Efficient Simultaneous Tumor Targeting Delivery of All-Trans

Sciences Department, Peking University, 38 Xueyuan Road, Haidian District, Beijing .... Hyaluronic acid (HA), a low toxic, biodegradable, and bioc...
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Efficient Simultaneous Tumor Targeting Delivery of All-Trans Retinoid Acid and Paclitaxel Based on Hyaluronic Acid-Based Multifunctional Nanocarrier Jing Yao,*,† Li Zhang,†,‡ Jianping Zhou,*,† Hongpan Liu,† and Qiang Zhang§ †

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China ‡ Department of Pharmacy, Nanhu Community Health Centre, 7 Nanhulu, Nanjing 210019, China § Pharmaceutical Sciences Department, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China ABSTRACT: An amphiphilic hyaluronic acid (HA)-g-all-trans retinoid acid (HRA) conjugate was successfully developed as a tumor-targeting nanocarrier for potentially synergistic combination chemotherapy of all-trans retinoid acid (ATRA) and paclitaxel (PTX). The HRA conjugate was synthesized by an imine reaction between HA-COOH and ATRA-NH2. PTX-loaded HRA nanoparticles possessed a high loading capacity, nanoscale particle sizes, and good biocompatible characteristics. Cell viability assays indicated that PTXloaded HRA nanoparticles exhibited concentration- and time-dependent cytotoxicity. Moreover, they displayed obvious superiority in inducing the apoptosis of tumor cells. Cellular uptake analysis suggested that HRA nanoparticles could be efficiently taken up by cells via endocytic pathway and transport into the nucleus, contributing to HA receptor-mediated endocytosis and ATRA-induced nuclear translocation, respectively. Moreover, in vivo imaging analysis indicated that the accumulation of DiR-loaded HRA nanoparticles in tumor was increased obviously after intravenous administration as compared to free DiR solution, which confirmed that the HRA nanoparticles could assist the drugs targeting to the tumor. Furthermore, PTX-loaded HRA nanoparticles exhibited greater tumor growth inhibition effect in vivo with reducing the toxicity. Therefore, HRA nanoparticles can be considered as a promising targeted codelivery system for combination cancer chemotherapy. KEYWORDS: tumor targeting, codelivery, combination chemotherapy, hyaluronic acid, all-trans retinoid acid

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INTRODUCTION Current combination chemotherapy of the cancer has attracted intensive attention to obtain optimal efficacy by avoiding the resistance of cancer cells to one kind of chemotherapeutic drug as well as overcoming the severe side effects during therapy. Paclitaxel (PTX) is known as the anticancer drugs with significant antitumor activity against a wide variety of tumors such as ovarian carcinoma, breast cancer, nonsmall cell lung cancer, and acute leukemias.1 At present, many drugs such as cisplatin, carboplatin, and 5-fluorouracil have combined with PTX for the combination chemotherapy against cancer.2,3 It has been reported that the retinoid acid (ATRA), one of the retinoids which inhibits cell proliferation and induces differentiation in a variety of tumor cells,4 could enhance PTXinduced death of tumor cells and the regression of tumor.5,6 In the study of Hong et al., the combination of PTX-incorporated pullulan acetate nanoparticles and ATRA-incorporated methoxy poly(ethylene glycol)-grafted chitosan copolymer nanoparticles showed a synergistic antiproliferative effect against CT26 cells. Furthermore, the activity of MMP-2, a key enzyme in tumor cell invasion, was significantly decreased in cells treated with the combination of PTX and ATRA.7 Moreover, © 2013 American Chemical Society

ATRA-conjugated polymeric carriers for nuclear import have also been noted,8 which contributes to enhance the proapoptotic action of PTX. However, some strategies are needed to overcome the obstacles which may prevent simultaneous delivery of PTX and ATRA, for example, the poor solubility of drugs, the aggregation and precipitation of drugs in aqueous medium, losing respective pharmaceutical activity, and raising a risk of embolisms.9 It is known that the conventional drug formulations such as a separate intravenous line cannot overcome these limitations. Currently, polymer−drug conjugates are considered as a promising delivery system for drug combination therapy due to their noticeable advantages, that is, increasing water solubility and stability, improving pharmacokinetic profile, reducing side effects, and sometimes, targeting disease site either by active or passive mechanisms.10,11 The combination therapy of cisplatin (75 mg/m2) with escalating doses of Received: Revised: Accepted: Published: 1080

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Scheme 1. Illustration of the Formation, Uptake by Tumor Cells, and Simultaneous Multiple Drug Delivery of PTX-Loaded HRA Nanoparticles

could also decrease the systemic toxicity and allow a higher relative dose. In this study, we synthesized a HA derivative coupled with hydrophobic ATRA (HRA conjugate). This amphiphilic HRA conjugate is used as the self-assembled targeting nanocarrier for potentially synergistic combination chemotherapy of PTX and ATRA. It is expected that this multifunctional nanocarrier will effectively achieve simultaneous targeting delivery of PTX and ATRA to tumor by a combination of EPR effect-based passive targeting mechanism and HA-mediated endocytosis action (as shown in Scheme 1). It also displays high drug-loaded capacity, good physical stability, and low toxic side effects just like general polymeric nanoparticles. The HRA nanoparticles were systemically characterized. To evaluate the potential of HRA nanoparticles as codelivery carriers of PTX and ATRA, the cytotoxicity, cell apoptosis, and cellular internalization of PTXloaded HRA nanoparticles were investigated. Moreover, the in vivo distribution and targetability of HRA nanoparticles were also investigated by the noninvasive near-infrared optical imaging technique. Furthermore, the in vivo antitumor activity of PTX-loaded HRA nanoparticles was evaluated in the melanoma tumor-bearing C57BL/6 mice.

poly(glutamic acid) (PGA)-PTX for the advanced solid tumor showed good activity in refractory patients.12 Lammers et al. reported that HPMA-based polymer−drug conjugate carrying 6.4 wt % of gemcitabine and 5.7 wt % of doxorubicin increased the antitumor activity without increasing the toxicity in the combination of gemcitabine and doxorubicin.13 It could also prolong the period time of circulation and localize to the tumor relatively selectively. Tumor cell targeting is a promising strategy for enhancing the therapeutic potential of chemotherapy agents.14 However, the polymer−drug conjugates often passively accumulate in the tumor as a result of the enhanced permeability and retention (EPR) effect. Therefore, it is essential to further improve the selectivity of polymer−drug conjugates for cancer cells. Hyaluronic acid (HA), a low toxic, biodegradable, and biocompatible polyanionic polysaccharide, is distributed widely in the extracellular matrix and the joint liquid of mammalians.15,16 It has been proved that the HA conjugates were specifically and efficiently internalized into the malignant cells that overexpressed CD44 receptor and LYCE-1 receptor.17 We have developed an amphoteric HA derivative (HA-g-PEI) for targeting gene delivery.15 Results indicated that the transfection efficiency of the HA-g-PEI with 500 kDa HA was 2.09 fold higher than that of PEI/DNA complex (p < 0.01). Moreover, the transgene expression of the complexes was remarkably reduced with the down-regulation of 75.2% by the pretreatment of the cells by 3.0 mg/mL HA. It was also reported that the PEI-HA conjugate could facilitate the intracellular delivery of anti-PGL3-Lic siRNA/PEI-HA complexes by the HA receptormediated endocytosis maintaining the endosomal escape capacity.18 In addition, HA-drug conjugates could increase cellular uptake and cytotoxicity in vitro, decrease tumor weight, and prolong the survival time of tumor-bearing mice compared to free drug.19,20 The conjugation of HA and hydrophobic drug

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EXPERIMENTAL SECTION Materials. Hyaluronic acid (10 KDa) was obtained from Shandong Freda Biochem Co. Ltd. (Shandong, China). Paclitaxel (PTX) and retinoid acid (ATRA) were purchased from Shanghai Zhongxi Sunve Pharmaceutical Co. Ltd. (Chongqing, China) and Wuhan Hezhong Bio-Chemical Manufacture Co. Ltd. (Hunan, China), respectively. Anhydrous dimethylformamide (DMF), anhydrous formamide, and 1ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC) were from Shanghai Lingfeng Chemical Reagent Co. Ltd. (Shanghai, China) and Sigma Chemical Co. (St. Louis, MO), respectively. N-Hydroxysuccinimide (NHS) and pyrene were from 1081

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Figure 1. (A) Synthetic scheme of hyaluronic acid-g-all-trans retinoic acid (HRA) conjugate. (B) 1H NMR spectra of hyaluronic acid (a) and HRA conjugate (b). (C) Hydrolysis of HRA conjugate under different pH and physiological conditions (n = 3).

mg of HRA conjugate was incubated in 1.5 mL of the medium at 37 °C with mild stirring. At predetermined times, 150 μL of the sample was taken and centrifuged. Then, 100 μL of supernatant solution was added 1 mL of ethyl acetate to extract ATRA. ATRA was analyzed using the LC/MS system with a Shimadzu LC-10AD high-performance liquid chromatography (HPLC) system and a Shimadzu LCMS-2010A quadrupole mass spectrometer. Quantitative analysis was operated in selected ion monitoring (SIM) and positive ion mode using target ions at m/z 301.15 ([M + H]+) for ATRA. Preparation and Characterization of PTX-Loaded HRA Nanoparticles. The PTX-loaded HRA nanoparticles were prepared by a dialysis method. Briefly, the PTX solution in ethanol was added into the HRA solution of 6 mg/mL with stirring, and the solution was ultrasonicated at 200 W for 30 min in an ice bath by an ultrasonicator (JY92-2D, Ningbo Scentz Biotechnology Co., Ltd., China). The resulting solution was dialyzed against the distilled water overnight followed by centrifugation at 3000 rpm for 15 min, filtering through a 0.8 μm microporous membrane, and lyophilization. The amount of PTX in the nanoparticles was measured by HPLC (Shimadzu LC-2010 system, Kyoto, Japan). The drug-loading (DL) and entrapment efficiency (EE) of PTX were calculated as described in previous studies.11 The particle size and zeta potential of PTX-loaded nanoparticles from three different batches were determined by dynamic light scattering measurements (BI-200SM, Brookhaven Instruments Corp., USA). The shape and surface morphology of the nanoparticles were observed by using a scanning probe microscope (SPM) (Nanoscope V, Veeco

Sinopharm Chemical Reagent Co. Ltd. (Nanjing, China). DeadEnd Fluorometric TUNEL System and mouse specific HRP/DAB detection IHC kit were purchased from Promega (Madison, WI) and Abcan (Cambridge, MA), respectively. All other chemicals were of analytical grade and were used without further purification. Synthesis of the HRA Conjugate. The HRA conjugate was synthesized by coupling HA with aminated ATRA (Figure 1). First, the aminated ATRA was prepared as described in our previous report.11 Second, HA was dissolved in formamide (10 mL) at 50 °C. The EDC and NHS solutions were mixed with HA solutions for 15 min under the presence of N2. The ratio of HA, NHS, and EDC was 1:1.2:1.2. Then, the ATRA-NH2 solution in DMF was added. The reaction was performed for 24 h under the condition of no light and the presence of N2, and the resulting mixture was precipitated in excess cold acetone. The precipitate was carefully washed with acetone to remove excess ATRA-NH2 and then dried after vacuum filtration. The dried HRA conjugate was dissolved in water and then dialyzed against deionized water for 48 h (MWCO 3500). The resulting solution was lyophilized and stored at 4 °C until use. The structure of products was analyzed by 1H NMR (Avace AV500, Bruker, Germany). The degree of substitution (DS), defined as the number of ATRA per HA molecule, was estimated by UV measurements (λ = 345 nm). The critical aggregation concentration (CAC) of HRA conjugate was estimated by the fluorescence spectroscopy using the pyrene as the probe as described previously.11 The hydrolysis study of HRA conjugate under different pH and physiological conditions was investigated. A sample of 15 1082

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FITC dye in these nanopaticles. The FITC-loaded HRA nanoparticles were prepared by the dialysis method. Confocal microscopy was used to internalize the intracellular location of nanoparticles. The B16F10 cells were seeded at 5 × 105 cells/ well in a 6-well plate at 37 °C. Then cells were incubated with the free FITC solution and the FITC-loaded HRA nanoparticle diluted with serum-free medium for 2 and 6 h, respectively. After incubated with a 4% paraformaldehyde solution for 30 min, Hochest33342 (Beyotime Biotechnology, China) was added and incubated for 20 min. Cells were then washed twice with PBS (pH 7.4) and observed using FV1000 focus drift compensating microscope (Olympus, Tokyo, Japan). Flow cytometry was also used for quantitative determination of cellular uptake of nanoparticles. The B16F10 cells were seeded at 5 × 105 cells/well in a 6-well plate and incubated in the growth medium at 37 °C for 24 h, respectively. The growth medium was removed and replaced with serum free media. Then cells were incubated with the free FITC solution and the FITC-loaded HRA nanoparticle diluted with serum-free medium for 6 h, respectively. The uptake efficiency of the nanoparticles was quantified for FITC-positive cells by flow cytometry (BD, Biosciences, USA). Cells incubated with equivalent amount of free FITC were used as a control. Furthermore, to investigate the interaction of HA and HA receptor, the HA of 5 mg/mL was added in the growth medium for 1 h to saturate the HA receptors of cell surface before adding nanoparticles. In Vivo Imaging Analysis. For in vivo optical imaging, nine female tumor-bearing BALB/c mice were randomly divided into three groups and injected intravenously with the saline solution (as the control), DiR solution (0.05 mg/mL), and DiR-loaded HRA nanoparticles (5 mg/mL) (n = 3 for each group), respectively. Imaging was performed at 0.5, 1, 4, 6, 8, and 12 h after injection by In-Vivo Imaging System (DXS4000PRO, Kodak, USA). Finally, the mice were sacrificed, and the tumors were excised and analyzed by the Kodak Molecular Imaging Software 5.X. In Vivo Antitumor Activity. In the subcutaneous B16F10 melanoma tumor model, when the treatment were started when the tumor volume was about 100 mm3, the tumor-bearing mice were divided into three groups (n = 6) and were intravenously administrated with normal saline (the control group), the PTX plus ATRA plus HA solution (10 mg PTX/kg, 2 mg ATRA/kg, and 18 mg HA/kg) and PTX-loaded HRA nanoparticles (10 mg PTX/kg), respectively. The ethanol with an equal volume of cremophor EL as well as 5% glucose solution was used as a solvent for free drug groups. The administration was continued six times at 2-day intervals through tail vein injection for 12 days. The lengths of the longest tumor axis (a(t), mm) and the vertical axis (b(t), mm) were measured with a caliper every other day, and the tumor volume (v(t), mm3) was calculated using the following equation: V = 0.5 × a × b2. The mice were sacrificed on the 12th day after the first administration, and the tumors were weighed. Furthermore, an TUNEL assay and an immunochemical analysis of proliferation cell nuclear antigen (PCNA) staining on the paraffin-embedded mice tumor were performed following the protocols provided by the manufacturers. The morphology of the tumor tissues after hematoxylin and eosin (HE) staining was observed by the microscope. Statistical Analysis. Data were expressed as mean ± standard deviation (SD). The statistical significance of group differences was analyzed using two-way unweighted mean

Instruments Inc., USA). Differential scanning calorimeter (DSC) analysis of the PTX, the HRA conjugate, the physical mixture of PTX and HRA, and the PTX-loaded HRA nanoparticles was carried out using NETZSCH DSC 204 equipment, respectively. The temperature range was 40−300 °C, and the heating rate was 10 °C/min. Hemolysis Test and Intravenous Irritation Assessment. The hemolysis test was performed as described previously.11 Briefly, the drug-free and PTX-loaded HRA nanoparticle solution at different volumes was added into 2% of rabbit red blood cells (RBC) suspension, respectively. Then 5% glucose injection solution was added in each tube to obtain a final volume of 5 mL. The positive (100% hemolysis) and negative control (0% hemolysis) were obtained by mixing 2.5 mL of water and 5% glucose injection solution with 2.5 mL of 2% RBC suspension to eliminate the effect of background. Samples were incubated at 37 °C for 1 h and centrifuged at 3000 rpm for 10 min to remove nonlysed RBC. The degree of hemolysis was calculated by the spectrophotometric method at 540 nm. Free drug solution was used as a positive control. Three rabbits, weighing 1.8−2.0 kg, were used for investigating the intravenous irritation of the nanoparticle. The rabbits were injected with PTX solution (0.4 mg/mL), HRA conjugate solution, and PTX-loaded HRA nanoparticle solution (0.4 mg/mL for PTX) into the vein at the edge of the left ear for 3 days, respectively. While 5% glucose solution at equivalent volume was injected into the right ear-border vein as the control. At 24 h after the last administration, the rabbits were sacrificed. Their ears were cut and fixed in 10% liquor formaldehyde for histological examination. In Vitro Cytotoxicity Studies. The cytotoxicity of PTXloaded HRA nanoparticles was assessed using the MTT assay. The B16F10 cells and HepG2 cells were seeded at a density of 1 × 104 cells/well in 96-well microtiter plates and incubated for 24 h, respectively. The cells were then treated with PTX-loaded HRA nanoparticles, blank HRA conjugate and PTX plus ATRA plus HA solution for 72 h. The ethanol with an equal volume of cremophor EL 35 was used as the vehicle of PTX and ATRA. After incubation, MTT solution (20 μL, 5 mg/mL in PBS) was then added to each well and the cells were incubated further for 4 h at 37 °C. The media were removed and the cells were dissolved in DMSO. Absorbance at 570 nm was measured with a microplate reader (SOFT max PRO, Molecular Devices Corporation, CA). Cell viability (%) was calculated as (OD of test group/OD of control group) × 100. Cell Apoptosis. Apoptotic cells were detected by Annxin VFITC/PI double-labeled flow cytometry. Exponentially growing B16F10 cells and HepG2 cells were seeded at a density of 5 × 105 cells/well in 6-well plates, respectively. Cells were treated with PTX plus ATRA plus HA solution (1 μg/mL of equivalent PTX) and PTX-loaded HRA nanoparticles (1 μg/mL of equivalent PTX) for 48 h, respectively. After treatment, the cells were harvested and washed twice with cold PBS buffer, followed by centrifugation. The cells were resuspended and stained with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) for 15 min in darkness. After washing with PBS buffer, samples were immediately analyzed using a flow cytometer(BD FACSCalibur flow cytometer, BD Biosciences, USA). In free HA competition studies, the HA of 5 mg/mL was added in the growth medium for 1 h to saturate the HA receptors of cell surface before adding nanoparticles. Cellular Uptake of HRA Nanoparticles. Cellular uptake studies were performed for HRA nanoparticles by loading 1083

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Figure 2. (A) Size distribution of PTX-loaded HRA nanoparticles. (B) Representative AFM images of PTX-loaded HRA nanoparticles. (C) DSC thennograms of PTX (a), HA-ATRA conjugate (b), physical mixture of PTX and HA-ATRA conjugate (c), and PTX-loaded HRA nanoparticles (d).

analysis of variance (ANOVA)-test, and a value of p < 0.05 was considered significant and specified in figures.

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HRA nanoparticles might have good dilution stability in the bloodstreams after intravenous injection.21 The hydrolysis of HRA conjugate under various conditions was investigated by determining the free ATRA in the mediums using the LC/MS system. The pH values of 7.4, 5.8, and 4.5 were chose to modulate physiological conditions of blood and microenvironments of the intracellular endosome and lysosome of tumors. The extent of hydrolysis was determined by monitoring the amount of ATRA. As illustrated in Figure 1C, the hydrolysis of HRA conjugate showed pH-dependence. The ATRA released from HRA conjugate were only approximately 0.1% in the PBS (pH 7.4) and the plasma within 5 days, suggesting that HRA conjugate was very stable at neutral pH and in blood cycle system. However, the amount of HRA hydrolysis significantly increased at the mildly acidic pH, which is consistent with release behavior of ATRA in the tumor homogenate. The faster release of ATRA in the tumor due to lower pH of tumors than normal tissues would contribute to reduced potential side effects as well as improved cancer therapy.22 Interestingly, it was found that HRA conjugate still held the significant activity of induction of HL-60 cell differentiation although the CD11b expression level in HRA group was slightly lower than that of free ATRA (data not shown). The release behavior of the drug as a rate-limiting step

RESULTS AND DISCUSSION

Preparation and Characterization of the HRA Conjugate. The HRA conjugate was prepared by an amine reaction between HA and ATRA-NH2. The synthetic scheme is shown in Figure 1 A. The composition of synthesized conjugate was analyzed by 1H NMR (Figure 1 B). The methyl peak of acetoamide group of HA appeared at 2.0 ppm (−NCOCH3), and proton peaks of C2−6 and C1 appeared at 3.3−3.9 ppm and 4.4−4.6 ppm, respectively. In the spectra of the products, two new amide linkages between HA and ATRA appeared at 8.01 ppm and 8.35 ppm, respectively. The characteristic peaks of ATRA appeared at 1.0−1.5 ppm. Moreover, the proton peaks of the product at 3.1−4.0 ppm were wider than that of HA. The results indicated that ATRA was grafted to the HA chain. The calculated DS of ATRA in HRA conjugate was 24.53 mol %. The amphiphilic HRA conjugate can self-assemble into the nanoparticles in aqueous solvent. The CAC of the conjugates in distilled water were determined by a fluorescence technique using pyrene as a probe. The CAC values of the HRA conjugate was 67.5 ± 9.5 mg/L. Such low CAC values indicated that 1084

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Figure 3. (A) Hemolysis as a function of vehicle concentration of HRA conjugate, PTX plus ATRA solution and PTX-loaded HRA nanoparticles. (B) Pathological section of vessel tissues after treated with 5% glucose injection (negative control, a), PTX solution (b), HRA conjugate (c), and PTX-loaded HRA nanoparticles (d).

Hemolysis Test and Intravenous Irritation Assessment. The adaptability of polymeric nanoparticles for intravenous (i.v.) administration should be considered seriously.23 Therefore, the hemolysis and i.v. irritation were investigated to evaluate the feasibility of HRA nanoparticles for i.v. administration in this study. As shown in Figure 3A, the hemolysis of PTX plus ATRA solution was dramatically increased by increasing the concentration and reached 30.9% at the concentration of 0.2 mg/mL. However, the hemolysis of PTX-loaded HRA naoparticles was only 3.5% at the investigated highest concentration, which was almost negligible as compared to PTX plus ATRA solution. In addition, the HRA conjugate also showed no hemolysis. The results suggested that HRA conjugate and PTX-loaded HRA naoparticles were not toxic toward erythrocytes after i.v. injection. Figure 3B shows the histopathologic section of the rabbit earborder vein after i.v. administration of nanoparticles for 3 days. After an administration of multidoses, pathological section in HRA conjugate and PTX-loaded HRA nanoparticles groups showed no significant difference as compared to the negative control (5% glucose solution). The vessel wall and endothelia cell structures kept integrity, and there were no angiectasia and

may result in the reduced CD11b expression of HRA compared to free ATRA. Preparation and Characterization of PTX-Loaded HRA Nanoparticles. The hydrophobic PTX was easily entrapped by HRA nanoparticles. The highest DL% and EE% were 29.3 ± 0.5% and 91.2 ± 1.3%, respectively. The particle size of PTXloaded HRA nanoparticles were 148.7 ± 10.2 nm with a polydisperse index of 0.187 ± 0.012 (seen in Figure 2A), which was smaller than that of unloaded nanoparticles. The representative AFM images of the nanoparticles were shown in Figure 2B. The images indicated that the nanoparticles had almost spherical shape. The DSC thermograms of PTX, HRA conjugate, physical mixture of PTX and HRA conjugate, and PTX-loaded HRA nanoparticles are shown in Figure 2C. PTX exhibited an endothermic melting peak at 217.7 °C and an exothermic decomposition peak at 238.8 °C, implying that PTX was in crystal state,11 while the melting and decomposition peaks of PTX in physical mixture of PTX and HRA conjugate were at 215.2 °C and 234.6 °C, respectively. However, there was no characteristic peak of PTX in the thermogram of PTXloaded HRA nanoparticles, suggesting that PTX existed in the nanoparticles with the state of amorphism or solid dispersion. 1085

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Figure 4. (A) Cell viability of B16F10 cells and HepG2 cells treated with PTX-loaded HRA nanoparticles and PTX plus ATRA plus HA solution. (B) Cytotoxicity of blank HRA nanoparticles and the vehicle of PTX plus ATRA plus HA solution against B16F10 cells (horizontal coordinate represents corresponding drug concentration of blank nanoparticles and the vehicle of PTX plus ATRA plus HA solution) at 37 °C for 72 h. (C) Effect of incubation time on cell viability of B16F10 cells. Cell survival fractions were assessed by MTT assay. Data represent mean ± SD (n = 5).

accumulated drug release.11,28 The equation of correction is as follows:

thrombus in the lumen of vein. The results demonstrated that HRA conjugate and PTX-loaded HRA nanoparticles had no simulative reaction in the ear vein of rabbit, indicating its good biocompatible characteristics. On the contrary, the PTX solution group showed serious irritation based on their obvious difference of the histopathology. In Vitro Cytotoxicity Studies. The cytotoxicity of HRA conjugate and PTX-loaded HRA nanoparticles were both investigated in B16F10 and HepG2 cells line by the MTT assay. As shown in Figure 4A, PTX-loaded HRA nanoparticles and PTX plus HA plus ATRA solution both exhibited concentration-dependent cytotoxicity as expected. Moreover, at high concentrations of >10 μg/mL, PTX-loaded HRA nanoparticles showed lower cytotoxicity as compared to PTX plus HA plus ATRA solution. It might be related to the sustained release of drugs from nanoparticles as suggested in the study by Min et al., 2008, where camptothecin (CPT)loaded hydrophobically modified glycol chitosan nanoparticales retained higher viability than free CPT due to sustained release of the drugs.24 Park et al. also described that doxorubicinloaded heparin-deoxycholic acid nanoparticles had a little bit lower than free doxorubicin against SCC cells.25 On the contrary, free drugs can be quickly transported into cells by passive diffuse due to high concentration gradient under in vitro conditions26,27 and instantly affect the cells growth without the drug release process. It was also found that PTX-loaded nanoaparticles (100 μg/mL of PTX) exhibited enhanced cytotoxicity for long incubation time (48 and 72 h), while mixtures of drugs show no significant difference in cell viability at different incubation time (Figure 4C), further suggesting that such delayed drug release behavior of nanoparticles might result in lower cytotoxicity of PTX-loaded nanoparticles than PTX plus HA plus ATRA solution. Furthermore, considering the sustainable drug-release feature of nanoparticles, some studies have described that the mortality of cells treated with nanoparticles should be corrected by the

Modified mortality = (measured mortality/accumulated drug release) × 2

In this study, PTX release from nanoparticles after incubation of 72 h in PBS buffer was investigated. The PTX amount released from HRA nanoparticles was 19% at pH of 7.4 after 72 h. The modified mortality of PTX-loaded HRA nanoparticles in B16F10 cells and HepG2 cells at equivalent PTX concentration of 10 μg/mL were 9.1 and 10.2 times higher than that PTX plus ATRA plus HA solution after correction through the drug release, respectively, indicating that PTX-loaded HRA nanoparticles could induce the death of tumor cells more effectively. More noticeably, the targeting ability of nanoparticles would increase the cellular uptake of drugs during circulation in vivo compared to free drugs.29 Thus, based on the fact that more amounts of drugs after encapsulated by nanoparticles would accumulate in the tumor rather than free drugs owing to tumor targeting effect of HA-based nanoparticles which was also confirmed through in vivo imaging analysis, it was expected that PTX-loaded HRA nanoparticles would have more potential to increase in vivo efficacy than PTX plus ATRA plus HA solution. In addition, a higher cytotoxicity of PTX plus HA plus ATRA solution also resulted from its solvent. As illustrated in Figure 4B, the reduction in cell viability after treatment with the mixture of cremophor EL and ethanol was observed, as suggested in previous studies, where the cell viability of the vehicle (cremophor EL and ethanol) at 200 μg/mL was less than 20% in HepG2 cells.11 However, blank HRA nanoparticles displayed high cell viability against B16F10 cells even at high concentrations, indicating its good biocompatible characteristics. Similar results were observed in HepG2 cells treated with blank HRA nanoparticles and mixed vehicles, respectively. Synthetically considering other factors such as the hemolysis 1086

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Figure 5. Flow cytometric analysis for detection of cell apoptosis in B16F10 cells and HepG2 cells induced by PTX plus GA plus HA solution (equivalent PTX of 1 μg/mL) and PTX-loaded HRA nanoparticles (equivalent PTX of 1 μg/mL) with and without the pretreatment of free HA. The apoptosis ratio represents the sum of the percentage of early apoptotic cells (Q4) and the percentage of apoptotic plus necrotic cells (Q2). **p < 0.01 and ***p < 0.001.

and intravenous irritation, HRA nanoparticles have a great potential in in vivo application as the carrier of antitumor drugs. Cell Apoptosis. Cell viability assay by MTT assay usually provides the information on cell death and survival.30 Therefore, to further assess the extent and mode of cell death, cell apoptosis induced by the nanoparticles was confirmed by Annxin V-FITC/PI double labeling assay. The investigated concentration of equivalent PTX was 1 μg/mL. As illustrated in Figure 5, PTX-loaded HRA nanoparticles could more significantly induce apoptosis than PTX plus ATRA plus HA solution in both tumor cells (p < 0.01). In particular, the percentages of early apoptotic cells were increased 2.0-fold for B16F10 cells and 1.5-fold for HepG2, respectively. In the competitive inhibitions studies, the cell apoptosis induced by PTX-loaded HRA nanoparticles was significantly reduced by the cell pretreatment with free HA. The down-regulation was 67.8% and 50.0% in B16F10 cells and HepG2 cells, respectively, demonstrating that high cell apoptosis induced by PTX-loaded HRA nanoparticles might contribute to HA receptor-mediated endocytosis. Noticeably, a number of studies have described that ATRA, a typical differentiation inducing agent, could also enhanced cytotoxic antitumor drugs-induced apoptosis in the differentiation-dependent or independent manner, for example, facilitating the down-regulation of Bcl-2 expression.31−34 Therefore, ATRA also played an important role in inducing cell apoptosis in this study, which will benefit the enhancement of antitumor efficacy of PTX. Cellular Uptake of the HRA Nanoparticles. A confocal microscopic study was conducted for the nanoparticles internalization. B16F10 cells were used to evaluate the selectivity of HA to tumor cells due to overexpressing CD44 receptor. Figure 6A showed fluorescence microscopy photographs of cells treated with free FITC and the FITC-loaded HRA nanoparticles. After 2 and 6 h of incubation with FITCloaded HRA nanoparticles, the green fluorescence was observed, and the fluorescence intensity increased correspondently with the incubation time, indicating that the nanoparticles could be internalized continuously.35 Moreover, the strong green fluorescence was observed in the nuclei of B16F10 after 6 h incubation with FITC-loaded HRA nanoparticles, which was also much higher than that of 2 h incubation. These results suggested that HRA nanoparticles could be taken up by cells via endocytic pathway and transport into the nucleus. Higher nuclear transport of HRA nanoparticles might be attributed to ATRA-induced nuclear translocation. Sessler et al. has described that ATRA could translocate into the nucleus by

Figure 6. Cellular uptakes without and with the pretreatment of 5 mg/ mL HA for 1.5 h. (A) Confocal microscopic images of B16F10 cells after 2 and 6 h incubation with free FITC and FITC-loaded HRA nanoparticles, respectively. (B) FACS graphs of FITC accumulation in B16F10 cells. (C) Positive cell percent by flow cytometry. Results were expressed as means ± SD (n = 3).

binding to specific cytosolic proteins including CRABP-II and FABP5.36 It was also associated with nuclear retinoid receptors 1087

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Figure 7. In vivo imaging analysis. (A) Typical in vivo noninvasive fluorescence images of tumor-bearing mice at different time. The NIR fluorescence images and X-ray images were fused together with Kodak Molecular Image Systems software V 5.0.1. (B) Representative ex vivo NIR fluorescence image of the tumor uptake of DiR-loaded HRA nanoparticles at 24 h after i.v. injection. Free DiR was as the control. (C) Tumor uptake at 24 h after i.v. injection obtained by the fluorescence intensity measurement method. All data are expressed as mean ± SD (n = 3). Arrows indicate the tumor sites.

in cells, which are ligand-activated transcription factors.37 On the other hand, the cells treated with free FITC showed little green fluorescence, indicating that free FITC could not be taken up by tumor cells. The quantitative fluorescence intensity in cells was determined by flow cytometry. The B16F10 cells were incubated with free FITC and the nanoparticles with equivalent FITC concentration, respectively. The results in Figure 6B,C showed that the positive cell percent of HRA nanoparticles was significant higher than that of free FITC (p < 0.05) (7.96 times higher). To further confirm the uptake of the nanoparticles by HA receptor-mediated endocytosis, a competitive inhibition experiment was performed by adding free HA in the growth medium to saturate the HA receptors on the surface of cancer cells. Results showed that the uptake efficiency of HRA nanoparticles was distinctly reduced through the pretreatment of the B16F10 cells by free HA (p < 0.01). The down-regulation was 72.8%, indicating that the HA receptor-mediated endocytosis facilitated the intracellular delivery of nanoparticle into B16F10 cell, as suggested in the study by Upadhyay et al. (2010), where the doxorubicin-loaded HA-based polymersomes showed successful uptake and high accumulation in the MCF-7 cells depending on HA receptor-mediated endocytosis.38 This HA-mediated endocytosis might also contribute to inverse the multidrug resistance (MDR) effect in tumor cells, which is a potential way to facilitate the transport of drugs into the tumor. In Vivo Imaging Analysis. To evaluate the biodistribution and tumor-targeting efficiency of the nanoparticles, a noninvasive near-infrared optical imaging technology was used in this study. The tumor cells were xenografted subcutaneously in the left armpit of BALB/c nude mice. In vivo fluorescent images were taken at different time points after administration. Figure 7A showed the real-time images of the free DiR (as the control)

and DiR-loaded HRA nanoparticles in the tumor-bearing mice. In vivo images showed that DiR-loaded HRA nanoparticles were obviously accumulated in the tumor, and the fluorescent signal at 0.5 h was the highest. Then the fluorescence signal gradually became weak as the time elapsed. However, the fluorescence signal still maintained stronger up to 12 h. On the contrary, little fluorescence of the free DiR in the tumor was observed. These results indicated that HRA nanoparticles could assist the drugs targeting to the tumor and prolong the circulation time of the drugs. As shown in Figure 7B which gave the ex vivo images, the tumor of mice treated with DiR-loaded HRA nanoparticles at 12 h after injection showed significant stronger NIR fluorescent signal than that treated with free DiR. The NIR fluorescent intensity from the tumors in HRA nanoparticles was 7.232 times (690.7 ± 34.49 versus 95.5 ± 12.3) greater than the control (p < 0.001), which further confirmed the availability of HRA nanoparticles for tumorspecific drug delivery. In Vivo Antitumor Activity. Owing to excellent tumor localization, prolonged circulation, coupled with good biocompatibility, PTX-loaded HRA nanoparticles was expected to possess superior antitumor efficacy and reduced side effects. Therefore, the in vivo antitumor efficacy was studied to provide more direct evidence for the antitumor potential of PTX-loaded HRA nanoparticles. As shown in Figure 8A,B, compared to the control, the tumor volumes in two treatment groups were significantly decreased after a schedule of multiple doses, indicating significantly effective inhibition of tumor growth. Notably, the PTX-loaded HRA nanoparticles yielded the most effective tumor growth inhibition activity. At 12 days after the first administration, PTX-loaded HRA nanoparticles had reduced tumor growth by 69.7% (258.4 ± 82.0 mm3 vs 852.9 ± 123.1 mm3, p < 0.005) while PTX plus ATRA plus HA solution was 35.4% (551.2 ± 108.4 mm3 vs 852.9 ± 123.1 mm3, 1088

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Figure 8. In vivo efficacy assay of PTX-loaded HRA nanoparticles. (A) Tumor grown curve of tumor-bearing mice after a schedule of multiple doses. Data were expressed as mean ± SD (n = 6). Tumors were measured every other day. ap < 0.05 and bp < 0.005 vs the control group, cp < 0.05 vs PTX plus ATRA plus HA solution. (B) Tumor weights of tumor-bearing mice after the treatment. Data were expressed as mean ± SD (n = 6). (C) Representative images of paraffin-embedded tumor sections after TUNEL (the apoptotic cells shown in green), PCNA (the proliferative cells shown in brown), and HE staining.

p < 0.05). Moreover, the tumor weights of mice treated with PTX-loaded HRA nanoparticles were significantly smaller than those in PTX plus ATRA plus HA group (p < 0.001), suggesting that the tumor targeting properties most likely contributed to the enhanced antitumor efficacy of the nanoparticles. Additional evidence of enhanced antitumor activity of PTXloaded HRA nanoparticles was obtained by observing DNA fragmentation in tumor cells after TUNEL and PCNA staining. As illustrated in Figure 8C, TUNEL assay and PCNA analysis revealed that PTX-loaded HRA nanoparticles resulted in greater induction of tumor cell apoptosis and inhibition of tumor cell proliferation. The tumor slides after the therapy were also stained with H.E. Compared to the control, some obviously necrotic regions distributed in the tumor slices of PTX plus ATRA plus HA solution. More notably, PTX-loaded HRA nanoparticle showed the bulk of necrosis, suggesting its outstanding antitumor efficacy once more. These results indicated that the therapy of PTX-loaded HRA nanoparticles was more efficacious in inducing the cell apoptosis, cell necrosis, and reducing the cell proliferation than the combination of free drugs, which might be attributed to nanoparticle-mediated greater uptake and accumulation in cancer cells. More importantly, the HRA nanoparticles, as a

successful codelivery system, can simultaneously deliver PTX and ATRA into the tumor, which will contribute to more efficiently additive or synergistic antitumor effects. Moreover, the characteristic properties of the nanoparticles also played a key role. These possible mechanisms were involved in several factors: (1) The particle sizes of HRA nanoparticles were about 160 nm. Some studies reported that nanostructure with less than 200 nm can passively target the tumor by the EPR effect as well as extended blood circulation time.39,40 (2) The HRA nanoparticles can keep stability without dissociation after i.v. injected into the larger volume of blood for systemic circulation due to lower CAC values of HRA.11 Finally, it has been reported that the HA even may be involved in tumor cell metastasis and inhibit the growth of cancer cells.41 Yin et al. have described that the combination of PTX and HA can produce additional or synergistic antimetastasis effects.42 Therefore, HA with the antitumor activity also contributed to higher antitumor effect besides the receptor-mediated endocytosis. The potential toxicity presents one of the major obstacles for cancer chemotherapy, especially during chronic administration. In this study, the potential toxicity of the formulations was determined by monitoring animal behavior and weight loss. No side effects, such as decreased body weight and noticeable 1089

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responsive and -resistant acute promyelocytic leukemia cells. Int. J. Hematol. 2005, 82, 215−223. (5) Pratt, M. A.; Niu, M. Y.; Renart, L. I. Regulation of survivin by retinoic acid and its role in paclitaxel-mediated cytotoxicity in MCF-7 breast cancer cells. Apoptosis 2006, 11, 589−605. (6) Karmakar, S.; Banik, N. L.; Ray, S. K. Combination of all-trans retinoic acid and paclitaxel-induced differentiation and apoptosis in human glioblastoma U87MG xenografts in nude mice. Cancer 2008, 112, 596−607. (7) Hong, G. Y.; Jeong, Y. I.; Lee, S. J.; Lee, E.; Oh, J. S.; Lee, H. C. Combination of paclitaxel- and retinoic acid-incorporated nanoparticles for the treatment of CT-26 colon carcinoma. Arch. Pharm. Res. 2011, 34, 407−417. (8) Park, K. M.; Kang, H. C.; Cho, J. K.; Chung, I. J.; Cho, S. H.; Bae, Y. H.; Na, K. All-trans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control. Biomaterials 2009, 30, 2642−2652. (9) Bae, Y.; Diezi, T. A.; Zhao, A.; Kwon, G. S. Mixed polymeric micelles for combination cancer chemotherapy through the concurrent delivery of multiple chemotherapeutic agents. J. Controlled Release 2007, 122, 324−330. (10) Garnett, M. C. Targeted drug conjugates: principles and progress. Adv. Drug Delivery Rev. 2001, 53, 171−216. (11) Hou, L.; Fan, Y.; Yao, J.; Zhou, J.; Li, C.; Fang, Z.; Zhang, Q. Low molecular weight heparin-all-trans-retinoid acid conjugate as a drug carrier for combination cancer chemotherapy of paclitaxel and alltrans-retinoid acid. Carbohydr. Polym. 2011, 86, 1157−1166. (12) Verschraegen, C. F.; Skubitz, K.; Daud, A.; Kudelka, A. P.; Rabinowitz, I.; Allievi, C.; Eisenfeld, A.; Singer, J. W. Oldham, F.B. A phase I and pharmacokinetic study of paclitaxel poliglumex and cisplatin in patients with advanced solid tumors. Cancer Chemother. Pharmacol. 2009, 63, 903−910. (13) Lammers, T.; Subr, V.; Ulbrich, K.; Peschke, P.; Huber, P. E.; Hennink, W. E.; Storm, G. Simultaneous delivery of doxorubicin and gemcitabine to tumors in vivo using prototypic polymeric drug carriers. Biomaterials 2009, 30, 3466−3475. (14) Shan, L.; Xue, J.; Guo, J.; Qian, Z.; Achilefu, S.; Gu, Y. Improved targeting of ligand-modified adenovirus as a new near infrared fluorescence tumor imaging probe. Bioconjugate Chem. 2011, 22, 567−581. (15) Yao, J.; Fan, Y.; Du, R.; Zhou, J.; Lu, Y.; Wang, W.; Ren, J.; Sun, X. Amphoteric hyaluronic acid derivative for targeting gene delivery. Biomaterials 2010, 31, 9357−9365. (16) Ito, T.; Iida-Tanaka, N.; Koyama, Y. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid. J. Drug Target. 2008, 16, 276−281. (17) Platt, V. M.; Szoka, F. Anticancer Therapeutics: Targeting Macromolecules and Nanocarriers to Hyaluronan or CD44, a Hyaluronan Receptor. Mol. Pharmaceutics 2008, 5, 474−486. (18) Jiang, G.; Park, K.; Kim, J.; Kim, K. S.; Oh, E. J.; Kang, H.; Kang, H.; Han, S. E.; Oh, Y. K.; Park, T. G.; Hahn, S. K. Hyaluronic acid −polyethyleneimine conjugate for target specific intracellular delivery of siRNA. Biopolymers 2008, 89, 635−641. (19) Auzenne, E.; Ghosh, S. C.; Khodadadian, M.; Rivera, B.; Farquhar, D.; Price, R. E.; Ravoori, M.; Kundra, V.; Freedman, R. S.; Klostergaard, J. Hyaluronic acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian carcinoma xenografts. Neoplasia 2007, 9, 479−486. (20) Luo, Y.; Ziebell, M. R.; Prestwich, G. D. A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells. Biomacromolecules 2000, 1, 208−218. (21) Zhang, Y.; Huo, M.; Zhou, J.; Yu, D.; Wu, Y. Potential of amphiphilically modified low molecular weight chitosan as a novel carrier for hydrophobic anticancer drug: Synthesis, characterization, micellization and cytotoxicity evaluation. Carbohydr. Polym. 2009, 77, 231−238. (22) Zhu, J.; Liao, L.; Bian, X.; Kong, J.; Yang, P.; Liu, B. pHcontrolled delivery of doxorubicin to cancer cells, based on small mesoporous carbon nanospheres. Small 2012, 8, 2715−2720.

change in activity, were observed in PTX-loaded HRA nanoparticles. On the contrary, compared to the control group, the PTX plus ATRA plus HA solution exhibited significant body weight loss (p < 0.05), while the motion of the mice became slowly with rapid breath after i.v. administration and lasted for 10−15 min. Moreover, the histopathological changes in liver, spleen, kidney, lung, and heart from PTXloaded HRA nanoparticles were examinated by HE staining (data not shown). No tissues damage in the investigated tissues was observed, suggesting better safety for clinical application. Overall, it was indicated that these nanoparticles with excellent therapeutic effects as well as lower toxicity would greatly improve the patient’s quality of life.

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CONCLUSIONS In this study, we synthesized an amphiphilic HRA conjugate by an imine reaction. It can self-assemble into the HRA nanoparticles which have extremely high PTX-loaded capacity, nanoscale particle size with narrow size distribution, and low toxicity. Based on HA receptor-mediated endocytosis and ATRA-induced nuclear translocation as well as EPR effects, HRA nanoparticles could be efficiently taken up by cells via endocytic pathway, transport into the nucleus and finally assist the drugs targeting to the tumor. Moreover, PTX-loaded HRA nanoparticles demonstrated greater inhibition of tumor growth in vivo with reduced toxicity. Therefore, HRA nanoparticles can be considered as a promising targeted codelivery system for combination cancer chemotherapy.

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AUTHOR INFORMATION

Corresponding Author

*J.Y.: e-mail: [email protected]. tel.: +86 25 83271102; fax: +86 25 83301606. J.Z.: e-mail: [email protected]; tel.: +86 25 83271102; fax: +86 25 83301606. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81173006), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGQ201107), the Qing Lan Project, and the National Basic Research Program of China (No. 2009CB930303).

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