Biotin-Tagged Polysaccharide Vesicular Nanocarriers for Receptor-Mediated Anticancer Drug Delivery in Cancer Cells

Article abstract of DOI:10.1021/acs.biomac.8b00833

Biotin-conjugated multistimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs to intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulfide and enzyme-biodegradable aliphatic ester chemical linkages, and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of <200 nm in size, which were characterized by dynamic and static light scattering, electron, and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles toward membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl)-loaded dextran vesicles exhibited stable formulation in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Redox-degradation by glutathione (GSH) showed 60% drug release, whereas lysosomal esterase enzyme enabled >98% drug release in 12 h. Confocal microscope and flow cytometry-assisted time-dependent cellular uptake studies revealed that the biotin-receptors overexpressed in cervical cancer cells (HeLa) exhibited larger drug accumulation through the receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pretreatment in cancer cells and energy-suppressed cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptor-overexpressed cervical cancer cells.

Full text of DOI:10.1021/acs.biomac.8b00833

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Article
Biotin-Tagged Polysaccharide Vesicular Nanocarriers for
Receptor-Mediated Anticancer Drug Delivery in Cancer Cells
Nilesh Umakant Deshpande, and Manickam Jayakannan
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00833 • Publication Date (Web): 15 Jun 2018
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Biotin-Tagged Polysaccharide Vesicular
Nanocarriers for Receptor-Mediated Anticancer
Drug Delivery in Cancer Cells
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Nilesh Umakant Deshpande and Manickam Jayakannan*
Department of Chemistry
Indian Institute of Science Education and Research (IISER) Pune
Dr. Homi Bhabha Road
Pune 411008, Maharashtra, India
*Corresponding Author: jayakannan@iiserpune.ac.in
Keywords: Receptor-mediated endocytosis, Polysaccharide vesicles, Multi-stimuli-
responsiveness, GSH redox stimuli, Enzymatic-biodegradation, Drug delivery.
Abstract
Biotin-conjugated multi-stimuli-responsive polysaccharide vesicular nanocarriers are
designed and developed, for the first time, to accomplish receptor-mediated endocytosis in
cancer cells and to deliver anticancer drugs at the intracellular compartments. For this
purpose, a new renewable hydrophobic unit was custom designed with redox-degradable
disulphide and enzyme-biodegradable aliphatic ester chemical linkages and it was conjugated
along with biotin on the dextran backbone. The dextran derivative self-assembled into
nanovesicles of < 200 nm in size which were characterized by dynamic and static light
scattering, electron and atomic force microscopes. Avidin-HABA assay established the high
affinity of biotin-tagged dextran vesicles towards membrane-receptors up to 25 nM
concentration. Doxorubicin-hydrochloride (DOX.HCl) loaded dextran vesicles exhibited
stable formulation in PBS and FBS. Redox-degradation by glutathione (GSH) showed 60 %
drug release whereas lysosomal esterase enzyme enabled > 98 % drug release in 12 h.
Confocal microscope and flow cytometry assisted time-dependent cellular uptake studies
revealed that the biotin-receptor over expressed cervical cancer cells (HeLa) exhibited larger
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drug accumulation through receptor-assisted endocytosis process. This process enabled the
delivery of higher amount of DOX and significantly enhanced the killing in cancer cells
(HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells).
Control experiments such as biotin pre-treatment in cancer cells and energy-suppressed
cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by
the biotin-tagged polymer vesicles. This report provides first insights into the targeted
polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in
biotin receptors over expressed cervical cancer cells.
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Introduction
Receptor-mediated endocytosis of nano-carriers is emerging as one of the important
approaches for targeted delivery of anticancer drugs or genes to cancer tissues under
physiological conditions.1-4 Cancer cells are over expressed by large number of signalling
receptors for metastasis and also enriching the nutrient supply to the fast and uncontrolled
cell growth. 5-7 These receptors exhibit high binding affinity towards targeting ligands such as
biotin (vitamin B7),8-9 folic acid10-13 and RGD peptide,14 tranSerrin15 etc. Nano-assemblies
tagged with these targeting ligands enhance the delivery of payload at the cancer tissue site
through receptor-ligand interaction.3 Biotin is one of the excellent targeting ligands for
streptavidin-type membrane proteins (avidin class of proteins) at the cell surface with high
association constant16 in the range of 1013 to1015 M-1. Various cancer cells such as cervical,
breast, lung, and ovarian are over expressed with biotin receptors.17 Biotin-conjugated nano-
assemblies from small molecules,18 block copolymers,19-21 dendrimers,12 gold nanoparticles,22
and carbon nano-tubes,23 etc were explored for delivering chemotherapeutic agents. The
polymer nano-carriers are particularly important since they have unique capability to passive
selectively target the cancer tissue via enhanced permeability and retention effect (EPR).24-25
Thus, the receptor-mediated endocytosis in macromolecular nano-carriers is an appropriate
choice for both active and passive selectively targeting the cancer tissues. Bio-resource based
polymer nano-carriers have recently gained significant importance in bio-medical field.26-27
Polysaccharide nano-carriers are unique bio-resource systems due to their excellent
biocompatibility associated with structural diversity,28-31 and also possess sugar-type
macromolecular backbone for enhanced internalisation via like-like interaction with lectins
(carbohydrate-binding proteins) at the cell membrane.32-34 Polysaccharide micelles,35
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nanoparticles,36capsules,37 hydrogels,38-40 and polymersomes (or vesicles)28-31 were reported
for drug and gene delivery. Among these nano-carriers; polysaccharide nanovesicles are very
unique for delivering both water soluble and water insoluble anticancer drugs from single
nano-carrier.41 Earlier dextran vesicles using stearyl chains as hydrophobic unit and folate as
targeting ligand were reported for delivering doxorubicin.42-43 From our group, new classes of
enzyme and pH responsive polysaccharide vesicles were reported based on dextran and
renewable resource hydrophobic units from cashew nut shell liquid.41, 44-45 Uptake studies
using caveolae (+) plus and caveolae (-) nul cells revealed that the dextran vesicles were
significantly taken up by the caveolae (-) nul type cells that resembled the membrane
architectures of breast cancer cells.46 Carboxylic functional dextran vesicles were designed to
administrate three different antagonistic drugs like doxorubicin (DOX), camptothecin, and
cisplatin together for synergistic combination therapy.47 Polymersomes or polymer vesicles
are one of the most important multipurpose nano-carriers for drug delivery; however, the
concept of targeted polysaccharide vesicle is not fully explored for better treatment
efficiencies in cancer. Despite biotin being known as excellent targeting ligand to cancer
cells; there is no report on biotin-tagged polysaccharide vesicles in the literature. The lack of
the non-availability of structural motifs for maintaining appropriate hydrophobic-hydrophilic
balance is one of the reasons with respect to the difficulty in producing biotin-tagged
polysaccharide vesicular assemblies. The present work is the first example to address this
important idea and the proof-of-concept is successfully demonstrated in cancer cell lines.
Here, we report biotin-tagged and multi-stimuli-responsive polysaccharide vesicles based on
dextran backbone and renewable resource hydrophobic unit. The receptor-mediated
endocytosis concept was successfully demonstrated in vitro by carefully designed time-
dependent cellular uptake studies in cancer cells. This new concept is shown in figure 1.
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Figure 1. Biotin-tagged polysaccharide nano-vesicular assemblies for receptor-mediated
endocytosis in cancer cells and delivery of drugs at intracellular levels by GSH and enzyme
multi-stimuli-responsiveness.
The present investigation is emphasised to make new biotin-conjugated and multi-
stimuli-responsive polysaccharide vesicles and demonstrate the proof-of-concept for water
soluble anticancer drug DOX.HCl in cancer cells. The polymer vesicles were tailor made
using redox and enzyme degradable chemical linkages containing renewable resource 3-
pentadecylphenol hydrophobic unit. The present polysaccharide (dextran) vesicle has
following features: (i) biotin-anchored at dextran backbone for receptor-mediated binding at
cancer cell membranes, (ii) the PDP hydrophobic units was connected on the hydrophilic
dextran backbone via disulphide (S-S) and aliphatic ester chemical linkages (see Figure 1),
(iii) the high levels of glutathione (GSH) at the intracellular level (1 mM) compared to
extracellular level (1 ꢀM) act as redox-trigger48 (stimuli-1) for the -S-S- bond cleavage
(marked green in Figure-1) and disrupt the vesicular assembly for drug delivery, and (iv) the
biodegradation of aliphatic ester linkages (marked yellow in Figure-1) by the esterase enzyme
in lysosomal compartments49-50 facilitated the drug release at the intracellular level (stimuli-
2). The polymer vesicles provided unique opportunity to encapsulate water soluble anticancer
drug doxorubicin-hydrochlroride (DOX.HCl) and deliver them to cancer cells using receptor-
mediated endocytosis. The nanovesicles produced were < 180 30 nm in size and Avidin-
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HABA assay established the biotin-receptor affinity up to 25 nM concentration. In vitro
studies revealed that the nanovesicles are stable under extracellular conditions and released
drugs in response to intracellular stimuli such as GSH and esterase enzyme. The receptor-
mediated endocytosis studies were performed in cervical cancer (HeLa) and WT-MEF
(normal) cell lines. Time-dependent uptake studies by confocal microscope and FACS
technique confirmed the occurrence of receptor-mediated endocytosis by biotin-tagged
dextran vesicles. This was further corroborated by the time-dependent cytotoxicity studies
wherein the cancer cells were found to be killed much better while delivering DOX.HCl from
biotin-tagged vesicular platform compared to biotin free nano-vesicles. In a nut shell, the
present investigation provides first insights into the administration of water soluble anticancer
drugs through receptor-mediated endocytosis based on biotin-tagged dextran vesicles and it
opens up new opportunities for the development of targeted polymer vesicular nano-carrier
platform for long term application in cancer treatment.
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Experimental Section
Materials and Methods: 3-Pentadecylphenol (PDP), 2-bromoethanol, triethylamine,
dextran (Mol. wt 6000), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
(EDC.HCl),
4-dimethylamino pyridine, dithiothreitol (DTT), glutathione (GSH),
doxorubicin hydrochloride (DOX.HCl), dicyclohexylcarbodidimide (DCC), 4-dimethyl
amino pyridine (DMAP), biotin and horse liver esterase enzyme were purchased from
Aldrich chemicals. Avidin was purchased from Alfa aesar and used as such. 4’-
hydroxyazobenzene-2-carboxylic acid (HABA) was purchased from Loba Chemie and used
as received. NaOH and all other necessary reagents and solvents like dimethylsulfoxide
(DMSO), N,N-dimethylformamide (DMF) and dichloromethane (DCM) were purchased
locally and purified following the standard procedures. The instrumentation details, structural
characterization, biological data collection for MTT assay, confocal imaging and Flow
cytometry analysis, tissue culture conditions, and the synthesis of compound (1) are given in
the supporting information.
Synthesis of 3-((3-oxo-3-(2-(3-pentadecylphenoxy) ethoxy) propyl) disulfanyl) propanoic
acid (2): 3, 3’-Dithiopropionic acid (DTPA) (5.0 g 24 mmol) and compound (1) (4.14 g 12
mmol) were dissolved in 100 mL dry THF and purged with nitrogen for 10 minutes. To the
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above mixture, DCC (3.19 g, 16 mmol) and DMAP (0.43g, 4 mmol) dissolved in dry THF
was added and the reaction continued for 12h at room temperature. Dicyclohexyl urea from
the reaction mixture was separated by filtration and the organic solvent was evaporated. The
product was purified by passing through silica gel column 60-120 mesh using 5 % ethyl
acetate: pet ether solvent system. Yield = 5.8 g (36.0 %) 1H NMR (400 MHz, d6-DMSO) δ =
12.36 (s, 1 H), 7.16 (t, J = 7.9 Hz, 1 H), 6.81 - 6.67 (m, 3 H), 4.36 (dd, J = 3.8, 5.5 Hz, 2 H),
4.21 - 4.08 (m, 2 H), 2.97 - 2.82 (m, 4 H), 2.78 - 2.71 (m, 2 H), 2.64 - 2.57 (m, 2 H), 1.60 -
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1.48 (m, 2 H), 1.34 - 1.18 (m, 24 H), 0.89 - 0.80 (m, 3 H).. C NMR (100 MHz, d6-DMSO)
δ: 172.4, 170.9, 158.0, 143.8, 128.9, 120.6, 114.3, 111.3, 65.2, 62.7, 39.9, 39.7, 39.5, 39.1,
38.9, 38.7, 35.0, 33.3, 33.1, 32.8, 32.4, 31.1, 30.6, 28.8, 28.8, 28.8, 28.7, 28.5, 28.5, 21.9,
13.7. MALDI TOF−TOF (MW: 540.27):m/z = 563.28 (M + Na) +. HR-MS (ESI+): m/z [M
+ H] + Calcd for C29H48O5S2 [M] +, 541.2978; found, 541.3019
Synthesis of DEX-SS-PDP: Dextran (Mw= 6,000, 1.0 g, 6.2 mmol of anhydroglucose unit),
compound (2) (1.0 g, 1.83 mmol) were dissolved in anhydrous DMSO+DMF+DCM solvent
mixture (15.0 mL: 8.0 mL: 2.0 mL). The formed reaction mixture was purged with dry
nitrogen. To the above content, DMAP (0.22 g, 1.86 mmol) in DMSO (3.0 mL) and
EDC.HCl (0.52 g, 2.72 mmol) in DMSO (3.0 mL) were added. The reaction mixture was
stirred for 24 hour at room temperature. DMSO was removed; the liquid was precipitated in
acetone. The purification was repeated at least twice to get pure dextran derivative which was
o
1
dried under vacuum at 60 C and stored. Yield = 0.65 g (65 %). H NMR (400 MHz, d6
DMSO ) δ: 7.17-7.01 ppm (s, 1H, Ar-H ), 6.76 - 6.72 ppm (m, 3H, Ar-H), 4.93, 4.85, 4.51
ppm (s) 4.68 ppm (s), 4.35ppm (t, 2H, Ar-O-CH2), 4.16 ppm (t, 2H, -CH2-OH), 3.14-3.69
ppm, 2.96 - 2.92 ppm (m, 4H, -CH2-CH2-S-S-), 2.76 - 2.72 (m,4H, -S-S-CH2-CH2-), 2.49
ppm (2H, Ar-CH2), 1.53 ppm (2H, Ar-CH2-CH2), 1.18-0.80 ppm.
Synthesis of DEX-SS-PDP-Biotin: Dextran (Mw= 6,000, 1.0 g, 6.2 mmol of anhydroglucose
unit), compound (2) acid (1.0 g, 1.83 mmol) and biotin (60.0 mg, 0.25 mmol) were dissolved
in anhydrous DMSO+DMF+DCM solvent mixture (15.0 mL: 8.0 mL: 2.0 mL). The formed
reaction mixture was purged with dry nitrogen. To the above content, DMAP (0.25 g, 2.04
mmol) in anhydrous DMSO (3.0 mL) and EDC.HCl (0.59 g, 3.08 mmol) in anhydrous
DMSO (3.0 mL) were added. The rest of the procedure is followed as described for DEX-SS-
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PDP. Yield =0.76 g( 70 %). H NMR (400 MHz, d6 DMSO ) δ: 7.17-7.01 ppm (s, 1H, Ar-H
), 6.76 - 6.72 ppm (m, 3H, Ar-H), 6.42-6.36 ppm (biotin –NH protons), 4.93, 4.85, 4.51 ppm
(s) 4.68 ppm (s), 4.35 ppm (t, 2H, Ar-O-CH2), 4.27- 4.15 ppm (biotin –CH protons) 4.16 ppm
(t, 2H, -CH2-OH), 3.14-3.69 ppm, 2.96 - 2.92 ppm (m, 4H, -CH2-CH2-S-S-), 2.76 - 2.72
(m,4H, -S-S-CH2-CH2-), 2.49 ppm (2H, Ar-CH2), 2.15 ppm (s, 2H, biotin –CH2 ), 1.60-1.30
(broad, 6H, biotin –CH2), 1.53 ppm (2H, Ar-CH2-CH2), 1.18-0.80 ppm.
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Lysosomal Tracking of the Nanovesicles: In a typical experiment, 25000 cells were seeded
in each well in live cell chamber and allowed to adhere for 16-18 h. Further these cells were
treated with required concentration of Fluorophore and incubated for another 6h. Finally the
media was aspirated and cells were washed two times with PBS (1X) and cells were treated
with 1.0 mL of DMEM media having 25.0 nM of Lysotracker green DND-26 and cells were
imaged immediately.
Cellular Uptake at 4 ºC: In a typical experiment, 50000 cells were seeded in each well of
six well plate and allowed to adhere for 16h. Media from the cells was then aspirated and
fresh media containing the Dox loaded nanovesicles was added and cells were incubated at 4
ºC (on Ice) for 0.5 h and finally fixed using paraformaldehyde. These cells were imaged
using the confocal microscopy.
Cellular Uptake using Biotin Pre-treatment: 100000 cells were seeded in each well of six
well plate and allowed to adhere for 16 h. After 16 h, cells were treated with fresh media
having 2.0 mM biotin concentration and incubated for another 1h at 37 ºC. Finally the media
containing biotin was aspirated and cells were treated with fresh media having required
concentration of Dox loaded nanovesicles and incubated for another 4 h at 37 ºC. Finally
cells were then fixed using paraformaldehyde and imaged using confocal microscopy.
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Results and Discussion
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Synthesis of Biotin-Conjugated Dextran
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Biotin conjugated and dual responsive dextran derivatives were synthesized as shown
in the scheme 1. A renewable resource and vesicular structure directing hydrophobic
molecule 3-pentadecyl phenol (PDP)41 from cashew nut shell liquid was coupled with 2-
bromoethanol to yield the compound (1). The compound (1) was coupled at one end of the
dithiopropionic acid through aliphatic ester linkages to yield compound (2). This hydrophobic
unit (2) has the disulfide bond (-S-S) which is redox-degradable by glutothione (GSH) at
intracellular compartmnets of cells (stimuli-1). The coupling of compound 2 in the dextran
backbone produced DEX-SS-PDP. The conjugation of both compound 2 and biotin in the
polysaccharide backbone in one-pot reaction yielded DEX-SS-PDP-Biotin. The reaction of
biotin and dextran produced DEX-Biotin and its structure is shown in the supporting
information scheme-S1. Both compound 2 and biotin were conjugated in the dextran
backbone via aliphatic ester linkages that are enzymatically-biodegradable (stimuli-2) at the
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1
intracellular compartments. The structure of dextran derivatives were confirmed by H-
NMR. In figure 2a, the PDP substituted unsymmetrical dithiopropionic acid molecule (2)
showed expected number of protons and they are alphabetically assigned in the chemical
structure. The carboxylic ester -SCH2CH2COOR (proton i) and carboxylic acid
SCH2CH2COOH (proton L) appeared at 2.73 and 2.59 ppm, respectively. In figure 2b, up on
anchoring onto the dextran backbone, the carboxylic acid (proton L) vanished and the new
ester peaks appeared together (proton i+L, double the intensity). Additionally, the DEX-SS-
PDP-Biotin dextran derivative showed peaks corresponding to PDP units at 6.8 to 7.3 ppm.
The broad peak at 6.5 ppm was assigned to N-H protons in biotin unit (proton m) and the new
ester peak with respect to biotin conjugation in the dextran backbone appeared at 2.43 ppm
1
(proton n). These peak assignments were further confirmed by comparing the H-NMR
spectra of DEX-SS-PDP (see S2) and DEX-Biotin (see S3) in supporting information. The
degree of substitution of disulfide PDP on dextran backbone was determined by comparing
the peak intensities of PDP aromatic protons at 6.8 ppm (proton e+f) or aliphatic protons of
PDP (protons j+k) with dextran anomeric proton 4.6 ppm (proton p). The degree of
substitution for biotin conjugation was determined by comparing the peak intensity of the
biotin N-H peak at 6.4 ppm (proton-m) or ester peak at 2.43 ppm (proton n) with anomeric
proton at 4.6 ppm (proton p). The degree of substitution of PDP and biotin were estimated to
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be 5.0 % and 2.0 %, respectively. The higher incorporation of PDP + biotin (more than 10 %
in total) in the dextran backbone produced water insoluble polymers; thus the total degree of
substitution was restricted to less than 10 %. TGA and DSC analysis of the polymer
derivatives showed their thermal stability up to 250 °C and they were found to be largely
amorphous polymers (see S4).
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Scheme1: Synthesis of enzyme and GSH dual responsive and biotin-conjugated dextran
derivatives.
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Figure 2: 1H-NMR spectra of compound 2 (a) and DEX-SS-PDP-Biotin (b) in d6-DMSO. The
solvent peaks are assigned by asterisk.
Dextran Nano-vesicles and DOX.HCl Encapsulation
In the present investigation, the hydrophilic polysaccharide backbone (dextran) was
conjugated with hydrophobic amphiphilic unit based on PDP; thus in aqueous medium, the
hydrophobic chain oriented away from the water to give amphiphilic sheet-like geometry.
The inter-digitations of PDP chain from the apmphiphilic dextran resulted in bi-layer self-
assemblies which folded into vesicular architectures through cooperative interactions. The
evidence for the PDP chains inter-digitations in the vesicular geometry was proved by single
crystal structure analysis of PDP units in our earlier work.51-52 Thus, it is anticipated that the
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PDP + dithiopropionic ester linkages in the present design is also expected to follow similar
inter-digitation to produce well-defined dextran vesicles (see Figure 3a). To study the self-
assembly behaviours of newly designed dextran derivatives DEX-SS-PDP-Biotin, DEX-SS-
PDP, and DEX-Biotin; they were subjected to dialysis in water. Typically, dextran
derivatives were dispersed in water + DMSO and dialysed using semi-permeable membrane
of MWCO=3500 in milli-Q water. The dialysate was replenished with fresh water
periodically to remove DMSO and at the end of 48 h of dialysis, a transparent solution was
produced (see Figure 3) which was lyophilized and stored at 4 °C. These dialysed dextran
derivatives were subjected to dynamic light scattering (DLS) and atomic force microscope
(AFM) analysis.
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Figure 3: (a) Self-assembly of dextran derivative into vesicles. (b) DLS histogram and AFM
images of DEX-SS-PDP vesicles (V). (c) DLS histogram and AFM images of DEX-SS-PDP-
Biotin vesicles (VBIOTIN). (d) DLS histogram, AFM, FESEM and HRTEM images of DOX.HCl
encapsulated DEX-SS-PDP-Biotin vesicles (VBIOTIN+DOX.HCl).
In figure 3b, DLS histograms of DEX-SS-PDP showed monomodal distribution with
size 180 20 nm. AFM is a very powerful tool to study the morphological features of soft-
materials such as vesicles created from aqueous medium.41 AFM images of DEX-SS-PDP in
figure 3b appeared as donut-shaped spherical objects with diameter 180 30 nm with height
of the nano vesicles around 20 10 nm (see Figure 3b). The sizes of the nano-vesicles in
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AFM image exactly matched with the DLS nano-aggregates in aqueous medium confirming
that vesicular self-assembly was indeed created and preserved in the aqueous medium
through the hydrophilic and hydrophobic interaction as represented in figure 3a. DLS
histograms and AFM images for DEX-SS-PDP-Biotin are shown in figure 3c. The size of the
assembly from DLS and AFM images were obtained as 190 30 nm and 200 20 nm,
respectively, indicating that the vesicular nano-assembly was perfectly preserved in the
biotin conjugated dextran derivative as well. DLS and AFM data for DEX-biotin (without
SS-PDP unit) revealed that the polymer structure produced nanoparticles rather than vesicles.
This emphasised the need of SS-PDP unit in the dextran backbone to produce vesicular
geometry (see S5) and the result was in accordance with our earlier observation41,47 that the
PDP unit was essential to self-assemble the dextran backbone into nano-vesicles in aqueous
medium. Further static light scattering (SLS) studies were carried out to establish the nano-
vesicle aggregates in solution state (see S6). SLS gives the radius of gyration (Rg) of the
polymer assemblies and DLS provides the hydrodynamic radius (Rh) of the polymer nano-
assemblies. The ratio of the Rg/Rh =1 for polymer nano-vesicles as reported in the
literature.53-54,41 In the present investigation, the Rg = 70 nm was obtained as from SLS and
Rh = 73 nm from DLS; thus the ratio Rg/Rh = 70 nm / 73 nm = 0.96 ≈ 1.0, which confirmed
the formation of polymer nanovesicles in the solution state. The size of the nano-vesicles is
matched very well with the DLS, AFM, FESEM and HRTEM images as shown in figure 3.
To study the role of concentration of polymer on the nanovesicle formation, the self-assembly
was carried out by dialysis method by varying the concentration from 0.001 to 1.0 mg/mL
(1000 time dilution). DLS data of these dialyzed solutions (see S7) revealed that the size of
the nanovesicles were almost retained in the same range of 220 20 nm irrespective of the
variation in the concentration of polymer solution. Thus, we believe that the polymer vesicles
formation was largely driven by the structure rather than concentration. Hence, the present
design could produce vesicles of 200 20 nm in size which is in the acceptable range for
EPR effect.55The above dextran nano-vesicles DEX-SS-PDP and its biotin conjugated
analogue DEX-SS-PDP-Biotin are hereafter referred as V and VBIOTIN, respectively. The
critical vesicular concentration of the dextran vesicles was determined using pyrene as
probe.41 A fixed amount of pyrene (0.6µM) was used and the concentrations of the dextran
conjugates were varied from 2.4× 10-7 to 4.8×10-5 M. From the ratio of the pyrene
characteristic peaks (I1 /I3), the critical vesicular concentration was determined as 2.4 × 10-6
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M (see S8). One of the unique features of vesicles or polymersomes is that they possess
hydrophilic core for encapsulation of water soluble fluorophore dye like Rhodamine B (RhB)
and water soluble anticancer drugs such as doxorubicin-hydrochloride DOX.HCl which are
not possible in micelles or nanoparticles.44 To study the encapsulation capability of newly
designed dextran vesicles, V and VBIOTIN were subjected for encapsulation of RhB and
DOX.HCl by dialysis method using semi-permeable membrane MWCO = 3500 Da as
described earlier. The DLS histogram of VBIOTIN+DOX.HCl showed the size of the nano-vesicles
as 200 ± 20nm (see Figure 3d). AFM morphology confirmed the donut-shaped features with
respect to vesicular morphology. Field mission scanning electron microscope (FESEM) and
transmission electron microscope (TEM) images further confirmed the spherical nature of the
vesicular nano-assemblies with difference in contrast with respect to the layer and core (see
Figure 3d). DLS, AFM, FESEM and HR-TEM images showed the sizes < 200 nm and
confirmed existence of the dextran nano-vesicles. VDOX.HCl (without biotin conjugation) and
VRhB and VBIOTIN+RhB were also found to exist as nano-vesicles (see S9). Using absorption
spectroscopy, the drug (or dye) loading content (DLC) was obtained in the range of 1.5 to 2.0
%. The drug (or dye) loading efficiency (DLE) was typically obtained in the ranges of 50 to
60 % (see S-9). The absorbance and fluorescence spectra for RhB and DOX.HCl
encapsulated nano-vesicles showed very good red-emission suitable for bio imaging process
(see S10). Other method like sonication was also explored for the encapsulation of DOX.HCl
in the vesicles. The sonication method gave much higher DLC = 37 %. However, further
dialysis of the sonication-assisted sample exhibited premature leaching of the drug. At the
end of the dialysis , the DLC was obtained as 1.8 % which is similar to that of direct dialysis
process (without prior sonication). Hence, the dialysis procedure is the one of the best way to
encapsulate water soluble drugs to produce stable Dox formulation in polysaccharide
vesicles.
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Avidin-Binding Assay and Stimuli-responsive Delivery
The dextran vesicles are designed with three components which are responsive to
cancer cells both at the extracellular and intracellular environments. These responsiveness
are: (i) receptor-assisted endocytosis process at the periphery of the cell membranes (see
Figure 4a), (ii) redox-cleavable disulfide (S-S) chemical linkages to disassemble the vesicular
assemblies by glutathione (GSH) residue in the cytosol, and (iii) lysosomal enzymatic-
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biodegradation of aliphatic ester linkage connecting the hydrophobic unit at the dextran
backbone (see Figure 4b). To study the receptor-mediated biological events, in vitro
experiments were designed based on Avidin-HABA assay 19,56-57 and the data is summarized
in Figures 4c and 4d. Avidin has four binding sites for HABA (4’-hydroxyazobenzene-2-
carboxylic acid); thus, 1:4 complex of Avidin-HABA was produced following the reported
procedure.19 Avidin-HABA complex showed unique absorption peak at 505 nm which is
completely distinguished from either HABA or avidin (see S11). This avidin-HABA complex
was titrated with biotin conjugated vesicles (VBIOTIN) and normal vesicles (V) and the data are
shown in figure 4c. As shown in figure 4c, there is a gradual decrease in the absorption of
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Avidin-HABA complex at 505 nm with the increase in the concentration of VBIOTIN
.
Interestingly, the non-biotin conjugated vesicles (V) did not show any changes in the
absorption maxima of Avidin-HABA complex (see red line in Figure 4c). This further
confirmed that the biotin free vesicles (V) do not possess any binding towards Avidin. This
trend clearly indicates that the HABA molecules are getting replaced by the VBIOTIN in the
Avidin-HABA complex. The reason for this replacement of HABA molecules by VBIOTIN
samples can be attributed to the higher association constant for Avidin-Biotin compared to
that of Avidin-HABA complex.8 Thus, the decrease in the absorbance maxima followed by
the additions of VBIOTIN is arisen followed by the complexation of Avidin-biotin at the
periphery of biotin conjugated (VBIOTIN) nanovesicles. This decrease in the absorption was
plotted as [(A0-AC)/A0] versus concentration of the biotin in the vesicles, where A0 is the
initial absorbance and AC is the absorbance at concentration ‘C’. As can be seen from the
figure 4d, the value of [(A0-AC)/A0] steadily increases and saturated at 25 nM. From the
break point in the plot, it can be assured that the avidin-biotin interactions saturated at 25 nM
of biotin at the periphery of vesicles. The number of biotin molecules per polymer chains
were determined by the method reported by Karen Wooley and co-workers.19 It was found to
be approximately one molecule of biotin present in each polymer chain ( see S12 for more
details) .The affinity of the biotin-nanovesicles towards Avidin is in accordance to the other
polymer systems.19,56-57 Thus, the current dextran vesicle possess significant amount of biotin
to bind streptavidin like membrane protein on cancer cells surface for targeted delivery. To
differentiate the circulating free biotin and biotin-conjugated vesicles towards their molecular
interaction with avidin like receptors under physiological conditions, competitive binding
assay was studied in fetal bovine serum (FBS) (see Figure S13). Interestingly, there was no
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replacement of HABA molecules from avidin-HABA complex by the residual biotin present
if at all in FBS. The addition of biotin conjugated vesicles (VBIOTIN) into this avidin-HABA
complex in FBS immediately showed decrease in the spectroscopic signal at 505 nm with
respect to the binding of biotin-conjugated vesicles (VBIOTIN vesicles) in the avidin pocket.
This control experiment provides direct evidence for the targeting ability of biotin-conjugated
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vesicles towards the membrane receptors under physiological conditions.
Figure 4. (a) Receptor mediated binding of biotin conjugated vesicles (VBIOTIN) on the cell
membrane. (b) GSH-assisted redox-degradation and enzymatic-biodegradation of
nanovesicles at the intracellular compartments. (c) Absorbance spectra of the Avidin-HABA
complex upon additions of biotin conjugated dextran vesicles (VBIOTIN) and biotin free
vesicles (V). (d) Plot of [(A0-AC)/A0] versus the concentration of VBIOTIN. (e) Cumulative drug
release plots showing the release of DOX. HCl from VBIOTIN+DOX.HCl vesicle in PBS at pH
7.4, 37°C, incubated in presence of 10 mM GSH, and incubated in presence of 10 U esterase
enzyme. (f) Drug release plot showing the DOX release from VBIOTIN+DOX.HCl in PBS at pH 7.4
in presence of 10 mM GSH for 24 h and subsequent addition of 10 U of esterase enzyme.
The dextran vesicles are designed with two intracellular stimuli to break nano-carriers
to release the drugs. Glutathione (GSH) is largely present in the cytosol of the cells and its
concentrations vary significantly from extracellular (1.0
ꢀM) to intracellular level (1.0
mM)48. The polymer nano-carriers built with disulphide chemical linkages are susceptible to
undergo redox-degradation by GSH48 (see Figure 4b). In the present case, the vesicles were
constructed with –S-S- linkages in the hydrophobic units; thus, in vitro drug release kinetic
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studies were carried out for the DOX.HCl loaded nano-vesicles by exposing them to GSH in
PBS. The GSH cleavage of the hydrophobic tail ruptured the nano-vesicular structure and the
DOX.HCl gets released. The cumulative drug release was estimated by measuring the
absorbance of DOX.HCl in the reservoirs and the release profile is shown in Figure 4e. In the
absence of GSH, about 20 % drug leaching was observed due to the presence of salts in PBS.
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2-
The anions like PO4 in PBS are well-known to influence on the stability of the polymer
nano-assemblies through Hofmeister effect and salting-out of macromolecules in solution.58
2-
The PO4 anion is typical salting-out type which induces the partial phase separation of the
polymer nano-assemblies in PBS which accounts for 20% leaching.59 The stability of the
dextran vesicles was further checked in FBS and since FBS are free of ions , DLS studies
showed that the vesicles are stable for more than 24h (see S14). The release was significantly
influenced by the presence of GSH and about 60 % drug was released in a controlled manner.
This suggested that once the nano-vesicles enter the cytosol, the GSH could partially
disassemble the vesicles to release about 60 % of the drugs. Another important process in
which the nano-vesicles are transported and digested across the cell membrane is mediated by
endosome-lysosome compartments. Lysosomes typically have large abundance of digestive
enzymes such as α-chymotrypsin, trypsin, esterase and phosphatase, etc60. Among this
esterase enzyme could readily biodegrade aliphatic ester chemical linkages49 to deliver the
drugs. Aliphatic ester-linkages are readily susceptible to undergo lysosomal enzymatic
biodegradation and this concept has been proven very well by us and few others in L-amino
acids based polyesters,49 poly(ester-amide)s,60 substituted polycaprolctone block
copolymers61 and PEG-PLLA copolymers.62 In the present design, the hydrophobic tail was
conjugated with dextran using aliphatic ester chemical linkages (see Scheme-1). Thus, the in
vitro drug release profile data shown in Figure 4e in the presence of esterase enzyme in PBS
is direct proof for the enzymatic biodegradation of the polysaccharide vesicles. Esterase
enzyme docking studies from our lab45 had shown that the dextran-PDP ester linkage readily
occupied the enzymatic pocket for biodegradation. Since the present vesicular structure has
been built with aliphatic ester linkages that connect the hydrophobic unit in the dextran
backbone, esterase enzyme was employed as stimuli to study the drug release kinetics. In
figure 4e, the drug release pattern was found much higher for esterase enzyme exposure and
almost 90 % DOX.HCl was released in 12 h. In order to study the combination effect of GSH
and esterase-enzyme action together, the nano-vesicles were initially subjected to GSH
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incubation for 24 h and subsequently esterase enzyme was added. In this process, the release
kinetics data in figure 4f showed GSH could release 60 % drugs and the remaining 40 %
drug was essentially released by esterase. Hence, the combination of GSH and esterase
enzyme stimuli are very good triggers to release water soluble DOX.HCl in controlled
manner at the intracellular compartments of cells. Similarly, the non-biotin conjugated
dextran vesicles were also subjected to drug release studies for GSH and enzyme
responsiveness (see S-14). The drug release patterns were found almost identical in both
biotin-conjugated (see Figure 4e) and biotin free dextran vesicles (see S15). To address the
possibility of disassembly of nano-carriers in the cytosol by GSH and their further migration
toward the endo-lysosomal compartment for biodegradation by enzymes; the drug release
study was carried out by (i) initially incubating the DOX loaded polymer vesicles in the
presence of esterase enzyme and then (ii) administrate glutathione (GSH, 10mM) in PBS.
The drug release was done by dialysis method and monitored by absorption spectroscopy
(S16). From the results, it is clear that the esterase enzyme predominantly released the drug
(65-70 %) and the remaining 30 % of the drugs released by GSH. This experiment along with
the data in Figure 4f suggests that enzyme and GSH responsiveness work together in the
intracellular compartment to maximize the digestion of nanovesicles for drug release rather
than in sequential manner. The above studies confirmed that the newly designed dextran
vesicles are efficient triple action nano-carriers to undergo receptor mediated interaction at
the cell membrane, readily cleavable by GSH in the cytosol, and also completely digested by
esterase enzyme at the lysosomal compartments.
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Figure 5. (a) Histogram showing the cytotoxicity profiles of dextran vesicles (V) and VBIOTIN
in HeLa cell line. (b) Histogram showing the cytotoxicity profile for free DOX.HCl, VDOX.HCl
and VBIOTIN+DOX.HCl in HeLa cell line. (c) Histogram showing the cytotoxicity profile for free
DOX.HCl, VDOX.HCl and VBIOTIN+DOX.HCl in WT-MEF cell line. Live cell confocal microscopic
images in HeLa cells for the uptake of VDOX.HCl (d) and VBIOTIN+DOX.HCl (e). The concentration
of the Lyso tracker green and drug used are 25nM and 2
incubated for 4 h at 37º C.
ꢀM respectively. The cells were
Cytotoxicity of the dextran vesicles was studied by MTT Assay. In cervical cancer
cells (HeLa) and wild type mouse embryonic fibrolast (WT-MEF) cells (normal cell line), the
dextran vesicles were found to be non-toxic to cells up to 80 µg/mL (see figure 5a and S17).
DOX.HCl loaded dextran vesicles VDOX.HCl and VBIOTIN+DOX.HCl were treated in HeLa cells by
varying the drug concentration ranging from 0.1 to 1.0 µg/mL. The vesicle assisted drug
administration seems to be better in accomplishing cytotoxicity in HeLa cells compared to
free drug (see Figure 5b). Cytotoxicity experiment was carried out in breast cancer cell line
(MCF 7) and the data is shown in S-17a. The effect of cell killing by the DOX loaded
nanovesicle in MCF 7 is almost identical to that of HeLa (cervical) cancer cell line (see
Figure 5b). This suggested that the approach is not restricted to one particular cancer cell
lines. Further among the vesicles, the biotin-conjugated vesicles VBIOTIN+DOX.HCl were able to
achieve much better IC50 at a concentration of 0.1 µg/mL which is almost 5 times better than
the IC50 of 0.5 µg/mL by free drugs. When DOX.HCl loaded dextran vesicles were tested in
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WT-MEF cell line (normal cells, see Figure 5c), they showed relatively less cytotoxicity
compared to free drugs. This observation is particularly important since the free drug is more
toxic to the WT-MEF cells compared to cancer cells. DOX-loaded vesicles largely kill the
cancer cells (> 90 %); however, the effect was much lower in the normal cells (< 50 %). To
compare the cytotoxicity effect induced by the GSH stimuli, two types of polymer vesicles
were employed for MTT assay. For this purpose (i) vesicles without disulfide chemical
linkages in the hydrophobic part reported in our earlier work46 and (ii) vesicles with disulfide
linkages (VBIOTIN, reported here) were employed. It is important to mention that both types of
vesicles have aliphatic ester linkages that connect the hydrophobic part in the dextran
backbone; thus, these vesicles are readily biodegradable by lysosomal enzymes. These
nanovesicles were encapsulated with DOX and their cytotoxicity data was carried out in
breast cancer cells (MCF 7) and the details are summarized in S18a and S18b. It is very clear
from the data that the disulfide containing vesicles exhibited comparatively better killing at
higher concentration compared to the non disulfide vesicles. The data validated that GSH
accelerated rupturing of the disulfide containing nanovesicles at the intracellular
compartments. To study the cellular internalization and localization of the DOX.HCl loaded
dextran vesicles and the digestion of dextran vesicles in the lysosomal compartments, live
cell imaging was done using lyso-tracker in confocal scanning electron microscopy (see
Figures 5d and 5e). The dextran vesicles VDOX.HCl and VBIOTIN+DOX.HCl showed significant
amount of drugs at the nucleus and cytoplasm in red channels with respect to DOX emission.
The lysosomes are stained in green colour and upon merging with the red channel, the
merged images showed yellow colour in the cytosol and red in the nucleus. The yellow
emission from the cytosol is an outcome of the co-localization of red emitting DOX and
green lyso-tracker. This study confirmed that the dextran vesicles are indeed digested in the
lysosomal compartments in cytosol. The DOX.HCl released from the vesicles accumulated in
the nucleus to exhibit strong red-emission. From the above in vitro results, it can be
concluded that the custom designed dextran vesicles can be used for targeting the biotin
receptor that are overexpressed in cancer cells as well as high cytotoxicity can also be
achieved through GSH and lysosomal-enzyme stimuli.
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The uptake of nano-carriers in cells is typically controlled by four independent
pathways: (i) Macropincytosis63, (ii) direct endocytosis through hydrophobic like-like
interactions between nano-carriers and membrane, (iii) endocytosis assisted by caveolae or
clathrin proteins64, and (iv) receptor-ligand interaction mediated endocytosis.1-4 Among all
these processes, the receptor-ligand mediated process seems to be very effective since many
cancer cells are over expressed with multiple receptors for folic acid, biotin and RGD
peptide, etc. To study the role of the receptor-mediated endocytosis process, two cell lines are
chosen that are reported to have large difference in their membranes for biotin-receptors.
Cervical cancer cells (HeLa) were found to be significantly over expressed with biotin-
receptors compared to WT-MEF cells (normal cells).17 Biotin receptor over expression is one
of the well proven concept in HeLa cell line (cervical cancer) and HeLa cells were widely
used by previous researchers to study the receptor-mediated uptake in biotin-tagged nano-
assemblies and biotin-tagged polymer nanoparticles.9,16-21,55,56 Hence, the HeLa cell line was
chosen in the present investigation for studying the biotin receptor-mediated endocytosis in
the newly designed biotin-tagged polysaccharide vesicles. The cellular uptake and
intracellular localisation of the dextran vesicles VDOX.HCl and VBIOTIN+DOX.HCl was monitored
by the confocal (CLSM) microscopy. Biotin conjugated and non-conjugated dextran vesicles
were treated in these two different cell lines by varying the incubation time from 30 minutes
to 360 minutes (6 h). The images in the figure 6a showed the cellular uptake of
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V
BIOTIN+DOX.HCl vesicles with increasing incubation time. It is very clear from the images that
the red fluorescence intensity from DOX.HCl gradually increased with the incubation time
indicating that more drugs are taken up by the cells over a period of time. The fluorescence
intensity from the images almost saturated at 6 h. The images for non-biotin conjugated
vesicles VDOX.HCl showed increment in the fluorescence intensity with incubation time (see
Figure 6b); however, the intensities of DOX emission were relatively low compared to the
biotin conjugated vesicles. Free DOX.HCl was used as control and the cellular uptake images
were recorded for 30, 120 and 360 minutes incubation. The free DOX uptake was relatively
poor when compared to their delivery from biotin-conjugated vesicles (S19).
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Figure 6: Confocal microscopic images of (a) VBIOTIN+DOX.HCl and (b) VDOX.HCl in HeLa Cells
at various incubation time. (c) The plot showing the corrected total cell fluorescence of DOX
estimated from the confocal images in the right side panels (a and b) for various incubation
time in HeLa cell line. Confocal microscopic images of (d) VBIOTIN+DOX.HCl and (e) VDOX.HCl in
WTMEF cells at different time intervals. (f) The plot showing the corrected total cell
fluorescence of DOX estimated from the confocal images in the right side panels (d and e) for
various incubation time in WTMEF cell line. The concentration of the DOX.HCl was
maintained as 2 µg/mL and the various incubation times are shown in the panel.
The DOX intensity in each panel in figure 6a (for biotin-conjugate vesicles), figure 6b
(biotin free vesicles) and free DOX (S-18) in HeLa cell lines was determined by image J
software and plotted and shown in Figure 6c for quantification and comparison. A similar
approach was carried for each panel in Figure 6d (for biotin-conjugate vesicles) and figure 6e
(biotin free vesicles) in WT-MEF cell lines as shown in Figure 6f. The plots in Figure 6c and
6f provide direct information on the uptake of the above vesicles in these two different cell
lines which are significantly different in the amount of membrane receptor concentration 8,9,16
In figure 6c, it can be seen that there is 3 fold more uptake of DOX in case of VBIOTIN+DOX.HCl
vesicles as compared to the VDOX.HCl vesicles in HeLa cell lines. The results revealed that in
case of VBIOTIN+DOX.HCl, the cellular internalization of drugs was significantly higher than
VDOX.HCl proving that the biotin conjugation in the dextran vesicles indeed enhanced the DOX
concentration at tumour cells. The healthy cells (WT-MEF cell line in Figure 6b) do not over
express the biotin receptors; thus, the cellular uptake by the biotin-functionalized vesicles and
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biotin-free vesicles do not show much difference in their cellular uptake. This is further
evident from the comparison of the uptake by the biotin-functionalized vesicles in Figures 6a
(in HeLa) and 6b (in WT-MEF) that the former one showed significant uptake compared to
the latter. A similar experiment was carried out for VBIOTIN+DOX.HCl and VDOX.HCl in normal
cell line (WT-MEF) and the images are showed in figures 6d and 6e. Both VBIOTIN+DOXHCl
and VDOX.HCl did not so much increment in the cellular uptake with increasing the incubation
period. This data was further supported by the plot of DOX intensity versus incubation time
in Figure 6f. The comparison of HeLa and WT-MEF cell lines data revealed that the cancer
cells have preferential uptake for Biotin conjugated dextran vesicles (VBIOTIN) compared to
biotin free vesicles (V). This observation is further supported by the enhanced cell killing by
the dextran vesicles in HeLa when compared to WT-MEF cells (see Figures 5b and 5c). The
lyso-tracker experiments indicated that the dextran vesicles were readily digested by both cell
lines at the intracellular level; thus, the difference in the cellular uptake (see Figures 6) and
cytotoxicity effect (see Figure 5) among HeLa and WT-MEF cells is attributed to the
difference in receptor-mediated uptake. The enhanced uptake of VBIOTIN+DOX.HCl in HeLa cells
is attributed to the enhanced receptor-mediated endocytosis process. It is important to
mention that the esterase enzyme concentrations could be similar in both WT-MEF and
cancer cells for biodegradation; however, the higher receptor-mediated uptake in cancer cells
allowed more uptake of biotin-targeted nanovesicles (see Figure 5). Thus, the selectivity
accomplished in cancer cell is assisted by receptor mediated endocytosis process for
increasing the drug payload in cancer cells (see Figure 6). The higher accumulation of drug
carrier in cancer cells enabled enhanced cell killing in cancer cells compared to WT-MEF
cells. Thus, the selectivity is accomplished in the present design by merging both intracellular
biodegradation along with receptor-mediated endocytosis. To validate the receptor-assisted
cellular uptake observation in confocal image, time-dependent flow cytometry analysis (uses
live cell counting) was carried out for free DOX, VDOX, and VBIOTIN+DOX.HCl in HeLa cell line.
In figure 7a, the VBIOTIN+DOX.HCl and VDOX.HCl were found to exhibit almost 1.5 and 2.0 times,
respectively, higher uptake than free DOX. The incubation time dependent flow cytometry
data in Figure 7b further indicates that the DOX was taken up by the cells in higher quantity
with increase in the incubation time. The plot of DOX intensity versus incubation time (see
Figure 7c) further validates this observation. The comparison of data at 9 h incubation for
biotin-conjugated dextran vesicles and biotin free dextran vesicles further established the
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enhanced uptake of VBIOTIN+DOX.HCl in HeLa cell line. The above results directly evidence that
the biotin-conjugated dextran vesicles are excellent nano-carriers for anticancer drugs like
DOX.HCl to selectively target cancer cells compared to WT-MEF cells. In vivo data is
further required to support the receptor-mediated approach for long-term application in
cancer therapy. Nevertheless, the present manuscript has demonstrated the receptor-mediated
endocytosis process in polymer vesicle and the proof-of-concept is successfully demonstrated
in cancer cell lines.
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Figure 7: (a) Histogram showing the cellular uptake by flow cytometry analysis for free
DOX.HCl, VDOX.HCl and VBIOTIN+DOX.HCl at 9h incubation. (b) Flow cytometry plots for
VDOX.HCl and VBIOTIN+DOX.HCl at various incubation time. (c) The plot of DOX intensity against
incubation time estimated from figures a and b and shown in bar diagram. The concentration
of the DOX.HCl was maintained as 2 µg/mL and 10000 cells were counted for each
experiment.
Evidence for Receptor-mediated Endocytosis
Three control experiments were carried out to support the receptor-mediated cellular
uptake in HeLa cell lines and they are: (i) cellular uptake studies of dextran vesicles in biotin
pre-treated cancer cells, (ii) cellular uptake of dextran vesicles at low temperature by
supressing the regular endocytosis process, and (iii) time-dependent cytotoxicty assay. To
study the biotin-receptor mediated endocytosis of dextran vesicles; cellular uptake
experiment was carried out in HeLa cells at 37 ºC using biotin pre-treatment. HeLa cells were
pre-treated with 2 mM biotin for 1 h65-66. The pre-treated cells and control cells (without
biotin pre-treatment) were subjected to VDOX.HCl and VBIOTIN+DOX.HCL and incubated for 4 h at
37 °C. Confocal microscope images in figure 8a showed the uptake of DOX from
VBIOTIN+DOX.HCl in biotin-pre-treated HeLa cells. The biotin pre-treatment in the HeLa cells
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were carried out to block all the receptors with free biotin; hence one would anticipate that
the biotin-functionalized vesicles uptake in cells will be reduced due to the non-availability of
the receptors on the cell membranes. The comparison of the uptake of the biotin-
functionalized vesicles in HeLa cells indeed showed lower uptake in the biotin pre-treated
cells due to the non-availability of receptors (see Figure 8a). This control experiment
validates the concept of receptor-mediated endocytosis in the polysaccharide vesicles. DOX
florescence intensity was estimated using image J software and shown in figure 8b. The
biotin-tagged vesicles VBIOTIN+DOX.HCl showed four-fold better uptake in normal cells
compared to biotin pre-treated cells. Further, this observation was validated by flow
cytometry analysis (see figure 8c) with biotin pre-treated and normal HeLa cells. The results
in flow cytometry plots in figure 8c showed relatively more uptake of VBIOTIN+DOX.HCl by
HeLa cells without biotin pre-treatment. In figure 8d, the DOX internalization from VDOX.HCl
did not show much difference between HeLa cells irrespective of their exposure to biotin pre-
treatment or not. This observation was also validated by DOX intensity plot and flow
cytometry data in Figures 8e and 8f. From the above discussion, it is evident that these biotin
conjugated dextran vesicles can be very good candidates for targeted drug delivery in cancer
cells. The cellular internalization of the foreign materials like nano-objects through
endocytosis is an active transportation process and hence it is an energy-dependent process.
The cellular uptake experiments at lower temperature would reduce or inactivate the
endocytosis process and thereby reduce the uptake of the nano-carriers.67-68 To evaluate the
role of the temperature on the dextran nanovesicles internalisation in cells, temperature
dependent cellular uptake experiment was carried out in HeLa cells at 4 °C and 37 °C. The
HeLa cells were incubated with VDOX.HCl and VBIOTIN+DOX.HCl at 4º C and 37 °C for 30 minutes
and subjected to imaging using confocal microscope. At 37 °C, the cellular uptake of
VBIOTIN+DOX.HCl was two times better than VDOX.HCl which supported the enhancement in the
endocytosis of biotin-tagged vesicles (see Figure 9b). The uptake of VDOX.HCl was found to
be almost similar in both temperatuers indicating that the biotin free vesicles do not have
impact towards the receptor mediated uptake process. The uptake of VBIOTIN+DOX.HCl was
found to reduce almost half at 4 °C indicating that the receptor-mediated endocytosis
primarily occurred under physiological temperature at 37 °C.
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Figure 8: (a) Confocal images showing the cellular uptake of VBIOTIN+DOX.HCl, in HeLa cells
with or without biotin pre-treatment. (b) Corrected total cell fluorescence of DOX determined
from images and plotted in bar diagram for VBIOTIN+DOX.HCl in HeLa cells. (c) Flow cytometry
plots showing the cellular uptake of VBIOTIN+ DOX.HCl in HeLa cells with or without biotin pre-
treatment. (d) Confocal images showing the cellular uptake of VDOX.HCl, in HeLa cells with or
without biotin pre-treatment. (e) Corrected total cell fluorescence of DOX determined from
images and plotted in bar diagram for VDOX.HCl in HeLa cells. (f) Flow cytometry plots
showing the cellular uptake of VDOX.HCl in HeLa cells with or without biotin pre-treatment.
For biotin pre-treatment, the cells were initially exposed to 2 mM biotin for 1h and then 2
ꢀg/mL DOX. HCl and incubated for 4h.
Figure 9: (a) Cellular uptake of dextran vesicles VDOX.HCl and VBIOTIN+DOX.HCl, and the plot of
corrected total cell fluorescence of DOX Intensity at 4 ºC as incubation temperature. (b)
Cellular uptake of dextran vesicles VDOX.HCl and VBIOTIN+DOX.HCl and plot of corrected total
cell fluorescence of DOX Intensity at 37 ºC as incubation temperature. HeLa cells are treated
with 2
ꢀg/mL of DOX concentration and incubated for 30 minutes.
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Figure 10: (a) Schematics showing the time dependent MTT experiment. (b) Histogram
showing the cytotoxicities for free DOX, VDOX.HCl and VBIOTIN+DOX.HCl in HeLa cells at various
incubation time intervals. (c) Histogram showing the cytotoxicities for free DOX, VDOX.HCl
and VBIOTIN+DOX.HCl in WTMEF cells at various incubation time intervals.
The time-dependent cellular uptake studies (in Figure 6), flow cytometry analysis (in
Figure 7) and the biotin pre-treatment experiment (in Figure 8) and temperature dependent
endocytosis process (in Figure 9) supported the receptor-mediated drug administration in
cancer cells. Since there is no significant difference between the HeLa and WT-MEF cells in
terms of GSH and esterase enzyme concentration; one would anticipate similar intracellular
digestion of the nano-vesicles. Thus, the difference in the cancer cell growth inhibition
should arise from significant difference in their receptor-mediated drug accumulation or
uptake. To validate this concept, time-dependent cytotoxicity studies were carried out for
DOX concentration of 0.8 ꢀg/ mL (based on data in Figure 5b) and subjecting the cells for
MTT assay at various time intervals as shown in figure 10a. In figure 10b, the IC50 value (for
50 % cell killing) was attained at 12 h by biotin-tagged vesicles VBIOTIN+DOX.HCl in cancer
(HeLa) cell. This value was thrice much faster compared to free DOX (36 h) and and VDOX.HCl
(more than 48 h) in HeLa cell lines. In WT-MEF cells (see Figure 10c), the free DOX killing
was much faster (< 36 h) compared to both biotin-tagged and biotin free dextran vesicles
(more than 48 h). The higher uptake of the nano-carriers results in the better cell killing due
to the increase in the drug concentration. This is further evident from the comparison of the
time dependent MTT assay in Figure 10a and time dependent cellular uptake in figure 6. The
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biotin conjugated vesicles showed enhanced uptake which further reflected on their
cytotoxicity trend in suppression of cell growth in HeLa cell line. Thus the receptor-
medicated endocytosis and their influence on cell growth inhibition are validated by these
time dependent MTT and uptake studies. This experiment suggetsed that the biotin-tagged
vesicles VBIOTIN+DOX.HCl provided two advantages; (i) it achieves faster rate of cell growth
inhibition in cancer cells owing to its targeting ability, and (ii) it is also relatively less toxic
to the WT-MEF cells. These control experiments validate the targeting ability of the biotin-
tagged dextran vesicles in HeLa cell lines. In the present investigation, the approach is
demonstrated exclusively for DOX.HCl delivery in HeLa cells; however, the concept is not
restricted only to this example and it may be applied to large number of other cancer cells
that are over expressed in biotin-receptors. Further, the dextran vesicles are capable of
loading both water soluble and water insoluble drugs; thus, the approach can be in principle
expanded to variety of other anticancer drugs.
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Conclusion
In summary, biotin-conjugated dextran vesicles were made with multi-stimuli-
responsiveness and employed them for receptor-mediated endocytosis to enhance the uptake
of water soluble anticancer drug doxorubicin-hydrochloride in cancer cell lines. Renewable
resource hydrophobic unit consisting of glutathione-responsive disulfide bond and
enzymatic-biodegrabale aliphatic ester linkage was tailor made and conjugated along with
biotin on the dextran backbone to yield amphiphilic dextran. The amphiphilic dextran self-
assembled into 180 ± 20 nm vesicular nano-assemblies in aqueous medium and the size and
morphology of the vesicles was characterized by dynamic light scattering, electron and
atomic force microscopes. The binding ability of biotin-tagged dextran vesicle with
streptavidin like receptors in the cancer cell membrane was confirmed by Avidin-HABA
binding assay. The biotin-tagged vesicles showed affinity up to 25 nM towards the membrane
protein indicating their excellent capability to selectively target the biotin receptors over
expressed cancer cells. The dextran vesicle provided appropriate geometry to encapsulate
water soluble doxorubicin-hydrochloride at the core of the vesicles. Under the intracellular
stimuli such as GSH and lysosomal esterase enzymes, the vesicles ruptured to release the
drug in 60 and 90 %, respectively. The combination of both GSH and esterase together
accomplished 100 % drug release at the intracellular conditions. The intracellular lysosomal
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degradation of these dextran vesicles was confirmed by lysosomal tracker experiments using
confocal microscope. Enhanced cellular uptake of the biotin-conjugated vesicles was proven
by time dependent cellular uptake studies which were validated by confocal microscopy
imaging and flow cytometry analysis. Receptor mediated endocytosis mechanism was
supported by control experiments such as endocytosis at lower temperature, endocytosis with
biotin pre-treated cervical (HeLa) cell lines, and time dependent MTT assay and flow
cytometry. All these results provide direct evidence for the targeting ability of biotin
conjugated dextran vesicles in drug delivery. Based on the above observations, it can be
concluded that the newly designed dextran vesicles are promising candidates for targeted
delivery of anticancer drugs. Currently, the research work is focussed in this direction to
explore the polysaccharide vesicles as targeted drug delivery vehicles to achieve better
therapeutic efficacy in cancer.
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Supporting Information: Experimental details, TGA, DSC of the polymers, plot for
1
Avidin-HABA complex in PBS, FBS, drug release plots , H-NMR of DEX-SS-PDP and
DEX-Biotin, 1H-NMR , 13C-NMR , Tissue culture Conditions, drug encapsulation
procedure, drug release protocol, HRMS, MALDI-TOF of compound 1 and 2 etc are
provided in supporting information. This material is available free of charge via Internet at
http://pubs.acs.org.
Acknowledgments: The authors thank research grant from Department of Science and
Technology (DST) for the project SB/S1/OC-37/2013 Authors thank the confocal
microscopy facilities at Venture Centre NCL Innovation Park Pune for all the bio-
imaging experiments. Authors thanks national chemical laboratory (NCL, Pune) for
HR-TEM facility. Nilesh thanks Mehak Malhotra and Sonashree Saxena for their help
in Bioimaging. Nilesh U. Deshpande thanks UGC for research fellowship.
Author Information: Corresponding Author: Prof. Manickam Jayakannan
[jayakannan@iiserpune.ac.in]. The authors declare no financial interest.
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