Comparative Assessment of Active Targeted Redox Sensitive

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Comparative Assessment of Active Targeted Redox Sensitive Polymersomes Based on pPEGMA-S-S-PLA Diblock Copolymer with Marketed Nanoformulation Chetan Nehate, Aji Alex Moothedathu Raynold, V. Haridas, and Veena Koul Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00178 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Biomacromolecules

Comparative Assessment of Active Targeted Redox Sensitive Polymersomes Based on pPEGMA-S-S-PLA Diblock Copolymer with Marketed Nanoformulation

Chetan Nehateab, Aji Alex Moothedathu Raynoldab, V. Haridasc, Veena Koulab* a

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

110016, India b

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

India. c

Department of chemistry, Indian Institute of Technology Delhi, New Delhi 110016, India

*Corresponding Author Tel: +91 11 26591041 Email: [email protected]

Keywords: Polylactide, Doxorubicin, Polymersomes, DOXIL, Glutathione.

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ABSTRACT In the present work, polymersomes based on self-assembled, folate targeted, redox responsive, ATRP based amphiphilic diblock copolymer poly(polyethylene glycol)-ss-polylactide with disulfide linkage were developed for efficient doxorubicin (DOX) delivery and compared with marketed DOXIL nanoformulation. The polymersomes formulation was optimized by quality by design approach providing monodisperse nanostructures of ~ 110 nm and enhanced DOX loading of ~ 20 %. Polymersomes showed excellent stability as per the ICH guidelines over the extended storage period of 3 months. The in vitro drug release profile confirmed the redox sensitive behavior of polymersomes providing ~ 80 % drug release in endosomal pH 5 with 10 mmol GSH as compared to ~ 20 % release at pH 7.4. The targeted polymersomes achieved enhanced cellular internalization in folate receptor over expressing cell lines, MDA-MB-231 and Hela, providing ~ 24 % higher tumor reduction than DOXIL in Ehrlich ascites tumor bearing Swiss albino mice.


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1. INTRODUCTION Today, cancer is the most prevailing threat to the humankind owing to its high mortality and increasing tendency year by year. As matter stand, the remedial procedure of tumor predominantly incorporates chemotherapy, radiotherapy, immunotherapy, and surgical resection.1, 2 Comparing all these therapeutics including recently advanced gene therapy with siRNA and CRISPR/Cas9, chemotherapy is a privileged methodology by virtue of its remarkable clinical curative impact.3 Be that as it may, this conventional therapy is nonspecific and has no capacity to differentiate between tumor cells and normal noncancerous cells, leading to severe systemic toxicity and other dire clinical manifestations.4 To address this issue, over the last two decades, nanotechnology, especially nanocarriers have attracted special attention for scientists as novel drug delivery systems. 5-10 Different types of polymeric nanocarriers such as nanocapsules, nanospheres, micelles, and polymersomes have been developed to overcome the cardiotoxicity and other clinical side effect of doxorubicin (DOX), a first line chemotherapeutic drug.11-14 Polymeric nanocarriers are easy to synthesize and after attaching specific targeting ligands can be set up for assessment of incorporated drug’s 5R framework: Right target, Right tissue exposure, Right commercial potential, Right patient and Right safety.15-17 The rise of DOXIL in 1995, a polyethylene glycol (PEG) modified liposomal DOX nanoformulation, drastically minimizes the cardiotoxicity and improves DOX accumulation in tumor tissues through EPR effect.18,

19

However, in the clinic, DOXIL only marginally

improves the therapeutic efficacy in solid tumors treatment due to its unfavorable DOX release profile.20-22 Multiple dosage regimens of DOXIL, give rise to dermatological reactions called Palmar-plantar erythrodysesthesia (PPE) or hand-foot syndrome. PPE is caused due to the accumulation of DOXIL particles at the periphery of blood vessels without further dispersal in interstitial space.23, 24 Moreover, one of the most debatable matter with repeated administration 3 ACS Paragon Plus Environment


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of DOXIL is its accelerated blood clearance (ABC) phenomenon, which induces IgM antibodies

against

its

1,2-distearoyl-sn-glycero-3-phosphoethanolaminen-

[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE) conjugate lipid, resulting in severe hypersensitivity reactions.25, 26 In addition, DOXIL particles cannot actively make their way to tumor tissue, instead, they tend to cluster at its perimeter leading to the hindrance for uptake of next dose.27, 28 Consequently, there is a key to develop smart, robust and actively targeted nanoformulation for enhanced therapeutic effect. PEGylated polymersomes have gained a lot of attraction for drug delivery applications as versatile nanocarriers on account of their tunable membrane properties, better colloidal stability, robustness, ability to encase variety of drugs and molecules.16,

29, 30

Moreover,

compared to liposomes, polymersomes have larger membrane thickness compared to liposomes.31 These unique attributes of polymersomes can conquer the greater part regarding stability issues (mainly disintegration of the membrane and drug leakage) experienced in lipidic nanocarriers because of high fluidity in their bilayer membrane. In addition, it has been established that polymersomes can be well tailor-made for controlled and optimum drug release behavior and kinetics in vitro and in vivo.32, 33 Depending on copolymer composition and chain length, polymersomes with the preferred size can be formulated which inclined towards the smart drug delivery frameworks.34 PEGylated polylactide (PLA) based amphiphilic block copolymers got broad consideration for developing polymeric nanocarriers with bringing about their fruitful section in clinical trials 35, or acquiring clinical endorsement (i.e. Genexol® PM, Samyang Genex Co., Seoul, Korea).36 In addition, PLA is being continuously used for treating prostate cancer in the form of depots suspension (Lupron Depot®, TAP Pharmaceutical Products Inc). 37 Ring Opening Polymerization (ROP)38,

39

and Atom Transfer Free Radical

Polymerization (ATRP)40 has been used to synthesize variety of amphiphilic copolymers with


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definite molecular weight and narrow polydispersity index. ATRP allows attaching multiple PEG chains to polymer in a controlled fashion which can be further conjugated with desired functionalities. Redox sensitive polymer based nanosystems, where disulfide linkage is incorporated in polymer chain have picked up fame for on-command intracellular drug delivery applications in cancer therapeutics.41, 42 In such nanocarriers, the redox potential of cancer cells, (2-10 mmol glutathione, GSH) is utilized as a triggering mechanism for enhanced intracellular drug delivery than normal cells and extracellular milieu (2-20 μmol GSH).43-45 To lessen the side effects of drugs used in cancer therapy in normal healthy cells and enhancing therapeutic efficiency, nanocarriers specifically targeted to tumor cells by employing active targeting through specific ligands present on the surface of cancer cells are being continuously explored. Actively targeted nanosystems acts like a guided rocket and displays particular affinity towards the over expressed receptors on cancer cells, which are generally not expressed on normal cells. Several targeting groups like vitamins, peptides, aptamers, hormones, sugars, growth factors and so on, have been envisaged for active targeting to tumor cells.46-49 Among all these ligands folic acid is most widely used targeting moiety for selective targeting of colloidal nanosystems to tumor tissues which binds specifically folate receptors, overexpressed on the surface of a variety of cancer cells.50 Normal cells show minimal expression of folate receptors, yet rather express less folate carrier for folic acid uptake. Folate receptors display enhanced binding affinity to folic acid (dissociation constant, Kd = 1 nmol), creating folic acid as an amazing ligand for targeted drug delivery in cancer therapy.51 Herein, we have introduced PLA based novel, biocompatible, folate targeted, PEGylated, redox sensitive polymersomes nanoplatform fabricated by ATRP with internal cell stimulus drug release for enhanced tumor suppression. We have incorporated disulfide bridge in polymer backbone which acts like a switchable gatekeeper for rapid drug release in cancer



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cells with high GSH level. To the best of our knowledge, this is the first study demonstrating the development of actively targeted polymersomes nanostructures by ATRP processed amphiphilic PLA polymer for enhanced DOX delivery. The biocompatible nature of investigated polymer was thoroughly carried out in vitro using hemolysis, protein adsorption, coagulation studies and MTT cell proliferation assay and in vivo in Swiss albino mice. The nanoformulation preparation was optimized by quality by design approach (QbD) to come up with best formulation process parameters providing enhanced DOX loading of ~ 20 %. In vitro drug release study was performed in pH 5 and pH 7.4 with and without 10 mmol GSH to investigate the effect of cancer cell redox milieu. During release study, we also accounted for blood GSH concentration level (20 μmol) to establish the safety of polymersomes during circulation. In vitro cellular uptake and cytotoxicity studies on folate overexpressing and normal cell lines were carried out to examine the folate targeting efficiency of polymersomes. While in vivo tumor regression effect of targeted polymersomes was compared with marketed DOXIL formulation in Ehrlich ascites tumor (EAT) bearing Swiss albino mice with histopathology and serum biochemistry analysis.

2. MATERIALS AND METHODS 3,6-dimethyl-1,4-dioxane-2,5-dione (Lactide), tin (II) octoate, poly(ethylene glycol) methacrylate

(PEGMA)

(Mn

~

360

Da),

2-hydroxyethyl

disulfide,

N,N'-

dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA),

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium

bromide

(MTT),

2′,7′-

dichlorofluorescin diacetate (H2DCFDA), glutathione (GSH) were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). GPC grade polystyrene standards were procured from Waters, U.S.A. Doxorubicin was obtained as a gift sample from Sun Pharmaceutical Industries Ltd., New Delhi, India as a gift sample. DOXIL was purchased from RPG life sciences Ltd. Mumbai, 6 ACS Paragon Plus Environment

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India. Leibovitz's L-15 Medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin antibiotic solution, trypsin-EDTA (0.25%) with phenol red and phosphate buffer saline (PBS) powder were procured from Thermo Fischer scientific, U.S.A. 4',6-diamidino-2-phenylindole (DAPI) containing fluoroshield mounting medium was purchased from Abcam U.S.A. Silica for column chromatography 60-120 mesh size, hematoxylin and eosin staining solutions and triethylamine was obtained from SISCO research laboratories (SRL) Ltd. India. Dialysis membrane (3.5 kDa) was purchased from spectrum labs, U.S.A. Sodium metal, sodium hydroxide, sodium hydrogen carbonate, anhydrous sodium sulfate, dry toluene, dimethyl sulfoxide (DMSO), dichloromethane (DCM), tetrahydrofuran (THF), chloroform, methanol, ethyl acetate, diethyl ether, hexane from petroleum, dimethyl sulfoxide-d6, chloroform-d and ultrapure water with resistivity 18 MΩ cm was acquired from (Merck, MA, U.S.A.). 2.1. Synthesis of folic acid conjugated poly[poly(ethylene glycol)methacrylate]-S-Spolylactide diblock copolymer [(PEGMA)n-S-S-PLA] The redox sensitive PEGyated PLA copolymer was consecutively synthesized by ROP and ATRP reactions. In the first step, PLA macroinitiator was developed by ROP of lactide with 2((2-hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl

propanoate.

The

synthesized

macroinitiator was further utilized for ATRP reaction with PEGMA to get poly[poly(ethylene glycol)methacrylate]-S-S-polylactide diblock copolymer. Furthermore, to provide the targeting ability to the copolymer, folic acid was conjugated to copolymer by DCC-NHS coupling reaction (Figure S1).



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2.1.1. Synthesis of 2-((2-Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate The 2-((2-Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate was synthesized by our previously published report 52. The purified compound was further characterized using 1H NMR and High Resolution Mass Spectrometry (HR-MS, MS630, Bruker, U.S.A.) Yield = 48.90 %. 1

H NMR spectra (400 MHz, CDCl3, δ (ppm)): 1.94 (s, Br-C(CH3)2-OC-O), 2.87-2.91 (t, -OC-

O-CH2-CH2-S-S-CH2-CH2-OH), 2.98-3.01 (t, -OC-O-CH2-CH2-S-S-CH2-CH2-OH), 3.903.94 (t, -OC-O-CH2-CH2-S-S-CH2-CH2-OH), 4.42-4.48 (t, -OC-O-CH2-CH2-S-S-CH2-CH2OH).

2.1.2 Synthesis of bromine terminated polylactide macroinitiator (Br-S-S-PLA) The PLA macroinitiator with disulfide linkage was synthesized by ROP of lactide with 2-((2hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate as initiator and tin(II) 2ethylhexanoate as a catalyst. In brief, 2-((2-Hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl Propanoate (100 mg, 0.33 mmol), lactide (3.29 g, 28.83 mmol, initiator/monomer ratio 1:87.62) were added in dry toluene (15 mL) in Schlenk flask and immersed in oil bath at a temperature of 120 °C. Intermediate cycles of N2 gas purging for 5 min were employed over the period of 30 min. The catalyst tin(II) 2-ethylhexanoate (5 mol % of initiator, 5.34 μL, 0.0164 mmol) was added to the solution, and polymerization was carried out at 120 °C for 24 h under continuous N2 atmosphere. After completion of 24 h, the viscous crude polymer was collected and toluene was removed under vacuum with rotavapor. The product was further precipitated three times in cold methanol to obtain the pure product. Yield 89 %. The polymer was characterized by 1H NMR (Bruker, U.S.A.) and Gel Permeation Chromatography (GPC) (Waters, U.S.A.). Waters 2414 refractive index detector was embedded with liquid 8 ACS Paragon Plus Environment

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chromatography system. Injection volume (20 μL) of polymer concentration 5 mg/mL in THF was injected in Rheodyne injector and passed through Styragel HR3 and Styragel HR4 columns in series mode. THF at a flow rate of 1 mL/min at 37 °C was used as a mobile phase. The molecular weight of the polymer was determined by correlating with a standard calibration curve obtained by running series of increasing molecular weights of polystyrene standards. 1

H NMR spectra (400 MHz, CDCl3, δ (ppm)): 1.55-1.59 (m, -OCO-CH(CH3)-OH), 1.94 (s,

Br-C(CH3)2-OC-O), 2.94 (m, -OC-O-CH2-CH2-S-S-CH2-CH2-OH), 2.39 (m, -OC-O-CH2CH2-S-S-CH2-CH2-OH), 5.14-5.23 (m, -OCO-CH(CH3)-OH).

2.1.3. Synthesis of poly[poly(ethylene glycol) methacrylate]-polylactide diblock copolymer by atom transfer radical polymerization (ATRP) [(PEGMA)n-ss-PLA] The weighed amounts of Copper (I) bromide (CuBr) (34.43 mg; 0.24 mmol) and N′′Pentamethyldiethylenetriamine (PMDETA) (50.11 μL; 0.24 mmol) were added to dry toluene (10 mL) in a Schleck flask under continuous N2 gas atmosphere and allowed to stir at 85 °C for 30 min to form a blue colored metal-complex. The PLA macroinitiator (2 g; 0.20 mmol) was dissolved in the metal-complex followed by addition of PEGMA (Mn = 360 Da, 0.78 ml; 2.38 mmol), and the reaction was allowed to continue at 85 °C for 24 h in the presence of N2 gas. CuBr, PMDETA and macroinitiator were added at a ratio of 1:1:1. After completion 24 h, the crude polymer was separated from metal-complex by passing through a basic alumina column using tetrahydrofuran as eluent. The eluate was concentrated over a rotavapor and further precipitated three times in cold methanol. The obtained purified product was dried on rotavapor and characterized by 1H NMR, CHNS analyzer (Elementar Analysensysteme GmbH, Vario EL III, Germany), and GPC. Yield = 78 %.



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H NMR spectra (400 MHz, CDCl3, δ (ppm)): 1.25 (m, Geminal -CH3 groups), 1.40 (s, -CH3

from PEGMA), 1.55-1.59 (m, -OCO-CH(CH3)-OH), 2.89-2.91 (m, -OC-O-CH2-CH2-S-SCH2-CH2-OH), 3.62-3.69 (m, O-CH2-CH2-O from PEGMA), 4.29-4.30 (m, -OC-O-CH2-CH2S-S-CH2-CH2-OH), 5.14-5.22 (m, -OCO-CH(CH3)-OH).

2.1.4. Conjugation of folic acid to poly[poly(ethylene glycol) methacrylate]-polylactide diblock copolymer [FA-(PEGMA)n-S-S-PLA] The folic acid was conjugated to [(PEGMA)n-S-S-PLA-OH] copolymer was sequentially synthesized. In the first step, DCC-NHS chemistry was used to activate the γ-carboxylic group of folic acid. Briefly, folic acid (37.05 mg, 0.08 mmol) was dissolved in dry DMSO (15 mL) in 100 mL Schlenk flask. DCC (17.33 mg, 0.08 mmol) and NHS (9.66 mg, 0.08 mmol) was added to the reaction mixture and was continued to stir at 37 °C for 24 h in the presence of N2 gas. Dicyclohexyl urea, a reaction by-product was seperated by filtration from activated folic acid. In the second step, the [(PEGMA)n-S-S-PLA-OH] copolymer (1 g, 0.07 mmol) was dissolved with activated folic acid in round bottom flask and allowed to stir at 37 °C for 24 h in N2 environment. The DMSO and unreacted folic acid was removed from the polymer by dialysing it against the polymer method distilled water for 24 h and further lyophilized. Yield = 96 %. The lyophilized copolymer was characterized by 1H NMR, GPC, CHNS analyzer, and UV-vis spectrophotometer (LAMBDA 650 UV-vis spectrophotometer, Perkin Elmer, U.S.A.)

2.2. Formulation and characterization of polymersomes DOX loaded polymersomes (PLA-DOX-FA) were prepared by simple nanoprecipitation method.31,

53

Nanoformulation was optimized by central composite design (CCD) using

Design-Expert® 11 software (Stat-Ease, Minneapolis, MN55413, U.S.A.). The drug:polymer ratio (w/w) was varied from 0.5-1.0, while solvent to the non-solvent ratio (v/v) was varied 10 ACS Paragon Plus Environment

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from 0.1 to 0.45 and used as independent factors in design. Twelve individual formulations were prepared and percent DOX loading, particle size, and polydispersity index (PDI) of polymersomes were considered as responses (Table S1). Four center points were included to evaluate the reproducibility of the design method. The optimized nanoformulation was prepared as per the predictor profile for maximum desirability to achieve targeted response goal. The drug:polymer ratio and solvent to non-solvent ratio were kept as 0.704 and 0.411. The weighed amount of DOX (7.04 mg),

triethylamine (equal molar of doxorubicin

hydrochloride) and 10 mg of polymer was dissolved in 6.17 mL DMSO and added dropwise to 15 mL of the water phase and allowed to stir at 800 rpm for 30 min. The DOX loaded polymersomes dispersion was dialyzed against water (Milli-Q) using dialysis cassette (MWCO, 3.5 kDa) under continuous stirring for 48 h to remove DMSO and unencapsulated drug. The outer water phase was changed thrice during the course of dialysis. The blank polymersomes were prepared in similar fashion excluding the addition of DOX and trimethylamine to the polymer solution. DOX loading and entrapment efficiency were determined by UV-vis spectrophotometer using following formulae.

Loading efficiency (%) =

(Weight of DOX detected in polymersomes) (Weight of polymersomes)

× 100

(Amount of DOX detected in polymersomes)

Entrapment efficiency (%) = (Amount of DOX taken in polymersomes preparation) × 100

The size and zeta potential of developed polymersomes were determined using dynamic laser scattering (DLS, Zetasizer Nano ZS, Malvern Instruments Ltd, U.K.). The morphological analysis of PLA-DOX-FA polymersomes was determined by Field Emission Scanning Electron Microscopy (FESEM) (SEM; Zeiss EVO 50, Carl Zeiss Microscopy GmbH, Germany), High Resolution Transmission Electron Microscopy (HR-TEM) (Technai G2, 200 11 ACS Paragon Plus Environment


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kV, FEI, U.S.A.), and Atomic Force Microscopy (AFM, MultiMode Nanoscope IIIA, Bruker, U.S.A.). The presence of folic acid in folate targeted polymersomes was also evaluated by using X-ray Photoelectron Spectroscopy (XPS, AP-XPS, SPECS surface nano analysis, GmbH Germany).

2.2.1. Freeze drying of optimized polymersomes nanoformulation DOX loaded or blank polymersomes dispersion was freeze dried using lyophilizer (VirTis, Wizard 2.0, New York, U.S.A. freeze dryer system). The polymersomes dispersion was taken into glass vials and mixed with 5 % trehalose as a cryoprotectant.54 The dispersion was vortexed for ~ 40 s to dissolve cryoprotectant. All the glass vials were subjected to freeze drying process as per the optimized cycle parameters (Table S1).

2.3. Powder X-ray diffraction technique (PXRD) study The physical status of DOX in polymersomes was evaluated by PXRD (Rigaku D/max 2500 diffractometer, Japan). PXRD scans of DOX, blank polymersomes, DOX and blank polymersomes physical mixture and PLA-DOX-FA polymersomes were recorded on X-ray diffractometer. All the measurements were performed at 25 ºC with the generator set at 45 kV and 40 mA. The scanning position was set from start to end as 10 º ≤ 2θ ≥ 70 º and scan step size was kept at 0.05 º with each step scan of 50 s. 2.4. Polymersomes-blood compatibility Hemolysis and coagulation studies were performed to assess the biocompatible nature of developed polymersomes through their interactions with blood components. A fresh human blood was provided by Biomedical Engineering Unit, All India Institute of Medical Science (AIIMS), Delhi, India. 12 ACS Paragon Plus Environment

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2.4.1. Hemolysis study The hemolytic behavior of blank polymersomes was evaluated in PBS 7.4. The fresh human blood samples collected in heparinized vials and RBCs were collected by centrifugation at 1500 rpm for 10 min. The adsorbed proteins were removed from RBCs by washing with PBS 7.4, three times. Then, 100 μL of RBC stock solution was further diluted with 20 mL of PBS 7.4. Polymersomes in PBS 7.4 were further incubated in RBCs stock so that to obtain final concentrations in the range of 0.1-2.0 mg/mL and further incubated for 1 h at 120 rpm. After completion of incubation time, the dispersion was centrifuged and the released hemoglobin in the supernatant was analyzed using UV-visible micro plate spectrophotometer (PowerWave XS2, BioTek Instruments, U.S.A.) at 540 nm. The percent hemolytic behavior of polymersomes was calculated by considering negative and positive controls as PBS 7.4 and Triton X-100 respectively as follows,

Hemolysis (%) =

(Sample540 nm − Negative control540 nm ) × 100 (Positive control540 nm − Negative control540 nm )

2.4.2. Coagulation studies The blank polymersomes dispersion in PBS 7.4 (100 μL) was incubated for 1 h with 900 μL of blood so that final concentration should vary between 0.1-2.0 mg/mL and kept in an incubator shaker at 37 °C, 120 rpm. Blood was centrifuged at 5000 rpm for 15 min to obtain platelet poor plasma and incubated at 37 °C. Phospholipid containing tissue factor was mixed with plasma and excess of calcium chloride (25 mmol) was added to the plasma and fibrin clot formation time (prothrombin time, PT) was calculated. The activated partial thromboplastin (aPTT) time was calculated by mixing cephalin and micronized silica in the separated plasma followed by addition of 25 mmol of calcium chloride. The PT and aPTT timing was determined considering PBS 7.4 as control using coagulation parameters analyzer (STA Max® Stago, Germany). 55 13 ACS Paragon Plus Environment


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2.5 Protein adsorption study Polymersomes were characterized for their protein adsorption by using Biuret test. In brief, 5 mg of blank polymersomes were incubated with physiological amount BSA, 4 % BSA solution (10 mL) and kept in shaking incubator at 37 °C for 24 h at 120 rpm. Polymersomes suspension was then centrifuged at 30000 rpm for 30 min and 1 mL of supernatant was mixed with biuret reagent (4 mL) and incubated in an incubator for 30 min with shaking at 120 rpm. Then, supernatant was analyzed for unabsorbed protein at 540 nm by UV-vis spectrophotometer. BSA standard calibration curve from 0.5-2.5 mg/mL was used to quantify the protein.52, 56 2.6. Drug release studies The drug release behavior of PLA-DOX-FA polymersomes was carried out in normal physiological condition (PBS 7.4) and with and without 20 μmol of GSH. The drug release was also explored at endosomal pH (citrate buffer pH 5) with and without 10 mmol GSH.30, 53, 57, 58 In brief, a weighed amount of PLA-DOX-FA polymersomes containing 1 mg of DOX were added to a dialysis tube (MWCO 3.5 kDa) and dialyzed against respective buffer solutions for 72 h at 37 ºC and 120 rpm. At different time intervals, an aliquot of 1 mL of buffer solution was removed and fresh 1 mL of respective buffer solution was added to the external media for simulating sink condition. The release samples were simultaneously analyzed by UV-vis spectrophotometer at 480 nm. The amount of DOX in each release sample was determined by comparing absorbance values with a standard calibration curve. 2.7. Colloidal suspension stability studies of polymersomes The colloidal suspension stability studies PLA-DOX-FA polymersomes was carried out in PBS 7.4 and DMEM media supplemented with 10 % FBS for 72 h at 37 °C.59, 60 The effect of GSH level on disulfide linkages in polymersomes was evaluated at physiological (20 μmol GSH) 14 ACS Paragon Plus Environment

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and cancer cell conditions (10 mmol GSH) in PBS 7.4. In brief, 100 μL of PLA-DOX-FA polymersomes (5 mg/mL) was resuspended in respective media and samples were periodically analyzed for change in size and PDI at different time intervals for a period of 72 h using DLS.61, 62

2.8. Long term stability study of lyophilized polymersomes The long term stability study of finished polymersomes dosage form was conducted as per the International Council for Harmonisation (ICH) of technical requirements (Q1A, R2). The PLADOX-FA polymersomes with 5 % trehalose were lyophilized in glass vials and kept in the stability chamber at 25 °C ± 2 °C, 60 % RH ± 5% RH over the period of 3 months. The lyophilized polymersomes cake was observed for its resuspendability and size, PDI and zeta potential were measured after 1, 2 and 3 months. 2.9. Cell Culture Studies The cell lines, MDA-MB-231 (human breast adenocarcinoma cell line), HeLa (human cervical cancer cell line) and L929 (mouse fibroblast cell line) were procured from National Centre for Cell Science (NCCS), Pune, India. Cells were cultured in T-75 cm3 culture flasks with media supplemented with 10 % FBS at 37 °C in CO2 incubator (BINDER Inc. Bohemia, U.S.A.). DMEM media was used to grow HeLa and L929 cells, while MDA-MB-231 cells were cultured in Leibovitz's L-15 medium. 2.9.1. Determination of reactive oxygen species (ROS) from polymersomes The ROS generated by polymersomes was evaluated by fluorescence spectrophotometer using 2,7-dichlorohydrofluorescein diacetate (H2DCFDA) dye. MDA-MB-231, HeLa, and L929 cells dispersion in media were added in 96 well plate at a density of 5 × 103 cells/well and further incubated in a CO2 incubator at 37 °C for 24 h to reach the confluency of 70-80 %. 15 ACS Paragon Plus Environment


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Blank polymersomes were added into wells so that the final concentration should vary from 0.1-2.0 mg/mL and further incubated for 48 h. After completion of incubation time, fresh media (150 μL) containing H2DCFDA dye (20 μmol) was added to each well and incubated for 30 min. To carry out the cell lysis, the media with dye was removed and 1 % Triton X-100 in PBS 7.4 was added for 30 min. Aliquot of 150 μL was removed from each well and added to black 96 well plate. The fluorescence produced by dichlorofluorescein (DCF, an oxidized product of H2DCFDA) was evaluated using fluorescence multi plate spectrometer (BioTek Synergy H1, U.S.A.) at excitation and emission of 485 nm and 530 nm respectively. The percent ROS generation was determined using untreated cells as negative control. 2.9.2. Cell viability assay The cytocompatibility of blank polymersomes was assessed by using MTT cytotoxicity assay on MDA-MB-231, HeLa, and L929 cells. In brief, cells dispersion was added to 96 well plate at a density of 5 × 103 cells per well and incubated in a CO2 incubator at 37 °C for 24 h to achieve 70-80 % confluency. Blank polymersomes were added to each well so that concentration should vary between 0.1-2.0 mg/mL and further incubated for 48 h. Moreover, to assess the cytotoxic potential of targeted PLA-DOX-FA polymersomes, non-targeted DOX loaded polymersomes (PLA-DOX) and DOXIL, these different nanoformulations were incubated with cells in varying concentration of DOX at a range of 0.025-6 μg of DOX for 48 h at 37 °C. The media with polymersomes were removed and replaced with fresh media (150 μL) containing 0.35 % of MTT dye and incubated for 4 h at 37 °C. The media was removed and 150 μL of DMSO solution was added to each well. The absorbance was measured at 540 nm on microplate spectrophotometer (PowerWave XS2, BioTek Instruments, U.S.A.). The cell viability was determined by considering negative and positive control as PBS 7.4 and 1 % Triton X-100 respectively as follows,


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Sample

540nm Cell viability (%) = Negative Control

−Positive Control540nm

540nm −Positive Control540nm

× 100

2.9.3. Qualitative evaluation of in vitro cellular internalization by confocal laser scanning microscopy (CLSM) CLSM was used to determine the cellular internalization of polymersomes, PLA-DOX-FA, PLA-DOX, and DOXIL in MDA-MB-231, HeLa, and L929 cells. Cells dispersion in media was added to 24 well plate containing 10 mm × 12 mm cover slips at a density of 5 × 104 cells per well and incubated in a CO2 incubator at 37 °C for 24 h to achieve the 70-80 % confluency. DOX loaded polymersomes and DOXIL were added to wells at a DOX concentration of 5 μg/mL and incubated for 2 h. The cells were washed thrice with PBS 7.4 and fixed with 4 % paraformaldehyde solution for 20 min. The fixative solution was removed and cells were washed three times with PBS 7.4. Cells nuclei were further stained with DAPI containing fluoroshield mounting medium and visualized under CLSM (FluoView FV1000 Olympus, U.S.A.) with respective emission wavelengths for DAPI (460 nm), DOX (560 nm) at 60 × magnification. The nuclear localization of DOX in MDA-MB-231, HeLa, and L929 cells was studied for PLA-DOX-FA polymersomes in a similar fashion as mentioned above by increasing the incubation time from 2 h to 8 h. 3.9.4 Competitive assay with free folic acid The cellular uptake of PLA-DOX-FA polymersomes was evaluated for competition assay with free folic acid on MDA-MB-231, HeLa, and L929 cells. Cells were seeded in 24 well plate containing 10 mm × 12 mm cover slips at a density of 5 × 104 cells per well and incubated in 17 ACS Paragon Plus Environment


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a CO2 incubator at 37 °C for 24 h to get the desired confluency of ~ 80 %. Prior to the addition of polymersomes, cells were incubated with free folic acid (1 mmol) for 2 h and kept at 37 °C to saturate the folate receptors of cells. PLA-DOX-FA polymersomes were added to cells at a DOX concentration of 5 μg/mL and incubated in a CO2 incubator at 37 °C for 2 h. After completion of 2 h, cells were washed three times with PBS 7.4 and fixed with paraformaldehyde and again washed with PBS 7.4 thrice. The nuclei of cells were stained with DAPI containing fluoroshield mounting medium and observed under CLSM at 60 × magnification. 2.9.5. Quantitative evaluation of in vitro cellular internalization by flow cytometry The quantitative cellular internalization of polymersomes and DOXIL was evaluated by flow cytometry in MDA-MB-231, HeLa, and L929 cells. Cells were seeded in 6 well plate at a density of 2 × 105 cells per well and incubated in a CO2 incubator at 37 °C for 24 h. After reaching the desired confluency PLA-DOX, PLA-DOX-FA polymersomes and DOXIL were added to wells at DOX concentration of 5 μg/mL and further incubated for 2 h in the CO2 incubator. Cells were washed three times with PBS 7.4 and harvested with 0.25 % trypsin solution, cells were further centrifuged at 3500 rpm for 5 min to obtain the cell pellet. The pellet was then resuspended in 250 μL of PBS 7.4 and analyzed using flow cytometry (BD accuri C6, U.S.A.). 2.10. Animal studies All animal studies were performed on female Swiss albino mice (7-8 weeks old, 25 ± 5 g) procured from the central animal facility, AIIMS, New Delhi, India. All the experimental procedures were carried out as per the animal ethical committee guidelines of AIIMS (786/IAEC/16). All animals were kept in polycarbonate cages at 25º C with appropriate


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humidity and fed with chow food and purified water ad libitum, during the entire experimental period. Animals were consistently acclimatized with uninterrupted 12 h light and dark cycle. 2.10.1. In vivo toxicity assessment of polymersomes Swiss albino mice were randomly divided into two groups (n = 6) for investigating repeated dose toxicity study of blank polymersomes. Lyophilized blank polymersomes were suspended in PBS 7.4 and 250 μL of the suspension was administered repeatedly through intraperitoneal route in one group at a dose of 100 mg/kg (on days 0, 3, 6, and 9). The second group was considered as a control with the repeated intraperitoneal administration of 250 μL of PBS 7.4. On the 11th day, blood from all animals was collected from retro orbital venous puncture and investigated for hematology and serum biochemistry analysis. All the readings were noted using fully automated biochemical parameter analyzer (TurboChem 100, Awareness Technology, U.S.A.). All animals were sacrificed by cervical dislocation to collect the organs and were stored in 10 % formalin buffered solution for histopathological assessment. 2.10.2. In vivo anticancer efficacy The in vivo efficacy of free DOX, PLA-DOX-FA polymersomes and DOXIL was evaluated by using Ehrlich ascites tumor (EAT) model. 36 Tumors were persuaded in Swiss albino mice by subcutaneous injection of 200 μL of EAT cell suspension (∼2 × 107 cells) on the dorsal side of mice. Tumor volume was checked periodically and when tumor volume reaches to 200-250 mm3, animals were randomly divided into four groups with six animals in each group. Each group was designated for respective formulation such as control (PBS 7.4), free DOX, PLADOX-FA polymersomes and DOXIL treated. All formulations with the volume of 100 μL were administered six times via tail vein at equivalent DOX dose of 5 mg/kg body weight at every repeating third day (Day 0, 3, 6, 11, 12 and 15). Tumor dimensions were measured at every



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third day using digital Vernier caliper and tumor volume was determined using following formula,

Tumor Volume =

L × W2 2

Where L and W symbolize the largest and perpendicular tumor diameter respectively. At the end point of the study (day 18), blood was collected from retro orbital venous plexus and further, mice were sacrificed by cervical dislocation. The collected blood from different groups’ animals was investigated for any changes in serum biochemical parameters. Vital organs including tumor were removed from each group mice and stored in 10 % formalin buffered solution which were later used for their histopathological evaluation. 2.10.3. Histopathological evaluation of the tumor and vital organs Histopathological assessment of tumor and vital organs was performed to evaluate the toxicity and antitumor activity of various formulations used during the course of tumor regression study. Tumor and vital organ sections were embedded in paraffin blocks and sliced into 5 μm thin and uniform sections by using fully automatic vibrating blade microtome (VT 1000 S, Leica, Germany). Hematoxylin-Eosin dye was used to stain the sections and further visualized under a light microscope in bright field illumination (Olympus IX73 microscope, U.S.A.). 2.11. Statistical Analysis All in vitro and in vivo data was represented as the mean ± standard deviation (SD), while the data from in vivo tumor regression and body weight changes during the tumor inhibition study were presented as mean ± standard error mean (SEM). Statistical analysis was performed by using Bonferroni multiple comparison test with GraphPad Prism (Version 7, GraphPad Software, Inc., La Jolla, U.S.A.). 20 ACS Paragon Plus Environment

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3. RESULTS AND DISCUSSION 3.1. Synthesis of folic acid conjugated poly[poly(ethylene glycol)methacrylate]-S-Spolylactide diblock copolymer [(PEGMA)n-S-S-PLA] The synthesis scheme for folic acid conjugated PEGylated PLA diblock copolymer was depicted in figure S1. Lactide ring opener substrate, 2-((2-hydroxyethyl)-disulfanyl)ethyl-2bromo-2-methyl propanoate (Supporting information S1) was used to get polylactide macroinitiator (Supporting information S2). This macroinitiator was further used to synthesize amphiphilic PEGylated PLA diblock copolymer through ATRP reaction between PEGMA and PLA macroinitiator in the presence of PMDETA and CuBr as a catalytic complex. One side ATRP reaction was proceeded with PLA macroinitiator: CuBr: PMDETA ratio 1:1:1. The PEGylation was carried out in a controlled fashion so as to get ~ 25 % hydrophilicity which can yield polymersomes nanostructures. The yielded product was characterized by 1H NMR and GPC. The 1H NMR, displayed the multiplet peak at δ 3.62-3.69 corresponding to the PEGMA units in PLA diblock copolymer (Figure S6). Based on 1H NMR integration it was calculated that ~ 6 units of PEGMA had been attached to PLA polymer providing ~ 24 % of hydrophilicity to the polymer backbone. 1H NMR depicts the Mn of the diblock copolymer as ~ 13000 Da while GPC showed Mn as ~ 17000 Da with PDI of 1.44 (Figure S7). Thus, a copolymer with uniform molecular weight distribution was successfully synthesized using ATRP process. Folic acid as a targeting moiety was conjugated to the polymer. Folic acid as targeting ligand has been enormously explored to target folate overexpress cell lines and tumor.16, 63, 64 The DCC-NHS coupling reaction was carried out between γ-carboxylic acid of folic acid and the terminal hydroxyl group of PEGMA-PLA diblock copolymer. The folic acid conjugated copolymer was characterized by various methods. The CHNS analysis depicted the percent 21 ACS Paragon Plus Environment


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nitrogen content of copolymer chain as ~ 4.5 % which must be due to the successful conjugation of folic acid to the (PEGMA)n-S-S-PLA chain (Table S2). Moreover, XPS scan of folic acid conjugated polymersomes depicted the increase in the nitrogen signal intensity at a binding energy of 401 eV as compared to non-conjugated polymersomes. Thus, XPS also indicates the conjugation of folic acid to the copolymer chain (Figure S8). This enhanced nitrogen content of folate conjugated copolymer is attributed to the folic acid attachment to the polymer backbone. The UV-visible spectroscopy scan of folate conjugated copolymer showed shift blue shift depicting the folic acid conjugation (Figure S9). FTIR spectra of PLA-PEGMAFA showed the characteristics stretching vibrational bands for aromatic ring and primary amine group of folic acid at 1576 cm-1 and 3300-3500 cm-1 (Figure S10), corroborating the observations obtained from CHNS and XPS analysis and UV-visible spectroscopy methods. 3.2 Optimization of drug loaded polymersomes by CCD The optimization of drug loaded polymersomes was carried out using quality-by-design (QbD) approach by applying design of experiments (DoE). DoE is a systematic approach which provides quality product using the minimum number of trials by systematically changing the processing parameters.65-67 The important variables affecting the quality of nanoformulation can be systematically varied using DoE which provides simultaneous effects of multiple variables and offers a significant advantage over the conventional optimization method.68 Response surface methodologies such as central circumscribed design (CCD) and BoxBehnken design are normally utilized for assessing the differences between variables.69 Herein, we have considered CCD to optimize polymersomes formulation process due to its suitability in the optimization of nanoformulation.69-71 Based on the prior research, we identified drug:polymer ratio and solvent:non-solvent ratio are critical variables in the optimization of polymersomes nanoformulation and hence were selected as independent variables for DoE (Table S3,S4). Statistical evaluation of each model was performed using Design-Expert 11 22 ACS Paragon Plus Environment

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software. All the four responses depicted goodness of fit to their respective model (Figure S11). Based on the suggested quadratic model, both the factors, significantly affected the tested responses (Table S3.1.1, S3.2.1, S3.3.1). As suggested in surface response 3D plots, when the drug:polymer ratio increases, drug loading get enhanced but above a ratio of 0.75 the particle size and PDI got compromised with steady increment. While the solvent:non-solvent ratio showed direct correlation for drug loading (Table S5, Figure 1A,B,C). Based on the optimum design space suggested by contour plots for each response, we have selected the desired variables given by software and formulated the optimized polymersomes nanoformulation, keeping in mind to maximize the drug loading and with minimal particle size and PDI (Figure 1A1,B1,C1).

Figure 1. Surface response plots showing the effect of Drug:Polymer ratio (w/w) and Solvent:Non-solvent ratio (v/v) on, (A) drug loading (%), (B) particle size (nm), (C) polydispersity index (PDI) and respective subtitle figures, (A1), (B1), and (C1) indicate their corresponding contour plots.



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3.3. Formulation and characterization of polymersomes Polymersomes were formulated by PLA-PEGMA-FA diblock copolymer, where the polymer could get spontaneously self-assembled in the aqueous phase. This self-assembly of developed polymer can be attributed to balanced hydrophilic/hydrophobic fractions of the polymer chain (~ 24 % hydrophilicity). The optimized formulation displayed uniform particle size of 113.31 ± 3.08 nm and low PDI of 0.04 ± 0.020 (Figure 2A), while the apparent zeta potential was found to be -30.30 ± 2.68 mV (Figure 2B). The DOX loading was improved by changing the pH of organic phase during the preparation of polymersomes, where triethylamine (equimolar concentration of doxorubicin hydrochloride) was used to for neutralization and its conversion into hydrophobic free base.30, 72 This unionized free DOX can be effectively loaded into the polymersomes with enhanced loading efficiency. The DOX loaded polymersomes depicted enhanced loading efficiency of 20.33 ± 1.20 % and entrapment efficiency of 76.55 ± 4.53 %. Since DLS gives the hydrodynamic size of polymersomes, we have determined the absolute size by using HR-TEM, where 200 particles were individually measured, depicting the average size of 58.45 ± 9.61 nm (Figure 2C). HR-TEM showed the spherical bilayer vesicular structures (Figure 2D). The other characterization techniques such as FESEM and AFM also showed the absolute particle as ~ 60 nm, supporting the HR-TEM observations (Figure 2E,F). Trehalose 5 % was used was used as cryoprotectant during lyophilization of polymersomes to prevent their aggregation during storage conditions. The developed polymersomes could readily redisperse in water without showing any aggregation. The resuspension time of lyophilized formulation in PBS 7.4 was found to be ~ 15 s.


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Figure 2. Characterization of PLA-DOX-FA polymersomes by, (A) dynamic light scattering (DLS) size, n = 3, (B) apparent zeta potential, n = 3, (C) Size by HR-TEM, n = 200, (D) HRTEM, (E) FESEM, and (F) AFM. Where arrows indicate their respective zoom images. 3.4. Powder X-ray diffraction technique (PXRD) study The physical nature of DOX in polymersomes was determined using PXRD technique. The PXRD scan of pure DOX depicted 13 major crystalline peaks at 2θ of 13.08°, 14.74°, 16.62°, 17.56°, 18.46°, 19.30°, 20.48°, 22.48°, 23.32°, 25.06°, 26.22°, 29.84° and 30.31° suggesting the crystalline nature of pure DOX (Figure S12). The blank polymersomes showed amorphous nature, while the physical mixture of pure DOX and blank polymersomes displayed characteristics peaks of pure DOX showing its crystalline nature in the physical mixture. The characteristics peaks of DOX were not observed in diffraction spectra of PLA-DOX-FA polymersomes and found to be more or less similar to that observed for blank polymersomes.



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This indicates loss of crystallinity of DOX, when it gets loaded into polymersomes in amorphous form. This change of crystalline to amorphous nature of DOX leads to the low crystalline lattice energy and increase in thermodynamic energy, which could further help the drug for its faster solubilization and release.16, 52, 73 3.5. Protein adsorption of polymersomes Protein adsorption is a paramount factor with respect to the stability of nanosystem in blood and its circulation half-life. The nanocarriers with a hydrophobic surface tend to adsorb proteins on their surface, thereby enhancing their macrophage recognition and opsonization which can lead to elimination from circulation and thus limiting their therapeutic efficacy. To minimize the protein adsorption, researchers have tried to make the surface of nanocarriers hydrophilic by using polyethylene glycol (PEG) or polyethylene oxide (PEO). These compounds render the “stealth” nature to the nanosystem thereby resisting interactions with components of the blood stream and result in increased circulation half-life and the enhanced probability of predisposing the drug to the target site.74-76 PEG and PEO has a long history of safety in humans and classified as Generally Regarded as Safe (GRAS) by the US FDA. 77 Herein, we have incubated the blank polymersomes with 4 % BSA, which is a physiological concentration of blood. Polymersomes showed protein adsorption of 5.93 ± 2.23 %. This low protein adsorption over the surface of polymersomes can be attributed to the PEG units in polymer chains of polymersomes. The hydrophilic chains of PEG over the polymersomes surface prevents the proteins from being adsorbed on their surface and thus can deceive the mononuclear phagocytic system (MPS) for their uptake.


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3.6. Polymersomes-blood compatibility The polymersomes-blood interactions were determined by hemolytic and coagulative behavior of polymersomes. 3.6.1. Hemolysis study Hemocompatible nature of nanosystem is an essential criterion towards the nontoxicity and safety profile of nanoplatforms intended for intravenous administration. Nanocarriers can cause disruption of RBCs after their interactions. According to the several studies based on in vitro and in vivo correlation reflects that the percent hemolysis of 5-25 % caused due to nanocarriers-blood interactions is considered as “no concern”.78,

79

Furthermore, in vivo

hemolysis caused due to nanocarriers interactions with RBCs is far less than their in vitro hemolytic effects. The released hemoglobin can be measured by UV-vis spectrophotometer at 540 nm. The amount of hemoglobin released has an inverse correlation with the hemocompatibility of nanosystem. Polymersomes displayed the concentration dependent hemolysis over the range of 0.1-2.0 mg/mL with a maximum hemolysis of 9.15 ± 1.39 % (Figure 3). This low percent hemolysis is attributed to the biocompatible nature of polymersomes reflecting their hemocompatible nature even over the highest tested concentration of 2 mg/mL.



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Figure 3. Percent hemolysis of blank polymersomes. (Mean ± SD, n = 3). 3.6.2. Coagulation studies When nanocarriers enter into circulation, they can activate the coagulation pathways which can cause dire consequences such as intravascular coagulation leading to deep vein thrombosis which can cause partial or complete blockage of vessels.80, 81 Moreover, their interaction with platelets can ameliorate the efficacy of nanosystem. Therefore, evaluation of thrombogenic parameters is an essential norm to assess the safety profile of nanosystem. 82-84 The activated partial prothrombin time (aPTT) and prothrombin time (PT) is related to intrinsic and extrinsic pathway respectively. The significant changes from their normal values after interactions of nanocarriers with blood can leads to activation of the coagulation cascade. The normal values for aPTT are in the range of 27-40 s, while for PT are in between 11-14 s.85 Polymersomes were incubated with blood for 1 h to evaluate the changes in aPTT and PT values. The aPTT and PT values did not show significant changes than control values, designating blood compatibility of developed polymersomes nanosystem with respect to coagulation pathways (Table 1). This coagulation compatibility of polymersomes must be attributed biocompatible nature of polymer used in polymersomes nanoformulation.


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Biomacromolecules

Table 1. Determination of coagulation parameters of blank polymersomesa.

Polymersomes concentration

aPTT (s)

PT (s)

Control

33.20 ± 2.30

12.74 ± 0.31

0.1

34.01 ± 1.28

12.44 ± 1.03

0.5

33.39 ± 1.72

11.77 ± 0.23

1.0

33.87 ± 1.22

12.84 ± 0.24

1.5

35.00 ± 1.63

12.45 ± 0.62

2.0

34.81 ± 0.98

12.55 ± 0.64

(μg/mL)

(aMean ± SD, n = 3) 3.7. Colloidal stability of DOX-PLA-FA polymersomes The colloidal stability of nanosystem is an indispensable aspect in clinical settings point of view. Nanocarriers with uniform dispersion in aqueous media without displaying any aggregation can facilitate the cellular uptake. Aggregation of nanocarriers in media can have adverse effects o cell-nanocarriers interactions and henceforth it is crucial to investigate the stability of nanocarriers in different media.86, 87 The cytosol of cancer cells have very high redox potential (100-1000 times higher) as compared to extracellular matrix and body fluids including blood. Moreover, this enhanced GSH concentration was constantly maintained due to high levels of NADPH and GSH reductase enzyme.88 The redox responsive nature of nanocarriers depends on the high reducing proficiency in the cytosol than extracellular milieu of tumor cells.41 The disulfide bonds in nanocarriers can be cleaved in such a high GSH concentration prompting the triggered drug release in tumor cells. Herein, we have examined



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the colloidal stability of PLA-DOX-FA polymersomes in different media such as PBS 7.4, DMEM with 10 % FBS, 20 μmol GSH and 10 mmol GSH over the period of 72 h and their size and PDI were measured using DLS. The PLA-DOX-FA polymersomes were found to be stable in PBS 7.4, DMEM with 10 % FBS and PBS 7.4 with 20 μmol GSH with average size and PDI below of 130 nm and 0.16 respectively (Figure 4). Stability of polymersomes in 20 μmol GSH designates their stability in blood circulation. Thus, the stable nature of polymersomes in blood GSH concentration suggested their stability during blood circulation. The abrupt increase of size and PDI in 10 mmol concentration was due to the reducing influence of GSH, where thiolates (R-S-) acts as reducing means and cause disruption of disulfide bonds with subsequent aggregation of polymersomes. 89

Thus, colloidal stable nature of polymersomes demonstrated their excellent stability in the

extracellular matrix and shall have abrupt intracellular drug disposition on account of their redox potential sensitiveness.


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Figure 4. Colloidal suspension stability study of DOX-PLA-FA polymersomes in PBS 7.4, DMEM with 10% FBS, PBS 7.4 with 20 μmol GSH and PBS 7.4 with 10 mmol GSH over the period of 72 h, determined by DLS: (A) size, (B) PDI, where arrows indicate the respective zoom images. (Mean ± SD, n = 3). 3.8. Long term stability study of PLA-DOX-FA polymersomes The long stability study of pharmaceutical finished nanocarriers dosage form is an important parameter to assess its resuspendability, syringeability, and efficacy.90-92 Long term stability for lyophilized PLA-DOX-FA polymersomes was conducted as per ICH guidelines. The size, PDI and zeta potential were measured for 3 months, where polymersomes did not display significant change compared to initial tested sample (Table 2, Figure S13). Moreover, during the studied storage period, the resuspendability for a lyophilized cake of polymersomes was below 1 min. Thus, the designed lyophilized formulation abide by the pharmaceutical speculations with the potential for its long term storage competency. 31 ACS Paragon Plus Environment


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Table 2. Long term stability study of lyophilized PLA-DOX-FA polymersomes as per the ICH guidelinesa. Long term stability study Sampling time

Storage conditions, 25 °C ± 2 °C, 60 % RH ± 5% RH

Size (nm)

Zeta potential

Resuspension

(mV)

time (s)

PDI

Initial

118 ± 2.30

0.08 ± 0.06

-28.16 ± 2.51

15

After 1 month

123.53 ± 3.15

0.06 ± 0.05

-30.33 ± 1.25

22

After 2 month

127.16 ± 2.81

0.09 ± 0.06

-28.93 ± 2.05

26

After 3 month

124.63 ± 3.95

0.03 ± 0.02

-29.10 ± 1.93

31

(aMean ± SD, n = 3) 3.9. In vitro drug release studies of DOX-PLA-FA polymersomes The in vitro release behavior of DOX-PLA-FA polymersomes depicted the highest DOX release at pH 5 with 10 mmol GSH (~ 82 %) followed by a release at pH 7.4 with 10 mmol GSH (~ 65 %), while cumulative drug release at pH 5 was around ~ 49 %. The release profiles at pH 7.4 and at pH 7.4 with 20 μmol GSH were comparable to each other and found to be ~ 21 % (Figure 5). The enhanced drug release in the GSH cytosolic concentration of cancer cells (10 mmol GSH) suggested the selective release pattern of redox sensitive polymersomes. This is credited to the degradation of polymersomes at this high GSH concentration. The decreased DOX release profile at pH 7.4 and pH 7.4 with 20 μmol GSH can be attributed to the slow and sustained drug release at normal physiological pH and GSH conditions. The less drug release at 20 μmol GSH is due to the stability of disulfide linkages in polymersomes at blood GSH level. The increased drug release at pH 5 is due to enhanced hydrophilicity of DOX on account 32 ACS Paragon Plus Environment

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of its protonation of glycosidic anime group at such low pH (DOX pKa = 8.24). This type of pH dependent release pattern of DOX was also demonstrated with other DOX loaded polymeric nanosystems.72, 93 This enhanced DOX release at pH 5 can definitely render an added advantage to the polymersomes for their improved cytosolic drug release within enndo/lysosomal compartments. The disruption of polymersomes by the breakdown of disulfide linkages in polymersomes at 10 mmol GSH concentration might have further increased the DOX release at pH 5. Thus, the developed polymersomes nanosystem revealed GSH and pH reliant improved DOX release pattern. To predict the drug release kinetics, release profiles were fitted in various kinetic models such as zero order, Hixon-Crowell, first order, and Higuchi. Korsmeyer peppas model (Table S6, Table S6.1, Supporting information S4).

Figure 5. The release profile of DOX-PLA-FA polymersomes at pH 7.4, pH 7.4 with 20 μmol GSH, pH 5, pH 7.4 with 10 mmol GSH and pH 5 with 10 mmol GSH. (Mean ± SD, n = 3).



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3.10. Cell culture studies 3.10.1. In vitro qualitative assessment of cellular internalization of polymersomes and DOXIL using confocal laser scanning microscopy (CLSM) The in vitro cellular uptake is an important aspect for nanosystem, as it can directly correlate with it's in vivo efficacy. The cellular uptake of polymersomes was evaluated in folate overexpressed cell lines such as MDA-MB-231 and HeLa, while negative folate expressing cell lines, L929 was used for comparative study. LysoTracker Green was used to observe colocalization and to investigate the internalization process of polymersomes, whereas DAPI was used to stain the nuclei of cells. PLA-DOX-FA polymersomes depicted the enhanced cellular uptake in MDA-MB-231 and HeLa cells than DOXIL and PLA-DOX polymersomes after 2 h incubation (Figure 6A, B). This can be attributed to the enhanced cellular uptake of targeted polymersomes through folate receptor mediated endocytosis than their non-targeted counterpart, PLA-DOX polymersomes. DOXIL being non-targeted nanoformulation, the DOX fluorescence was also found to be similar as obtained with PLA-DOX polymersomes in MDAMB-231 and HeLa cells. The non-folate overexpressing cells, L929 depicted the less DOX fluorescence for MDA-MB-231 and HeLa cells for PLA-DOX-FA polymersomes which can be due to their nonspecific cellular uptake of polymersomes and less folate expressing capability of L929 cells. Moreover, the observed fluorescence in these normal cells for PLADOX-FA polymersomes was more or less similar to the DOXIL and PLA-DOX polymersomes (Figure 6C). Thus, after 2 h incubation, the DOX fluorescence of polymersomes can be visualized in the cytoplasm co-localizing with LysoTracker suggesting their effective entry in lysosomal compartments. The nuclear localization of DOX was clearly observed after incubation of DOX-PLAFA for 8 h (Figure S14). A minimal amount of drug was found in the cytoplasm and maximum


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extent of the drug reached to the nucleus. This can be attributed to the diffusion DOX into the nucleus through endo/lysosomal compartments, resulting in nuclear accrual of DOX.94


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.

Figure 6. Cellular internalization of polymersomes by confocal laser scanning microscopy (CLSM) in (A) MDA-MB-231, where (A1) PLA-DOXFA polymersomes, (A2) DOXIL, (A3) PLA-DOX polymersomes, (B) HeLa, where (B1) PLA-DOX-FA polymersomes, (B2) DOXIL, (B3) PLADOX polymersomes and (C) L929, where (C1) PLA-DOX-FA polymersomes, (C2) DOXIL, (C3) PLA-DOX polymersomes. All the images were taken at 60 × magnification. 36 ACS Paragon Plus Environment

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3.10.2. Competitive assay with free folic acid In the competitive binding assay, after addition of free folic acid, the uptake of PLA-DOX-FA polymersomes was evidently reduced in folate receptors over expressing cell lines, MDA-MB231 and HeLa (Figure S15). The results suggested that decreased intensity of DOX fluorescence must be due to complete saturation of available folate receptors on cell surface so that there was reduced receptor mediated endocytosis of folate targeted polymersomes. Such type of competitive inhibition with free folic acid was also demonstrated with other folate targeted nanosystems.95,

96

Therefore, it can be concluded that cellular internalization of

targeted polymersomes could be competitively inhibited by free folic acid ligand, suggesting their uptake process by folate receptor mediated endocytosis. 3.10.3. In vitro quantitative assessment of cellular internalization of polymersomes and DOXIL using flow cytometry The quantitative evaluation of cellular internalization for PLA-DOX-FA, PLA-DOX polymersomes, and DOXIL was carried out with MDA-MB-231, HeLa, and L929 cells to validate the enhanced internalization of folate targeted polymersomes as perceived in confocal microscopy. The folate receptor overexpressing cells, MDA-MB-231 and HeLa depicted enhanced cellular uptake for PLA-DOX-FA polymersomes compared to non-targeted nanocarriers, PLA-DOX and DOXIL. The uptake of PLA-DOX-FA polymersomes in MDAMB-231 and HeLa cells was observed to be ~ 9 and ~ 4 folds higher than their non-targeted counterpart (PLA-DOX polymersomes), while with respect to DOXIL ~ 4 and ~ 2 folds increment was witnessed, respectively (Figure 7). This superior cellular uptake demonstration of PLA-DOX-FA polymersomes can be credited to the targeting efficacy of attached folic acid units. In case of L929 cells, targeted and non-targeted polymersomes and DOXIL had depicted low DOX fluorescence, nonetheless of attached targeting ligand to polymersomes. This



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specifies about their non-specific internalization by L929 cells, which was also demonstrated in confocal microscopy visualization. Hence, flow cytometry study harmonizes with confocal microscopy outcomes that folate decorated polymersomes brought about an improved cell uptake while sparing their uptake in non-cancerous cells.

Figure 7. Quantitative cellular internalization assessment of PLA-DOX-FA, PLA-DOX polymersomes and DOXIL in (A) MDA-MB-231, (B) HeLa and, (C) L929 cells. 3.10.4. In vitro toxicity evaluation of polymersomes The cytocompatibility of developed polymersomes formulation was assessed by MTT cytotoxicity assay. The assay is based on the cell metabolic efficiency NADPH dependent dehydrogenase enzyme. The MTT dye in viable cells gets reduced by this enzyme giving purple colored formazan crystals. These crystals can be dissolved in DMSO or 10 % sodium dodecyl sulfate (SDS) in 0.01 M HCl and absorbance can be measured at 540 nm.97 Blank polymersomes were tested for their cytotoxic level at a concentration range of 0.1-2.0 mg/mL on MDA-MB-231, HeLa, and L929 cell lines. Polymersomes were found to be cytocompatible in the tested concentration range (Figure 8A). Moreover, we have also evaluated the reactive oxygen species (ROS) produced by developed polymersomes nanoformulation in 38 ACS Paragon Plus Environment

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aforementioned cell lines. The polymersomes did not display any significant ROS production compared to the control samples (Figure S16). This must be due to the biocompatible nature of PEGylated polymer. Thus, the developed nanosystem did not keep any perspective to enhance intracellular free radicals. Therefore, this non-toxic nature of polymersomes designates their biosafety profile for safely employing them in biological and drug delivery applications. As far as the DOX loaded nanocarriers are concerned, dose dependent cytotoxicity was observed in all three cell lines with significant cytotoxicity for PLA-DOX-FA polymersomes in folate receptor overexpressing cell lines, MDA-MB-231 and HeLa (Figure 8B,C). Excitingly, PLA-DOX-FA polymersomes were able to spare the normal L929 cells, resulting in less cytotoxicity. This can be attributed to the presence of minimal folate receptors on their surface (Figure 8D). Herein, we have compared the designed nanoformulation with DOXIL and IC50 for PLA-DOX-FA polymersomes and DOXIL on MDA-MB-231 was found to be 0.537 μg/mL and 1.286 μg/mL respectively, while for HeLa cells it appeared as 1.075 μg/mL and 1.447 μg/mL respectively. This less IC50 value for PLA-DOX-FA polymersomes is attributed to the selective folate receptor targeting, while DOXIL does possess folate targeting. The cytotoxicity of non-folate targeted polymersomes, PLA-DOX was more or less comparable to the DOXIL formulation. The IC50 values for the PLA-DOX polymersomes in MDA-MB-231 and HeLa cells were overserved to be 2.242 μg/mL and 4.398 μg/mL respectively. The lower IC50 values of DOXIL in MDA-MB-231 and HeLa cells than non-targeted polymersomes can be attributed to its phospholipids content used in DOXIL preparation which mimics the cellular bilayer structures. Interestingly, after folate targeting, polymersomes displayed superior cellular cytotoxicity in folate overexpressing cell lines which must be on account of their higher folate receptor mediated endocytosis. We found, DOXIL with L929 cells, depicted significant 39 ACS Paragon Plus Environment


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toxicity at a high concentration of DOX as compared to PLA-DOX-FA polymersomes, which can be due to its non-targeting capability.

Figure 8. Effect of cell proliferation of polymersomes determined by MTT cytotoxicity assay, (A) Cytocompatibility of blank polymersomes on MDA-MB-231, HeLa, and L929 cells. The cytotoxicity profile of DOXIL and PLA-DOX-FA polymersomes on, (B) MDA-MB-231, (C) HeLa and (D) L929 cells. (Mean ± SD, n = 4). Where *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, “ns” indicates not significant, p > 0.05).


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3.11. Animal studies 3.11.1. In vivo toxicity evaluation of polymersomes Biocompatibility and safety profile of developed polymersomes was evaluated in female Swiss albino mice for eleven days by their repeated dose administration as per the previously published reports.52, 98, 99 The blank polymersomes at a dose of 100 mg/kg were dispersed in 200 μL of PBS 7.4 and repeatedly administered four times, intraperitoneally at an interval of three days. Polymersomes treated group did not display any significant changes in body weights compared to control group, treated with PBS 7.4 (Figure S17). The histopathological assessment of vital organs like heart, kidneys, liver, spleen, and lungs was comparable to the control (Figure S18). Moreover, we have also analyzed the serum biochemical hematological parameters of treated groups, where the parameters had the negligible difference between polymersomes treated group and control. (Table S7, S8). Thus, as per the obtained results developed polymersomes nanoformulation is considered to be safe for in vivo use. 3.11.2. In vivo studies for evaluation of antitumor effect of polymersomes The Ehrlich ascites tumor (EAT) is a transplantable tumor model originating form mammary carcinoma of a mouse. EAT cells are well known for their high transplantable efficiency and quick multiplication rate. It is postulated that EAT cells express a high amount of folic acid receptors on their surface, and henceforth, we have decided to explore this folate receptor overexpressing decent model for assessing antitumor activity of our targeted polymersomes nanosystem.100-103



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3.11.2.1. Tumor suppression study in EAT tumor model The antitumor effect of free DOX, PLA-DOX-FA polymersomes and DOXIL was assessed in EAT tumor bearing Swiss albino mice. Free DOX and all DOX loaded nanocarriers were administered intravenously six times with a dosage comparable to 5 mg/kg of DOX, while the control group was administered with PBS 7.4 for 15 days and reduction in tumor volume was inspected. The PBS 7.4 treated (control) group demonstrated a dynamic increment in tumor volume with roughly ~ 6 fold increment on the 18th day compared to the initial value at day 0 (Figure 9A). During the course of the experiment, the body weight of control group animals was found to be continuously increasing than other treatment groups depicting significant variations in their body weights (p < 0.001). This increase in the body weight of control animals must be credited to their expanding tumor volume day by day (Figure S19). Free DOX showed moderate antitumor effect with a decrease in tumor volume at a level of ~ 25 %, whereas DOXIL nanoformulation depicted significant tumor reduction of ~ 70 % tumor reduction compared to the control (p < 0.001). Interestingly, PLA-DOX-FA polymersomes displayed significant and drastic tumor suppression compared to the control (p < 0.001) and free DOX (p < 0.001) having tumor volume declining of 96 % comparing to the introductory tumor volume of the control group (Figure 9A,A1). Additionally, we found that PLA-DOX-FA polymersomes have significantly reduced the tumor ~ 24 % with respect to DOXIL (p = 0.023). Interestingly, out of six mice, three mice did not show tumors in PLA-DOX-FA polymersomes treated group. DOXIL being a non-targeted and PEGylated liposomal formulation, can only take an advantage of EPR effect for tumor inhibition. The predominant antitumor effectiveness of our PLA-DOX-FA polymersomes can be attributed to the folate receptor mediated active targeting with an additional EPR effect. Moreover, developed polymersomes have minimal protein adsorption and therefore longer half-life, which must be providing their increased 42 ACS Paragon Plus Environment

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tumoral uptake with the added benefit of quickened DOX release in high redox potential of tumor cells. The tumor images of mice treated with different DOX formulations also corroborates the above findings (Figure 9B). Thus, our developed polymersomes nanoformulation showed relatively better antitumor efficiency than free drug and marketed PEGylated liposomal formulation, DOXIL. The survival of animals in different groups was observed for 45 days. Kaplan-Meir survival plot designated the significant increment in the survival of mice in DOX-PLA-FA treated groups compared to PBS 7.4 (control), free DOX and DOXIL treated groups (Figure 9C). The median survival time (MST) for PBS 7.4, free DOX, DOXIL and PLA-DOX-FA polymersomes was observed to be 25, 34, 39.5 and 45 days respectively. Thus, when one sees towards the lifespan of animals during cancer treatment, the designed polymersomes nanoformulation displayed promising anticancer therapy than free DOX and DOXIL.



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Figure 9. In vivo tumor suppression study on Ehrlich ascites tumor (EAT) bearing Swiss albino mice, (A,A1) Percent tumor growth curves after intravenous administration of PBS 7.4 (Control), free DOX, DOXIL and PLA-DOX-FA polymersomes. An arrow indicates the respective zoom image for free DOX, DOXIL and PLA-DOX-FA polymersomes, (Mean ± SEM, n = 6; where *** indicates p < 0.001). (B) Tumor images of mice taken at the end point of the study, (B1) PBS 7.4 (control), (B2) free DOX, (B3) DOXIL and, (B4) PLA-DOX-FA polymersomes. (C) Kaplan-Meier survival plot of tumor bearing mice.


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3.11.2.2. Histopathological evaluation for antitumor efficacy Histopathological evaluation for tumor suppression effect was assessed by observing the tumor cells necrosis behavior. Tumors from all treated groups were excised and analyzed for viable and dead/necrotic cells using Hematoxylin-Eosin staining of tumor sections. The tumor of control group mice treated with PBS 7.4 depicted irregular necrotic portions (Figure 10). The tumor of free DOX treated mice displayed necrotic areas alongside the prominence of viable neoplastic patches, signifying limited tumor suppression. In case of PLA-DOX-FA polymersomes, tumor demonstrated complete necrotic areas as compared to a tumor of free DOX treated mice. Tumors of DOXIL treated mice also evidently showed necrosis with a very few viable neoplastic cells. The EPR effect could be the reason for this observation providing sufficient accumulation of these PEGylated liposomes in tumor mass. 3.11.2.2.1. Histopathological studies for toxicity evaluation of vital organs The cardiotoxic effect of DOX therapy was evaluated by examining the heart histology of mice treated with different formulations (Figure 10). Free DOX treatment depicted cardiotoxicity with swelling in the cardiomyocytes, loss in myofibrils, and focal necrosis of cardiac myofibrils with cytosolic vacuolization. Heart from DOXIL treated group suggested minimal toxicity with a very less distorted myofibrils pattern and lower swelling in cardiomyocytes. Interestingly, developed PLA-DOX-FA polymersomes system did not display any significant cardiac damage and found to be better than free DOX and DOXIL. Thus, this negligible toxicity of DOX in polymersomes nanosystem was conquered by folate receptor targeting and PEGylation “stealth” effect with improved antitumor effect. Other vital organs such as kidneys, liver, spleen, and lungs were also studied for their toxicity assessment. The kidneys from free DOX treated group, showed markedly degenerative changes with nephrotoxicity and substantial damage to renal tubules with loss of glomeruli, which was evidenced by white off nature of



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kidney at the time of harvesting. Such a kidney dysfunction was explainable due to fluid accumulation in the abdomen in mice treated with free DOX. The liver sections for free DOX treatment showed spotty focal necrosis with lobular injury and confluent necrosis. In addition, granulomatous abrasions and Kupffer cells were found diffusely distributed in the sinusoids all through the lobules in free DOX treatment, indicating its hepatotoxic potential. The disordered pattern of thin wall alveoli of lungs and pigmentation in marginal zones of spleen firers were also observed in free DOX treatment group. DOXIL and PLA-DOX-FA polymersomes did now show any significant damage to the vital organs such as liver, kidney, lungs, and spleen. Thus, this nontoxic nature of developed polymersomes formulation can be ascribed to their biocompatible and nontoxic nature. The safety profile of PLA-DOX-FA polymersomes must be due to its totally biodegradable behavior. The redox triggered polymersomes can only be degraded in the presence of high GSH concentration in tumor region, delivering the DOX at the site of action without compromising its leakage during circulation of polymersomes. Moreover, the designed polymersomes have dual passive (EPR) and active targeting effect, providing better disposition of the drug in tumor cells and causing lower accrual in vital organs. Thus, developed biocompatible polymersomes nanocarriers presented high therapeutic index compared to the free DOX with negligible toxicity.


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Figure 10. Histopathological assessment of tumors and vital organs of EAT tumor bearing Swiss albino mice used in tumor suppression study. (A) PBS 7.4 treated (Control) (B) free DOX treated (C) DOXIL treated and, (D) PLA-DOX-FA polymersomes treated. All images were taken at 20 × magnification under bright field illumination. 3.11.2.2.2. Analysis of serum biochemical parameters The serum biochemical parameters of mice treated with free DOX, PLA-DOX-FA polymersomes and DOXIL were evaluated to emphasize the aforesaid histopathological manifestation (Table 3). Serum biochemistry is an essential parameter to assess the biocompatibility and safety behavior of developed nanoformulation.104 As per the well-known cardiotoxic nature of DOX, it is meaningful to evaluate the cardiotoxic potential of developed nanoformulation to support our postulate that folate targeting has insignificant cardiotoxicity in comparison with free DOX. The cardiotoxicity of developed polymersomes was assessed by 47 ACS Paragon Plus Environment


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measuring creatine kinase MB (CK-MB) level, a specific cardiac toxicity biomarker, which is generally elevated in blood at considerably high level after cardiotoxicity.105 The free DOX treated group displayed significantly elevated levels of CK-MB as compared to control, DOXIL and PLA-DOX-FA polymersomes treated groups (p < 0.001), depicting the cardiac damage potential of free DOX treatment. The CK-MB levels for DOXIL and PLA-DOX-FA polymersomes were comparable with a control group and were below the toxicity threshold. Nevertheless, we found that the cardio protective nature of PLA-DOX-FA was quite better than DOXIL (p = 0.0017). This cardio protective nature of developed nanoformulation can be credited to targeted, biocompatible and stimuli sensitive degradability of polymersomes in tumor mass so that maximum amount to the drug can be deposited into tumor cells. Liver damage biomarkers such as aspartate transaminases (AST/SGOT), alanine transaminases (ALT/SGPT), were analyzed to observe liver toxicity. Free DOX treated group displayed significant elevations of both these biomarkers compared to control, suggesting the liver injury (p < 0.001). PLA-DOX-FA polymersomes presented insignificant increment in liver damage markers, signifying their nontoxic nature (p > 0.05). The DOXIL treated group depicted a slight increase in ALT level compared to control (p = 0.0354). This can be due to sequestration of these non-targeted liposomes in liver and change in liver functions, nonetheless, this increment is in the below liver toxicity range. Nephrotoxicity assessment was done by measuring renal function biomarkers such as urea and creatinine, where in free DOX treatment, both the markers got elevated compared to control (p < 0.001). The PLA-DOX-FA polymersomes depicted safer drug delivery approach with insignificant enhancement of renal biomarkers corresponding to the control group (p >


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0.05). While in case of DOXIL treatment, renal biomarkers assessment was comparable to the PLA-DOX-FA polymersomes. Thus, the serum biochemistry analysis corroborated the histopathological observations and displayed the safety and biocompatible behavior of developed polymersomes nanosystem. This superior and fascinating drug delivery carrier presented insignificant toxicity with respect to cardiac, hepatic and kidney injuries compared to free DOX treatment (Table S9). This can be attributed to the specific folate receptor targeting, providing maximum accumulation of nanocarriers in tumor and minimal sequestration in vital organs. Accordingly, with polymersomes nanoformulation, we were able to achieve enhanced antitumor efficacy with minimum toxicity issues associated with free DOX treatment. Table 3. Serum biochemistry parameters of animals used in tumor regression study treated with PBS pH 7.4 (control), free DOX, DOXIL and, PLA-DOX-FA polymersomes at a dose of 5 mg/kg body weighta. Biochemical Control

DOX

DOXIL

PLA-DOX-FA

CK-MB (U/L)

3.22 ± 0.23

17.42 ± 2.75

6.51 ± 0.78

4.18 ± 0.14

AST (U/L)

55.63 ± 5.20

118.31 ± 7.26

61.50 ± 1.68

54.03 ± 4.08

ALT (U/L)

31.56 ± 0.88

87.14 ± 7.38

37.44 ± 3.14

36.97 ± 3.80

Urea (mg/dl)

36.49 ± 1.78

65.93 ± 6.12

37.33 ± 6.22

37.88 ± 2.23

Creatinine (mg/dl)

0.03 ± 0.03

0.39 ± 0.04

0.07 ± 0.01

0.03 ± 0.01

Parametersb

a

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

aspartate aminotransferase.



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4. CONCLUSIONS In summary, we have demonstrated the efficacy of folate receptor targeted, PLA based ATRP fabricated amphiphilic polymer system for enhanced DOX delivery. The polymer can be easily self-assembled into polymersomes with excellent colloidal stability and depicted safety with respect to its cytocompatibility, low protein adsorption, and hemocompatibility. The targeted polymersomes exhibited enhanced cellular uptake in folate receptor overexpressing cells than normal cells. Importantly, in in vitro drug release studies, augmented DOX release in 10 mmol GSH can postulate the facile drug release from polymersomes in cancer cells. The synthesized polymer was found to be biocompatible and biodegradable with excellent tolerability with high dose, in vivo. The targeted polymersomes depicted great superiority in tumor suppression as compared to free drug and marketed DOXIL formulation in EAT tumor bearing Swiss albino mice, without displaying significant toxicity to heart, kidneys, liver, spleen and, lungs. Thus, the developed folate targeted, redox responsive polymersomes in cancer therapy can certainly hold promise towards clinical settings.

ASSOCIATED CONTENT Supporting information Characterization of polymer, including 1H NMR, HR-MS, GPC, predicted versus actual data points graphs for polymersomes formulations, UV-visible spectra, confirming folic acid conjugation to PLA diblock copolymer, DLS data for accelerated stability of lyophilized PLADOX-FA polymersomes, Nuclear localization study of PLA-DOX-FA polymersomes, competition assay for PLA-DOX-FA polymersomes in the presence of free folic acid, reactive oxygen species (ROS) determination of blank polymersomes, percent relative body weight changes of animals used during repeated dose toxicity study, histopathological evaluation of vital organs of mice treated with blank polymersomes, Serum biochemistry parameters of 50 ACS Paragon Plus Environment

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animals for toxicity evaluation of blank polymersomes, hematological parameters analysis of mice treated with blank polymersomes, percent relative body weight changes observed during tumor regression study and statistical P values for serum biochemical parameters of mice used in tumor regression study.

AUTHOR INFORMATION Corresponding Author *Tel.: +91 11 26591041. E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful for to Department of Biotechnology (DBT) for financial support (BT/PR10191/NNT/28/717/2013). We are thankful to Dr. Amit Kumar Dinda form department of pathology, AIIMS, Delhi for his help in histopathology analysis. We are also thankful to Mrs. Archana Bansal form Cardio-Thoracic Sciences Centre-AIIMS for her valuable support in performing serum biochemistry and hematological analysis. We are grateful to Anil Pandey from Centre for Biomedical Engineering, Indian Institute of Technology, Delhi for his fruitful assistance in performing animal experimentation. We truly acknowledge Central Research Facility (CRF) and Nanoscale Research Facility (NRF) of Indian Institute of Technology, Delhi for FESEM and AFM study. We are thankful to Sophisticated Analytical Instrumentation Facility (SAIF), AIIMS for HR-TEM analysis. Chetan Nehate is additionally appreciative of the Indian Institute of Technology, Delhi, for granting him an Institute Fellowship.



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Comparative Assessment of Active Targeted Redox Sensitive

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