Cisplatin-Stitched Polysaccharide Vesicles for Synergistic Cancer Therapy of Triple Antagonistic Drugs

Article abstract of DOI:10.1021/acs.biomac.6b01411

New cisplatin-stitched polysaccharide vesicular nanocarrier is developed for combination therapy of three clinical important antagonistic drugs together to accomplish synergistic cancer therapy in breast cancer treatment. Carboxylic functionalized dextran was tailor-made for the chemical conjugation of cisplatin, and a renewable hydrophobic unit was anchored in the backbone to interdigitize the chains to self-assemble as cisplatin-stitched polysaccharide nanovesicles. Water-soluble DNA-intercalating drug doxorubicin·HCl (DOX) and water insoluble topoisomerase type I inhibitor drug camptothecin (CPT) were encapsulated in these vesicles to produce dual or triple drug-loaded vesicular nanocarrier. This unique cisplatin, DOX and CPT triple drug-loaded dextran vesicles were stable in aqueous medium, and the vesicular geometry acted as a shield for Pt-polymer drug conjugate against glutathione (GSH) detoxification under physiological conditions. Lysosomal enzymes ruptured the nanovesicle exclusively at the intracellular compartments to deliver the combination of all three drugs simultaneously to maximize the therapeutic efficacies. In vitro cytotoxicity studies revealed that free cisplatin was highly detoxified by the GSH in breast cancer cells, whereas the enhanced stability of Pt-stitched dextran vesicle against GSH facilitated ～99% cell killing in breast cancer cells. Combination therapy studies revealed that the free cisplatin, DOX, and CPT were found to be antagonistic to each other. Dual drug-loaded vesicles exhibited synergistic cancer cell killing while delivering these antagonistic drugs from a dextran vesicular platform. Remarkable synergistic cell killing was accomplished in cisplatin, DOX, and CPT triple drug-loaded vesicles at nanogram concentrations in breast cancer cells. The internalization of drugs and cellular uptake were confirmed by confocal microscope and flow cytometry analysis. The drugs were taken by the cancer cells in large amounts while delivering them from dextran vesicles compared to their free form. These spectacular results opened new opportunities for synergistic cancer therapy for GSH-overexpressed breast cancer using triple drug-loaded polysaccharide vesicular nanocarriers.

Full text of DOI:10.1021/acs.biomac.6b01411

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Article
Cisplatin-Stitched Polysaccharide Vesicles for
Synergistic Cancer Therapy of Triple Antagonistic Drugs
Nilesh Umakant Deshpande, and Manickam Jayakannan
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01411 • Publication Date (Web): 22 Nov 2016
Downloaded from http://pubs.acs.org on November 24, 2016
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Cisplatin-Stitched Polysaccharide Vesicles for
Synergistic Cancer Therapy of Triple
Antagonistic Drugs
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1
Nilesh Umakant Deshpande and Manickam Jayakannan*
Department of Chemistry
Indian Institute of Science Education and Research (IISER)ꢀPune
Dr. Homi Bhabha Road
Puneꢀ411008, Maharashtra, India
Keywords: Polymer vesicles, Combination Therapy, Synergistic effect, Ptꢀdrug delivery and
Polysaccharides.
Abstract
New cisplatinꢀstitched polysaccharide vesicular nanoꢀcarrier is developed for combination
therapy of three clinical important antagonistic drugs together to accomplish synergistic
cancer therapy in breast cancer treatment. Carboxylic functionalized dextran was tailorꢀmade
for the chemical conjugation of cisplatin and renewable hydrophobic unit was anchored in the
backbone to interꢀdigitize the chains to selfꢀassemble as cisplatinꢀstitched polysaccharide
nanovesicles. Water soluble DNAꢀintercalating drug doxorubicin.HCl (DOX) and water
insoluble topoisomerase type I inhibitor drug camptothecin (CPT) were encapsulated in these
vesicles to produce dual or triple drug loaded vesicular nanoꢀcarrier. This unique cisplatin,
DOX and CPT triple drugs loaded dextran vesicles were stable in aqueous medium and the
vesicular geometry acted as a shield for Ptꢀpolymer drug conjugate against glutathione (GSH)
detoxification under physiological conditions. Lysosomal enzymes ruptured the nanoꢀvesicle
exclusively at the intracellular compartments to deliver the combination of all three drugs
simultaneously to maximize the therapeutic efficacies. In vitro cytotoxicity studies revealed
that free cisplatin was highly detoxified by the GSH in breast cancer cells whereas the
1
Corresponding Author: jayakannan@iiserpune.ac.in
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enhanced stability of Ptꢀstitched dextran vesicle against GSH facilitated ~ 99 % cell killing in
breast cancer cells. Combination therapy studies revealed that the free cisplatin, DOX, and
CPT were found to be antagonistic to each others. Dual drug loaded vesicles exhibited
synergistic cancer cell killing while delivering these antagonistic drugs from dextran
vesicular platform. Remarkable synergistic cell killing was accomplished in cisplatin, DOX
and CPT triple drug loaded vesicles at nanogram concentrations in breast cancer cells. The
internalization of drugs and cellular uptake were confirmed by confocal microscope and flow
cytometry analysis. The drugs were taken by the cancer cells in large amounts while
delivering them from dextran vesicles compared to their free form. This spectacular results
opened new opportunity for synergistic cancer therapy for GSHꢀover expressed breast cancer
using triple drug loaded polysaccharide vesicular nanoꢀcarriers.
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Introduction
Combination therapies are emerging as important treatment protocol to overcome the
Ptꢀdrug resistance in breast and ovarian cancers at the second and third levels of
1,2
chemotherapy to improve the patient compliance and survival against metastasis. The
inherent ability of breast cancer tissues to develop resistance against Ptꢀdrugs directly
associated with various biochemical processes including glutathione (GSH) over expression,
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poor uptake of the anticancer drugs, and DNA autoꢀrepair mechanism and so on so forth.
Breast (also ovarian) cancer tissues are estimated to be over expressed with glutathione
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ꢀ5
(GSH, 20 mM) compared to normal tissues (1.0 mM); as a result, the highly reactive Ptꢀ
drugs were readily detoxified by the GSH through the formation of stable PtꢀS bond and
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excreted from the cytoplasm prior to their action at the nucleus. The absence or low
expression of CTR1 protein in the cell membrane was also responsible for the poor
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transportation of Ptꢀdrug. DNA autoꢀrepairing process at the PtꢀGuanineꢀN position by NER
proteins was also accounted for the cell proliferation and increasing the chances of
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metastasis. To overcome some of these limitations, combination therapy were developed for
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cisplatin (CP) along with DNA specific drugs such as doxorubicin (DOX)
and
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paclitaxel,
etc. The cellular uptakes of the Ptꢀdrugs were improved using small molecular
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11ꢀ13,22ꢀ35
nanoꢀvectors
and polymeric nanoꢀcarriers
to accumulate them at the cancer
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tissues via enhanced permeability and retention (EPR) effect.
Polymer nanovesicles
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(
or polymersomes) and liposomes are excellent choice for this purpose since they are
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capable of loading both water insoluble and water soluble anticancer drugs simultaneously in
the hydrophobic layer and hydrophilic core, respectively. Cisplatin loaded liposomes were
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developed to enhance the membrane protein trafficking, efficacy in triple negative breast
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cancer tumors, and in glioma cells. It was found that cisplatin encapsulation induced usual
rigidity in the liposome layers; however, they were successfully taken up by the cancer cells
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compared to free drug. Cell penetrating peptide anhored liposomes were also developed for
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loading and delivering of DOX in brain glioma in animals.
Few attempts were also made
on the coꢀdelivery of cisplatin with other anticancer drugs to accomplish combination
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therapy.
Synthetic polymer vesicles are excellent nanoꢀcarriers with enhanced stability in
the blood plasma and also has structural feasibility for chemical conjugation of drugs such as
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cisplatin in cancer treatment. However, there is no effort has been taken to deliver cisplatin
along with other anticancer drugs in synthetic polymer vesicles. Thus, by designing polymer
vesicular nanoꢀscaffold with appropriate provision for cisplatin conjugation would facilitate
the coꢀdelivery of clinical important multiple anticancer drugs together from single polymer
dose. Further, the combination therapy of anticancer drugs encounter several challenges prior
to their application in clinical studies and few of these important problems are: (i) Ptꢀdrug
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detoxification by GSH in the cytoplasm,
(ii) antagonism of cisplatin drug towards DNA
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intercalating drugs like DOX, (iii) lack of appropriate nanoꢀcarrier design for chemical
conjugation of Ptꢀdrug along with the encapsulation of water soluble and insoluble drugs, etc.
To solve this multiꢀtask problem; it is necessary to design and develop new biodegradable
and biocompatible polymer nanoꢀcarriers having competence to overcome GSH
detoxification, provision for cisplatin conjugation and ability to load multiple anticancer
drugs of different solubility parameters. Among the polymer structures, polysaccharides are
excellent candidates due to their biocompatibility, ability to interact with the cell membrane
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receptors, and also provide opportunity for chemical modification and so on so forth. Thus,
in the present investigation polysaccharideꢀdextran is chosen and the new triple drug
delivering polysaccharide nanoꢀvesicle concept is shown in figure 1.
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Figure 1. A novel cisplatin-stitched polysaccharide vesicular nano-carrier design for the
synergistic combination therapy of antagonistic drugs: cisplatin, doxorubicin.HCl and
camptothecin against GSH detoxification in breast cancer treatment.
To accomplish this goal, here we created new “cisplatin-stitched polysaccharide
vesicle” nanoꢀcarrier and demonstrated the combination therapy of triple antagonistic drugs
together from single nanoꢀscaffold to accomplish synergistic breast cancer treatment. The
new carboxylic polysaccharide vesicular design was conceived based on our previous
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experience in designing pH and enzymeꢀresponsive
dextran vesicles in drug delivery
applications. Earlier studies from our laboratory using caveolae knock out (ꢀ) and caveolae
(+) cell models revealed that these dextran vesicles are unique in transporting anticancer
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5
drugs in caveolae (ꢀ) cells similar to that of breast cancer cells. Thus, the selection of
dextran vesicular design is appropriate choice for exploring the triple drug delivery concept
to improve the efficacies of drugꢀresistance breast cancer cells. Polysaccharide nanoparticles
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(not vesicles) based on hyaluronic acid;
heparin, alginate and modified dextran
were explored for cisplatin conjugation; however, up to our knowledge, no effort has been
made to explore polysaccharide vesicles for the triple drug combination therapy of anticancer
drugs. In the present investigation, a new class of carboxylic dextran vesicle was tailor made
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using renewable resource 3ꢀpentadecyl phenol (produced from cashew nut shell liquid) as
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vesicle director and succinic acid unit was introduced for cisplatin chemical conjugation.
These cisplatinꢀstitched vesicles were capable of encapsulating water soluble DOX.HCl and
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water insoluble CPT to obtains cisplatin, DOX and CPT triple drug loaded polymer nanoꢀ
vesicle. In vitro drug release kinetics confirmed that the cisplatin drug is preserved in the
active form in the dextran vesicular scaffold against the GSH detoxification. These drug
loaded vesicles were stable at extracellular conditions and they exclusively biodegraded by
lysosomal enzymes at the intracellular compartments to release all three drugs. In breast
cancer cells, the free cisplatin drug became ineffective due to GSH detoxification whereas the
cisplatin loaded vesicle VCP exhibited very good cell killing. Combination therapies were
designed for the cocktail of free drugs, cocktail of individual drug loaded vesicles and dual or
triple drug loaded vesicles. It was found that the free drugs were antagonistic to each other
whereas the dual or triple drug loaded vesicles exhibited excellent synergistic combination
therapy in breast cancer cells. Confocal microscopic imaging and flow cytometry analysis
were employed to study the drug internalization and accumulation at the perinuclear
environment. The present investigation opens new polysaccharide vesicular concept for the
combination therapy of antagonistic drugs together to accomplish synergistic breast cancer
treatment.
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Experimental Section
Meterials: Dextran (M
(DCC), 4ꢀdimethylamino pyridine, 3ꢀpentadecylphenol (PDP), triethylamine, 20ꢀ(S)ꢀ
camptothecin (CPT), doxorubicin hydrochloride (DOX), RhodaminꢀB,
w
= 6,000), succinic anhydride, dicyclohexylcarbodiimide
cisdiaminedichloroplatinum (II) (cisplatin, CP), silver nitrate, glutathione (GSH) and horse
liver esterase enzyme were purchased from Aldrich chemicals. Wildꢀtype mouse embryonic
fibroblasts (WtꢀMEFs), and human breast cancer cells (MCFꢀ7) were maintained at 37 °C
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under a 5% CO
2
atmosphere as described earlier. All other instrumentation details, cell
viability assay, confocal microscope imaging, flow cytometry analysis procedures are given
in the supporting information. Compound (1) in the synthetic scheme was synthesized using
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the procedure from our earlier report.
Synthesis of 4-(tert-butoxy)-4-oxobutanoic acid (2) : Succinic anhydride (5.0 g, 50 mmol),
Nꢀhydroxy succinimide (1.72 g, 15 mmol), triethylamine (2.0 mL, 15 mmol), tertiary butanol
(8.0 mL,80 mmol) and DMAP (0.62 g, 6 mmol) were dissolved in 50 mL of dry toulene and
refluxed for 24 h. Then solution was cooled and diluted with ethyl acetate (50 ml), washed
with 10 % citric acid (2X50 ml), brine and finally dried over anhydrous Na SO and
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concentrate to give brown oil. The product was purified passing over silica (100ꢀ200 mesh)
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column using 10% ethyl acetate:pet ether as a eluent. Yield = 2.80 (30 %). HꢀNMR (400
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MHz, d
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ꢀDMSO) δ: 2.56 (m, 4H), 1.36 (s, 9H). CꢀNMR (100 MHz, CDCl
3
) δ 178.80 (C=O
acid), 171.44 (C=O ester), 81.52 (tert. carbon), 30.20, 28.90, 27.56 (primary carbons). FTꢀIR
KBr, cm−1), 3380 (carboxyl –OH Stretch), 2990, 2854 (aliphatic C−H stretch), 1762 (ester
C=O stretch), 1208 (C(=O)ꢀO stretch). MALDI TOFꢀTOF, (MW: 174.09), m/z = 213 (M +
K+). HRꢀMS (ESI+): m/z [M + H+], calculated for C [M+]: 175.09; found: 175.09.
(
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14 4
H O
w
Synthesis of DEX-PDP-C20 tert. butyl ester: Dextran (M = 6,000, 1.0 g, 6.2 mmol of
anhydroglucose unit), PDP acid (0.54 g, 1.50 mmol) and tert. butyl succinate (0.56 g, 3.22
mmol) were dissolved in anhydrous DMSO (25.0 mL) and the solution was purged with dry
nitrogen. To the above content DMAP (0.106 g, 0.87 mmol) in anhydrous DMSO (3.0 mL)
and DCC (1.20 g, 5.81 mmol) in anhydrous DMSO (3.0 mL) were added. The reaction
mixture was stirred for 24 hour at room temperature. The solution was filtered to remove
dicyclohexyl urea and the solvent was removed under reduced pressure. The thick viscous
liquid was precipitated by adding into acetone (100 mL). The solid was filtered and washed
several times with acetone. It was dissolved again in DMSO and the purification via the
precipitation technique was done at least twice. The product filtered out and dried under
o
1
vacuum at 60 C to get yellowish white solid as product. Yield = 45 %. HꢀNMR (400 MHz,
ꢀDMSO ) δ: 7.12 ppm (s, 1H, ArꢀH ), 6.72ppm (m, 3H, ArꢀH), 4.47, 4.82, 4.88 ppm (s,
d
6
hydroxyl of dextran) 4.63 ppm (s, dextran anomeric proton), 3.14ꢀ3.69ppm (dextran
glucosidic protons), 2.6 ppm (m,4H) methylene protons of SA, 2.49 ppm (2H, ArꢀCH2),
1
13
.48ppm (2H, ArꢀCH
2
ꢀCH
CꢀNMR (100 MHz, d6ꢀDMSO) δ: 178.2 (ester C=O), 171.41 (tert butylester C=O),170.92
(ester C=O), 157.60, 143.97, 129.00, 121.19 (Arꢀ5C), 98.10 (dextran anomeric Carbon),
9.86 (tertiary carbon of tertiary butyl group),73.21,71.13,70.25,69.99,65.95 (dextran
glucosidic carbons),65.46 (ꢀO=CꢀCH ), 35.09, 31.23, 30.99, 22.58, 13.27(PDP aliphatic
2
),1.34ppm (s, 9H) tert. butyl, 1.18ꢀ0.80ppm (aliphatic protons).
7
2
carbons), 28.90, 28.66, 27.60 (aliphatic carbons of tertiary butyl succinate).
A similar procedure was followed to make DEXꢀPDPꢀC5 tert. butyl ester , DEXꢀPDPꢀ
C10 tert. butyl ester and the details are given in supporting infromation.
Deprotection of DEX-PDP-C20 tert. butyl ester: 0.5 g (1.57 mmol) of DEXꢀPDPꢀC20 was
dissolve in 4ml of dried DMSO to which 0.39 mL (5.04 mmol) of TFA was added and stirred
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for 4 h. After removing TFA on rota vap, the polymer was further purified by dialyzing
against distilled water using dialysis membrane (MWCO 3500) for 24 h, and then sample was
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lyophilized to get brownish colored powder. Yield = 72 %. HꢀNMR (400 MHz, d
6
DMSO)
δ: 7.12 ppm (s, 1H, ArꢀH ), 6.72 ppm (m, 3H, ArꢀH), 4.47, 4.82, 4.88 ppm (s, hydroxyl of
dextran) 4.63 ppm (s, dextran anomeric proton), 3.14ꢀ3.69ppm (dextran glucosidic protons),
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2
.6 ppm (m,4H) methylene protons of SA, 2.49ppm (2H, ArꢀCH
2
), 1.48 ppm (2H, ArꢀCH
2
ꢀ
13
CH
2
), 1.18ꢀ0.80 ppm (Aliphatic protons). CꢀNMR (100 MHz, d ꢀDMSO) δ: 176.2 (ester
6
C=O),169.41 (tert.butylester C=O),170.92 (ester C=O), 155.60, 145.97,130.00, 121.32 (Arꢀ
C), 98.20 (dextran anomeric Carbon), 73.26, 71.58, 70.85, 69.29, 65.66 (dextran glucosidic
carbons), 65.48 (ꢀO=CꢀCH ), 35.23, 31.58, 30.70, 22.58, 13.80 (PDP aliphatic carbons),
8.74 and 28.31 (aliphatic carbons of tertiary butyl succinate). A similar procedure was
5
2
2
followed to deprotect DEXꢀPDPꢀC5 tert. butyl ester and DEXꢀPDPꢀC10 tert. butyl ester and
the details are given in supporting information.
2+
3 2 2 2
Preparation of aquated cisplatin [Pt (NH ) (OH ) ] : The aquated cisplatin was
3
5
synthesized following the reported procedure. For the synthesis of aquated cisplatin, 20 mg
0.066 mmol, 1equiv) of cisplatin was dissolved in 20.0 mL of H O. To this solution, 22.5
(
2
mg (0.132 mmol, 2 equiv) of silver nitrate was added with continuous stirring at 37 °C for 24
h. The resulting milky white silver chloride precipitate confirmed the aquated cisplatin
formation. The precipitate was removed by centrifuging at 10,000 rpm for an hr followed by
filtration using a 0.45 ꢁm filter. The obtained solution was then lyophilized to get light green
colored powder which was stored at 4 °C.
Synthesis of polymer-cisplatin Conjugate: The procedure for cisplatin conjugation to
DEXꢀPDPꢀC20 is explained in detail. DEXꢀPDPꢀC20 (20 mg) polymer was dissolved in 4
o
mL NaOH (1 mg / mL) solution and left for stirring at 37 C for 30 minutes. This was
followed by addition of the aquated cisplatin (3.4 mg, 0.012 mmol) to the prepared activated
o
polymer solution and stirred it for another 48 hours at 37 C in the dark. The solution was
dialyzed (MWCO=3500) against distilled water for 2 days with periodically changing water
to remove the unꢀencapsulated molecules. The solution from the dialysis bag was filtered
through 0.4 µm filter, lyophilized and stored at 4 °C. FTꢀIR (KBr, cm−1): 3300, 2920, 2880,
1660, 1560, 1395, 1360, 1090, 1050, 930, 830 and 548.
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A similar procedure was followed to make cisplatin stitched DEXꢀPDPꢀC5 and DEXꢀPDPꢀ
C10 derivatives. The drug loading efficiency (DLE) and drug loading content (DLC) were
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determined by using OPD colorimetric assay from the equations as reported earlier .
Preparation of Drug encapsulated Dextran nanovesicles: Typically 0.2 mg of the drug
and 10 mg of DEXꢀPDPꢀC20 polymer were dissolved in 1ml of DMSO and 1ml of milliꢀq
water was added slowly for 30min. The resulting solution was stirred for 12h and then
extensively dialysed (MWCO:3500) against milliq water to remove any unencapsulated drug
molecule. Dialysed solution was then filtered through 0.4µm filter,lyophilised and used for
the further experiments. To determine drug loading content and encapsulation efficiency, 1
mg of freezeꢀdried drug loaded polymer was dissolved in 1.0 mL of DMSO. A total of 100
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L of this solution was diluted to 3.0 mL with DMSO and the absorbance at 480 nm was
measured on UV−visible spectrophotometer. The amount of drug encapsulated in the vesicles
was determined using the respective molar extinction coefficients of the drugs. Molar
Extinction coefficient for doxorubicin and Camptothecin in water are 11500 and 11250 L
ꢀ1
ꢀ1
47
mol cm respectively for the determination of DLC and DLE. For making double and
triple loaded samples a similar procedure was followed.
In Vitro Drug release studies for Cisplatin: Cisplatin loaded vesicles were taken in a
dialysis bag (in 3 mL) and they were immersed in 100 ml beaker and dialyzed at 37 °C with
constant stirring. At specific time intervals, 1.0 ml of the dialysate was withdrawn and
replaced with an equal volume of fresh buffer (or) saline. The amount of molecule (or drug)
released in each aliquot was measured using OPD colorimetric assay by using absorption
35
spectroscopy to quantify their percentage of cumulative release. Cumulative release (%) =
th
C
n
x V
o
/ m x 100, where C
n
is amount of loaded cargo in n sample, V
o
is total volume and m
is total amount loaded in vesicles. For esterase assisted release studies 10 units of enzyme
was used, above mentioned procedure was followed for calculation of cumulative release.
In Vitro Drug Release Studies for DOX.HCL and CPT: Dialysis method was adopted to
monitor the in virto release profile of DOX and CPT from DEXꢀPDPꢀ20 nanoꢀvesicles.
Briefly,3.0 mg DEXꢀPDPꢀ20/ DEXꢀPDPꢀC20ꢀCP vesicles encapsulated with DOX.HCL or
CPT was dispersed in 3.0 mL of PBS and placed in a dialysis tube. The tube was dipped in
100 mL of phosphate buffer saline maintained at pH 7.4 were taken in a beaker and incubated
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at 37 °C. At predetermined time intervals, 3.0 mL of solution was taken out and replaced with
equal volume of fresh PBS. Absorbance of the each of the solution taken out has been
measured and amount of the DOX.HCl and CPT released were calculated using Beer’s law.
For esterase assisted drug release studies, the same protocol was followed with the addition
of 10 U esterase enzyme into the dialysis bag.
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Results and Discussion
Dextran-Cisplatin Vesicular Conjugates
Amphiphilic dextran was synthesized using renewable hydrophobic carboxylic
functionalized 3ꢀpentadecyl phenol (PDP) derivative (1) and mono tꢀbutyl ester of succinic
acid (SA) (2) as shown in Figure 2. The compounds 1 and 2 were substituted in the dextran
backbone via aliphatic ester linkage so that these chemical linkages could be readily cleaved
3
5,56
at the intracellular level by lysosomal enzymes to deliver the drugs.
The deꢀprotection of
succinic tꢀbutyl ester yielded amphiphilic dextran having hydrophobic PDP unit along with
carboxylic functional groups, DEXꢀPDPꢀCx, where Cx represents the amount of carboxylic
units. The substitution of the PDP and SA units on the dextran backbone was confirmed by
1
13
HꢀNMR, CꢀNMR and 2D NMR HSQC experiments and these details are given supporting
information (see SFꢀ1 to SFꢀ3). The PDPꢀacid (1) was fixed and the amount of SAꢀderivative
(2) was varied to produce different amount of carboxylic units in the dextran backbone. From
1
the HꢀNMR spectra, the degree of substitution in dextran was estimated as 5 % of PDP
(fixed in all samples) and the SA contents were varied as 5, 10 and 20 %. These samples were
named as DEXꢀPDPꢀC5, DEXꢀPDPꢀC10 and DEXꢀPDPꢀC20. At higher PDP (> 5 %) and SA
contents (> 20 %); the dextran samples were found to be insoluble or partially soluble in
water and not useful for drug delivery. Additionally two dextran derivatives were also
prepared having 5 % PDP alone (named DEXꢀPDP) or 20 % SA alone (named as DEXꢀC20)
to study the role of the substitution on the selfꢀassembly of dextran derivatives (see scheme
Sꢀ1 and figures SFꢀ4 and SFꢀ5). The modified dextran was found to be thermally stable up to
250 °C (see SFꢀ6) and they were also found to be amorphous (see SFꢀ7). The cisꢀdiamineꢀ
diaquoꢀplatinum (II) complex was produced by treating cisplatin with AgNO in MilliꢀQ
3
3
5
water and removed the resultant AgCl precipitate by filtration (see Figure 2). The cisplatin
aqua complex was reacted with sodium salt of DEXꢀPDPꢀCꢀX (X= 5, 10 and 20) in deionised
water for 48 h in dark. The ratio of cisplatin aqua complex to carboxylic acid groups in DEXꢀ
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PDPꢀCx was kept as 1.0: 1.1 (in mole %) to ensure the complete Ptꢀdrug conjugation in the
dextran derivative. The resultant dextranꢀcisplatin conjugate was filtered through 0.45 µm
o
filter and lyophilized and stored at 4 C under dark. The cisplatin conjugation at the dextran
carboxylic unit was confirmed FTꢀIR analysis (for details, see SFꢀ8). The drug loading
content (DLC) and the drug loading efficiencies (DLE) of the polymerꢀcisplatin conjugates
were determined using orthophenylenediamine colorimetric assay (OPDꢀassay, see SFꢀ9).
The DLC for dextranꢀcisplatin conjugates were obtained as 3.5 %, 5.2 % and 9.3 % for DEXꢀ
PDPꢀC5, DEXꢀPDPꢀC10, DEXꢀPDPꢀC20, respectively (DLEs are obtained as 46 %, 61 %
and 55 % for DEXꢀPDPꢀC5, DEXꢀPDPꢀC10, DEXꢀPDPꢀC20, respectively). Statically, two
carboxylic acid units can be coordinated at each Ptꢀcentre; thus, the DLC values indicate that
almost all the carboxylic acids are coordinated to the cisplatin residues.
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Figure 2. Synthesis of PDP and carboxylic substituted amphiphilic dextran derivatives and
cisplatin conjugation to produce Pt-stitched polysaccharide vesicles.
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Figure 3. DLS histograms of DEX-PDP-C5 (a), DEX-PDP-C20 (b), and cisplatin conjugated
DEX-PDP-C20 (c). AFM images of DEX-PDP-C5 (d), DEX-PDP-C20 (e), and cisplatin
conjugated DEX-PDP-C20 (f). FESEM image of DEX-PDP-C20 (g), HR-TEM image of
DEX-PDP-C20 (h), and HR-TEM image of cisplatin conjugated DEX-PDP-C20 (i). The
photographs of vials showed the dextran vesicles. Concentration of the samples was
maintained as 0.1mg/mL for DLS and 0.05mg/ml for AFM, FESEM and TEM imaging.
To study the selfꢀassembly of the dextran derivatives, the polymer samples were
dissolved in DMSO + water and subjected to dialysis using semiꢀpermeable membrane with
MWCO = 3000 g/mol. The dextran samples DEXꢀPDPꢀC5 and DEXꢀPDPꢀC20 were found to
produce stable aqueous clear polymer solution (see the figures 3a and 3b). Dynamic light
scattering (DLS) analysis of these aqueous polymer solution exhibited monoꢀmodel
distribution with good autocorrelation with respect to the formation of uniform size
distribution of nanoꢀaggregates of diameter < 200 nm (see Figures 3a and 3b). The cisplatin
stitched DEXꢀPDPꢀC20 sample also showed monoꢀmodal distribution with 180 nm size
nanoꢀobjects indicating that the cisplatin conjugation did not alter the size of the nanoꢀ
assemblies in water (see Figure 3c). AFM is a very powerful tool to differentiate the
57
polymers vesicles from other nanoꢀobjects. AFM images of DEXꢀPDPꢀC5 and DEXꢀPDPꢀ
C20 are shown in Figures 3d and 3e (see SFꢀ10 for DEXꢀPDPꢀC10). The PDP and
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carboxylic substituted dextran samples showed donutꢀshaped images with respect to the
formation of vesicles. The tendency for the vesicular scaffold formation was not disturbed by
the various amounts of SA units from 5 to 20 %. The dextranꢀcisplatin conjugate DEXꢀPDPꢀ
C20 also exhibited the formation of vesicles confirming that the new dextran design is very
unique in preserving the vesicular geometry even after the chemical conjugation of drugs (see
Figure 3f). The height profiles of the images showed typical two hump profiles with respect
to the vesicles (see Figures 3d to 3f). The average sizes of the vesicles were calculated from
AFM images and they were determined to be 160 ± 10 nm; 170 ± 10 nm and 180 ± 10 nm for
DEXꢀPDPꢀC5; DEXꢀPDPꢀC20 and cisplatin conjugated DEXꢀPDPꢀC20, respectively. AFM
images SA alone substituted dextran derivative DEXꢀC20 (without PDP) was found to be
nanoparticle rather than vesicles (see SFꢀ11). AFM images of PDP alone substituted dextran
sample DEXꢀPDP (without SA unit) exhibited vesicular geometry (see SFꢀ12). This trend
clearly supports that the introduction of the PDP units is very much crucial for the formation
of carboxylic dextran vesicles as shown in figure 2. During the cisplatin conjugation, the
smaller succinic acid units present in the hydrophobic layer behave as anionic ligands to
stitch the cisplatin aquoꢀcomplex without disturbing the vesicular geometry (see Figure 3c).
The FESEM image for DEXꢀPDPꢀC20 showed the formation of spherical nano objects of
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170 ± 20 nm (see figure 3g). HRꢀTEM image of DEXꢀPDPꢀC20 sample showed formation of
spherical nanoꢀvesicles with distinct layer and core and directly evident for the formation of
5
5
vesicular assemblies (see figure 3h). HRꢀTEM image of cisplatin conjugated of DEXꢀPDPꢀ
C20 sample also showed spherical shapes and dark contrast for the presence of cisplatin
metalꢀparticles (see figure 3i). Based on the DLS studies and morphological characterization
by AFM, FESEM and HRTEM; it can be conformed that the newly designed carboxylic
dextran vesicles are very unique in producing vesicular geometry and retaining its shape and
size even after the cisplatin conjugation. The DEXꢀPDPꢀC20 has highest amount of
carboxylic acid substitution and it also showed higher DLC values for cisplatin content
compare to all other samples. Thus for all further discussion, the cisplatin conjugated dextran
vesicle DEXꢀPDPꢀC20 is used and it is referred as VCP (Vꢀ vesicle and CPꢀ cisplatin).
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Figure 4. DLS histograms, AFM morphology and photographs of the vials of drug loaded
vesicles VDOX (a), VCPT (b), VDOX-CPT (c), VCP-DOX (d), and VCP-CPT (e). (f) Absorption and
emission spectra of DOX loaded vesicles (λex = 490 nm). Concentration of the samples was
maintained as 0.1mg/mL for DLS and 0.05mg/ml for AFM imaging.
The polymer vesicular assemblies are very unique nanoꢀstructures that they can
encapsulate water soluble dye like RhodaminꢀB in the inner hydrophilic cavity unlike other
48
nanoꢀobjects such as nanoparticles or micelles. The photographs of vials in SFꢀ13 clearly
indicate that the dextran vesicles DEXꢀPDPꢀCx could able to load both RhꢀB whereas the
DEXꢀSA (without PDA substitution) nanoparticles did not load RhꢀB. Further, the vesicles
DEXꢀPDPꢀCx were also found to load water insoluble dye Nile red in the hydrophobic layer
(see SFꢀ13). The size of the RhꢀB loaded DEXꢀPDPꢀC20 showed monoꢀmodel distribution
with diameters < 200 nm and its AFM images confirmed the retaining of the vesicular
geometries (see figure SFꢀ13). Among all the samples the DEXꢀPDPꢀC20 seems to be
excellent candidates for loading both water soluble and insoluble dyes; thus, it was chosen for
further studies. As evident from the loading capabilities of dextran vesicular structures for
both water soluble and water insoluble molecules; two anticancer drugs with different
solubility are chosen for encapsulation. Water insoluble drug camptothecin (CPT, typeꢀI
topoisomerase inhibitor) and water soluble drug doxorubicin.HCl (DOX.HCl, typeꢀII
topoisomerase inhibitor and DNA intercalating agent) were chosen to load in DEXꢀPDPꢀC20
vesicle. These vesicles are referred as VDOX and VCPT for DOX and CPT loading,
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respectively. Dual drug loaded vesicles VCPꢀDOX and VCPꢀCPT were prepared by encapsulating
DOX and CPT in VCP. Dual drug loaded VDOXꢀCPT was prepared by loading DOX and CPT
together in DEXꢀPDPꢀC20. The DLCs of individual drug loaded vesicles VDOX and VCPT
were obtained as 1.2 % and 0.8 %, respectively (DLEs = 68 % and 43 % for DOX and CPT).
DLCs of dual drug loaded vesicles VCPꢀDOX were obtained as 9.0 % and 1.3 % for CP and
DOX, respectively (DLE = 54 % and 73 % for CP and DOX); VCPꢀCPT were obtained as 9.5 %
and 0.6 % for CP and CPT, respectively (DLE = 56 % and 43 % for CP and CPT); and VDOXꢀ
CPT were obtained as 1.8 % and 0.5 % for DOX and CPT, respectively (DLE = 65 % and 43
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for DOX and CPT). From the DLC values, cisplatin to DOX ratio was obtained as 7.0 :
1.0; cisplatin to CPT ratio as 16 : 1.0; and DOX to CPT as 3.6 : 1.0 in dual loaded dextran
vesicles. Since DOX and CPT are luminescent drugs, the drug loaded vesicles were subjected
to photophysical studies (see Figure 4f). Absorbance and emission spectra of VDOX and VCPꢀ
were almost identical whereas VDOXꢀCPT exhibited features of both CPT and DOX
DOX
chromophores. The photographs of vials containing VDOX and VCPꢀDOX exhibited redꢀcolor
and VDOXꢀCPT showed magenta color with respective to their photophyical characteristics.
Similarly the vesicular samples VCPT and VCPꢀCPT were also exhibited CPT absorption and
emission spectra (see SFꢀ14). DLS histograms of these drug loaded samples showed the
formation of vesicles of < 150 nm (see figure 4). The dual drug loaded VDOXꢀCPT did not
exhibit any energy transfer process from CPT to DOX upon excitation at the CPT confirming
that the optical purity of drugs were retained in the vesicular scaffold (see SFꢀ14). AFM
images confirmed their vesicular geometry with respect to the donut shaped spherical objects.
This revealed that the custom designed carboxylic dextran vesicles are unique classes nanoꢀ
carriers: (i) it has ability to conjugate cisplatin drug, (ii) capable of loading water soluble drug
DOX.HCl, (iii) able to encapsulate water insoluble drug CPT, and (iv) it also exhibited
excellent capability of dual drug loading of these three drugs in a single vesicularꢀcarrier.
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GSH Detoxification and Enzyme responsiveness
Ptꢀbased proꢀdrugs encounter three important challenges under the physiological
conditions as shown in figure 5a: (i) undesirable cleavage of PtꢀOOC bond by the chloride
and other ions present in the plasma or saline, (ii) Ptꢀdrug detoxification by GSH, and (iii)
lysosomal enzymeꢀresponsiveness to release the Ptꢀdrugs. The in vitro stability of the
cisplatinꢀpolymer drug conjugates were tested in phosphate buffer saline (PBS) and
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water at 37
°C by dialysis method. The cisplatin is conjugated at the polymer
backbone through PtꢀOOCꢀpolymer bond which is susceptible to cleave by ions such
ꢀ
2ꢀ
4
as Cl in saline (or PO
in PBS) to produce free cisplatin drug (destabilization of
68
ꢀ
drug). The extracellular conditions estimated to have [Cl ] = 100 mM which is much
ꢀ
6
higher compared to the intracellular environment, [Cl ] = 3 ꢀ 20 mM. Hence, it is
ꢀ
important to test the stability of VCP in saline ([Cl ] = 154 mM) which is the regular
carrier medium for the intracellular administration of drugs in chemotherapy. To test
this process, the polymerꢀdrug conjugate was dispersed in saline (also in PBS) and it
was subjected to incubation in semiꢀpermeable membrane of MWCO= 3500 g/mol.
Cisplatin aliquots were collected in the reservoir and subjected to
OPD) colorimetric assay to estimate the cumulative drug release. The cisplatin
stitched dextran vesicles VCP showed > 90 % stability both in saline and water at 37
oꢀphenylenediamine
(
°
C
(
see figure 5b). In PBS, the dextranꢀcisplatin conjugates were found to break < 20 %
ꢀ
60
4
of the cisplatin due to dechelation by phosphate ions (PO ) in the PBS. This study
confirmed that the VCP preserved the Ptꢀpolymer conjugation in saline for intravenous
administration against the attack of the ions. Reaction of GSH with cisplatin is known to
produce the S–Pt (see Figure 5a) bond that could be monitored by absorption
6
,35
spectroscopy.
Both free cisplatin and oxaliplatin were employed as references to study the
GSH reaction with ptꢀresidues. Oxaliplatin is represents the small molecular prodrug counter
part for polymerꢀcisplatin conjugates. The GSH reaction with dextranꢀcisplatin conjugate (or
with cisplatin and oxaliplatin) was carried out in 0.1 mM TrisꢀHCl buffer by incubating at 37
3
5
°C for 12 h under dark. The UV–Vis absorbance spectra of the reaction product of free
cisplatin with GSH are shown in figure 5c (for oxaliplatin and VCP, see SFꢀ15). Both
cisplatin and oxaliplatin reacted with GSH and produced a new absorbance peak at 260 nm
with respect to the formation of the Pt–S bond. Interestingly, the reaction product of GSH
with VCP vesicles showed negligible absorption at 260 nm for PtꢀS bond (see figure SFꢀ15).
The extent of the reaction of GSH with cisplatin, oxaliplatin and VCP were determined form
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absorbance spectra and plotted in figure 5c as inset. These plots showed that both free
cisplatin and oxliplatin readily detoxified by GSH and more than 95 % of the drugs became
inactive. On the other hand, the dextranꢀcisplatin conjugate VCP was protected from
detoxification against GSH and only < 20 % drug was detoxified. This experiment is clearly
evident for the unique capability of dextran vesicular structure for shielding the cisplatinꢀ
polymer conjugate from the GSH detoxification under physiological conditions.
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Figure 5. (a) Schematic representation of fate of dextran vesicle in the extracellular and
intercellular level. (b) Cumulative release of cisplatin from VCP in PBS (pH= 7.4), saline
(
pH= 6.9), distilled water (pH = 7.0) at 37
PBS (pH= 7.4) at 37 C. (c) Absorbance spectra of reaction product of cisplatin with 10 mM
GSH in tris-buffer (pH = 7.4) at 37 C. The figure in the in-set showed the plot of extent of
reaction of free cisplatin and oxaliplatin and VCP over time. (d) Cumulative drug release of
DOX and VCPT in PBS (pH= 7.4) at 37 C in the absence and in the presence of esterase
enzyme 10 U. (e) Cumulative drug release of dual loaded VCP-DOX and VCP-CPT in PBS (pH=
.4) at 37 C in the absence and in the presence of esterase enzyme 10 U. Concentration of
°C and in the presence of esterase 10 U in PBS
°
°
V
°
7
°
the free cisplatin and VCP used for GSH detoxification is 30 µg. Concentration of the
doxorubicin and Camptothecin used for enzyme responsive release experiments 42 ± 10 µg
and 24 ± 10 µg, respectively.
The PDP and SA units in the dextran vesicle were connected via aliphatic ester
linkages in the dextran backbone. Aliphatic ester linkages are biodegradable in enzyme rich
57,35
lysosomal compartment;
thus, the enzymeꢀresponsiveness of dextran vesicles in releasing
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drugs was investigated in pH = 7.4 at 37 °C. The in vitro kinetics were performed both in the
presence of 10 U of esterase enzyme and in the absence of enzyme and their release profiles
are shown in figures 5b, 5d and 5e. The amount of the cisplatin released was monitored by
OPD colorimetric assay and the release of DOX and CPT were estimated directly by
measuring their absorption. In figure 5b, the dextranꢀcisplatin conjugate exhibited good
stability and exhibited ~ 20 % leaching in PBS. Up on exposure to esterase enzyme, the
vesicles were ruptured to release the cisplatin drug more than 70 % (see Figure 5b). A similar
trend was observed for DOX and CPT release from VDOX and VCPT in PBS (~ 30 %) and more
than 85 % release in the presence of enzyme (see figure 5d). The DOX release from the dual
loaded vesicle VCPꢀDOX (see figure 5e) seems to be same as that of the VDOX indicating that
the reputing of dextran vesicles by the enzyme followed almost identical kinetics in both
dextran vesicles and well as dextranꢀcisplatin vesicles. The release for the CPT trend from
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CPT and VCPꢀCPT (see figures 5e) was also appeared to be almost identical to that of the DOX
release in the presence of esterase enzyme. Based on the above in vitro release studies; it may
be concluded that the enzymatic release of the physically loaded drugs like CPT and DOX as
well as the chemically conjugated cisplatin followed identical release kinetics for single and
dual drug loaded forms. This observation proves that these dextran vesicles can be cleaved
exclusively in the presence of enzyme to release the drugs at the intracellular compartments.
Combination Therapy and Synergistic Effect
Two cell lines having large difference in the GSH content were chosen to test
ability of the cisplatin conjugated dextran vesicles against GSH detoxification. Breast
cancer cells (MCF 7) have 20 times overꢀexpression of GSH (in 20 mM) compared to
wildꢀtype mouse embryonic fibroblasts (WTꢀMEFs, normal cells, [GSH] = 0.5 to 1.0
4ꢀ5
mM). The cytotoxicity of the nascent dextran vesicles were checked in WTꢀMEF
and MCF 7 cell lines and the data exhibits > 95 % cell viability up to 80 µg/ mL (see
SFꢀ16). Cytotoxicity of free cisplatin, DOX and CPT drugs and their drug loaded
vesicles
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CP, VDOX and VCPT were tested and the data is shown in Figures 6a to 6f. In
CP was found to be less toxic (< 50 %) compared
WTꢀMEF (normal cell) cell line, the
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to the free cisplatin that exhibited > 90 % cell killing (see Figure 6a). Similarly VDOX
and VCPT were also found to be relatively less toxic (< 60 %) to normal cells compared to
their free drugs (see Figures 6b and 6c). This is an important observation since vesicle
loaded drugs are less toxic to normal cells which is more desirable for cancer therapy. In
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breast cancer cell line (MCF 7), free cisplatin drug was highly detoxified by GSH
content in the cytoplasm and only < 50 % killing was achieved. On the other hand, VCP
was very stable against GSH attack and this property facilitated > 95 % cell growth
inhibition killing by
VCP with IC50 at 1.0 ꢀg/ mL. Similarly, the VDOX and VCPT also
exhibited better cell killing in MCFꢀ7 while delivering them from dextran vesicles compared
to their free form. These studies emphasized the need for the encapsulation of DOX and CPT
and also the chemical conjugation of cisplatin in the dextran vesicles for higher cell growth
inhibition in breast cancer cells.
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Figure 6: Histograms depicting the cytotoxicity of free drugs along with VCP (a), VDOX (b) ,
VCPT (c) in Wt-MEF cells and cytotoxicity of VCP (d), VDOX (e), VCPT (f) in MCF 7 cells.
To study the dextran vesicles in cisplatin combination therapy; the dual drug loaded
samples VCPꢀDOX, VCPꢀCPT and VDOXꢀCPT were employed. The cytotoxicity experiments were
tested in cisplatin resistant human breast cancer MCFꢀ7 cells. Cytotoxicity studies for the
dual loaded vesicles i.e. VCPꢀDOX, VCPꢀCPT and VDOXꢀCPT were carried out at various
concentrations and the data is summarized in Figures 7a to 7f. Since, the weight ratios of the
drugs in the dual drug loaded vesicles have inherent compositions of 7.0 : 1.0 for CP vs
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DOX; 16 : 1.0 for CP vs CPT ; as 3.6 : 1.0 for and DOX vs CPT; a similar drug ratios were
retained for the cocktails of individual drug loaded vesicles and for free drugs. The
combination drug treatment was tested for the dual loaded vesicles along with cocktail of
individual drug loaded vesicles and free drugs and the data is shown in figures 7a to 7f. The
comparison of data in figures 6a and 6d revealed that IC50 was accomplished by dual loaded
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CPꢀDOX much better compared to their cocktails VDOX + VCP and also that of free CP+DOX.
Similar results were obtained for the VCPꢀCPT versus VCPT + VCP (see Figures 7b and 7e) and
DOXꢀCPT versus VDOX+VCPT (see Figures 7c and 7f). The results obtained from the
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cytotoxicity data clearly revealed that the dual loaded vesicles better in exhibiting cell killing
compared to their cocktails.
Figure 7. Histograms depicting the cytotoxicity of cocktails of free drugs and dual drug
loaded vesicles for CP and DOX (a), CP and CPT (b) and DOX and CPT (c) in MCF 7 cell
line. Histograms depicting the cytotoxicity of cocktails of individual drug loaded vesicles for
CP and DOX (d), CP and CPT (e) and DOX and CPT (f) in MCF 7 cell line.
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Figure 8. Combination index (CI) plotted against the IC values for free drugs, cocktails of
X
the individually loaded vesicles and dual loaded vesicles for cisplatin and DOX system (a),
cisplatin and CPT system (b) and DOX and CPT system (c). Triangle models are showing the
synergistic, antagonistic and additive effects in the combination therapy of free drugs (d),
individual drug loaded vesicles (e) and dual drug loaded vesicles (f).
To analyse the synergism of the drug action in the combination delivery of two drugs,
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1 2 1 2
the following formula was employed: CI = D /Dm1 + D /Dm2, where, D and D are the
concentrations of drug 1 and drug 2 that in combination produce a certain level of
cytotoxicity, while Dm and Dm are the concentrations of the single drugs, administered
1
2
separately, which produce the same effect. CI < 1.0 indicate synergism, CI =1.0 indicate an
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additive effect, and CI values > 1.0 indicate antagonism. In Figures 8a to 8c, the plots
provide quantitative information about the extent of drug interactions. From the plot, it is
very clear that the dual loaded VCPꢀDOX exhibited values well below 1.0 suggesting the
synergistic effect in MCFꢀ7 cell line (see Figure 8a). On the other hand, the cocktails of free
drugs and cocktails of individual drug loaded vesicles VDOX+VCP showed antagonism (see
Figure 8b). In case of VCPꢀCPT, the dual loaded system showed additive effect whereas its
cocktails exhibited antagonism. In the combination of DOX and CPT, the dual loaded VDOXꢀ
and individual drug loaded vesicles (VDOX + VCPT) exhibited synergistic effect compared
CPT
the antagonistic effect of cocktail of the free drugs (see Figure 8c). For better clarity and easy
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understanding; a triangle diagram is drawn to summarize the synergistic, additive and
antagonise effect of three important clinical drugs in combination therapy. The following
conclusion may be drawn from the diagrams. All three drugs cisplatin, DOX and CPT were
antagonistic to each other while administrating them in free forms (see Figure 8d). The
cocktails of individual drug loaded vesicles exhibited diverse results: for example,
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antagonistic effect for VCP + VDOX; additive effect for VCP +VCPT; and synergistic for VDOX
+
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CPT (see figure 8e). Exceptionally, both DOX loaded dual vesicles VCPꢀDOX and VDOXꢀCPT
showed synergist effect in combination therapy (see Figure 8f). The dual drug loaded vesicle
of VCPꢀCPT still showed additive effect and need additional improvement. Hence, the custom
dextran vesicles are unique classes of nanoꢀcarriers for accomplishing synergistic cancer
therapy of clinical antagonistic drugs cisplatin plus DOX and DOX plus CPT for breast
cancer treatment.
Cellular uptake and Flow Cytometry
The cellular uptake studies were done by confocal microscopic imaging and flow
cytometry. Since the cisplatin is a nonꢀfluorescent drug, cellular uptake studies were done for
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CPT,
DOX, VCPꢀDOX, V VCPꢀCPT and VDOXꢀCPT in MCF 7 cells. The red fluorescence from
doxorubicin at 520 nm was monitored through the red channel (λ = 568 nm) and the blue
fluorescence produced by the cell nuclei by CPT or by DAPI staining (λ = 461 nm)
depending upon the requirement. The images corresponding to free DOX and VDOX and VCPꢀ
DOX are shown in figure 9a. The merged image corresponding to the free DOX in the first
panel clearly indicates that the free DOX accumulation in the nucleus is relatively less. On
the other hand, the VDOX taken up cells at much higher drug concentration and the DOX was
accumulated both in the cytoplasm and at the nucleus. The imaged images appeared as
magenta colour corresponding to perfect accumulation of DOX at the nucleus long with
DAPI. In order to rule out the background signal from the cells in red and blue channels;
controls cells were exposed to the red and blue channels using parameters employed for drug
loaded samples. These control did not show any significant background emission (See SFꢀ
18); thus, coming that the images red and blue images were observed as the result of drugs
accumulated in the cytoplasm and nuclei. The cellular uptake of VDOX and VCPꢀDOX were
found to be almost identical which suggesting that the DOX release was not disturbed by the
cisplatinꢀstitching. The cellular internalisations for free CPT and VCPT were done (nucleus
was not stained with DAPI). CPT is blue luminescent drug and the images in Figure 9b and
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9c showed that the CPT accumulation at the nucleus better while delivering it from dextran
vesicles. Similar results were obtained for VCPꢀCPT (see SFꢀ17). Further, the CLSM images of
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DOXꢀCPT in figure 9d clearly showed the CPT and DOX emission in the blue and red
channels and the merged image showed magenta colour corresponding mixed emission.
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Figure 9. CLSM images of MCF 7 cells incubated with free DOX, VDOX and VCPꢀDOX (a);
and free CPT (b), VCPT (c), and VDOXꢀCPT (d) at 37°C. For each panel, the images from the left
to right show differential interference contrast (DIC), staining of cell nuclei by DAPI, DOX
fluorescence in the cells, and an overlay of the three images, in case of free CPT and VCPT
nuclei is not stained with DAPI. Flow cytometry plots for control, free DOX , VDOX and
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CP0DOX (e) in MCF 7 cell line after 9 h incubation (DOX concentration = 2 ꢁg/ mL and
10000 cells used for measurements).
To further confirm the cellular uptake of the vesicles, flow cytometry analysis
was carried out for free DOX and DOX loaded vesicles VDOX and VCPꢀDOX (see figure
9e). The histograms clearly indicate that the DOX internalization by VDOX and VCPꢀDOX
is twice and 1.5 times better than the free DOX in MCFꢀ7 cells. The higher uptake of
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DOX and VCPꢀDOX suggested that the drugs were taken up by the cells while delivering
from vesicular nanoꢀscaffold much better compared to their free forms. The
comparisons of the cytotoxicity data in figures 6dꢀ6e (also 7aꢀ7b) with the CLM
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images 9a and flow cytometry (9e) confirmed that the higher amount of DOX uptake
by the cells exhibited better cell killing. From the above observations, it can be concluded
that dextran vesicles has ability to internalise both water soluble (DOX) and water insoluble
(CPT) anticancer drugs to exhibit its antitumor efficacy.
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Cisplatin, DOX and CPT Triple Drug Loaded Vesicles
A unique triple drug loaded dextran vesicle VCPꢀCPTꢀDOX was produced by conjugating
cisplatin and loading both water soluble DOX.HCl and water insoluble CPT in single dose as
shown in figure 10 (a). The DLCs in VCPꢀDOXꢀCPT were obtained as 9.3 % , 2.2 % and 1.5 %
for cisplatin, DOX and CPT, respectively (DLE= 52 %, 50 % and 40 % for cisplatin, DOX
and CPT, respectively). The average size and the morphology of these triple loaded vesicles
were determined by DLS and AFM as 200 ± 10 nm (see figures 10b and 10c). The enzymeꢀ
responsiveness of this triple loaded dextran vesicles in releasing drugs was investigated in
PBS pH = 7.4 at 37 °C. The in vitro kinetics revealed that the vesicles ruptured exclusively in
the presence of esterase enzyme to release the drugs (see SFꢀ18). Confocal microscope
images confirmed the cellular internalisation of both DXO and CPT from the dextran vesicles
(see SFꢀ19). The cytotoxicity profiles of the triple loaded dextran vesicle in MCF 7 cell line
is shown in figure 10c. The triple loaded vesicles could able accomplish 100 % cell death at
very low concentrations of drugs with the IC50 values of 0.1µg/mL of cisplatin, 0.02 µg/mL
of DOX and 0.02 µg/mL of CPT. These IC values are much lesser compared to their dual
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and individual loaded vesicles (see ST1). The cytotoxicity profiles for the individually loaded
vesicles and their cocktail form were shown in figure 10d. The cocktail of individual drug
loaded vesicles showed better cell killing compared to their individual components. To
evaluate its combination therapy effect, the combination index values were calculated using
1 2 3 x
the equation CI = D /Dm1 + D /Dm2 + D /Dm3 and the values are plotted against IC in figure
10e. The results obtained for the triple loaded vesicles, their cocktail form were depicted
using a triangle model as shown in figure 10f. These data emphasized the synergistic effect
for triple drug loaded vesicles and cocktails of individual loaded vesicles. On the other hand,
the physical mixture of the free drugs showed the antagonistic or additive effect. Thus, it may
summarized that the cisplatin stitched dextran vesicles could able to load and deliver both the
hydrophilic and hydrophobic drugs together to achieve the synergistic cell killing in human
breast cancer cell lines (MCF 7) at very low drug concentration. In the present investigation,
the carboxyl dextran vesicle was successfully demonstrated as efficient multiple drug nanoꢀ
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carrier for the intracellular internalisation of cisplatin, DOX and CPT to boost the synergistic
breast cancer therapy.
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Figure 10. (a) Schematic representation of triple drug loaded vesicles VCP-DOX-CPT. DLS
histograms (b) and AFM images (c) of triple drug loaded vesicles VCP-DOX-CPT. (c) Histograms
depicting the cytotoxicity of the free drugs and triple drug loaded vesicles. (d) Histograms
depicting the cytotoxicity of individually loaded vesicles and cocktails of individual drug
X
loaded vesicles in MCF 7 cell line. (e) Combination index (CI) plotted against the IC values
for free drugs, cocktails of the individually loaded vesicles and triple loaded vesicles. (f)
Triangle models are showing the synergistic, antagonistic and additive effects in the
combination therapy of triple antagonistic drug.
Conclusion
In conclusion, the present investigation demonstrates one of the first examples of
cisplatinꢀstitched polysaccharide vesicles for combination therapy of three antagonistic
anticancer drugs together to accomplish synergistic cancer therapy. Dextran was anchored
with selfꢀassembly directing renewable hydrophobic unit 3ꢀpentadecylphenol (PDP) along
with carboxyl functionality which enables the vesicle formation as well as cisplatin
conjugation. The vesicles were found to retain their size and shape in the cisplatinꢀstitched
form and also exhibited excellent encapsulation capability for water soluble DOX and water
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insoluble CPT in the core and layer, respectively. The cisplatinꢀpolymer conjugate was
stabilized by the dextran vesicular geometry against the GSH drug detoxification which was
very crucial for the delivery of the drugs in the active form in the cytoplasm. In vitro drug
release kinetics confirmed that the dextran vesicles were readily cleaved by lysosomal
esterase enzymes at the intracellular conditions to deliver the drugs to the nucleus.
Cytotoxicity studies in breast cancer cells (MCF 7) revealed that of the cisplatin stitched
vesicles accomplished > 90 % cell killing whereas the free cisplatin was ineffective. Both
CPT and DOX was also exhibited better cell killing breast cancer cell lines while
administrating them from the vesicle compare to free form. Combination therapy of the drug
loaded vesicles and free drugs were studied in cisplatin resistant breast cancer cells MCF 7
cells. The results from the combination index values showed that the physical mixture of the
free drugs cisplatin, DOX and CPT were turned out to be antagonistic in all the three
combination. Exceptionally, the dual loaded vesicles showed synergistic cell killing at lower
concentration than that of their free drugs or cocktails of individual drug loaded vesicle
counterparts. The vesicles are very unique to load all three drugs together in single platform
to produce triple drug loaded nanoꢀcarriers. This triple drug loaded vesicle exhibited
excellent synergistic killing in breast cancer cells at nanogram concentration level which is
very remarkable for future therapeutic applications. Thus, the dextran vesicle nanoꢀcarrier
overcome the antagonistic effect of three important anticancer drugs like cisplatin,
doxorubicin and camptothecin in combination therapy to accomplish synergistic cell killing
in breast cancer cells. Confocal microscopic. Flow cytometry analysis revealed that these
vesicles were taken up by the cells and they are accumulated in nucleus and periꢀnucleuar
region. The polysaccharide vesicles are proven as excellent biocompatible nanoꢀcarrier for
multiple drug delivery irrespective of physical loading or chemical conjugation of drugs, their
water solubility or insolubility and antagonistic effect. Thus, the dextran vesicular scaffold is
not restricted only to these anticancer drugs, and in principle, it can be expanded to wide
ranges of other water soluble and water insoluble anticancer drugs to achieve synergistic
effect in other cancer diseases.
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Supporting information: Methods, Instrumentation, cell viability studies, confocal
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microscopy imaging, flow cytometry analysis procedures, HꢀNMR, CꢀNMR, TGA, DSC,
of the polymers, AFM image of polymer nanoparticles in water, and cytotoxicity of
polymers. Cellular uptake of free CPT and VCPꢀCPT in MCF 7 cells etc. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgments: The authors thank research grant from Department of Science and
Technology (DST) for the project SB/S1/OCꢀ37/2013. Nilesh thanks, UGC, New
Delhi for Ph.D. research fellowship. The authors thank National Chemical laboratory,
Pune, India for HRꢀTEM facility. AFM facility is sponsored by the DSTꢀFIST Project.
We thank Ms. Mehak Malhotra, Ms. Sonashree Saxena and Dr. Bapurao Surnar from
our laboratory for CLSM images and discussion.
Author Information: Corresponding Author: Prof. Manickam Jayakannan
[
jayakannan@iiserpune.ac.in]. The authors declare no financial interest.
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