Article pubs.acs.org/molecularpharmaceutics

Chitosan‑g‑TPGS Nanoparticles for Anticancer Drug Delivery and Overcoming Multidrug Resistance Yuanyuan Guo,†,‡ Min Chu,†,‡ Songwei Tan,† Shuang Zhao,† Hanxiao Liu,† Ben Oketch Otieno,† Xiangliang Yang,§,⊥ Chuanrui Xu,*,† and Zhiping Zhang*,† †

Tongji School of Pharmacy, §National Engineering Research Center for Nanomedicine, ⊥College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430030, P.R. China S Supporting Information *

ABSTRACT: To overcome the P-glycoprotein (P-gp)-induced multidrug resistance (MDR) of cancer cells, a novel copolymer, chitosan-graf t-D-α-tocopheryl polyethylene glycol 1000 (TPGS) (CT) was synthesized for doxorubicin (DOX) delivery by the Pgp inhibiting virtue of TPGS. DOX-loaded CT nanoparticles (NPs) were fabricated by a modified solvent extraction/evaporation method combined with ionic cross-linking to form a uniform particle size of 140−180 nm with ∼40% DOX loading efficiency. These drug-loaded CT NPs demonstrated a pH-responsive release behavior, and DOX was released more quickly under low pH values. Significant cell cytotoxicity was observed on the human hepatocarcinoma cells (HepG2 and BEL-7402) and human breast adenocarcinoma cells (MCF-7). The cell cytotoxicity and apoptosis of drug-resistant cells (MCF-7/DOX and BEL-7402/5-Fu), was greatly enhanced as compared to Adriamycin. The IC50 value showed that DOX-loaded CT NPs could be 1.5−199-fold more effective than Adriamycin. This can be attributed to the P-gp blocking and down-regulation of ATP levels by the CT NPs. The potential of these NPs to act as an oral delivery system was also investigated. Both the pharmacokinetic properties and in vivo antitumor activity of DOX-loaded CT NPs were improved compared with Adriamycin. KEYWORDS: nanoparticles, chitosan, TPGS, doxorubicin, multidrug resistance

1. INTRODUCTION Cancer is one of the leading causes of death and almost one out of every four deaths is from cancer. Current treatment mainly employs a combination of chemotherapy, radiotherapy, and surgical resection. However, the high toxicity of naked drugs, adjuvants used as solubilizers, or the drug leakage from traditional drug delivery systems before reaching the tumor site poses a serious challenge to effective treatment. Furthermore, occurrence of multidrug resistance (MDR) is one of the important reasons for treatment failure. The MDR happens through a variety of mechanisms. Among them, P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) transporter, by acting as an efflux pump for most hydrophobic anticancer drugs causes MDR and thus limits the efficacy in treatment.1 Nanotechnology has provided an innovative and promising strategy for circumventing MDR by encapsulating or conjugating chemotherapeutic drugs to nanocarriers.2 Nanoparticles (NPs) can passively target via the enhanced © 2013 American Chemical Society

permeability and retention (EPR) effect or actively target by conjugating the ligand on the surface of NPs to tumors. Codelivery of different functional therapeutic agents in NPs has also been developed to overcome MDR. For example, coadministration of paclitaxel and curcumin can increase cancer cell cytotoxicity by inhibition of NF-κB activity and down regulation of P-gp expression.3 Coadministration of P-gp inhibitor can also induce the synergistic cell apoptosis in MDR cells.4 To enhance effective drug accumulation in tumors, stimuli-responsive polymeric NPs were also applied in the reversal of MDR through sensitive drug release in tumor tissues or cell microenvironment.5−8 The development of nanoReceived: Revised: Accepted: Published: 59

February 4, 2013 November 13, 2013 November 14, 2013 November 14, 2013 dx.doi.org/10.1021/mp400514t | Mol. Pharmaceutics 2014, 11, 59−70

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Scheme 1. Synthetic Scheme of CT Copolymer

cytotoxicity of these DOX-loaded NPs against human hepatocarcinoma cell lines (HepG2 and BEL-7402), breast adenocarcinoma cell line MCF-7, and drug-resistant cell lines (BEL-7402/5-FU and MCF-7/DOX) were investigated, and the mechanism for overcoming MDR was also studied. In vivo pharmacokinetics and the tumor inhibition effect of the NPs were also evaluated in animal models.

formulations with polymer which can act as an inhibitor of P-gp may be more promising in drug delivery carriers.9 Chitosan (CS) is a natural cationic polymer of 2-amino-2deoxy-β-D-glucan linked through glycosidic bonds. The excellent biocompatibility, biodegradability, and low immunogenicity of CS makes it useful in pharmaceutical applications as a drug delivery carrier for small molecular drugs and macromolecular drugs such as doxorubicin (DOX), DNA, and proteins.10−13 Its cationic property causes it to approach cell membranes more easily and allows ionic cross-linkage with multivalent anions. Moreover, its mucoadhesive property also extends its retention onto targeted substrates.14,15 However, CS NPs show limitation in the stability and entrapment efficiency of anticancer reagents.16 Increased investigations have to date been reported on CS hydrophilic/hydrophobic modifications in a bid to overcome its drawbacks in drug delivery systems.17−19 D-α-Tocopheryl polyethylene glycol succinate (TPGS) is a water-soluble derivative of natural vitamin E. It has an amphiphilic structure with a lipophilic alkyl tail and a hydrophilic polar head of polyethylene glycol 1000. It has been approved as a safe pharmaceutical adjuvant in drug formulation by the FDA. TPGS has been widely used in NPs formulations as emulsifier, additive, stabilizer, and permeation enhancer.20−22 TPGS-related copolymers of poly(lactide)TPGS (PLA-TPGS), and poly(lactide-co-glycolide)-TPGS have been developed. The copolymers exhibited significant drug circulation time, up to 360 h, after intravenous administration and enhanced therapeutic efficiency on the breast cancer MCF-7 xenograft model.23,24 It is worth noting that TPGS has been used as an inhibitor of P-gp in overcoming MDR. The supposed mechanism is modulating P-gp efflux transport via P-gp ATPase inhibition.25−27 Wang et al. applied the TPGS property of P-gp inhibition and synthesized a starshaped copolymer of ditocopherol PEG 2000 succinate for DOX delivery. The DOX-loaded micelles can have a combined effect as a P-gp inhibitor, anticancer efficacy enhancer, and nanocarrier.9 Li et al also found that PLA-TPGS copolymer micelles can enhance the cytotoxicity of DOX in drug-resistant MCF-7 cancer cells.28 This investigation was aimed at developing CS-g-TPGS (CT) NPs drug delivery system, particularly designed for overcoming MDR. The DOX-loaded CT NPs were fabricated via a modified solvent extraction/evaporation method combined with an ionic cross-linking method. The cell

2. MATERIALS AND METHODS 2.1. Materials and Cells. Chitosan (low molecular weight, 75−85% deacetylated), TPGS, succinic anhydride (SA), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), propidium iodide (PI), RNase A and trypsin-EDTA were purchased from Sigma Aldrich (St. Louis, MO, USA). 4-Dimethylamino pyridine (DMAP) was purchased from Aladdin, China. Doxorubicin hydrochloride was obtained from Beijing Huafeng United Technology Co., China. Adriamycin (doxorubicin hydrochloride for injection) was procured from Actavis (S.p.A, Italy). RPMI-1640 medium was purchased from Gibco BRL (Gaithersberg, MD, U.S.A.). Penicillin−streptomycin, fetal bovine serum (FBS) and trypsin without EDTA were purchased from Hyclone, U.S.A. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and Hoechst 33342 were purchased from Biosharp, South-Korea. The solvents used were of analytical grade and included dichloromethane (DCM, anhydrous), dimethyl sulfoxide (DMSO anhydrous), methanol, diethyl ether, and chloroform from Sinopharm, China, and distilled water from ULUP system, China. Annexin V-FITC/PI double staining assay kit was supplied by KeyGEN, China. The human hepatocarcinoma cell lines (HepG2 and BEL7402), 5-fluorouracil(5-FU)-selected drug-resistant BEL-7402/ 5-FU cell line, the human breast adenocarcinoma cell line MCF-7 and doxorubicin-resistant cell line MCF-7/DOX were donated by Dr. Yaping Li (Shanghai Institute of Materia Medica, Chinese Academy of Sciences). The immortalized mouse brain cerebral endothelial cell line (bEnd.3) were purchased from the American Type Culture Collection (ATCC). 2.2. Synthesis and Characterization of Chitosan-gTPGS copolymer. Chitosan-g-TPGS (CT) copolymer was synthesized by a two-step amidation reaction (Scheme 1).29 TPGS was first functionalized with a carboxylic acid group by 60

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pellet was washed twice to remove unloaded DOX and then resuspended in water before lyophilization to get NPs powder. 2.4. Characterization of the DOX-Loaded NPs. 2.4.1. Particle Size, Surface Charge and Morphology. The size and ζ potential of DOX-loaded NPs was measured by dynamic light scattering (Zeta Plus, Brookhaven Instruments, U.S.A.) at 25 °C. The result was based on the average from six runs. The morphology of the DOX-loaded NPs was observed by field emission scanning electron microscopy (FESEM, SIRION 200, Netherlands) at an accelerating voltage of 15 kV. The suspension of NPs was added onto metal studs, dried at room temperature, and then coated with a platinum layer by the Auto Fine Platinum Coater for 120 s. 2.4.2. Drug Loading Efficiency. DOX entrapped in the CT or CS NPs was determined by fluorospectrophotometry (F4600, Shimadzu, Japan) with a slit width of 5 nm and excitation and emission wavelengths at 479 and 590 nm, respectively. The drug loading efficiency (DLE) was calculated on the basis of the following eq 1:

esterification with succinic anhydride (SA). Briefly, 3.00 g of predried TPGS (1.95 mmol), 0.39 g of SA (3.90 mmol) and 0.24 g of DMAP (1.95 mmol) were dissolved in 30 mL of anhydrous DCM and then reacted for 24 h at room temperature (RT) under N2 atmosphere. The product was precipitated in cold diethyl ether followed by dialyzing (Spectra/Por, MWCO 1000, U.S.A.) against 1:1 (v/v) water/ ethanol for 72 h to remove unreacted SA. The second step was the formation of amide bond between the primary amine groups of CS and carboxylic acid functions of activated TPGS-SA was activated by EDC/NHS (TPGS-SA/ NHS/EDC = 1/1.2/1.2 in stoichiometric molar ratio) in anhydrous DCM for 24 h at RT. After evaporation of the solvent, the product was added into CS acetic acid solution (1% v/v, pH = 4). Twenty-four hours later, the reaction was terminated by dialyzing against distilled water (Spectra/Por, MWCO 10,000). After freeze-drying and extracting in a Soxhlet extractor with methanol, the final product was obtained. CT copolymers with different graft degrees (GD, defined as the ratio of molar number of TPGS side chain to monosaccharide units of CS) were prepared with the feeding molar ratio of TPGS to monosaccharide units of CS of 1:5 and 1:10 (Table1).

DLE (%) = weight of incorporated DOX in nanoparticles /the feed amount of DOX in fabricating nanoparticles

Table 1. Properties of Chitosan-TPGS Copolymers polymer

feeding ratio

Mwb

PDI

CS CT3 CT9

− 1:5 1:10

12467 15466 22026

1.335 1.493 1.924

a

b

× 100%

theoretical GD (%)

GD (%)

− 10 20

− 3.58 9.04

a

GD (%)

2.4.3. In vitro Drug Release. To simulate the tumor microenvironment and endosome environment, in vitro drug release experiments were carried out at pHs 7.4, 6.8, and 5.5 buffer, respectively. The DOX-loaded NPs were dispersed into 1 mL of phosphate buffered saline (PBS, pH 7.4, 6.8) or sodium acetate buffer (pH 5.5) and then transferred into an inner tube (Spectra/Por Float-A-Lyzer G2, U.S.A.) with the outer tube filled with 5 mL of similar release media. The device was placed in an incubator shaker (THZ-C, Huamei Biochemistry Instrument Factory, China) at 120 rpm at 37 °C. At predetermined intervals, the whole media was removed and replaced with equal volumes of fresh media to maintain sink conditions. The amount of released DOX was assayed by fluorometry and calculated according to the relative DOX standard curve in different release media. The in vitro drug release assay was performed thrice. 2.5. In vitro Cellular Uptake Studies. A confocal microscope was used to compare the cellular uptake and intracellular distribution of nanoparticles incubated with MCF7 and MCF-7/DOX cells. The cells were seeded in 24-well plate which kept coverslips in each well. After the cells reached 70−80% confluence, they were incubated with Adriamycin (or) DOX-loaded NPs at 4.5 μg/mL concentration for 2 h at 37 °C. The sample wells were carefully washed twice with 500 μL of cold PBS to remove the excess NPs not taken up by the cells. After fixing with 100 μL of 4% paraformaldehyde for 15 min, the cells were washed twice again with cold PBS. The cell nuclei were then stained with Hoechst 33342 (5 μg/mL in PBS) for 8 min and rinsed with PBS to remove the free dye. The cells were viewed and imaged under a confocal laser scanning microscope (CLSM, Leica TCSNT1, Germany). 2.6. In vitro Cytotoxicity. In vitro cytotoxicity of Adriamycin and DOX-loaded NPs was investigated on HepG2, BEL-7402, MCF-7, BEL-7402/5-Fu, and MCF-7/ DOX cells. The cytotoxicity of the blank nanoparticles was determined using bEnd.3 cells. All the cells were grown in RPMI 1640 medium in a humidified atmosphere incubator with

− 2.68 8.54

Calculated by 1H NMR results via the following equation GD (%) =

(1)

b

I0.86/12 × 100% I4.8

where I0.86 and I4.8 represent the integrated peak area at 0.86 ppm and 4.8 ppm, respectively. bCalculated by GPC results.

TPGS-SA and CT copolymer were characterized by 1H NMR (Bruker AM-400 spectrometer, Switzerland). TPGS-SA and TPGS were dissolved in CDCl3, while CT and CS were dissolved in D2O:CF3COOD (5:1, v/v). Fourier Transform Infrared Spectroscopy (FTIR) was also carried out to confirm the structure of the products using a Bruker VERTEX 70 spectrophotometer (Germany). The molecular weight and molecular weight distribution of CS and final products were determined by gel permeation chromatography (GPC) on a Waters GPC system. 2.3. Preparation of DOX-Loaded Nanoparticles. The DOX-loaded NPs of the CS and CTs were prepared by a modified solvent extraction/evaporation method combined with an ionic cross-linking method;30 30 mg of CS or CT was dissolved in 0.2% (v/v) acetic acid solution and the pH adjusted to 6.0 with sodium hydroxide; 1.5 mg of DOX was dissolved in 1.5 mL of chloroform in the presence of TEA (molar ratio of DOX/TEA = 1:2). This was mixed with the copolymer solution to form a primary emulsion using a probe sonicator. The emulsion was stirred overnight to evaporate chloroform, and then 2 mL of 0.5% sodium tripolyphosphate solution was added as a cross-linking reagent. The NPs dispersion were first centrifuged at 3000 rpm for 10 min to remove large particles and aggregations and then recovered by centrifugation at 11,500 rpm for 20 min at 4 °C. The resulting 61

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fluorescence intensity was measured by a flow cytometer (Becton Dickinson, San Jose, CA). 2.9. Intracellular ATP Level Assay. MCF-7/DOX cells were seeded into 12-well plates (10,000 cells per well). After the cells reached 90% confluence, they were treated with Adriamycin (or) DOX-loaded NPs at 4.5 μg/mL DOX concentration and blank CS NPs (or) blank CT NPs at the same particle concentration for 24 h. Intracellular ATP levels were determined by the luciferrin−luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, China) as instructed by the manufacturer. The ATP level assay was performed in order to confirm the potential apoptosis effect of the formulations. 2.10. In Vivo Studies. Sprague−Dawley rats and Kunming mice were purchased from the Animal Center of Huazhong University of Science and Technology Wuhan, China (Certificate No. SCXK 2010-0009). The animals were maintained at 25 ± 1 °C and 60% ± 10% humidity under a 12 h light−dark cycle during the experiments. 2.10.1. Pharmacokinetic Study. Female Sprague−Dawley rats (200−220 g) were fasted overnight and randomly distributed into four groups each containing four animals. Adriamycin, DOX-loaded CS NPs, CT3 NPs, and CT9 NPs, at an equivalent drug dose of 10 mg/kg body weight, were administered to the rats by oral gavage, respectively. Blood samples (about 0.5 mL) were collected from the retro-orbital plexus at 0.5, 1, 2, 4, 12, 24, 48, 72 h in microcentrifuge tubes containing 20 μL of 1000 U heparin/mL of blood. These were centrifuged at 3500 rpm for 10 min to separate the plasma. Methanol (0.1 mL) was added to the plasma (0.1 mL), and the resulting mixture was vortexed for 3 min. Trichloromethane (1 mL) was added, and the samples were further vortexed and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and dried at 37 °C using an N-EVAP MTN-2800D (AutoScience, Tianjin, China). The residue was diluted with acetonitrile to prepare samples for injection. The concentration of DOX in plasma was determined by high-performance liquid chromatography (HPLC) using a reverse phase column (Restek C18 5 μm, 150 mm × 4.6 mm). KH2PO4 buffer (1/ 15 mM, pH 4.21, adjusted with H3PO4) and acetonitrile (75:25 v/v) were used as the mobile phase with a flow rate of 1.0 mL/ min. The injection volume was 20 μL, and the retention time of DOX was 5.6 min. The fluorimetric detector was operated at 470 nm (excitation), and 585 nm (emission) was used. The calibration curve was linear between the concentration ranges of 25−5000 ng/mL in plasma (R2 = 0.9991). 2.10.2. Tumor Inhibition Assay. Tumor inhibition activity against a solid tumor model was evaluated using female Kunming mice (6 week, 18−22 g). H22 cells (1 × 107) suspended in 0.2 mL physiological saline were injected subcutaneously (s.c.) at the right armpit of each mouse. After 48 h of transplantation, all the tumor-bearing mice were divided randomly into saline, Adriamycin, DOX-loaded CS NPs, DOXloaded CT3 NPs, DOX-loaded CT9 NPs, respectively (6 mice per group). All the formulations were given at an equivalent drug dose of 5 mg/kg body weight by oral gavage (0.1 mL/10 g body weight) for 5 days. Treatment started when the tumor volume of the mice reached 100−150 mm3 on average, and this was designated as day 1. The tumor sizes were measured (length and width) using a caliper for 10 days and then were excised and observed by HE staining and electron microscopy. The tumor volume was

5% CO2 at 37 °C. MCF-7, HepG2, and BEL-7402 were supplemented with 10% fetal bovine serum, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. BEL-7402/5-FU cells were cultured in complete medium supplemented with 20 μg/mL 5-FU. MCF-7/DOX cells were also cultured in complete medium supplemented with 1 μg/mL DOX. The cells (5 × 103 cells/well) were seeded in 96-well plates and supplemented with 100 μL of culture medium to attach overnight for establishment of a monolayer at 37 °C in a humidified environment of 5% CO2. The medium was replaced by RPMI 1640 medium containing serial dilutions of Adriamycin and DOX-loaded NPs with similar DOX concentrations of 20, 10, 2, 0.2, 0.02, 0.002 μg/mL (n = 8), and the cell viability was determined using MTT assay. At designed time intervals (24, 48, and 72 h), the medium was removed, and the wells were washed twice with PBS. The cells were then incubated with 100 μL of medium and 10 μL of MTT (5 mg/mL in PBS) for 4 h at 37 °C, and each precipitant was dissolved in 150 μL of DMSO using an automated shaker. The absorbance of each well was read at 570 nm by a microplate reader (Multiskan MK3, Thermo, U.S.A.). Relative cell viability was calculated as the percentage in relation to untreated control cells. IC50 (concentration resulting in 50% inhibition of cell growth) value for drugs was determined by SPSS software (version 19.0). The experiment was repeated thrice. 2.7. Apoptosis Analysis. 2.7.1. Hoechst 33342 Staining. Apoptotic cells were measured by Hoechst 33342 staining. MCF-7/DOX cells were plated onto the wells of a 24-well culture plate for 24 h. DOX-loaded NPs or Adriamycin (final concentration of 4.5 μg/mL) was added and further incubated for 24 h. Hoechst 33342 solution (10 μg/mL) was then added to the wells, the plates were kept in the dark for 8 min at 37 °C, and then the wells were triple washed with PBS. The cells were finally observed by a fluorescence microscope (IX71, Olympus, Tokyo, Japan). 2.7.2. Flow Cytometry Analysis-Annexin V-FITC/PI Double Staining. The quantitative apoptosis of DOX-loaded NPs was then studied by flow cytometry analysis. Typically, MCF-7 and MCF-7/DOX cells were seeded into 6-well plates at a density of 5 × 103 cells/well and cultured at 37 °C for 24 h. Prior to the experiment, the cells were washed twice with PBS and then incubated in RPMI 1640 medium containing Adriamycin or DOX-loaded NPs at 4.5 μg/mL DOX concentration. Untreated cells were used as control. At the end of the incubation period, the cells were trypsinized, collected, and resuspended in 500 μL of binding buffer. Thereafter, 5 μL of annexin V-FITC and 5 μL of PI were added and mixed for 30 min in the dark. The stained cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA). 2.8. Inhibition of P-gp Efflux Test. To evaluate the potential P-gp inhibitory effects by CT copolymer, P-gpmediated efflux inhibitory trials were carried out on P-gp overexpressing MCF-7/DOX cells, with blank cells as control. Verapamil was used as a standard P-gp inhibitor. MCF-7/DOX cells were seeded in 6-well plates (1 × 105 cells/well) and incubated overnight. The medium was then replaced with fresh medium containing blank CS NPs or blank CT NPs at the same particle concentration (45 μg/mL) and 20 μM Verapamil for 24 h. The cells were washed and treated with 5 μg/mL rhodamine 123 (Rh123), then incubated for an additional 30 min. After that, the cells were harvested and washed twice with PBS and then resuspended in 500 μL of PBS. The Rh123 62

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calculated as 0.5 × length × width2. The inhibition ratio against the growth of tumor (%) was determined by the following eq 2: the inhibitory rate (%) =

TWcontrol − TWtest × 100% TWcontrol

(2)

TWcontrol = the average weight of the model control group TWtest = the average weight of treated groups 2.11. Statistical Analysis. All experiments were repeated at least three times. Data were expressed as mean ± standard deviation in tables and figures. Statistical analysis was performed using Student’s t test. The differences were considered statistically significant with p < 0.05. Pharmacokinetic calculations were performed on each individual set of data using the drug and statistics (DAS) software (version 2.1.1, Mathematical Pharmacology Professional Committee, Shanghai, China).

3. RESULTS AND DISCUSSION 3.1. Characterization of Synthesized Copolymer. The structure of TPGS-SA and CT was confirmed by 1H NMR (Figure 1) and FTIR spectra (Figure 2). The newly appearing

Figure 2. In vitro drug release behaviors of DOX-loaded NPs under pH 7.4 (solid), 6.8 (dash) and 5.5 (dot) solutions.

NMR results was 9.04% and 3.58% (denoted as CT9 and CT3), respectively (1). The FTIR spectra of TPGS, TPGS-SA, CT, and CS are shown in Supporting Information. Compared with TPGS, the stronger absorption of νc=o in TPGS-SA at 1736 cm−1 proved the occurrence of successful esterification. In the cases of CT copolymer, new absorption bands at 1736 and 1100 cm−1 were observed, which can be assigned to the CO and C−O−C of TPGS, respectively. The peaks at 1634 and 1541 cm−1, which corresponded to the amide I (νc=o) and amide II (δN−H), indicated that the linkage between CS and TPGS was of an amide group. GPC was performed to further investigate the TPGS content in CT copolymer. The GD of the CT9 and CT3 copolymers calculated by GPC results were 8.54% and 2.68%, respectively, which were close to the 1H NMR results. This also confirmed the successful grafting of TPGS onto CS. 3.2. Characterization of DOX-Loaded NPs. 3.2.1. Particle Size, Surface Charge and Morphology. DOX was first

Figure 1. 1H NMR of TPGS, TPGS-SA, CS and CTs copolymers.

signals at 2.65−2.72 ppm were assigned to the −CH2CH2− part of succinyl group of TPGS-SA, which verified the successful esterification of TPGS as compared to TPGS spectrum.31 The synthesized CT copolymer exhibited a combination of spectra at 0.86 ppm for the methyl protons of the long-chain alkyl group of TPGS and 4.8 ppm for the signal of the anomeric carbon proton of CS (C1). The GD of the CT copolymers with different feeding ratio calculated by 1H 63

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DOX-loaded NPs was observed by SEM, and they had a spherical shape as shown in Figure 2a. The particles were slightly aggregated due to the evaporation of moisture during the preparation process of the sample. There is a relatively good correlation on particle size observed from the SEM image and measurement from particle size analysis. 3.2.2. DOX Encapsulation and in Vitro Release. Compared with CS NPs (DLE = 35.0 ± 2.2%), CT3 NPs exhibited an improved DLE of 43.5 ± 5.0%, while for CT9 NPs, the DLE was slightly decreased. This may be attributed to the amphiphilic property of TPGS. TPGS has exhibited an enhanced drug entrapment efficiency in fabricating polymeric NPs.20,33 As a result, the DLE of CT3 NPs increased compared with that of CS NPs. However, when more TPGS got involved in the matrix, CT NPs became more hydrophilic. This may reduce the entrapment capability on DOX as reported in previous investigations.23 The release profiles of DOX from CT NPs in different pH conditions, pH 7.4 (corresponding to the environment of blood), pH 6.8 (the pH at tumor tissue), and pH 5.5 (simulating the pH in mature endosomes of tumor cells), are depicted in Figure 2b. All CS, CT3, and CT9 NPs had a pHresponsive release profile. CT9 NPs had the accumulated amount of DOX released over 7 days being approximately 5.71 ± 0.40%, 19.3 ± 1.4%, and 23.9 ± 0.8% at pH 7.4, pH 6.8, and pH 5.5, respectively. The pH-responsive release behavior may be attributed to the protonation of the amino groups on CS and DOX at lower pH value, and thus the enhanced electrostatic repulsion effect made the diffusion of DOX from NPs easier. Although the CS and CT NPs exhibited a low release rate of entrapped drug, the intracellular drug release amounts of DOXloaded NPs were high enough to efficiently kill cancer cells by

deprotonated by adding an excessive amount of triethylamine to make it hydrophobic before fabricating NPs. DOX was then encapsulated in copolymer NPs by the modified solvent extraction/evaporation method combined with ionic crosslinking, which was applied to increase the particles stability and prevent drug leakage from the core of NPs. The particle size and ζ potential of DOX-loaded NPs are detailed in Table 2. The fabricated DOX-loaded NPs exhibited Table 2. Particle Size and ζ Potential of DOX-Loaded Nanoparticles sample

particle size (nm)

PDI

ζ potential (mv)

DLE (%)

CS NPs CT3 NPs CT9 NPs

121.8 ± 4.5 144.6 ± 12.8 183.1 ± 21.1

0.139 ± 0.025 0.120 ± 0.010 0.193 ± 0.009

29.6 ± 3.2 22.5 ± 0.7 19.9 ± 0.9

35.0 ± 2.2 43.5 ± 5.0 31.2 ± 0.9

particle sizes less than 200 nm with narrow distribution (PDI < 0.2). As the introduced quantity of TPGS and the GD increased, the NPs exhibited increased particle size and reduced surface charge. The particle size of CS NPs was increased from 121.8 ± 4.5 nm to 183.1 ± 21.1 nm for CT9 NPs, and the ζ potential was reduced from 29.6 ± 3.2 mv to 19.9 ± 0.9 mv. The CT NPs were stable, and the particle sizes remained unchanged within 72 h (data not shown). It has been previously reported that the PEG chain of TPGS could interact with CS to form a CS/TPGS semi-interpenetrating network through intermolecular hydrogen bonding between the electropositive amino hydrogen of CS and electronegative oxygen atom of polyethers.32 Therefore, the presence of TPGS could enlarge the size and decrease the ζ potential due to a reduction in the amino groups substituted by TPGS. The morphology of

Figure 3. Confocal laser scanning microscope (CLSM) images of Adriamycin (a), DOX-loaded CS NPs (b), DOX-loaded CT3 NPs (c) and DOXloaded CT9 NPs (d) in MCF-7 cells and MCF-7/DOX cells after 2 h incubation. 64

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Figure 4. In vitro cytotoxicity of DOX-loaded NPs and Adriamycin against bEnd.3 (a), HepG2 (b), Bel-7402 (c), Bel-7402/5-Fu (d), MCF-7 (e) and MCF-7/DOX (f) cancer cells after treatment for 24, 48, and 72 h (*NPs vs Adriamycin p < 0.05; ζ CT3 and CT9 NPs vs CS NPs p < 0.05; δ CT9 NPs vs CT3 NPs p < 0.05).

h of exposure. For CT NPs, the intracellular fluorescence was exhibited in the perinuclear region of MCF-7 cells, which was in a manner consistent with the results of previous investigations.34 Compared with MCF-7 cells, free DOX accumulated throughout the cytoplasm of MCF-7/DOX cells as observed after 2 h of exposure. This may be the reason for the MCF-7/ DOX cells reduced sensitivity to Adriamycin even after 72 h. DOX-loaded CT9 NPs showed significant red fluorescence throughout the cytoplasm, which is closely located around the nuclei (blue). It also exhibited enhanced cell uptake as compared with CS NPs. This observation indicated that TPGS could effectively enhance drug accumulation in cancer cells, which may be attributed to the TPGS property as an inhibitor of P-gp. It demonstrated that CT NPs could be

the specific synergistic effect between TPGS and DOX as shown in the following cell cytotoxicity experiment. Furthermore, the different release properties of DOX-loaded NPs at different pH were beneficial in treating tumors. Reduced amounts of DOX got released into the blood (pH 7.4) while a majority of the active drug was released after reaching the tumor tissue and tumor cells as a result of a pH decrease especially after endocytosis. The side effects of the drug could thus be reduced, and the therapeutic efficacy could also be greatly enhanced, in addition to overcoming MDR. 3.3. In vitro Cellular Uptake Studies. The cellular uptake of DOX and NPs was qualitatively analyzed on MCF-7 and MCF-7/DOX cells by CLSM, as shown in Figure 3. Free DOX was mainly located in the nucleus of MCF-7 cells while DOXloaded CS NPs accumulated throughout the cytoplasm after 2 65

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Figure 5. Nucleus apoptosis assay of MCF-7/DOX cells treated with blank medium (a), Adriamycin (b), DOX-loaded CS NPs (c), DOX-loaded CT3 NPs (d), and DOX-loaded CT9 NPs (e) for 24 h.

in MCF-7/DOX cells. However, NPs exhibited the overcoming of MDR as manifested by the decreased IC50 values in Figure 4f. The IC50 values of CS NPs, CT3 NPs, and CT9 NPs were 21.7 ± 2.70 μg/mL, 7.48 ± 1.20 μg/mL, and 2.51 ± 0.65 μg/ mL, respectively, after treatment for 48 h. These results suggested that DOX-loaded CT NPs maintained the pharmacological activity of DOX and efficiently delivered DOX to drug-resistant cells. This may be a result of the pHresponsive release behavior. Furthermore, the cell cytotoxicity of CT NPs was greatly increased in comparison with that of CS NPs. This could be attributed to the synergistic effect of pHsensitive release and P-gp inhibiting activity of TPGS, which maintained a high intracellular drug concentration. As for the BEL-7402/5-FU cells, the IC50 values of Adriamycin were increased 4.25-fold in comparison with those of BEL-7402 cells. They also showed a drug-resistant property against DOX. For CS NPs, CT3 NPs, and CT9 NPs, the increased values were 3.24-fold, 1.46-fold, and 2.86-fold, respectively, after treatment for 72 h. These NPs overcame the MDR of BEL-7402/5-FU cells to a certain extent with the help of TPGS. In summary, DOX-loaded CT NPs maintain the pharmacological activity of DOX and efficiently deliver it to cancer cells. DOX-loaded CT NPs overcame MDR of drug resistant cancer cells (MCF-7/DOX and BEL-7402/5-FU) by the employment of P-gp inhibition effects of TPGS, which have great potential to kill drug resistant cancer cells. 3.5. Cell Apoptosis Analysis. The nuclei of MCF-7/DOX cells treated with Adriamycin and DOX-loaded NPs for 24 h were stained with Hoechst 33342 and their morphologies observed by fluorescence microscopy. The observations were consistent with the in vitro cytotoxicity results (Figure 5). The nuclei, when treated with the control and Adriamycin showed a good integrity. In comparison, some nuclei shrank, and apoptosis bodies began to appear in the groups of DOXloaded CS NPs and CT NPs, which was much more obvious after treatment with DOX-loaded CT9 NPs.

potentially used in cancer therapy as carriers for hydrophobic anticancer drugs against drug-resistant tumors. 3.4. In vitro Cytotoxicity. The in vitro cytotoxicity of the blank and DOX-loaded NPs was determined by MTT assay using several different cell lines. The cytotoxicity of blank NPs was first tested by using immortalized mouse brain cerebral endothelial cells (bEnd.3) treated with different NP concentrations (200, 100, 20, 2, 0.2, and 0.02 μg/mL). As can be seen in Figure 4a, the cell growth curves of nanoparticle-treated groups were similar to that of the control. This result suggested that the NPs at the tested concentrations exhibited nontoxicity on normal cells. In order to evaluate the cytotoxicity of DOX-loaded NPs, as well as the role of TPGS in overcoming MDR, the cytotoxicity of DOX-loaded NPs against drug-sensitive cells, HepG2 (Figure 4b), BEL-7402 (Figure 4c), MCF-7 (Figure 4e), and drug-resistant cells, BEL-7402/5-FU (Figure 4d) and MCF-7/ DOX cells (Figure 4f), was assessed and compared with Adriamycin. The cell viability of NP-treated groups was similar to that of Adriamycin in HepG2 cell lines. The DOX-loaded NPs did not exhibit obvious superiority over Adriamycin even with the help of the grafted TPGS and the NP formulation. DOX-loaded CT9 NPs exhibited better inhibition effects on BEL-7402 cells than Adriamycin (p < 0.01). The cell cytotoxicity was enhanced with an increase of TPGS GD in CT copolymer. CT9 NPs exhibited enhanced cell cytotoxicity on BEL-7402 cells as shown by the decreased IC50 values after treatment for 24 and 48 h compared to other formulations. As for MCF-7 cells, DOX-loaded NPs exhibited significantly lower cell cytotoxicity compared with that of Adriamycin. Irrespective of the high IC50 of CS NPs and CT NPs, CT NPs still exhibited higher cell cytotoxicity compared to CS NPs, which is a tendency similar to that of the above two cell lines. The cells exhibited significant drug resistance to Adriamycin with the drug incubation concentration of 0.002−20 μg/mL and the IC50 values being too high after treatment for 24−72 h 66

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In order to verify the MDR cell apoptosis rate induced by the DOX-loaded NPs, annexin V-PI staining assay was performed to carry out a quantitative analysis of the early apoptosis (Figure 6). The lower right quadrant (annexin V-positive and

Figure 7. P-gp inhibition assay of Rh123 intracellular accumulation in MCF-7/DOX cells that were untreated (control) or treated with verapamil served as positive control and blank CS and CTs NPs.

level. As the most important energy origin, ATP molecules play a crucial role on physiological processes of cells especially the drug efflux pump of P-gp which overexpresses on the membranes of drug resistant cells. Under the conditions of apoptosis, necrosis, or toxicity, the intracellular ATP level often decreases. Adriamycin resulted in a slight reduction of ATP level (81.7 ± 1.9%), which showed very little influence on the apoptosis of MCF-7/DOX cells (Figure 8a). However, DOX-

Figure 6. Cell apoptosis analysis of MCF-7/DOX cells by flow cytometry using staining of annexinV-FITC and PI treated with blank medium (a), Adriamycin (b), DOX-loaded CS NPs (c), DOX-loaded CT3 NPs (d), and DOX-loaded CT9 NPs (e) for 24 h.

PI-negative) is a symbol of the percentage of early apoptosis. The percentage of early apoptosis of MCF-7/DOX cells was 7.92% under controlled conditions, while after 24 h incubation with free DOX, DOX-loaded CS NPs, CT3 NPs and CT9 NPs, cell apoptosis was increased to 6.57%, 7.90%, 9.81%, and 15.16%, respectively. This was consistent with the results of cell apoptosis qualitative assays. Both the quantitative and qualitative results demonstrated that the DTX-loaded CT9 NPs significantly enhanced DOX-induced apoptosis. The apoptotic rates of cells treated with the CT NPs were higher than with any other treatments. This also proved the ability of CT copolymer to overcome MDR in MCF-7/DOX tumor cells. CT NPs can enhance anticancer effects to a greater degree with a collaborative or additive antitumor effect with anticancer drugs and overcome MDR in cancer chemotherapy. 3.6. Inhibition of P-gp Efflux Assay. To investigate the potential of P-gp efflux inhibition of CT NPs, a study was conducted in P-gp overexpressing MCF-7/DOX cells using Rh123 as a substrate of P-gp. Cell accumulation of Rh123 with verapamil treatment (P-gp efflux inhibitor) was applied as positive control. As can been seen from Figure 7, Rh123 cell accumulation in untreated cells as negative controls was significantly lower than that in the cells treated with CT NPs. The efficacy of CS NPs in intracellular Rh123 retention was compared with that of positive control. However, CT3 and CT9 NPs treatment showed Rh123 accumulation was 1.3- and 1.5-fold higher than the accumulation obtained with verapamil. This result confirmed the function of TPGS in CT copolymers as a P-gp inhibitor. 3.7. Intracellular ATP Level Assay. The potential apoptosis effect of the formulations was confirmed by measurement of the intracellular ATP (adenosine triphosphate)

Figure 8. Intracellular ATP levels in MCF-7/DOX cells treated with blank CS and CT NPs and DOX-loaded NPs after 24 h incubation.

loaded CS NPs significantly reduced the intracellular ATP level to 55.6 ± 5.6% (p < 0.05). DOX-loaded CT NPs induced a greater reduction in the intracellular ATP level than free DOX (p < 0.01) and DOX-loaded CS NPs (p < 0.05). It may be owed to the inhibition of P-gp and increase of intracellular drug concentration through CT NPs. TPGS and blank NPs effects on ATP level were demonstrated in Figure 8b. TPGS exhibited significant decrease on ATP levels to 49.1 ± 12.1%. Blank CT9 NPs significantly decreased ATP levels (32.1 ± 4.1%) as compared with CS NPs (60.3 ± 14.1%) and CT3 NPs (45.8 ± 2.9%). Therefore, DOX-loaded CT NPs can induce MCF-7/ DOX cell apoptosis by a synergistic apoptosis-accelerating effect between accumulated release of DOX and carrier. 3.8. In Vivo Studies. 3.8.1. Pharmacokinetic Study. TPGS-based NPs have been reported as a kind of oral bioavailability enhancer.35 The property of CT NPs as oral delivery system was investigated here. The plasma concentration−time profiles of different DOX-loaded formulations are 67

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described in Figure 9, and the corresponding pharmacokinetic parameters are summarized in Table 3. All plasma profiles were

Figure 9. DOX plasma concentration versus time curves for various DOX formulations. Formulations were administered by oral gavage into SD rat at dose of 10 mg/kg DOX. Data are shown as mean ± SD (n = 4).

found to be in line with the two-compartment model. The pharmacokinetic properties of DOX-loaded CS NPs were almost the same as Adriamycin, while these were slightly enhanced by CT3 NPs. For CT9 NPs, the plasma concentration of DOX was significantly improved. The AUC (area under curve) of the DOX-loaded CT9 NPs was 3.439 ± 0.943 mg/mL·h, which was 2.36-, 2.34-, and 2.13-fold higher than the one for Adriamycin, DOX-loaded CS NPs, and DOXloaded CT3 NPs, respectively. The MRT and half-life (t1/2) for the DOX-loaded CT9 NPs were 12.168 ± 2.756 h and 10.939 ± 4.208 h, which are 2.02 and 2.53 times longer than for Adriamycin, respectively. The clearance of DOX-loaded CT9 NPs was lower than other formulations. The increased plasma circulation time and concentration would have a positive and increased antitumor effect. 3.8.2. Tumor Inhibition Assay. The potential in vivo antitumor activity of DOX formulations was further validated in mouse hepatoma H22-bearing mice. Both CS and CT3 NPs groups showed no measurable effect on tumor inhibition compared with saline due to the low bioavailability. Only CT9 NPs exhibited significant therapeutic efficacy and showed a similar tendency in the pharmacokinetic results. Compared to saline, DOX-loaded NPs reduced the tumor weight (Figure 10b). The rate of tumor growth inhibition from Adriamycin, DOX-loaded CS NPs, DOX-loaded CT3 NPs, and DOXloaded CT9 NPs was 16.10%, 23.41%, 37.94%, and 46.90%, respectively.

Figure 10. In vivo antitumor effects of the NPs against saline and Adriamycin. (a) Tumor growth curves of H22 tumor-bearing mice model that received the different treatments indicated. (b) Weights of tumors in different groups.

Tumor tissue was investigated by HE staining (Figure 11). In the control group, the tumor tissue was spindle and round, with a rich cytoplasm and more nuclear division. It was clear that cell nuclei apoptosis and vacuoles of DOX-loaded CT9 NPs group were more severe as compared to other groups. A few necrotic cells were observed in tumors from the DOX and other NPs groups, while only viable tumor cells were observed in tumors from the control group. The above results showed that DOXloaded CT9 NPs exhibited the strongest antitumor efficacy, which was consistent with the in vitro cell experiment.

4. CONCLUSION This study presented the fabrication and application of CT NPs as DOX carriers in overcoming MDR. We successfully synthesized CT copolymers with different graft degrees of

Table 3. Pharmacokinetic Parametersa of DOX Formulations in Rat Following Oral Administration formulation DOX CS CT3 CT9 a

AUC (0−∞) (mg/L*h) 1.459 1.467 1.613 3.439

± ± ± ±

0.034 0.206 0.204 0.943

MRT(0−t) (h)

Tmax (h)

± ± ± ±

2 2 2 2

6.029 6.076 7.762 12.168

0.425 0.626 2.081 2.756

Cmax (hμg/mL) 0.390 0.350 0.319 0.451

± ± ± ±

0.050 0.045 0.021 0.094

t1/2z (h) 4.332 4.470 6.435 10.939

± ± ± ±

0.389 1.703 3.803 4.208

CLz/F (L/h/kg) 6.856 6.482 5.852 2.890

± ± ± ±

0.161 0.339 1.141 0.634

The values are shown as mean ± SD (n = 4). 68

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Figure 11. Images of HE-stained sections tumor excised from subcutaneous tumor-bearing mice on 12th day after different treatment: saline (a), Adriamycin (b), DOX-loaded CS NPs (c), DOX-loaded CT3 NPs (d) and DOX-loaded CT9 NPs (e). Images were obtained under Leica microscope using a 40× objective.

China (973 Program, 2012CB932500), NSFC (21204024 and 81373360), Doctoral Fund of Ministry of Education of China (20120142120093), China Postdoctoral Science Foundation funded project (2013T60722), Chutian Scholar of Hubei province and Innovative Research Fund.

TPGS, fabricated DOX-loaded NPs with good drug-loading properties, and demonstrated that DOX could be efficiently released in a pH-dependent manner. In the drug-resistant cells, CT NPs, as compared with Adriamycin, significantly enhanced cytotoxicity of DOX and increased cell apoptosis by the virtue of TPGS as a P-gp inhibitor. In vivo investigations of DOXloaded CT9 NPs significantly exhibited the enhanced DOX pharmacokinetic properties and antitumor activity as compared with Adriamycin and DOX-loaded CS and CT3 NPs. These findings indicate that CT NPs may be an appropriate carrier for anticancer drug delivery in tumors especially drug-resistant cancer cells. We believe that this novel copolymer would chart a new way of treating MDR solid tumors. We intend to carry out further investigations to ascertain the superiority of CT NPs over other TPGS-modified polymer NPs.

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ABBREVIATIONS P-gp P-glycoprotein MDR multidrug resistance TPGS D-α-tocopheryl polyethylene glycol 1000 DOX doxorubicin NPs nanoparticles ABC ATP-binding cassette EPR enhanced permeability and retention CS chitosan PLA-TPGS poly(lactide)-TPGS CT CS-g-TPGS SA succinic anhydride EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide NHS N-hydroxysuccinimide PI propidium iodide DMAP 4-dimethylamino pyridine MTT 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide DMSO dimethyl sulfoxide DCM dichloromethane ATP adenosine triphosphate AUC area under the concentration−time curve MRT mean residence time

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Fax and telephone: +86-27-83601832. E-mail: zhipingzhang@ mail.hust.edu.cn. *E-mail: [email protected]. Author Contributions ‡

These authors contributed equally.

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Notes

The authors declare no competing financial interest.

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REFERENCES

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ACKNOWLEDGMENTS We acknowledge Prof. Li-Qun Wang and Mr. Fang Yuan in Department of Polymer Science & Engineering, Zhejiang University for the assistance in GPC measurement. This research is supported by National Basic Research Program of 69

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