Enhanced Antitumor Efficacy and Reduced Toxicity of Docetaxel

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Enhanced antitumor efficacy and reduced toxicity of docetaxel loaded estradiol functionalized stealth polymeric nanoparticles Sanyog Jain, Gollapalli Spandana, Ashish Kumar Agrawal, Varun Kushwah, and Kaushik Thanki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00281 • Publication Date (Web): 16 Sep 2015 Downloaded from http://pubs.acs.org on September 22, 2015

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Molecular Pharmaceutics

Enhanced antitumor efficacy and reduced toxicity of docetaxel loaded estradiol functionalized stealth polymeric nanoparticles Sanyog Jain*, Gollapalli Spandana, Ashish Kumar Agrawal, Varun Kushwah, Kaushik Thanki Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S Nagar, Mohali160062, Punjab, India. * Corresponding author. Tel.: +91- 172 2292055, Fax: +91-172 22914692, E-mail address: [email protected], [email protected]

ACS Paragon Plus Environment


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Abstract In spite of extensive research over the decades, breast cancer treatment is still a major challenge due to non-specific distribution of the chemotherapeutics. This void can be filled by restricting the distribution of chemotherapeutics towards the cancerous cells. In the present report estradiol (E2) functionalization of docetaxel (DTX) loaded PLGA nanoparticles was supposed to have specific distribution of DTX to cancerous cells simultaneously avoiding the non-specific distribution towards the normal cells. In line, E2-PEG-PLGA conjugate was synthesized and characterized by FTIR and NMR spectroscopy. Extensive optimization of different process variables resulted in the formation of spherical E2-PEGPLGA NPs in the size range of 228.5 ± 11.8 nm and entrapment efficiency of 94.25 ± 2.49. Trehalose (5% w/v) resulted in the formation of fluffy, easy to redisperse freeze dried cake of nanoparticles. PXRD analysis revealed the amorphous nature of DTX encapsulated with in the nanoparticles. X-ray photoelectron spectroscopy confirmed the presence of E2 over the surface of nanoparticles. In line with our hypothesis, cellular uptake studies on ER positive MCF-7 cells revealed relatively higher uptake and efficient localization into the nuclear region of E2-PEG-PLGA NPs in comparison with plain counterparts, while in case of ER negative HeLa cells E2-PEG-PLGA NPs showed no difference in fluorescence pattern as compared to MCF-7 cells incubated with unmodified nanoformulation, indicating nonspecific delivery of DTX. Moreover, MTT assay revealed relatively higher cytotoxicity of E2PEG-PLGA NPs in comparison with free DTX. Furthermore, in vivo pharmacokinetic studies revealed 9.36 and 4.79 fold enhancement in circulation half-life and AUC(0-∞), respectively, of E2-PEG-PLGA NPs in comparison with Taxotere®. In vivo antitumor efficacy in DMBA induced rat model, demonstrated significant reduction in tumor volume in comparison with plain counterpart (PLGA-NPs) and marketed formulation, Taxotere®. Moreover, the safety of


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the estradiol functionalized PLGA NPs was confirmed by hepato- and nephro-toxicity studies. Key words: - Estrogen, breast cancer, nanoparticles, docetaxel, PLGA Abbreviations: AUC

Area under the curve

ALT

Alanine transaminase

AST

Aspartate transaminase

BUN

Blood urea nitrogen

DAPI

4',6-diamidino-2-phenylindole

DCC

Dicyclohexylcarbodiimide

DMBA

7, 12-Dimethylbenz(a)anthracene

DTX

Docetaxel

E2

17β-Estradiol

EPR

Enhanced permeability and retention

ER

Estradiol receptor

FDA

Food and drug administration

FTIR

Fourier transform infrared spectroscopy

HeLa

Human cervical cancer

MCF-7

Michigan Cancer Foundation-7



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MDA

Malondialdehyde

MDR

Multidrug resistance

MEM

Minimum Essential Medium Eagle

MTT

[3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]

NHS

N-hydroxysuccinimide

NMR

Nuclear magnetic resonance

NPs

Nanoparticles

PDI

Polydispersity index

PEG

Poly ethylene glycol

PLGA

Poly(lactic-co-glycolic acid)

PXRD

Powder X-Ray Diffraction

SEM

Scanning electron microscopy


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1.

Introduction

The major challenge of the conventional chemotherapy is deleterious side-effects associated with non-specific tissue distribution of anticancer agents. Systemically administered bolus doses of powerful chemotherapeutics often result in intense side effects due to their action on sites other than the intended target sites. With such non-specific drug action, the concentration of the drug administered to the patient is a vicious predicament between choosing a near-toxic effective dose and a comfortable ineffective dose. In addition, the efficacy of treatment is often obscured by the poor bioavailability of anticancer agents as well as multidrug resistance (MDR), acquired upon repeated chemotherapeutic cycles.1 Emergence of nanotechnology is definitely a medical boon for the diagnosis, treatment and prevention of cancer as it offers a less invasive alternative compared to the conventional therapeutic cocktails (e.g. chemotherapy, radiation-therapy, surgery etc.). Docetaxel (DTX) is a clinically-established, anti-mitotic chemotherapy medication, highly effective as monotherapy and/or combination therapy across a variety of tumor types including breast, lung, stomach, ovarian, prostate as well as head and neck cancers.2 Docetaxel works by interfering the normal working of tubulin protein by hyper stabilizing the microtubule assembly after binding with β subunit of tubulin protein of the microtubules.3 As of now, the only available formulation of DTX for clinical use (Taxotere®) consists of a solution of DTX (40 mg/mL) in a vehicle containing high concentration of Tween 80® as solubilizer and ethanol as co-solvent. However, Tween 80® interferes with the protein binding of docetaxel which ultimately results into hepatotoxicity, fluid retention, neurotoxicity, musculoskeletal toxicity and neutropenia.4 In addition to these toxicities, anaphylactic (hypersensitivity) toxicity is also associated by the virtue of solvent system.5 Unfortunately, this vehicle is not only associated with severe hypersensitivity reactions but also shows incompatibility with common poly vinyl chloride intravenous administration sets.6 It also



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interferes with the normal binding of DTX to serum proteins in a concentration dependentmanner and may even alter the pharmacokinetics of DTX in vivo.7 In order to eliminate the Tween 80®-based vehicle and increase the drug solubility, alternative nanoformulations have been suggested, including liposomes and cyclodextrins.8, 9 As a therapeutic vector, biodegradable polymeric nanoparticles offer multiple advantages as compared to other drug delivery systems. These include higher stability, good biocompatibility, non-toxicity, biodegradability, increased accumulation at the tumor site via enhanced permeability and retention (EPR) effect and controlled drug release.10 In addition, polymeric nanoparticles (NPs) can also reduce the multi-drug resistance associated with many anticancer drugs, including DTX by altering their cellular internalization mechanism or reducing their cellular efflux mediated by the P-glycoprotein.11 Amongst various biodegradable polymer, poly (lactic-co-glycolic acid) (PLGA) is the most widely studied and FDA approved polymer. Beside this, ease of chemical coupling to fabricate the smart polymer having predesigned fate is another advantage.12 We reasoned that introduction of a tumor-targeting module onto the surface of polymeric nanoparticles will augment the sitespecificity as well as intracellular uptake of the loaded drugs through ligand-receptor binding interaction. Estradiol is a potent human estrogen that can specifically bind to estrogen receptors (ER) over-expressed on a variety of cancer types including breast, ovarian or endometrial.13 Being an endogenous molecule, Estradiol is also biocompatible and nonimmunogenic, which makes it a promising ligand for targeted cancer therapy.14 Thus in the present work 17β-Estradiol (E2) functionalized, DTX loaded PLGA nanoparticles (E2PEG-PLGA NPs) were hypothesized for efficient tumor targeting and ultimately for improved therapeutic outcomes.


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2.

Materials and Methods

2.1. Materials DTX was obtained as gift sample from Fresenius Kabi Oncology Limited, Gurgaon, India. PLGA 50:50 (MW 48,000 inherent viscosity 0.45-0.60 dl/g in chloroform at 25°C) was procured

from

Boehringer

Ingelheim,

Germany.

17β-Estradiol

(E2),

7,

12-

dimethylbenz[α]anthracene (DMBA), 4,6-diamidino-2-phenylindole (DAPI), Coumarin-6, succinic anhydride (SA) and anhydrous benzene were purchased from Sigma Aldrich, USA. Polyethylene glycol bisamine (MW 3000 Da) was procured from JenKem Technology, USA. Poly-(vinyl alcohol) (MW 30000-70000 Da), Dicyclohexylcarbodiimide (DCC) and Nhydroxysuccinimide (NHS) were purchased from Fluka. Pyridine, dichloromethane and diethyl ether were procured from Merck while hydrochloric acid, sodium bicarbonate, disodium hydrogen phosphate and sodium acetate were purchased from Loba Chemie Pvt. Ltd., Mumbai. Dichloromethane and pyridine were dried over phosphorus pentoxide and potassium hydroxide respectively and distilled prior to use. Ultrapure deionized water (SG water purification system, Barsbuttel, Germany) was used for all the experiments. All other solvents and reagents, unless otherwise stated, were of analytical grade and procured from local suppliers. 2.2. Methods 2.2.1. Synthesis and spectral characterization of E2-PEG-PLGA bioconjugate The E2-PEG-PLGA bio-conjugate was synthesized in three steps: (i) Preparation of active NHS ester of PLGA; (ii) PEGylation of PLGA with homobifunctional PEG-bisamine (NH2PEG-NH2) and (iii) Covalent conjugation of estradiol with activated ester of PEG-PLGA diblock copolymer by using standard carbodiimide chemistry. All synthesized compounds were characterized by FTIR and NMR. By considering readers interest a detailed description



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about the synthesis and characterization of conjugates has been provided in supplementary material. 2.2.2. Preparation of DTX loaded E2-PEG-PLGA NPs DTX loaded E2-PEG-PLGA NPs were prepared by emulsion diffusion evaporation method as reported earlier

15

with slight modifications. The method involves the dissolution of both

PLGA and DTX in dichloromethane and its subsequent injection into stabilizer aqueous solution under magnetic stirring. PVA was selected based on our previous experience.10, 16 Different process variables viz. surfactant concentration (0.5% w/v), volume of aqueous phase in primary emulsion (10 ml) and theoretical drug loading (15% w/w were found optimum in producing particles with desired particle size, PDI, zeta potential and entrapment efficiency. Estradiol functionalized PLGA nanoparticles were prepared by using E2-PEGPLGA in place of PLGA by keeping other optimized parameters constant. Readers are suggested to refer supplementary material for detailed optimization. 2.2.3. Particles size, zeta potential, entrapment efficiency and morphology analysis DTX NPs were evaluated for their mean particle size, polydispersity index (PDI) and zeta potential by using Zeta Sizer (Nano ZS, Malvern Instruments, UK). Zeta potential was estimated on the basis of electrophoretic mobility under an electric field, as an average of 20 measurements.17 Final values are presented as the average of 6 measurements. Entrapment efficiency was determined by centrifuging the nanoparticle suspension at 21,000 rpm for half an hour. The pellet, formed following the centrifugation, was dissolved in acetonitrile. The content of DTX was determined by using validated HPLC method calculated by using the following formula;


18

and

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The shape and surface morphology of the NPs were determined by scanning electron microscopy (SEM) (S-3400N, Hitachi, Japan). A drop of colloidal dispersion of DTX NPs was placed on a glass coverslip previously adhered to a metallic stub by a bi-adhesive carbon tape. The drop was air-dried and coated with gold so as to obtain a conducting surface and analysed by using SEM.19, 20 2.2.4. Freeze drying DTX loaded NPs were freeze-dried (Vir Tis, Wizard 2.0, New York, USA) by following an optimized universal stepwise freeze-drying process.21 Briefly, 2 mL of DTX NPs dispersion were added to glass vials containing 2.5, 5 and, 10% w/v of different cryoprotectants viz. mannitol, sucrose and trehalose. The samples were then subjected to freezing until −60°C in 8 h through an 8 step process, followed by primary drying until 20°C in 36 h through 8 steps. The secondary drying was carried out at 25°C for 6 h. The condenser temperature was −60°C, and the pressure applied in each step was 200 Torr. The freeze-dried DTX NPs were then characterized for the appearance of the cake, reconstitution time, Sf/Si ratio (ratio of particle size obtained after lyophilization to particle size before lyophilization), and PDI following the reconstitution of the freeze dried cake. 2.2.5. XRD analysis The X-ray diffraction pattern of pure DTX, plain PLGA and DTX loaded freeze dried nanoparticles was obtained by using X-ray diffractometer (Bruker D8 advance, Bruker, Germany). Measurements were performed at a voltage of 40 kV and 25 mA. The scanned angle was set from 3°≤ 2ϴ ≥40°, and the scanned rate was 2 min. 2.2.6. Surface chemistry and elemental composition Surface chemistry and elemental composition was determined by using X-ray Photoelectron Spectroscopy (XPS, AXIS Ultra DLD, Kratos, UK). XPS analyses were discriminated for C 1s, O 1s and N 1s peaks using Al Kα excitation source (ESCA-2000 Multilab VG microtech).



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The fixed transmission mode was used with a passing energy of 80 eV and the binding energy spectrum was analyzed from 0 to 1100 eV. 2.2.7. In vitro release study All nanoformulations were characterized for their in vitro release profile at (i) phosphate buffer saline (pH 7.4) (ii) phosphate buffer saline (pH 5.5), to mimic the pH of tumor microenvironment and (iii) Plasma, respectively. The in vitro release of DTX from various nanoformulations (PLGA, PEG-PLGA, and E2-PEG-PLGA) was carried out by dialysis membrane method. Briefly, freeze dried formulations (equivalent to 500 µg of DTX) were dispersed in 200 µL of release media and filled into the dialysis bag. The dialysis bags were then suspended in 15 mL phosphate buffer (pH 7.4 and 5.5) containing 0.1% Tween 80. Tween 80 was added to solubilize the drug entering the release medium and thereby maintain sink conditions. To study the release in plasma, freeze dried NPs (equivalent to 500 µg of DTX) were dispersed in 200 µL of plasma and suspended in vials containing 15 mL plasma. The vials containing suspending dialysis bags were then kept in shaker bath at 37°C and 100 rpm. At predetermined time points (0.5, 1, 3, 6, 12, 24, 48, 96, 120 h) 1 mL sample was withdrawn and replaced with equal quantity of fresh buffer/plasma. The samples were analyzed by HPLC and cumulative % drug release was calculated. 2.2.8. In vitro targeting and cytotoxicity studies 2.2.8.1. Cell culture Estrogen receptor (ER) negative Human cervical cancer (HeLa) and Estrogen receptor (ER) positive Human breast adenocarcinoma cells (MCF-7; ATCC, Manassas, VA, USA) were grown in tissue culture flasks (75 cm2) and maintained at 5% CO2 atmosphere at 37°C. The growth medium comprised Minimum Essential Medium Eagle (MEM, Sigma) supplemented with Earle’s salts, L-glutamine, nonessential amino acids, sodium bicarbonate, sodium pyruvate, 10% FBS, 100 U/mL penicillin, and 100µg/mL streptomycin (PAA Laboratories


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GmbH, Austria) and was changed on every alternate day. The cultured cells were trypsinized once 90% confluent with 0.25% trypsin EDTA solution (Sigma, USA). MCF-7 and HeLa cells were seeded at a density of 50,000 cells/well in 6-well culture plate (Costars, Corning Inc., NY, USA) for qualitative cell uptake analysis by the CLSM. Furthermore, MTT assay was also employed to determine the viability of MCF-7 cells by seeding in 96-well cell culture plates (Costars, Corning Inc., NY, USA) at a density 10,000 cells/well. 2.2.8.2. Cell uptake and intracellular localization After the cells reached 90% confluence, the cell culture medium was removed, and cells were washed with Hank’s Buffered Salt (HBS) Solution (PAA Laboratories GmbH, Austria) three times. For cell uptake studies coumarin 6 loaded nanoparticles were prepared by following the same method except using coumarin 6 in place of DTX. The cells were then incubated with coumarin 6 loaded nanoparticles (equivalent to 1µg/mL of free C-6) for 3 h, and extracellular particles were removed by washing with HBSS (5×). The cells were then fixed with 3% paraformaldehyde (Merck, India) and permeabilized with 0.2% Triton X-100. The nuclei of the cells were stained with 10 µg/mL DAPI (Sigma, USA) and were observed under a confocal laser microscope (CLSM) (Olympus FV1000). In separate set of experiments, the nanoformulations loaded with DTX were also incubated with HeLa and MCF-7 cells at concentration equivalent to DTX, 5 µg/ml for 3 h. Post incubation, cells were washed twice with PBS, pH 7.4, lysed with Triton X 100 and recovered in microcentrifuge tubes. The cell lysates were then centrifuged at 21000 rpm for 10 min at 4 °C and supernatant analyzed for DTX content using validated HPLC method.22 2.2.8.3. Cytotoxicity studies Standard MTT assay was employed to determine the cell cytotoxicity of DTX-NPs in MCF-7 cells.23 MCF-7 cells (4 × 105) were seeded to 96-well tissue culture plates in a total volume of 180 µl of complete media and kept for 18 h, following the attachment of MCF-7 cells, fresh



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medium containing free DTX, Taxotere® and various NP preparations was added to the different wells so as to achieve net concentrations of 0.1, 1, 5, and 10 µg/mL (equivalent to free DTX) and incubated for 24 h and 48 h. Upon completion of the incubation period, the medium containing formulation was aspirated, and cells were washed with PBS, pH 7.4. Subsequently, 150 µL of MTT solution (500 µg/mL in PBS) was added to each well and reincubated for 3−4 h to facilitate formation of formazan crystals. The excess solution was then aspirated carefully, and MTT formazan crystals were dissolved in 200 µL of DMSO. The optical density (OD) of the resultant solution was then measured at 550 nm using an ELISA plate reader (BioTek, USA). 2.2.9. In vivo studies 2.2.9.1. Animals Female Sprague Dawley (SD) rats (200-250 g) and female Swiss mice (20-25 g) were procured from the central animal facility, NIPER, India and used for antitumor and toxicity studies, respectively. Animals were kept in the plastic cages and maintained at a temperature of 25 ± 2°C and relative humidity of 50-60% under 12 h light/dark cycles having free access to water and food. All the experimental protocols were dually approved by Institutional Animal Ethics Committee (IAEC), and performed in accordance with guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. 2.2.9.2. Pharmacokinetics The animals were randomly distributed into four groups each containing 5 animals (n=5). First group of animals received Taxotere while the second, third and fourth groups received DTX loaded PLGA, PEG-PLGA and E2-PEG-PLGA NPs, respectively. All the formulations were administered via intravenous injection at a DTX dose of 2 mg/Kg of body weight. The


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blood samples (approximately 0.2 ml) were collected from the retro orbital into the heparinized micro-centrifuge tubes. Plasma was separated by centrifuging the blood samples at 10000 rpm for 10 min at 4°C. To 100 µl of plasma, 200 µl of acetonitrile and 25 µl of internal standard (1-amino 4-nitro naphthalene) were added to precipitate the plasma proteins. The samples were vortexed and centrifuged at 10,000 rpm for 10 min. The supernatant was separated and analyzed for drug content by validated RP-HPLC.24 2.2.9.3. Pharmacokinetic data analysis The pharmacokinetic analysis of plasma concentration-time data was analyzed by biphasic clearance kinetics using Kinetica Software (Version 5.0, Thermo scientific). Required pharmacokinetics parameters like total area under the curve (AUC)0-∞, terminal phase halflife (t1/2) and plasma concentration after 30 min (C30min) were determined. 2.2.9.4. In vivo antitumor efficacy Chemically induced breast cancer model was used to measure the antitumor efficacy of the developed formulations and the cancer was induced by following our previous protocol.25, 26 Briefly, DMBA in soya bean oil was administered orally to the rats at 45 mg/kg dose at weekly interval for three consecutive weeks. After 10 weeks of the last dose of DMBA, tumor bearing animals were separated and divided randomly into different treatment groups each containing 5 animals (n=5). A single dose of Taxotere® and DTX loaded PLGA, PEGPLGA and E2-PEG-PLGA NPs (each normalized to 2 mg/kg of free DTX) was administered to the first four groups of animals via intravenous injection. The last group, kept as control, received normal saline in a similar manner. The tumor volume was measured on every alternate day by measuring the tumor width (w) and length (l) with an electronic digital caliper by using the following formula; (V) = l*w2/2 up to 15 days.



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2.2.9.5. Toxicity studies The mice were divided into different treatment groups, each containing six animals (n=6). Taxotere® and DTX loaded PLGA, PEG-PLGA and E2-PEG-PLGA NPs (each normalized to 2 mg/kg of free DTX) were administered through a single dose of intravenous injection via tail vein. After 7 days, the animals were humanely sacrificed and blood was collected via cardiac puncture to analyze the various biochemical parameters. Individual organs (viz. liver, spleen and kidney) were also excised and weighed to determine the organs indices. Serum biochemical parameters like Aspartate transaminase (AST), Alanine transaminase (ALT), Creatinine, Serum Albumin and Blood urea nitrogen (BUN) level in serum were analysed using commercially available kits (Accurex, Biomedical Pvt. Ltd). Oxidative stress parameter Malondialdehyde (MDA) content and) level were estimated in liver tissue homogenate. 2.2.10. Statistical analysis All the data are expressed as mean ± standard deviation (SD) for all in vitro and mean ± standard error of mean (SEM) for all in vivo results. Statistical analysis was performed using Sigma Stat (version 3.5) utilizing one-way ANOVA followed by Tukey-Kramer multiple comparison test. p0.05). The release pattern, observed at pH 7.4 and plasma, was not significantly different (p>0.05) while the percentage of drug released was slightly higher (p PEG-PLGA NPs>free DTX. It is however, worthy to mention that DTX-deprived formulations presented little or practically no toxicity (EC-50>>10), even at concentrations greater than 100 µg/ml. To further validate the ER targeting capability, cytotoxicity was also determined by preexposing the cells to excess (50 µg/ml) of free E2, followed by incubation with E2-PEGPLGA NPs. In presence of E2, the cytotoxicity of the NPs was significantly depreciated and IC-50 value was even higher than that of free drug. Furthermore, the cell cytotoxicity potential of the developed nanoformulations were also noted in recovery experiments which revealed 1.72 fold, 2.07 fold, 6.50 fold and 6.61 fold increase in cell cytotoxicity of E2-PEGPLGA NPs as compared to that of PLGA, PEG-PLGA, free DTX and E2-PEG-PLGA NPs in presence of excess E2, respectively. Among all nanoformulations, the IC50 of the ER-targeted conjugate in MCF-7 cells was significantly lower (P

Enhanced Antitumor Efficacy and Reduced Toxicity of Docetaxel

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