Article pubs.acs.org/Biomac
Galactose-Decorated pH-Responsive Nanogels for HepatomaTargeted Delivery of Oridonin Cunxian Duan,†,⊥ Jian Gao,‡,⊥ Dianrui Zhang,*,† Lejiao Jia,† Yue Liu,† Dandan Zheng,† Guangpu Liu,† Xiaona Tian,† Fengshan Wang,§ and Qiang Zhang∥ †
Department of Pharmaceutics, ‡Department of Natural Product Chemistry, and §National Glycoengineering Research Center, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China ∥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, P. R. China ABSTRACT: Nanogels based on the polymers of galactosylated chitosan-graf t-poly (N-isopropylacrylamide) (Gal-CS-gPNIPAm) were used as carriers of oridonin (ORI) for tumor targeting. Three ORI-loaded nanogels with various degrees of galactose substitution were prepared, and their characteristics were evaluated. The release behavior of ORI from these nanogels was pH-dependent, and the release could be accelerated under mildly acidic conditions. The cytotoxicity of ORI-loaded nanogels was pH-sensitive. ORI-loaded nanogels exhibited a higher antitumor activity than drug-loaded nanogels without galactosylation, and the anticancer activity increased in relation to increases in the number of galactose moieties of the nanogels in HepG2 cells. In contrast, the cytotoxicity of ORI-loaded nanogels against MCF-7 cells decreased compared with that of drug-loaded nanogels without galactosylation. Results demonstrated that these nanogels could enhance the uptake of ORI into HepG2 cells via asialoglycoprotein receptor-mediated endocytosis. These galactose-decorated pHresponsive nanogels were well-suited for targeted drug delivery to liver cancer cells.
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targeting because the nanoparticles often encountered difficulties in accessing cancer cells or in interacting with the targeted cells after accumulation.12−14 For improving targeting efficiency, introducing target-specific ligands into nanoparticles is necessary because the ligands can recognize and bind to specific receptors that are unique to cancer cells. For achieving the effectively targeted delivery of nanoparticles into the liver cancer cells, carbohydrates are commonly conjugated to the surface of the nanoparticles because they can selectively bind to the asialoglycoprotein receptors that are specifically overexpressed on the surface of hepatoma cells.15−19 Therefore, galactosylated nanogels, as carriers of an anticancer drug, can enhance drug delivery to the HCC cells through receptormediated endocytosis. In this research, Gal-CS-g-PNIPAm nanogels were utilized for the hepatoma-targeted delivery of ORI. ORI (Figure 1), a potential anticancer agent against liver cancers in Chinese traditional medicine, was chosen as the hydrophobic model drug.20,21 A family of nanogels with varied degrees of galactosylation was synthesized, and ORI was loaded into the nanogels. ORI-loaded nanogels were prepared by a selfassembly method, and their physicochemical properties were
INTRODUCTION Primary liver cancer with a high mortality rate is one of the major health problems in the world. Chemotherapy has so far been the main treatment. However, the chemotherapeutic treatment of hepatocellular carcinoma (HCC) is limited because of the poor specificity of chemotherapeutic agents. Therefore, it is of great importance to develop drug delivery systems that can effectively and selectively target the liver, especially HCC cells.1−3 Nanogels, which are nanosized polymeric networks, have many advantages including relatively simple formulation procedure, high drug loading capacity, excellent stability, and stimuli-responsive behavior.4−6 In particular, these nanogels can be delivered to tumor sites via a size-dependent passive targeting or the EPR (enhanced permeability and retention) effect.6,7 Among kinds of nanogels, chitosan-based ones have gained much attention owing to their biodegradability and biocompatibility.8−10 In our previous studies, we developed the chitosan-based nanogels and succeeded in encapsulating the hydrophobic anticancer drug in the nanogels.11 The drugloaded nanogels exhibited a pH-dependent cytotoxicity and better antitumor activity than free drug. Therefore, these chitosan-based nanogels might be developed as potential carriers for tumoral acidic extracellular pH targeting. Nevertheless, recent studies revealed that only a small amount of nanoparticles could be taken up by tumor cells by passive © 2011 American Chemical Society
Received: September 12, 2011 Revised: November 9, 2011 Published: November 11, 2011 4335
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(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), and N,N,N′,N′-tetramethylethylenediamine (TEMED) were supplied by Alfa Aesar Chemical (Tianjin, China). Oridonin was from Nanjing Zelang Pharmaceutical (Nanjing, China). All other solutions and chemicals were obtained from local commercial suppliers and were of analytical reagent grade. Synthesis of Gal-CS-g-PNIPAm. As illustrated in Scheme 1, the synthesis involved two steps: (1) synthesis of CS-g-PNIPAm; (2) galactosylation. In the first step, CS-g-PNIPAm was polymerized by using our previous method.11 CS (0.25 g) was dissolved in 100 mL of aqueous acetic acid (0.60% by v/v), and the solution was heated to 80 °C under nitrogen. APS (1.5 mL, 1.0 × 10−2 mol/L) was then introduced. Under a N2 atmosphere, the solution was stirred for 10 min before NIPAAm (1.0 g) and MBA (0.010 g) were added. The polymerization proceeded for 3 h under nitrogen. The final product was isolated by dialysis against distilled water and freeze-drying. In the final step, Gal-CS-g-PNIPAm was synthesized according to the method described by Park.22 We dissolved 170 mg of CS-g-PNIPAm in 20 mL of aqueous acetic acid (0.6% by v/v). Subsequently, LA, EDC (140 mg), and NHS (60 mg) were added to this solution. The pH of reaction solution was finally regulated at 4.7 by TEMED. The reaction was allowed to proceed under stirring for 72 h. The product was isolated by dialysis against the distilled water for 4 days and freezedrying. Determination of Chemical Composition. Fourier transform infrared (FT-IR, Bruker Vertex 70) spectrometer and 1H nuclear magnetic resonance (1H NMR, Inova-600, Varian) spectrometer were utilized to determine the chemical structure of Gal-CS-g-PNIPAm. Preparation of ORI-Loaded Nanogels. ORI-loaded nanogels were prepared according to a previously reported procedure. 11,23 Gal-
Figure 1. Chemical structure of ORI.
studied. The in vitro release behaviors of ORI from these nanogels were determined at different pH values. Finally, the in vitro antitumor activities of these ORI-loaded nanogels against HepG2 and MCF-7 cells were studied under different pH values. The objectives of this investigation were to ascertain whether the decoration of galactose could enhance the antitumor efficacy of these ORI-loaded nanogels and to optimize the number of galactose for the substantially enhanced internalization of the ORI-loaded nanogels in the HCC cells. This research would be beneficial to the future design and preparation of carriers for the tumor-targeted drug delivery.
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EXPERIMENTAL SECTION
Materials. N-Isopropylacrylamide (NIPAAm, Sigma) was purified by repeated recrystallization from a 1/5 (v/v) mixture of toluene and hexane. Chitosan (CS, MW ≈ 2.0 × 105, degree of deacetylation of 96%), N,N-methylenebisacrylamide (MBA), and ammonium persulfate (APS) were all purchased from Sigma and used without further purification. Lactobionic acid (4-O-β-D-galactopyranosyl-D-gluconic acid, LA) was obtained from J&K Chemical (Logan, UT). 1-Ethyl-3-
Scheme 1. Synthesis of Gal-CS-g-PNIPAm Polymers
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of Gal-CS-g-PNIPAm was determined by FT-IR and 1H NMR measurements. The spectra of FT-IR and 1H NMR are shown in Figures 2 and 3, respectively. As shown in Figure 2, the
CS-g-PNIPAm (100 mg) and ORI (10 mg) were dispersed in 10 mL of distilled water. The dispersion was sonicated at 100 W in an ice bath for 2 min. The drug-loaded nanogels were collected by centrifugation. The encapsulation efficiency and drug loading efficiency were calculated by the same method, as we previously reported.11 Characterization. The particle size distributions of ORI-loaded nanogels were estimated by the dynamic light scattering (DLS) method using a Dawn Heleos, Wyatt QELS, and Optilab DSP instrument (Wyatt Technology, Santa Barbara, CA). The morphology of the ORI-wrapped nanogels was observed with a transmission electron microscope (TEM, Hitachi, Japan), and zeta potentials were measured with a ZATA Instruments apparatus (Malvern, U.K.). An Xray diffractometer (Rigaku, Japan) was applied to estimate the crystalline state of nanogels. In Vitro Drug Release. The release properties of ORI from the ORI-loaded nanogels were determined in the phosphate-buffered saline solution (PBS) at pH 5.0, 6.0 and 7.4, respectively. 11 The ORIencapsulated nanogels (0.5 mg of ORI) dispersed in 1 mL of the PBS were placed in a dialysis membrane bag (MWCO 12 000−14 000). The whole bag was then immersed in 15 mL of the same PBS solution. The release studies were performed at 37 °C with continuous magnetic stirring at 100 rpm. After a predetermined amount of time, 1 mL of the release media was taken for HPLC measurement and replenished with an equal volume of fresh media. The amount of ORI was finally determined using the HPLC method.11 Cell Culture. HepG2 cells and MCF-7 cells, kindly supplied by the Department of Pharmacology, Shandong University, were routinely cultured with RPMI 1640 supplemented with 10% fetal bovine serum, 100 unit/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator with an atmosphere of 5% CO2 at 37 °C. In Vitro Cytotoxicity. To evaluate the cytotoxic activity of the ORI-loaded nanogels, we performed MTT assay to measure cell inhibitory rate; to assess their pH-sensitive cytotoxicity, we measured the inhibitory rate under different pH conditions. Two types of cancer cells (HepG2 cells and MCF-7 cells) were each seeded in 96-well culture plates at a cell density of (3 to 4) × 104 cells/well. After 12 h of conventional cultivation, the cells were further incubated for 24 h in fresh culture media containing pure ORI or various ORI-encapsulated nanogels with different degrees of galactosylation at pH 6.5 or 7.4, respectively. For comparison, four concentrations levels of oridonin were utilized for ORI and the nanogels. We added 15 μL of MTT solution (5 mg/mL) to each well, and the plates were cultured for another 4 h at 37 °C. After that, the supernatant was discarded and 150 μL of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the optical density (OD) was measured at 570 nm with a Microplate Reader. Inhibitory rate was calculated as follows: inhibitory rate (%) = (A570control cells − A570treated cells)/ A570control cells × 100. IC50 value, a concentration at which 50% of cells were killed, was used here to quantify the cytotoxicity of drugloaded nanogels. Statistical Analysis. All experimental data were analyzed by Student’s t tests to evaluate the significance of differences between the subgroups considered. P values GCN-2 > GCN-1, and GCN-3 showed the highest cytotoxicity against HepG2 cells after 24 h treatment. The results suggested that the antitumor activity of ORI-loaded nanogels was more effective than that of drug-loaded nanogels without galactosylation, and galactose could improve the cytotoxic ability of ORI-loaded nanogels to cancer cells because the cytotoxicity appeared to be positively related to the amount of galactose moieties. This might be because the binding of galactose moieties to asialoglycoprotein receptors with high affinity mediated the cellular uptake of drug-loaded nanogels via receptor-mediated endocytosis.17 In addition, the cytostatic effects of ORI-loaded nanogels were found to be pH-sensitive, and this pH-responsive cytotoxicity might be relevant to pH-dependent ORI release from the ORI-
Figure 6. XRD patterns of ORI (A), LA (B), CS-g-PNIPAm (C), GC3 (D), and GCN-3 (E).
though the concentration of the ORI-free nanogels reached 5 mg/mL. Drug-loaded Gal-CS-g-PNIPAm nanogels were further investigated to evaluate the potential therapeutic efficacy. HepG2 cells were treated with the solutions of free ORI and the ORI-entrapped nanogels (GCN-1, GCN-2, and GCN-3) at different ORI concentrations ranging from 5.06 to 40.0 μg/mL for 24 h, respectively. As shown in Figure 11, the cell viability was dose-dependent, and ORI-entrapped nanogels exhibited much higher cytotoxicity compared with free ORI under otherwise the same conditions. The higher cytotoxicity might result from the enhanced internalization of nanoparticles via endocytosis or phagocytosis and the increased uptake of the nanogels via the receptor-mediated mechanism. 17,38 The antitumor activities of three drug-loaded nanogel formulations (GCN-1, GCN-2, and GCN-3) were different and evaluated using the values of IC50. At pH 7.4, the IC50 values were 11.07,
Figure 7. Release profiles of oridonin from the drug-loaded nanogels in PBS: GCN-1 (A), GCN-2 (B), and GCN-3 (C). Samples performed in triplicate; data shown were mean ± standard deviation. 4340
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Figure 9. In vitro cytotoxicity of drug-free Gal-CS-g-PNIPAm nanogels against HepG2 cells at different pH values: the cytotoxicity of drug-free nanogels at pH 7.4 (A) and the cytotoxicity of drug-free nanogels at pH 6.5 (B). Data were mean ± SD (n = 4).
Figure 10. The in vitro cytotoxicity of drug-free Gal-CS-g-PNIPAm nanogels against MCF-7 cells at different pH values: the cytotoxicity of drug-free nanogels at pH 7.4 (A) and the cytotoxicity of drug-free nanogels at pH 6.5 (B). Data were mean ± SD (n = 4).
Figure 11. Cytotoxic activities of ORI and ORI-loaded nanogels on HepG2 cells after 24 h of treatment at different pH values: the cytotoxic activities at pH 7.4 (A) and the cytotoxic activities at pH 7.4 (B). Data were mean ± SD (n = 4).
Figure 12. Cytotoxic activities of ORI and ORI-loaded nanogels on MCF-7 cells after 24 h of treatment at different pH values: the cytotoxic activities at pH 7.4 (A) and the cytotoxic activities at pH 7.4 (B). Data were mean ± SD (n = 4).
loaded nanogels. This pH-sensitive cytostatic effect could enhance the therapeutic efficacy in vivo, which was due to the tumoral extracellular slightly acidic environments. To confirm that galactose-decorated ORI-loaded nanogels were taken up by HepG2 cells via the receptor-mediated mechanism, we performed control experiments using MCF-7 cells (without asialoglycoprotein receptors). As shown in Figure 12, the cytotoxicity of drug-entrapped nanogels in MCF-7 cells was much higher than that in HepG2 cells under otherwise the same conditions. This was because MCF-7 cells were more vulnerable than HepG2 cells under otherwise the same conditions (p < 0.01). The IC50 values were 6.32, 5.84, and
5.90 μg/mL for GCN-1, GCN-2, and GCN-3, respectively, at pH 7.4; the values were 7.17, 6.34, and 6.78 μg/mL for GCN-1, GCN-2, and GCN-3, respectively, at pH 6.5. Meanwhile, the values of IC50 for ORI-loaded CS-NG were 7.07 μg/mL at pH 7.4 and 6.02 μg/mL at pH 6.5. (Data were not listed.) Interestingly, the cytotoxicity of GCN-1, GCN-2, and GCN-3 decreased with a decrease in pH of culture media on MCF-7 cells. The cytotoxicity of ORI-loaded Gal-CS-NG against MCF7 cells was higher than that of ORI-loaded CS-NG at pH 7.4, whereas the cytotoxic activity of drug-entrapped Gal-CS-NG was significantly inhibited as compared with that of drugentrapped CS-NG at pH 6.5. The ORI-entrapped CS-NG 4341
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showed the lower cytotoxicity than ORI-entrapped Gal-CS-NG due to time-consuming ORI release from ORI-loaded CS-NG in comparison with ORI-loaded Gal-CS-NG at the same ORI concentration, proved by the in vitro ORI release at pH 7.4. 11 However, considering the similar drug release profiles at pH 6.5, the reduced cytotoxicity of ORI-entrapped Gal-CS-NG in comparison with that of the ORI-entrapped CS-g-PNIPAm nanogels might result from the unsuccessful cellular uptake of ORI-entrapped Gal-CS-NG nanogels because the binding between the positively charged nanogels and the negatively charged surface was reduced as the number of free amino groups in the Gal-CS-g-PNIPAm decreased. The pH-responsive cytotoxicity of ORI-entrapped Gal-CS-NG against MCF-7 cells displayed markedly different compared with that against HepG2 cells, which might be attributed to the unsuccessful drug delivery to cancer cells via the receptor-mediated endocytosis. Taken together, the results of MTT assay indicated that ORI-loaded Gal-CS-g-PNIPAm nanogels could enhance the uptake of ORI into HepG2 cells through the mechanism of galactose-specific receptor-mediated endocytosis.
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CONCLUSIONS In this research, we synthesized Gal-CS-g-PNIPAm polymers with different levels of galactose substitution. They could selfassemble into nanogels, and ORI was successfully encapsulated in the nanogels. Drug release studies indicated that ORI-loaded nanogels exhibited a pH-responsive drug release behavior. Moreover, MTT assay revealed that ORI-loaded nanogels exhibited an enhanced anticancer activity against HepG2 cells under a slightly acidic environment (pH 6.5), and the anticancer efficiency enhanced as the degrees of galactose substitution increased. However, distinct phenomena were observed when MCF-7 cells were cultivated with the drugloaded nanogels. Although the antitumor efficiency of galactose-decorated nanogels was enhanced compared with that of nondecorated nanogels when MCF-7 cells were treated at pH 7.4, the antitumor efficiency of galactose-decorated nanogels was inhibited at pH 6.5. Besides, nanogels without ORI exhibited no cytotoxicity. The results indicated that drugloaded Gal-CS-g-PNIPAm nanogels could be effectively absorbed by HepG2 cells through the mechanism of galactose-specific receptor-mediated endocytosis. These nanogels, which were implemented with galactose-mediated cancer cell targeting and pH-triggered drug releasing properties, would be promising carriers for specific delivery into liver cancer cells.
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AUTHOR INFORMATION
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ACKNOWLEDGMENTS
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REFERENCES
Corresponding Author *Tel/Fax: +86-531-88382015. E-mail: zhangdianrui2006@163. com. Author Contributions ⊥ Both authors contributed equally.
This work was supported by the National Nature Science Foundation of China (no. 81073054) and the National Basic Research Program of China (no. 2009CB930300).
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