeted Mitoxantrone
encapsulated liposomes is contingent on lipid composition and fluidity of the membranes.18
Zhu and coworkers studied the aggregation properties of PEGylated bile acids using
lithocholic acid, deoxycholic acid, and cholic acid conjugated with different
polyethyleneglycol chains; and their ability to encapsulate hydrophobic drugs.19,20
Earlier studies suggested that the inclusion of hydroxyl groups in the hydrophobic domains
of lipids modulate the rigidity of the liposomes,21 which might thereby regulate drug
encapsulation efficacy and release kinetics from these liposomes (Fig. 1). Differential drug
release kinetics from liposomes may impact anticancer treatment efficiency and survivability
of tumorꢀbearing mice. Therefore, we synthesized three bile acidꢀphosphocholine (BPC)ꢀ
derived phospholipids using lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid
(CA), namely, LCA-PC, DCA-PC, and CA-PC, which differ in the number of hydroxyl groups
on their backbone (Fig. 1). These phospholipids were subsequently used to form liposomes
for encapsulation of Doxorubicin, a model anticancer drug. Studies on anticancer activity
and survival in a murine tumor model, along with pharmacokinetic and bioꢀdistribution
studies of the liposomes were performed to determine the impact of rigidity and drug
release kinetics on tumor regression and mice survivability. This study will provide new
insights on impact of membrane rigidity using bile acid derived phospholipids on doxorubicin
delivery.
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Experimental Section:
Materials and Methods: All solvents and chemicals used are of ACS grade. Lithocholic
acid (LCA), deoxycholic acid (DCA), cholic acid (CA), benzyl bromide, Lꢀαꢀ
Phosphatidylcholine (EggPC), 2ꢀchloroꢀ1,3,2ꢀdioxaphospholaneꢀ2ꢀoxide, Doxorubicin.HCl,
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and Diphenylhexatriene (DPH) was purchased from SigmaꢀAldrich. 1,2ꢀdistearoylꢀsnꢀ
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glyceroꢀ3ꢀphosphoethanolamineꢀNꢀ[amino(polyethyleneglycol)ꢀ2000]
ammonium
salt
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(DSPEꢀPEG2000ꢀAmine) was purchased from Avantiꢀpolar lipids. Unless mentioned, all
compounds were purified using Combiꢀflash chromatography (Teledyne ISCO) using silica
gel rediꢀsep or C18 silica rediꢀsep columns. NMR spectra were recorded on BruckerꢀAvanceꢀ
400 MHz FTꢀNMR spectrometer. Chemical shifts are reported in δ ppm with reference to
respective deuterated solvent. Mass spectra were recorded with AB SCIEX Triple TOF 5600
system. Synthesis and characterization of bile acidꢀphosphocholine (BPC)ꢀderived
phospholipids is reported in supporting information.
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Determination of critical miceller concentration:22 Stock solutions of LCAꢀPC, DCAꢀPC
and CAꢀPC were prepared in ethanol; and serial dilutions were done in ethanol at 4 °C.
Ethanol from the samples was evaporated in speedꢀvac for 1 hour at 37 °C. Samples were
then suspended in 1 mL of pyrene solution (1.0 µg/mL), followed by vortexing, and
fluorescence was recorded at 375 and 395 nm with excitation of 334 nm. Fluorescence ratio
(395 nm/375 nm) was plotted against the concentration of compounds.
Liposome preparation and doxorubicin encapsulation: Doxorubicin.HCl was
encapsulated in liposomes using remote drug loading method.23 Typically Egg PC (5 mg),
BPC (5 mg), and DSPEꢀPEG2000ꢀAmine (1 mg) were taken in chloroform (0.2 mL) in 1: 1:
0.2 weight ratio in a round bottom flask. Chloroform is evaporated by rotary evaporation
followed by vacuum pump to form a dry thin film. Thin films were hydrated with 250 mM
ammonium sulfate solution (2 mL) at 60 ˚C for two hours with intermittent sonication at 60
˚C for 1 min at 15 minutes interval. Lipid suspension was sequentially extruded through 200
(14 times) and 100 nm polycarbonate (14 times) membranes at 60 ˚C. Free ammonium
sulfate was removed by dialysis (Spectrapor/CE, molecular weight cut off 100 KD,
Spectrum laboratories Inc. USA.) of liposomes against 25 volumes of 5% glucose for 4
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times in 24h. Liposomes were then incubated with Doxorubicin.HCl (5mg) in 5% glucose (1
mL) at 60 ˚C. Liposomal suspension was cooled down to room temperature, and mixed with
activated Dowex 50WXꢀ4 200 ion exchange to remove free or surface bound doxorubicin,
rotated for 20 minutes, and filtered to get doxorubicin encapsulated liposomes. All these
experiments were performed at least three times.
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Encapsulation and entrapment efficiency: Encapsulation efficiency (concentration of
drug present in liposomes) was determined using absorbance method24 where liposomes
(10 ꢁL) were treated with 90% isopropyl alcohol containing 0.075 N HCl (190 ꢁL), followed
by measurement of Doxorubicin absorbance at 480 nm. Percentage of encapsulated
Doxorubicin content was calculated by formula (I)
Encapsulation efficiency (% Doxorubicin encapsulated) = (Ad/Ai) × 100ꢂꢂꢂꢂ(I)
where Ai = initial absorbance of the sample where 5 mg of Doxorubicin was added.
Ad = final absorbance after Dowex treatment.
To determine the entrapment efficiency (drug to lipid weight ratio), we calculated the amount
of phospholipids retained after extrusion of liposomes by colorimetric method.25 Different
linear concentrations of phospholipids and sample (10 ꢁL) were diluted with chloroform (2
mL) and then treated with 1 mL ferrothiocyante reagent. Samples were vortexed for 20 sec.
and spun for 10 min at 1000 r.p.m to remove out the lower layer. Absorbance of standards
and samples was read at 488 nm; and concentration of phospholipids present in the test
sample was computed using standard curve. Percentage of phospholipid content retained
was plotted using following formula (II)
% Phospholipids retained = [PL]d/[PL]i × 100
ꢂꢂ(II)
whereas [PL]i = concentration of total phospholipids used for liposome preparation before
extrusion.
[PL]d = concentration of phospholipids retained after extrusion
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Entrapment efficiency was calculated using following formula (III)
Entrapment efficiency (%) = [Dox]d /[PL]d × 100
ꢂꢂ....(III)
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[DOX]d = Doxorubicin concentration after dowex treatment.
[PL]d = Phospholipid concentration in liposomes after extrusion.
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In vitro drug release experiments: In vitro drug release experiments were conducted
using dialysis method in three different conditions.26 Typically doxorubicin encapsulated
liposomes (1 mL) were placed in dialysis tubing (Spectrapor/CE, molecular weight cut off
100 KD, Spectrum laboratories Inc. USA.) and dialyzed against 30 mL of different release
buffer conditions (pH 7.4, PBS; pH 5.0, PBS; 10% FBS in PBS) at 37 ˚C away from bright
light. At various time points; aliquots (1 mL) were withdrawn from outer aqueous solution
and replaced by fresh release medium (1 mL). Samples were stored at 4 ˚C until analysis
were performed spectrofluorimetrically using Molecular devices M5 instrument with λex of
480 nm and λem of 590 nm. Rate and percentage of drug released was computed using
following formula (IV)
Percentage of drug released = [Dox]t/[Dox]i × 100
ꢂꢂ.(IV)
where [Dox]t = Doxorubicin concentration released at time t.
[Dox]i = Doxorubicin concentration in doxorubicin entrapped liposomes in dialysis
tube.
We then computed release rate constants using Higuchi model27 by fitting release data in
following equation (V)
Amount of doxorubicin released at time t
[Dox]t = KH x (t)1/2
ꢂꢂꢂ.(V)
where KH = Higuchi rate constant.
t = corresponding time t.
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Dynamic light scattering and zeta potential: Mean particle size (Zꢀavg. diameter),
polydispersity index (pdI), and zeta potential of liposomes were measured by Dynamic Light
Scattering method using Zetasizer NanoZS90 (Malvern, UK). Liposomal suspension (100
ꢃL) was diluted to 1 mL using PBS and 3 sets of 11 measurements were performed at 90˚
scattering angle to get average particle size and pdI for each liposome. Zeta potential of
liposomes was measured using zeta cuvette at 173˚ scattering angle.
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Transmission electron microscopy: For negative stain transmission electron microscopy
(TEM), bile acid suspension (~5 mM) was deposited on to a sample grid. Samples were
then stained with 1% phosphotungstic acid for 10 minutes and dried with whatman filter
paper. Samples were observed under FEI Tecnai TF20 transmission electron microscope,
and images were processed using Tecnai G2 software.
Membrane rigidity studies: Hydrated thin films of EggPC, BPC, and DSPEꢀPEG2000ꢀ
Amine (1:1:0.2 weight ratio) (as prepared above) were processed for 4ꢀ5 freeze thaw cycles
from 70 ˚C to 4 ˚C with intermittent vortexing followed by sonication at 70 ˚C for 15 min. For
anisotropy studies, 1% Diphenyl hexatriene (DPH) was incorporated during thin film
preparation.21 Steady state DPH anisotropy measurements were carried out at 37 ˚C in 96ꢀ
well plate and anisotropy was calculated as per published protocol.21
Cell culture: Murine (4T1) and human (MDAꢀMBꢀ231) breast cancer cells were maintained
as monolayers in RPMIꢀ1640 (for 4T1 cells) or DMEM (Hyclone, USA) (for MDAꢀMBꢀ231
cells) media containing 10% (v/v) fetal bovine serum, penicillin (100 ꢁg/mL), streptomycin
(100 U/mL), and gentamycin (45 ꢁg/mL) at 37 ˚C in a humidified atmosphere with 5% CO2;
and subcultures were made by trypsinization.
MTT assay: Cytotoxicity of bile acids, doxorubicin, BPCs, and doxorubicin entrapped BPC
liposomes was estimated using MTT {3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyltetrazolium
bromide} assay. MTT was dissolved in PBS at a concentration of 5 mg/mL and filtered.
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Approximately ~3000 cells/well were seeded in 96 well plates containing 200 ꢁL of complete
medium. After 24h of preꢀculture period to ensure cell attachment, medium was replaced with
fresh medium supplemented with different concentrations of BPCs, doxorubicin, or doxorubicin
entrapped liposomes. After 48 h of treatment, 20 ꢃL of MTT solution was added to each well
and incubated for 4 h. Formazan crystals formed were then dissolved in 1:1 mixture of
DMSO and methanol and optical density (OD) of each well was measured using a micro
culture plate reader at 540 nm. Percentage viability was then calculated as [{A540 (treated
cells) ꢀ background]/[A540 (untreated cells) ꢀ background}] x 100.
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In vivo anticancer and toxicological studies:28 All animal experiments were done with
approval of Institutional Animal Ethical Committee (IAEC), National Institute of Immunology,
New Delhi. Murine breast cancer (4T1) cells (1.5 x 106) in 200 ꢁL FBS were injected
subcutaneously into right flanks of 5ꢀweekꢀold female Balb/c mice. Mice were randomized
into six groups (9 mice in each group) after they develop tumor of ~75 mm3. Groups were
then subjected to different treatments of a) 5% glucose (control group); b) free doxorubicin;
and doxorubicin encapsulated c) LCAꢀPC liposomes, d) DCAꢀPC liposomes, e) CAꢀPC
liposomes, f) AdrisomeTM (Positive control) with an equivalent doxorubicin dose of 5 mg/kg,
intravenously through lateral tail vein on alternate day (total three doses). Tumor
measurements were made every alternate day using digital caliper, and tumor volume was
calculated using formula L×B2/2 where L and B are length and breadth of tumor. Survival
study was continued till the last mouse was found live in all the groups. To test the effect of
neat liposomes without doxorubicin, 4T1ꢀtumor bearing mice were randomized into four
groups a) untreated, b) LCAꢀPC, c) DCAꢀPC, and d) CAꢀPC liposomes treated group, where
mice were treated with three doses of 20 mg/kg (phospholipid content) of liposomes on
alternate day; and tumor volumes and body weights were measured on alternate days.
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For toxicological studies, two groups (n = 3) of tumor (4T1) bearing mice (200 mm3) were
administered with a single intravenous dose (equivalent to 5 mg/kg of doxorubicin) of free
doxorubicin and doxorubicin entrapped LCAꢀPC liposomes respectively. Bleeds were taken
post 24 h treatment and plasma/serum samples were processed for estimation of cardiac
muscle specific toxicity marker (CKꢀMB), muscular toxicity marker lactate dehydrogenase
(LDH), and liver specific toxicity marker alkaline phosphatase (ALP). Estimations were
carried out at certified Newmax care pathological laboratory at New Delhi.
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Pharmacokinetics and bio-distribution studies: Tumor (4T1) bearing mice (200 mm3)
were randomized into three groups a) control group (n= 3 mice), b) doxorubicin treated
group (n = 3 per time point), c) doxorubicin entrapped LCAꢀPC liposomes treated group (n =
3 per time point). Mice in group b and c were administered with a single intravenous dose
(equivalent to 5 mg/kg of doxorubicin) of free doxorubicin and doxorubicin entrapped LCAꢀ
PC liposomes respectively. Serial bleeds were collected in ACDꢀmicro centrifuge tube
through orbital sinus under mild ketamineꢀxylazine anesthesia at 0.08, 0.25, 0.5, 1, 5, 10,
and 24 h after intravenous injection. Plasma was separated from samples and stored under
ꢀ80 ˚C until analysis. At time points of 1, 5, and 10 h; mice were sacrificed; and tumor, liver,
spleen, kidneys, lungs, and heart were removed after blood withdrawal, washed with ice
cold PBS, weighed, and stored in ꢀ80 ˚C until analysis.
We extracted drug content present in 100 ꢁL of plasma and 100 mg of respective tissue
samples using 500 ꢁL acidified alcohol (90% isopropanol with 0.75 N HCl). Tissue samples
with extracting solvent were placed in 2 mL homogenization tubes and homogenized with
zirconia beads by bead beating at 5000 rpm. Plasma samples were transferred to 2 mL
tubes and mixed with 500 ꢁL of acidified alcohol. Homogenized tissue samples and plasma
samples were stored in 4 ˚C overnight to extract the drug. Samples were then centrifuged at
14,000 rpm for 5 min at 4 ˚C. Supernatant liquid was transferred to fresh tube and solvent
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was completely evaporated using speedꢀvac. Final residue was dissolved in 200 ꢁL
acidified alcohol and transferred to 96ꢀwell plates. Analysis was performed
spectrofluorimetrically using Molecular devices M5 instrument with λex of 480 nm; and λem of
590 nm. A calibration curve was prepared for doxorubicin using concentrations from 5ꢀ5000
ng/mL in acidified alcohol. Final fluorescence values were obtained by subtracting
background fluorescence from each tissue type. A graph was plotted against concentration
of doxorubicin versus different time points for pharmacokinetics and concentration of
doxorubicin versus each tissue type at different time points for bioꢀdistribution studies.
Plasma drug concentration data was fitted and pharmacokinetic analysis were performed
using WinNonLin (Version 6.4).29
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Results and Discussion:
Design and synthesis of bile acid-phospholipids: Bile acids are interesting scaffolds for
biomaterials as their biocompatible nature and adaptability for bioconjugation chemistry can
be applied in biomedicine30 for drug delivery,31 as potential inhibitors,32 antimicrobial
agents,33 siRNA delivery,34 as hydrogels,35 and sensors.36 Natural bile acids differ from
each other in the number and stereochemistry of free hydroxyl groups present on the
concave side of the hydrophobic backbone.37 The membrane properties of liposomes
depend on the amphiphilic nature of bile acids and their unique structural features that can
impact the biomedical applications.38 Therefore, we synthesized bile acids with varying
number of hydroxyl groups for liposome preparation and explored their use for drug
delivery.
We selected three bile acids—LCA, DCA, and CA—and designed three bile acidꢀ
phosphocholine (BPC)ꢀderived phospholipids, where the phosphocholine head group was
appended to the 3'ꢀhydroxyl group of bile acids (Fig. 1). The C24ꢀcarboxyl group of bile acids
(4-6) was initially protected by benzyl esters39 (7-9) that were reacted with 2ꢀchloroꢀ1,3,2ꢀ
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dioxaphospholaneꢀ2ꢀoxide to produce unstable intermediates (10-12) and a triethylamine
hydrochloride (NEt3.HCl) salt (Scheme 1). The reaction mixture was filtered through celite
and immediately reacted with trimethylamine in an Ace pressure tube to generate
phospholipid conjugates (1-3).40 Purification of these conjugates by reverse phase C18 silica
combiflash column chromatography yielded 60ꢀ65% of the final phospholipids (1-3)
(Scheme1), which were characterized by 1HꢀNMR, 13CꢀNMR, 31PꢀNMR, and HRMS
(Supporting information).
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Liposome preparation and characterization: We first, determined the critical miceller
concentration (CMC) of the LCAꢀPC, DCAꢀPC and CAꢀPC using PyreneꢀFluorescence
based method.22 CMC of LCAꢀPC, DCAꢀPC and CAꢀPC were found to be 12, 22 and 51 µM
(Figure S1) suggesting that increase in number of hydroxyl groups enhances the CMC as
reported in earlier studies as well.41 We then tested the ability of BPCs to form selfꢀ
assembled aggregates without addition of any helper lipid. Transmission electron
microscopy (TEM) of the negativeꢀstained aggregates revealed their spherical nature of 90ꢀ
120 nm, where the LCAꢀPC aggregates were smaller in size than the formed by DCAꢀPC
and CAꢀPC (Fig. 2A-2C). Next, we used a mixture of BPC, EggPC, and DSPEꢀPEG 2000ꢀ
amine in a weight ratio of 1:1:0.2 to form liposomes, where the Egg PC and DSPEꢀPEG
2000ꢀamine promoted efficient encapsulation42 and stealthing of liposomes43, respectively.
Hydrated films of the lipid mixture were then subjected to 14 cycles of extrusion to obtain
desired liposomes. Quantification of phospholipid content analysis showed an average 73,
47, and 44 % retention of phospholipids after extrusion in case of LCAꢀPC, DCAꢀPC and
CAꢀPC liposomes respectively. Dynamic light scattering (DLS) studies showed a
monomodal size distribution of 100ꢀ110 nm (Fig. 2D-2G) with a polydispersity index of 0.1ꢀ
0.2 (Fig. 2H) and zeta potential ranging from 12ꢀ22 mV (Fig. 2I).
Drug encapsulation efficacy: Next, we tested the doxorubicin (a model water soluble
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anticancer drug) encapsulation ability of BPC liposomes using a remote drug loading
method,23 and observed ~58% drug encapsulation for LCAꢀPC and ~75% drug
encapsulation for both DCAꢀPC and CAꢀPC liposomes (Fig. 3A). The presence of free
hydroxyl groups increased the fluidity of DCAꢀPC and CAꢀPC liposomes, which allowed
them to entrap additional doxorubicin molecules in the lipid bilayer than the nonꢀ
hydroxylated LCAꢀPC liposomes. Estimation of the phospholipids retained by liposomes
revealed that LCAꢀPC liposomes had higher lipid content (73%) than DCAꢀPC and CAꢀPC
liposomes (~40%) because of better packing of LCAꢀPC in the membranes (Fig. 3B).
Therefore, the entrapment efficiency of DCAꢀPC and CAꢀPC liposomes was around 38%,
whereas it was ~18% for LCAꢀPC liposomes (Fig. 3C).
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Drug release kinetics: We investigated the release kinetics of doxorubicin from BPC
liposomes under the following conditions: (a) physiological pH using phosphate buffer saline
(PBS, pH 7.4), (b) acidic tumor microenvironment (acetate buffer, pH 5.0), and (c) blood
serum (10% fetal bovine serum (FBS) in PBS, pH 7.4) at 37 oC (Fig. 3D-3F; Fig. S2). The
initial burst release of doxorubicin was observed from all the three liposomes at pH 7.4, and
the cumulative doxorubicin release was below 50%. Although we observed a slight
increased rate of doxorubicin release at both pH 7.4 and pH 5.0, there were no distinct
differences in drug release among the three liposomes. Marked differences in the rates of
doxorubicin release in FBS were observed among these liposomes as serum proteins are
known to interact with liposomes and cause burst release of the encapsulated contents.44
We observed faster release rate and enhanced doxorubicin release content (~80%) from
the hydroxylated DCAꢀPC and CAꢀPC liposomes, whereas the LCAꢀPC liposomes
maintained a sustained release of doxorubicin with a maximum ~60% release of contents.
The Higuchi release rate constants45 with R2 > 90% confirmed the differential drug release
kinetics of BPC liposomes in blood serum conditions, and LCAꢀPC liposomes showed the
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slowest release of doxorubicin (Fig. 3G). We believe that the differential drug release
patterns of BPC liposomes might arise from the distinct packing of phospholipids in the
lipidated liposomes, which allows faster release of the loosely encapsulated doxorubicin
from DCAꢀPC and CAꢀPC liposomes. Therefore, we probed the effect of BPCs on
membrane fluidity using diphenylhexatriene (DPH)ꢀbased membrane probe.
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Membrane rigidity studies: The anisotropy of DPH, a rigid fluorophore, is reduced when it
transits from a tightly packed environment to fluidic conditions as perturbations in the
internal packing of membranes allow random movement of DPH.21 Anisotropy studies
revealed marked differences in the rigidity of hydroxylated and nonꢀhydroxylated
phospholipid liposomes (Fig. 3H). Membranes containing LCAꢀPC were highly rigid due to
better packing efficiency of LCAꢀPC lipids in membranes, whereas membranes containing
DCAꢀPC and CAꢀPC were comparatively fluid in nature. Rigid packing of LCAꢀPC causes
slow release of doxorubicin, whereas the fluidic nature of hydroxylated phospholipids (DCAꢀ
PC and CAꢀPC) induces enhanced encapsulation with faster drug release kinetics (Fig. 3H).
Therefore, distinct differences in membrane rigidity of lipidated liposomes that vary in
number of free hydroxyl groups are responsible for marked differences in encapsulation and
drug release from BPC liposomes.
In vitro cytotoxicity studies: We first tested the cytotoxicity of phospholipids and
compared with corresponding bile acids against murine (4T1) and human (MDAꢀMBꢀ231)
breast cancer cell lines (Fig. S3, Table S1). Among bile acids, LCA, DCA and CA;
hydrophilic CA is nonꢀtoxic to both the cells at concentrations tested whereas hydrophobic
LCA has IC50 of ~182 and 309 µM against 4T1 and MDAꢀMBꢀ231 cells respectively. DCA in
contrast was nonꢀtoxic to MDAꢀMBꢀ231 cells but has IC50 of ~261 µM against murine cell
lines. Phosphatidylcholine conjugation to bile acids make them more toxic for both the cells
where LCAꢀPC and DCAꢀPC has IC50 of ~73 and ~150 µM against 4T1 and MDAꢀMBꢀ231
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cells respectively; whereas CAꢀPC was toxic only to murine cancer cells. The toxic
concentrations of bile acid phospholipids are much higher than the concentrations used for
doxorubicin delivery.
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Anticancer activities of doxorubicinꢀencapsulated BPC liposomes were then studied in
murine (4T1) and human (MDAꢀMBꢀ231) breast cancer cell lines. Interestingly, we observed
that human MDAꢀMBꢀ231 cells were less susceptible to liposomal formulation as compared
to murine 4T1 breast cancer cells (Fig. 4, S3). As expected 48h exposure was more toxic to
the cells as compared to 24h exposure. After 24h of exposure to 4T1 murine cancer cells,
drug entrapped CAꢀPC liposomes were more toxic as compared to drug encapsulated LCAꢀ
PC liposomal formulations due to fast drug release from drugꢀentrapped CAꢀPC liposomes.
All drugꢀentrapped BPC liposomal formulations in general were found to induce cell death in
both the cell lines after 48 h, indicating effective release of doxorubicin from the liposomes
(Fig. 4).
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In vivo antitumor activities: We first tested the effect of neat LCAꢀPC, DCAꢀPC and CAꢀ
PC liposomes without Doxorubicin encapsulation on tumor regression, where mice were
treated with three doses of liposomes. As shown in Fig. S4, none of the liposomal
formulation induce any effect of tumor proliferation thereby concluding that these liposomal
formulations are safe and does not induce any cytotoxicity,
The frequency of chemotherapy schedules depends on the elicited toxicity, tumor
regression, and survival response of the patients. Frequent chemotherapeutic schedules
enhance tumor regression, albeit with high toxicity and poor survival. Therefore, to
understand the impact of BPC liposomes on anticancer activity, toxicity, and survival, we
evaluated the potency of doxorubicin encapsulated BPC liposomes against 4T1 murine
breast cancer model in Balb/c mice. Tumorꢀbearing animals were randomized into six
groups of nine animals each when the average tumor size reached a volume of ~75 mm3;
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the animals were administered with (a) 5% glucose (control group), (b) free doxorubicin, and
doxorubicin entrapped in (c) LCAꢀPC, (d) DCAꢀPC, (e) CAꢀPC; and f) AdrisomeTM
liposomes at an equivalent doxorubicin dose of 5 mg/kg for three alternate days. We
observed a ~40ꢀ50% reduction in tumor volume upon treatment with doxorubicin loaded
BPC liposomes as compared to untreated control whereas AdrisomeTM treated mice did not
show any progression of tumor growth (Fig 5A). Drug release from the liposomal
formulations impacts the tumor regression, toxicity, and survival of the mice as tumor
regression with enhanced toxicity of poor survival is not recommended. We therefore,
compared the mice survivability of tumor bearing mice on different treatments. Mice treated
with doxorubicin alone without any carrier could not enhance the mice survivability due to its
toxic nature. We observed a differential impact on mice survival on treatment with drug
encapsulated liposomes irrespective of the differences in body weights of the drugꢀtreated
mice (Fig 5B). Mice treated with LCAꢀPC liposomes showed significant improvement in
survival than that observed with DCAꢀPC and CAꢀPC liposomes (Fig. 5C) whereas mice
treated with drugꢀloaded CAꢀPC liposomes died before the untreated tumorꢀbearing mice in
spite of 50% tumor regression due to the high toxicity of the free drug present in the blood
circulation. We observed the similar effect during in vitro studies where doxorubicinꢀ
entrapped CAꢀPC liposomes are more toxic to murine cancer cells as compared to drugꢀ
entrapped LCAꢀPC liposomes. Therefore, rigid LCAꢀPC liposomes triggered a significant
increase in mice survival compared to the poor survival rate of the DCAꢀPCꢀ and CAꢀPCꢀ
liposomes treated mice. The unꢀentrapped doxorubicin treated mice showed sudden weight
loss and decline in survival.
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To investigate this, we estimated and compared the levels of lactate dehydrogenase (LDH;
muscle tissue marker), cardiac muscleꢀspecific creatinine phosphokinase (CKꢀMB), and
alkaline phosphatase (ALP for liver toxicity) in mice serum after 24 h of treatment with free
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doxorubicin and doxorubicinꢀentrapped LCAꢀPC liposomes. We observed a significant
decrease in ALP levels (Fig. 5D); and an increase in circulatory levels of LDH (Fig. 5E) and
CKꢀMB (Fig. 5F) upon doxorubicin treatment, whereas the doxorubicinꢀentrapped LCAꢀPC
liposomes did not elicit any toxic response in the tumorꢀbearing mice. Higher circulatory
levels of these markers in mice treated with free doxorubicin could possibly explain the
cardiac toxicity and loss in muscle mass of these animals.
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Pharmacokinetics and bio-distribution studies: To estimate the doxorubicin
concentration in plasma and other body organs, we performed pharmacokinetic and bioꢀ
distribution studies in 4T1 tumor bearing Balb/c mice that were randomized into three
groups: (a) untreated control (n = 3), (b) free doxorubicin (n = 3 per time point), and (c)
doxorubicinꢀentrapped LCAꢀPC liposomes (n = 3 per time point). Mice in groups (b) and (c)
were treated with equivalent doses of doxorubicin (5 mg/kg) and pharmacokinetic
parameters were estimated by the twoꢀcompartment single intravenous bolus model. We
observed ~1.5ꢀfold increase in plasma concentration of Doxorubicin on treatment with drug
entrapped LCAꢀPC liposomes as compared to doxorubicin alone (Fig. 5G, 5H) with similar
mean resident time (MRT) for LCAꢀPC liposomes and doxorubicin. There was ~1.5ꢀfold
increase in drug availability using doxorubicin entrapped liposomes as compared to
doxorubicin alone (Fig. 5H).
Bioꢀdistribution studies revealed a threeꢀfold increase in doxorubicin concentration at the
tumor site upon treatment with LCAꢀPC liposomes than that observed with doxorubicin
alone (Fig. 5I-5K). We observed negligible doxorubicin concentration in vital organs such as
the heart and lungs. Treatment with doxorubicinꢀentrapped LCAꢀPC liposomes lowered the
accumulation of doxorubicin in the kidneys, implying enhanced blood circulation compared
to that observed upon treatment with free doxorubicin (Fig. 5I-5K).
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Conclusions: In summary, we developed three bile acidꢀphosphocholineꢀderived
phospholipids that differ in the number of free hydroxyl groups. Drug encapsulation and
release studies revealed that increased fluidity of membranes enhanced drug encapsulation
efficacy of liposomes. The highly fluid liposomes allowed burst release of drugs, whereas
the rigid liposomes maintained a sustained release of drugs. In vivo studies in murine
models confirmed that survival of tumorꢀbearing mice is contingent on the drug release
kinetics, where the hydrophobic liposomes caused tumor regression with increased survival
rates. Therefore, this study provides new insights into designing of bile acidꢀderived
lipidated liposomes, which will assist in the development of future cancer therapies.
Acknowledgements: We thank RCB and NII for intramural funding, and Department of
Biotechnology (DBT) for supporting this project. VS, NM, SP, and SK thank RCB, UGC and
CSIR for research fellowship. We thank Dr. Aniruddha Sengupta and Mr. Mallik Samarla for
helping us in analyzing pharmacokinetic data.
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Author contributions: AB conceived idea. VS and AB designed the experiments. VS
synthesized molecules. VS and NM carried out liposomal drug encapsulation and release
studies. SP and VS carried out in vivo studies. SK performed biophysical studies. SS
supervised the animal studies. VS and AB wrote manuscript.
Supporting information: Synthesis of Bile acidꢀphospholipids, cytotoxicity of bile acids
1
and Bile acidꢀPhospholipids, CMC of amphiphiles, Drug release studies till 48h, HꢀNMR,
13CꢀNMR, 31PꢀNMR and HRMS of final compounds is available in supporting information.
References:
1) Torchillin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev.
Drug. Discov. 2005, 4, 145ꢀ160.
2) Chang, H.; Yeh, M. Clinical development of liposomeꢀbased drugs: formulation,
characterization, and therapeutic efficacy. Int. J. Nanomed. 2012, 7, 49–60.
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1
2
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5
6
3) Sharma, A.; Sharma, U. S. Liposomes in drug delivery: Progress and limitations. Int. J.
Pharm. 1997, 154, 123ꢀ140.
7
8
9
4) Alavizadeh, S. H.; Badiee, A., Golmohammadzadeh, S.; Jaafari, M. R. The influence of
phospholipid on the physicochemical properties and antiꢀtumor efficacy of liposomes
encapsulating cisplatin in mice bearing C26 colon carcinoma. Int. J. Pharm. 2014, 473,
326ꢀ333.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5) Oussoren, C.; Eling, W. M.; Crommelin, D. J.; Storm, G.; Zuidema, J. The influence of
the route of administration and liposome composition on the potential of liposomes to
protect tissue against local toxicity of two antitumor drugs. Biochim. Biophys. Acta.
1998, 1369, 159ꢀ172
6) Mayer, L. D.; Tai, L. C.; Ko, D. S.; Masin, D.; Ginsberg, R. S.; Cullis, P. R.; Bally, M. B.
Influence of vesicle size, lipid composition, and drugꢀtoꢀlipid ratio on the biological
activity of liposomal doxorubicin in mice. Cancer Res. 1989, 49, 5922ꢀ5930.
7) Johnston, M. J.; Edwards, K.; Karlsson, G.; Cullis, P. R. Influence of drugꢀtoꢀlipid ratio
on drug release properties and liposome integrity in liposomal doxorubicin formulations.
J. Liposome Res. 2008, 18, 145ꢀ157.
8) Bhargava, P.; Singh, M.; Sreekanth, V.; Bajaj, A. Nature of the charged headgroup
determines the fusogenic potential and membrane properties of lithocholic acid
phospholipids J. Phys. Chem. B 2014, 118, 9341–9348.
9) Bhattacharya, S.; Dileep, P.V. Membraneꢀforming properties of cationic lipids bearing
oxyethyleneꢀbased linkages. J. Phys. Chem. B, 2003, 107, 3719–3725
10) Kumar, S.; Bhargava, P.; Sreekanth, V.; Bajaj, A. Design, synthesis, and physicoꢀ
chemical interactions of bile acid derived dimeric phospholipid amphiphiles with model
membranes. J. Colloid Interface. Sci. 2015, 448, 398ꢀ406.
19
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1
2
3
4
5
6
7
8
11) Huang, Z.; Szoka, Jr. F. C. Sterolꢀmodified phospholipids: Cholesterol and phospholipid
chimeras with improved biomembrane properties. J. Am. Chem. Soc. 2008, 130,
15702–15712.
9
12) Huang, Z.; Jaffari, M. R.; Szoka, Jr. F. C. Disterolphospholipids: nonexchangeable
lipids and their application to liposomal drug delivery. Angew. Chem. Int. Ed. 2009, 48,
4146–4149.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13) Vares, L.; Koulov, A. V.; Smith, B. D. Highly impermeable vesicles composed of
conformationally restricted phosphatidylethanolamine. Chem. Commun. 2002, 14, 1482ꢀ
1483.
14) Wang, D.; Tu, C.; Su, Y.; Zhang, C.; Greiser, U.; Zhu, X.; Yana, D.; Wang, W.
Supramolecularly engineered phospholipids constructed by nucleobase molecular
recognition: upgraded generation of phospholipids for drug delivery. Chem. Sci. 2015, 6,
3775ꢀ3787.
15) Zhou, C. Y.; Wua, H.; Devaraj, N. K. Rapid access to phospholipid analogs using thiolꢀ
yne chemistry. Chem. Sci. 2015, 6, 4365–4372.
16) Bhattacharya, S.; Biswas, J. Understanding membranes through the molecular design
of lipids. Langmuir, 2010, 26, 4642ꢀ4654.
17) Shimanouchi, T.; Ishii, H.; Yoshimoto, N.; Umakoshi, H.; Kuboi, R. Calcein permeation
across phosphatidylcholine bilayer membrane: Effects of membrane fluidity, liposome
size, and immobilization. Colloids Surf. B Biointerfaces. 2009, 73, 156ꢀ160.
18) Kawano, K.; Onose, E.; Hattori, Y.; Maitani, Y. Higher liposomal membrane fluidity
enhances the in vitro antitumor activity of folateꢀtargeted liposomal mitoxantrone. Mol
Pharm. 2009, 6, 98ꢀ104.
19) Dévédec, F. L.; Fuentealba, D.; Strandman, S.; Bohne, C.; Zhu, X. X. Aggregation
behavior of pegylated bile acid derivatives. Langmuir, 2012, 28, 13431ꢀ13440.
20
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Molecular Pharmaceutics
1
2
3
4
5
6
7
8
20) Dévédec, F. L.; Strandman, S.; Hildgen, P.; Leclair, G.; Zhu, X. X. PEGylated bile acids
for use in drug delivery systems: enhanced solubility and bioavailability of Itraconazole.
Mol. Pharm. 2013, 10, 3057ꢀ3066.
9
21) Sreekanth, V.; Bajaj, A. Number of free hydroxyl groups on bile acid phospholipids
determines the fluidity and hydration of model membranes. J. Phys. Chem. B 2013, 117,
12135ꢀ12144.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22) Basu Ray, G.; Chakraborty, I.; Moulik, S. P.; Pyrene absorption can be convenient
method for probing critical miceller concentration (cmc) and indexing miceller polarity, J.
Colloid Interface Sci. 2006, 294, 248ꢀ254.
23) Bolotin, E. M.; Cohen, R.; Bar, L. K.; Emanuel, N.; Ninio, S.; Lasic, D. D.; Barenholz, Y.
Ammonium sulfate gradients for efficient and stable remote loading of amphipathic weak
bases into liposomes and ligandoliposomes. J. Liposome Res. 1994, 4, 455ꢀ479.
24) Amselem, S.; Cohen, R.; Druckmann, S.; Gabizon, A.; Goren, D.; Abra, R. M.; Huang,
A.; New, R.; Barenholz, Y. Preparation and characterization of liposomal doxorubicin for
human use. J. Liposome Res. 1992, 2, 93ꢀ123.
25) Sterwart, J. C. M. Colorimetric determination of phospholipids with ammonium
ferrothiocyanate. Anal. Biochem. 1980, 104, 10ꢀ14.
26) Hua, S. Comparison of in vitro dialysis release methods of loperamide encapsulated
liposomal gel for topical drug delivery. Int. J. Nanomedicine. 2014, 9, 735ꢀ744.
27) Siepmanna, J.; Peppas, N. A. Higuchi equation: derivation, applications, use and
misuse. Int. J. Pharm. 2011, 418, 6ꢀ12.
28) Sengupta, P.; Basu, S.; Soni, S.; Pandey, A.; Roy, B.; Oh, M. S.; Chin, K. T.; Paraskar,
A. S.; Sarangi, S.; Connor, Y.; Sabbisetti, V. S.; Kopparam, J.; Kulkarni, A.; Mutoc, K.;
Amarasiriwardena, C.; Jayawardene, I.; Lupoli, N.; Dinulescu, D. M.; Bonventre, J. V.;
Mashelkar, R. A.; Sengupta, S. Cholesterolꢀtethered platinum IIꢀbased supramolecular
21
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2
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5
6
nanoparticle increases antitumor efficacy and reduces nephrotoxicity. Proc Natl Acad.
Sci. USA 2012, 109, 11294ꢀ11299.
7
8
9
29) Nders, C. K.; Adamo, B.; Karginova, O.; Deal, A. M.; Rawal, S.; Darr, D.; and
Schorzman, A. Pharmacokinetics and efficacy of PEGylated liposomal doxorubicin in an
intracranial model of breast cancer, PLOS One, 2013, 8, e61359.
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30) Nonappa; Maitra, U. Unlocking the potential of bile acids in synthesis,
supramolecular/materials chemistry and nanoscience. Org. Biomol. Chem. 2008, 6, 657ꢀ
669.
31) Luo, J.; Xiao, K.; Li, Y.; Lee, J. S.; Shi, L.; Tan, Y. H.; Xing, L.; Cheng, H. R.; Liu, G. Y.;
Lam, K. S. Wellꢀdefined, sizeꢀtunable, multifunctional micelles for efficient paclitaxel
delivery for cancer treatment. Bioconjugate Chem. 2010, 21, 1216ꢀ1224.
32) Park, J.; AlꢀHilal, T. A.; Jeong, J. H.; Choi, J. U.; Byun, Y. Design, synthesis, and
therapeutic evaluation of Poly(acrylic acid)ꢀtetraDOCA conjugate as bile acid transporter
inhibitor. Bioconjugate Chem. 2015, 26, 1597ꢀ1605.
33) Lai, X.; Feng, Y.; Pollard, J.; Chin, J. N.; Rybak, M. J.; Bucki, R.; Epand, R. F.; Spand,
R. M.; Savage, P. B. Ceragenins: Cholic acidꢀbased mimics of antimicrobial peptides.
Acc. Chem. Res. 2008, 41, 1233ꢀ1240.
34) Amjad, M. W.; Amin, M. C.; Katas, H.; Butt, A. M.; Kesharwani, P.; Iyer, A. K. In Vivo
antitumor activity of folateꢀconjugated cholic acidꢀpolyethylenimine micelles for the coꢀ
delivery of doxorubicin and siRNA to colorectal adenocarcinomas. Mol. Pharm. 2015,12,
4247ꢀ4258.
35) Mukhopadhyay, S.; Maitra, U,; Krishnamoorthy, G.; Schmidt, J.; Talmon, Y. Structure
ad dynamics of a molecular hydrogel derived from a tripodal cholamide, J. Am. Chem.
Soc. 2004, 126, 15905ꢀ15914.
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36) Khatri, V. K.; Chahar, M.; Pavani, K.; Pandey, P. S. Bile acidꢀbased cyclic
bisbenzimidazolium receptors for anion recognition:ꢄ highly improved receptors for
fluoride and chloride ions. J. Org. Chem., 2007, 72, 10224ꢀ10226.
9
37) Mukhopadhyay, S.; Maitra, U. Chemistry and biology of bile acids, Curr. Sci. 2004, 87,
1666ꢀ1683.
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55
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38) Singh, M.; Singh, A.; Kundu, S.; Bansal, S.; Bajaj, A. Deciphering the role of charge,
hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile
acid based facial amphiphiles. Biochim. Biophys. Acta 2013, 1828, 1926ꢀ1937.
39) Qian, S.; Wu, JꢀB.; Wu, XꢀC.; Li, J.; Wu Y. Synthesis and characterization of new liver
targeting 5ꢀfluorouracilꢀcholic acid conjugates. Arch. Pharm. Chem. Life, Sc. 2009, 342,
513ꢀ520.
40) Sandbhor, M.S.; Key, J. A.; Strelkov, I. S.; Cairo, C. W. A Modular synthesis of alkynylꢀ
phosphocholine head groups for labeling sphingomyelin and phosphatidylcholine. J.
Org. Chem. 2009, 74, 8669ꢀ8674.
41) Monte, M. J.; Marin, J. JG.; Antelo, A.; Vazquezꢀtato, J. Bile acids: Chemistry,
physiology, and pathophysiology, World J. Gastroenterol. 2009, 15, 804ꢀ816.
42) Harasym T. O.; Cullis, P. R; Bally, M. B. Intratumor distribution of doxorubicin following
i.v. administration of drug encapsulated in egg phosphatidylcholine/cholesterol
liposomes. Cancer Chemother Pharmacol. 1997, 40, 309ꢀ317.
43) Palvai, S.; More, P.; Mapara, N.; Basu, S. Chimeric nanoparticle: A platform for
simultaneous targeting of phosphatidylinositolꢀ3ꢀkinase signaling and damaging DNA in
cancer cells, ACS Appl. Mater. Interfaces, 2015, 7, 18327ꢀ18335.
44) Agarwal, K.; Bali, A.; Gupta, C. M. Influence of the phospholipid structure on the
stability of liposomes in serum. Biochim. Biophys. Acta 1986, 856, 36ꢀ40.
23
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45) Paul, D. R. Elaborations on the Higuchi model for drug delivery. Int. J. Pharm. 2001,
418, 13ꢀ17.
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Figure 1. Design of BPCs: Molecular structures and schematic presentation of Lithocholic
Acid (LCA), Deoxycholic Acid (DCA), and Cholic Acid (CA) derived phospholipids LCA-PC,
DCA-PC and CA-PC; and schematic representation depicting the impact of membrane
packing on encapsulation efficacy and drug release kinetics of bile acidꢀphospholipid
derived liposomes;
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Figure 2: Liposome formation and characterization: A-C) TEM images of neat LCAꢀPC
(A), DCAꢀPC (B), and CAꢀPC (C) liposomes; D-F) Representative dynamic light scattering
graphs of BPC liposomes developed from BPC:EggPC:DSPEꢀPEG using LCAꢀPC (D),
DCAꢀPC (E), and CAꢀPC (F); G-I) Average hydrodynamic diameter (G), Polydispersity index
(H), and Zeta potential (I) of LCAꢀPC, DCAꢀPC, and CAꢀPC liposomes from three
independent experiments.
Figure 3: Doxorubicin encapsulation and biophysical characterization: A-C)
Doxorubicin encapsulation efficiency (A), percentage of phospholipid retained (B),
entrapment efficiency (C) of BPC liposomes suggesting high entrapment efficacy of
hydroxylated DCAꢀPC and CAꢀPC liposomes; D-F) Doxorubicin release from BPC
liposomes against PBS, pH = 7.4 (D), PBS, pH = 5.0 (E), 10% FBS (F); and G) Average
Higuchi release rate constants from two independent experiments with R2 > 90% of BPC
liposomes in different release media implying slow release of doxorubicin from LCAꢀPC
liposomes; H) DPHꢀbased membrane rigidity of BPC liposomes revealing hydrophobic
nature of LCAꢀPC liposomes.
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Figure 4: In vitro activities: In vitro cytotoxicity of doxorubicin entrapped BPC liposomes:
LCAꢀPC, DCAꢀPC, and CAꢀPC against murine (4T1) (A, B) and human (MDAꢀMBꢀ231) (C,
D) breast cancer cell lines after 24 (A, C) and 48h (B, D).
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Figure 5: In vivo activities: A) Change in tumor volume, B) body weight, and C) survival of
4T1 tumor bearing Balb/c mice treated with doxorubicin and doxorubicin entrapped BPC
liposomes; D-F) Hepatic alkaline phosphatase (ALP) (D); skeletal muscle lactate
dehydrogenase (LDH) (E); and cardiac muscle specific toxicity marker (CKꢀMB) (F) toxicity
markers post 24h of treatment with doxorubicin and doxorubicin encapsulated LCAꢀPC
liposomes in 4T1 tumor bearing mice; G-H) Pharmacokinetic profile (G) and analysis (H) of
doxorubicin and doxorubicin entrapped LCAꢀPC liposomes in 4T1 tumor bearing Balb/c
mice; I-K) Bioꢀdistribution of doxorubicin and doxorubicin entrapped LCAꢀPC liposomes in
different organs in 4T1 tumor bearing Balb/c mice after 1h (I), 5h (J), and 10 h (K) of dosing.
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