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Folic Acid and Trastuzumab Functionalized Redox Responsive Polymersomes for Intracellular Doxorubicin Delivery in Breast Cancer Shantanu V. Lale, Arun Kumar, Shyam Prasad, Alok Chandra Bharti, and Veena Koul Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00244 • Publication Date (Web): 28 Apr 2015 Downloaded from http://pubs.acs.org on May 2, 2015

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Folic Acid and Trastuzumab Functionalized Redox Responsive Polymersomes for Intracellular Doxorubicin Delivery in Breast Cancer Shantanu V. Lale,1,2 Arun Kumar,1,2 Shyam Prasad,3 Alok C. Bharti3 and Veena Koul*,1,2 1

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

India 2

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

3

Division of Molecular Oncology, Institute of Cytology and Preventive Oncology, Noida

201301, India *Corresponding Author Tel: +91 11 26591041 Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Redox responsive biodegradable polymersomes comprising of poly(ethylene glycol)-polylactic acid-poly(ethylene glycol) [PEG-s-s-PLA-s-s-PLA-s-s-PEG] triblock copolymer with multiple disulfide linkages were developed to improve intracellular delivery and to enhance chemotherapeutic efficacy of doxorubicin in breast cancer with minimal cardiotoxicity. Folic acid and trastuzumab functionalized monodispersed polymersomes of size ~150 nm were prepared by nanoprecipitation method while achieving enhanced doxorubicin loading of ~32% in the polymersomes. Multiple redox responsive disulfide linkages were incorporated in the polymer in order to achieve complete disintegration of polymersomes in redox rich environment of cancer cells resulting in enhanced doxorubicin release as observed in in vitro release studies where ~90% doxorubicin release was achieved in pH 5.0 in presence of 10 mM glutathione (GSH) as compared to ~20% drug release in pH 7.4. Folic acid and trastuzumab mediated active targeting resulted in improved cellular uptake and enhanced apoptosis in in vitro studies in breast cancer cell lines. In vivo studies in Ehrlich ascites tumor bearing Swiss albino mice showed enhanced antitumor efficacy and minimal cardiotoxicity of polymersomes with ~90% tumor regression as compared to ~38% tumor regression observed with free doxorubicin. The results highlight therapeutic potential of the polymersomes as doxorubicin delivery nanocarrier in breast cancer therapy with its superior antitumor efficacy and minimal cardiotoxicity.

KEYWORDS Cancer therapy, intracellular drug delivery, polymersomes, redox sensitivity, trastuzumab


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INTRODUCTION Polymeric nanoparticles have been extensively studied for intracellular doxorubicin delivery in cancer therapy because of several advantages. They achieve passive targeting of tumor by enhanced permeation and retention effect (EPR).1 They minimize renal clearance of doxorubicin and also prevent its extravasation into healthy cells. Nanoparticles with hydrophilic surface minimize protein adsorption and macrophage uptake resulting in improved circulation half-life of nanoparticles. They also overcome P-glycoprotein mediated multidrug resistance of cancer cells.1-3 Thus, nanoparticles improve the bioavailability and thus efficacy of doxorubicin while minimizing its side effects on healthy cells. Polymers can be designed depending on the need of drug delivery applications such as side chain functionality, enhanced stability and responsiveness. Various types of polymeric nanoparticles have been developed for drug delivery applications which include micelles, nanospheres, nanocapsules and polymersomes.4, 5 Polymersomes have been extensively studied as drug delivery carriers. PEG based amphiphilic block-copolymers can self assemble to form polymersomes when its hydrophilic fraction (f) is 10-40%.6 Polymersomes offer several advantages as compared to other polymeric nanoparticles including better stability, higher robustness and prolonged circulation time. Also, polymersome’s unique combination of hydrophobic shell and aqueous lumen allows simultaneous loading of hydrophilic as well as hydrophobic drugs in large quantities.7, 8 Researchers have explored various ways to improve doxorubicin delivery efficiency of polymersomes in cancer cells. Cancer cells/tumor exhibit an environment which is different than that exist in normal healthy cells. Incorporation of stimuli responsive linkages in the polymer, specific to cancer cell environment, can achieve enhanced drug release and improved


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biodegradation of the polymeric nanosystem. Various stimuli responsive polymeric nanosystems like pH responsive, temperature responsive and redox responsive polymersomes have been developed for doxorubicin delivery applications in cancer.9-12 Redox responsive nanosystems have gained popularity for intracellular drug delivery applications in cancer therapeutics. It utilizes redox potential as a stimulus for intracellular delivery of anticancer drugs in cancer. Cell cytosol exhibit 2-3 orders high glutathione (GSH) concentration (2-10 mM), a tripeptide responsible for reduction of disulfide linkages, as compared to extracellular compartment.13 Physiological body fluids such as blood and normal extracellular matrices have low GSH concentration (2-20 µM). This significant difference in GSH level in intracellular and extracellular compartment has made redox responsive nanosystems a promising and appealing platform for intracellular drug delivery in tumor. Cancer tumor tissues consist of about four-fold higher GSH levels as compared to normal healthy tissues.14,

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Higher GSH levels in cancer cells are associated with chemotherapeutic drug

resistance of cancer cells due to GSH mediated phase II detoxification mechanism.16 Endolysosomal compartment is also redox-active with high concentration of reducing agents such as cysteine and reducing enzymes such as gamma interferon inducible lysosomal thiol reductase (GILT).17 Nanosystem can be tailor-made to be redox responsive by incorporation of disulfide linkages which are cleavable in reductive environment.17, 18 Nanoparticles can selectively target cancer cells using active targeting via cancer cell specific ligands. Active targeting acts like a guided missile and exhibits specific affinity towards receptors present on the cancer cells which are not generally expressed on normal healthy cells. Various ligands such as antibodies, antibody fragments, aptamers, folic acid, hormones, growth factors, sugars etc. have been studied for active targeting of cancer cells.19-24 Dual targeting


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approach is widely used for targeting cancer cells since it involves targeting of two different molecular targets on cancer cells. Thus, it helps to improve cellular uptake of nanoparticles as well as helps in overcoming chemo-resistance exhibited by cancer cells. Thus, even if cancer cell down-regulates expression of certain receptor on its cell surface, presence of other ligand on nanoparticles assures cellular uptake of NPs in the cancer cell.25-27 Folic acid is extensively studied as a ligand for active targeting of variety of cancers. Folate receptors FRα are often overexpressed on cancer cells to fulfil the higher folate requirement for DNA synthesis. Normal healthy cells show minimal expression of FRα receptor, but instead express reduced folate carrier (RFC) for folate uptake. FRα receptor exhibits high binding affinity for folic acid with dissociation constant (Kd) < 1 nM, making folic acid an excellent ligand for active targeting of variety of cancer cells including breast cancer cells.28, 29 Human epidermal growth factor receptor 2 (EGFR2/HER2 receptor) are overexpressed in most breast cancers. Trastuzumab (Herceptin®), a humanized IgG1 kappa monoclonal antibody exhibits high affinity and selectivity towards HER2 receptors (Kd = 0.1 nM) and hence is widely used for targeting HER2 positive breast cancer cells.30, 31 In the present work, dual targeted redox responsive polymersomes comprising of PEG-s-sPLA-s-s-PLA-s-s-PEG triblock copolymer with multiple disulfide bonds were developed for targeted doxorubicin delivery in breast cancer (Scheme 1). Redox responsive disulfide linkages were incorporated in the center of PLA unit and two disulfide linkages were incorporated between peripheral PEG units and central PLA units. These disulfide linkages will be cleaved in presence of 10 mM GSH resulting in disintegration of hydrophobic PLA core and removal of hydrophilic units of polyethylene glycol, leading to enhanced doxorubicin release. PLA has high biocompatibility and biodegradability and is approved by US FDA for drug delivery


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applications.32 Polyethylene glycol is also biocompatible and non-toxic and is approved by US FDA.33 Nanoparticles based on PLA-PEG are easily removed from the body due to their biodegradable nature and thus do not cause toxicity.34 Hydrophilic PEG units improve circulation half-life of nanoparticles with minimal macrophage uptake bestowing ‘stealth’ nature to the nanoparticles.35-37

Scheme 1. Schematic representation of redox responsive PEG-s-s-PLA-s-s-PLA-s-s-PEG drug delivery nanocarriers for intracellular doxorubicin delivery in breast cancer. (Graphic adapted from ref 22. Reproduced

by

permission

of

The

Royal

Society

of

Chemistry.

http://pubs.rsc.org/en/Content/ArticleLanding/2015/PY/C4PY01698J#!divAbstract)


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Folic acid and trastuzumab monoclonal antibody were conjugated with the polymersomes for active targeting of breast cancer cells which often overexpress folate FRα and HER2 receptors. Biocompatibility studies of polymer were carried out using protein adsorption (Supporting Information), hemolysis, coagulation studies (Supporting Information) and MTT cytotoxicity assay. In vitro drug release was carried out in pH 5.0 and pH 7.4 in presence and absence of 10 mM GSH to study the effect of redox environment on doxorubicin release from polymersomes. In vitro studies in breast cancer cell lines and in vivo studies in Ehrlich ascites tumor bearing mice were carried out to evaluate cellular uptake, apoptosis and antitumor efficacy of the polymersomes while toxicity of the formulation was evaluated using histopathology and serum biochemistry. All the studies were carried out to evaluate the potential of the nanosystem as drug delivery nanocarrier for efficient doxorubicin delivery in breast cancer.

MATERIALS & METHODS Materials 3,6-Dimethyl-1,4-dioxane-2,5-dione (DL-Lactide), 2-hydroxyethyl disulfide, tin (II) 2ethylhexanoate,

2-bromoisobutyryl

bromide,

thioglycolic

acid,

poly(ethylene

glycol)

methacrylate (PEGMA) (Mn ~525 Da), glutathione (GSH), 4-dimethylaminopyridine (DMAP), folic acid, bovine serum albumin (BSA), trehalose, N,N′-dicyclohexylcarbodiimide (DCC), Nhydroxysuccinimide (NHS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used as received without further purification. Doxorubicin was obtained from Ranbaxy Laboratories Ltd., New Delhi as a gift sample. The marketed product HerclonTM (Trastuzumab for Injection, Roche, India) was purchased and dialyzed with phosphate buffer saline pH 7.4 (PBS) to obtain pure


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trastuzumab. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin antibiotic solution were obtained from Cellclone, Genetix Biotech Ltd. (New Delhi, India). Dialysis membrane (12-14 kDa) and DAPI were purchased from Himedia Laboratories (Mumbai, India). Amicon ultracentrifugal filters (30 kDa) were obtained from Merck Millipore (Billerica, MA, USA). Annexin V-FITC assay kit was obtained from BD Biosciences (San Jose, USA). Ultrapure water (18 MΩ cm resistivity) was obtained from Milli-Q system (Merck Millipore, Billerica, MA, USA). Synthesis of PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer was synthesized in four steps as show in Scheme 2. Synthesis of disulfide core polylactide (PLA-s-s-PLA) polymer Ring opening polymerization of DL-lactide was carried our using 2-hydroxyethyl disulfide as initiator in presence of tin (II) 2-ethylhexanoate as catalyst. Weighed amount of DL-lactide (7 g; 48.6 mmol) was added in a Schlenk flask kept at 140 °C. 2-Hydroxyethyl disulfide (0.2 mL; 1.62 mmol) and tin (II) 2-ethylhexanoate (0.065 mL; 0.162 mmol) was added to the Schlenk flask and reaction mixture was allowed to stir at 140 °C for 24 h under N2 flow. Resulting viscous solution was diluted with chloroform, precipitated thrice in diethyl ether and dried overnight in oven. The product thus obtained (PLA-s-s-PLA polymer) was characterized using 1H-NMR (300 MHz, Brukers, USA) and gel permeation chromatography (GPC) (Waters, USA). Yield = 83%. 1

H-NMR (300 MHz, CDCl3) δ (ppm): 1.55 (m, -CO-CH(CH3)-O-), 2.88 (t, -CH2-CH2-S-S-

CH2-CH2-), 4.36 (t, -CH2-CH2-S-S-CH2-CH2-), 5.16 (m, -CO-CH(CH3)-O-).


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Synthesis of 2-((2-hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate 2-Hydroxyethyl disulfide (4.76 mL; 38.9 mmol) and triethylamine (10.75 mL; 77.8 mmol) were dissolved in 50 mL dry THF in a Schlenk flask on ice bath. 2-Bromoisobutyryl bromide (4.8 mL; 38.9 mmol) was then slowly added over a period of 30 min with continuous stirring under N2 flow. After complete addition, reaction temperature was slowly raised to 30 °C and reaction was continued for 24 h. Reaction mixture was then filtered to remove quaternary ammonium salts, concentrated and extracted with dichloromethane (DCM) and saturated sodium bicarbonate solution thrice. DCM layer was dried on anhydrous sodium sulfate and concentrated. The product was purified by silica column chromatography by slowly increasing the elution gradient from

100%

hexane

to

70/30

hexane/ethyl

acetate.

The

product

2-((2-

hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate was collected and characterized using 1H-NMR. Yield = 51%. 1

H-NMR (300 MHz, CDCl3) δ (ppm): 1.94 (s, -O-CO-C(CH3)2-Br), 2.89 (t, OH-CH2-CH2-S-

S-CH2-CH2-O-CO-), 2.97 (t, OH-CH2-CH2-S-S-CH2-CH2-O-CO-), 3.89 (t, OH-CH2-CH2-S-SCH2-CH2-O-CO-), 4.45 (t, OH-CH2-CH2-S-S-CH2-CH2-O-CO-). Synthesis of OH-s-s-PLA-s-s-PLA-s-s-OH polymer PLA-s-s-PLA polymer (2 g; 0.5 mmol) and triethylamine (0.275 mL; 2 mmol) were dissolved in dry THF (50 mL) in a Schlenk flask and stirred at 60 °C under N2 flow. 2-((2hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate (0.455 g; 1.5 mmol) was then slowly added to the reaction mixture over a period of 30 min and reaction was continued for 96 h. Reaction mixture was then concentrated and precipitated thrice in diethyl ether. Product thus obtained (OH-s-s-PLA-s-s-PLA-s-s-OH polymer) was characterized using 1H-NMR. Yield = 81%.


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H-NMR (300 MHz, CDCl3) δ (ppm): 1.55 (m, -CO-CH(CH3)-O- and -CO-CH(CH3)-O-

C(CH3)2-CO-O-), 2.89 (t, -CH2-CH2-S-S-CH2-CH2-), 3.12 (t, -CH2-CH2-S-S-CH2-CH2-OH), 3.75 (q, -CO-CH(CH3)-O-C(CH3)2-CO-O-), 4.36 (t, -CH2-CH2-S-S-CH2-CH2- and -CH2-CH2-SS-CH2-CH2-OH), 5.16 (m, -CO-CH(CH3)-O-).

Scheme 2. Synthesis scheme for PEG-s-s-PLA-s-s-PLA-s-s-PEG copolymer


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Synthesis of SH-s-s-PLA-s-s-PLA-s-s-SH polymer In a Schlenk flask, OH-s-s-PLA-s-s-PLA-s-s-OH polymer (0.5 g; 0.125 mmol) was dissolved in 50 mL DCM followed by DCC (0.258 g; 1.25 mmol) and DMAP (0.003 g; 0.025 mmol). Thioglycolic acid (0.087 mL; 1.25 mmol) was then added and reaction was stirred at 30 °C for 24 h. Reaction mixture was then concentrated on rotary evaporator and product was purified by precipitation in diethyl ether. Product thus obtained (SH-s-s-PLA-s-s-PLA-s-s-SH) was characterized by 1H-NMR. Yield = 85%. 1


C(CH3)2-CO-O-), 2.93 (t, -CH2-CH2-S-S-CH2-CH2-), 3.08 (t, -CH2-CH2-S-S-CH2-CH2-O-COCH2-SH), 3.27 (s, -O-CO-CH2-SH), 3.50 (q, -CO-CH(CH3)-O-C(CH3)2-CO-O-), 4.41 (t, -CH2CH2-S-S-CH2-CH2-), 5.18 (m, -CO-CH(CH3)-O-). Michael addition reaction of PEGMA with SH-s-s-PLA-s-s-PLA-s-s-SH polymer SH-s-s-PLA-s-s-PLA-s-s-SH polymer (0.25 g; 0.0625 mmol) was dissolved in 50 mL DCM in a Schlenk flask followed by triethylamine (0.086 mL; 0.3125 mmol) and DMAP (0.006 g; 0.05 mmol). To this reaction mixture, PEGMA (Mn ~525 Da; 0.179 mL; 0.1875 mmol) was added and reaction was stirred at 40 °C for 72 h. Reaction mixture was concentrated and product was obtained by precipitation in diethyl ether. Triblock copolymer PEG-s-s-PLA-s-s-PLA-s-s-PEG thus obtained was characterized by 1H-NMR and GPC. Yield = 82%. 1


C(CH3)2-CO-O), 2.75 (d, -S-CH2-C(CH3)-CO), 2.88 (t, -CH2-CH2-S-S-CH2-CH2-), 3.12 (t, CH2-CH2-S-S-CH2-CH2-O-CO-CH2-S-), 3.24 (s, O-CO-CH2-S-), 3.63 (m, -O-CH2-CH2-O- ), 3.71 (q, -CO-CH(CH3)-O-C(CH3)2-CO-O-), 4.36 (t, -CH2-CH2-S-S-CH2-CH2-), 5.16 (m, -COCH(CH3)-O-).


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Folic acid conjugation with PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer Folic acid (0.039 g; 0.088 mmol) was dissolved in 50 mL dry DMSO in a Schlenk flask followed by DCC (0.036 g; 0.176 mmol) and NHS (0.02 g; 0.176 mmol). The reaction mixture was stirred at 30 °C under N2 atmosphere overnight. PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer (0.5 g; 0.176 mmol) was then reacted with activated folic acid for another 24 h at 30 ºC under N2 atmosphere. The reaction mixture was filtered, dialyzed and lyophilized. Folate conjugated triblock polymer (PEG-s-s-PLA-s-s-PLA-s-s-PEG-FA polymer) thus obtained was characterized by CHN analysis (Vario EL III Element Analyzer, Elementar Analysensysteme GmbH, Germany) and UV-visible spectroscopy (Perkin Elmer, USA). Yield = 85%. Polymersomes preparation and characterization Polymersomes (NPs) were prepared using nanoprecipitation method developed by Fessi et al.38 Doxorubicin was loaded in the polymersomes during nanoprecipitation using combination of techniques developed by Kataoka et al.39 and Sanson et al.40 to improve doxorubicin loading in the polymersomes. Doxorubicin hydrochloride (35 mg) was dissolved in 2 mL DMSO containing triethylamine (100 µL; 5 mole equivalent to doxorubicin hydrochloride) to deprotonate doxorubicin molecules and to make them hydrophobic in nature. Weighed amount of polymer (50 mg) was dissolved in 3 mL DMSO and both solutions were mixed with constant stirring. Carbonate buffer pH 10.5 (45 mL; 50 mM) was then quickly added to the organic phase under magnetic stirring and the solution was dialyzed for 6 h against water (1 L, changed every 2 h) to remove DMSO and excess doxorubicin. The dialyzed nanoparticles were then lyophilized with 10% trehalose as cryoprotectant. Doxorubicin loading and encapsulation efficiency was calculated using following formula-


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Loading content % Encapsulation efficiency %

Weight of doxorubicin loaded in polymersomes Weight of polymersomes

100

Amount of doxorubicin loaded in polymersomes Total amount of doxorubicin taken for loading

100

Polymersomes were characterized using dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd, UK), atomic force microscopy (AFM) (Nanoscope Multimode AFM, Digital Instruments, USA), high-resolution transmission electron microscope (HRTEM) (Technai G2, 200 kV, FEI, USA), cryo-transmission electron microscopy (cryo-TEM) (Tecnai G2, 200 kV, FEI, USA) and scanning electron microscope (SEM) (Zeiss EVO 50, Carl Zeiss Microscopy GmbH, Germany). Reconstitution time for the polymersomes was determined using 10 mL water. Trastuzumab conjugation on polymersomes Trastuzumab (3 mL; 1 mg/mL in phosphate buffer saline pH 7.4 (PBS)) was incubated with EDC (200 µL; 200 mM) and NHS (150 µL; 200 mM) at 4 °C for 30 min with continuous stirring at 50 rpm. Doxorubicin loaded polymersomes (PLA-Dox NPs and PLA-Dox-FA NPs) in PBS (10 mg/mL concentration) were then incubated with trastuzumab for 4 h at 4 °C with gentle stirring. Sample was then washed with water using Amicon ultracentrifugal filter. Trastuzumab conjugated polymersomes (PLA-Dox-Her NPs and PLA-Dox-FA-Her NPs) were then lyophilized with 10% trehalose and stored at 4 °C. Unconjugated trastuzumab was quantified from the filtrates by Biuret assay. Concentration of conjugated trastuzumab on nanoparticle surface was determined by subtracting unconjugated trastuzumab concentration from initial trastuzumab concentration taken for the reaction. The composition of each polymer is given in Table 1.


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Table 1. Composition of synthesized polymersomes with attached targeting ligand Polymersomes notations

Composition

PLA NPs

Native PEG-s-s-PLA-s-s-PLA-s-s-PEG polymersomes

PLA-Dox NPs

Non-targeted doxorubicin loaded PLA NPs

PLA-Dox-FA NPs

Folate targeted doxorubicin loaded PLA NPs

PLA-Dox-Her NPs

Trastuzumab targeted doxorubicin loaded PLA NPs

PLA-Dox-FA-Her NPs

Folate and trastuzumab (dual) targeted doxorubicin loaded PLA NPs

Characterization of trastuzumab conjugation Zeta potential measurement and X-ray photoelectron spectroscopy (XPS) were employed to determine trastuzumab conjugation on the surface of polymersomes. Binding energy spectrum of dried NPs suspension on silicon substrate was recorded on XPS (SPECS, Germany) from 0 to 1100 eV with pass energy of 80 eV under fixed transmission mode. Stability studies of polymersomes Polymersomes stability was studied in water, saline, DMEM media and fetal bovine serum (FBS) for 72 h. Briefly, 0.1 mL of 5 mg/mL polymersomes were mixed with 5 mL of respective media at 37 °C and particle size of the polymersomes was determined periodically by DLS. Effect of GSH on disulfide linkages was established by evaluating nanoparticle aggregation in presence of 10 mM GSH using DLS. Drug release studies Doxorubicin release was carried out in phosphate buffer pH 7.4 (cytoplasm/serum pH) and acetate buffer pH 5.0 (endo-lysosomal pH) in presence and absence of 10 mM GSH to simulate reducing conditions present in the cytoplasm/endo-lysosomes. Known concentrations of NPs were dialyzed against 10 mL buffer solutions of pH 7.4 and pH 5.0 with and without 10 mM


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GSH at 37 °C at 120 rpm. Samples were periodically analyzed using UV-visible spectrophotometry at 481 nm. Doxorubicin standard calibration curve was used to determine the concentration of released doxorubicin. Hemolysis assay Blood was obtained from Blood bank, All India Institute of Medical Sciences (AIIMS), New Delhi. One millilitre blood was centrifuged at 1500 rpm for 10 min to obtain RBCs which were washed thrice with PBS to remove any adsorbed proteins. Then, 50 µL of RBCs were diluted to 10 mL with PBS to prepare RBC stock solution. PLA NPs in PBS (100 µL) were incubated with RBC stock solution (100 µL) at 37 °C for 1 h at 120 rpm with final concentration in the range 0.125-2 mg/mL. After 1 h, the mixture was centrifuged at 1500 rpm for 5 min and released hemoglobin in the supernatant was analyzed using UV-visible spectrophotometer at 540 nm. Percent hemolysis was calculated with respect to hemolysis caused by negative control (PBS) and positive control (1% Triton X-100) as shown in following equationHemolysis %

Sample Positive control

Negative control Negative control

100

Cell culture studies MCF-7 (breast cancer cells), L929 (mouse fibroblast cells) and BT-474 cell line (breast cancer cells) were obtained from NCCS, Pune, India. Cells were grown in 25 cm2 tissue culture flasks in 5% CO2 atmosphere at 37 °C with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cell viability study Biocompatibility of polymersomes was evaluated in L929 and MCF-7 cell lines using MTT cytotoxicity assay. Briefly, 10,000 cells were seeded per well in a 96 well plate and incubated in


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CO2 incubator at 37 °C for 24 h. Polymersomes with final concentration in the range 0.125 to 2 mg/mL were incubated with cells for 24 h at 37 °C. Media was then replaced and 10 µL of 5% MTT solution was added to the wells and incubated for 4 h followed by addition of 200 µL DMSO. Absorbance was measured at 540 nm on Microplate spectrophotometer (PowerWave XS2, BioTek Instruments, USA). Cell viability was calculated with respect to PBS (negative control) and 1% Triton X-100 (positive control) as shown in following equationCell viability %

Sample Negative control

Positive control Positive control

100

Qualitative assessment of in vitro cellular uptake using confocal laser scanning microscopy (CLSM) Confocal laser scanning microscope was used to visualize cellular uptake of polymersomes in MCF-7, L929 and BT-474 cells. Cells at density of 1×104 cells per well were seeded in a 6 well plate containing a cover slip and kept at 37 °C for 24 h in CO2 incubator. Polymersomes with concentration equivalent to 10 μg/mL of doxorubicin were added to the wells and were incubated at 37 °C for 4 h. Cells were then rinsed two times with PBS, fixed with paraformaldehyde (4%) and stained with 50 µL DAPI. Cells were then observed under CLSM (FluoView FV1000 Olympus, USA) for DAPI (460 nm) and doxorubicin (560 nm) at 100× magnification. Quantitative assessment of in vitro cellular uptake using flow cytometry Flow cytometry was used for quantitative assessment of cellular uptake in MCF-7, L929 and BT474 cells. Cells were seeded in a 6 well plate (1×105 cells/well) and kept at 37 °C for 24 h in CO2 incubator. Polymersomes with a concentration equivalent to 10 μg/mL of doxorubicin were added to each well and incubated at 37 °C for 4 h. Cells were then rinsed two times with PBS, trypsinized, and centrifuged at 3500 rpm for 5 min. Cell pellet was then resuspended in PBS and


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analyzed in Flow Cytometer (BD FACSAria III, USA) for cellular uptake studies of polymersomes. Flowing Software (Version 2.5.1, Turku Centre for Biotechnology, Finland) was used for data analysis. Annexin V-FITC apoptosis assay Effect of polymersomes on apoptosis and necrosis in MCF-7, L929 and BT-474 cells was evaluated using Annexin V-FITC apoptosis assay. Cells were seeded in a 6 well plate (1×105 cells/well) and incubated at 37 °C for 24 h in CO2 incubator. Polymersomes (concentration equivalent to 10 μg/mL of doxorubicin) were incubated with cells at 37 °C for 7 h. Cells were then washed two times with PBS, trypsinized, and centrifuged at 3500 rpm for 5 min. Cell pellet was then resuspended in 100 µL Annexin V binding buffer and cells were incubated with 5 μL each of Annexin V-FITC and propidium iodide solution for 20 min in dark. Annexin V binding buffer (400 µL) was further added in FACS tube and cells were analyzed in Flow Cytometer. Flowing Software (Version 2.5.1, Turku Centre for Biotechnology, Finland) was used for data analysis. Animal studies Antitumor efficacy of PLA nanosystem was evaluated using Ehrlich ascites tumor (EAT) bearing Swiss albino mice. EAT cell line was obtained as a gift from Dr. B. S. Dwarakanath, Institute of Nuclear Medicine and Allied Sciences (INMAS), New Delhi. Mice (25 ± 5 g; 55-65 days of age) were procured from Central Animal Facility, All India Institute of Medical Sciences (AIIMS) and maintained in their enclosure. All the experiments were conducted according to the guidelines of CPCSEA and the Animal Ethical Committee (796/IAEC/14) of AIIMS, New Delhi. Animals were housed in an animal house facility at a controlled environment of 25 °C with


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suitable humidity and 12 h light/dark cycles. Mice were kept in polycarbonate cages with supply of chow food and purified water ad libitum throughout the study. In vivo antitumor activity EAT cell suspension (150 μL; ~20 million cells) was injected subcutaneously on the dorsal side of mice for tumor onset. Drug treatment was started when tumor volumes reached ~200-250 mm3 and the day was designated as day 0. Three groups with 6 animals in each group were formed with group I as Control i.e. PBS treated mice, group II consisted of free doxorubicin treated mice and group III consisted of PLA-Dox-FA-Her NPs treated mice. Formulations were given via tail vein injection at a dose equivalent to doxorubicin dose of 5 mg/kg body weight of mice every third day for 15 days (i.e. day 0, 3, 6, 9, 12 and 15). Vernier caliper was used for measuring tumor dimensions and tumor volume was calculated using the formula-

Tumor volume

Length

Width 2

Where, length symbolizes the largest tumor diameter while width depicts the perpendicular tumor diameter. On last day of experiment (day 18), blood was collected from venous plexus of eyes of anesthetized mice and mice were euthanized by cervical dislocation. Tumor and vital organs such as heart, liver, kidneys, lungs and spleen were removed for toxicity determination using histopathology studies. Histopathological analysis of organs and tumor Histopathological analysis of tumor and vital organs- heart, liver, kidneys, lungs and spleen was studied to determine antitumor efficacy and toxicity of doxorubicin formulations. Tumor and organs collected from euthanized mice were fixed in 10% buffered formalin solution at 25 °C and were embedded in paraffin. They were cut into 5 µm sections using a rotary microtome


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(Leica, Germany). Hematoxyline and eosin (H&E) stains were used to stain the slides and slides were observed under light microscope to determine damage to the tissues. Blood biochemistry analysis Blood biochemistry analysis was carried out to determine toxicity of the formulations on heart, liver and kidney. Creatine kinase MB (CK-MB) was analyzed for cardiac damage determination using CK-MB kit (Audit Diagnostics, Ireland) on Automated Analyzer (Cobas e411, Roche Diagnostics, USA). Liver and kidney function tests were also carried out using Automated Biochemical Analyzer (TurboChem 100, Awareness Technology Inc., USA) for assessing hepatic and renal toxicity. Statistical analysis All in vitro data are expressed as mean ± standard deviation (SD). In vivo data are expressed as mean ± standard error of mean (SEM). Sigma Stat (Version 3.5, Systat Software Inc., San Jose, CA, USA) was used for statistical analysis of the data. One-way analysis of variance (one-way ANOVA) with Bonferroni multiple comparison test was used for determining statistical significance between the groups. Statistical significance was considered at p value < 0.05.

RESULTS & DISCUSSION Synthesis of PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer was synthesized as shown in Scheme 2. First, ring opening polymerization of DL-lactide was carried out using 2-hydroxyethyl disulfide as initiator and tin (II) 2-ethylhexanoate as catalyst to synthesize PLA with disulfide core (PLAs-s-PLA polymer). The synthesized polymer was characterized using 1H-NMR (Supporting Information Figure S1A). The 1H-NMR spectra of synthesized PLA-s-s-PLA polymer showed


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peaks at δ 1.53 and δ 5.17 corresponding to methyl and methine protons of synthesized polylactide respectively, whereas triplets at δ 2.89 and δ 4.36 corresponds to methylene groups from 2-hydroxyethyl disulfide. Based on 1H-NMR integration, molecular weight of synthesized polymer was found to be ~4500 Da. GPC showed molecular weight Mn and Mw to be 4823 Da and 6742 Da respectively with polydispersity of 1.398 (Supporting Information Figure S2). In the next step, one hydroxyl group of 2-hydroxyethyl disulfide was functionalized by reacting with one equivalent of 2-bromoisobutyryl bromide to synthesize mono-functionalized 2hydroxyethyl

disulfide

which

is

2-((2-hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl

propanoate with one free hydroxyl group and one bromo-functionalized group. The product was purified and isolated using silica column chromatography. The purified product thus obtained was characterized using 1H-NMR (Supporting Information Figure S1B). It was observed in 1HNMR that the mono functionalized 2-hydroxyethyl disulfide was free from any possible impurities of di-functionalized 2-hydroxyethyl disulfide or unreacted 2-hydroxyethyl disulfide. Further, terminal hydroxyl groups of synthesized PLA polymer were reacted with 2-((2hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate to synthesize OH-s-s-PLA-s-sPLA-s-s-OH polymer with disulfide linkages ‘in and around’ the PLA polymer. The said polymer was characterized using 1H-NMR (Supporting Information Figure S1C). 1H-NMR spectrum showed peak at δ 3.11 which corresponds to methylene protons attached to terminal hydroxyl group of newly attached hydroxyethyl disulfide whereas peak at δ 3.75 corresponds to methine protons of PLA attached to 2-((2-hydroxyethyl)disulfanyl)ethyl-2-bromo-2-methyl propanoate. This confirmed successful conjugation of 2-((2-hydroxyethyl)disulfanyl)ethyl-2bromo-2-methyl propanoate on the hydroxyl groups of PLA-s-s-PLA polymer to synthesize OHs-s-PLA-s-s-PLA-s-s-OH polymer.


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Thioglycolic acid was then reacted with terminal hydroxyl groups of OH-s-s-PLA-s-s-PLA-ss-OH polymer to introduce thiol groups in the polymer. The synthesized SH-s-s-PLA-s-s-PLA-ss-SH was characterized by 1H-NMR (Supporting Information Figure S1D). Peak at δ 3.27 corresponding to methylene protons of thioglycolic acid confirmed the conjugation reaction and formation of thiol functionalized PLA (SH-s-s-PLA-s-s-PLA-s-s-SH) polymer. In the next step, polyethylene glycol was introduced in the polymer by Michael addition reaction of PEGMA with terminal thiol groups of SH-s-s-PLA-s-s-PLA-s-s-SH polymer. The synthesized triblock polymer PEG-s-s-PLA-s-s-PLA-s-s-PEG was characterized by 1H-NMR (Supporting Information Figure S1E). Peak at δ 3.61 corresponding to -OCH2 groups from polyethylene glycol in 1H-NMR confirmed the successful conjugation of PEGMA with SH-s-sPLA-s-s-PLA-s-s-SH polymer. Molecular weight- Mn and Mw of the triblock copolymer were determined using GPC and found to be 5847 and 7636 Da, respectively, with a polydispersity index of 1.306 (Supporting Information Figure S3). DCC-NHS chemistry was utilized for conjugating folic acid to the triblock polymer. One hydroxyl group from the triblock copolymer was conjugated with γ-carboxylic acid of folic acid while other hydroxyl group of the triblock copolymer was kept free for trastuzumab conjugation after polymersomes preparation. Folic acid conjugation on the triblock copolymer was characterized using CHN analysis and UV-visible spectroscopy. CHN analysis showed C/N ratio of folate conjugated polymer to be 28.37. Based on calculations from C/N ratio, ~1 molecule of folic acid was found to be conjugated to the polymer. UV-visible spectroscopy (Supporting Information Figure S4) also showed blue shift in absorption spectrum of folic acid indicating successful conjugation of folic acid with the triblock copolymer.41


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Polymersomes preparation and characterization Nanoprecipitation method developed by Fessi et al. was used for preparation of polymersomes.38 The triblock copolymer self assembles to form polymeric vesicles/polymersomes due to its hydrophilic fraction (fPEG) of ~18%. Polymersomes showed uniform particle size and low polydispersity index in DLS as shown in Table 2. The nanoparticle size was increased to ~150 nm after trastuzumab conjugation due to high molecular weight and size of trastuzumab. Table 2. Polymersomes characterization by DLS Before lyophilization Polymersomes

After lyophilization

Particle size

Polydispersity index

Particle size

Polydispersity index

PLA NPs

97.2 ± 4.5 nm

0.192 ± 0.006

115.6 ± 5.1 nm

0.103 ± 0.024

PLA-Dox NPs

107.4 ± 3.7 nm

0.204 ± 0.211

132.3 ± 6.7 nm

0.087 ± 0.025

PLA-Dox-FA NPs

117.2 ± 1.0 nm

0.096 ± 0.011

137.7 ± 2.9 nm

0.114 ± 0.017

PLA-Dox-Her NPs

120.6 ± 8.2 nm

0.312 ± 0.054

141.3 ± 1.1 nm

0.085 ± 0.014

PLA-Dox-FA-Her NPs

126.2 ± 7.1 nm

0.097 ± 0.019

147.1 ± 5.5 nm

0.091 ± 0.018

Mean ± SD, n=3

Doxorubicin loading in the polymersomes was enhanced by varying the pH of solutions during nanoprecipitation using combination of techniques developed by Kataoka et al.39 and Sanson et al.40 First, water soluble doxorubicin hydrochloride was converted to water insoluble free doxorubicin base using triethylamine which deprotonates the doxorubicin. Next, carbonate buffer pH 10.5 was used as dispersing media during nanoprecipitation since increasing the pH of solution above the pKa of doxorubicin (8.25) results in decreased ionization of doxorubicin with majority of doxorubicin molecules in the unionized state. This causes decreased solubility of doxorubicin resulting in increased loading efficiency of polymersomes.40 In order to prevent doxorubicin degradation in alkaline solution, the polymersomes suspension was immediately neutralized by dialyzing against water. With this procedure, we were able to obtain high


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doxorubicin loading of ~36% in our polymersomes (362 µg of doxorubicin/mg of polymer) with encapsulation efficiency of ~75%. However, during trastuzumab conjugation step, small amount of doxorubicin gets released in the media resulting in lowering of final doxorubicin content to ~32% (324 µg of doxorubicin/mg of polymer) in the polymersomes.

1000 nm

B

A

C

200 nm

100 nm

D

E

Figure 1. PLA-Dox-FA-Her polymersomes characterization using A) DLS, B) AFM, C) SEM, D) conventional TEM and E) Cryo-TEM showing core-shell morphology of polymersomes with uniform particle size and narrow size distribution.

Absolute particle size of polymersomes was determined using AFM, SEM, TEM and cryoTEM since DLS gives hydrodynamic size of the polymersomes. Spherical bi-layered vesicular

structures of polymersomes with inner hydrophilic core and outer hydrophobic shell were observed in TEM and cryo-TEM. Average size of polymersomes was observed in the range 120140 nm in AFM, SEM, TEM and cryo-TEM as shown in Figure 1.


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Trehalose at concentration of 10% was used as a cryoprotectant to prevent polymersomes aggregation during lyophilization and to increase their stability during storage. Polymersomes quickly re-dispersed in water with uniform dispersion without any aggregation. The reconstitution time of lyophilized polymersomes was found to be ~30 s. Trastuzumab conjugation Trastuzumab was conjugated with hydroxyl groups of PEG in the polymersomes using EDCNHS chemistry. Hydroxyl groups of PLA-Dox and PLA-Dox-FA NPs were conjugated with activated carboxyl groups of trastuzumab antibody to form trastuzumab conjugated NPs- PLADox-Her NPs and PLA-Dox-FA-Her NPs, respectively. Trastuzumab concentration on PLADox-Her and PLA-Dox-FA-Her polymersomes surface was found to be 63.8 μg/mg and 58.4 μg/mg respectively based on Biuret assay of unconjugated trastuzumab from the filtrate. Trastuzumab conjugation on polymersomes surface was also determined using zeta potential measurement and XPS. The zeta potential of PLA-Dox NPs and PLA-Dox-FA NPs before and after trastuzumab conjugation is shown in Table 3. The shift in zeta potential towards positive side can be ascribed to positive charge of trastuzumab,42 which indicates successful conjugation of trastuzumab on polymersomes surface. Table 3. Zeta potential measurements of polymersomes before and after trastuzumab conjugation Polymersomes

Zeta potential (mV) Before conjugation

After conjugation

PLA-Dox NPs

-15.3 ± 1.5

-5.5 ± 0.2

PLA-Dox-FA NPs

-2.9 ± 0.2

26.6 ± 0.5

Mean ± SD, n=3


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XPS was used for analyzing the surface chemistry of polymersomes for further confirmation of trastuzumab conjugation on polymersomes surface. Trastuzumab gives stronger nitrogen signal in XPS because of its 1726 nitrogens.43 As shown in Figure 2, XPS wide scan spectra of unconjugated polymer showed very weak nitrogen 1s signal in the binding energy range of 398402 eV, whereas in case of conjugated polymer, strong nitrogen 1s signal was observed at 401 eV which corresponds to nitrogens from trastuzumab molecule, indicating successful conjugation of trastuzumab on the polymersomes. 3000

CPS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

2500 2000 1500 410

405

400

395

390

Binding Energy (eV)

Figure 2. XPS wide scan spectra of PLA NPs before and after trastuzumab conjugation. Stronger nitrogen 1s peak (398-402 eV) in trastuzumab conjugated polymersomes confirmed successful trastuzumab conjugation.

Thus, with the help of zeta potential and XPS, trastuzumab conjugation on polymersomes surface was successfully established.


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Stability studies of polymersomes Nanoparticle stability is very essential for nanoparticles intended for intravenous administration. Aggregated nanoparticles can clog the capillaries or blood vessels resulting in severe complications. Hence, stability of polymersomes was determined in water, saline, DMEM media and FBS at 37 °C. As shown in Figure 3, particle size of polymersomes suspended in water, saline and DMEM media did not show any significant change up to 72 h at 37 °C indicating good stability of the polymersomes. Polymersomes stability was also carried out in presence of fetal bovine serum (FBS). No significant change in polymersomes size was observed in presence of FBS and the average nanoparticle size remained below 190 nm up to 72 h. This can be attributed to presence of polyethylene glycol units in the polymersomes which renders ‘stealth’ nature to the polymersomes.36,

37

This reduces protein adsorption on the polymersomes rendering them

stability. The robust stability of polymersomes allows longer circulation half-life of NPs in the blood with higher chances of tumor accumulation leading to better therapeutic efficacy. Effect of 10 mM GSH on polymersomes was also studied at 37 °C for 72 h to evaluate redox responsive nature of disulfide linkages in the polymersomes. As shown in Figure 3, nanoparticle size increased steadily with respect to time in presence of 10 mM GSH as compared to that in absence of 10 mM GSH. This can be attributed to the reducing effect of GSH on disulfide linkages present in the polymersomes. Thiolates (R-S-) from glutathione (GSH) act as reducing agents and causes cleavage of the disulfide linkages leading to polymer degradation and subsequent aggregation of the polymersomes with increase in particle size.44


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Water

DMEM

Saline

10 mM GSH

FBS

Particle Size (nm)

2000 1500 1000 500 0 0

6

12 18 24 30 36 42 48 54 60 66 72 78

Time (h) Water

190

Particle Size (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

DMEM

Saline

FBS

180 170 160 150 140 0

6

12 18 24 30 36 42 48 54 60 66 72 78

Time (h) Figure 3. Stability studies of PLA-Dox-FA-Her polymersomes in water, saline, DMEM media, fetal bovine serum (FBS) and 10 mM GSH. Aggregation of NPs was observed in presence of 10 mM GSH due to GSH mediated breaking of disulfide linkages in the NPs. (Mean ± SD, n = 3)

Drug release studies Drug release studies of polymeric nanosystem was carried out in pH 5.0 and pH 7.4 in presence and absence of 10 mM GSH to simulate endocytic, cytosolic and physiological (blood, extracellular matrix) environments. Cancer cells consist of about four-fold higher GSH concentration as compared to normal healthy cells.45, 46 Hence, GSH was chosen as the reducing agent for evaluating redox responsive nature of polymersomes. Doxorubicin release curve is shown in Figure 4.


27

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Cumulative Doxorubicin Release (%)

Biomacromolecules

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pH 7.4

pH 5.0

pH 7.4 + 10 mM GSH

pH 5.0 GSH + 10 mM GSH

100.00 80.00 60.00 40.00 20.00 0.00 0

6

12

18

24

30

36

42

48

54

60

66

72

Time (h) Figure 4. Drug release studies of PLA-Dox-FA-Her NPs in acetate buffer pH 5.0 and phosphate buffer pH 7.4 with and without 10 mM GSH at 37 °C. (Mean ± SD, n = 3)

Doxorubicin release was found to be highest in pH 5.0 in presence of 10 mM GSH (~91%) followed by in pH 7.4 in presence of 10 mM GSH (~67%), whereas it was only ~46% in pH 5.0 and ~20% in pH 7.4 in absence of 10 mM GSH, indicating selective drug release in redox rich environment of cancer cells. This clearly demonstrates effect of GSH on drug release behavior of redox responsive polymersomes. Higher doxorubicin release in pH 5.0 (~46%) as compared to that in pH 7.4 (~20%) in absence of GSH can be attributed to increased hydrophilicity of doxorubicin due to complete protonation of its amine moiety in acidic pH (doxorubicin pKa = 8.25). Increased hydrophilicity leads to higher solubility which causes higher diffusion of doxorubicin from polymersomes to outer aqueous phase. This results in higher drug release in acidic media (pH 5.0) as compared to neutral pH (pH 7.4). Similar observations have been observed by many researchers.40, 47, 48 The low release at pH 7.4 in absence of GSH (~20%) can be attributed to stability of disulfide bonds in the polymer backbone at physiological conditions. High doxorubicin release in presence of 10 mM GSH can be attributed to the redox triggered breaking of disulfide linkage resulting in


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degradation of polymeric chains in the polymersomes which leads to faster drug release. Thus, disulfide linkages from the polymersomes minimized drug release at physiological conditions but allowed faster and higher release in presence of 10 mM GSH, predicting low doxorubicin release during NPs circulation and faster drug release in the redox environment of cancer cells after NPs uptake. Hemolysis study Hemocompatibility is an essential criterion for intravenously administered nanoparticles. Circulating nanoparticles can interact with RBCs and damage their membranes causing hemolysis. Hemoglobin released from RBCs is inversely proportional to the hemocompatibility of the nanoparticles which can be measured spectrophotometrically at 540 nm. Hemolysis value for hemocompatibility varies from 10% to 25%. Generally, hemolysis less than 20% is considered to be hemocompatible.49, 50 Surface charge and surface hydrophilicity of NPs play major role in interaction of NPs with RBCs. Hemocompatibility of NPs can be improved by reducing the surface charge and hydrophilic surface coating of NPs.51, 52 Polymersomes in the concentration range 0.125 to 2.0 mg/mL were incubated with RBCs for 1 h at 37 °C to determine their hemocompatibility. Polymersomes at maximum concentration of 2.0 mg/ml showed ~12% hemolysis, while lower concentrations showed lower hemolysis as depicted in Figure 5, indicating hemocompatibility of PEG-s-s-PLA-s-s-PLA-s-s-PEG triblock copolymer. This can be attributed to presence of hydrophilic polyethylene glycol and low surface charge resulting in minimal hemolysis.53, 54


29

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20

Hemolysis (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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15 10 5 0 0.125

0.25

0.5

1

2

Polymer Conc. (mg/mL) Figure 5. Hemocompatibility of PLA NPs (Mean ± SD, n = 3)

Cell culture studies Receptor over-expression on the cell surface was the criterion for cell line selection for the cellular uptake and apoptosis studies. The expression of FRα and HER2 receptors on selected cell lines is given in Table 4. Table 4. Expression of FRα and HER2 receptors on cell lines Cell Line

Folate Receptor (FRα)

HER2 Receptor

References

BT-474

+

+++

55, 56

MCF-7

+++

+

43, 57, 58

L929





59, 60

+++: Over-expression, +: Medium to low expression, : No expression

Cell viability studies Cytotoxicity of polymersomes was determined in MCF-7 and L929 cells using MTT assay. MTT assay is dependent on cellular metabolic activity of NADPH dependent dehydrogenase enzyme. This enzyme reduces MTT reagent to purple colored formazan crystals in viable cells. The crystals when dissolved in DMSO can be measured spectrophotometrically at 540 nm.61 L929 cells treated with PLA NPs showed cell viability ~90% at all concentrations (0.125 to 2 mg/mL) indicating biocompatible and non-toxic nature of the PEG-s-s-PLA-s-s-PLA-s-s-PEG


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triblock copolymer towards non-cancerous cells as shown in Figure 6. Doxorubicin loaded NPs showed dose dependent cytotoxicity in L929 cells irrespective of ligands attached. This can be attributed to non-specific uptake of NPs by the cells.

120

PLA NPs PLA-Dox-Her NPs PLA-Dox-FA-Her NPs

PLA-Dox NPs PLA-Dox-FA NPs

100 80 60 40 20 0 0.125

MCF-7

120

Cell Viability (%)

Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

0.25

0.5

1

NP Concentration (mg/ml)

2

PLA NPs PLA-Dox-Her NPs PLA-Dox-FA-Her NPs

PLA-Dox NPs PLA-Dox-FA NPs

100 80 60 40 20 0

L929

0.125

0.25

0.5

1

2

NP Concentration (mg/ml)

Figure 6. Effect of polymersomes on viability of cancer cell lines (MCF-7 cells) and normal noncancerous cells (L929 cells) using MTT cytotoxicity assay (mean ± SD, n = 3).

In case of MCF-7 cells, as shown in Figure 6, PLA NPs showed ~90% cell viability at all concentrations (0.125 to 2 mg/mL), showing biocompatibility and non-toxicity of PEG-s-s-PLAs-s-PLA-s-s-PEG triblock copolymer. Single and dual targeted NPs showed dose dependent cytotoxicity in MCF-7 cells with cytotoxicity in the order - dual targeted NPs > folate targeted NPs > trastuzumab targeted NPs > non-targeted NPs. This can be attributed to overexpressed folate FRα receptors and few HER2 receptors in MCF-7 cells leading to higher cellular uptake of these NPs in MCF-7 resulting in enhanced cytotoxicity. The IC50 values of all the nanoparticle formulations in MCF-7 cells are given in Table 5. Folate targeted NPs showed higher toxicity as compared to non-targeted NPs as well as trastuzumab targeted NPs (IC50= 63.83 μg/mL vs. 1866.47 and 1354.21 μg/mL, respectively). This can be attributed to over-expressed folate FRα receptors and few HER2 receptors in MCF-7 cells leading to higher cellular uptake of folate targeted NPs in MCF-7 cells as compared to nontargeted and trastuzumab targeted NPs resulting in enhanced cytotoxicity. Dual targeted NPs


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exhibited even higher cytotoxicity as compared to non-targeted as well as single targeted NPs (IC50= 46.22 μg/mL) due to both FRα and HER2 receptor mediated enhanced cellular uptake of NPs in MCF-7 cells. Free doxorubicin also showed higher cytotoxicity in MCF-7 cells as indicated by lower IC50 value of 1.99 μg/mL. Table 5. In vitro cytotoxicity of doxorubicin encapsulated nanoparticle formulations against MCF-7 cells Doxorubicin formulation

IC50 (μg/mL)

Free doxorubicin

1.99 ± 0.38

PLA-Dox NPs

1866.47 ± 144.08

PLA-Dox-FA NPs

63.83 ± 6.31

PLA-Dox-Her NPs

1354.21 ± 101.80

PLA-Dox-FA-Her NPs

46.22 ± 5.35

Mean ± SD, n=3

Higher cytotoxicity of free doxorubicin over polymersomes formulations can be attributed to doxorubicin’s rapid diffusion in cells due to its smaller size resulting in higher accumulation and thus higher toxicity in MCF-7 cells. Polymersomes, however, are slowly internalized in the cells via receptor mediated endocytosis. Moreover, time consuming doxorubicin release from the polymersomes as observed in in vitro drug release studies is also responsible for low in vitro potency of PLA-Dox-FA-Her NPs as compared to free doxorubicin. Similar observations have been reported by many researchers where free drug has showed higher cytotoxicity as compared to sustained release nanoparticle formulation.62-66 Even though free doxorubicin showed lower IC50 value as compared to PLA-Dox-FA-Her NPs in MCF-7 breast cancer cells in in vitro cell culture studies, results of in vivo studies provides us realistic comparison of their antitumor efficacy. Thus, IC50 values can be used to compare cytotoxicity efficacy of different nanoparticle drug delivery systems, while in vivo studies are


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necessary for complete evaluation of the therapeutic efficacy of free drug and nanoparticle formulations. The results demonstrate biocompatible and non-toxic nature of PEG-s-s-PLA-s-s-PLA-s-sPEG triblock copolymer. CLSM based qualitative assessment of cellular uptake Cellular uptake determination is essential for evaluation of efficacy of drug delivery nanosystems since it can predict their in vivo performance. Cellular uptake of polymersomes was observed in MCF-7, L929, and BT-474 cell lines using confocal microscopy to study the targeting efficiency of ligands conjugated with the polymersomes. As shown in Figure 7, enhanced doxorubicin fluorescence intensity was observed in MCF-7 and BT-474 cells treated with folate targeted NPs (PLA-Dox-FA NPs) and trastuzumab targeted NPs (PLA-Dox-Her NPs) as compared to non-targeted NPs (PLA-Dox NPs). MCF-7 showed higher doxorubicin fluorescence with folate targeted NPs as compared to trastuzumab targeted NPs owing to presence of overexpressed folate FRα receptors, whereas BT-474 showed higher doxorubicin fluorescence with trastuzumab targeted NPs as compared to folate targeted NPs owing to presence of overexpressed HER2 receptors on the cell surface. This higher cellular uptake of folate and trastuzumab conjugated NPs in the cancer cells can be ascribed to folate and HER2 receptor mediated endocytosis of NPs, respectively. Dual targeted NPs (PLA-Dox-FAHer NPs) showed superior fluorescence intensity than folate or trastuzumab targeted NPs in both the cell lines indicating higher cellular uptake of dual targeted NPs in MCF-7 & BT-474 cells. The enhanced cellular uptake is facilitated by both folate and trastuzumab mediated endocytosis causing higher doxorubicin concentration inside cancer cells and thus showing higher targeting efficiency of dual targeted NPs in cancer cells.


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BT-474

Bright Field

DAPI

MCF-7

Doxorubicin

Merged

Bright Field

DAPI

Doxorubicin

Merged

A

A

B

B

C

C

D

D

Bright Field

DAPI

L929

Doxorubicin

Merged

A

B

C

Scale bar

D

Figure 7. Cellular uptake of polymersomes in BT-474, MCF-7 and L929 as observed by confocal laser scanning microscopy (A) PLA-Dox NPs, (B) PLA-Dox-FA NPs, (C) PLA-Dox-Her NPs, and (D) PLADox-FA-Her NPs. (100× magnification)


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In case of L929 cells which lack over-expressed folic acid or HER2 receptors, very low doxorubicin fluorescence was observed in the cells treated with polymersomes irrespective of their attached ligands. The low doxorubicin fluorescence observed in L929 cells can be indicative of non-specific cellular uptake of polymersomes by L929 cells.

Flow cytometry based quantitative assessment of cellular uptake Cellular uptake was analyzed using flow cytometry to validate and quantify the enhanced cellular uptake of dual targeted NPs as observed in confocal microscopy. As shown in Figure 8, BT-474 cells treated with trastuzumab targeted NPs (PLA-Dox-Her NPs) showed higher fluorescence intensity as compared to non-targeted NPs (PLA-Dox NPs) (~4 fold) and folate targeted NPs (PLA-Dox-FA NPs) (~1.5 fold) owing to HER2 receptor overexpression in BT-474 cells. Dual targeted NPs (PLA-Dox-FA-Her NPs) showed even higher doxorubicin fluorescence as compared to folate targeted NPs (~3 fold), trastuzumab targeted NPs (~2 fold) and non-targeted NPs (~9 fold). Enhanced cellular uptake observed with dual targeted NPs can be ascribed to presence of folate as well as trastuzumab mediated cellular uptake of NPs in cancer cells. MCF-7

BT-474

Untreated Cells

PLA-Dox NPs

PLA-Dox-FA NPs

PLA-Dox-Her NPs

L929

PLA-Dox-FA-Her NPs


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Figure 8. Cellular uptake of polymersomes in BT-474, MCF-7 and L929 cell lines as observed in flow cytometry

MCF-7 cells treated with folate targeted NPs showed enhanced doxorubicin fluorescence as compared to non-targeted NPs (~7 fold) and trastuzumab targeted NPs (~2 fold) due to overexpressed folate FRα receptors on its surface. Dual targeted NPs showed even higher doxorubicin florescence as compared to folate targeted NPs (~1.5 fold), trastuzumab targeted NPs (~3.5 fold) and non-targeted NPs (~11 fold). This demonstrates higher cellular uptake efficiency of NPs decorated with folate and trastuzumab in cancer cells. L929 cells treated with targeted as well as non-targeted NPs showed similar low doxorubicin fluorescence irrespective of attached targeting ligand. This indicate non-specific uptake of NPs by L929 cells which corroborate results obtained in the confocal studies. Thus, flow cytometry study agrees with confocal microscopy results that NPs decorated with two ligands resulted in enhanced cellular uptake and higher nanoparticle internalization in cancer cells while limiting its uptake in non-cancerous cells. Annexin V-FITC apoptosis assay Annexin V-FITC apoptosis assay is rapid and reliable method for determination of apoptosis and necrosis in the cells. Apoptosis is characterized by exposure of phosphatidylserine on the outer cell surface, which is normally present on the inner side of plasma membrane. Annexin V, a calcium dependent phospholipid binding protein, has high affinity towards phosphatidylserine exposed on the apoptotic cells and thus detects apoptotic cells. Propidium iodide intercalates DNA of necrotic cells whose membrane is compromised and thus detects necrotic cells. Apoptotic cells having intact plasma membrane do not take up propidium iodide and thus can be differentiated from necrotic cells. Thus, in Annexin V-FITC apoptosis assay, viable cells


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(AnnexinV-FITC−ve PI−ve) are differentiated from early apoptotic cells (AnnexinV-FITC+ve PI−ve) and late apoptotic/ necrotic cells (AnnexinV-FITC+ve PI+ve).67, 68

Control

PLA-Dox

PLA-Dox-FA

PLA-Dox-Her

PLA-Dox-FA-Her

0.13%

0.21%

0.56%

17.06%

2.81%

64.09%

1.94%

75.74%

2.46%

84.59%

98.76%

0.90%

59.04%

23.33%

15.19%

17.92%

11.59%

10.73%

5.56%

7.39%

BT474

0.21%

0.24%

6.09%

32.01%

1.02%

52.36%

1.62%

43.19%

2.75%

85.31%

99.25%

0.31%

49.42%

12.47%

16.93%

29.69%

26.49%

28.69%

9.06%

2.88%

MCF-7

0.53%

0.25%

0.15%

7.02%

0.85%

98.55%

0.67%

65.13%

27.70%

57.39%

12.53%

0.57%

11.65%

0.21%

66.25%

21.53%

58.75%

11.61%

L929 29.24%

29.43%

Figure 9. Therapeutic efficacy of polymersomes in BT-474, MCF-7, and L929 cells as determined by Annexin V-FITC apoptosis assay using flow cytometry

As observed in Figure 9, in case of BT-474 and MCF-7 cells, folate targeted NPs (PLA-DoxFA NPs) and trastuzumab targeted NPs (PLA-Dox-Her NPs) treatment resulted in higher percent of early apoptotic cells (17.92% and 10.73% in BT-474 cells; 29.69% and 28.69% in MCF-7 cells, respectively) and late apoptotic/necrotic cells (64.09% and 75.74% in BT-474 cells; 52.36% and 43.19% in MCF-7 cells, respectively), as compared to that in non-targeted NPs (PLA-Dox NPs). Non-targeted NPs (PLA-Dox NPs) showed 23.33% and 12.47% early apoptotic cells and 17.06% and 32.01% late apoptotic/necrotic cells in BT-474 and MCF-7 cells, respectively. Dual targeted polymersomes (PLA-Dox-FA-Her NPs) showed even higher


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apoptosis with higher percent of early and late apoptotic/necrotic cells (7.39% and 84.59% in BT-474 cells; 2.88% and 85.31% in MCF-7 cells, respectively) as compared to non-targeted and single targeted NPs, suggesting superior therapeutic efficacy due to presence of two ligands as compared to NPs decorated with single ligand. In case of L929 cells, they lack folate FRα and HER2 receptors and hence majority of cells remained healthy (AnnexinV-FITC−ve PI−ve). Polymersomes, irrespective of attached ligand, showed minimal apoptosis/necrosis in L929 cells indicating their lower cellular uptake and lower internalization in non-cancerous cells. The higher apoptosis in dual targeted NPs is in good agreement with confocal and FACS cellular uptake study. It can be attributed to presence of two ligands (folic acid and trastuzumab) on nanoparticle surface which mediated enhanced cellular uptake of NPs in cancer cells. This resulted in higher amount of doxorubicin being delivered in the cancer cells resulting in higher apoptosis. However, in case of non-cancerous L929 cell line, due to lack of folate FRα and HER2 receptors, cellular uptake of NPs was minimal resulting in low apoptosis. Thus, cancer cells were selectively targeted for delivery of doxorubicin with the help of dual targeted NPs, while sparing normal non-cancerous cells from doxorubicin toxicity. Animal studies The Ehrlich ascites tumor (EAT) is a transplantable tumor model originating from mouse mammary carcinoma. EAT cells are characterized by high transplantable capacity and rapid proliferation rate. EAT cells express folate as well as HER2 receptors and hence it is a good model for evaluation of antitumor efficacy of PLA-Dox-FA-Her NPs.69-71


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In vivo tumor regression study Antitumor efficacy of PLA-Dox-FA-Her NPs was evaluated in EAT bearing Swiss albino mice. Mice were treated with free doxorubicin and PLA-Dox-FA-Her NPs with dose equivalent to 5 mg/kg doxorubicin every 3rd day for 15 days and tumor volume were measured. As shown in Figure 10, mice treated with PBS (Control group) showed progressive increase in tumor volume with approximately 10 fold increase in tumor volume observed on 18th day as compared to initial volume on day 0. Free doxorubicin showed moderate antitumor activity with tumor volume reduction of ~38% on 18th day as compared to initial tumor volume, whereas PLA-Dox-FA-Her polymersomes showed statistically significant enhanced antitumor activity as compared to Control (p < 0.001) as well as free doxorubicin (p < 0.001) with tumor volume reduction of ~90% with respect to initial tumor volume. Control Relative Tumor Volume (%)

1200

Doxorubicin

1000

Relative Tumor Volume (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PLA-Dox-FA-Her NPs

800 600

*

400

PBS (Control)

200 0 0

3

6

9

12

15

18

Time (Days)

Doxorubicin

100

PLA-Dox-FA-Her NPs

Doxorubicin

80 60 40

*

20 0 0

3

6

9

12

Time (Days)

15

18

PLA-Dox-FA-Her NPs

Figure 10. In vivo tumor regression study of PLA-Dox-FA-Her NPs and free doxorubicin with respect to Control in Ehrlich ascites tumor (EAT) bearing Swiss albino mice. (Mean ± SEM, n=6), *p < 0.001.


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Tumor images of Ehrlich ascites tumor bearing Swiss albino mice treated with PBS (Control), free doxorubicin and PLA-Dox-FA-Her NPs on 18th day of study.

This superior antitumor efficacy of PLA-Dox-FA-Her NPs can be ascribed to folate and trastuzumab mediated active targeting as well as enhanced permeation and retention effect (EPR effect) mediated passive targeting of tumor, accelerated doxorubicin release in redox environment of cancer cells, low protein adsorption and longer circulation half-life leading to enhanced cellular internalization of doxorubicin loaded NPs in cancer cells. This resulted in higher doxorubicin accumulation in tumor cells leading to higher therapeutic efficacy of doxorubicin. Antitumor efficacy determination based on histopathology Antitumor efficacy of the nanosystem was further analyzed based on tumor histopathology studies. Tumors were removed from the mice from each group on 18th day and sections were cut for H&E staining for histology analysis as shown in Figure 11. In PBS treated mice, tumor section showed viable neoplastic cells with intermittent necrotic areas. In case of free doxorubicin treated mice, tumor section showed presence of necrotic cells along with patches of viable neoplastic cells in the tumor tissue indicating partial tumor regression. In case of PLADox-FA-Her NPs treated mice, necrotic cells with very few viable neoplastic cells were observed in the tumor region. Thus, histology results agree with tumor regression study that PLA-DoxFA-Her NPs showed improved antitumor efficacy as compared to free doxorubicin treatment. Determination of organ toxicity using histopathology Cardiotoxicity of PLA-Dox-FA-Her NPs and free doxorubicin was evaluated by studying histology of heart of treated mice as shown in Figure 11. Heart from mice treated with free doxorubicin showed cardiac damage/cardiotoxicity with mild cardiomyocyte swelling,


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disordered patterns of myofibrils, myofribillar loss and high cytoplasmic vacuolization. Heart of mice treated with PLA-Dox-FA-Her NPs showed minimal disordered patterns of myofibrils with mild interstitial edema and lower cardiomyocyte swelling. This indicates minimal cardiac damage suggesting non-toxicity of the polymersomes to cardiac tissue. Thus, cardiotoxicity of doxorubicin was conquered by dual targeted polymersomes with enhanced antitumor activity. Vital organs such as liver, kidneys, lungs and spleen were also evaluated for any toxicity. As shown in Figure 11, liver section of free doxorubicin treated mice showed liver fibrosis with multiple focal cellular granulomatous lesions, kupffer cell prominence and foci of spotty necrosis indicating hepatotoxicity of doxorubicin. Kidney section of free doxorubicin treated mice also showed nephrotoxicity with significant tubular damage. Sections of vital organs of mice treated with PLA-Dox-FA-Her NPs showed no substantial organ toxicity as compared to that of control group indicating non-toxic and biocompatible nature of polymersomes. Tumor

Heart

Liver

Kidney

Lungs

Spleen

A

B

C

Figure 11. H&E staining of histopathological sections of tumor and vital organs-heart, liver, kidney, lungs and spleen after treatment with free doxorubicin and PLA-Dox-FA-Her NPs in Ehrlich ascites


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tumor bearing Swiss albino mice. A) Control (PBS treatment), B) Free doxorubicin treatment, C) PLADox-FA-Her NPs treatment. (20× magnification).

The non-toxicity of PLA-Dox-FA-Her NPs can be ascribed to its low molecular weight and biodegradable nature. Redox responsive disulfide linkages in the polymer which breaks only in the cancer cells due to presence of high concentration of glutathione (10 mM) also helps in biodegradation of polymer and release of doxorubicin specifically in the cancer cells and prevents drug release during NPs circulation. Polymersomes are passively targeted via EPR effect and actively targeted because of folate and trastuzumab ligands, together leading to higher accumulation in tumor and lower accumulation in other tissues/organs causing minimal organ toxicity. Thus, the developed nanosystem showed strong potential as a doxorubicin delivery nanocarrier with high biocompatibility and better therapeutic index as compared to free doxorubicin with minimal toxicity. Serum biochemistry analysis Serum biochemistry of mice treated with PLA-Dox-FA-Her NPs and free doxorubicin was carried out to determine biocompatibility and safety of the formulations and reinforce the histopathology results as shown in Table 6. Due to known cardiotoxic nature of doxorubicin, it is essential to evaluate cardiotoxicity of doxorubicin loaded NPs to support our hypothesis that dual targeted NPs will have minimal cardiotoxicity as compared to free doxorubicin.


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Table 6. Serum biochemical analysis of Ehrlich ascites tumor bearing Swiss albino mice treated with PLA-Dox-FA-Her NPs and free doxorubicin Biochemical parameters CK-MB (IU/L) AST (SGOT) (IU/L) ALT (SGPT) (IU/L) Alkaline Phosphatase (IU/L)

Control (PBS) 3.67 ± 0.33

Free Doxorubicin 11.00 ± 1.73*

PLA-Dox-FA-Her NPs 4.50 ± 0.50

55.67 ± 4.59

101.80 ± 5.15*

56.03 ± 6.93

31.70 ± 4.44 118.20 ± 7.13

*

75.80 ± 6.47

202.47 ± 9.47 *

**

35.77 ± 5.65 133.67 ± 9.13

Total Bilirubin (mg/dL)

0.25 ± 0.02

0.62 ± 0.06

0.34 ±0.05

Total Protein (g/dL)

6.20 ± 0.61

5.37 ± 0.76

6.53 ± 0.50

Albumin (g/dL)

3.87 ± 0.38

3.40 ± 0.46

3.33 ± 0.52

Globulin (g/dL)

1.87 ± 0.19

1.73 ± 0.35

1.70 ± 0.26

A:G Ratio

2.08 ± 0.12

1.96 ± 0.07

1.96 ± 0.02

113.63 ± 5.67

125.57 ± 5.18

Total Cholesterol (mg/dL)

*

114.20 ± 6.35

Urea (mg/dL)

35.83 ± 4.57

66.50 ± 6.94

42.63 ± 3.35

Creatinine (mg/dL)

0.35 ± 0.05

0.59 ± 0.09

0.35 ± 0.06

Uric Acid (mg/dL)

2.67 ± 0.43

*

6.53 ± 0.69

3.17 ± 0.61

Calcium (mg/dL)

7.57 ± 0.48

7.37 ± 0.72

7.37 ± 0.71

Phosphorus (mg/dL)

2.90 ± 0.32

3.60 ± 0.46

3.57 ± 0.35

*

**

All values expressed as mean ± SEM (n=3). Significant (P < 0.05); Highly significant (P < 0.001)

Cardiotoxicity of PLA-Dox-FA-Her NPs was determined using cardiac specific biomarker creatine kinase MB (CK-MB) which is widely used to evaluate cardiac damage/toxicity. After cardiac muscle damage, CK-MB is released in the blood which can be detected using immunochemical methods.72 Significantly high levels of CK-MB were observed in serum of free doxorubicin treated mice as compared to control group (p < 0.001) indicating cardiac damage/cardiotoxicity, whereas CK-MB levels observed in PLA-Dox-FA-Her NPs treated mice were statistically similar as that of control group (p > 0.05) showing cardiac biocompatibility of PLA-Dox-FA-Her NPs. Liver specific biomarkers- alanine transaminases (ALT/SGPT), aspartate transaminases (AST/SGOT), alkaline phosphatase (ALP) and total bilirubin were analyzed to determine liver damage. AST, ALT, ALP and total bilirubin levels in serum increases in liver damage and hence


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are considered reliable markers for liver toxicity. Free doxorubicin treated mice showed significantly higher levels of ALP as compared to Control group (p < 0.001) indicating liver toxicity. Higher levels of ALT, AST and total bilirubin were also observed in free doxorubicin treated mice as compared to Control group (p < 0.05). PLA-Dox-FA-Her NPs treated mice showed statistically insignificant elevation in serum ALT, AST, ALP and total bilirubin levels (p > 0.05) with respect to control group suggesting non-toxicity to liver. Nephrotoxicity was also observed after free doxorubicin treatment with significant increase in renal function biomarkers- blood urea, creatinine and uric acid levels as compared to Control group (p < 0.05). PLA-Dox-FA-Her NPs showed statistically insignificant increase in these parameters with respect to Control group (p > 0.05) indicating biocompatibility of NPs with respect to kidneys. Thus,

serum biochemistry

analysis

supported

histopathology results

and showed

biocompatibility of PLA-Dox-FA-Her NPs with respect to heart, liver and kidney when compared with significant cardiotoxicity, hepatotoxicity and nephrotoxicity exhibited by free doxorubicin treatment. This can be attributed to folate and trastuzumab mediated active targeting and EPR mediated passive targeting of NPs which resulted in higher tumor accumulation of NPs causing minimal exposure to other organs resulting in lower toxicity. Also, redox responsive nature of polymersomes prevented doxorubicin release during the circulation of NPs leading to lower toxicity to vital organs. Thus, we were able to minimize toxicities associated with doxorubicin (cardiotoxicity, hepatotoxicity and nephrotoxicity) using dual targeted PLA-DoxFA-Her NPs while achieving enhanced antitumor efficacy.


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CONCLUSIONS The present study demonstrated combined activity of dual targeting and redox responsiveness for improved intracellular doxorubicin delivery in breast cancer. The polymeric nanosystem was found to be biocompatible with respect to protein adsorption, hemolysis, coagulation and cell viability studies. Dual targeting with folate and trastuzumab resulted in higher cellular uptake leading to higher apoptosis in breast cancer cells. It also showed superior tumor regression in in vivo studies with EAT bearing Swiss albino mice as compared to free doxorubicin. In summary, dual functionalized redox responsive biocompatible polymeric nanosystem shows great potential as a chemotherapeutic drug delivery nanocarrier for breast cancer therapy and holds promise towards further development for clinical applications.

Supporting Information Available [Polymer characterization data including 1H-NMR, GPC, UV-visible spectra and FTIR spectra; X-ray powder diffraction (XRD) study; protein adsorption study and coagulation studies]. This material is available at free of charge via the Internet at http://pubs.acs.org

ACKNOWLEDGEMENTS Authors are thankful to the Department of Biotechnology (DBT), India for the research grant (BT/PR13341/NNT/28/467/2009). Shantanu V. Lale is grateful to the Indian Institute of Technology, Delhi for awarding him an Institute Fellowship. He is thankful to Dr. Farhat Naz, AIIMS and Dr. Nadeem Tanveer, University College of Medical Sciences, Delhi for their help with the histopathology analysis. He is also thankful to Prof. Renu Saxena and Dr. Archana


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Bansal from the All India Institute of Medical Sciences (AIIMS), New Delhi for their help with the coagulation studies and CK-MB determination, respectively.

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