Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery

BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 371 – 381

Original Article www.nanomedjournal.com

Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery Qin Wang, MMeda , Liang Zhang, PhDb , Wei Hu, MMeda , Zhan-Hong Hu, MMeda , Yong-Yan Bei, BSa , Jing-Yu Xu, BSa , Wen-Juan Wang, BSa , Xue-Nong Zhang, PhDa,⁎, Qiang Zhang, PhDc a

Department of Pharmaceutics, School of Pharmacy, Soochow University, Suzhou, China Department of Biopharmaceutics, School of Pharmacy, Soochow University, Suzhou, China c Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Received 14 May 2009; accepted 13 July 2009 b

Abstract In this study a new chitosan (CS) derivative, galactosylated chitosan (GC), was synthesized and used to prepare norcantharidin-associated GC nanoparticles (NCTD-GC NPs) by taking advantage of the ionic cross-linkage between the molecules of the anti-hepatocarcinoma medicine NCTD and of the GC as carrier. NCTD-GC NPs were obtained with average particle size of 118.68 ± 3.37 nm, entrapment efficiency of 57.92 ± 0.40%, and drug-loading amount of 10.38 ± 0.06%. Several important factors influencing the entrapment efficiency, drug-loading amount, and particle size of NCTD-GC NPs were studied. The characteristics of sustained and pH-sensitive release of NCTD from NCTD-GC NPs in vitro were studied. In addition, in vitro cellular uptake and cytotoxicity of nanoparticles to hepatoma cell lines SMMC-7721 and HepG2 were also investigated. In vitro, and compared to CS-based NCTD-CS NPs, NCTD-GC NPs demonstrated satisfactory compatibility with hepatoma cells and strong cytotoxicity against hepatocellular carcinoma cells. In vivo antitumor activity of NCTD-GC NPs was evaluated in mice bearing H22 liver tumors. NCTD-GC NPs displayed tumor inhibition effect in mice, better than either the free NCTD or the NCTD-CS NPs. As a hepatocyte-targeting carrier, GC NPs are potentially promising for clinical applications. From the Clinical Editor: In this paper, a galactosylated chitosan (GC), was synthesized and norcantharidin (NCTD)-associated galactosylated chitosan nanoparticles (NCTDGC NPs) were generated by coupling NCTD - an anti-hepatocarcinoma drug - and GC as carrier. Compared to chitosan nanoparticles, NCTD-GC-NPs demonstrated satisfactory compatibility with hepatoma cells and strong cytotoxicity against the cells. © 2010 Elsevier Inc. All rights reserved. Key words: Galactosylated chitosan; Nanoparticles; Cellular uptake; Hepatocyte-targeted delivery; Norcantharidin

The work was supported by National Key Technology R&D Program in the 11th Five Year Plan of China (2006BAI09B00), the Innovation Fund of Technology-based Small and Medium-sized Enterprises by Ministry of Science and Technology (07C26223201333), “the Project of High and New Technology Research” from the Education Department of Jiangsu Province (JHB05-46) and grant from the Health Department of Jiangsu Province (H200630). ⁎Corresponding author: Department of Pharmaceutics, School of Pharmacy, Dushuhu Campus of Soochow University, Suzhou City, Jiangsu Province 215123, China. E-mail address: [email protected] (X.-N. Zhang).

Norcantharidin (NCTD), a demethylation derivative of cantharidin, is one of the new chemotherapy agents that have been shown to be very effective against cancer, especially against primary hepatic carcinoma.1 Administered clinically by the oral or the intravenous route, NCTD is effective against primary carcinoma of the liver as an inhibitor of protein phosphatase 1 and protein phosphatase 2A.2,3 However, this clinical application of NCTD has been limited by a serious side effect: intense irritation at the injection site and the urinary organs.4,5 The maximum solubility of NCTD in water is 2.5 mg mL–1 at pH 6.0, and it increases to 9.5 mg mL–1 at pH 9.5. Clinically, NCTD is mostly given by injection of its sodium salt (5 mg mL–1, 2 mL) at a pH of about 9.0. This high pH of the NCTD solution is an important cause of the irritations. To

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2009.07.006 Please cite this article as: Q., Wang, et al, Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery. Nanomedicine: NBM 2010;6:371-381, doi:10.1016/j.nano.2009.07.006

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improve the safety and efficacy of NCTD treatment, many new alternative formulations, such as microspheres, microemulsions, liposomes, and nanoparticles (NPs), have been studied to improve the targeted delivery of NCTD.6-8 Chitosan (CS), as a nontoxic and biodegradable polycationic polymer with low immunogenicity, has been extensively investigated as a formula carrier and in delivery systems for therapeutic molecules, such as genes, protein molecules, vaccines, and cyclosporine A.9,10 One of the reactions used to modify CS is the quaternization of the amino group at C-2 position or its reaction with a –CHO via reductive amination.11,12 Morimoto et al reported the synthesis of sugar-bound CS, such as with D- and L-fucose, and the specific interactions with cells.13,14 Stredanska et al synthesized lactosemodified CS for a potential application in the repair of the articular cartilage.15 Lactosaminated N-succinyl-CS was found to be a good drug carrier for mitomycin C in treatment of liver metastasis.16 Moreover, Kato et al also prepared lactosaminated N-succinyl-CS and its fluorescein thiocarbanyl derivative as a liver-specific drug carrier in mice through the asialoglycoprotein receptor (ASGP-R).17 NPs with moderate particle size can be delivered to specific sites by size-dependent “passive” targeting. It has been reported that NPs injected intravenously were taken up by the reticuloendothelial system in liver after only a few minutes as a result of the opsonization process. When the diameter is less than 200 nm, these NPs can be captured easily by the reticuloendothelial system, especially by the Kupffer cells in the liver. CS-NPs have been prepared by many methods, such as emulsion cross-linking, coacervation, spray-drying, emulsiondroplet coalescence, ionic gelation, and self-assembled methods.18 A large number of research studies on CS-NPs for targeting and controlled drug release have been reported.19,20 The ASGP-R, located on the hepatocellular carcinoma cell membrane, can specifically recognize β-D-galactose or Nacetylgalactosamine residue of desialylated glycoproteins.21,22 For this reason, the ASGP-R has been exploited as a hepatocyte-specific targeting marker for drug and gene delivery.23-25 Considerable effort has been directed to the use of galactose as a ligand of hepatocyte-specific targeted agents. To obtain higher selectivity and enhance the ligand-mediated endocytosis and NP uptake by the targeted cells, active targeting can be integrated with the passive targeting to enhance hepatocyte-specific delivery.26 We have synthesized galactosylated chitosan (GC), and NCTD-GC NPs to be tested as a hepatocyte-targeted carrier for NCTD. In addition, in vitro release characteristics of NCTD from the NCTD-GC NPs have been investigated in various media. Both in vitro and in vivo antitumor activity of NCTD-GC NPs have been studied, including cellular uptake and cytotoxicity on human hepatoma cell lines SMMC-7721 and HepG2 in vitro, and in vivo tumor-inhibitory activity was investigated in mice bearing H22 hepatoblastoma tumors. The two hepatoma cell lines SMMC-7721 and HepG2 have been selected as models, because both of them are known to express ASGP-R.27,28 The aim of this study is to develop a hepatocyte-targeted delivery for NCTD and confirm its targeting characteristics.

Methods Materials Human hepatoblastoma SMMC-7721, HepG2 cell lines were obtained from Jiangsu Province Key Laboratory of Biotechnology and Immunology (Suzhou, China). SMMC-7721 and HepG2 cell lines were maintained in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum, heatinactivated (56°C, 30 minutes), and 100 U·ml–1 penicillin G– streptomycin at 37°C in a humidified incubator (5% CO2). CS (molecular weight [MW] 8–10 kDa, deacetylation degree [DD] 93.1%) was purchased from Jiangsu Nantong Xingcheng Biological Product Factory (Nantong, China); 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N,N,N',N'-tetramethylethylenediamine (TEMED), 5-isothiocyanato fluorescein, fetal bovine serum, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Nhydroxysuccinimide, and dimethylsulfoxide were purchased from Sigma-Aldrich (St. Louis, Missouri); NCTD (lot no. 20060508) was from Suzhou Surui Pharmaceutical and Chemical Co. (Suzhou, China); sodium tripolyphosphate (TPP) was supplied from Sinopharm Chemical Reagent Co. (Shanghai, China); acetonitrile was obtained from Merck Co. (Chromatographic Grade; Darmstadt, Germany). All other chemicals were of analytical grade and used without further purification. Six- to eight-week-old Kunming mice weighing 18–22 g were supplied by the Experimental Animal Center, Soochow University (Suzhou, China). Mice were kept at 40% humidity at room temperature (25°C). All the animal experiments adhered to the principles of care and use of laboratory animals and were approved by the Experimental Animal Center, Soochow University. Mice bearing the H22 hepatoblastoma cell line (supplied by Fudan University, China) were established as follows: H22 cells were reanimated and centrifuged, and the supernatant was discarded. Cells were then resuspended in physiological saline and injected subcutaneously into the mice for serial subcultivation. All chemicals, reagents, and solvents in the present study were the highest grade available and used as directed. Synthesis of GC GC was synthesized as described by Chung et al.29 Briefly, a solution of sodium lactobionate was passed through a cation exchange resin column (Model 732, TianDiao ShuZhi Co., Shanghai, China) to convert the salt to free lactobionic acid. The eluted free acid was vacuum-dried. CS (108 g, 115.38 mmol) was dissolved in 2.5 L of 1% HCl (vol/vol) solution. Subsequently, lactobionic acid (41.4 g, 115.38 mmol), EDC·HCl (26.46 g, 134.82 mmol), and N-hydroxysuccinimide (13.32 g, 115.38 mmol) were added into the solution. Then the pH of the reaction solution was regulated to 4.5 by TEMED. The reaction was performed for 72 hours at room temperature. The resulting product was purified by using a dialysis tube (3500 MW cutoff) against distilled water for 4 days. An excess of acetone was added dropwise into the dialysate, and then the product precipitated. After lyophilization, a light-yellow powder was obtained. The chemical structure of GC was confirmed through

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Figure 1. Synthesis scheme of galactosylated chitosan (GC).

Fourier transform–infrared (FT-IR) (Varian, Palo Alto, California) and 1H–nuclear magnetic resonance (1H-NMR) spectroscopy (Varian; 400 MHz) (Figure 1). The degree of substitution of galactose residues in GC (DSGC) estimated by 1H-NMR was calculated as follows:

remove insoluble aggregate residues to give a solution of NCTDGC NPs. Four factors (DD of CS, pH, temperature, and weight proportion of NCTD to GC) were constantly monitored to optimize the technique for preparation of NCTD-GC NPs.

  DSGC = HGC ðd3:37f4:16Þ  HCS ðCSd3:32f3:89Þ =12  100k

Evaluation of NCTD-GC NPs

where HGC(δ3.37∼4.16) was the number of hydrogen atoms of GC at δ3.37∼4.16, and HCS(δ3.32∼3.89) was the number of hydrogen atoms of CS at δ3.32∼3.89. Preparation of NCTD-GC NPs NCTD-GC NPs were prepared according to the procedure first reported by Calvo et al,30 which was based on the ionic gelation of GC with TPP anions and slightly modified. Briefly, GC was dissolved in acetic acid aqueous solution (0.2% vol/vol) containing NCTD, and 20 mL of 1.2 mg mL–1 TPP aqueous solution were added to the GC solution drop by drop under magnetic stirring (500 rpm), resulting in cross-linkage. Finally, the opalescent suspension was filtered through a 0.45-μm filter to

The particle distribution and zeta potential of NCTD-GC NPs was measured by Zetasizer nanoparticle analyzer (HPP-5001; Malvern Instruments, Worcestershire, United Kingdom), which works on the principle of laser diffraction analysis. The shape characteristic of NCTD-GC NPs was determined by transmission electron microscopy (TEM) analysis, which was performed using a Hitachi instrument (Hitachi; Tokyo, Japan). The drug entrapment efficiency (EE) and drug-loading (DL) amount of NCTD-GC NPs were determined as described by Zhang et al. 31 Generally, NCTD-GC NPs solution was ultracentrifuged by Optima MAX Centrifuge (Beckman Coulter, Fullerton, California) at 40,000 rpm for 45 minutes. The supernatant was sampled, and the concentration of NCTD in the supernatant was determined by a reversed-phase highperformance liquid chromatography (HPLC) method. A 20-μL

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Figure 2. Fourier transform–infrared spectra of (A) chitosan, (B) galactosylated chitosan (GC), and (C) norcantharidin (NCTD)-GC nanoparticles.

aliquot of supernatant was injected into a chromatograph equipped with an ultraviolet detector (LC-10AT, SPD-M10A; Shimadzu, Kyoto, Japan) and reversed-phase column (Hypersil ODS-C18, 4.6 × 250 mm, 5 μm; Thermo, Waltham, Massachusetts). The mobile phase was a mixture of acetonitrile and H2O (ratio 10:90, adjusted to pH 3.1 by adding phosphoric acid), and the flow rate was 0.8 mL min–1 with the wavelength of 210 nm at 40°C. The NCTD EE and DL amount were calculated as follows:

EE =

T F T L  100k DL =  100k T W

where T was total NCTD in the colloid, F was free NCTD in the supernatant, and W was the weight of NCTD-GC NPs. In vitro drug release assay The drug release from NCTD-GC NPs was measured by dialysis. Briefly, 10 mL NCTD-GC NP solution was placed into a dialysis tube (8000–14,000 MW cutoff) and the tube sealed at both ends with clips. The NP-loaded dialysis tube was then placed into a beaker containing 60 mL of medium and stirred for 6 hours at 37 ± 0.5°C. At various time points, aliquots were withdrawn from the beaker and replaced with equal volumes of the medium. Ten-milliliter aliquots of NCTD-GC NP solution were placed into a measuring flask, mixed with the same amount of medium, and then digested by 88% methanoic acid as total dosage. The NCTD concentrations were then measured by HPLC. The release medium contained 0.1 M HCl, phosphate-

buffered saline (PBS) pH = 5.4, PBS pH = 6.8, PBS pH = 7.4, or physiological saline, for comparison to NCTD. Fluorescein isothiocyanate labeling of GC NCTD-GC NPs were fluorescence-labeled with fluorescein isothiocyanate (FITC) so that their cellular uptake could be detected by flow cytometry. The FITC-labeled GC was prepared by the reaction between the isothiocyanate group of FITC and the primary amino group of GC, which was described by Huang et al.32 In this study, GC was dissolved in 10 mL of 0.1 M acetic acid aqueous solution, and 10 mL of carbinol was added to the solution. This was mixed with 20 mL of FITC-carbinol solution, and the reaction was performed for 3 hours under magnetic stirring in darkness at room temperature. The product was precipitated by adding 0.2 M sodium hydroxide into the reaction mixture dropwise. The precipitated solid was obtained by centrifugation at 3000 rpm for 10 minutes and was washed with cold carbinol-aqua mix solution (70:30 vol/vol). The centrifugation and washing steps were repeated several times, until fluorescence (λex = 485 nm, λem = 535 nm) could not be detected in the supernatant. The fluorescence-labeled product FITC-GC was dehydrated with pure carbinol and freeze-dried in dark. Similarly, FITC-CS, NCTD-GC NPs, FITC-labeled NCTD-GC NPs, and NCTD-CS NPs were also prepared in a dark room. In vitro cellular uptake The exponential-growth-phase SMMC-7721 and HepG2 cells were incubated in culture flasks to a density of 105 cells

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per flask and divided into the following groups: control group (blank culture medium), FITC-labeled NCTD-CS NPs, and FITC-labeled NCTD-GC NPs in various concentrations. The cells were incubated (37°C, 5% CO2) for several hours, then washed three times with PBS, resuspended in fresh culture medium, and placed in an incubator for 30 minutes to allow the cell membranes to recover. After washing twice with cold PBS, cells were transferred into tubes and immediately placed on ice. The intracellular fluorescence intensity was determined on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, California). Approximately 10,000 event cells were evaluated to determine the mean fluorescence intensity of FITC-labeled NPs taken up by cells. Measurements were repeated three times. In vitro cytotoxicity of NCTD-GC NPs The in vitro antineoplastic activity of NCTD-GC NPs was determined by their cytotoxicity on SMMC-7721 and HepG2 cells using MTT assay. The cells in their logarithmic growth were seeded at a density of 104 cells per well onto 96-well plates and incubated for 24 hours, then exchanged for fresh medium. Cells were then treated with 100-μL sterile samples (NCTD, NCTD-CS NPs, and NCTD-GC NPs) at gradient molarities (0.8, 4, 20, 100, and 500 µg/mL). The cells were next incubated for 48 hours. After incubation, 20 μL of 5 mg·mL–1 MTT solution in PBS were added to each well, and the plate was incubated for another 4 hours, supernatant fluid was removed carefully, then 100 μL dimethylsulfoxide were added to each well. After the formazan crystal dissolved completely, the optical density at 490 nm (A490nm) was determined with a Model ELx808 microplate reader (Bio-Tek, Winooski, Vermont). The inhibition rate (IR) of the treated cells was defined as follows:  IR = 1 

A490ðtreatedÞ A490ðnon−treatedÞ

  100k

A490nm(treated) and A490nm(nontreated) were A490nm of the treated cells and untreated cells, respectively. In vivo antineoplastic activity of NCTD-GC NPs The in vivo antineoplastic study followed the methods of Shimura et al and Xiong et al with a few modifications.33,34 Briefly, after serial subcultivation of the H22 cells for 7 days, the mice with viable H22 ascites tumors were killed by cervical dislocation under the condition of sterility. The ascites were withdrawn and diluted with physiological saline to modulate the cell density at l × 107 mL–1. The ascites tumor cells were injected subcutaneously into the right hindlimb of mice at a dose of approximately 0.1 mL 10 g–1. After inoculation the mice were assigned at random to different experimental groups, including control group (physiological saline) and medication administration groups (NCTD, NCTD-CS NPs with low dose, NCTD-CS NPs with moderate dose, NCTD-CS NPs with high dose, NCTDGC NPs with low dose, NCTD-GC NPs with moderate dose, NCTD-GC NPs with high dose). They were given an intraperitoneal injection of the various formulations 24 hours after tumor cell inoculation daily for 8 days, and tumor size was measured every day with a vernier calipers. Individual tumor

Figure 3. 1H-nuclear magnetic resonance spectra of (A) chitosan and (B) galactosylated chitosan.

volumes (V) were calculated by the formula: V = [length × (width)2]/2. Then the mice were weighed before being killed by cervical dislocation the day after administration termination, and the subcutaneous tumor, spleen, and thymus were minced carefully and weighed. The inhibition rate on tumor weight (IRw) and immune organ coefficient (OC) were tested as follows:   WM WO ðmgÞ IRw = 1   100k OC = WC WB ð gÞ where WM and WC were tumor weight of the drug-administered group and control group, respectively. WO and WB were weight of organ and body, respectively. Statistical methodology Values are expressed as mean ± SD. Data were analyzed by one-way analysis of variance with the post hoc Tukey's test applied for paired comparisons (SPSS 16; SPSS, Chicago, Illinois), and the criteria for statistical significance were taken as *P b .05, **P b .01, or #P b .05, ##P b .01 (see Figures 9 and 10). Results Synthesis of GC Figure 2 shows the FT-IR spectra of CS, GC, and NCTDGC NPs. CS has characteristic amide absorptions at 1665 and 1592 cm–1. The very weak amide I band and the remarkably strong amide II band indicates the presence of abundant free

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Figure 4. The influence of diverse factors on entrapment efficiency (EE, —□—), drug-loading amount (DL, —○—), particle size (—△—), and polydispersity index (PDI, —▽—) of nanoparticles, including (A) degree of deacetylation (DD) of chitosan (CS), (B) pH, (C) temperature, and (D) weight proportion of norcantharidin (NCTD) to galactosylated chitosan (GC) (n = 3).

amino groups in the CS molecule. In GC the amide bands II and I shift to 1658 cm–1 and 1593 cm–1, respectively, indicating the new amide bond formation. For NCTD-GC NPs (Figure 2, C), changes in the amine absorption bands were detected. These spectral changes were attributed to the electrostatic interaction between GC amine and TPP phosphoric groups.35 Figure 3 shows the 1H-NMR spectra of CS and GC (D2O/F3CCOOD, 400 MHz). CS (Figure 3, A), δ (ppm): 4.59 (H1), 2.91 (H2), 3.32–3.89 (H3, H4, H5, H6), 1.92 (NHCOCH3); GC (Figure 3, B), δ (ppm): 4.72 (H1), 4.43 (H1′, Hc), 3.37– 4.16 (H3, H4, H5, H6, H2′, H3′, H4′, H5′, H6′, Ha, Hb, Hd, He), 3.02 (H2), 1.92 (NHCOCH3) (mark of H shown in Figure 1). The degree of substitution of galactose residues in GC estimated by 1H-NMR was about 8.92%. Preparation and evaluation of NCTD-GC NPs NCTD-GC NPs were prepared through an ionic cross-linking process and evaluated by the EE, DL, particle size, and

Figure 5. Particle size distribution of norcantharidin–galactosylated chitosan nanoparticles.

polydispersity index (PDI) of NPs. The four most important factors (DD of CS, pH, temperature, weight proportion of NCTD to GC) were investigated in detail.

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Figure 6. TEM micrograph of norcantharidin–galactosylated chitosan nanoparticles.

Figure 8. Cellular uptake of SMMC-7721 and HepG2 cell lines at (A) different incubation times with 6 μg mL–1 nanoparticles (NPs). (B) Different NP concentrations after 5 hours' incubation (n = 3). SMMC-7721 with norcantharidin-chitosan (NCTD-CS) NPs (—●—), HepG2 with NCTD-CS NPs (—○—), SMMC-7721 with norcantharidin–galactosylated chitosan (NCTD-GC) NPs (—■—), HepG2 with NCTD-GC NPs (—□—).

Figure 7. In vitro drug release of norcantharidin–galactosylated chitosan nanoparticles (NCTD-GC NPs) in various media with comparison to NCTD including 0.1 M HCl for NCTD (—●—), 0.1 M HCl for NCTD-GC NPs (—○—), physiological saline for NCTD (—■—), physiological saline for NCTD-GC NPs (—□—), phosphate-buffered saline (PBS) pH 5.3 for NCTD (—▲—), PBS pH 5.3 for NCTD-GC NPs (—△—), PBS pH 6.8 for NCTD (—▼—), PBS pH 6.8 for NCTD-GC NPs (—▽—), PBS pH 7.4 for NCTD (—◀—), and PBS pH 7.4 for NCTD-GC NPs (—◁—).

Chitosan's DD is a pivotal quality, which had an important influence on its application. In fact, we purchased two kinds of CS with different DD values, but the same mean MW. The DDs of CS were in ranges from 77.79 ± 3.34% to 93.06 ± 2.38% (n = 6). GC was synthesized and NPs were prepared by using the two different CS materials as drug carriers, and the three indexes of NCTD-GC NPs were evaluated. The preparation conditions were identical (100 mg of GC or CS, 20 mg of NCTD, temperature 40°C), and the results are shown in Figure 4, A. It is thus evident that the CS DD value has a marked impact on the EE of NPs and no effect on the particle size. The higher the DD of CS used, the higher the EE value of the NPs we obtained. This characteristic arose because CS or GC with high DD values contained more free amino groups, which dissociated to more cationic amidogen for cross-linking with anionic NCTD in acid

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Figure 10. Tumor growth in H22 tumor-bearing mice at 2.0 mg/kg-1 dosage of norcantharidin (NCTD) (n = 10). Physiological saline (—□—); NCTD (—○—); NCTD-CS NPs middle dose (—△—); NCTD-GC NPs middle dose (—▽—). **P b 0.01 vs NCTD group; *P b 0.05 vs NCTD group; ##P b 0.01 vs NCTD-CS NPs group; #P b 0.05 vs NCTD-CS NPs group.

Figure 9. In vitro cytotoxicity of norcantharidin (NCTD) (▨), NCTDchitosan nanoparticles (NCTD-CS NPs) (▧), and NCTD–galactosylated chitosan NPs (NCTD-GC NPs) (▩) on (A) SMMC-7721 and (B) HepG2 cell lines (n = 3). **P b .01 vs NCTD group; *P b .05 vs NCTD group; ##P b .01 vs NCTD-CS NPs group; #P b .05 vs NCTD-CS NPs group.

aqueous solution. However, the EE value of NCTD-GC NPs was lower than NCTD-CS NPs because of the introduction of galactose residues. CS and GC were not so stable when pH value was higher than 5.0 in aqueous acetic acid solutions (Figure 4, B). The results with GC were mainly matched to CS, and there was no variance among the particle sizes at various pH values. Nevertheless, EE of NCTD-CS NPs reached its highest value at pH = 4. Consequently, the EE of NCTD-GC NPs cannot be increased further because of the competition between acetic acid and NCTD for the primary amine groups of GC. Temperature always has an important role in the preparation of CS NPs, as Wu et al pointed out.36 In our study, NCTD-GC NPs were prepared at temperatures ranging from 25°C to 40°C, and particle size and PDI were evaluated. Figure 4, C shows that at 100 mg of GC, 20 mg of NCTD, pH = 4, the medium

temperature is optimal for the preparation of NCTD-CS NPs, and that larger NCTD-GC NPs form at higher temperature. Also studied was the influence of NCTD-to-GC weight proportion on NCTD-CS NPs with the weight proportion changed from 10% to 25%. The conditions were 100 mg of GC, temperature 40°C, pH = 4 (results are shown in Figure 4, D). It is obvious that the weight proportion of NCTD to GC has an important effect on DL, and no clear impact on particle size. However, the weight proportion has no obvious influence on EE of NCTD-GC NPs at lower level, and the EE is actually reduced when the proportion is high enough to lead to saturation of ion cross-linking between GC-NH+3 and the anionic NCTD. To sum up the above arguments, we prepared NCTD-GC NPs with high DD of CS and under optimized conditions: (100 mg of GC, pH = 4, 30°C temperature, and weight proportion of NCTD to GC of 20%). All experiments were repeated five times. The EE and DL amount of NCTD-GC NPs were 57.92 ± 0.40% and 10.38 ± 0.06%, respectively. As a comparison, the EE of NCTD-CS NPs was 54.64 ± 3.71%. The NCTD-GC NPs had a mean particle size of 118.7 ± 8.84 nm and PDI of 0.180 ± 0.051, as shown in Figure 5. The zeta potential of NCTD-GC NPs was 27.37 ± 3.62 mV, whereas that of NCTD-CS NPs was 30.08 ± 3.95 mV because of the decreased number of surface positive charges after CS modification. However, there was no statistically relevant difference between the zeta potential results of NCTD-CS NPs and NCTD-GC NPs. A TEM image of NCTD-GC NPs is shown in Figure 6. Most of the NPs appear uniform and round under TEM, and the particle size is similar to the results obtained by dynamic light scattering. In vitro drug release of NCTD-GC NPs The in vitro NCTD release data are presented in Figure 7. NCTD is released quickly and completely in 0.1 M HCl, and

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Table 1 Body weight, inhibition rate (IRw) on tumor weight and immune organ coefficient in H22 tumor-bearing mice⁎ Groups

Dose (mg·kg–1)

Body weight (g) Start

Normal saline NCTD NCTD-CS NPs

NCTD-GC NPs

– 2.0 0.5 2.0 4.0 0.5 2.0 4.0

23.5 ± 23.4 ± 23.1 ± 24.0 ± 23.1 ± 23.6 ± 23.6 ± 22.9 ±

Sacrifice 2.1 2.5 2.2 2.5 1.4 3.4 1.9 2.8

32.9 ± 3.2 28.8 ± 2.6 30.9 ± 2.0 30.1 ± 2.8 27.4 ± 2.2 30.6 ± 3.7 28.8 ± 2.5 27.2 ± 1.4

Tumor weight (g)

IRw (%)

Spleen coefficient (10–3)

Thymus coefficient (10–3)

1.688 ± 0.354 1.179 ± 0.386⁎⁎ 1.313 ± 0.292⁎ 1.191 ± 0.375⁎⁎ 0.777 ± 0.268⁎⁎ 1.289 ± 0.385⁎ 0.940 ± 0.297⁎⁎,# 0.661 ± 0.325⁎⁎

– 30.15 22.21 29.47 53.98 23.64 44.33 60.81

8.64 ± 1.10 8.44 ± 1.02 8.43 ± 1.14 8.88 ± 0.84 8.82 ± 1.02 8.95 ± 1.29 8.79 ± 1.09 8.10 ± 1.26

1.81 ± 0.52 2.20 ± 0.67⁎ 2.54 ± 0.38⁎⁎ 2.05 ± 0.59 1.94 ± 0.53 2.23 ± 0.34⁎ 2.18 ± 0.43⁎ 1.80 ± 0.46

CS, chitosan; GC, galactosylated CS; NCTD, norcantharidin; NPs, nanoparticles. ⁎ P b .05 vs normal saline group. ⁎⁎ P b .01 vs normal saline group # P b .05 vs NCTD group at 2.0 mg·kg–1.

slower and incompletely in physiological saline and phosphate-buffered saline (PBS) pH 5.3. The effect of sustained drug release of NCTD-GC NPs in various media was evident with comparison to NCTD in PBS pH 6.8 and PBS pH 7.4. We found that in vitro release of NPs in diverse media followed the Higuchi equation37,38 (data not shown) by data fitting (r N 0.9900). In vitro cellular uptake of NCTD-GC NPs The FITC-labeled NCTD-GC NPs had a mean particle size of 124.6 ± 9.92 nm and 26.91 ± 3.32 mV of zeta potential, and the difference between NCTD-GC NPs and FITC-labeled NCTD-GC NPs was statistically insignificant. Figure 8 shows the in vitro cellular uptake for various incubation times using different tumor cells treated with diverse concentrations of FITC-labeled NPs. The mean fluorescence intensity of NCTDGC NPs was higher than the control NCTD-CS NPs at any concentration and incubation time. Specifically, Figure 8, A shows the cellular uptake as a function of incubation time using the two studied cell lines treated with 6 μg/mL of NCTD-GC NPs and NCTD-CS NPs. The mean fluorescence intensity of NCTD-GC NPs increased sharply within the initial 5 hours of incubation, and the process became saturated after 16 hours, whereas the uptake of the control NCTD-CS NPs increased progressively as the incubation time was extended. As shown in Figure 8, B, the mean fluorescence intensity of NCTD-GC NPs was higher than that of NCTD-CS NPs at each concentration. Accordingly, the cellular uptake of NCTD-GC NPs (calculated from the mean fluorescence intensity) increased sharply at low concentration values, whereas it increased slowly at higher concentrations of NCTD-GC NPs. However, the uptake of NCTD-CS NPs increased nearly linearly through all the experimental concentrations. In vitro cytotoxicity of NCTD-GC NPs Cytotoxicity was experimentally determined with MTT at different concentrations of NCTD (1000, 100, 10, 1, and 0.1 μg mL–1), and the half-maximal inhibitory concentration (IC50) of NCTD to various cell lines was calculated by linear regression. The IC50 values to SMMC-7721 and HepG2 were 31.43 ± 6.12

μg mL–1 and 57.88 ± 9.54 μg mL–1, respectively. In vitro cytotoxicity results at different dosages of NPs (500, 100, 20, 4, and 0.8 μg mL–1) are shown in Figure 9. It is obvious that NCTD and both types of NPs inhibited the growth of hepatocarcinoma cells in vitro, which was in direct proportion to the dosage, and statistically significant differences of inhibition rates among the various formulations were shown. NCTD-GC NPs display the highest inhibition level, followed by NCTD-CS NPs. Galactosylation of CS-NPs increases the in vitro cytotoxicity of NCTD to hepatoma cells, as a result of the better availability of NCTDGC NPs to hepatoma cells through receptor-mediated endocytosis, as is illustrated above under “In vitro cellular uptake of NCTD-GC NPs”. In vivo antineoplastic activity of NCTD-GC NPs The in vivo antineoplastic activity of NCTD-GC NPs was evaluated in mice bearing H22 hepatoblastoma tumors. The tumor growth curve measured with a vernier calipers is shown in Figure 10. All experimental materials were effective in preventing tumor growth as compared with the treatment with physiological saline. Treatment with NCTD-GC NPs displayed stronger tumor inhibition than treatment with either NCTD or NCTD-CS NPs. The body weights of mice after killing (Table 1) show that all mice gained weight during the experiment, although high doses resulted in less weight gain due to the toxicity of the antitumor drugs. Inhibition rate in relation to tumor weight in response to the above treatments was also determined. The results (Table 1) corroborate the tumor growth curve showing NCTD-GC NPs to be the most effective drug in vivo followed by NCTD-CS NPs. Compared to free NCTD, only NCTD-GC NPs express a statistically significant difference at 2.0 mg kg–1. We conclude that the delivery of NCTD in galactosylated CS-NPs improves tumor-inhibitory activity in vivo. There was no significant difference among spleen coefficients of various formulations, as is shown in Table 1. It was apparent that NCTD and NPs had no effect of immunological suppression on spleen. However, there are significant statistical differences between thymus coefficients of the control group and those of low-dose groups. The immunoregulation effect of NCTD and its NP formulations preserved thymus immunity and enhanced immunity at a low dose.

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Discussion Lactobionic acid is a weak acid that cannot acylate CS directly. In this study we used carbodiimide chemistry, which is popular in the field of peptide chemistry.39 The water-soluble EDC·HCl was selected because of the hydrophilicity of both the CS and lactobionic acid. In addition, NHS was used to protect the reactive intermediate O-acylurea and depress its transformation into N-acylurea, and to prevent the racemization of the product. The main effect of TEMED was to form a buffer system with the HCl in the aqueous solution, and promote the reaction. In acidic water, CS dissolves and forms the cation CS-NH+3 , which is in dynamic equilibrium with the neutral form of CS-NH2. The galactosylation reaction takes place between the CS-NH2 and the reactive intermediate O-acylurea, but O-acylurea tends to produce N-acylurea as a byproduct at neutral or slightly alkaline pH values; therefore, we chose 4.5 as the pH of the reaction system, as was used in the study by Park et al.40 Liang et al reported that the majority of the fenestrae of the liver sinusoid were usually smaller than 200 nm in diameter.41 Besides, the permeability of vascellum endothelium was greatly enhanced in the liver tumor body.42 Thus, NCTD-GC NPs, with mean particle size of 118.7 ± 8.84 nm, could pass through the fenestrate sinusoid and accumulate in the tumor sites of liver. As a result, the small particle size reduced the adverse reaction of NCTD-GC NPs on normal hepatic cells by reducing the passive targeting in the liver. The cellular uptake of NCTD-GC NPs indicated that receptormediated endocytosis was initiated by ligand binding to hepatoma cell membrane receptors. In contrast, the cellular uptake of NCTD-CS NPs indicated that adsorptive endocytosis existed as a nonspecific interaction of the cytomembrane with the NCTD-CS NPs. It was reported by Meschini et al that a higher intracellular content of a drug via endocytosis of a carrier may remarkably increase the therapeutic effect against the target cells.43 The excessive uptake of NCTD-CS NPs by hepatoma cells as opposed to the control NCTD-GC NPs could hold promise for the specific, receptor-mediated cellular endocytosis of these galactosylated NPs displaying active liver-targeting characteristics and more satisfactory compatibility with hepatoma cells. In the case of hepatocellular carcinoma therapy, even though hepatocellular carcinoma cells overexpress ASGP-Rs, the help of NCTD-GC NPs is also required for selective delivery; this is because the NCTD-GC NPs can deliver incorporated drugs not only to hepatocellular carcinoma cells but also to normal liver cells. Although the conjugate was selectively taken up by the liver, the pharmacological activity of NCTD was not restricted to this organ. This was due to NCTD release from liver cells into the bloodstream and redistribution. NCTD-GC NPs could be taken up by hepatoma cells more quickly, so that NCTD could be released from NCTD-GC NPs, reducing its toxicity to normal liver cells. A similar release was observed after administration of hepatotropic conjugates of other antitumor drugs.44,45 It has been reported that CS itself has antitumor effects on mice bearing tumor. Previous studies by Suzuki and Matsumoto showed that CS itself had obvious antitumor activity in vivo at an extremely high dose of more than 100 mg kg–1.46,47 In our experiments the dosage of CS or GC was only about 35 mg kg–1

in the high-dose group; therefore, the effect of the carrier itself was negligible in our study. In summary, we have synthesized a new liver-targeting CS derivative with a high yield. In addition, the NCTD-GC NPs with stable EE and high drug-loading efficacy were prepared. Compared with the NCTD-CS NPs, the NCTD-GC NPs show better cytotoxicity and compatibility with various hepatoma cells and significant antitumor effect in hepatoma-bearing mice. Galactosylated CS-NPs carrying NCTD and targeting hepatocytes by both active and passive mechanisms, have a great potential for potential clinical applications.

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