Reduction Responsive Nanovesicles Derived from Novel α

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Reduction Responsive Nanovesicles Derived from Novel #Tocopheryl-Lipoic Acid Conjugates for Efficacious Drug Delivery to Sensitive and Drug Resistant Cancer Cells Bappa Maiti, Krishan Kumar, Parikshit Moitra, Paturu Kondaiah, and Santanu Bhattacharya Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00497 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Glutathione Responsive Drug Delivery to Sensitive and Drug Resistant Cancer Cells S

S S S

S

S

S S

S S

S

S S

S S

S S

S S

S

S

HS HS HS HS SH SH

SH

SH

HS

S

S

S S

S S S

SH SH SHSH

S

S

HS SH HS S

SH

S S

S

S

S S

SH

HS

HS

S

S S

SH SH SH

SH

S

S

S

S S

S S

S

S S

S

S S S

S

S S

S S

S

TL1

Cytoplasm

DOX-loaded liposome inside HeLa-DOXR Cells DOX

Nucleus

GSH S S

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TL1

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Reduction Responsive Nanovesicles Derived from Novel αTocopheryl-Lipoic Acid Conjugates for Efficacious Drug Delivery to Sensitive and Drug Resistant Cancer Cells Bappa Maiti,†, §, ǁ Krishan Kumar,†, ǁ Parikshit Moitra,∆ Paturu Kondaiah,‡ Santanu Bhattacharya†, §, ∆, * †

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India Director’s Research Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India ∆ Technical Research Centre, Indian Association for the Cultivation of Science, Kolkata 700032, India ‡ Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India *Corresponding author at: Director’s Research Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India. E-mail: [email protected], Fax: +91 33 2483 6561, Tel: +91 33 2473 4688. §

S

Supporting Information

ABSTRACT: Two novel α-tocopheryl-lipoic acid conjugates (TL1 and TL2) were synthesized for the anticancer drug, doxorubicin (DOX) delivery. Both conjugates were able to form stable nanovesicles. The critical aggregation concentration (CAC) was determined using 4-(N, N-dimethylamino) cinnamaldehyde (DMACA) as a fluorescence probe. Formation of highly packed nanovesicles was characterized by 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) fluorescence anisotropy and microviscosity measurements. The morphologies of nanovesicles were visualized by transmission electron microscope (TEM) and atomic force microscope (AFM). The response of nanovesicles to reducing environment of cells was probed by the addition of dithiothreitol (DTT), which was followed by the increase in hydrodynamic diameter in dynamic light scattering (DLS) measurements. The encapsulation efficiency of a commonly used anticancer drug, doxorubicin (DOX) in nanovesicles was found to be ~60% and ~55% for TL1 and TL2 respectively (TL1-DOX and TL2-DOX). Also, the cumulative drug (DOX) release from DOX-encapsulated nanovesicles in response to biological reducing agent glutathione (GSH) was ~50% and ~40% for TL1-DOX and TL2-DOX respectively over a period of 10h. Both TL1-DOX and TL2-DOX delivered the anticancer drug, doxorubicin (DOX) across the DOX-sensitive and DOX-resistant HeLa (HeLa-DOXR) cells in an efficient manner and significantly more efficaciously than the drug alone treatments especially in HeLa-DOXR cells. The nanovesicle mediated DOX treatment also showed significantly higher cell death when compared to DOX alone treatment in HeLa-DOXR cells. Blood compatibility of the nanovesicles was supported from clotting time, hemolysis and red blood cells (RBCs) aggregation experiments for their potential in vivo applications. Concisely, we present biocompatible and responsive nanovesicles for efficacious drug delivery to drug sensitive and drug-resistant cancer cells.

INTRODUCTION The chemotherapeutic treatments are indispensable tools in the treatment of cancer.1-4 Such therapies involve the use of drug molecules that inhibit the cellular proliferation leading to cell death. In the traditional chemotherapy, the efficacy levels are often compromised due to the drug overload, which leads to undesirable side effects. Among such cases, doxorubicin (DOX) is a widely known chemotherapeutic drug, which is currently used clinically to treat different types of cancers like breast cancer, acute leukemia, small cell lung carcinoma, gastric cancer and ovarian cancer. But such treatments also account for severe sideeffects such as cardiotoxicity, hepatotoxicity, nephrotoxicity and damage to healthy cells due to the presence of the free drug.5-8 Another obstacle arises during the chemotherapeutic drug treatments is drug resistance. After repeated treatment of chemotherapeutic drug like DOX, cancer cells become drug resistant.9 The drug treatment under such cellular resistance requires higher dose, which in turn gives rises to severe and unwanted side-effects. Therefore, it is necessary to develop vehicles for delivering the drugs efficiently without using an excess of them by considering the factors restraining the delivery efficacy levels in both drug sensitive and resistant cancer cells. Nanobiomaterials comprise an important class of such delivery systems, which show promise in delivering different anticancer

Figure 1. Molecular and energy optimized (CPK space filling model; C: cyan, H: white, N: blue, O: red, S: yellow) structures of the tocopheryl-lipoic acid conjugates, TL1 and TL2.

drugs to various cancer cells.10, 11 These increase biocompatibility and decrease unwanted side effects.12 Various nanobiomaterials available, which include micelles, vesicles and self-assembled prodrugs.13-20 Tocopherol or vitamin E is an important naturally occurring membrane-soluble molecule well known for its antioxidant properties.21 Tocopherol or their derivatives have been used to co-

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Bioconjugate Chemistry Scheme 1a: S S

HO

OH

O O

α-Tocopherol

Lipoic Acid

O

HO

O

TsO

i O

O 1

2-Tocopheroloxyethanol ii

HO

N

iii

HO

O

O

N

O

O

2

3

OH

v

iv

S S O O

N

O

O

N

O

O

O

O O

TL1

O

TL2

S S S S

a

Reagents, Conditions and Yields: i) TsCl, Py, CH2Cl2, DMAP(cat), 6h, rt, 85%; ii) N-methyl ethanolamine, CH3CN, EtOH, 80 °C, reflux, 24h, N2-atm, 70%; iii) Diethanolamine, CH3CN, EtOH, 80 °C, reflux, 24h, N2-atm, 71%; iv) Lipoic acid, DIPC, DMAP (cat), DCM, rt, 66%; v) Lipoic acid, DIPC, DMAP (cat), DCM, rt, 72%.

-nstruct both gene and drug delivery vehicles.22-26 Further vitamin E derivatives like tocopherol succinate (α-TOS) and tocopherol polyethylene glycol succinate (TPGS) have inherent anti-cancer activities.27, 28 Such systems either by themselves or as prodrugs are used as components in micellar or vesicular formulations, which may accommodate different anticancer drugs and efficiently deliver the same to cancer cells.29-34 Another naturally occurring stimuli-responsive molecule, lipoic acid has been used in the polymeric micellar system to introduce sensitivity toward cellular reducing environment mainly to exploit high concentrations of reducing GSH present in intracellular compartments (1-11 mM) compared to the extracellular tissues (10 µM in plasma).35, 36 The disulfide bond, which is stable in plasma, however, in the presence of high concentration of intracellular GSH breaks and perturbs the molecular structure. The resulting organization at the aggregate level of the carrier will then triggers a quick release of the drug.37-46 To fulfill such objectives, we introduce herein two new tocopherol-based molecular entities, which are covalently connected to lipoic acid via a successive scissile ester linkage to form the final tocopheryl lipoic acid conjugates (TL1 and TL2, Figure 1). Here we disclose the remarkable properties of these molecules, which spontaneously respond to a chemical stimuli of cell redox potential (concentration gradient of GSH). Upon dispersion in aqueous media these newly synthesized molecules by hydration method afforded nanovesicles. The CAC was measured using DMACA as a fluorescent probe. The aggregates were further characterized by TEM and AFM measur-

-ements. DPH fluorescence anisotropy and microviscosity indicated bilayer like packing in such aggregates. A nonbiological reducing agent such as DTT induced changes in vesicular sizes, which was followed by DLS measurements. The encapsulation of DOX inside nanovesicles and release under the influence of external stimuli were measured by the change in UVVis spectra. These nanovesicles were able to encapsulate DOX efficiently and release the drug in response to the treatment of GSH. The DOX encapsulated nanovesicles were used to treat both DOX-sensitive HeLa cells and DOX-resistant HeLa cells and the activities were compared to that DOX alone treatments. The DOX encapsulated nanovesicles had comparable efficiency to that of the drug alone treatment in terms of cell viability loss in sensitive cancer cells but more efficacious in resistant cancer cells. These behaviors of nanovesicles present them as promising nanocarriers for the delivery of chemotherapeutic drug molecules in cancer treatments. For the use of these nanovesicles for in vivo studies, the stability in the bloodstream is essential. The basic blood compatibility was evaluated by clotting time, hemolysis and RBCs aggregation measurements. The observations revealed that the nanovesicles could be potential candidates for in vivo applications as well.

RESULTS AND DISCUSSION Synthesis of Tocopherol-lipoic Acid Conjugates. Tocopherollipoic acid conjugates (TL1 and TL2) were prepared by the following a procedure described here and summarized in Scheme 1. We had synthesized both of these compounds from (±) α-tocop-

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Figure 2. (A-B) Normalized (w.r.t DMACA in H2O) fluorescence intensity at 485 nm of doped DMACA (5 µM) at different conjugates concentration. (C) The morphological features of nanovesicles of TL1 (1 and 3) and TL2 (2 and 4) respectively as revealed from the TEM (1 and 2) and AFM (3 and 4) studies.

-herol. Firstly, 2-tocopheryloxyethanol was prepared from (±) α tocopherol by following a reported procedure in literature.47, 48 Tosylation of 2-tocopheryloxyethanol was carried out with TsCl and pyridine in dichloromethane to get 2-tocopheryloxyethyl tosylate. 2-(methylamino)ethanol was reacted with 2tocopheryloxyethyl tosylate in CH3CN and EtOH (v/v 1:1) to give 2-(N-(2-tocopheryloxyethyl)-N-methylamino)ethanol (2). 2Tocopheryloxyethyl tosylate (1) was further reacted with diethanolamine to form 2(N-(2-tocopheryloxyethyl)-N, Naminodiethanol (3). The final compound TL1 and TL2 were produced via esterification with lipoic acid in presence of N, N'diisopropylcarbodiimide (DIC) and a catalytic amount of DMAP (Scheme 1). Detailed synthetic procedures and characterizations are given in the material and method section. Characterization of Nanovesicles. Critical aggregation concentrations (CAC) for TL1 and TL2 was measured using DMACA as a probe (Figure S1A-B, SI).49, 50 The break point of I485 vs concentration plot i.e CAC for TL1 and TL2 were 0.1 mM and 0.06 mM respectively (Figure 2A-B). So, we kept the concentrations for both the conjugates as ~1 mM, which were higher than the CAC values. The conjugates, TL1 and TL2 were cast into the thin film, which was subjected to hydration to prepare stable nanovesicular formulations.51, 52 TEM analysis revealed that the aggregates derived from both TL1 and TL2 were well-defined spherical vesicles of nanometer sizes (~100 nm) (Figure. 2C 1-2).53, 54 AFM analysis on these nanoaggregates in mica surface also showed the presence of spherical particles of 100-120 nm sizes (Figure 2C 3-4 and S2A-

Conjugate

Ƭf = (ns)

Anisotropy (r) ƬR(ns)

ɳ (cP)

TL1

3.68

0.27

11.12

146

TL2

2.49

0.30

12.05

158

Table 1. Fluorescence lifetime (τf), rotational correlation time (τR) and anisotropy (r) of DPH probe molecules and microviscosity (ƞ) around DPH doped in the aggregates of conjugates (TL1 and TL2).

B, SI).22 The hydrodynamic diameters of the nanovesicular suspensions observed for both the nanovesicular formulations were in the range of 140-170 nm (Figure 3A and S5A-B, SI) on the basis of DLS measurements. The sizes observed under DLS were relatively larger compared to that of TEM presumably due to the differences in the methods of sample preparation, which might have led to certain degree vesicle shrinkages.51-54 Fluorescence anisotropy (FA) measurements using DPH doped TL1 and TL2 aggregates (at higher concentration than CAC) (Table 1) gave high fluorescence anisotropy values (0.27 for TL1and 0.30 for TL2) for both the aggregates indicating that the probe was confined in a ‘rigid’ bilayer-like environment.55, 56 To further ascertain this, we also determined the microviscosity around DPH in the aggregate from the fluorescence lifetime measurements of DPH molecule (Figure S3A-B, SI). The values of microviscosity for TL1 and TL2 were 146 and 158 cP respectively, suggesting highly packed bilayer like arrangements

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in such aggregates (Table 1).57 From these experiments we assume that in both conjugates (TL1 and TL2) the tertiary ‘N’ atom, which is expected to be hydrated in water acts as a hydrophilic headgroup. The both α-tocopherol and lipoic acid backbone act as hydrophobic part of the vesicular bilayer. Also, due to the hydrophobicity of disulfide bond in lipoic acid, helps it to be embedded in the bilayer. The structural differences of TL1 (one lipoic acid moiety) and TL2 (two lipoic acid moieties) make their bilayer packing little different. The plausible molecular packing in the bilayer is given in Figure S4. Subsequently, we attempted a reduction triggered destabilization of the nanovesicles, which was monitored by DLS measurements using DTT (10 mM). This showed a significant increase in the size of vesicles (>500 nm) upon incubation for a period of 1h (Figure 3A and S5A-B, SI). This increase in size was manifested most probably due to the reduction of disulfides and cross-linking, which triggered a damage to the organization of the nanovesicular aggregates. Such reduction sensitive transformations are useful in the scenario of biological application wherein intracellular reductive environment (presence of GSH) assists in the release of loaded cargoes.58-60 DOX Encapsulation and Stimuli-Responsive Release from Nanovesicles. To exploit the very nature of these nanovesicles for a relevant application, the commonly used chemotherapeutic drug, doxorubicin (DOX) was encapsulated into the nanovesicles and encapsulation efficiency were ~60% for TL1 and ~55% for TL2 (Figure S6A, SI) respectively as described in the material and method section.61 The amount of DOX entrapped inside vesicles were calculated from the calibration plot of DOX (Figure S6B, SI). Hydrodynamic diameters (size) of the encapsulated nanovesicles were found to be 160±5 nm for TL1-DOX and 200±10 nm for TL2-DOX respectively (Figure S7, SI). The morphology of the encapsulated nanovesicles, observed from AFM were also nearly spherical like the unencapsulated nanovesicles (Figure S8A-B, SI) and DOX fluorescence was clearly visible in their fluorescence microscopic images (Figure S9A-B, SI).

nonbiological reducing molecules, such as DTT induced a significant release (TL1-DOX; ~60% and TL2-DOX;~50%) of DOX from nanovesicles within a period of one hour (Figure 3B) demonstrating the reduction responsive nature of the vesicles for the release of the drug. The remaining drug was subsequently released within a period of ~10h. The phenomenon of drug release in response to the intracellular reducing environment was also checked using the natural reducing agent of cells, GSH (5 mM) and the released DOX was measured over a period of 10h (Figure S11A-C, SI). The TL1-DOX and TL2-DOX nanovesicles showed a distinct response to GSH treatment. TL1-DOX formulations released ~50% of the drug and relatively more responsive than TL2-DOX formulations, which released ~40% of the encapsulated drug (Figure 3C). This might be attributed to the differences in the molecular packing during aggregation. Doxorubicin Delivery across DOX-Sensitive and DOXResistant HeLa Cells. Such reduction responsive nanocarriers are often useful in terms of their application towards the effective drug delivery in vitro. 64-66 For the realization of full potential of nanocarriers towards chemotherapeutic applications, there are growing efforts toward developing the nanocarriers that are devoid of any cytotoxic response and efficacious enough in order to become beneficial at practical grounds. Therefore, we first examined the cell viability of HeLa cancer cells treated with nanovesicles, TL1 and TL2 alone. Interestingly, the cellular treatments of both the vesicular formulations did not indicate any decrease of the cell viability counts and profiled both the nanovesicles to be biocompatible (Figure S12, SI). This was probably due to the fact that the conjugate was derived from the naturally occurring molecules, i.e., tocopherol and lipoic acid.67 Subsequently, we performed flow cytometry experiments to examine the DOX cellular transport to HeLa cells in the presence of serum by means of TL1-DOX and TL2-DOX formulations and compared the data with that of DOX alone treatments. The nanovesicular formulations could efficiently deliver DOX to cell and DOX cellular internalization was even found to be higher

For any vesicular drug delivery system to be successful, it should also manifest significant stability in the presence of serum.62, 63 Interestingly, both the DOX encapsulated nanovesicular formulations were quite stable and did not show any significant drug release (TL1-DOX; ~10% and TL2-DOX; ~20%) even after 10h of incubation in presence of 10% serum (FBS) in 10 mM phosphate buffer at pH 7.4 (Figure 3C and S10 A-B, SI). But, the

-DTT +DTT

400 200 0

% DOX Release

A

90

TL2

1h 10 h

60 30 0

TL1

B

TL1-DOX

TL2-DOX

% DOX Release

100

600 Size (nm)

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Bioconjugate Chemistry

80

C

FBS GSH

60 40 20 0

TL1-DOX

TL2-DOX

Figure 3 (A) The size of nanovesicles of TL1 and TL2 and the effect of DTT addition (1h incubation) on the hydrodynamic diameters of nanovesicles under DLS measurements. (B) The drug (DOX) release from TL1-DOX and TL2-DOX formulations in the presence of DTT (10 mM) after 1 and 10h respectively. (C) The stability of TL1-DOX and TL2-DOX formulations in the presence of 10% serum (FBS) and GSH (5 mM) induced DOX release from nanovesicles after 10h.

Figure 4. (A) The cellular internalization of DOX in HeLa cells at different concentrations after 4h incubation. Statistical significance when compared to DOX alone treatment (***P < 0.001). (B) Cell viability of HeLa Cells (MTT assay) after 24h of DOX treatments mediated by TL1-DOX, TL2-DOX and DOX alone. Representative confocal microscopic images for DOX internalization by means of TL1-DOX (C2) as compared to DOX alone treatment (C1). Scale bar is 10 µm.

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than the cells treated with DOX alone (Figure 4A). The higher efficacy in cellular transport of the drug by nanovesicles was further substantiated and also quantified with the help of confocal microscopy imaging. The confocal microscopic analysis revealed that the DOX fluorescence intensity was higher in the cells treated with DOX-encapsulated nanovesicles compared to those treated with DOX alone (Figure 4C, Figure S13, SI). This observation, in turn, demonstrated the potential of these nanovesicles to effectively deliver drugs into cells. The drug resistance acquired by the cells during the drug treatments is one of the serious impediments in chemotherapeutic treatment.9, 68 The chemotherapy under such cellular resistance against drugs lead to the requirement of repeated treatments with higher drug doses which in turn gives rise to severe and unwanted side-effects. The stimuli-responsive drug nanocarriers have shown a considerable promise for being a tool to overcome the barrier of drug resistance while improving the drug accumulation inside the cells.69-73 Accordingly, we used TL1-DOX and TL2-DOX nanovesicles for the treatment of DOX-resistant HeLa cells (HeLa-DOXR). It was observed that the nanovesicles effectively delivered the drug inside HeLa-DOXR cells and the intracellular DOX concentration was much higher and more effective than DOX alone treatment as revealed from the confocal microscopic analysis (Figure 5C, Figure S13, SI). We also performed flow cytometry to distinguish the intracellular DOX accumulation in HeLa-DOXR cells after DOX treatments by means of nanovesicles and DOX alone, which corroborated the events observed under confocal microscopy (Figure 5A). We determined the cytotoxicity level of DOX encapsulated nanovesicles in HeLa and HeLa-DOXR cells and compared the same with that of DOX alone treatments (Figure 4B, 5B and S14, SI). In DOX sensitive HeLa cells, the IC50 value of DOX-encaps-

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-ulated nanovesicles (TL1-DOX; 9.79±1.83 µg/mL and TL2DOX; 12.71±1.44 µg/mL) were almost comparable to DOX alone (14.59±1.28 µg/mL) after 24h treatment. It was also observed that TL1-DOX nanovesicles were slightly more efficacious than TL2DOX nanovesicles probably due to their structural packing differences around DOX in encapsulated nanovesicles, which contributes to their drug release profile inside cancer cells. The IC50 values were also calculated in DOX-resistant HeLa cell (HeLa-DOXR) in presence of DOX-encapsulated nanovesicles and DOX alone after 72h treatment. Due to DOX resistive nature of HeLa-DOXR cells, free DOX had significantly higher IC50 value (66.75±2.12 µg/mL). However, the treatment using DOXencapsulated vesicles led to a significant decrease in IC50 values (TL1-DOX; 32.81±1.26 µg/mL and TL2-DOX; 42.99±2.36 µg/mL). Compatibility of Nanovesicles for in vivo Experimentation. For the use of these nanovesicles for in vivo application, the concentration-dependent stability of nanovesicles was evaluated. The concentration of the vesicular conjugates (TL1 and TL2) was studied upto 6 mM. It was observed that upto 3 mM concentration, the nanovesicular formulations were stable enough without any sign of precipitation. The hydrodynamic diameters (sizes) of both the nanovesicles (~1 mM concentration) were almost consistent for one week without any sign of agglomeration (Figure S15, SI). One of the important criteria for in vivo applications is the blood compatibility, which was evaluated by clotting time measurements, hemolysis and hemagglutination (RBCs aggregation) studies.74, 75 The clotting time, which is platelets related phenomenon, is an indicator of hemostasis. The clotting of blood on a glass surface in presence of normal saline took ~6.8 min. Any deviation from this time was taken as abnormal behavior. The polyethylenimine (PEI, MW ~25 kDa) was used as positive control and had clotting time of ~1.3 min. The clotting time measurements were performed with a series of nanovesicular concentrations from 0.5-2 mM and have been presented in the supporting information (Table S1). A very little deviation of clot time from negative control was observed even with the treatment of concentration as high as 2 mM. Hemolysis is another phenomenon where hemoglobin leaks from ruptured RBC in presence of non-compatible compounds. Percentage of hemolysis was calculated for different concentrations of nanovesicles and presented in the supporting information (Table S2). As evident from the data, only ~5 % of hemolysis occurred for concentrations as high as 2 mM of the nanovesicles. Also, binding of nanovesicles with RBC membrane could cause circulatory disorders. When these nanovesicles were incubated with RBCs, little aggregations were observed, which were almost comparable to the negative control (normal saline). On the other hand, in presence of PEI (positive control), the aggregated RBCs were clearly visible in the phase contrast microscopic image (Figure S16, SI).

Figure 5. (A) The cellular internalization of DOX (10 µg/mL) in DOX-resistant HeLa (HeLa-DOXR) cells at different time points. Statistical significance when compared to DOX alone treatment (*P < 0.05, **P < 0.01 and ***P < 0.001). (B) Cell viability of HeLa-DOXR cells (MTT assay) after 72h of DOX treatments mediated by TL1-DOX, TL2-DOX and DOX alone. Representative confocal microscopic analyses for DOX internalization by means of TL1-DOX (Cb) as compared to the free DOX alone treatment (Ca). Scale bar is 10 µm.

CONCLUSION AND REMARKS Taken together from all of the above observations, it may be inferred that these reduction responsive, serum tolerant and biocompatible nanovesicles derived from tocopheryl-lipoic acid conjugates are promising tools for improving the efficacy of drug treatment in both drug sensitive and drug-resistant cells. Such stimuli-responsive nanovesicles should be of great interest in

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Bioconjugate Chemistry

MDR (multi-drug resistant) cell lines wherein the intracellular GSH concentration is relatively higher compared to that of normal cell lines. However, the relatively high CAC values of the nanovesicles could be addressed by doping these conjugates with natural or synthetic lipids with inherently low CAC values without compromising their properties for practical applications. Concisely, the stability and blood compatibility experiments reveal that the drug formulations derived from these conjugates could be ideal candidates for drug delivery applications.

MATERIALS AND METHODS Materials. All solvents and reagents were purchased from the best-known sources with high purity. Lipoic acid and (±) αtocopherol were purchased from Sigma-Aldrich. Silica gel of mesh size 60-120 was used for open column chromatography to purify all compounds. All compounds were fully characterized and the final compounds, TL1 and TL2, are analyzed by 1H NMR, 13 C NMR, mass spectra, FT-IR and elemental analysis. 1H NMR of compounds was recorded on a Bruker-400 Avance NMR spectrometer at 400 MHz and for 13C NMR at 100 MHz. Mass spectra were measured on a MicroMass ESI-TOF spectrometer. Infrared spectra were recorded on a Jasco FT-IR 410 spectrometer. Elemental analysis was recorded on a Carlo Erba elemental analyzer model 1106. The absorbance of DOX has measured on Shimadzu model 2100 spectrophotometer. Dulbecco’s modified Eagle’s medium (DMEM, Sigma), Dulbecco’s phosphate-buffered saline (DPBS, Sigma) and fetal bovine serum (FBS, Gibco) were used for the cell culture. Synthetic Procedure. 2-Tocopheryloxyethanol was prepared from (±) α-tocopherol by following a reported procedure in literature.48 2-Tocopheryloxyethyl tosylate (1). To a solution of 2tocopheryloxyethanol (1.2 g, 2.53 mmol) in dichloromethane (20 mL), pyridine (1 mL) and p-toluenesulfonyl chloride (0.72 g, 3.8 mmol) were added at 0 °C. The reaction mixture was stirred at room temperature for 6h. Then solvent was removed in vacuum to get residual product, which was dissolved in ethyl acetate (100 mL). The ethyl acetate layer was further washed with 1N HCl (2 X 50 mL), saturated NaHCO3 (2 X 50 mL) and water (2 X 50 mL).The ethyl acetate layer was filtered over anhydrous sodium sulphate and evaporated to get crude product, which was purified by column chromatography over silica gel (mesh size 60-120) with eluting n-hexane and ethyl acetate (v/v 100:4). The isolated yield of 2-tocopheryloxyethyl tosylate was 1.35 g, 85%. FT-IR (Neat, cm-1) 3054, 2962, 2866, 1597, 1457, 1417, 1262, 1175, 1091, 1017, 921, 814, 734, 704, 663; 1H NMR (400 MHz, CDCl3, ppm) δ 0.83-0.87 (m, 12H, -CH-CH3, phytyl chain), 1.07-1.83 (m, 26H), 2.05 (s, 9H, -CH3), 2.08 (s, 3H, -CH3), 2.44 (s, 3H), 2.54 (t, 2H, J = 6.8 Hz), 3.85 (t, 2H, J = 4.8 Hz), 4.33 (t, 2H, J = 4.4 Hz), 7.33-7.35 (d, 2H, J = 8 Hz), 7.83-7.85 (d, 2H, J = 8 Hz); 13C NMR (100 MHz, CDCl3, ppm) δ 11.72, 11.77, 12.63, 19.55, 19.65, 19.72, 20.57, 21.00, 21.62, 22.60, 22.69, 23.81, 24.40, 24.77, 27.97, 31.13, 31.18, 32.64, 32.66, 32.76, 37.25, 37.30, 37.42, 37.54, 39.33, 39.99, 40.03, 69.09, 69.93, 74.84, 117.59, 122.95, 125.71, 127.61, 127.96, 129.81, 132.99, 144.79, 147.37, 148.04; HRMS (ESI) m/z calcd for [C38H60O5S + Na]+: 651.4059; found: 651.4059. 2-(N-(2-tocopheryloxyethyl)-N-methylamino)ethanol (2). To a solution of 2-tocopheryloxyethyl tosylate (0.67 g, 1.067 mmol) in dry acetonitrile (15 mL), ethanol (3 mL) and N-methyl

ethanolamine (2 mL) were added and the resulting solutions refluxed for 12h. After this the reaction mixture was isolated, evaporated and diluted with chloroform (200 mL). The chloroform was washed with water (2 X 50 mL), saturated brine (2 X 50 mL) and dried over anhydrous sodium sulphate. Then chloroform layer was evaporated in vacuum to get a crude product. The compound was purified by column chromatography over silica gel (mesh size 60-120) upon eluting with a mixture of chloroform and methanol (v/v 100:4). The isolated yield was 0.39 g, 70%. FT-IR (Neat, cm-1) 3420, 3053, 2925, 2865, 1475, 1414, 1376, 1262, 1159, 1088, 1037, 894, 734, 705; 1H NMR (400 MHz, CDCl3, ppm) δ 0.83-0.87 (m, 12H, -CH-CH3, phytyl chain), 1.07-1.83 (m, 26H, tocopheryl protons), 2.07 (s, 9H, -CH3), 2.13 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 2.56 (s, 3H), 2.56 (t, 2H, J = 6.8 Hz), 2.68 (t, 2H, 5.2 Hz), 2.87 (t, 2H, J = 5.6 Hz), 3.64 (t, 2H, J = 5.2 Hz), 3.75 (t, 2H, J = 5.2 Hz); 13C NMR (100 MHz, CDCl3, ppm) δ 11.75, 11.88, 12.75, 19.59, 19.65, 19.71, 20.62, 20.99, 22.59, 22.69, 23.83, 24.40, 24.78, 27.94, 31.02, 31.25, 32.64, 32.66, 32.74, 37.25, 37.35, 37.42, 37.54, 39.33, 39.97, 40.01, 42.38, 57.09, 58.56, 59.22, 70.07, 74.76, 117.53, 122.87, 125.67, 127.65, 147.79, 148.16; HRMS (ESI) m/z calcd for [C34H61NO3 + H]+: 532.4731; found: 532.4731. 2(N-(2-tocopheryloxyethyl)-N, N-aminodiethanol (3). To a solution of 2-tocopheryloxyethyl tosylate (0.67 g, 1.067 mmol) in acetonitrile (15 mL), ethanol (5 mL) and diethanolamine (3 mL) were added together. Then resulting solution was refluxed for 12h. After cooling the solvent was evaporated from the reaction mixture. The residue was diluted with chloroform (200 mL) and the chloroform layer was washed with water (2 X 50 mL), saturated brine (2 X 50 mL) and dried over anhydrous sodium sulfate. The chloroform was removed to a get crude product. Then the compound was purified by column chromatography over silica gel (60-120 mesh size) upon eluting with a mixture of chloroform and methanol (v/v 100:4). The isolated yield was 0.5 g, 71%. FTIR (Neat, cm-1) 3426, 3016, 2925, 2866, 1457, 1413, 1374, 1256, 1213, 1172, 1086, 922, 750; 1H NMR (400 MHz, CDCl3, ppm) δ 0.83-0.87 (m, 12H, -CH-CH3, phytyl chain), 1.07-1.83 (m, 26H, tocopheryl protons), 2.07 (s, 9H, -CH3), 2.13 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 2.56 (t, 2H, J = 6.8 Hz), 2.81 (t, 4H, J = 5.2 Hz), 3.00 (t, 2H, J = 5.2 Hz), 3.13 (s, 2H), 3.68 (t, 4H, J = 4.8 Hz), 3.74 (t, 2H, J = 5.2 Hz); 13C NMR (100 MHz, CDCl3, ppm) δ 11.75, 11.96, 12.83, 19.62, 19.66, 19.77, 20.63, 21.01, 22.60, 22.69, 23.80, 24.41, 24.77, 27.94, 31.25, 32.65, 32.67, 32.74, 32.76, 37.26, 37.35, 37.42, 39.34, 39.36, 40.02, 54.57, 57.09, 59.84, 71.17, 74.84, 77.21, 117.58, 122.96, 125.57, 127.54, 147.91, 147.96; HRMS (ESI) m/z calcd for [C35H63NO4 + H]+: 562.4835; found: 562.4835. Mono-Lipoic acid Conjugate (TL1). To a solution of lipoic acid (0.15 g, 0.733 mmol) in 10 mL dichloromethane, N,N′diisopropylcarbodiimide (0.09 g, 0.73 mmol) and DMAP (8 mg, 0.073 mmol) were added and the mixture was stirred for 30 minute at room temperature. Then the compound 2 (0.39 g, 0.733 mmol) in dichloromethane (5 mL) was added to the above mixture and stirred together at room for 12h. After that it was diluted with dichloromethane (100 mL) and washed with water (2 X 20 mL), saturated brine (2 X 20 mL) and dried over anhydrous sodium sulphate. The organic solvent was then evaporated to get a crude product, which was purified by column chromatography over silica gel (mesh size 60-120) by eluting with pet ether and ethylacetate (v/v 65:35) as solvent mixtures. The isolated yield of TL1 was 0.35 g, 66%. FT-IR (Neat, cm-1) 2923, 2856, 1733, 1633, 1598, 1529, 1456, 1373, 1309, 1252, 1216, 1168, 1088, 1013, 914, 803, 753; 1H NMR (400 MHz, CDCl3, ppm) δ 0.830.87 (m, 12H, -CH-CH3, phytyl chain), 1.07-1.7 (m, 26H), 1.6-1.7

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(m, 4H), 1.83-1.90 (m, 2H), 1.90-1.95 (m, 1H), 2.08 (s, 9H, -CH3), 2.13 (s, 3H, -CH3), 2.17 (s, 3H, -CH3) 2.34 (t, 2H, J = 7.2), 2.42 (s, 3H), 2.44 - 2.54 (m, 1H), 2.57 (t, 2H, J = 6.8 Hz), 2.81 (t, 2H, J = 6 Hz), 2.88 (t, 2H, J = 6 Hz), 3.1-3.2 (m, 2H), 3.54 – 3.6 (m, 1H), 3.76 (t, 2H, J = 6 Hz) 4.23 (t, 2H, J = 5.6 Hz); 13C NMR (100 MHz, CDCl3, ppm) δ 11.70, 11.94, 12.08, 19.50, 19.65, 19.71, 20.62, 21.00, 22.60, 24.76, 27.93, 28.71, 29.66, 30.27, 31.18, 31.23, 32.64, 32.73, 33.99, 35.56, 37.23, 37.34, 37.53, 38.43, 39.32, 40.02, 40.06, 40.16, 43.07, 55.93, 56.28, 57.42, 62.08, 70.69, 74.74, 117.47, 122.78, 125.64, 127.63, 147.72, 148.28, 173.46. HRMS (ESI) m/z calcd for [C42H73NO4S2 + H]+: 720.5059; found: 720.5059; Anal. Calcd for C42H73NO4S2: C 70.05, H 10.22, N 1.94. Found: C 70.10, H 10.29, N 1.98. Di-Lipoic acid Conjugate (TL2). To a solution of lipoic acid (0.183 g, 0.89 mmol) in dichloromethane (10 mL), N,N′diisopropylcarbodiimide (0.112 g, 0.89 mmol) and DMAP (10 mg, 0.089 mmol) were added and stirred for 30 min at room temperature. Then the compound 3 (0.5 g, 0.89 mmol) in dichloromethane (5 mL) was added to the resulting solution and stirred at room for 12h. After that the mixture was diluted with dichloromethane (100 mL). Then dichloromethane layer was washed with water (2 X 20 mL), saturated brine (2 X 20 mL) and dried over anhydrous sodium sulphate. This solution was evaporated to a get crude product, which was purified by column chromatography over silica gel (mesh size 60-120) by eluting with n-hexane and ethyl acetate (v/v 50:50) as solvent mixtures. The isolated yield of TL2 was 0.6 g, 72%. FT-IR (Neat, cm-1) 3019, 2926, 1729, 1457, 1375, 1262, 1213, 927, 749; 1H NMR (400 MHz, CDCl3, ppm) δ 0.83-0.87 (m, 12H, -CH-CH3, phytyl chain), 1.07-1.7 (m, 28H), 1.7-1.8 (m, 8H), 1.83-1.90 (m, 2H), 1.90-1.95 (m, 2H), 2.07 (s, 9H, -CH3), 2.12 (s, 3H, -CH3), 2.16 (s, 3H, -CH3) 2.3 (t, 4H, J = 7.2), 2.42 - 2.47 (m, 2H), 2.56 (t, 2H, J = 6.4 Hz), 2.93 (t, 4H, J = 6 Hz), 3.00 (t, 2H, J = 6 Hz), 3.11-3.15 (m, 4H), 3.53 – 3.57 (m, 2H), 3.70 (t, 2H, J = 6 Hz) 4.17 (t, 4H, J = 6 Hz); 13 C NMR (100 MHz, CDCl3, ppm) δ 11.73, 11.91, 12.77, 19.51, 19.55, 19.61, 19.68, 20.58, 20.94, 22.56, 22.65, 23.75, 24.34, 24.55, 24.71, 27.88, 28.67, 31.15, 31.20, 32.58, 32.60, 32.66, 33.93, 34.51, 37.18, 37.28, 37.35, 37.48, 38.27, 39.93, 39.98, 40.13, 53.48, 54.84, 56.23, 62.59, 71.44, 74.69, 117.45, 122.76, 125.55, 127.53, 147.67, 148.15. HRMS (ESI) m/z calcd for [C51H87NO6S4 + H]+: 938.5495; found: 938.5495; Anal. Calcd for C51H87NO6S4: C 65.27, H 9.34, N 1.49. Found: C 65.35, H 9.39, N 1.42.

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vortexed for at least of 15 min while subjecting them to repeated freeze-thaw cycles and sonicated to produce the nanoaggregates. Transmission Electron Microscopy (TEM).53, 54 The vesicular suspension was drop coated on a Formvar-coated, 400 mesh copper grid. The sample was allowed to stand for at least of 15 min and the excess of the volume was removed with the help of a filter paper. Subsequently, 20 µl of 0.1% uranyl acetate was added for negative staining. The excess stain was removed and the grid was air-dried overnight. Finally, the samples were vacuum dried for 4h and examined under TEM (JEOL2100F), with an acceleration voltage (DC voltage) of 200 keV. Atomic Force Microscopy (AFM).22 In the experiment, lipid suspensions were drop-cast over freshly cleaved mica sheets and dried at room temperature for a minimum period of 24h. The measurements were obtained using tapping mode (JPK Instruments, NanoWizard JPK00901 software) at room temperature. Fluorescence Microscopy. The DOX-encapsulated vesicular formulations were photographed under a fluorescence microscope (Olympus IX 81). Fluorescence Anisotropy.51-54 Fluorescence anisotropy measurements were performed on vesicular suspensions of TL1 and TL2 (1 mM) doped with DPH (Vesicles:DPH = 1000:4). Vesicular suspensions were obtained following the vesicle preparation protocol as discussed above. The steady-state fluorescence anisotropy (r) value of DPH was measured at excitation wavelength 360 nm and emission wavelength 430 nm. Slit width for both excitation and emission were 2 nm. Time-resolved Fluorescence Lifetime Measurements.55, 56 The fluorescence lifetime of DPH probe was measured in FluoroHub instrument from HORIBA Jobin Yvon. The 375 nm diode laser source was used. The decay curves were fitted tri-exponential curve. The best fit was obtained by the χ2 value 0.99-1.1. Measurement of Microviscosity.57 The microviscosity (ηm) was calculated using DPH fluorescence, environment sensitive probe. ηm value was calculated using Debye-Stokes-Einstein equation. ηm = kTτR/vh

Measurement of CAC using DMACA as a Fluorescent Probe.49, 50 Dissolved DMACA in 1, 4-dioxane was added into 0.5 mL amphiphilic (TL1 and TL2) suspension and mixed with a final concentration of DMACA at 5 µM. For this experiment, DMACA was excited at 398 nm and the emission spectrum was recorded from 450 to 550 nm. The fluorescence intensities of twisted-intramolecular-charge-transfer (TICT) band at 485 nm (I485) at different concentration of TL1 and TL2 were plotted and the breakpoints of the curves were taken as the respective CAC values. Preparation of nanovesicular aggregates.51, 52 Nanovesicles were prepared using a thin film hydration procedure. (±) αtocopheryl-lipoic acid conjugates (TL1 or TL2) were dissolved in chloroform in Wheaton glass vials, followed by solvent evaporation under steady a stream of nitrogen gas to give rise to thin films. Thin films were dried further in vacuum for 4h to remove the trace amounts of organic solvent. Autoclaved Milli-Q water was added to the glass vials while obtaining the desirable lipid concentration (1 mM) and kept at 4 °C for a minimum of 12h for hydration of the lipid thin films. These samples were then

The hydrodynamic volume (vh) of DPH was 313 Å3 and τR is the rotational correlation time of DPH. Perrin’s equation was used to calculate τR. τR = τf (r0/r –1)-1 The steady-state fluorescence anisotropy of DPH, r0 and r were the values in a highly viscous solvent (0.362) and amphiphilic solution, respectively. τf was the measured fluoresce lifetime of DPH probe in amphiphilic solution. Dynamic Light Scattering. The particle sizes of the nanovesicles and DOX-encapsulated nanovesicles were measured at room temperature using Malvern Zetasizer Nano ZS (Malvern Instruments) with a laser beam of 633 nm. All experiments were performed in dust-free conditions in deionized water with refractive index of 1.59 and viscosity of 0.89. DOX-Encapsulation into Nanovesicles.61 To a vesicular suspension of TL1 or TL2 (1 mM), DOX solution (stock

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Bioconjugate Chemistry

concentration 3 mM) was added and kept for hydration at 4 °C for 12h. The sample was heated to room temperature with vortexing to form DOX-encapsulated suspension (TL1-DOX and TL2DOX). The solution was centrifuged at a rate of 15000 rpm and a residue was formed at the bottom. Then a small volume of supernatant was taken and UV-Vis spectra were recorded to measure the DOX content. Encapsulation Efficiency (%) = (absorbance of bound DOX molecules/absorbance of total DOX molecules) × 100. Here, the absorbance of bound DOX molecules = (absorbance of total DOX molecules – absorbance of supernatant DOX molecules). The amount of DOX encapsulated in nanovesicles was quantified from the standard calibration plot (obtained by linear fitting) of DOX in an aqueous solution, which was plotted using the absorbance of DOX (at λmax = 485 nm) at varying concentrations of DOX (from 1 to 35 µg/mL). The unknown concentration of DOX in each system was determined from the calibration plot. There is no observable shift in the wavelength between the free DOX and the DOX encapsulated in the vesicles. Thus it was considered that the concentrations were comparable between the free DOX and the DOX entrapped in vesicles under our experimental conditions. GSH Triggered Drug Release from DOX-encapsulated Nanovesicles.61 The drug encapsulated nanovesicles (TL1-DOX or TL2-DOX) were dispersed in 10 mM phosphate buffer solution at pH 7.4. Then the solution was treated either with 10 mM DTT, 5 mM GSH or 10% FBS to study release or leakage of the drug. The release of drug from the nanovesicles was investigated by UV-Visible spectroscopy at the different time points (1h, 5h, and 10h). The following formula was used to calculate the release. The release of the drug in presence of 0.15% Triton X-100 was taken as 100%. Release (%) = [{Absorbance of DOX (t) - Absorbance of DOX (t0)}/{Absorbance of DOX in presence of Triton X-100 {Absorbance of DOX (t0)}] × 100. Here t0 was the initial time and t was the time at which the absorbance was measured in presence of DTT, GSH or FBS. Cytotoxicity Assay. Cytotoxicity of nanovesicles (with or without DOX) and free DOX was evaluated in HeLa and HeLaDOXR cell lines by conventional 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. In a typical experiment, cells were seeded at the density of 10000 cells/well in a 96 well microplate. After 24h, cells were treated with nanovesicles (with or without DOX) or free DOX. For HeLa cells, the medium was removed after 6h and replaced with 200 µL of fresh medium and the readings were taken at the end of 24h. For HeLa-DOXR cells, the readings were taken at the end of 72h. All the experiments were terminated while adding MTT (20 µL, 5 mg/mL in PBS) for 4h at the end of the experiments. Finally, the whole medium was removed from the wells, DMSO (200 µL) was added and absorbance was read on a microplate reader. Experiments were performed in triplicates for each concentration and the results expressed are from three such independent experiments. The IC50 values were determined using Graphpad Prism software following nonlinear regression model {log (inhibitor) versus Response; variable slope}. Flow Cytometry. Evaluation of internalization of free DOX and TL1-DOX/TL2-DOX was performed by FACS analysis. Cells were first seeded at the density of 60000/well in a 24 well plate.

Cells were treated with free DOX and DOX encapsulated nanovesicles when cells were at about 80% confluent. After the treatments of specified concentrations and required time points, the media were removed and then cells were properly washed with PBS three times followed by trypsinization to harvest the cells. Determination of the fluorescence intensities of the internalized DOX was performed using flow cytometry (BD FACS Calibur, BD Biosciences, USA). Cells without any treatment served as control. Analysis of flow cytometry data was performed using WinMDI software where ten thousand events were collected for analysis. Confocal Microscopy. For visualization of the intracellular delivery of DOX, cells were cultured on cover slips placed in 12well cell culture plates. Drug treatment was performed using required concentration and time points (HeLa cells; 5 µg/mL, 4h, and HeLa-DOXR; 20 µg/mL, 24h). After treatment, cells were washed with PBS buffer three times and fixed in 4% paraformaldehyde solution for 10 min. Cells were then rinsed again with PBS buffer thrice and incubated with nuclear staining dye DAPI (4,6-diamidino-2-phenylindole) for 10 min. Wells were washed properly again to remove excess dye and control overstaining. The glass coverslips were taken out and mounted on glass slides over ProLong Gold antifade reagent (Molecular probes) and viewed under confocal laser scanning microscope (LSM meta, Zeiss). The relative intracellular DOX concentration was quantified and plotted based on at least five images of three independent experiments. Clotting Time.74 The swiss albino mice (5-6 weeks of age) were maintained in a laminar air flow cabinet under specific pathogenfree conditions. All the experiments were performed in accordance with the institutional guidelines established for the Animal Facility at IACS and the study had approval from the Animal Ethics Committee of IACS. Mice blood was taken to find out the blood clotting time. Briefly, 100 µL of citrated blood samples were incubated with the nanovesicular samples for 5 minutes on a clean glass slide. After addition of 25 µL CaCl2 (50 mM), time recording was started. The appearance of the visible clot was recorded as the clotting of blood. The normal saline served as the negative control for this experiment. PEI was used as the positive control. The nanovesicular concentration was varied from 0.5 mM to 2 mM. Hemolysis Assay.75 The nanovesicular samples were added to 100 µL of RBC and incubated for 2h at 37 °C. Then all the samples were centrifuged at 1500 rpm for 5 min. The supernatant was taken and checked for released hemoglobin in UV-Vis spectrophotometer at 541 nm. Triton X100 and normal saline buffer were taken as positive and negative control respectively. Based on absorbance measurements, the percentage of hemolysis was calculated. % of hemolysis = {(Absorbance of supernatant with nanovesicles) – (Absorbance of supernatant with negative control)}/{( Absorbance of supernatant with positive control) – Absorbance of supernatant with negative control)} * 100. The concentration range of nanovesicles ranges 0.5-2 mM. RBC Aggregation.74 RBCs were collected from mice blood following reported protocol and diluted with normal saline (1:10). The nanovesicular samples with various concentrations were incubated with RBC samples for 30 min at 37 °C. Aggregation properties of RBC were visualized in a phase contrast microscope wherein PEI was used as positive control.

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(5) Carvalho,

ASSOCIATED CONTENT S 1

Supporting Information (SI).

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H and C NMR spectra of TL1 and TL2, Emission spectra of aggregate doped DMACA, Fluorescence life time of DPH, Effect of DTT on hydrodynamic diameter of TL1 and TL2, UV-Vis spectra of DOX alone and encapsulated nanovesicles, AFM images DOX-encapsulated nanovesicles, Fluorescence microscopy images of DOX-encapsulated nanovesicles, UVVis spectra of DOX-encapsulated nanovesicle in presence of FBS and GSH, and Cell viability of TL1 and TL2, Clotting time, Percentage of hemolysis and RBCs’ aggregation images are given in SI. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

(7) (8)

(9) (10)

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Corresponding Author E-mail: [email protected], *Fax: +91 33 2483 6561, Tel: +91 33 2473 4688

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Author Contributions ǁ

These authors contributed equally (13)

NOTES The authors declare no competing financial interest.

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ACKNOWLEDGEMENT This work was supported by J. C. Bose Fellowship (to SB), Department of Science and Technology, Government of India (DST), New Delhi, India and Council of Scientific and Industrial Research (CSIR) grant via “Advanced Drug Delivery” project. BM and KK thank CSIR for awarding CSIR senior research fellowships and PM thanks DST for the Technical Research Centre (Project No. AI/1/62/IACS/2015) at IACS.

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ABBREVIATIONS DMACA; 4-(N, N-dimethylamino) cinnamaldehyde, DOX; Doxorubicin, DPH; 1,6-Diphenyl-1,3,5-hexatriene, DTT; dithiothreitol, GSH; glutathione, DOX; doxorubicin, FBS, fetal bovine serum, GMFI; geometric mean of fluorescence intensity.

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REFERENCES (1) Chabner, B. A., Roberts Jr., T. G. (2005) Chemotherapy

and the war on cancer. Nat. Rev. Cancer 5, 65-72.

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(2) Misra, S. K., Naz, S., Kondaiah, P., Bhattacharya, S. (2014)

A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells. Biomaterials 35, 1334-1346. (3) Misra, S. K., Kondaiah, P., Bhattacharya, S. (2012) Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins. Small 8, 131-143. (4) Yousefpour, P., Chilkoti, A. (2014) Co-opting biology to deliver drugs. Biotechnol. Bioeng. 111, 1699-1716.

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Bioconjugate Chemistry

Glutathione Responsive Drug Delivery to Sensitive and Drug Resistant Cancer Cells

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