Enhanced Tumor Targeting and Antitumor Efficacy via Hydroxycamptothecin-Encapsulated Folate-Modified N-Succinyl-N′-Octyl Chitosan Micelles

Enhanced Tumor Targeting and Antitumor Efficacy via Hydroxycamptothecin-Encapsulated Folate-Modified N-Succinyl-N -Octyl Chitosan Micelles HONGYAN ZHU,1,2 JIE CAO,1 SISI CUI,1 ZHIYU QIAN,3 YUEQING GU1 1

Department of Biomedical Engineering, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China 2

Department of Pharmacology, Medical College, Nantong University, Nantong 226001, China

3 Department of Biomedical Engineering, School of Automation, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, China

Received 21 June 2012; revised 14 December 2012; accepted 18 January 2013 Published online 11 February 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23470 ABSTRACT: 10-Hydroxycamptothecin (HCPT) is an effective anticancer drug against various types of solid tumors. But the antitumor efficacy of HCPT is far from satisfactory because of its poor physicochemical properties, short circulating half-life, low stability, and nonspecific toxicity to normal tissues. Therefore, a targeted delivery strategy for HCPT to pathological sites is eagerly needed to overcome these limitations. The folate-modified N-succinylN -octyl chitosan (folate-SOC) micelle was chosen in this study and served as the targeted delivery system for HCPT to improve the antitumor efficacy. The water-insoluble anticancer drug HCPT was encapsulated into the folate-SOC micelles by the dialysis method. The nearspherical HCPT-loaded folate-SOC (HCPT/folate-SOC) micelles were formed in aqueous media with diameter of about 100–200 nm. The HCPT/folate-SOC micelles displayed a good stability, reasonable drug-loading content (about 10%), and sustained release behavior for the waterinsoluble HCPT. Compared with free HCPT, HCPT/folate-SOC micelles exhibited a significant enhancement of cellular uptake, higher cytotoxicity against folate receptor positive tumor cell (Bel-7402), excellent tumor-targeting capability and substantially better antitumor efficacy on the nude mice bearing Bel-7402 xenografts. These results demonstrate the potential of folateSOC micelles as long-term stable and effective drug delivery systems in cancer therapy. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1318–1332, 2013 Keywords: 10-hydroxycamptothecin; micelle; folate; chitosan; cancer; targeted drug delivery; cancer chemotherapy

INTRODUCTION Nowadays, chemotherapy is still a commonly used strategy for cancer treatment. But chemotherapy is far from satisfactory because of poor physicochemical properties, short circulating half-life, low stability, or the toxicity associated with the anticancer drugs to normal tissues. 10-Hydroxycamptothecin (HCPT), the natural camptothecin (CPT) analogue, has shown to have a broad spectrum of antitumor activity against various types of solid tumors, such Correspondence to: Yueqing Gu (Telephone: +86-2583271046; Fax: +86-25-83271046; E-mail: guyueqingsubmission@ hotmail.com) Journal of Pharmaceutical Sciences, Vol. 102, 1318–1332 (2013) © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association

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as gastric carcinoma, hepatoma, leukemia, and tumor of head and neck in clinical practice.1 However, effective delivery of HCPT to tumor targets is extremely challenging because of its insolubility in water (3.9 :g/mL),2 structural instability, short half-life, and high toxicity to normal tissue cells. Under physiological conditions, that is, pH equal to or above 7, HCPT undergoes lactone ring-opening hydrolysis to form the inactive carboxylate form as shown in Figure 1.3 In the plasma of mice at pH 7.4, the two structures have a ratio of 50%–55%.4 The carboxylate form of camptothecin has no antitumor activity, but has potential toxic effects.5–7 Additionally, human serum albumin in the blood has a high affinity for binding to the carboxylate form of HCPT, thus driving the above lactone–carboxylate equilibrium toward the

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Figure 1. Chemical structure of lactone and carboxylate forms of HCPT.

formation of the inactive carboxylate form.8 As a result, the potency of the drug is reduced substantially when administrated to humans. In addition, because of its toxic side effects, HCPT, like most other antitumor drugs, needs to be frequently administered with limited doses to achieve desirable drug efficacy. Several different approaches have been taken to address these problems in HCPT delivery. One of the solution is to use drug delivery devices such as polymer conjugates,9–11 lipid-based micelles,12,13 liposomes,14,15 and polymeric micelles.16–19 These drug delivery systems can solve the water insolubility and the poor lactone stability problems associated with HCPT, as well as provide sustained release of controllable amount of drugs over a prolonged period of time. Recently, an increasing attention has been paid to use polymeric micelles, which possess attractive characteristics such as higher bearing capacity for hydrophobic drugs, sustained-release property, a propensity to evade scavenging by the mononuclear phagocyte system (MPS), higher stability, passive targeting ability on tumor tissues,20–22 and so on. In addition, polymeric micellar drug delivery systems could be further improved by active targeting for tumor therapy, for example, by using a ligand coupled to the surface of micelles, which could be actively taken up via a receptor-mediated endocytic pathway. The overexpression of folate receptor (FR) on tumor cells makes this glycoprotein an attractive and effective target for site-specific delivery of antitumor drug into proliferating cells.23 Folate–drug conjugates, folate–polyplex, folate-modified liposomes, micelles, and dendrimers have been widely investigated in recent years.24–28 Chitosan, an abundant natural biopolymer, has shown many advantages such as high biocompatibility, biodegradation, hydrophilicity, and low toxicity toward mammalian cells.29 Active hydroxyl and amine groups in the backbone of chitosan allow simple chemical modification to form a large variety of multifunctional structures including micelles. In our previous studies, folate-modified Nsuccinyl-N -octyl chitosan (folate-SOC) micelles were confirmed with enhanced tumor-targeting ability.30 Folate-SOC micelles demonstrated a promising clinical candidate for the targeted therapy to FR positive tumors.

DOI 10.1002/jps

In this paper, the HCPT-loaded folate-SOC (HCPT/ folate-SOC) micelles were prepared and characterized for targeted therapy. The storage stability of the HCPT/folate-SOC, in vitro release behavior, in vivo dynamic distribution, and tumor-targeting ability were investigated. Importantly, the antitumor efficacy was determined on tumor-bearing nude mice.

MATERIALS AND METHODS Materials Chitosan was purchased from Aoxing Biotechnology Company Ltd. (Zhejiang, China), with deacetylation degrees of 90% (100 kDa, 95%). HCPT (99%) and CPT (99.7%) were provided by Longxiang Biological Medicine Development Company Ltd. (Shanghai, China). The injection of HCPT was purchased from Hubei Huangshi Feiyun Pharmaceutic Company Ltd. (Wuhan, China). Folic acid (or folate, 97%), N, (DCC, 99%), NN -dicyclohexylcarbodiimide hydroxysuccinimide (NHS, 98%), and methyl thiazolyl tetrazolium (MTT, 98%) were all purchased from Sigma–Aldrich (St. Louis, Missouri). Indocyanine green (ICG) derivative (Cypate, 85%) was prepared in our laboratory referring to the previously reported method.31 Fluorescein isothiocyanate (FITC, 96%) was purchased from Aladdin reagent (Shanghai, China). 1,1 -Dioctadecyl-3,3,3 ,3 tetramethylindocarbocyanine perchlorate (DiI, 97%) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Roswell Park Memorial Institute (RPMI) 1640 medium, folate-free RPMI 1640 medium, calf serum, penicillin, streptomycin, trypsin, and ethylenediaminetetraacetic acid (99%) were all purchased from Invitrogen-Life Technologies (Carlsbad, California). The other chemical reagents used in the study were all commercially acquired from Shanghai Chemical Reagent Company (Shanghai, China) with analytical reagent grade. Preparation of HCPT/folate-SOC Micelles

Preparation of Folate-SOC Micelles Folate-SOC was synthesized by following the procedure reported in our previous work.30 First, succinic anhydride and octaldehyde, serving respectively as

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hydrophilic and hydrophobic groups, were conjugated to backbone of chitosan to form the SOC micelles. Briefly, chitosan (1 g) was reacted with 3 g succinic anhydride dissolved in 17 mL acetone solution. After constant stirring for 48 h at room temperature, the resultant N-succinyl-chitosan was precipitated with 5% (w/v) NaOH. The yield of N-succinyl-chitosan obtained was about 92%. Then N-succinyl-chitosan (1 g) was further reacted with octaldehyde (1.02 g). After constant stirring for 4 h, 1.6 mL NaBH4 solution (10 mg/mL) was dropwise added to the reaction liquid and followed by continuous stirring for 12 h. The product solution (SOC) was neutralized with 5% (w/ v) NaOH and then purified by dialysis [10,000 molecular weight cutoff (MWCO)] to form SOC micelles. Second, folate, as a tumor-targeting ligand, was modified to the SOC micelles to enhance the tumor active targeting delivery. Here, DCC/NHS catalyst system was employed for the conjugation of folate and SOC in anhydrous DMSO (molar ratio of folate–DCC–NHS = 1:1.2:2). To compare the properties of SOC micelles with different folate conjugation, different feed ratios of folic acid and SOC micelles (folate–SOC = 1:3 or 1:1) were investigated. The quantity of folate in the folate-SOC micelles was detected at 362 nm by 754PC UV–vis spectrophotometer (Jinghua Instruments Ltd., Shanghai, China).

Fabrication of HCPT/Folate-SOC Micelles HCPT/folate-SOC micelles were prepared using the dialysis method. Briefly, HCPT (5 mg) was firstly dissolved in 0.5 mL dimethylsulfoxide (DMSO) and then dropwise added into 5 mL phosphate-buffered saline (PBS) solution containing 25 mg folate-SOC, under magnetic stirring at room temperature for 20 min. Then, the solutions were placed into a dialysis tube (10,000 MWCO) against 2 L distilled water for 8 h to encapsulate the HCPT into the micelles. After dialysis, the micellar solution was centrifuged at 3000 g for 10 min to remove the free HCPT. HCPT/folate-SOC micelles were collected, further lyophilized, and kept at 4◦ C until further evaluations. Characterization of HCPT/Folate-SOC Micelles

on the surface of copper grid with carbon film and dried at room temperature.

Stability of HCPT/Folate-SOC Micelles To investigate the stability of HCPT/folate-SOC micelles under physiological conditions, HCPT/folateSOC micelles were suspended in PBS (pH 7.4, 4◦ C) for 1 month. The micellar size, polydispersity, and zeta potential were also measured following the protocol described in section Size Distribution, Zeta Potential, and Morphology. Drug Loading and In Vitro Drug Release

Entrapment Efficiency and Drug-Loading Content To determine the entrapment efficiency (EE) of folateSOC for HCPT, the amount of HCPT in the resulting micellar systems was measured using Agilent 1100 high-performance liquid chromatograph (HPLC) (Agilent, Palo Alto, California), with a Agilent Zorbax SB-C18 column (4.6 × 250 mm2 , 3.5 :m particle size). The lyophilized powder of HCPT/folate-SOC micelles (10 mg) was dissolved in 10 mL PBS. Then, 1 mL HCPT/folate-SOC micellar solution (1 mg/mL) was diluted with 9 mL methanol to disrupt the structures of micelles. HCPT was released from the exploded folate-SOC micelles in the methanol solution. After vortexed for 2 min, the mixture was separated by ultracentrifugation at 10,000 g for 10 min and the HCPT in the supernatant was detected by HPLC. The HPLC was performed with a flow rate of 1.0 mL/min, a column temperature of 35◦ C, and a detection wavelength of 384 nm. The mobile phase contained methanol and 20 mmol/L ammonium acetate buffer at a ratio of 55:45 (v/v). The lower limit of quantitation [signalto-noise ratio (S/N) is 10:1] for HCPT was 35 ng/mL. The EE and drug-loading content (DLC) were calculated according to the following Eqs. 1 and 2: EE =

mass of drug loaded in micelles × 100% (1) mass of drug fed initially

DLC =

mass of drug loaded in micelles × 100% (2) mass of drug − loaded micelles

Size Distribution, Zeta Potential, and Morphology

In Vitro Release Studies

The size distribution and zeta potential and morphology of HCPT/folate-SOC micelles were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer 3000 system (Malvern Instruments Ltd., Malvern, UK). The morphology of HCPT/folate-SOC micelles were characterized by transmission electron microscope (TEM) using a FEI Tecnai G2 20s-TWIN microscrope (Philips, Eindhoven, The Netherlands) with accelerated voltage of 200 kV. For TEM, the micellar suspension without being stained was dropped

The release profiles of HCPT from micelles in vitro were studied by a dialysis method. HCPT/folateSOC micellar solution (1 mg/mL) was dialyzed (10,000 MWCO) against 150 mL PBS (pH 7.4) in dark environment at 37◦ C. The released drug in the incubation buffer was collected and the aliquots taken from the dialysate were replaced with fresh PBS at predetermined time intervals to keep the volume constant during the assay. The concentration of the released HCPT was determined by HPLC, referring to the method

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in section Entrapment Efficiency and Drug-Loading Content.

served using the Olympus FV10i confocal laser scanning microscope (Olympus, Tokyo, Japan).

In Vitro Cytotoxicity of HCPT/Folate-SOC Micelles

Pharmacokinetic Experiments in Rats

Human hepatoma cells (Bel-7402) were purchased from American Type Culture Collection (Manassas, Virginia) and cultured continuously at 37◦ C in a humidified atmosphere containing 5% CO2 in folate free RPMI 1640 medium, supplemented with 10% (v/v) calf serum, penicillin (100 U/mL), and streptomycin (100 :g/mL). To evaluate the antitumor activity of HCPT/folate-SOC micelles, MTT assays were conducted on Bel-7402 cell lines by following standard protocol.32 Two hundred microliters of RPMI 1640 medium containing HCPT injection (free HCPT), HCPT-loaded SOC (HCPT/SOC), or HCPT/ folate-SOC with particular concentrations of HCPT (3.125, 6.25, 12.5, 25, 50, and 100 :g/mL) was added into each well in a 96-well plate with Bel-7402 cells (5 × 103 cells/well). After incubation for 24, 48, or 72 h, the RPMI 1640 medium with drugs was replaced by 180 :L fresh RPMI 1640 medium and 20 :L MTT solution (5 mg/mL). After incubation for another 4 h, culture medium was removed and 150 :L DMSO was added into each well. The optical density (OD) was measured at 490 nm with a Microplate Reader Model 550 (BIO-RAD, Hercules, California). Cell viability was calculated base on the formula given below (Eq. 3), in which ODtreated was obtained in the presence of HCPT, HCPT/SOC, or HCPT/folate-SOC, and ODcontrol was obtained in the absence of HCPT, HCPT/ SOC, or HCPT/folate-SOC.

To obtain preliminary parameters about the pharmacokinetic properties of the HCPT-loaded SOC micelles, 15 Wistar rats were divided into three groups at random. Group 1 was treated with HCPT injection (free HCPT), group 2 with HCPT/SOC micelles, and group 3 with HCPT/folate-SOC micelles solution, via the tail vein injection. All groups were given a single dose of 2.5 mg HCPT/kg. After intravenous (i.v.) administration, blood samples were collected at 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, and 4 h from the orbital plexus. Then the plasma was obtained by centrifuging immediately at 4000 g for 10 min and stored at −20◦ C. HCPT in 200 :L plasma sample and 50 :L internal standard (CPT, 200 ng/ mL) were extracted twice with 6 mL of acetic ether and 2 mL dichloromethane. The drug residue was obtained by evaporation and reconstituted in 100 :L methanol solution. After centrifugation at 14,000 g for 10 min, 20 :L aliquots of the supernatant were injected into the HPLC system. The HPLC method was same as the previous method in section Entrapment Efficiency and Drug-Loading Content. The HCPT concentration was determined based on the standard curves prepared with HCPT and CPT. The lower limit of quantitation (S/N = 10) for HCPT in plasma-spiked calibration curves was 50 ng/mL. Pharmacokinetic parameters were calculated using the Practical Pharmacokinetic Program DAS 2.1.1 (Shanghai University of Chinese Medicine, Shanghai, China).

Cell viability (%) =

ODtreated × 100% ODcontrol

(3)

Cellular Uptake of the HCPT/folate-SOC Micelles Bel-7402 cells were cultured in the plates for the cell internalization study of HCPT/folate-SOC micelles. FITC [excitation/emission (ex/em): 488/525 nm] was labeled to the micelles. The fluorescence of FITC was measured to trace the dynamics of the micelles. Fresh folate-free RPMI 1640 medium (1.5 mL) containing 100 :g FITC-labeled HCPT/SOC or HCPT/folate-SOC micelles was added into each plate. For folate competition assay, Bel-7402 cells were firstly incubated with 1 mM free folic acid for 30 min before adding 1.5 mL medium containing FITC-labeled HCPT/folate-SOC (100 :g). After incubation for 30 min and 2 h, the medium was removed. Then, fluorescence dye DiI (ex/ em: 549/565 nm) was added and stained on the membrane of Bel-7402 cells to display the cell profiles. After dyeing for 10 min, the cells were washed with PBS solution three times. The excitation and emission wavelengths for HCPT are about 364 and 454 nm, respectively. The fluorescent images of the cells were obDOI 10.1002/jps

In Vivo Targeting and Distribution Study in Tumor-Bearing Mice The athymic nude mice (BALB/C), 4–6 weeks old and weighed at about 18–22g, were purchased from SLAC Laboratory Animal Company Ltd. (Shanghai, China). Sterile PBS (0.1 mL) containing 3 × 106 tumor cells (Bel-7402 cells) were injected subcutaneously under left armpit of each mouse. After the tumor volume reached 100 mm3 , the mice were anesthetized with an intraperitoneal injection of 1 mg/g of ethyl carbamate for in vivo targeting and distribution studies. To evaluate the tumor-targeting capability of folate-modified SOC micelles, near infrared dye ICG derivative (ex/em: 765.9/813 nm) was encapsulated into the micelles by following the HCPT loading procedure. The saturated concentration of ICG derivative in physiological saline is about 2.4 :g/mL, which approaches the aqueous solubility of HCPT (3.9 :g/ mL). ICG derivative, ICG derivative-loaded SOC micelles, and ICG derivative-loaded folate-SOC micelles (0.2 mL, containing 10 :g ICG derivative) were administered into the blood stream of the subject mice groups through tail vein (n = 5, each group). Then, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

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the nude mice bearing Bel-7402 tumor were immobilized in a Lucite jig after being anesthetized and used for the investigation of the dynamics and targeting ability of the samples. The fluorescence imaging was acquired by our near infrared (NIR) imaging system using the method reported previously.30 Briefly, for the fluorescence images, the subject was illuminated with a semiconductor laser (λ = 765.9 nm) (nLight, Shanghai, China) defocused to provide a broad spot with even OD on the skin of the mouse. An 800 nm long pass filter (Princeton Instruments, Trenton, New Jersey) was used for capturing the emitted fluorescence from the micelle-trapped dye. All images were taken by a high sensitivity PIXIS 512B NIR CCD camera (Princeton Instruments). Besides, another HLU32F400 808 nm laser (LIMO, Dortmund, Germany) was supplied as background light to obtain the profile of the subjected animal. (Otherwise, the imaging was completely black except the fluorescence spot.) To quantitatively measure the targeting ability of HCPT-loaded micelles, the tumor-to-background ratio (TBR) was calculated with definition as Eq. 4. The NIR fluorescence signal per selected region (5 mm2 ) was measured based on the analysis of region of interest (ROI, tumor or normal tissue) from the obtained NIR fluorescence images. TBR =

tumor signal − background signal background signal

(4)

To further compare the distribution of HCPTloaded SOC micelles with and without folate modification, 15 athymic nude mice bearing Bel-7402 tumor were evenly and randomly divided into three groups. Each mouse was injected via tail vein with HCPT injection (free HCPT), HCPT/SOC, and HCPT/folateSOC micelles. The mice were sacrificed at 1 h postinjection. Major organs including liver, spleen, kidney, and tumors were snap frozen in liquid nitrogen and stored at −80◦ C for further analysis. The organs and tumors were cut into 8 :m sections on a Leica CM1900 cryostat (Leica, Nussloch, Germany). Fluorescence of HCPT was acquired with a confocal laser scanning microscope. Antitumor Efficacy on Bel-7402 Tumor-Bearing Mice In vivo antitumor efficacy of HCPT/folate-SOC was investigated on Bel-7402 tumor-bearing nude mice. The treatment started after the tumor volume reached about 100 mm3 . Twenty four tumor-bearing mice were randomly divided into four groups. Groups A was used as control treated with saline. And the groups B, C, and D received HCPT injection (free HCPT), HCPT/SOC, and HCPT/folate-SOC micelles, at a dose of 5.0 mg HCPT/kg i.v. administration. Each group of mice was treated every other day for six times. Tumor JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

size was measured and tumor volume was calculated by the formula: (W2 × L)/2, where W is the tumor measurement at the widest point, and L is the tumor dimension at the longest point. Relative tumor volume (RTV) was calculated by the formula: Vi /V0 , where V0 is the tumor volume at day 0, and Vi is the tumor volume at the measurement date. Antitumor activity was estimated by relative tumor growth rate [T/C (%)], which was calculated by the formula (Eq. 5): T/C(%) =

RTV(treatmentgroup) × 100% RTV(negativecontrolgroup)

(5)

In addition, the body weight was weighed. And the behaviors related to the toxicity, such as breathing problems, failure to eat and drink, lethargy, or abnormal posture, were observed. Statistical Analysis To find out the difference between two independent samples, t test and one-factor analysis of variance were performed by SPSS Version 11.5 (SPSS Inc., Chicago, Illinois). The means were judged to be different when p < 0.05 and significantly different when p < 0.01.

RESULTS AND DISCUSSION Characterization of HCPT/Folate-SOC Micelles N-succinyl-N -octyl chitosan micelles with different number of folic acid molecules were prepared by following the above procedures (in section Preparation of Folate-SOC Micelles). Folate-SOC-1 and folate-SOC2 were the conjugations of folic acid and SOC with the different feed ratios of 1:3 and 1:1 (folate–SOC), respectively. The amount of folate in these two conjugations was measured by UV absorption at 362 nm and calculated based on the standard curves prepared with pure folate. Results showed that the amounts of folate associated with SOC-1 and SOC-2 were 99 and 190 :g/mg, respectively. The two folate-modified SOC micelles will be used to optimize the antitumor effect of HCPT-loaded chitosan micelles. The sizes and zeta potentials of these HCPT-loaded micelles in solution were studied by DLS. Results showed that the average diameters by intensity for HCPT/SOC, HCPT/folate-SOC-1, and HCPT/folateSOC-2 micelles were about 185, 145, and 122 nm, respectively. The presence of folate residues seemed to reduce the size of the micelles. In particular, more folate conjugation rendered smaller size of the particles, which was in accordance with the result of our previous report.28 Activation of both "- and (-carboxyl groups of folate could result in a cross-linking between the two activated carboxyl of folate and the amino DOI 10.1002/jps

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group of chitosan. The crisscross on the surface of micelles could tighten the structure and reduce the diameter of the micelles. Dynamic light scattering measurements revealed that HCPT-loaded SOC, folate-SOC-1, and folateSOC-2 micelles had a negative surface charge of −8.36, −20.03, and −34.65 mV, respectively. HCPT/ folate-SOC micelles exhibited a much more negative surface charge than HCPT/SOC micelles. As we know, folate acid has two negative carboxy groups. The conjugation of folate to chitosan could consume one positive “ NH2 ” and bring more “ COO− ” to the micelles, and thus, enhance the negative charge on the surface of micelles. The higher density of folate, the more negative surface charge micelles had. The morphology of HCPT and HCPT-loaded micelles was displayed by TEM imaging, as shown in Figure 2. Self-assembled micelles were well dispersed as individual micelles with a near-spherical shape. Figures 2b and 2c show that the black spots of HCPT accumulated in the grey micelles. The result further confirmed the successful loading of HCPT into the SOC micelles. However, the average size of HCPTloaded micelles was about 70 nm in TEM photo, which was not in good agreement with the value determined by DLS. The dehydration process during the sample preparation of TEM likely resulted in the shrinkage of the particles, thus a reduced diameter of the micelles. The TEM image of HCPT/SOC was similar with that of HCPT/folate-SOC micelles (see Figs. 2b and 2c). The TEM image of HCPT shows that the grain of HCPT was rod. The length and width of grain were about 0.6 and 0.2 :m, respectively (see Fig. 2a). Stability of HCPT/Folate-SOC Micelles The stability of HCPT-loaded SOC, folate-SOC-1, and folate-SOC-2 micelles PBS solutions (pH 7.4) at 4◦ C were investigated for 30 days. The time profiles of average size, polydispersity, and zeta potentials were measured at designated time points and displayed in Figure 3. Results implied that HCPT/folate-SOC displayed better storage stability than that of HCPT/ SOC. The mean diameters of HCPT/ folate-SOC-1 and HCPT/folate-SOC-2 micelles in PBS determined at 1, 3, 7, 15, and 30 days displayed a stable tendency, with the polydispersity about 0.2 (Figs. 3a and 3b). The size, polydispersity of folate-SOC exhibited a stable curve within 30 days, indicating the thermodynamic stability of HCPT/folate-SOC micelles in the aqueous condition. And as the amount of folate increased, the physical stability of HCPT/folate-SOC increased, which indicate folate conjugation could stabilize the SOC micelles by cross-linking. In contrast, the HCPT/ SOC solutions displayed unstable properties. The particle size of HCPT/SOC increased significantly after 15 days storage, and large particles with the diameter about 600 nm were observed at 30 days (Fig. DOI 10.1002/jps

Figure 2. Transmission electron microscope pictures of (a) HCPT, (b) HCPT/SOC, and (c) HCPT/folate-SOC micelles.

3a). The polydispersity of HCPT/SOC significantly increased from 7 days, about 0.452 at 7 days, 0.55 at 15 days, and 1 at 30 days (Fig. 3b). It means that HCPT-encapsulated SOC micelles could remain stable storage at 4◦ C for 7 days. In addition, the zeta potentials of all the micelles within 30 days were also JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

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Table 1. The DLC and EE of HCPT/SOC, HCPT/Folate-SOC-1, and HCPT/Folate-SOC-2 Sample HCPT/SOC HCPT/folate-SOC-1 HCPT/folate-SOC-2

DLC (%)

EE (%)

10.37 ± 0.19 10.05 ± 0.21 10.55 ± 0.55

57.87 ± 1.21 55.83 ± 1.29 58.93 ± 3.45

Data were given as mean ± SD (n = 3).

negative charges on the surface of micelles rendered more stable particles due to the repulsion interaction among the surface charges. The results of zeta potentials further proved that poor physical stability of HCPT-encapsulated SOC micelles could be surmounted by introducing folate into the SOC. Drug Loading and In Vitro Release Studies The hydrophobic drugs, HCPT, could be incorporated into the micelles by the hydrophobic interaction between HCPT and octaldehyde segments of SOC micelles. The HCPT loading into micelles occurred simultaneously with self-assembly of amphiphilic folate-SOC micelles during dialysis process. When the weight ratio of HCPT and chitosan micelles was 1:5, SOC and folate-SOC micelles had optimal drug loading about 10% and entrapped efficiency close to 60% for HCPT, as listed in Table 1. Results implied that folate-SOC micelles had reasonable DLCs and entrapment efficacy for HCPT. This relative high DLC and entrapped efficiency were due to the hydrophobicity of octaldehyde-rich chains and the extremely low water solubility of HCPT. The release profiles of HCPT-loaded chitosan micelles in PBS at 37◦ C were investigated and compared with the commercial HCPT lyophilized powder. Figure 4 shows that HCPT was released rapidly from lyophilized powder formulation and almost completely released within 12 h (Fig. 4). In comparison, there was no obvious burst effect of HCPT releasing from SOC and folate-SOC micelles. It took about 168 h to release 50% of the drug loaded, and took about 720 h to reach the maximum release of HCPT (Fig. 4). Data demonstrated that folate-SOC micelles provided hydrophobic drugs a practical delivery system for high drug loading and long-term sustained release.

In Vitro Cytotoxicity Figure 3. The stability of HCPT/SOC, HCPT/folateSOC-1, and HCPT/folate-SOC-2 micelles: (a) particle size, (b) polydispersity, and (c) zeta potential. Data were given as mean ± SD (n = 3).

investigated, as shown in Figure 3c. The zeta potentials of the micelles were between 0 and −10 mV for HCPT/SOC, −20 and −23 mV for HCPT/folate-SOC1, and −29 and −38 mV for HCPT/folate-SOC-2. More JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

To compare the anticancer activity of SOC and folateSOC micelles-encapsulated HCPT with HCPT injection (free HCPT), human hepatoma cells (Bel-7402) were used here for the MTT assay. The cell viability under the treatment with different formulation of HCPT at 24, 48, and 72 h are depicted in Figures 5a, 5b, and 5c, respectively. The increased concentrations of free HCPT or HCPT-loaded micelles resulted in a significant increase in cell killing in all the DOI 10.1002/jps

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Figure 4. (a) In vitro release of HPCT from commercial HCPT lyophilized powder, HCPT/SOC, HCPT/folate-SOC1, and HCPT/folate-SOC-2 micelles in PBS (0.1 M, pH 7.4, 37◦ C) within 720 h. (b) The release kinetics of the initial 12 h. Data were given as mean ± SD (n = 3).

cases. And the cytotoxicity displayed more severe with the increasing of the exposured time. In comparison, SOC-encapsulated HCPT showed similar cytotoxicity to free HCPT (p > 0.05), and HCPT/folate-SOC-1 with targeted modification induced a significant cytotoxic effect to free HCPT at each HCPT concentration point after incubation of 48 h (p < 0.05). Particularly, it was found that the higher density of folate, the stronger inhibition of HCPT/folate-SOC-2 had on Bel-7402 cells at 48 h (p < 0.01). This enhanced cytotoxic effect was mostly due to the FR-mediated endocytosis of drugloaded micelles. The stronger cytotoxicity of HCPT/folate-SOC micelles depended on not only the quantity of HCPT entering cytoplasm but also the release rate of HCPT from micelles within the cells. Although the release of HCPT from the micelles was much slower than free HCPT in PBS, the HCPT/folate-SOC micelles showed much stronger cytotoxic effect than free HCPT. This was because the cell uptake of HCPT/folate-SOC micelles could be increased by FR-mediated endocytosis, which contributed to the enhanced cytotoxicity of DOI 10.1002/jps

Figure 5. Cell viability assay of HCPT, HCPT/SOC, HCPT/folate-SOC-1, and HCPT/folate-SOC-2 micelles at (a) 24 h, (b) 48 h, and (c) 72 h. Bel-7402 cells were treated with the indicated concentrations of HCPT, HCPT/SOC, HCPT/folate-SOC-1, and HCPT/folate-SOC-2 micelles, respectively. Data were given as mean ± SD (n = 3).

HCPT/folate-SOC micelles. The following cell uptake data would further confirm this uptake mechanism. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

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Figure 6. Confocal microscopic images of Bel-702 cells were incubated with HCPT/SOC, HCPT/folate-SOC micelles, and HCPT/folate-SOC micelles with free folate for competition assay. Micelles and cell membrane were visualized in the blue, green, and red channels.

The above results showed that HCPT/folate-SOC-2 micelles had smaller particle size, better stability, and greater cytotoxicity. All things considered, HCPT/ folate-SOC-2 micelles were selected as the best candidate for the next experiments.

In Vitro Cellular Uptake of HCPT/Folate-SOC Micelles To visualize the cellular uptake of HCPT/folate-SOC micelles to FR positive tumor cells, the chitosan micelles were labeled with FITC, which emitted green fluorescence, the membranes of Bel-7402 cells were marked with red fluorescent DiI, and the HCPT was excited to produce the blue fluorescence. As displayed in Figure 6, the SOC micelles without folate conjugation were hardly captured by the FR positive Bel7402 cells and no HCPT signal was observed inside the cells. In contrast, the folate-modified chitosan micelles were found to stick to the cell membrane at JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

about 30 min and gradually internalized into the cells at 2 h. The encapsulated HCPT was accordingly observed in the cells. More importantly, because of the competitive binding of folic acid to FR, the endocytosis of Bel-7402 cells for folate-SOC micelles was distinctly blocked after treated with free folate, and thus, much weaker fluorescence of HCPT (compared with the group of nonpretreatment with free folate) was observed in the cells. Results indicated that folate modification to the SOC micelles produced a significantly higher uptake of micelles to the FR positive tumor cells, thus a higher drug accumulation within the cells, as evidenced by the increase of fluorescence intensity of HCPT. In addition, the endocytosis of HCPT/folateSOC could be prevented by the competitive binding of free folic acid to FR. The uptake results were consistent with our designed goal for the drug delivery system. DOI 10.1002/jps

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Pharmacokinetics of HCPT/Folate-SOC Micelles To investigate the pharmacokinetics process in vivo, HCPT injection (free HCPT), HCPT/SOC, and HCPT/ folate-SOC micelles were administrated into different groups of rats by tail vein. Then, blood samples were collected at the designated time points and HCPT concentrations were measured. The plasma clearance curves of free HCPT, HCPT/SOC, and HCPT/folateSOC micelles in rats are shown in Figure 7a. Higher initial plasma levels of free HCPT were observed compared with HCPT/SOC and HCPT/folate-SOC micelles. Clearance of free HCPT from the blood circulation after i.v. administration was very rapid and the plasma concentration decreased to about 0.12 mg/L at 1 h. Compared with free HCPT, the HCPT releasing from HCPT/SOC and HCPT/folate-SOC micelles displayed slower clearance from the circulating system in vivo. The plasma concentrations of HCPT in SOC and folate-SOC micelles groups were about 0.75 mg/ L even at 4 h postinjection. No obvious difference was observed between SOC and folate-SOC micelles groups. The concentration–time curve of free HCPT was fitted well with the two-compartmental model. And the three-compartmental model completely accorded with the concentration–time curves of HCPT/ SOC and HCPT/folate-SOC micelles. Compare the characteristics of the in vitro release and in vivo pharmacokinetics of the free HCPT, HCPT/SOC, and HCPT/folate-SOC micelles. The release and elimination rates of free HCPT were much faster than that of HCPT/SOC and HCPT/folate-SOC micelles either in vitro or in vivo. However, HCPT released from SOC and folate-SOC micelles in the PBS at 37◦ C more slowly than that eliminated in the plasma of the rats. It was determined by the different physiological conditions in vitro and in vivo. In addition, the change of plasma concentrations of HCPT/SOC and folate-SOC micelles groups was related with the process of elimination of HCPT-loaded micelles in vivo more than the release rate of HCPT from micelles. The pharmacokinetic parameters of HCPT in the three formulations after i.v. administration are shown in Table 2. For free HCPT, HCPT/SOC, and HCPT/ folate-SOC micelles, the values of area under the plasma concentration–time curve (AUC0–∞ ) were 1.068, 5.936, and 6.414 (mg/L) h, respectively; the corresponding total body clearance (CL) were 2.355, 0.422, and 0.409 L/(h kg), respectively; the half-lives (t1/2 ) were 0.161, 1.867, and 2.346 h, respectively; the apparent volumes of distribution (V) were 0.546, 1.138, and 1.31 L/kg, respectively. From the above data, we can come to the conclusion that the plasma AUC of HCPT-loaded micelles was lower than that of the free HCPT and the V, t1/2 , and CL of HCPT after being entrapped by micelles were increased.

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Figure 7. The dynamic behaviors and tumor-targeting capability of ICG derivative and ICG derivative-loaded micelles in nude mice bearing Bel-7402 tumor xenograft monitored by NIR images system. Fluorescence images of the mice (a) after administration of ICG derivative, (b) after administration of ICG derivative-loaded SOC micelles, (c) after administration of ICG derivativeloaded folate-SOC micelles. (d) Time courses of tumor-tobackground ratio (TBR) ratio in Bel-7402 tumor for ICG derivative and ICG derivative-loaded micelles. Statistical analysis indicates that there was significant difference of TBR ratio in the Bel-7402 tumors between the SOC micelles and folate-SOC micelles groups (∗ p < 0.01). The data were represented as mean ± SD (n = 5).

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Table 2.

Pharmacokinetics Parameters of Free HCPT, HCPT/SOC, and HCPT/Folate-SOC in Rats

Parameters AUC(0–t) AUC(0–∞) MRT(0–t) MRT(0–∞) t1/2 Tmax CL V Cmax

Unit

Free HCPT

HCPT/SOC

HCPT/Folate-SOC

(mg/L) h (mg/L) h h h h h L/(h kg) L/kg mg/L

1.053 ± 0.088 1.068 ± 0.09 0.202 ± 0.007 0.216 ± 0.019 0.161 ± 0.039 0.033 2.355 ± 0.213 0.546 ± 0.132 4.351 ± 0.324

4.71 ± 0.262 5.936 ± 0.269 1.568 ± 0.015 2.629 ± 0.089 1.867 ± 0.062 0.033 0.422 ± 0.02 1.138 ± 0.087 3.172 ± 0.038

4.276 ± 0.331 6.414 ± 1.521 1.582 ± 0.039 3.479 ± 1.014 2.346 ± 0.658 0.033 0.409 ± 0.1 1.31 ± 0.126 3.109 ± 0.105

Data were given as mean ± SD (n = 5). AUC(0–t) , area under the plasma concentration–time curve from 0 h to the last measurable time point; AUC(0–∞) , area under the concentration–time curve from 0 h to infinity; MRT(0–t) , mean residue time from 0 h to the last measurable time point; MRT(0–∞) , mean residue time from 0 h to infinity; t1/2 , elimination half-life; Tmax , the time at which the maximum concentration of drug is present in blood; CL, total body clearance; V, apparent volume of distribution; Cmax , the maximum concentration of drug in blood.

These data suggested that folate-SOC micelles would be a new potential drug delivery system for HCPT with longer circulation time and sustained release behavior. Tumor Targeting and Biodistribution of HCPT/Folate-SOC Micelles To investigate the dynamic biodistribution of folateSOC micelles in Bel-7402 tumor-bearing mice, near infrared dye (ICG derivative) was entrapped into the micelles and the fluorescence images in different time

points were recorded by the self-built NIR fluorescence imaging system. The depth resolution of the self-built NIR fluorescence imaging is about 2–4 mm. Representative fluorescence images of mice after administration of ICG derivative-loaded micelles are shown in Figure 8. The fluorescent signals from the entrapped ICG derivative indicated that ICG derivative, SOC and folate SOC micelles initially accumulated in liver, gradually moved to gastrointestinal organ and evacuated from the body through the liver–intestine system after 12 h postinjection. The

Figure 8. Laser scanning confocal microscopy images of HCPT in tissue sections of Bel-7402 tumor-bearing mice at 1 h after injection with HCPT, HCPT/SOC micelles, or HCPT/folate-SOC micelles. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

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Figure 9. (a) Concentration–time curve of HCPT in rat plasma after i.v. administration of HCPT, HCPT/SOC, and HCPT/folate-SOC (2.5 mg/kg). Data were given as mean ± SD (n = 5). (b) Tumor volumes of Bel-7402-bearing mice as a function of time. Data were given as mean ± SD (n = 6). (c) Tumor weights of Bel-7402-bearing mice as a function of time. Treatments beginning indicated with arrows. Data were given as mean ± SD (n = 6).

fluorescence was also observed in the feces (data not shown), which confirmed the liver–intestine clearance pathway of the chitosan micelles. Images of control group (Fig. 8a) show that free ICG derivative had little targeting ability toward Bel-7402 tumor tissue. For the ICG derivative-loaded SOC micelles, the fluorescence appeared in the tumor site at about 12 h postinjection and reached the peak at around 24 h (Fig. 8b). In comparison, the fluorescence from the ICG derivative-loaded folate-SOC micelles was detectable in the tumor site just 6 h postinjection (Fig. 8c), which was much faster than that of nonfolate-modified SOC micelles. In addition, the fluorescence intensity of ICG derivative-loaded folate-SOC micelles in the tumor site was much higher than that of nonfolate-modified DOI 10.1002/jps

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SOC micelles at the corresponding time points (1, 6, 12, 24, 48, and 72 h). To quantify the targeting ability of the micelles in tumors, fluorescence intensities in the obtained NIR fluorescence images were measured by analyzing the ROI. The NIR fluorescence signals from the three samples (ICG derivative-loaded SOC and folateSOC micelles) in tumors were normalized to surround muscles tissues and depicted in Figure 8d. The time profiles of TBR for folate-modified micelles was 1.02 ± 0.35 at 1 h postinjection, reaching a peak at 24 h postinjection (15.49 ± 1.55), and slowly declining to 4.32 ± 0.72 by 72 h (Fig. 8c). As expected, the nonfolate-modified SOC micelles exhibits relatively lower TBR, with 0.16 ± 0.09 at 1 h, 6.47 ± 1.34 at the 24 h peak uptake time point and declining to 2.49 ± 0.26 by 72 h (Fig. 8c). Statistical analysis indicated a significant difference of TBR ratio in Bel7402 tumors between ICG derivative-loaded SOC and folate-SOC micelles (p < 0.01). These results indicate that folate-modified SOC micelles could improve the targeting ability to FR positive tumors. Above all, the folate-SOC micelles could enhance the tumortargeted drug delivery, prolong drug retention time, and maintain its concentration in tumor tissue. To further demonstrate whether the folate-SOC micelles could enhance the quantities of anticancer drugs in the tumor, HCPT injection (free HCPT), HCPT/SOC, HCPT/folate-SOC micelles were injected through tail vein into Bel-7402 xenograft tumorbearing mice. After 1 h postinjection, the tumor and other organs were excised and sliced into sections with 8 :m thickness and then visualized under the confocal laser scanning microscope. The fluorescence intensity images from HCPT are shown in Figure 9. The bright fluorescence signals were displayed in liver, which was consistent with the in vivo imaging (Fig. 8). Also, strong fluorescence signal appeared in lung, which could not be in vivo detected by NIR fluorescence imaging system from the obverse side due to the deep location of lung inside the body and the shelter of ribs. Relative lower fluorescence signals were exhibited in kidney, which might be due to the smaller particles. The nanoparticles above 200 nm from the blood stream after their injection can be rapidly swallowed by macrophages of the MPS. On the contrary, the particles below 10 nm can be easily filtered and eliminated through the kidney. Therefore, the particles of 30–150 nm were considered to mainly accumulate in the body (including kidney). HCPT/SOC (30%) and HCPT/folate-SOC (50%) micelles had the average diameters below 150 nm by volume. So, the kidney displayed a little fluorescence intensity of HCPTloaded SOC and folate-SOC micelles after 1 h postinjection, as shown in Figure 9. Almost no fluorescence was observed in heart and spleen, indicating no micelles entering these organs. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

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Table 3. The Mean Tumor Size (Mean ± SD) and Relative Tumor Growth Rate T/C (%) of Bel-7402-Bearing Mice After Being Treated with Physiological Saline, Free HCPT, HCPT/SOC, and HCPT/Folate-SOC Micelles (n = 6) Formulation F1 F2 F3 F4

Dose (mg/kg)

Mean Tumor Size

Relative Tumor Growth Rate (%)

5 5 5 5

967.21 ± 2.48 643.99 ± 4.72 489.26 ± 2.92 376.79 ± 2.34

100 65.68 50.3 38.43

F1, physiological saline; F2, free HCPT; F3, HCPT/SOC; F4, HCPT/folate-SOC.

For the tumor sites at 1 h postinjection, the HCPT/ folate-SOC micelles-treated mice displayed relative higher fluorescence signal compared with the mice treated with free HCPT and HCPT/SOC micelles, indicating the enhanced active tumor-targeting ability of folate-modified micelles. Further, HCPT/SOC micelles showed a little higher accumulation in the tumor compared with free HCPT (Fig. 9), which might be attributed to the well-known enhanced permeation and retention (EPR) effect.33–34 The nanoparticles of size <200 nm with hydrophilic surfaces tend to extravagate through disorganized and defective vascular architecture and preferentially accumulate in the tumor via its EPR effect.35 This phenomenon is known as passive tumor-targeting effect. Therefore, HCPT/ SOC micelles with the size of 185 nm by intensity can target tumors via EPR effect. The organ distributions implied that the antitumor ability of HCPT might be enhanced by the tumortargeting deliver system of HCPT/folate-SOC micelles.

In Vivo Antitumor Efficacy Although the cytotoxicity of the drug-loaded micelles in the tumor cells was demonstrated, the antitumor efficacy in a mouse xenograft model needed to be further investigated. The therapy of antitumor began on day 7 after the subcutaneous xenotransplanted model was prepared, when tumor volume of group A, group B, group C, and group D was 101.67 ± 2.48, 103.08 ± 2.77, 102.26 ± 2.79, and 103.05 ± 2.41 mm3 , respectively. The antitumor effect of micelles-encapsulated HCPT on the tumorbearing mice was compared with that of HCPT injection (free HCPT) by measuring tumor volume of the subject mice. As shown in Figure 7b, the tumor volumes in micelles-encapsulated HCPT-treated group were significantly smaller than that of mice treated with either normal saline or free HCPT (p < 0.01). For the groups of mice treated with micellesencapsulated HCPT, the tumor volume in HCPT/ folate-SOC micelles-treated mice was smaller than that of mice treated with HCPT/SOC micelles (p < 0.01). The relative tumor growth rates were calculated based on the Eq. 5. As listed in Table 3, the T/ C (%) for groups of mice treated with HCPT, HCPT/ JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

SOC, and HCPT/folate-SOC micelles were 65.68%, 50.3%, and 38.43% on day 22 postinjection, respectively. The HCPT/folate-SOC micelles markedly inhibited the growth of human hepatoma solidity (Bel7402) at dose of 5 mg/kg. Moreover, the body weight loss was only observed in free HCPT-treated group. Body weight in free HCPT-treated groups decreased 29.53% after 2 weeks treating (Fig. 7c), whereas increased 7.34% and 15.35% in groups of mice treated with HCPT/SOC and HCPT/folate-SOC micelles, respectively. Nonsignificant weight changes of HCPTloaded micelles groups indicated the lower toxic effects of micelles-encapsulated HCPT compared with free HCPT. Application of HCPT is hampered because of its extreme insolubility in water, structural instability, short half-life and unpredictable side effect. To address these problems, many efficient and safe HCPTcontaining drug delivery systems based on chitosan have been developed, such as HCPT-loaded stearic acid and poly (lactic-co-glycolic acid) grafted chitosan oligosaccharide micelles,36 amphiphilic N-alkyl-Ntrimethyl chitosan micelles,37 N-succinyl-chitosan micelles38 , and so on. SOC micelles as doxorubicin carriers have been reported showing effective antitumor activity.39 However, no report has been found to conjugate folate to amphiphilic chitosan micelles for targeted delivery of HCPT. To further improve the antitumor efficacy, HCPT-loaded folate-modified SOC micelles were prepared in this study. The SOC micelles modified by folate could be easily internalized into the FR-positive tumor cells and showed much higher abilities of tumor targeting in vivo. In addition, folate-SOC micelles were able to modulate the in vitro release of HCPT and improve its pharmacokinetic properties in vivo, which was highly conducive to the enhanced antitumor activity.

CONCLUSIONS Polymeric micelles enhanced the drug’s solubility and stability by encapsulating the insoluble HCPT into the hydrophobic cores of the micelle carriers. The pharmacokinetics data indicated that nanosized SOC micelles-encapsulated HCPT exhibited a prolonged circulation time in vivo, which allows the DOI 10.1002/jps

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encapsulated HCPT to extravasate and accumulate into the tumor tissue. Moreover, the disorganized and defective vascular architecture in tumors features an EPR effect for the passive tumor targeting of micelles, which was observed in our study. FR is an important target since it is overexpressed in many cancer cells. The folate-modified SOC micelles showed significant targeting to the FR positive Bel-7402 tumor by distinguishing and binding to the FRs on cell membranes. Compared with SOC micelles, folate-SOC micelles increased HCPT cytotoxicity toward Bel-7402 tumor with minimized host organ toxicity by passive and active tumor-targeting effect. This study demonstrated an effective drug delivery system for hydrophobic antitumor drug to targeted tumor sites, with enhanced antitumor efficacy and reduced toxicity.

ACKNOWLEDGMENTS This research was supported by the Natural Science Foundation Committee of China (NSFC 81202467, 30672015, 30700779, 30800257, 30970776, 81000666, 81071194, 31050110123, and 81171395), the major project from the Ministry of Science and Technology for new drug development (2009ZX09310-004), the Natural Science Foundation of Jiangsu Province in China (BK2012232, BK2011389), the Natural Science Research Project of Universities in Jiangsu Province of China (11KJB350004), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

REFERENCES 1. Pu X, Sun J, Wang Y, Wang Y, Liu X, Zhang P, Tang X, Pan W, Han J, He Z. 2009. Development of a chemically stable 10-hydroxycamptothecin nanosuspension. Int J Pharm 3799(1):167–173. 2. Wang S, Shang D, Li X, Jiang T. 2009. Preparations and properties of hydroxycamptothecin emulsion and its tissue distribution in mice. Asian J Pharm Sci 4(5):299–307. 3. Giovanella BC, Stehlin JS, Wall ME, Wani MC, Nicholas AW, Liu LF, Silber R, Potmesil M. 1989. DNA topoisomerase Itargeted chemotherapy of human colon cancer in xenografts. Science 246(4933):1046–1048. 4. Giovanella BC, Hinz HR, Kozielski AJ, Stehlin JS Jr, Silber R, Potmesil M. 1991. Complete growth inhibition of human cancer xenografts in nude mice by treatment with 20-(S)camptothecin. Cancer Res 51(11):3052–3055. 5. Ertl B, Platzer P, Wirth M, Gabor F. 1999. Poly (D,L-lactic-coglycolic acid) microspheres for sustained delivery and stabilization of camptothecin. J Control Release 61(3):305–317. 6. O’Leary J, Muggia FM. 1998. Camptothecins: A review of their development and schedules of administration. Eur J Cancer 34(10):1500–1508. 7. Shenderova A, Burke TG, Schwendeman SP. 1997. Stabilization of 10-hydroxycamptothecin in poly (lactide-co-glycolide) microsphere delivery vehicles. Pharm Res 14(10):1406–1414. 8. Mi ZH, Burke TG. 1994. Differential interactions of camptothecin lactone and carboxylate forms with human blood components. Biochemistry 33(34):10325–10336. DOI 10.1002/jps

1331

9. Conover CD, Greenwald RB, Pendri A, Gilbert CW, Shum KL. 1998. Camptothecin delivery systems: Enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker. Cancer Chemother Pharmacol 42(5):407–414. 10. Fleming AB, Haverstick K, Saltzman WM. 2004. In vitro cytotoxicity and in vivo distribution after direct delivery of PEGcamptothecin conjugates to the rat brain. Bioconjug Chem 15(6):1364–1375. 11. Ying V, Haverstick K, Page RL, Saltzman WM. 2007. Efficacy of camptothecin and polymer-conjugated camptothecin in tumor spheroids and solid tumors. J Biomater Sci Polym Ed 18(10):1283–1299. 12. Eichhorn ME, Luedemann S, Strieth S, Papyan A, Ruhstorfer H, Haas H, Michaelis U, Sauer B, Teifel M, Enders G, Brix G, Jauch KW, Bruns CJ, Dellian M. 2007. Cationic lipid complexed camptothecin (EndoTAG-2) improves antitumoral efficacy by tumor vascular targeting. Cancer Biol Ther 6(6):920–929. 13. Peer D, Karp JM, Hong S, FaroKhzad OC, Margalit R, Langer R. 2007. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2(12):751–760. 14. Watanabe M, Kawano K, Toma K, Hattori Y, Maitani Y. 2008. In vivo antitumor activity of camptothecin incorporated in liposomes formulated with an artificial lipid and human serum albumin. J Control Release 127(3):231–238. 15. Yang H, Lou C, Xu M, Wu C, Miyoshi H, Liu Y. 2011. Investigation of folate-conjugated fluorescent silica micelles for targeting delivery to folate receptor-positive tumors and their internalization mechanism. Int J Nanomed 6:2023–2032. 16. Kawano K, Watanabe M, Yamamoto T, Yokoyama M, Opanasopit P, Okano T, Maitani Y. 2006. Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles. J Control Release 112(3):329–332. 17. Watanabe M, Kawano K, Yokoyama M, Opanasopit P, Okano T, Maitani Y. 2006. Preparation of camptothecin-loaded polymeric micelles and evaluation of their incorporation and circulation stability. Int J Pharm 308(1–2):183–189. 18. Yang X, Li L, Wang Y, Tan Y. 2009. Preparation, pharmacokinetics and tissue distribution of micelles made of reverse thermo-responsive polymers. Int J Pharm 370(1–2):210–215. 19. Zhang C, Ding Y, Yu LL, Ping Q. 2007. Polymeric micelle systems of hydroxycamptothecin based on amphiphilic N-alkyl-Ntrimethyl chitosan derivatives. Colloids Surf B Biointerfaces 55(2):192–199. 20. Kataoka K, Kwon GS, Yokoyama M, Okano T, Sakurai Y. 1993. Block copolymer micelles as vehicles for drug delivery. J Control Release 24(1–3):119–132. 21. Kwon GS, Naito M, Kataoka K, Yokoyama M, Sakurai Y, Okano T. 1995. Physical entrapment of adriamycin in AB block copolymer micelles. Pharm Res 12(2):192–195. 22. Yokoyama M, Miyauchi M, Yamada N, Okano T, Sakurai Y, Kataoka K, Inoue S. 1990. Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer. Cancer Res 50(6):1693–1700. 23. Antony AC. 1996. Folate receptors. Annu Rev Nutr 16:501– 521. 24. Gabizon A, Shmeeda H, Horowitz AT, Zalipsky S. 2004. Tumor cell targeting of liposome-entrapped drugs with phospholipidanchored folic acid-PEG conjugates. Adv Drug Deliv Rev 56(8):1177–1192. 25. Lee D, Lockey R, Mohapatra S. 2006. Folate receptor-mediated cancer cell specific gene delivery using folic acid-conjugated oligochitosans. J Nanosci Nanotechnol 6(9–10):2860–2866. 26. Lee RJ, Low PS. 1995. Folate-mediated tumor cell targeting of liposome-entrapped doxorubicin in vitro. Biochim Biophys Acta 1233(2):134–144. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

1332

ZHU ET AL.

27. Li Q, Liu C, Zhao X, Zu Y, Wang Y, Zhang B, Zhao D, Zhao Q, Su L, Gao Y, Sun B. 2011. Preparation, characterization and targeting of micronized 10-hydroxycamptothecin-loaded folate-conjugated human serum albumin micelles to cancer cells. Int J Nanomed 6:397–405. 28. Yang J, Ni B, Liu J, Zhu L, Zhou W. 2011. Application of liposome-encapsulated hydroxycamptothecin in the prevention of epidural scar formation in New Zealand white rabbits. Spine J 11(3):218–223. 29. Felt O, Buri P, Gurny R. 1998. Chitosan: A unique polysaccharide for drug delivery. Drug Dev Ind Pharm 24(11):979–993. 30. Zhu H, Liu F, Guo J, Xue J, Qian Z, Gu Y. 2011. Folatemodified chitosan micelles with enhanced tumor targeting evaluated by near infrared imaging system. Carbohydrate Polym 86(3):1118–1129. 31. Ye Y, Bloch S, Kao J, Achilefu S. 2005. Multivalent carbocyanine molecular probes: Synthesis and applications. Bioconjug Chem 16(1):51–61. 32. Mueller H, Kassack MU, Wiese M. 2004. Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines. J Biomol Screen 9(6):506–515. 33. Fang J, Nakamura H, Maeda H. 2011. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev 63(3):136–151.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 102, NO. 4, APRIL 2013

34. Maeda H. 2001. The enhanced permeability and retention (EPR) effect in tumor vasculature: The key role of tumorselective macromolecular drug targeting. Adv Enzyme Regul 41:189–207. 35. Danhier F, Feron O, Preat V. 2010. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 148(2):135–146. 36. Zhou YY, Du YZ, Wang L, Yuan H, Zhou JP, Hu FQ. 2010. Preparation and pharmacodynamics of stearic acid and poly (lactic-co-glycolic acid) grafted chitosan oligosaccharide micelles for 10-hydroxycamptothecin. Int J Pharm 393(1–2):143–151. 37. Zhang C, Ding Y, Yu LL, Ping Q. 2007. Polymeric micelle systems of hydroxycamptothecin based on amphiphilic N-alkyl-Ntrimethyl chitosan derivatives. Colloids Surf B Biointerfaces 55(2):192–199. 38. Hou ZQ, Han J, Zhan CM, Zhou CX, Hu Q, Zhang QQ. 2010. Synthesis and evaluation of N-succinyl-chitosan nanoparticles toward local hydroxycamptothecin delivery. Carbohydr Polym 81(4):765–768. 39. Xu XY, Li L, Zhou JP, Lu SY, Yang J, Yin XJ, Ren JS. 2007. Preparation and characterization of N-succinylN -octyl chitosan micelles as doxorubicin carriers for effective anti-tumor activity. Colloids Surf B Biointerfaces 55:222– 228.

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