Phosphatidyl Ethanolamine Conjugated

May 15, 2017 - *Phone.: 040-6630-3630. Mobile: 0970-377-0857. ... hydrodynamic diameter of 141.0 ± 0.94 nm with low critical micelles concentrations ...
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D‑α-Tocopheryl

Succinate/Phosphatidyl Ethanolamine Conjugated Amphiphilic Polymer-Based Nanomicellar System for the Efficient Delivery of Curcumin and To Overcome Multiple Drug Resistance in Cancer Omkara Swami Muddineti,† Preeti Kumari,† Balaram Ghosh,† Vladimir P. Torchilin,‡ and Swati Biswas*,† †

Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Shameerpet, Hyderabad 500078, Telangana, India ‡ Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: Nanomedicines have emerged as a promising treatment strategy for cancer. Multiple drug resistance due to overexpression of various drug efflux transporters and upregulation of apoptotic inhibitory pathways in cancer cells are major barriers that limit the success of chemotherapy. Here, we developed a D-α-tocopherol (α-TOS)/lipid-based copolymeric nanomicellar system (VPM) by conjugating phosphatidyl ethanolamine (PE) and α-TOS with poly(ethylene glycol) (PEG) via an amino acid linkage. The synthesized polymers were characterized by Fourier transform IR, gas-phase chromatography, and 1H and 13C NMR spectroscopy. VPM exhibited mean hydrodynamic diameter of 141.0 ± 0.94 nm with low critical micelles concentrations (CMC) of 15 μM compared to plain PEG−PE micelles (PPM) with size of 23.9 ± 0.34 nm and CMC 20 μM. The bigger hydrophobic compartment in VPM resulted in improved loading of a potent chemotherapeutic drug, curcumin (Cur), and increased encapsulation efficiency (EE) (% drug loading 98.3 ± 1.92, and 85.3 ± 3.29; EE 14.8 ± 0.16 and 12.8 ± 0.09 for VPM and PPM, respectively). Curcumin loaded Vitamin E based micelles exhibited higher cytotoxicity compared to Curcumin loaded PEG-PE micelles in tested cancer cell lines. C-VPM demonstrated ∼3.2 and ∼2.7-fold higher ability to reverse multiple drug resistance compared to PPM and verapamil (concentration used 30 μM), respectively. In the in vivo study by using B16F10 implanted C57Bl6/J mice, C-VPM reduced the tumor volume and weight more efficiently than C-PPM by inducing apoptosis as analyzed by TUNEL assay on tumor cryosections. The newly developed polymeric micelles, VPM with improved drug loadability and ability to reverse the drug resistance could successfully be utilized as a nanocarrier system for hydrophobic chemotherapeutic agents for the treatment of drug-resistant solid tumors. KEYWORDS: α-TOS, multidrug resistance, P-glycoprotein, curcumin, cancer, phosphatidyl ethanolamine

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INTRODUCTION Cancer is one of the most dreadful diseases with increasing rate of morbidity and mortality worldwide every year.1,2 According to a report by world Health organization (WHO), ∼14 million new cases with 8.2 million patients died in 2012, which is predicted to rise to 22 million new cancer cases within the next two decades. Even though various cytotoxic chemotherapeutic drugs are discovered over the decades, however, the drugs are toxic to normal cells, which leads to nonspecific adverse side effects. Nonspecific toxicity has been one of the major drawbacks of conventional chemotherapy, where drugs upon administration get accumulated to various organs according to their tissue preference.3,4 Therefore, accumulation to tumor region is extremely low. Another major drawback that limits the success of chemotherapy is the poor pharmacokinetics of the chemotherapeutic agents, including poor aqueous solubility and © XXXX American Chemical Society

absorption. The excipients added to the formulation to improve the solubility of these drugs lead to severe hypersensitivity reactions that reduces patient’s compliance manifold. Another important barrier of conventional chemotherapy is multiple drug resistance developed by the cells eventually, which efflux out the internalized drugs by up-regulated drug efflux transporters, thereby reducing the intracellular drug concentration at subtherapeutic level.5,6 Use of nanocarriers for the delivery of chemotherapeutic agents has been emerging as a powerful strategy to overcome various drawbacks of conventional therapy.7−9 Because of the nanosize, nanocarriers eventually get accumulated to the tumor Received: January 21, 2017 Accepted: March 16, 2017

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DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Synthesis scheme for the preparation of the vitamin E-based amphiphilic polymer mPEG-α-TOS-Lys-DOPE.

α-Tocopheryl succinate (α-TOS), commonly referred to as vitamin E succinate, is a well-known hydrophobic vitamin analogue that has been utilized extensively in preclinical studies either as micellar forms by conjugating it to PEG or as a component of a mixed micellar system to solubilize poorly soluble drugs.15 The lipophilic portion is relatively bulky, which may allow it for enhanced solubilization.16 Apart from the unique solubilizing property, α-TOS has demonstrated anticancer activities against a wide variety of human cancer cells.17−20 α-TOS produced apoptosis in more than 50 types of cancer cell lines, including lung, breast, prostate, cervical, colon, lymphoma, leukemia, and melanoma.21−23 In addition to inducing apoptotic effect in various cell lines, α-TOS caused inhibition of cell proliferation by arresting the cell cycle at G0/ G1 cell cycle block in MDA-MB-435 breast cancer cell line by upregulating the key cell cycle regulatory protein p21.24 Apart from this, the treatment with α-TOS caused downregulation of the nuclear transcription factor, NF-κB, that leads to induction of apoptosis.25 NF-κB acts as an antiapoptotic agent by activating various inhibitors of apoptosis proteins (IAPs). Another major advantage of utilizing vitamin E analogues in nanomedicines is that this could act as an inhibitor of drug efflux transporter P-glycoprotein (P-gp), which can sensitize

microenvironment by enhanced permeability and retention (EPR) effect, thereby reducing the adverse side effects of conventional therapy. Nanocarriers improve pharmacokinetic properties of the hydrophobic drugs by solubilizing them in the core. Even though various nanocarrier systems, including liposomes, polymeric micelles, and inorganic nanoparticles, have been emerged with potential for targeted delivery of chemotherapeutic drugs to the tumor, the use of nanocarrier systems is usually restricted by the biocompatibility of the nanocarrier forming materials.10 Polymeric micelles are considered as widely acceptable nanocarrier system for drug delivery mainly due to the advantages such as favorable biodistribution, long circulation, higher therapeutic effects, and lower side effects of the loaded drugs.11 However, polymeric micelles could disassemble during systemic circulation if the critical micelles concentration (CMC) is not optimum for retaining the self-assembled structure, which could cause premature drug release and low encapsulation efficiency.12 Polymeric micelles composed of amphiphilic polymer poly(ehtylene glycol)-phosphatidyl ethanolamine (PEG−PE) were widely explored as a carrier for insoluble drugs due to their excellent biocompatibility and solubilizing ability.13,14 B

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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for 4 h before addition of PEG-5K-amine dropwise. The reaction mixture was stirred overnight at room temperature. After thorough solvent evaporation, the crude reaction mixture was dialyzed by using cellulose ester membrane (molecular weight cutoff (MWCO) 1000 Da) in 4 L of water for 2 d with intermittent change of water. The dialysate was lyophilized to obtain white fluffy solid (102.4 mg). Synthesis of mPEG-Fmoc-Lys. The Boc-deprotection was performed using 4 N HCl/dioxane mixture. The solid obtained from previous reaction was suspended in equal volume of 4 N HCl and dioxane. The reaction mixture was stirred at room temperature for 4 h. The solvent was evaporated by using rotary evaporator, and the crude reaction mixture was used in the next step without further purification. Synthesis of mPEG-α-TOS-Lys. Into the solution of α-TOS (14.88 mg,18.7 μmol) in chloroform and triethylamine was added EDC (10.75 mg, 56.1 μmol) and NHS (6.45 mg, 56.1 μmol), and the reaction condition for acid-amine coupling as stated above was followed to couple α-TOS with mPEG-Fmoc-Lys. The dialyzed product was subjected to Fmoc-deprotection by stirring with excess piperidine in dimethylformamide (DMF) at room temperature for 4 h. Synthesis of mPEG-α-TOS-Lys-DOPE. DOPE-glutaryl (18.72 mg, 21.27 μmol) in chloroform and 20 μL of triethylamine was stirred in the presence of EDC (10.19 mg, 53.18 μmol) and NHS (6.12 mg, 53.18 μmol) at 0 °C for 4 h. Fmoc-deprotected mPEG-α-TOS-Lys was added dropwise in the activated acid. The reaction mixture was stirred at room temperature overnight. After solvent evaporation, crude reaction mixture was dialyzed in deionized (DI) water against cellulose ester membrane (MWCO 12−14 kDa). The outer phase was replaced with fresh DI water every 6 h for 72 h.34,35 The dialysate was lyophilized to yield the final amphiphilic polymer of 94.9 mg. The lyophilized final product was dissolved in chloroform and kept refrigerated at concentration of 10 mg/mL. Characterization of the Copolymers. Polymers were characterized by using Fourier transform infrared (FTIR), 1H NMR (Bruker AMX 300, Lindlar, Germany), and 13C NMR (Bruker AMX 300, Lindlar, Germany) spectroscopies and gas-phase chromatography (GPC; Waters, Breeze 2, Massachusetts, USA). The polymers were dissolved in CDCl3 at concentration 4 mg/mL (data shown in Supporting Information, Figure S1). In addition, final polymer was analyzed for 1H NMR in D2O for confirmation of its ability to form micelles. Formulation of Micelles (VPM and C-VPM). The micelles were formed by solvent evaporation technique.29,36 For VPM, mPEG-αTOS-Lys-DOPE in chloroform was evaporated to dryness by using rotary evaporator as shown in Figure S6. The thin film composed of the polymer was hydrated using phosphate-buffered saline (PBS), pH 7.4. To prepare C-VPM/C-PPM, a preweighed amount of Cur was dissolved in methanol (1% acetic acid) and was added to mPEG-αTOS-Lys-DOPE/PEG−PE in chloroform. After hydration and vortexing, the drug-loaded micelles were subjected to centrifugation (13 500g, 4 °C), and the supernatant was preserved for further studies. Characterization of Micelles. Size, Surface Charge, and Morphological Analysis. The hydrodynamic radius and surface charge were measured by zetasizer (Nano ZS90, Malvern Instruments Ltd., U.K.). The micelles were diluted, and hydrodynamic radius was obtained at room temperature. All samples were subjected to equilibration for 10 min, and data were analyzed in triplicates. Further, morphological analysis was performed under transmission electron microscopy (TEM, JEM-1200EX, JEOL, Tokyo, Japan). Sample was dropped onto a copper grid coated with a carbon membrane. The grid was allowed to dry before characterization. Determination of Critical Micelles Concentrations (CMC). CMC of VPM and PEG−PE micelles (PPM) were assessed by using pyrene incorporation method as mentioned in previously reported procedure.37,4 Briefly, pyrene solution (10 mg/mL) was prepared in chloroform, and 50 μL of pyrene was transferred to 5 mL glass vials. The solutions were subjected to evaporation, and predetermined concentrations of PPM and VPM were transferred to the tubes containing pyrene. These polymeric solutions were shaken (150 rpm) at 25 °C for overnight. All incubated solutions were filtered using polycarbonate membranes (0.45 μm) to remove insoluble pyrene.

multidrug resistant (MDR) tumors to various anticancer drugs, including curcumin (Cur), doxorubicin, paclitaxel, and vinblastine.26,27 It has been found that PEGylated α-TOS inhibited ATPase activity of P-gp, which is the energy source of P-gp for active transport that leads to decreased binding of the intracellular cytotoxic agents. Dioleoylphosphatidyl ethanolamine (DOPE) has been regarded as a fusogenic or endosomedisrupting lipid.28 The incorporation of DOPE in the nanocarrier would reduce their lysosomal degradation. Cur, a naturally occurring polyphenolic derivative, is widely explored as a potent drug in traditional Asian medicine. Despite potent anticancer activity in preclinical studies, the clinical application of Cur is limited due to its poor aqueous solubility (0.6 mg/mL).29 Cur demonstrates anticancer and antiinflammatory properties by inhibiting NF-κB signaling pathway.30 As a well-known JAK-STAT3 pathway inhibitor, Cur inhibited myeloid-derived suppressor cells in spleen, and tumor tissues in 3LL Lewis lung carcinoma and breast cancer models.31 However, as stated earlier, poor aqueous solubility acts as a barrier to achieve optimum therapeutic efficacy of Cur. An obvious approach to improve the poor biopharmaceutical properties of Cur is to improve its aqueous solubility by using nanocarriers, including polymeric micelles.29,32,33 On the basis of these approaches, it will be helpful to study the merits of combination chemotherapy by using vitamin E analogue and Cur packed in a fusogenic, polymeric micellar cargo to deliver the drugs in target cancer site. To the best of our knowledge, two biocompatible lipophilic moieties, α-TOS and PE, have been conjugated for the first time in an amphiphilic polymer and are utilized as a bigger hydrophobic compartment for improved drug encapsulation and micellar stability with the potential of inducing fusogenicity and MDRinhibitory activity. Co-delivery of this vitamin E analogue and loaded Cur would increase the intracellular concentration of cytotoxic Cur by P-gp inhibition resulting in improved chemotherapeutic response.

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MATERIALS AND METHODS

Materials. Fmoc-Lys(boc)−OH, N-(3-(dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), polyethylene glycol amine phospatidyl ethanolamine (PEG5kPE), anhydrous chloroform, α-tocopheryl succinate, fluorescence-free glycerol-based mounting medium (fluoromount-G), Cur, rhodamine 123, verapamil, TPGS, eosin, hematoxylin, and pyrene were procured from Sigma-Aldrich Chemicals (Bangalore, India). Dialysis membranes (1, 2, and 12−14 kD) were obtained from Spectrum Laboratories, Inc. (California, USA). Methoxy PEG (5K) amine hydrochloride (mPEGamine·HCl) was purchased from Jenkem technologies, (Texas, USA). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (DOPEglutaryl) and rhodamine-PE were purchased from Avanti polar, (Alabaster, USA). FragEL DNA Fragmentation kit was procured from Merck Millipore, (Massachusetts, USA). Trypan blue and 3-(4, 5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Himedia Laboratories (Mumbai, India). Organic solvents and chemicals procured commercially were of analytical grade or higher. Synthesis of Vitamin E-Based Amphiphilic Copolymer (VP). Synthesis of the vitamin E-based copolymer DOPE-PEG-α-TOS (VP) was performed via multistep procedures described below as per the synthetic scheme shown in Figure 1. Synthesis of mPEG-Boc-Fmoc-Lys. Into the solution of FmocLys(boc)-COOH (11.2 mg, 24 μmol) and 20 μL of triethylamine in chloroform was added 1-(ethyl3-(3-(dimethylamino)propyl) carbodiimide (EDC; 11.5 mg, 60 μmol) and N-hydroxysuccinimide (NHS; 6.9 mg, 60 μmol) at room temperature. The reaction mixture was stirred C

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Solubilized pyrene was estimated by using fluorescence microscope (Spectramax, microplate reader, Molecular Devices, California, USA) at wavelengths λex 339 and λem 390 nm. CMC was obtained from the concentration of the polymeric solution, where sharp rise in fluorescence was observed. Entrapment Efficiency (EE) and Drug Loading (DL). EE and DL were determined for C-PPM and C-VPM by diluting the sample in 80% (v/v) ethanol to release Cur from micellar assembly.38 Cur concentration was determined by using HPLC equipped with a UV detector (Shimadzu, Kyoto, Japan) at a wavelength of 420 nm. The supernatant was drained, and the pellet was resuspended with mobile phase consisting of acetonitrile (mixed with 0.1% formic acid) and water (80:20 v/v). The amount of drug released was determined. EE and DL of Cur-loaded micelles were calculated using the following equations.

ratories (Mumbai, India). Other solvents and chemicals were of analytical grade and were purchased from Sigma-Aldrich (Bangalore, India). Cell lines were grown in DMEM supplemented with 10% FBS, 100 IU/mL of penicillin, streptomycin at 37 °C, and 5% CO2. Cellular Uptake of Cur-Loaded Micelles. Fluorescence Microscopy. Both B16F10 and MDA-MB-231 cells (5 × 104 cells/ well) were seeded in 12-well tissue culture plates in complete media; coverslips were placed in all the wells as reported in earlier study.4 Briefly, both cells were treated with free Cur, C-VPM, and C-PPM at Cur concentration of 50 and 100 μg/mL for 1 and 4 h in serum-free media. After completion of the time points, media containing formulations were discarded, and cells adhered to coverslips were stained with Hoechst 33342 (5 μg/mL for 5 min). Then, the cells were washed thoroughly with PBS, treated with 4% w/v paraformaldehyde for 10 min at room temperature for fixation. Coverslips were collected, washed thoroughly with PBS, and mounted on microscope slides using Fluoromount-G (Sigma-Aldrich, Missouri, USA). Finally, coverslips were visualized under the fluorescence microscope (Leica DMi8 inverted fluorescence microscope, Hitachi, Japan) using FITC filter (λex 495 and λem 520 nm). The fluorescence images obtained were analyzed by using Image J software. Flow Cytometry. Quantification of Cur uptake was performed in a time- and concentration-dependent manner using flow cytometry in both B16F10 and MDA-MB-231 cell lines as reported in earlier study.4 Initially, cells (4 × 105 cells/well) were seeded in six-well tissue culture plates and treated with C-VPM and C-PPM for 1 and 4 h at the Cur concentration of 50 and 100 μg/mL (as used in fluorescence microscopy study). After incubation, growth medium was discarded and washed thoroughly with PBS. Then cells were trypsinized, transferred to 15 mL centrifuge tubes, and subjected to centrifugation for 5 min at 1000 rpm at 4 °C. Then the pellets formed were resuspended in PBS, pH 7.4 (200 μL) and analyzed in flow cytometer (Amnis Flowsight, Washington, USA) after excitation with a 488 nm argon laser. For all the samples, fluorescence was measured for 1 × 104 cells, data were generated as a histogram, and geo mean fluorescence was obtained for all the samples. Data were collected, and IDEAS Analysis application (Version 5.0) was used for data analysis. Cytotoxicity of the Cur-Loaded Micelles. Cytotoxicity of free Cur, VPM, PPM, C-VPM, and C-PPM in B16F10, B16F10-R, and MDA-MB-231 was evaluated by the MTT assay as mentioned in earlier rerports.4 Briefly, B16F10, B16F10-R (1 × 104 cells/well), and MDA-MB-231 (5 × 103 cells/well) cells were seeded in sterile 96-well tissue culture plates in 100 μL growth medium and incubated overnight. The following day, both the plates were treated with Curmicelles at different Cur concentrations (0−50 μg/mL) for 6 and 24 h. In case of cells incubated for 6 h, growth media were discarded, and further, incubation was continued for 24 h in fresh complete media. Cells without any treatment were used as control at both the time points. Finally, the incubating media were discarded, and 50 μL of MTT solution (5 mg/mL in PBS) in serum-free media was added. After 4 h, MTT in serum-free media was discarded, and dimethyl sulfoxide (DMSO; 150 μL) was added to all the wells and incubated for 30 min under shaking at 25 °C. Absorbance for all the samples was measured by using UV−visible spectroscopy (Spectramax, microplate reader, Molecular Devices, California, USA) at λmax of 590 nm with a reference λmax of 620 nm. Cell viability was determined by the following equation

EE(%) = (amount of drug in micelles/amount of drug input)× 100

(1)

DL(%) = (amount of drug in micelles /amount of drug and polymer)× 100

(2)

Storage Stability. Stability is an important property for drug-loaded micelles. It can be expected that compared to PPM, the intramicellar hydrophobic interactions will be strengthened by the presence of αTOS in the VPM. To evaluate the micellar stability, different PPM and VPM formulations (P1−P6 and V1−V6, respectively) were evaluated at 4 and 25 °C. In addition, storage stability for liquid formulation and lyophilized powder was evaluated for three months at 4 °C for particle size, zeta potential, and drug content. In Vitro Drug Release. C-VPM and free Cur solutions were placed in a dialysis bag (MWCO 12−14 k Da) with 0.1% w/v Tween-80. The dialysis bags were incubated in PBS (0.1 M) dispersed in different pH (pH 7.4 and 5.5) containing 0.1% (w/v) Tween-80 and were shaken at 90 rpm and 37 °C. At predetermined time points, samples were collected and replaced by fresh medium. The samples were centrifuged at 13 000 rpm for 20 min at 4 °C. Cur release was determined by the HPLC equipped with UV detector (Shimadzu, Kyoto, Japan) following the procedure described in earlier section. Safety of the Micelles: Hemolysis Study. Hemolysis assay was performed to evaluate the safety of the micelles in vivo by using a method formerly elaborated.36,39 Briefly, heparinized rat red blood cells (RBC) were isolated from 5 mL of rat blood by centrifugation (3000 rpm for 30 min at 4 °C). Plasma supernatant was discarded, and RBC was resuspended in saline (2.5% v/v suspension). 100 μL of the resulting RBC suspension was added to 900 μL of VPM at varied concentration range in normal saline prior to shaking incubation for 30 min at 37 °C. Triton X-100 (1% v/v) was used as positive control, and physiological saline was used as negative positive control in the experiment, respectively. All samples were subjected to centrifugation at 8000 rpm for 20 min to separate damaged erythrocytes and their membranes. Hemoglobin (Hb) present in supernatant was analyzed at 500−600 nm using UV−visible spectro-photometer (Spectramax, microplate reader, Molecular Devices, California, USA). The degree of hemolysis was determined on the basis of absorbance at 576 nm and calculated from the following formulation.40

hemolysis(%) = (As − Ao)/(A100 − Ao)x100

cell viability(%) = Abssample /Abscontrol × 100

(3)

where As = absorbance of the sample, Ao = absorbance of unlysed sample treated with saline, and A100 = absorbance of fully lysed sample (Triton X-100) treated with DI water. In Vitro Studies. Murine melanoma (B16F10) and human breast cancer cells (MDA-MB-231) were procured from the National Center for Cell Science (NCCS, Pune, India). Cur-resistant B16F10 (B16F10R) cells were generated by continuously culturing the parental tumor cells in growth media containing 5 μM of Cur.41 Roswell Park Memorial Institute medium (RPMI-1640), Dulbecco’s modified Eagle’s media (DMEM), heat-inactivated fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Himedia Labo-

(4)

where Abssample was the absorbance of the cells treated with either free Cur, VPM, PPM, C-VPM, or C-PPM, while Abscontrol was the averaged absorbance of the cells without any treatment. Data were represented as the mean absorbance ± standard deviation (SD) for three replicates. Effects of Micellar Nanocarrier on P-gp Efflux: Assessment of Accumulation of P-gp Substrate (Rh-123) in Resistant Melanoma Cells. Fluorescence Spectroscopy. B16F10 and B16F10R cells were seeded in 12-well tissue culture plates (60−70%) and washed thoroughly with Hank’s buffer. Then, 5.0 μM of Rh-123 in 1% methanol in the Hank’s buffer was added in all the wells. Further, various treatments like D-α-tocopheryl polyethylene glycol 1000 D

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Characterization of final polymer using 1H NMR spectroscopy in (A) CDCl3 and (B) D2O. Level of Apoptosis in Tumor Cryosections. For apoptosis detection, optimal cutting temperature (OCT)-media preserved tumors isolated from animals treated with either Cur, VPM, C-PPM, or C-VPM were cryosectioned to 4 μm thickness slices by using cryostat (Leica CM 1520 cryotome, Leica biosystems, Germany). Then, the tissue slices were subjected to terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (Merck Millipore, USA) following manufacturer’s protocol. The TUNEL-positive cells emit green fluorescence, which emanated from the nuclei of the apoptotic cells.45 Histopathological Analysis. For this study, 5 μm thick cryosections of tumor tissues embedded in OCT media were stained with Harris’s hematoxylin−eosin (H and E) reagent (Sigma-Aldrich, USA) following manufacturer’s protocol. The tumor tissues were mounted in fluoromount G medium for histological observations under light microscope46 Statistical Analysis. All data were analyzed using the one-way Student’s t test with unequal variances. All numerical in vitro data are expressed as mean ± SD, n = 5. In vivo data are represented as mean ± standard error of mean (SEM), n = 5 in each group. Two-way ANOVA followed by the Bonferroni’s post-hoc test was applied for all paired groups using Graphpad prism (GraphPad Software, Inc.; San Diego, CA, USA). P value less than 0.05 was considered to be significant statistically.

succinate (TPGS, a known vitamin E-based P-gp inhibitor), VPM, and PPM (0.5%w/v) in Hank’s buffer with or without treatments were added to all the wells by following previously mentioned protocol.42 All treatments were incubated for 120 min at 37 °C, 5% CO2, and cells were washed with the Hank’s buffer followed by incubation with or without the treatments for 2 h at 37 °C, and 5% CO2. Cells were washed with sterile PBS, lysed, and made up to 1 mL using 1% w/v DMSO in PBS. Fluorescence intensity of the treatments was analyzed at λex and λem at 485 and 530 nm, respectively, using fluorescence microplate reader (Spectramax, microplate reader, Molecular Devices, California, USA). Fluorescence Microscopy. B16F10 and B16F10-R cells were seeded in 12-well tissue culture plates (60−70%) on coverslips and washed thoroughly with Hank’s buffer. Then, 5.0 μM of Rh-123 in 1% methanol in the Hank’s buffer was added in all the wells. As mentioned earlier various treatments, including TPGS (a known vitamin E-based P-gp inhibitor), VPM, and PPM (0.5%w/v) in Hank’s buffer with or without treatments were added to all the wells.12,42,43 All treatments were incubated for 120 min at 37 °C, 5% CO2, and cells were washed with Hank’s buffer after incubation period. The coverslips were washed thoroughly with sterile PBS and mounted on fresh glass slides using fluoromount G. Mounted slides were observed with Leica DMi8 inverted fluorescence microscope under FITC filter. Determination of Therapeutic Efficacy in Vivo. Female C57BL/6J mice (18 ± 2 g) of 6−8 weeks age were used for in vivo studies. The animals were procured from the National Center for Laboratory Animal Science (NCLAS, Hyderabad, India), which were kept at controlled temperature of 20−22 °C, relative humidity of 50−60%, and maintaining nocturnal and diurnal cycles. All the animals were supplemented with food and tap water ad libitum. Animals were subjected to acclimatization for one week before initiating the study. Experimental procedures were performed following the protocol approved by the Institutional Animal Ethics Committee. Tumor Inhibition Study. The therapeutic efficacy of free Cur, VPM, C-VPM, and C-PPM was assessed by using B16F10 xenografted C57BL/6J mouse model. In vivo studies performed were in accordance with the guidelines by Institutional animal ethics committee of BITS Pilani Hyderabad Campus. Approximately one million freshly cultured cells resuspended in 100 μL of Hanks buffer saline solution was injected subcutaneously in the right flank of the animal.44 Tumor volume was measured every 2 d, which was measured in two perpendicular dimensions taken with vernier calipers by using the formula (L × W2)/2, where L is the length of longer axis, and W is the length of shorter axis. Once the tumors reached ∼50 mm3 (12−14 d postinoculation), mice were randomly split into five different groups and were administered the formulations (∼0.4 mL) intraperitoneally at a dose of 25 mg/kg Cur at every alternate day. All animals were sacrificed when the tumor volume in the control group reached 1500 mm3. The animal weights were monitored throughout the study.

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RESULTS AND DISCUSSION Synthesis and Characterization of Polymers. The design of this amphiphilic polymer for drug delivery application was based on the rationale that, while α-TOS would improve the hydrophobicity of the core to solubilize the poorly watersoluble drugs and sensitize the multiple drug-resistant cancer cells toward treatment, biomolecules, PE, and α-TOS as hydrophobic moieties would impart biocompatibility to the vehicle polymer. Inclusion of PE, a cell membrane-building block as hydrophobic component in amphiphilic polymer, would have superior biocompatibility, biodegradability, and nonimmunogenicity compared to other synthetic block copolymers. In addition, PEG is a widely utilized polymer to impart hydrophilicity to any nanocarrier system. The synthetic route for VPM is illustrated in Figure 1. Amine-protected lysine was coupled to PEG5k-amine by following a standard procedure of acid-amine coupling reaction. The boc-deprotection was performed in the next step by using 4 N HCl/dioxane mixture at room temperature. α-TOS was coupled with the deprotected amine group by using standard coupling cross-linker, EDC, and NHS. Next, Fmoc was E

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Table 1. Characteristics of Drug-Loaded Micelles micelles

sample

polymer/ druga

particle sizeb (mean ± SD)

PDI (mean ± SD)

PPM

P1 P2 P3 P4 P5 P6 V1 V2 V3 V4 V5 V6

10:0 9.5:0.5 9:1 8.5:1.5 8:2 7:3 10:0 9.5:0.5 9:1 8.5:1.5 8:2 7:3

21.6 ± 0.42 22.8 ± 0.36 23.1 ± 0.47 23.9 ± 0.34 24.6 ± 0.68 c 114.2 ± 0.64 127.5 ± 0.87 134.6 ± 1.20 141.0 ± 0.94 144.2 ± 0.62 c

0.214 ± 0.023 0.341 ± 0.028 0.231 ± 0.018 0.415 ± 0.036 0.423 ± 0.042 c 0.214 ± 0.024 0248 ± 0.046 0.344 ± 0.034 0.276 ± 0.064 0.421 ± 0.042 c

VPM

a

Zeta potential (mV) (mean ± SD) −18.3 −21.4 −20.1 −22.8 −21.1 c −28.8 −32.2 −34.4 −36.2 −35.8 c

± ± ± ± ±

1.03 1.24 1.32 0.62 0.74

± ± ± ± ±

0.94 1.26 1.15 1.74 0.92

DL (%) (mean ± SD) 0 4.9 ± 0.59 9.4 ± 0.18 12.8 ± 0.09 13.2 ± 0.32 c 0 4.9 ± 0.72 9.6 ± 0.42 14.8 ± 0.16 15.1 ± 0.28 c

EE (%) (mean ± SD) 0 97.2 94.1 85.3 66.8 c 0 98.4 96.4 98.3 75.6 c

± ± ± ±

1.04 1.45 3.29 2.18

± ± ± ±

0.98 2.14 1.92 2.42

The weight ratio of polymer to drug. bThe size and size distribution of micelles determined by DLS. cCannot be determined.

Figure 3. Characterization of micelles (PPM and VPM). (A) Particle size and zeta potential of VPM by DLS measurement. (B) Transmission electron microscopy of VPM and C-VPM. (C) Pyrene incorporation in VPM and PPM to assess critical micelles concentrations. (D) Stability of Cur-loaded PPM (P2 to P5) and VPM (V2 to V5) at 4 °C and at room temperature. (E) In vitro release studies for free Cur and Cur-loaded PPM and VPM. (F) Hemolysis study for the VPM-forming polymer.

F

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ACS Applied Materials & Interfaces

Particle size and surface charge for PPM and VPM were measured by using DLS. The measured values were shown in Figure 3A and represented in Table 1. Significant increase in the particle size was observed for VPM when compared to PPM. As stated earlier, this could be due to enhanced hydrophobicity due to presence of additional TOS moiety in the micelles core.12,49 Morphological analysis of TEM showed spherical shape of 110 to 130 nm as shown in Figure 3B. Further, self-assembly of the synthesized polymer was confirmed by determination of critical micelles concentration (CMC) using a fluorescent method employing pyrene as the fluorescent probe. CMC for PPM and VPM were measured and found to be 20 and 15 μM, respectively. The sharp rise in fluorescence at CMC confirms the micellar solubilization of pyrene and its corresponding increase in the signal (Figure 3C). This study indicated the ability of the synthesized polymer to form micelles at lower concentration compared to the standard PPM. Aggregation behavior of amphiphiles is influenced by their chemical structures and also on the nature of media. Steric forces between the charged polar head groups and hydrocarbon chains of the amphiphiles are liable for aggregation in aqueous medium. Reduced electrostatic repulsions between the polar head groups of amphiphiles having two alkyl chains reduce vesicle formation.50 As we conjugated long chain PEG(5k) as a polar headgroup, it facilitates the formation of micelles rather than vesicles due to presence of higher steric forces between polar groups. It confirms the formation of micelles instead of vesicles. The chromatography conditions followed the protocol reported by Jose et al.51 Briefly, HPLC (Shimadzu, Kyoto, Japan) analysis was performed using a C8 column (Phenomenex) with mobile phase consisting of acetonitrile/0.1% formic acid (80:20% v/v). Sample (10 μL) was injected at a flow rate of 0.5 mL/min, and Cur was detected at 420 nm wavelength. A calibration curve with acceptable linearity (R2 = 0.9981) was constructed by plotting the peak area versus Cur concentration within the range of 2.5−25 ppm as shown in Figure S5. Notably, most VPM (V1−V4) demonstrated high drug encapsulation efficiencies (EE) of 96.5−98.8% (Table 1). The DL was increased with enhanced drug feeding, and the DL was found to be higher for sample V5 (Table 1). The encapsulation efficiency of prepared VPM (V2) was as high as 97.6%. Particle sizes were less than 145 nm for VPM and 25 nm for PPM. With increase in the amount of Cur in the micelles, the DL, PDI, and particle size increased, while EE and stability reduced. As P6 and V6 were not stable and formed precipitate immediately, EE and DL could not be measured. In regard to drug loading and stability, V4 (drug/polymer = 1.5/8.5) was chosen for further studies. DL and EE were 14.75 ± 0.16% and 98.3 ± 1.92%, respectively. Moreover, the average particle size, PDI, and zeta potential for VPM and PPM were found to be 23.9 ± 0.34, 0.415 ± 0.036, and −22.8 ± 0.62 and 141 ± 0.94, 0.276 ± 0.064, and −36.2 ± 1.74, respectively. As per the particle size distribution spectrum, the C-VPM was monodisperse and had a very narrow particle size distribution. The diameter of the C-VPM found by TEM was matching with the results of hydrodynamic radius by DLS measurements, which represents C-VPM were stable and dispersed in aqueous medium. PEG is a nonionic molecule present in outer layer of micellar system, which shields the surface charge. However, shielding effect of PEG was not sufficient to hide the anionic charge attributed by phosphate monoester group of DOPE as depicted in many research reports.52−55

deprotected by using excess piperidine in DMF at room temperature. Final conjugation was the acid-functionalized lipid (DOPE-glutaryl) with mPEG-α-TOS-Lys to yield mPEG-αTOS-Lys-DOPE. The synthesized polymers were characterized by FTIR, 1H NMR, and 13C NMR spectroscopies (represented in Supporting Information, Figures S1−S3). After PEGylation of lysine in step 1, characteristic signals contributed by PEG were 3.6 ppm in mPEG-boc-Fmoc-Lys (Figure S1a). Other signals from Fmoc and Boc were retained, which indicated that the protection of amine was intact. However, in the next step, upon deprotection of Boc under acidic condition, the characteristic peaks of protons associated with Boc group at 1.38 ppm disappeared indicating successful deprotection (Figure S1b). Next, free amine of mPEG-Fmoc-Lys was coupled to acid group of α-TOS. The signals from α-TOS were represented at 1.29, 2.35, 2.62 ppm (Figure S1c). In the next step, deprotection of Fmoc disappeared the Fmoc proton peaks between 7.2 and 8.5 ppm (Figure S1d). Finally the free amine group present in the mPEG-α-TOS-Lys was coupled to the acid group of DOPE-glutaryl. Among many other peaks, the peak of methyl protons of the long chain of DOPE at 1.26 ppm indicated successful conjugation. In CDCl3, resonance peaks corresponding to the hydrophilic and hydrophobic parts, including PEG, DOPE, and vitamin E of the polymer, were clearly observed at 3.56 ppm (PEG), 1.26 ppm (DOPE), and 1.29, 2.35, and 2.62 ppm (vitamin E) (Figure 2A). Further, the 1H NMR was performed by dissolving the polymer in D2O to assess micelle-forming ability of the polymer. The resonance peaks obtained at 4.78 ppm represent PEG blocks solvated in D2O, that is, micelles shells (Figure 2B). The resonance peaks of DOPE and vitamin E blocks, which constitute the core, were not observed due the lack of solvent within the micelle core.13,47,48 This NMR spectroscopic observation clearly supports rationale against micelle-forming ability of the newly synthesized amphiphilic polymer, mPEG-α-TOS-Lys-DOPE. Further, VPM was characterized using gel permeation chromatography (GPC) as shown in Figure S4. Formulation and Characterization of Micelles. The micelles were formulated by thin-film hydration technique.32 Cur was loaded in VPM and plain PEG−PE micelles, while PPM was loaded by following the scheme represented in Supporting Information, Figure S2. In the mixed solvent of chloroform and methanol mixture, Cur was uniformly distributed in copolymers as amorphous substance, which was hydrated in buffer to obtain Cur-loaded C-VPM and C-PPM. To optimize the process parameters, we studied the effect of drug/polymer ratio on micellar properties. As listed in Table 1, C-VPM and C-PPM ratios from 0/10 to 3/7 were prepared. It was observed that the prepared micelles P6 and V6 were extremely unstable in feed ratio of 3/7, which aggregated in less than 10 min. Hence, particle size and polydispersity index (PDI) of P6 and V6 could not be determined. As expected, the C-VPM was large with bigger hydrophobic compartment compared to plain PPM, varying in size from 128.4 to 171.0 nm depending upon the drug loading content (LC), and had a narrow size distribution (PDI, 0.118 ± 0.134). In comparison, under the same preparation conditions, the C-PPM yielded micelles with much smaller sizes (17.1 to 25.5 nm). The size of VPM was clearly larger than that of PPM for all the drug-topolymer ratios, which was consistent with the result obtained by using dynamic light scattering (DLS). Narrow size distribution indicated stability of the formed micelles. G

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 4. Cellular uptake of Cur loaded in C-PPM and C-VPM. (A) Fluorescent images at 1 h. (B) Fluorescent images at 4 h and (C) flow cytometry data of B16F10 and MDA-MB-231 cells incubated with C-VPM and C-PPM at Cur concentration of 50 and 100 μg/mL for 1 and 4 h. For each panel in (A) and (B), the images from left to right showed the bright field image of cells, cell nuclei stained by using Hoechst 33342 (blue), Cur fluorescence in cells, and the overlay images of both the fluorescence images. For Figure 4C, geo mean fluorescence data are mean ± SD, averaged from three separate experiments. The significance of difference between the means was analyzed by Student’s t test, ***p < 0.001.

Storage stability of micelles (P2−P5 and V2−V5) were evaluated at 4 and 25 °C for 72 h (Figure 3D). P6 and V6 were

eliminated due to the appearance of turbidity within 10 min as explained earlier. Contrary to P4 versus P5, there was no H

DOI: 10.1021/acsami.7b01087 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

micelles.60 In addition, cancer cells, compared with normal cells, depend profoundly on glycolysis, which leads to generation of lactic acid that results in decreased interstitial pH of 6.3−6.6. Further, α-TOS (pKa of 5.64) can exist in solution in two forms, that is, deprotonated charged species and its protonated uncharged counterpart. Out of two, the uncharged counterpart can enter cells by free diffusion. As αTOS is a weak acid, it acts effectively at the acidic pH of tumor environment due to the formation of major proportion of uncharged counterpart.61 In Vitro Cell-Based Assays. For assessing the uptake of Cur by cancer cells B16F10 and MDA-MB-231, cells were treated with C-PPM and C-VPM for the time period of 1 and 4 h. The cell-associated Cur fluorescence was observed by visualizing them under fluorescence microscope and by flow cytometer using FITC channel. Fluorescence images of B16F10 and MDA-MB-231 cells treated with C-PPM and C-VPM for 1 and 4 h are shown in Figure 4A,B, respectively. No significant difference in fluorescence intensity was found when cells treated with free Cur for 1 and 4 h at concentrations of 50 and 100 μg/mL compared to the control group (Figure S8), which was similar to earlier reports. Though, C-VPM and C-PPM could quickly accumulate in B16F10 and MDA-MB-231 cells after 4 h of incubation as indicated by the bright green fluorescence. In addition, intensity of fluorescence was enhanced significantly following a 4 h treatment in both the formulations compared to that of 1 h. Similarly, dosedependent increase in fluorescence intensity was observed for both C-PPM and C-VPM compared to 50 μg/mL in both the time points of 1 and 4 h. These dose- and time-dependent increases in uptake were confirmed quantitatively by the geo mean fluorescence generated using flow cytometer in both B16F10 and MDA-MB-231 cell lines as shown in Figure 4C. The fluorescence intensity of B16F10 cells at 4 h treated with C-PPM and C-VPM is much stronger (Geo means of fluorescence 297.4 ± 5.2, and 294.7 ± 3.7 for PPM, and VPM, respectively) than the treated cells (Geo mean 245.4 ± 5.8, and 248.7 ± 4.2, for PPM and VPM, respectively) after incubation for 1 h (similar trend was observed for MDA-MB231 cells also). There was no significant difference in the cellular uptake of Cur by C-PPM and C-VPM at fixed Curconcentration. This is obvious, as there is no difference in their micellar surface (both have PEG corona) that would cause change in their interaction with cellular surface for association. The flow cytometry analysis supported the results obtained from fluorescence image analysis. In addition, colocalization study was performed by treating B16F10 cells with VPM and Lysotracker (Deep red, 75 nM) at various time pointes (1, 4, and 8 h). The cells were visualized under confocal fluorescence microscopy (shown in Figure S9). Effects of Micelles on P-gp Efflux. Development of multidrug resistance (MDR) is one of the major drawbacks behind the failure of conventional chemotherapy. Inhibition of P-gp efflux pump causes accumulation of drug molecules inside the cells that leads to higher therapeutic activity of the chemotherapeutic drugs in multidrug resistant tumors as shown in Figure S11. For this study, resistant B16F10 cells (B16F10R) were developed for overexpression of P-gp on cellular membranes to study multidrug resistance. Rh-123, a fluorescent molecule, is a well-known P-gp substrate present on the cancer cells. Hence, intracellular accumulation of Rh-123 can be used as a marker for P-gp activity in cells.41 Instead, verapamil and TPGS are known inhibitors of P-gp, which prevents the efflux

significant change in Cur retention for V4 compared to V5 at room temperature and at 4 °C. Higher stability achieved for VPM might be due to the strong hydrophobic compartments constituted of α-TOS and DOPE that caused higher drug entrapment efficiency.56 As discussed earlier, with respect to drug loading and stability, V4 showed optimistic results; therefore, V4 was finalized to use in subsequent studies. Further, storage stability of all micellar formulations (P2 to P5 and V2 to V5) were monitored for three months at room temperature. There was no significant change in percentage retention of drug for both P4 and V4 within this time period. At the end of third month, 94.8% of Cur was retained by V4, whereas 84.5% drug retention was found in P4. Therefore, the freeze-dried form of V4 showed better storage stability than P4 at experimental condition. As shown in Table S1, particle size and zeta potential of freeze-dried VPM were not significantly affected when VPM was rehydrated. Further, C-VPM micelles were found to be stable in PBS and serum containing DMEM medium as shown in Table S2 and Table S3. The polydispersity of the formulations was increased slightly, but no significant change in size was found for the formulations, which supported earlier works.57,58 This means that these formulations protected the loaded drug up to the moment of their uptake (certainly taking place in less than in 72 h). Since Cur is insoluble in buffer solution, 0.1% w/v Tween 80 was added to simulate the sink condition.13 As shown in Figure 3E, C-PPM and C-VPM exhibited a much slower rate of cumulative release compared to that of free Cur. Almost ∼88% of the Cur from free Cur was released within 4 h, while only ∼25% and ∼35% of the loaded Cur was released from C-VPM and C-PPM, respectively, as reported in earlier studies.4 After 48 h, the rates of cumulative drug release in C-micelles were much lower than that of the free Cur. Initial burst release from C-VPM and C-PPM (>20% of the initial loaded amount) followed by sustained release pattern was observed. The hydrophobic interactions between the hydrophobic drug molecules and hydrophobic segments of the micelles caused sustained drug release. Remaining amount of the Cur was entrapped in the strong hydrophobic segments present in the micellar system.13,33 Initial faster release may be due to location of curcumin with in hydrophilic shell or at interface between the micelle core and shell.59 Contrary to the results attained from the release of free Cur, the rate of drug release from CVPM and C-PPM was sustained significantly. Almost similar results were obtained when the release studies were conducted at pH 5.5 as shown in Figure S7, which was due to absence of typical pH-sensitive functional moiety in the VPM-based micellar system. The results provide strong rationale for the use of VPM as a controlled-release drug delivery system for Cur. Hemolysis study assesses biocompatibility of the polymers for in vivo application. The hemolytic curve for VPM at different concentrations (0.5−10 mg/mL) was shown in Figure 3F. The results suggested that VPM exhibited dramatically lower hemolysis (1.10 to 2.81%) at the tested concentration range. The hemolytic agent Triton X-100 showed strong damage to the red blood cells. As VPM showed no obvious hemolytic activity (hemolysis percentage