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Redox Responsive Polymersomes for Enhanced Doxorubicin Delivery Chetan Nehate, Aradhana Nayal, and Veena Koul ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00238 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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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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ACS Biomaterials Science & Engineering

Redox Responsive Polymersomes for Enhanced Doxorubicin Delivery Chetan Nehateabɸ, Aradhana Nayalabɸ, Veena Koulab* aCentre

for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi 110016,

India. bBiomedical ɸThese

Engineering Unit, All India Institute of Medical Sciences, New Delhi 110029, India.

authors contributed equally.

*Corresponding Author Prof. Veena Koul Mailing address: Corresponding author: Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi. India Pin: 110016 Tel: +91 11 26591041.

Email: [email protected]

Mailing address of author: Chetan Nehate Centre for Biomedical Engineering, Block III, Indian Institute of Technology Delhi, Hauz Khas, New Delhi. India Pin: 110016. Email: [email protected] Mailing address of author: Aradhana Nayal Centre for Biomedical Engineering, Block III, Indian Institute of Technology Delhi, Hauz Khas, New Delhi. India Pin: 110016. Email: [email protected]

Keywords: Redox sensitive, Doxorubicin, Polymersomes, Polycaprolactone, Polyethylene glycol. 1 ACS Paragon Plus Environment

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ABSTRACT In the present investigation, the potential of novel, self-assemble, biocompatible and redox sensitive copolymer system with disulfide bond was explored for doxorubicin (DOX) delivery through polymersomes nanostructures of ~ 120 nm. The polymer system was synthesized with less number of steps, providing a high yield of 86 %. The developed polymersomes showed admirable biocompatibility with high dose tolerability in vitro and in vivo. The colloidal stability of DOX loaded polymersomes depicted a stable and uniform particle size over the period of 72 h. The cellular internalization of polymersomes was assessed in HeLa and MDA-MB-231 cell lines, where enhanced cellular internalization was observed. The dose dependent cytotoxicity was observed for DOX loaded polymersomes by MTT cytotoxicity assay in above cell lines. The tumor suppression studies were assessed in Ehrlich ascites tumor (EAT) carrying Swiss albino mice, where polymersomes exhibited 7.16 fold reduction in tumor volume correlated with control and 5.39 fold higher tumor inhibition capacity compared to conventional chemotherapy (free DOX treatment). The developed polymersomes gave safer insights concerning DOX associated toxicities by histopathology and serum biochemistry analysis. Thus, results focus the potential of redox responsive polymersomes for efficacious and improved DOX therapy with enhanced antitumor activity and insignificant cardiotoxicity which can be translated for clinical settings.

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INTRODUCTION Nanocarriers developed from biocompatible polymers have been comprehensively investigated for drug delivery applications in cancer treatment by virtue of their numerous benefits.1-2 Nanocarriers can make their way to tumor mass using enhanced permeability and retention (EPR).3-6 The nanocarriers with hydrophilic groups reduce the protein adsorption and thereby avoid the mononuclear phagocytic system (MPS) and thus provide enhanced circulation half-life.79

Consequently, nanocarriers improve the bioavailability of encapsulated drug with a reduction in

drug-associated side effects.10-11 An expanded consideration has been paid in recent years to create vesicular nanocarriers based on diblock copolymers, namely polymersomes, as proficient nanocarriers in drug delivery applications with attractive, remarkable and feasible characteristics.12-15 In comparison to liposomes, polymersomes nanostructures have several leads like extraordinary membrane robustness and insignificant permeability which can be reasonably fine-tuned by varying copolymer block length.

Moreover, membrane of polymersomes have higher thickness in

comparison to liposomes (5-20 nm vs 3-5 nm).15 These exclusive features of polymersomes can overcome stability concerns experienced in lipidic nanostructures which have high fluidity in their membrane, predisposing higher rate of membrane breakdown and drug leakage problem. Furthermore, polymersomes nanostructures can be efficiently tailored for optimum and controlled drug release kinetics in vitro and in vivo.16-17 Moreover, by tuning chain length of copolymer and composition, intelligent polymersomes of optimum size can be developed.18-19



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Herein, we have investigated the novel, scalable, biocompatible and biodegradable redox sensitive polymersomes nanosystem based on polyethylene glycol (PEG) modified polycaprolactone copolymer with disulfide linkage in between. As the cancer cells exhibit high glutathione (GSH) levels in their cytosol (2-10 mmol), the disulfide linkage in the developed copolymer acts like manageable porter for prompt drug disposition in tumor cells on account of fast degradation of the polymer chain. Based on our earlier study by Arun et al.20, we had developed nanosystem based on atom transfer free radical polymerization (ATRP) fabricated poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol) [PEG-PCL-SS-PCL-PEG] triblock copolymer with less yield using hazardous metal catalyst such as copper bromide and thus system can have less translational scope. In our another report by Shantanu et al.21, where nanosystem based on PEG modified polylactide copolymer (PEG-SS-PLA-SS-PLA-SS-PEG) with multiple disulfide bonds was developed by multiple steps in the synthesis process. In the present study, the developed the copolymer system has a commercial scope, as it can be synthesized with less number of steps and high yield of ~ 86 %. Also, the copolymer was developed with simple conjugation step without using a hazardous metal catalyst, copper bromide. We have used doxorubicin (DOX) as a model drug to establish the effectiveness of developed copolymer system as smart drug delivery paradigm through polymersomes nanoformulation. To the extent of our knowledge, this is the first work validating the polymersomes nanoformulation by using such a simple and comparatively easy processible copolymer system than polymer systems existing in the literature. The biocompatibility of polymersomes nanoformulation was validated by its high dose tolerability in vitro and in vivo. DOX release profile was studied in 10 mmol GSH to validate the redox responsive nature of polymersomes in cancer cell environment. Cellular internalization and 4 ACS Paragon Plus Environment

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cytotoxic profile of polymersomes was examined in HeLa and MDA-MB-231 cells. In vivo tumor suppression study for DOX loaded polymersomes was executed in Swiss albino mice bearing EAT tumor and compared with conventional chemotherapy of free DOX treatment.

EXPERIMENTAL SECTION Materials ε-Caprolactone, Methoxypoly(ethylene glycol) mPEG5000 acetic acid (mPEG), tin (II) 2ethylhexanoate,

Cystamine

mono-boc,

(EDC.HCl), Triflouro acetic acid (TFA),

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Glutathione reduced (GSH), 3-(4, 5-Dimethyl-2-

thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), Phosphotungstic acid (PTA), and Bovine serum albumin (BSA) were bought from Sigma-Aldrich USA. Anhydrous sodium sulfate, Triton X-100, Sodium acetate, Sodium bicarbonate, Acetic acid, Dimethyl sulfoxide (DMSO), Hexane from petroleum, Sodium metal, Toluene, Diethyl ether, Benzophenone, Dichloromethane (DCM), Tetrahydrofuran (THF) and chloroform-d were purchased from Merck, India. Mounting medium with DAPI was obtained from Abcam U.S.A. Dialysis membrane (3.5 kDa) was obtained from spectrum labs, USA. LysoTracker green, Penicillin-streptomycin solution, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Phosphate buffer saline (PBS) powder, Trypsin-EDTA (0.25%) with phenol red, and Leibovitz's L-15 Medium were obtained from Gibco Life Technologies USA. Milli-Q water was used for all experiments.

Methods Synthesis of methoxypoly(ethylene) glycol polycaprolactone diblock copolymer (mPEG-SSPCL-OH) 5 ACS Paragon Plus Environment


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The synthesis scheme for mPEG-SS-PCL-OH diblock copolymer is depicted in Figure 1. In the first step, cystamine mono-boc was reacted with the carboxyl group of mPEG5000 acetic acid to give methoxypolyethylene glycol conjugated with cystamine mono-boc. Further, in the second step, TFA was used for removal of boc group from boc protected mPEG. Finally, ring opening polymerization (ROP) of ε-caprolactone was carried out by using the mPEG with free amine group as an initiator to obtain mPEG modified polycaprolactone diblock copolymer.

H3C

O

OH

O

n O Methoxypoly(ethylene) glycol 5,000 acetic acid

+

EDC.HCl

O H2N

S

S

N H

Dry DCM, N2 atm, 37 OC

O

Cystamine mono-boc

O

H3C

O

O

O

O n

S

N H

O

O Methoxypoly(ethylene) glycol conjugated with cystamine mono-boc Dry DCM, TFA 37 OC N2 atm

O

Dry Toluene, 110o C, Tin(II) 2-ethylhexanoate, N2 atm

H3C

S

n

-Caprolactone

H N

O

H N

H3C

O

H N

O

S

S

NH2

O Methoxypoly(ethylene) glycol conjugated with cystamine n

O S

S

NH

O

H m

O

Methoxypoly(ethylene) glycol polycaprolactone diblock copolymer

Figure 1. Synthesis scheme for methoxypoly(ethylene) glycol polycaprolactone diblock copolymer (mPEG-SS-PCL-OH). 6 ACS Paragon Plus Environment

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Synthesis of methoxypoly(ethylene) glycol conjugated with cystamine mono-boc (mPEG-SSboc) Methoxypolyethylene glycol5000 acetic acid (800 mg, 0.16 mmol) was added in 25 mL of dry DCM in Schlenk flask with subsequent addition of EDC.HCl (0.04 g, 0.19 mmol). The reaction mixture was stirred for 15 min at 37 ºC under the presence of N2 atmosphere. After 15 min, cystamine mono-boc (0.80 mg, 0.32 mmol) was added to the reaction with further addition of triethylamine (88 µL, 0.63 mmol) and was preceded to 24 h in continuous N2 gas supply. After completion of 24 h, solvent was removed under vacuum and recovered polymer was precipitated in cold petroleum ether three times. The purity of product was examined by gel permeation chromatography (GPC, Waters, USA) and 1H NMR (Bruker, USA) GPC was performed with binary pump system fitted with refractive index detector (Waters 2414) using THF based Styragel HR3 column. THF was used as mobile phase with flow rate of 1 mL/min. Column heater temperature was kept at 40 °C. Number average molecular weight (Mn) and polydispersity index (PDI) of polymer was determined using standard calibration curve of polystyrene standards. Yield = 95 %.

1H

NMR (400 MHz, CDCl3, δ ppm): 1.45 (s, Boc CH3), 2.73 (m, -CH2SSCH2), 3.02-3.19 (m, -

NHCH2), 3.19 (m, -CH2NHCOCH2), 3.38 (s, -CH3 of mPEG), 3.43-3.88 (m, -OCH2-CH2O- of mPEG unit), 4.01(s, CH2CONHCH2). Mn (GPC) = 5033 Da, PDI = 1.1



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Deprotection of boc group from methoxypoly(ethylene) glycol conjugated with cystamine mono-boc (mPEG-SS-NH2) Methoxypoly(ethylene) glycol conjugated with cystamine mono-boc (0.70 g, 0.13 mmol) was added in dry DCM in a Schlenk flask with further addition of trifluoroacetic acid (TFA) (0.60 g, 5.28 mmol) under ice bath. The reaction was continued for 12 h under the presence N2 gas. After 12 h, DCM with TFA was evaporated under vacuum, recovered polymer was precipitated in cold petroleum ether three times and characterized by 1H NMR. Yield = 94 %.

1H

NMR (400 MHz, CDCl3, δ ppm): 2.85-2.96 (m, -CH2-NH2, -CH2SSCH2), 3.03-3.05 (m, -

CH2NHCOCH2), 3.37 (s, -CH3 of mPEG), 3.47-3.81 (m, -OCH2-CH2O- of mPEG unit), 4.50 (s, CH2CONHCH2).

Synthesis of mPEG-SS-PCL-OH diblock copolymer mPEG-SS-PCL-OH diblock copolymer was synthesized by ring opening polymerization of εcaprolactone using mPEG-SS-NH2 as an initiator in the presence of tin (II) 2-ethylhexanoate as a catalyst. In brief, ε-caprolactone (1.61 gm, 14.11 mmol) was added in 10 mL of dry toluene in Schlenk flask and mPEG-SS-NH2 (0.50 mg, 0.10 mmol) was added at 110 ºC under N2 atmosphere. The resultant mixture was continued to stir for 15 min with the further addition of tin (II) 2ethylhexanoate (5 mol % of the initiator) and continued for 24 h at 110 ºC under continuous N2 gas supply. After 24 h, toluene was evaporated under vacuum and recovered polymer was precipitated in frosty petroleum ether three times and characterized using 1H NMR and GPC. Yield 86 %.


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1H-NMR

(400 MHz, CDCl3, δ ppm): 1.36-1.40 (m, -OCH2CH2CH2CH2CH2CO-), 1.62-1.66 (m,

-OCH2CH2CH2CH2CH2CO-), 2.29-2.32 (m, NHCOCH2CH2CH2CH2), 2.81 (m, -CH2SSCH2,), 3.38 (s, -OCH3 of mPEG), 3.42-3.48 (m, -CH2NHCOCH2, -CH2NHCOCH2 ), 3.64-3.68 (m, OCH2-CH2O- of mPEG unit) 4.04-4.4.07 (m, -OCH2CH2CH2-CH2CH2CO-). Mn (GPC) = 24579 Da, PDI = 4.08.

Formulation and characterization of polymersomes Nanoprecipiation technique was used for formulation of DOX loaded polymersomes.22 Briefly, copolymer (30 mg) and DOX (7.5 mg) with an equimolar concentration of triethylamine was added in DMSO (1 mL) and slowly added to Milli-Q water (10 mL) and stirred for 30 min. Then, the resultant nanosuspension was kept for dialysis upto 48 h using dialysis tubing to remove organic solvent and unentrapped DOX. During course of dialysis, external water was replaced with fresh water three times. Blank polymersomes nanoformulation was prepared similarly as mentioned above without using DOX and triethylamine. The hydrodynamic size and PDI of the DOX loaded polymersomes were determined using dynamic light scattering, DLS (Zetasizer Nano ZS, Malvern Instruments Ltd), while their absolute size was determined by high resolution transmission electron microscopy (HR-TEM, Technai G2, 200 kV, FEI, USA) and field emission scanning electron microscopy (FESEM; Zeiss EVO 50, Carl Zeiss Microscopy GmbH, Germany). Loading and encapsulation of DOX in polymersomes were determined using following equations (1) and (2) respectively,

Weight of DOX in polymesomes

Loading efficiency (%) = Weight of DOX loaded polymersomes × 100

(1)



Amount of DOX loaded in polymesomes

Encapsulation efficiency (%) = Amount of DOX taken for loading in polymersomes × 100

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(2)

Stability of polymersomes The stability of DOX loaded polymersomes was assessed in DMEM containing 10 % FBS. In short, 100 μL of DOX loaded polymersomes (5 mg/mL) were added in 10 mL of DMEM supplemented with 10 % FBS and samples were intermittently examined by using DLS for alteration of size and PDI at varying times over 72 h time period.

Evaluation of drug release behavior Drug release from the DOX loaded polymersomes was explored in PBS pH 7.4 and with 10 mmol GSH (cancer cells GSH concentration) and 20 µmol GSH (extracellular milieu and blood GSH concentration).23 The release behavior of DOX was also evaluated at endolysosomal pH (pH 5.0) and with 10 mmol GSH.24-26 DOX loaded polymersomes equivalent to 1.0 mg of DOX were dialyzed against buffer solutions (10 mL) for 72 h. The 1.0 mL of external medium was collected at varying time, and sink condition was maintained by adding equal volume of fresh buffer solution at each time. The samples were periodically studied for DOX absorbance at 481 nm by UV-vis spectrophotometer (LAMBDA 650, Perkin Elmer, USA). DOX in released samples was calculated from the standard calibration curve.

In vitro cell culture studies 10 ACS Paragon Plus Environment

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HeLa (human cervical cancer cell line) and MDA-MB-231 (human breast adenocarcinoma cell line) were acquired from NCCS, Pune, India and grown in cell culture flasks.

In vitro cell cytotoxicity HeLa and MDA-MB-231 cells dispersed in media were added in 96 well plates (5 × 103 cells/well) and kept for 24 h in CO2 incubator at 37 ºC to reach ~ 75 % confluency. Toxicity profile of blank polymersomes was examined to access their cytocompatible nature at a concentration of 0.05-1.0 mg/mL for 48 h incubation. In addition, the cytotoxicity of DOX in above cells lines was determined with free DOX and DOX loaded polymersomes at 0.025-8.0 μg/mL of DOX concentration for 48 h incubation. After 48 h, media was removed, and 200 μL of fresh media containing 0.35 % MTT dye was added and kept for 4 h at 37 ºC in incubator. After completion of 4 h, media with dye was replaced with and 200 μL DMSO. The absorbance readings were recorded at 540 nm on plate reader (BioTeck, USA). The percent cell proliferation was calculated using following equation (3),

Cell proliferation =

Sample540nm ― Positive Control (Triton X ― 100)540nm Negative Control(PBS pH 7.4)540nm ― Positive Control(Triton X ― 100)540nm

× 100

(3)

Confocal Microscopy The uptake of DOX loaded polymersomes and free DOX was evaluated by CLSM in HeLa and MDA-MB-231 cells. Cells with media were added over cover slips of 10 mm × 12 mm size in 24 well plate (8 × 104 cells/well) and incubated for 24 h at 37 °C. Free DOX and DOX loaded polymersomes (equivalent to 5 μg/mL) were added to the wells and further kept in incubator for 2 h. After 2 h, cells were rinsed with PBS 7.4 thrice and further fixed with 4 % paraformaldehyde. 11 ACS Paragon Plus Environment


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The fixed cells were again given three washes with PBS pH 7.4. DAPI and LysoTracker green were used to stain nuclei and lysosomes of cells respectively and further observed under CLSM (FV1000 Olympus, USA) at 60 × magnification.

Flow cytometric analysis Cellular uptake of DOX loaded polymersomes was determined using flow cytometric analysis in HeLa and MDA-MB-231 cells, quantitatively. The media containing cells (2 × 105 cells/well) were cultured in 6 well plate and incubated for 24 h at 37 °C. After 24 h, Free DOX and DOX loaded polymersomes (equivalent to 5 μg/mL) were added to wells and kept for 2 h in incubator. After washing the cells with PBS pH 7.4 three times, cells were further harvested with 0.25 % trypsin. The cells pellet was obtained by centrifuging the dispersion at 3500 rpm for 5 min and further resuspended in 300 μL of PBS pH 7.4. Cell suspensions were examined using flow cytometry (BD accuri C6, USA).

In vivo animal experimentation The anticancer effectiveness of developed nanoformulation was evaluated using EAT tumor carrying Swiss albino mice (25 ± 5 g; 7-8 weeks old; n = 6 for each group). Ethical review for animal experimentation was finalized by the Institutional Animal Ethics Committee (IAEC), All India Institute of Medical Sciences (AIIMS), New Delhi, India (approval number 952/IAEC/16). All animal experimental protocols were followed under the guidelines put up by the committee for control and supervision of experiments on animals (CPCSEA), India for the correct implementation of animal care and use in experiments.


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In vivo toxicity studies for high dose tolerability The blank polymersomes suspension in PBS pH 7.4 (250 μL) was administered intraperitoneally (100 mg/kg) in Swiss albino mice for four doses (on days 0, 3, 6, and 9).27-28 The control group was administered with 250 μL of PBS pH 7.4. On 12th day, blood was collected from the mice through retro-orbital puncture, and serum biochemistry parameters and hematological factors were assessed. Vital organs were collected by sacrificing animals by cervical dislocation for histopathology assessment.

Tumor suppression evaluation Ascites form of tumor was developed by injecting EAT cells in intraperitoneal cavity of mice. After 7 days, EAT cells were removed form intraperitoneal cavity of mice and ~ 2 × 107 cells/mice were injected subcutaneously at the dorsal site of each mice for development of solid tumors. Drug treatments were started when tumor size reached to 200-250 mm3. Different formulations such as PBS pH 7.4 (control), free DOX, and DOX loaded polymersomes were injected by intravenous route to individual mice in each group (n = 6 mice) at 5 mg/kg of DOX. Six doses were injected at an interval of three days. The tumor dimensions were measured by using digital Vernier caliper at each third day, and tumor volume was determined using following equation (4),

Tumor volume = 0.5 × (Length) × (Width)2

(4)

Where length and width are longest and perpendicular diameter in mm respectively. At the end, animals were sacrificed by cervical dislocation and tumor, and vital organs were collected for toxicity evaluation using histopathology serum biochemistry assessment. 13 ACS Paragon Plus Environment


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Toxicity evaluation using histopathological serum biochemistry analysis Toxicity assessment was carried out as per our earlier published report.26 The serum was separated from collected blood and was further evaluated for cardiac toxicity using creatine kinase MB, a cardiotoxicity marker using chemical analyzer (Awareness technology, TurboChem 100, Germany). In addition, liver and kidney function tests markers such as alanine aminotransferase (AST) and aspartate aminotransferase (ALT) were also assessed for toxicity profile of DOX containing formulations. Statistical Analysis All experiments were performed with at least three replicates. The statistical significance is considered at p value < 0.05.

RESULTS AND DISCUSSION Synthesis of diblock polymer (mPEG-SS-PCL-OH) Cystamine mono-boc was reacted with methoxypolyethylene glycol5000 acetic acid to obtain methoxypolyethylene glycol conjugated with cystamine mono-boc. The purified polymer was characterized by 1H NMR (Figure S1). The presence of a peak at δ 1.45 ppm corresponds to the methyl groups of boc. The methylene groups of cystamine conjugated unit, (CH2SSCH2), (14 ACS Paragon Plus Environment

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NHCH2) and (CH2NHCOCH2) appeared as multiplet at δ 2.73, δ 3.02-3.19 and δ 3.19 ppm, respectively. Singlet peaks at δ 3.38 ppm, and δ 4.01 ppm corresponds to the methyl end group of mPEG and methylene protons of (CH2CONHCH2), respectively. The protons for PEG repeating units (-O-CH2-CH2-) were observed at δ 3.43-3.88 ppm as multiplet. GPC spectra of polymer displayed molecular weight as Mn 5033 Da and PDI 1.1 (Figure S2). In the next step deprotection of boc group from methoxypolyethylene glycol conjugated with cystamine mono-boc (mPEG-SS-boc) was carried out by using TFA treatment to obtain mPEG-SS-NH2. The successful deprotection of boc group was confirmed by 1H NMR, where a peak conforming to methyl protons of boc group at δ 1.45 ppm disappeared (Figure S3). In the final step, mPEG-SS-PCl-OH diblock copolymer was synthesized by using ROP of ε-caprolactone with mPEG-SS-NH2 as an initiator and characterization was performed using 1H NMR (Figure S4). The methylene protons of caprolactone unit appeared as multiplet in the range of δ 1.36-1.40 ppm (-CO-CH2-CH2-CH2-CH2-CH2-OH), δ 1.62-1.66 ppm (-CO-CH2-CH2-CH2CH2-CH2-OH), δ 2.29-2.32 ppm (-CO-CH2-CH2-CH2-CH2-CH2-OH), and δ 4.04-4.4.07 ppm (CO-CH2-CH2-CH2-CH2-CH2-OH). The peak at δ 2.81 ppm relates to the methylene protons neighboring the disulfide linkage (-CH2SSCH2-). PEG repeating unit protons appeared at δ 3.643.68 ppm (-O-CH2-CH2-). Accordingly, 1H NMR depicted the successful formation of mPEG-SSPCL-OH copolymer. Based on 1H NMR the percent hydrophilicity in copolymer chain was found to be ~ 23 %, which is in a good agreement for polymersomes nanostructures. The absolute Mn of mPEG-SS-PCl-OH copolymer was calculated by 1H NMR and observed to be 22427 Da. The GPC spectra displayed Mn as 24579 Da and PDI 4.08 (Figure S5).

Loading, size and morphological evaluation of DOX loaded polymersomes 15 ACS Paragon Plus Environment


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The developed copolymer could easily self-assemble in aqueous phase providing uniform and monodispersed polymersomes with enhanced loading efficiency and entrapment efficiency of 16.13 ± 1.05 % and 60.74 ± 3.95 % respectively. This improved loading of DOX in polymersomes must be attributed to the long hydrophobic chain of caprolactone unit. The average hydrodynamic size of DOX loaded polymersomes was appeared to be 120.26 ± 2.51 nm with PDI 0.11 ± 0.04 (Figure 2A). Zeta potential for polymersomes was appeared to be -7.23 ± 0.24 mV (Figure 2B). HR-TEM analysis depicted polymersomes bilayer structures with an absolute size of ~ 100 nm (Figure 2 C,C1). Moreover, we have investigated the morphological characteristics of DOX loaded polymersomes by FESEM, where uniformly dispersed spherically shaped nanocarriers were observed (Figure 2 D,D1). The size of polymersomes obtained by FESEM analysis (~ 100 nm) corroborates the observations seen in HR-TEM analysis.


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Figure 2. Size and morphological characterizations of DOX loaded polymersomes, (A) DLS size (n = 3), (B) zeta potential (n = 3), (C) HR-TEM analysis, (D) FESEM. Where, subtitle figures, (C1) and (D1) represent their respective zoom images.

Stability of polymersomes The stability of DOX loaded polymersomes was analyzed in DMEM media supplemented with 10 % FBS over the period of 72 h. Polymersomes did not show any aggregation and were found to be a stable and uniformly dispersed in media with a particle size 120-126 nm (Figure 3). PDI of nanocarriers’ dispersion designates about the uniform distribution of nanocarriers; low PDI is 17 ACS Paragon Plus Environment


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essential and acceptable for effective nanoformulation in terms of its uniform distribution in aqueous solvent and syringeability. The designed polymersomes nanoformulation depicted PDI below 0.14 over the period of 72 h, signifying their stable nature in the aqueous phase.

Figure 3. Stability study of DOX loaded polymersomes in DMEM with 10 % FBS over the period of 72 h (Mean ± SD, n= 3).

Evaluation of drug release behavior In vitro DOX release in PBS pH 7.4 supplemented with 10 mmol GSH showed abrupt DOX release indicating the effect of GSH on destabilization of polymer chains in polymersomes. This was expected on account of reducing the effect of GSH, where thiolates (R-S-) plays an essential role in disruption of disulfide linkages in the polymersomes29, providing higher drug release of ~ 60 % 18 ACS Paragon Plus Environment

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over the period of 72 h (Figure 4). In case of physiological conditions, PBS pH 7.4 and PBS pH 7.4 containing 20 μmol GSH (blood GSH concentration), drug release of ~ 20 % was observed over 72 h, indicating the stability of drug loaded polymersomes during circulation. In addition, we have also studied the drug release profile at pH 5.0 (endolysosomal pH) with and without 10 mmol of GSH. The increase in drug release at acidic pH (~ 42 %) must be due to the protonation of glycosidic amine group of DOX (DOX pKa = 8.24) and thereby enhancing its hydrophilicity. Such a pH dependent drug release behavior for DOX was also revealed by other DOX encapsulated polymeric nanocarriers.30-32 The presence of GSH could have further boosted the DOX release at pH 5.0 in 10 mmol of GSH giving ~ 74 % release. Henceforth, it can be concluded that the designed polymersomes with disulfide linkage were able to release drug rapidly in reducing atmosphere similar to that of the cancer cells cytosol. The drug release kinetics was determined by using several kinetic release models such as zero order, Hixon-Crowell, first order, Higuchi and Korsmeyer peppas model (Table S1,S1.1, supporting information S1). The observed kinetics was in a good agreement with the existing literature demonstrating release behavior for DOX loaded polymersomes.33



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Figure 4. In vitro DOX release behavior of DOX loaded polymersomes over the period of 72 h (Mean ± SD, n = 3).

In vitro cell cytotoxicity The cytotoxic effect of blank polymersomes was measured in HeLa and MDA-MB-231 cells. The polymersomes depicted cytocompatible nature at concentration of 0.05-1 mg/mL (Figure 5A). This exciting cytocompatibility of polymersomes over the high dose must be ascribed to the generally regarded as safe (GRAS) and U S Food and Drug Administration (US FDA) endorsement attributes of PEG and polycaprolactone, used in designing redox responsive copolymer system.34-35 Dose dependent cytotoxicity was observed for free DOX and DOX loaded polymersomes at 0.025-8 μg/mL of DOX (Figure 5B,C). Indeed, free DOX was more cytotoxic in both cells lines than DOX loaded polymersomes with IC50 in HeLa and MDA-MB-231 as 0.84 and 0.66 μg/mL respectively. This could be due to its fast passive diffusion in cells, while polymersomes showed sustained release.36-38 The IC50 of DOX loaded polymersomes in HeLa, 20 ACS Paragon Plus Environment

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and MDA-MB-231 cell lines were observed to be 1.98 and 1.11 μg/mL, respectively. Such low IC50 for DOX loaded polymersomes must be due to their fast internalization into cancer cells with the added advantage of stimuli responsive behavior in the GSH enriched cancer cells.

Figure 5. In vitro cell cytotoxicity, (A) Testing for cytocompatible nature of blank polymersomes on HeLa and MDA-MB-231 Cells, and DOX loaded polymersomes and free DOX on, (B) HeLa and (C) MDA-MB-231 cells (Mean ± SD, n = 3). Where, *** designates P < 0.001, ** designates P < 0.01, * designates P < 0.05 and “ns” designates not significant. 21 ACS Paragon Plus Environment


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Confocal microscopy Cellular uptake was determined by CLSM in HeLa and MDA-MB-231 cells. DOX loaded polymersomes could easily get internalized after 2 h incubation in both cells (Figure 6). This can be accredited to the increased cell trafficking of polymersomes nanostructures through endocytosis process.39-40 This was validated by using LysoTracker green, where co-localization of DOX loaded polymersomes was visualized providing yellow color which indicated their trafficking in endolysosomal compartments. The free DOX mainly localized in the nucleus of both the cell lines, which must be due to its fast passive diffusion.24


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Figure 6. Confocal microscopic images for in vitro cellular internalization of DOX loaded polymersomes for 2 h incubation in (A) HeLa and (C) MDA-MB-231 cells. While free DOX internalization is represented as in (B) HeLa and (D) MDA-MB-231 cells. All images were taken at 60 × magnification. Scale bars are 20 μm.

Flow cytometric analysis The cellular internalization of DOX loaded polymersomes was assessed in HeLa and MDA-MB231 cells. Polymersomes could be internalized easily into both the cell lines in 2 h incubation providing enhanced intensity with respect to DOX fluorescence (Figure 7A,B). However, the uptake of free DOX in both cell lines is more which must be ascribed to the quick and passive diffusion of free DOX in cells.24-25, 41 (Figure 7C). As shown in CLSM, polymersomes internalized through endocytosis and gets further trapped in endosomes and lysosomes in cytoplasm.42 The increased uptake of DOX loaded polymersomes ~ 3 and ~ 5 fold increment was observed in HeLa and MDA-MB-231 cells in comparison to untreated cells respectively, demonstrating their significant uptake. This must be attributed to the uniformly suspended nano-sized polymersomes in cell culture media.



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Figure 7. Cellular internalization of free DOX and DOX loaded polymersomes in, (A) HeLa and (B) MDA-MB-231 cells. Figure C represents the mean fluorescence intensity for DOX loaded polymersomes and free DOX in HeLa and MDA-MB-231 cells. Where, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05.

In vivo animal experimentation In vivo toxicity studies for high dose tolerability Eleven days repeating dose toxicity study was executed in Swiss albino mice to evaluate the biocompatible profile of developed polymersomes. Body weight changes of polymersomes treated group did not show significant changes in comparison to normal control (Figure 8A). Histopathological analysis of vital organs of mice for polymersomes treated group was almost comparable with the control group without showing any toxicity concerns (Figure 8B). Moreover, the hematological and serum biochemistry parameters estimation did not display notable changes compared to control group (Table 1). Consequently, the developed polymersomes were safe and 24 ACS Paragon Plus Environment

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biocompatible in nature which can be further employed for in vivo studies of drug loaded polymersomes nanoformulation.



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Figure 8. In vivo toxicity studies for high dose tolerability, (A) body weight changes, and (B) histopathological analysis of vital organs, for control group treated with PBS pH 7.4 and blank polymersomes treated group. Images were taken at 40 × magnification. Scale bars are 25 μm. (Figure 8 A, control group data was reproduced with permission from ref 26. Copyright 2018 American Chemical Society)


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Table 1. Analysis of hematological and serum biochemistry parameters of mice used in toxicity studies for high dose tolerability. (Control group data was reproduced with permission from ref. 26. Copyright 2018 American Chemical Society).

Hematological parametersa Sr. No. 1

Parameters with units 3

Control group (PBS) Blank polymersomes

3

7.40 ± 0.95

7.17 ± 3.51

2

White blood cells (10 /mm ) Lymphocytes (%)

46.00 ± 3.15

63.40 ± 12.61

3

Monocytes (%)

34.63 ± 3.46

27.30 ± 6.77

4

Granulocytes (%)

19.70 ± 3.10

9.30 ± 5.89

5

Lymphocytes (10 /mm )

3.37 ± 0.55

4.80 ± 3.18

6

Monocytes (10 /mm )

2.50 ± 0.10

1.77 ± 0.59

7

Granulocytes (10 /mm )

1.53 ± 0.40

0.60 ± 0.36

8

6.50 ± 0.53

5.98 ± 0.21

9

Red blood cells (10 /mm ) Hemoglobin (g/dl)

9.07 ± 0.58

9.57 ± 1.27

10

Hematocrit (%)

28.77 ± 1.27

30.10 ± 3.42

44.33 ± 2.08

50.67 ± 5.86

13.97 ± 0.72

16.03 ± 2.29

31.53 ± 0.72

31.70 ± 0.79

17.90 ± 1.25

20.17 ± 4.84

1427.00 ± 34.60

1121.33 ± 79.10

7.97 ± 1.78

6.77 ± 2.23

17

Mean platelet volume (µm ) Procalcitonin (%)

1.14 ± 0.28

0.76 ± 0.26

19

Platelet distribution width (%)

11.13 ± 3.32

8.8 ± 1.25

11 12 13 14 15 16

3

3

3

3

3

3

3

3

3

Mean corpuscular volume (µm ) Mean corpuscular hemoglobin (pg) Mean corpuscular hemoglobin concentration (g/dl) Red cell distribution width (%) 3

3

Platelets (10 /mm ) 3

Serum biochemistry parametersb 1

CK-MB (U/L)

3.46 ± 0.63

3.22 ± 0.29

2

AST (U/L)

54.64 ± 7.03

59.13 ± 2.53

3

ALT (U/L)

30.71 ± 1.75

37.33 ± 3.37

4

Urea (mg/dl)

36.51 ± 3.24

37.56 ± 4.47

5

Creatinine (mg/dl)

0.05 ± 0.04

0.1 ± 0.02



a,bMean

Page 28 of 44

± SD, n = 3. bCK-MB: creatine kinase-MB, AST: alanine aminotransferase, and ALT: aspartate

aminotransferase.

In vivo tumor suppression studies The antitumor efficiency of free DOX, DOX loaded polymersomes, and PBS pH 7.4 (control) was assessed in EAT tumor carrying Swiss albino mice. Each drug containing formulations at a DOX dose of 5 mg/kg of body were injected 6 times intravenously to each mice at 3 days interval (Figure 9A). On the 18th day of the study, control group mice displayed a progressive increase in tumor volume with 5.93 fold increase in comparison to introductory tumor volume (Figure 9B). In case of free DOX treatment, moderate tumor suppression was observed with 1.33 fold reduction in tumor volume. The developed DOX loaded polymersomes nanosystem depicted remarkable tumor reduction of 7.16 fold in comparison to initial tumor volume (P < 0.001) (Figure 9B1). This superior antitumor effectiveness of designed polymersomes nanoformulation must be ascribed to their small particle size of ~ 120 nm. Such small size nanostructures could have easily taken advantage of EPR effect of tumor environment and thereby providing enhanced accrual of polymersomes in tumor mass through passive targeting.43-45 Moreover, the boosted drug disposition in tumor cells due to the redox triggered degradation of polymersomes could be the possible cause for such fascinating tumor suppression efficacy of DOX loaded polymersomes.27 A considerable increment in the body weight of the control group was observed as compared to free DOX, and DOX loaded polymersomes during the indicated experimental period (p < 0.001) (Figure 9C). This is attributed to daily increment in tumor volume of control group. In the treatment of free DOX and DOX loaded polymersomes, no significant difference in body weight was witnessed.


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Survival of treated mice was studied over the period of 45 days and determined using Kaplan-Meier survival method (Figure 9D). The lifespan of mice after treatment with DOX loaded polymersomes was observed to be significantly improved during the course of treatment. The median survival time for control, free DOX and DOX loaded polymersomes was appeared as 25, 34 and 42.5 days, respectively. Accordingly, our designed polymersomes nanoformulation displayed significant improvement with promising therapeutic efficacy for DOX treatment in cancer therapy.

Figure 9. In vivo anticancer activity of DOX loaded polymersomes compared with free DOX treatment. (A) Treatment regimen for in vivo study. (B) Tumor growth suppression curves for the tumor suppression study after intravenous administration of PBS pH 7.4 (control), free DOX and 29 ACS Paragon Plus Environment


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DOX loaded polymersomes. Subtitle figure (B1), denoted by arrow, represent respective tumor growth suppression curves for free DOX and DOX loaded polymersomes. Figure (C) represent body weight changes of mice employed for tumor suppression study. Figure (D) represent the Kaplan-Meier survival plot. ( in B, B1, C and D, control and free DOX treated groups data was reproduced with permission from ref. 26. Copyright 2018 American Chemical Society).

Toxicity evaluation using histopathological analysis DOX is well known to produce cardiotoxicity, and therefore it is essential to study the safety profile of DOX loaded nanocarriers regarding its cardiac damage potential. The cardiotoxicity of free DOX and DOX loaded polymersomes was assessed by studying histology of vital organs (Figure 10). Free DOX treated mice showed significant cardiac toxicity with cardiac myofibrils necrosis with a high cytoplasmic vacuolization, loss in myofibrils, and swelling in the cardiomyocytes. Interestingly, the heart of DOX loaded polymersomes treated mice did not display any cardiac toxicity features, which can be due to the accumulation of a maximum quantity of DOX loaded polymersomes in tumor tissues. It is well reported that PEGylation has an encouraging impact for effective EPR effect of nanocarriers46-47, and hence, hereinto, this “stealth” characteristic of developed polymersomes must be playing a crucial role in enhanced accretion of these nanostructures in tumor mass. DOX containing formulations treated groups except control showed significant tumor necrosis. Nevertheless, we found that our DOX loaded polymersomes exhibited complete tumor necrosis, while in free DOX treatment few viable neoplastic cells were observed. The liver section of free DOX treated mice showed kupffer cell prominence and liver fibrosis, signifying hepatotoxicity caused in free DOX treatment. Kidney of mice treated with free DOX also displayed significant tubular damage demonstrating nephrotoxicity. The DOX loaded 30 ACS Paragon Plus Environment

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polymersomes treated mice did not show notable toxicity to liver and kidney. Consequently, the developed polymersomes nanoformulation have shown nontoxic and biocompatible nature concerning the vital organ toxicity issues in DOX therapy.

Figure 10. Hematoxylin and Eosin staining of tumor and other vital organs of mice treated with PBS pH 7.4 (control), free DOX, and DOX loaded polymersomes. Images were taken at 40 × magnification. Scale bars are 25 μm.

Toxicity evaluation using serum biochemistry parameters analysis The serum biochemistry parameters analysis was also performed for DOX containing formulations treated groups to assess the cardiotoxicity caused due to DOX treatment, where a control group (PBS pH 7.4 treatment) was used for comparison (Table 2). After cardiac damage, creatine kinase CK-MB (cardiac injury biomarker) is released into the blood which can be analyzed by in vitro methods.48 Significant increased amount of CK-MB was detected in free DOX treatment in comparison to control and DOX loaded polymersomes (p < 0.001), showing cardiac injury during treatment. In case of DOX loaded polymersomes, CK-MB levels were comparable to the control 31 ACS Paragon Plus Environment


Page 32 of 44

group and were perceived to be below toxicity threshold. The liver specific markers such as AST and ALT were also analyzed hepatotoxicity. The mice treated with free DOX showed significantly high levels of AST and ALT comparison to control and DOX loaded polymersomes (p < 0.001) indicating its hepatotoxic effect. In addition, the significant renal damage was also witnessed in free DOX treatment with an increment in renal function biomarkers as urea and creatinine. The liver and renal biomarkers for DOX loaded polymersomes were comparable to the control group. Thus, the serum biochemical parameter analysis complements histopathological evaluation, signifying the safety profile of developed polymersomes nanoformulation concerning free drug associated toxicity issues.

Table 2. Serum biochemistry parameters of animals used during tumor suppression studya. (Control and free DOX treated group data was reproduced with permission from ref. 26. Copyright 2018 American Chemical Society). Control

DOX

DOX loaded polymersomes

CK-MB (U/L)

3.22 ± 0.23

17.42 ± 2.75#

6.26 ± 1.14

AST (U/L)

55.63 ± 5.20

118.31 ± 7.26#

63.77 ± 5.05

ALT (U/L)

31.56 ± 0.88

87.14 ± 7.38#

42.97 ± 2.12

Urea (mg/dl) Creatinine (mg/dl)

36.49 ± 1.78 0.03 ± 0.03

65.93 ± 6.12# 0.39 ± 0.04

37.50 ± 4.68 0.06 ± 0.03

Biochemistry Parametersb

aMean

± SD, n = 3. bCK-MB, creatine kinase-MB; AST: alanine aminotransferase; and ALT,

aspartate aminotransferase. Where, # indicates highly significant, P < 0.001.

CONCLUSIONS 32 ACS Paragon Plus Environment

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In summary, we have developed new PEGylated, redox responsive copolymer system for polymersomes nanoformulation with less steps and high yield. The nanosystem was found to be biocompatible with high dose tolerability in vitro and in vivo. Tumor suppression studies using DOX as a model drug shown promising antitumor activity for designed nanosystem in EAT tumor carrying Swiss albino mice. Being a capability to yield monodispersed polymersomes nanostructures, this robust and smart copolymer system can certainly be of interest as a recent nanomedicine paradigm to encapsulate various other hydrophilic and hydrophobic anticancer drugs as well. Thus, the developed polymersomes nanosystem positively holds a promise to be translated further in clinical applications.

ASSOCIATED CONTENTS Supporting Information 1H

NMR spectra for mPEG-SS-boc, mPEG-SS-NH2, and mPEG-SS-PCL-OH. GPC spectra for

mPEG-SS-boc and mPEG-SS-PCL-OH. Drug release kinetics for in vitro drug release profiles.

AUTHOR INFORMATION Chetan Nehate ORCID: 0000-0001-6124-5210

Aradhana Nayal ORCID: 0000-0002-7402-3542

Corresponding Author Veena Koul 33 ACS Paragon Plus Environment


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*Phone: +91 (11) 2659 1041. E-mail: [email protected] ORCID: 0000-0001-8293-4349 Notes: The authors declare no competing financial interest. Contributions: The authors, Chetan Nehate and Aradhana Nayal contributed equally. Funding sources: All authors have received funding from Department of Biotechnology, Ministry of Science and Technology. Award number: BT/PR14132/NNT/28/855/2015

ACKNOWLEDGEMENTS We are thankful for to Department of Biotechnology (DBT) for the research grant (BT/PR14132/NNT/28/855/2015). We are grateful to Dr. A. K. Dinda form AIIMS, Delhi for support in histopathology analysis.


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Manuscript Title: Redox Responsive Polymersomes for Enhanced Doxorubicin Delivery. Authors: Chetan Nehate, Aradhana Nayal, Veena Koul



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Redox Responsive Polymersomes for Enhanced ... - ACS Publications

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