环境响应型肝靶向纳米凝胶给药系统研究
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摘要
本课题首先合成了环境响应型壳聚糖聚合物,并首次将此聚合物与乳糖酸缩合制备具有肝细胞靶向作用的半乳糖化壳聚糖材料。以冬凌草甲素(Oridonin, ORI)为模型药物,采用自组装法制备半乳糖化及未半乳糖化的冬凌草甲素纳米凝胶制剂,并对上述两种纳米制剂的体内外抗肿瘤活性进行考察,为肝癌靶向制剂的开发提供理论和实验依据。主要研究内容及结果如下：1.壳聚糖-g-聚(N-异丙基丙烯酰胺)纳米凝胶作为药物载体的研究
以壳聚糖和N-异丙基丙烯酰胺为原料,采用自由基聚合法制备具有环境pH响应特性的壳聚糖-g-聚(N-异丙基丙烯酰胺)(CS-g-PNIPAm),并采用FTIR,1H-NMR和XRD等方法对该聚合物结构进行确证。以ORI为模型药物,采用自组装法制备载药纳米凝胶(ORI-CS-NG),并考察各种制备条件对纳米凝胶中药物包封率的影响；结果表明,ORI-CS-NG中药物包封率受到载体材料用量和载体聚合物制备条件的影响。透射电镜和激光光散射分析显示ORI-CS-NG为类球形、表面光滑圆整、粒径分布窄、平均粒径在100nm左右。ORI-CS-NG的XRD分析结果表明ORI在纳米凝胶中以分子、微晶或无定形状态存在。以透析法考察ORI-CS-NG中ORI的体外释放行为,结果表明制剂中药物的释放具有明显的pH敏感特性：在pH7.4的释放介质中,12h的药物累积释放量低于50%；而在弱酸性的释放介质中(pH6.5、6.0和5.0),12h时药物累积释放量超过80%。采用MTT比色法考察空白纳米凝胶(CS-NG)及ORI-CS-NG对人肝癌HepG2细胞的细胞毒作用。结果表明CS-NG无明显的细胞毒性,而ORI-CS-NG对HepG2细胞的抗肿瘤活性随药物浓度的增加和作用时间的延长而增强；ORI(?)(?)ORI-CS-NG对HepG2细胞的细胞毒作用均具有pH敏感特性,在弱酸性条件下这两种制剂的抗肿瘤活性增强；且ORI-CS-NG的抗肝癌活性明显高于ORI。 ORI-CS-NG在pH7.4和6.5时对HepG2细胞的IC50值分别为13.18(?)(?)8.86μg/mL,而ORI在pH7.4和6.5时的IC50值分别为16.94和12.00μg/mL。对HepG2细胞形态考察结果表明CS-NG无明显的细胞毒性,而ORI-CS-NG可显著提高ORI体外对HepG2的抗肿瘤活性。
2.半乳糖化壳聚糖-g-聚(N-异丙基丙烯酰胺)纳米凝胶作为药物载体的研究
利用壳聚糖结构中的氨基和乳糖酸结构中的羧基在EDC作用下进行缩合,制备半乳糖化壳聚糖-g-聚(N-异丙基丙烯酰胺)(Gal-CS-g-PNIPAm)聚合物,采用FTIR、1H-NMR和XRD等方法对该产物的结构进行表征。Gal-CS-g-PNIPAm中半乳糖的取代度采用1H-NMR进行测定,结果表明随着反应体系中乳糖酸用量的增加,Gal-CS-g-PNIPAm中半乳糖的取代度先增加后降低。选择取代度分别为7.26%,11.95%和14.06%的Gal-CS-g-PNIPAm作为载体进行实验,采用自组装法制备冬凌草甲素Gal-CS-g-PNIPAm(?)内米凝胶(ORI-GC-NG),三种载药纳米凝胶均为类球形,表面光滑,粒径分布均匀,平均粒径在200nm左右。随着聚合物中半乳糖取代度的增加,ORI-GC-NG中药物包封率与Z-电位均逐渐降低。对ORI-GC-NG的XRD分析结果说明ORI在此三种纳米凝胶制剂中均以分子、微晶或无定形状态存在。以透析法测定上述三种ORI-GC-NG中ORI的体外释放行为,结果表明ORI-GC-NG中药物的释放均具有pH敏感的特性,随着释放介质pH的降低,释药速率增加。采用MTT法分别考察空白Gal-CS-g-PNIPAm纳米凝胶(GC-NG)及三种ORI-GC-NG在不同pH条件下对HepG2和人乳腺癌MCF-7细胞的细胞毒作用,结果表明GC-NG对这两种肿瘤细胞均无明显的毒性；ORI-GC-NG中随着药物浓度的增加,两种肿瘤细胞的存活率均降低；pH相同时ORI-GC-NG对HepG2和MCF-7细胞的抗肿瘤活性比ORI强。ORI-GC-NG的抗肿瘤活性具有明显的pH敏感特性,对HepG2的细胞毒作用随着pH的增加而降低,而对MCF-7细胞体外生长的抑制作用随着pH的增加而增加；pH7.4时,ORI-GC-NG对HepG2和MCF-7细胞的抗肿瘤作用均高于ORI-CS-NG;而在pH6.5时,ORI-GC-NG对HepG2G勺增殖抑制效果要显著高于ORI-CS-NG,但对MCF-7细胞的抑制作用要明显低于ORI-CS-NG。
3.冬凌草甲素纳米凝胶组织分布、药动学及其体内抗肿瘤作用研究
以ORI为对照,研究了半乳糖化及未半乳糖化的载药纳米凝胶制剂(ORI-GC-NG和ORI-CS-NG)尾静脉注射后在正常及荷瘤小鼠体内各主要组织、器官中的经时变化规律和分布情况,及其对荷肝癌H22小鼠的抑瘤效果。结果表明,在正常小鼠体内,ORI-CS-NG的相对摄取率从高到低依次为肝3.260、血1.914、脾1.294、肺1.112、心0.879和肾0.489；GCN-3(半乳糖取代度为14.06%的ORI-GC-NG)的相对摄取率依次排列为肝6.335、血3.639、脾1.083、心0.992、肺0.727和肾0.434。而在荷瘤小鼠体内,ORI-CS-NG的相对摄取率从高到低依次为肝2.538、肿瘤2.280、血2.106、脾1.27I、肺1.212、肾0.706和心0.518；GCN-3的相对摄取率依次排列为肿瘤5.672、肝4.171、血2.966、脾1.038、肾0.790、肺0.779和心O.708.GCN-3和ORI-CS-NG均具有明显的肿瘤靶向性,而GCN-3肿瘤靶向效果更佳。荷瘤小鼠尾静脉注射ORI、ORI-CS-NG和GCN-3后血药浓度-时间曲线均符合二室模型。ORI的主要药动学参数为：半衰期t12α=0.721h,t1/2β=6.806h,血药浓度-时间曲线下面积AUC0～∞=18.112(h·μg/mL),清除率CLs=0.442(mg/kg/h/(μg/mL))；ORI-CS-NG的主要药动学参数为：半衰期t1/2α=3.273h,t1/2β=69.315h,血药浓度-时间曲线下面积AUC0～∞=21.721(h·μd/mL),清除率CLs=0.243(mg/kg/h/(μd/mL))；GCN-3的主要药动学参数为：半衰期t1,2α=3.755h,t1/2β=69.315h,血药浓度-时间曲线下面积AUC0～∞=46.373(h·μd/mL),清除率CLs=0.171(mg/kg/h/(μg/mL))。由结果可知,上述两种载药纳米凝胶可以显著延长药物ORI在体内的消除半衰期t1/2β,明显增加AUC,降低药物体内清除率。采用动物移植性肿瘤的实验方法,将小鼠肝癌H22细胞接种于昆明种小鼠皮下,进行体内抑瘤实验；结果表明,在相同药物剂量时,ORI的抗肿瘤活性最差,ORI-CS-NG和GCN-3的抑瘤率均有所提高,且GCN-3的抑瘤效果更强。在给药量为8mg/(kg·d)(?)寸,ORI、ORI-CS-NG和GCN-3的抑瘤率分别为32.16%、51.80和70.76%,且此三种制剂的体内毒性均较小。
本研究选用具有环境pH响应型智能聚合物材料作为抗肿瘤药物冬凌草甲素的载体,首次将具有肝靶向性的半乳糖基团链接在载体材料中,得到同时具备pH敏感和主动靶向这两种特性的纳米凝胶给药输送体系,以实现抗肿瘤药物冬凌草甲素的肝癌靶向输送。本文的研究成果为肝癌靶向给药新剂型的开发提供了思路,并为药物冬凌草甲素的进一步临床治疗应用提供了实验和理论依据。
In this study, pH-responsive and biocompatible chitosan-based copolymers, chitosan-graft-poly (N-isopropylacrylamide)(CS-g-PNIPAm) were synthesized and conjugated with lactobionic acid to provide hepatoma-targeted drug delivery carriers. Oridonin was chosen as a model drug and was capsulated in galactose-decorated and non-decorated CS-g-PNIPAm nanogels by the self-assembly method. This paper includes three parts as follows:
1. Research of CS-g-PNIPAm copolymers as drug delivery carriers
The CS-g-PNIPAm copolymers were synthesized via free radical copolymerization and characterized for their chemical structure by FTIR,1H-NMR and X-ray diffraction (XRD) study. Oridonin (ORI) was loaded into the nanogels by the self-assembly method as a model drug. The influence of different factors such as the amount of copolymers and the synthesis procedure of the preparation of copolymers on the drug encapsulation efficiencies was investigated. TEM indicated that unloaded and drug-loaded CS-g-PNIPAm nanogels were approximately spherical and regular. The average hydrodynamic diameter of the ORI-loaded nanogels (ORI-CS-NG) was about100nm. XRD demonstrated that ORI was either molecularly dispersed or distributed in an amorphous state in the nanogels. The release behavior of ORI from ORI-CS-NG was assayed in vitro by the dialysis method and the results displayed that pH had an effect on the drug release. The drug release was slow at pH7.4while it was accelerated at low pH. The cumulative release rates drastically increased from about50%at pH7.4to more than80%at pH6.5,6.0and5.0. The MTT tests for black nanogels indicated that the nanogels with the concentrations from0.025to5.0mg/mL had no apparent harm on the proliferation of HepG2cells after24h incubation. The cytostatic activities of both ORI-CS-NG and ORI solution increased in parallel with drug concentrations and incubation times. Besides, ORI-CS-NG showed a higher cellular cytotoxicity relative to the ORI at the same pH. In addition, the anticancer cytotoxic activity of ORI-CS-NG and ORI solutions against HepG2cells was found to be pH-dependent. The IC50value was also pH-sensitive. The IC50value for ORI-CS-NG was8.86μg/mL at pH6.5compared with that of13.19μg/mL at pH7.4, whereas the values of ORI solution were16.94and12.00μg/mL at pH7.4and6.5, respectively. The cellular morphological analysis demonstrated that ORI-CS-NG could enhance the anti-tumor activity and no significant cytotoxicity, however, was observed with the blank carriers themselves.
2. Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery
Gal-CS-g-PNIPAm was prepared by direct coupling LA with CS-g-PNIPAm via carbodiimide chemistry. The chemical structure of Gal-CS-g-PNIPAm was determined by FTIR,1H-NMR and XRD measurements. The degree of substitution of galactose (DSGc(%)) in Gal-CS-g-PNIPAm estimated by'H-NMR was calculated and the results indicated that the degree of galactose substitution increased and then decreased with increasing the amount of LA in the coupling reaction. Three polymers with7.26,11.95, and14.06%of degrees of galactose substitution were chosen for the next study. ORI-loaded nanogels (ORI-GC-NG) were readily prepared via the self-assembly method. TEM revealed that three drug-loaded nanogels had regularly spherical morphology with narrow distributions. ORI-GC-NG displayed slightly positive surface charges. When the degrees of galactose substitution increased, the zeta potential values and drug encapsulation efficiency slightly decreased. XRD measurement demonstrated that ORI was either molecularly dispersed or distributed in an amorphous state in the nanogels. The drug release profiles from three dosage forms were pH-dependent and the release rate of ORI from ORI-GC-NG was relatively slow at pH7.4while it was accelerated under acidic conditions. The cytotoxicity of nanogels without ORI against HepG2and MCF-7cells was measured by MTT assay. All nanogels without ORI exhibited no cytotoxicity at pH7.4or6.5. The antitumor activities of ORI-GC-NG were dose-dependent and pH-sensitive, and ORI-GC-NG exhibited much higher cytotoxicity compared with free ORI under otherwise the same conditions. The cytostatic effects of ORI-GC-NG against HepG2and MCF-7cells increased with decreasing the pH values of culture media and the anticancer efficiency enhanced as the degrees of galactose substitution increased. Interestingly, the cytotoxicity of ORI nanogels decreased with a decrease in pH of culture media on MCF-7cells. The cytotoxicity of ORI-GC-NG against MCF-7cells was higher than that of ORI-loaded non-decorated nanogels at pH7.4, whereas the cytotoxic activity of ORI-GC-NG was significantly inhibited as compared to that of drug-entrapped non-decorated nanogels at pH6.5.
3. Research of biodistribution, pharmacokinetics and in vivo antitumor activity
To study the biodistribution of GCN-3(ORI-GC-NG with14.06%of degrees of galactose substitution) and ORI-CS-NG in normal and tumor-bearing mice, we utilized the HPLC method to determine and compare the content of oridonin following the tail intravenous injection of free ORI and these ORI-loaded nanogels. The results of biodistribution in the normal mice showed that the relative efficiencies of ORI-CS-NG in the liver, blood, spleen, lung, heart, and kidney were3.260,1.914,1.294,1.112,0.879and0.489. The relative efficiencies of GCN-3in the liver, blood, spleen, lung, heart, and kidney were6.335,3.369,1.083,0.727,0.992and0434. While the results of biodistribution in the tumor-bearing mice showed that the relative efficiencies of ORI-CS-NG in the tumor, liver, blood, spleen, lung, heart and kidney were2.280,2.538,2.106,1.271,1.212,0.518and0.706; and these relative efficiencies of GCN-3were5.672,4.171,2.966,1.038,0.779,0.708and0.790, respectively. CS-g-PNIPAm and Gal-CS-g-PNIPAm nanogels can improve the tumor targeting of oridonin, and the Gal-CS-g-PNIPAm nanogels possessed better tumor-targeted capability.
The results of pharmaceutics in tumor-bearing mice showed that the encapsulation of oridonin in nanogels was remarkably effective in prolonging its blood circulation time. The major pharmacokinetic parameters of free ORI group were as follows:t1/2α=0.721h, t1/2β=6.806h, AUC=18.112h·μg/mL, CLs=0.442mg/kg/h/(μg/mL); the major parameters of the ORI-CS-NG group were:t1/2α=3.273h, t1/2β=69.315h, AUC=21.721h·μg/mL, CLs=0.243mg/kg/h/(μg/mL); the parameters of the GCN-3group were: t1/2α=3.755h, t1/2β=69.315h, AUC=46.373h·μg/mL, CLs=0.171mg/kg/h/(μg/mL). The results indicated that nanogels could be a potential carrier for oridonin to obtain prolonged elimination half life.
H22mouse hepatoma carcinoma cells were transplanted subcutaneously in mice to evaluate the effect of GCN-3, ORI-CS-NG and free oridonin on tumor cells in vivo. Tumor weight inhibition was detected and the results indicated that GCN-3showed a stronger anticancer effect than ORI-CS-NG and free oridonin. The inhibition rates of GCN-3, ORI-CS-NG and free oridonin were32.16%、51.80%and70.76%at the dose of8mg/(kg·d), respectively. The results of growth curve of body weight in the tumor-bearing mice displayed that GCN-3, ORI-CS-NG and free oridonin have little influence in the growth of body weight of the tumor-bearing mice during the seven days of drug administration.
This is the first report on the preparation of galactose-decorated pH-sensitive nanogels as the carriers of oridonin. 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. Our studies contribute to the development of tumor-targeted delivery of anticancer drugs and play a very important role in clinical application of oridonin.
以壳聚糖和N-异丙基丙烯酰胺为原料,采用自由基聚合法制备具有环境pH响应特性的壳聚糖-g-聚(N-异丙基丙烯酰胺)(CS-g-PNIPAm),并采用FTIR,1H-NMR和XRD等方法对该聚合物结构进行确证。以ORI为模型药物,采用自组装法制备载药纳米凝胶(ORI-CS-NG),并考察各种制备条件对纳米凝胶中药物包封率的影响；结果表明,ORI-CS-NG中药物包封率受到载体材料用量和载体聚合物制备条件的影响。透射电镜和激光光散射分析显示ORI-CS-NG为类球形、表面光滑圆整、粒径分布窄、平均粒径在100nm左右。ORI-CS-NG的XRD分析结果表明ORI在纳米凝胶中以分子、微晶或无定形状态存在。以透析法考察ORI-CS-NG中ORI的体外释放行为,结果表明制剂中药物的释放具有明显的pH敏感特性：在pH7.4的释放介质中,12h的药物累积释放量低于50%；而在弱酸性的释放介质中(pH6.5、6.0和5.0),12h时药物累积释放量超过80%。采用MTT比色法考察空白纳米凝胶(CS-NG)及ORI-CS-NG对人肝癌HepG2细胞的细胞毒作用。结果表明CS-NG无明显的细胞毒性,而ORI-CS-NG对HepG2细胞的抗肿瘤活性随药物浓度的增加和作用时间的延长而增强；ORI(?)(?)ORI-CS-NG对HepG2细胞的细胞毒作用均具有pH敏感特性,在弱酸性条件下这两种制剂的抗肿瘤活性增强；且ORI-CS-NG的抗肝癌活性明显高于ORI。 ORI-CS-NG在pH7.4和6.5时对HepG2细胞的IC50值分别为13.18(?)(?)8.86μg/mL,而ORI在pH7.4和6.5时的IC50值分别为16.94和12.00μg/mL。对HepG2细胞形态考察结果表明CS-NG无明显的细胞毒性,而ORI-CS-NG可显著提高ORI体外对HepG2的抗肿瘤活性。
2.半乳糖化壳聚糖-g-聚(N-异丙基丙烯酰胺)纳米凝胶作为药物载体的研究
利用壳聚糖结构中的氨基和乳糖酸结构中的羧基在EDC作用下进行缩合,制备半乳糖化壳聚糖-g-聚(N-异丙基丙烯酰胺)(Gal-CS-g-PNIPAm)聚合物,采用FTIR、1H-NMR和XRD等方法对该产物的结构进行表征。Gal-CS-g-PNIPAm中半乳糖的取代度采用1H-NMR进行测定,结果表明随着反应体系中乳糖酸用量的增加,Gal-CS-g-PNIPAm中半乳糖的取代度先增加后降低。选择取代度分别为7.26%,11.95%和14.06%的Gal-CS-g-PNIPAm作为载体进行实验,采用自组装法制备冬凌草甲素Gal-CS-g-PNIPAm(?)内米凝胶(ORI-GC-NG),三种载药纳米凝胶均为类球形,表面光滑,粒径分布均匀,平均粒径在200nm左右。随着聚合物中半乳糖取代度的增加,ORI-GC-NG中药物包封率与Z-电位均逐渐降低。对ORI-GC-NG的XRD分析结果说明ORI在此三种纳米凝胶制剂中均以分子、微晶或无定形状态存在。以透析法测定上述三种ORI-GC-NG中ORI的体外释放行为,结果表明ORI-GC-NG中药物的释放均具有pH敏感的特性,随着释放介质pH的降低,释药速率增加。采用MTT法分别考察空白Gal-CS-g-PNIPAm纳米凝胶(GC-NG)及三种ORI-GC-NG在不同pH条件下对HepG2和人乳腺癌MCF-7细胞的细胞毒作用,结果表明GC-NG对这两种肿瘤细胞均无明显的毒性；ORI-GC-NG中随着药物浓度的增加,两种肿瘤细胞的存活率均降低；pH相同时ORI-GC-NG对HepG2和MCF-7细胞的抗肿瘤活性比ORI强。ORI-GC-NG的抗肿瘤活性具有明显的pH敏感特性,对HepG2的细胞毒作用随着pH的增加而降低,而对MCF-7细胞体外生长的抑制作用随着pH的增加而增加；pH7.4时,ORI-GC-NG对HepG2和MCF-7细胞的抗肿瘤作用均高于ORI-CS-NG;而在pH6.5时,ORI-GC-NG对HepG2G勺增殖抑制效果要显著高于ORI-CS-NG,但对MCF-7细胞的抑制作用要明显低于ORI-CS-NG。
3.冬凌草甲素纳米凝胶组织分布、药动学及其体内抗肿瘤作用研究
以ORI为对照,研究了半乳糖化及未半乳糖化的载药纳米凝胶制剂(ORI-GC-NG和ORI-CS-NG)尾静脉注射后在正常及荷瘤小鼠体内各主要组织、器官中的经时变化规律和分布情况,及其对荷肝癌H22小鼠的抑瘤效果。结果表明,在正常小鼠体内,ORI-CS-NG的相对摄取率从高到低依次为肝3.260、血1.914、脾1.294、肺1.112、心0.879和肾0.489；GCN-3(半乳糖取代度为14.06%的ORI-GC-NG)的相对摄取率依次排列为肝6.335、血3.639、脾1.083、心0.992、肺0.727和肾0.434。而在荷瘤小鼠体内,ORI-CS-NG的相对摄取率从高到低依次为肝2.538、肿瘤2.280、血2.106、脾1.27I、肺1.212、肾0.706和心0.518；GCN-3的相对摄取率依次排列为肿瘤5.672、肝4.171、血2.966、脾1.038、肾0.790、肺0.779和心O.708.GCN-3和ORI-CS-NG均具有明显的肿瘤靶向性,而GCN-3肿瘤靶向效果更佳。荷瘤小鼠尾静脉注射ORI、ORI-CS-NG和GCN-3后血药浓度-时间曲线均符合二室模型。ORI的主要药动学参数为：半衰期t12α=0.721h,t1/2β=6.806h,血药浓度-时间曲线下面积AUC0～∞=18.112(h·μg/mL),清除率CLs=0.442(mg/kg/h/(μg/mL))；ORI-CS-NG的主要药动学参数为：半衰期t1/2α=3.273h,t1/2β=69.315h,血药浓度-时间曲线下面积AUC0～∞=21.721(h·μd/mL),清除率CLs=0.243(mg/kg/h/(μd/mL))；GCN-3的主要药动学参数为：半衰期t1,2α=3.755h,t1/2β=69.315h,血药浓度-时间曲线下面积AUC0～∞=46.373(h·μd/mL),清除率CLs=0.171(mg/kg/h/(μg/mL))。由结果可知,上述两种载药纳米凝胶可以显著延长药物ORI在体内的消除半衰期t1/2β,明显增加AUC,降低药物体内清除率。采用动物移植性肿瘤的实验方法,将小鼠肝癌H22细胞接种于昆明种小鼠皮下,进行体内抑瘤实验；结果表明,在相同药物剂量时,ORI的抗肿瘤活性最差,ORI-CS-NG和GCN-3的抑瘤率均有所提高,且GCN-3的抑瘤效果更强。在给药量为8mg/(kg·d)(?)寸,ORI、ORI-CS-NG和GCN-3的抑瘤率分别为32.16%、51.80和70.76%,且此三种制剂的体内毒性均较小。
本研究选用具有环境pH响应型智能聚合物材料作为抗肿瘤药物冬凌草甲素的载体,首次将具有肝靶向性的半乳糖基团链接在载体材料中,得到同时具备pH敏感和主动靶向这两种特性的纳米凝胶给药输送体系,以实现抗肿瘤药物冬凌草甲素的肝癌靶向输送。本文的研究成果为肝癌靶向给药新剂型的开发提供了思路,并为药物冬凌草甲素的进一步临床治疗应用提供了实验和理论依据。
In this study, pH-responsive and biocompatible chitosan-based copolymers, chitosan-graft-poly (N-isopropylacrylamide)(CS-g-PNIPAm) were synthesized and conjugated with lactobionic acid to provide hepatoma-targeted drug delivery carriers. Oridonin was chosen as a model drug and was capsulated in galactose-decorated and non-decorated CS-g-PNIPAm nanogels by the self-assembly method. This paper includes three parts as follows:
1. Research of CS-g-PNIPAm copolymers as drug delivery carriers
The CS-g-PNIPAm copolymers were synthesized via free radical copolymerization and characterized for their chemical structure by FTIR,1H-NMR and X-ray diffraction (XRD) study. Oridonin (ORI) was loaded into the nanogels by the self-assembly method as a model drug. The influence of different factors such as the amount of copolymers and the synthesis procedure of the preparation of copolymers on the drug encapsulation efficiencies was investigated. TEM indicated that unloaded and drug-loaded CS-g-PNIPAm nanogels were approximately spherical and regular. The average hydrodynamic diameter of the ORI-loaded nanogels (ORI-CS-NG) was about100nm. XRD demonstrated that ORI was either molecularly dispersed or distributed in an amorphous state in the nanogels. The release behavior of ORI from ORI-CS-NG was assayed in vitro by the dialysis method and the results displayed that pH had an effect on the drug release. The drug release was slow at pH7.4while it was accelerated at low pH. The cumulative release rates drastically increased from about50%at pH7.4to more than80%at pH6.5,6.0and5.0. The MTT tests for black nanogels indicated that the nanogels with the concentrations from0.025to5.0mg/mL had no apparent harm on the proliferation of HepG2cells after24h incubation. The cytostatic activities of both ORI-CS-NG and ORI solution increased in parallel with drug concentrations and incubation times. Besides, ORI-CS-NG showed a higher cellular cytotoxicity relative to the ORI at the same pH. In addition, the anticancer cytotoxic activity of ORI-CS-NG and ORI solutions against HepG2cells was found to be pH-dependent. The IC50value was also pH-sensitive. The IC50value for ORI-CS-NG was8.86μg/mL at pH6.5compared with that of13.19μg/mL at pH7.4, whereas the values of ORI solution were16.94and12.00μg/mL at pH7.4and6.5, respectively. The cellular morphological analysis demonstrated that ORI-CS-NG could enhance the anti-tumor activity and no significant cytotoxicity, however, was observed with the blank carriers themselves.
2. Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery
Gal-CS-g-PNIPAm was prepared by direct coupling LA with CS-g-PNIPAm via carbodiimide chemistry. The chemical structure of Gal-CS-g-PNIPAm was determined by FTIR,1H-NMR and XRD measurements. The degree of substitution of galactose (DSGc(%)) in Gal-CS-g-PNIPAm estimated by'H-NMR was calculated and the results indicated that the degree of galactose substitution increased and then decreased with increasing the amount of LA in the coupling reaction. Three polymers with7.26,11.95, and14.06%of degrees of galactose substitution were chosen for the next study. ORI-loaded nanogels (ORI-GC-NG) were readily prepared via the self-assembly method. TEM revealed that three drug-loaded nanogels had regularly spherical morphology with narrow distributions. ORI-GC-NG displayed slightly positive surface charges. When the degrees of galactose substitution increased, the zeta potential values and drug encapsulation efficiency slightly decreased. XRD measurement demonstrated that ORI was either molecularly dispersed or distributed in an amorphous state in the nanogels. The drug release profiles from three dosage forms were pH-dependent and the release rate of ORI from ORI-GC-NG was relatively slow at pH7.4while it was accelerated under acidic conditions. The cytotoxicity of nanogels without ORI against HepG2and MCF-7cells was measured by MTT assay. All nanogels without ORI exhibited no cytotoxicity at pH7.4or6.5. The antitumor activities of ORI-GC-NG were dose-dependent and pH-sensitive, and ORI-GC-NG exhibited much higher cytotoxicity compared with free ORI under otherwise the same conditions. The cytostatic effects of ORI-GC-NG against HepG2and MCF-7cells increased with decreasing the pH values of culture media and the anticancer efficiency enhanced as the degrees of galactose substitution increased. Interestingly, the cytotoxicity of ORI nanogels decreased with a decrease in pH of culture media on MCF-7cells. The cytotoxicity of ORI-GC-NG against MCF-7cells was higher than that of ORI-loaded non-decorated nanogels at pH7.4, whereas the cytotoxic activity of ORI-GC-NG was significantly inhibited as compared to that of drug-entrapped non-decorated nanogels at pH6.5.
3. Research of biodistribution, pharmacokinetics and in vivo antitumor activity
To study the biodistribution of GCN-3(ORI-GC-NG with14.06%of degrees of galactose substitution) and ORI-CS-NG in normal and tumor-bearing mice, we utilized the HPLC method to determine and compare the content of oridonin following the tail intravenous injection of free ORI and these ORI-loaded nanogels. The results of biodistribution in the normal mice showed that the relative efficiencies of ORI-CS-NG in the liver, blood, spleen, lung, heart, and kidney were3.260,1.914,1.294,1.112,0.879and0.489. The relative efficiencies of GCN-3in the liver, blood, spleen, lung, heart, and kidney were6.335,3.369,1.083,0.727,0.992and0434. While the results of biodistribution in the tumor-bearing mice showed that the relative efficiencies of ORI-CS-NG in the tumor, liver, blood, spleen, lung, heart and kidney were2.280,2.538,2.106,1.271,1.212,0.518and0.706; and these relative efficiencies of GCN-3were5.672,4.171,2.966,1.038,0.779,0.708and0.790, respectively. CS-g-PNIPAm and Gal-CS-g-PNIPAm nanogels can improve the tumor targeting of oridonin, and the Gal-CS-g-PNIPAm nanogels possessed better tumor-targeted capability.
The results of pharmaceutics in tumor-bearing mice showed that the encapsulation of oridonin in nanogels was remarkably effective in prolonging its blood circulation time. The major pharmacokinetic parameters of free ORI group were as follows:t1/2α=0.721h, t1/2β=6.806h, AUC=18.112h·μg/mL, CLs=0.442mg/kg/h/(μg/mL); the major parameters of the ORI-CS-NG group were:t1/2α=3.273h, t1/2β=69.315h, AUC=21.721h·μg/mL, CLs=0.243mg/kg/h/(μg/mL); the parameters of the GCN-3group were: t1/2α=3.755h, t1/2β=69.315h, AUC=46.373h·μg/mL, CLs=0.171mg/kg/h/(μg/mL). The results indicated that nanogels could be a potential carrier for oridonin to obtain prolonged elimination half life.
H22mouse hepatoma carcinoma cells were transplanted subcutaneously in mice to evaluate the effect of GCN-3, ORI-CS-NG and free oridonin on tumor cells in vivo. Tumor weight inhibition was detected and the results indicated that GCN-3showed a stronger anticancer effect than ORI-CS-NG and free oridonin. The inhibition rates of GCN-3, ORI-CS-NG and free oridonin were32.16%、51.80%and70.76%at the dose of8mg/(kg·d), respectively. The results of growth curve of body weight in the tumor-bearing mice displayed that GCN-3, ORI-CS-NG and free oridonin have little influence in the growth of body weight of the tumor-bearing mice during the seven days of drug administration.
This is the first report on the preparation of galactose-decorated pH-sensitive nanogels as the carriers of oridonin. 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. Our studies contribute to the development of tumor-targeted delivery of anticancer drugs and play a very important role in clinical application of oridonin.
引文
1. Yallapu MM, Jaggi M, Chauhan SC. Design and engineering of nanogels for cancer treatment[J]. Drug Discovery Today 2011; 16(9-10):457-63.
2.熊微.温度/pH双重敏感纳米凝胶的构建、药物偶联与新型血管栓塞材料的研究[D].华中科技大学,2011.
3. Tamura M, Ichinohe S, Tamura A, Ikeda Y, Nagasaki Y. In vitro and in vivo characteristics of core-shell type nanogel particles:optimization of core cross-linking density and surface poly(ethylene glycol) density in PEGylated nanogels[J]. Acta Biomater 2011;7(9):3354-61.
4. Vinogradov SV, Kohli E, Zeman AD. Comparison of nanogel drug carriers and their formulations with nucleoside 5'-triphosphates[J]. Pharm Res 2006;23(5):920-30.
5.刘永,崔英德,尹国强等.快速响应水凝胶在给药系统中的应用进展[J].高分子材料科学与工程2009(06)：158-62.
6. Galmarini CM, Warren G, Kohli E, Zeman A, Mitin A, Vinogradov SV. Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines[J]. Mol Cancer Ther 2008;7(10):3373-80.
7. Quan CY, Sun YX, Cheng H, Cheng SX, Zhang XZ, Zhuo RX. Thermosensitive P(NIPAAm-co-PAAc-co-HEMA) nanogels conjugated with transferrin for tumor cell targeting delivery[J]. Nanotechnology 2008;19(27):275102.
8. Oishi M, Sumitani S, Nagasaki Y. On-off regulation of 19F magnetic resonance signals based on pH-sensitive PEGylated nanogels for potential tumor-specific smart 19F MRI probes[J]. Bioconjug Chem 2007; 18(5):1379-82.
9. Bae B-c, Na K. Self-quenching polysaccharide-based nanogels of pullulan/folate-photosensitizer conjugates for photodynamic therapy[J]. Biomaterials 2010;31(24):6325-35.
10. Xiong W, Wang W, Wang Y, Zhao Y, Chen H, Xu H, et al. Dual temperature/pH-sensitive drug delivery of poly(N-isopropylacrylamide-co-acrylic acid) nanogels conjugated with doxorubicin for potential application in tumor hyperthermia therapy[J]. Colloids Surf B Biointerfaces 2011;84(2):447-53.
11. Fan T, Li M, Wu X, Li M, Wu Y. Preparation of thermoresponsive and pH-sensitivity polymer magnetic hydrogel nanospheres as anticancer drug carriers[J]. Colloids and Surfaces B-Biointerfaces 2011;88(2):593-600.
12. Stubbs M, McSheehy PM, Griffiths JR, Bashford CL. Causes and consequences of tumour acidity and implications for treatment[J]. Mol Med Today 2000;6(1):15-9.
13. Karve S, Bandekar A, Ali MR, Sofou S. The pH-dependent association with cancer cells of tunable functionalized lipid vesicles with encapsulated doxorubicin for high cell-kill selectivity[J]. Biomaterials 2010;31(15):4409-16.
14. Lee I, Akiyoshi K. Single molecular mechanics of a cholesterol-bearing pullulan nanogel at the hydrophobic interfaces[J]. Biomaterials 2004;25(15):291-18.
15. Yallapu MM, Jaggi M, Chauhan SC. Poly(beta-cyclodextrin)/curcumin self-assembly:a novel approach to improve curcumin delivery and its therapeutic efficacy in prostate cancer cells[J]. Macromol Biosci 2010;10(10):1141-51.
16. Oh NM, Oh KT, Baik HJ, Lee BR, Lee AH, Youn YS, et al. A self-organized 3-diethylaminopropyl-bearing glycol chitosan nanogel for tumor acidic pH targeting:in vitro evaluation[J]. Colloids Surf B Biointerfaces 2010;78(1):120-6.
17. Akiyoshi K, Kobayashi S, Shichibe S, Mix D, Baudys M, Kim SW, et al. Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs:complexation and stabilization of insulin[J]. J Control Release 1998;54(3):313-20.
18. Liu SQ, Tong YW, Yang YY. Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glyco lide) with varying compositions[J]. Biomaterials 2005;26(24):5064-74.
19. Bhuchar N, Sunasee R, Ishihara K, Thundat T, Narain R. Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release[J]. Bioconjugate Chemistry 2011;23(l):75-83.
20. Cai G, Jiang H. pH-sensitive nanoparticles self-assembled from a novel class of biodegradable amphiphilic copolymers based on chitosan[J]. J Mater Sci Mater Med 2009;20(6):1315-20.
21. Vinogradov S, Batrakova E, Kabanov A. Poly(ethylene glycol)-polyethyleneimine NanoGelTM particles:novel drug delivery systems for antisense oligonucleotides[J]. Colloids and Surfaces B:Biointerfaces 1999;16(1-4): 291-304.
22. Choi SH, Lee SH, Park TG. Temperature-sensitive pluronic/poly(ethylenimine) nanocapsules for thermally triggered disruption of intracellular endosomal compartment[J]. Biomacromolecules 2006;7(6):1864-70.
23. Li M, Huang Q, Wu Y. A novel chitosan-poly(lactide) copolymer and its submicron particles as imidacloprid carriers.
24. Zhang C, Qineng P, Zhang H. Self-assembly and characterization of paclitaxel-loaded N-octyl-O-sulfate chitosan micellar system[J]. Colloids and Surfaces B: Biointerfaces 2004;39(1-2):69-75.
25. Duan C, Gao J, Zhang D, Jia L, Liu Y, Zheng D, et al. Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery of oridonin[J]. Biomacromolecules 2011;12(12):4335-43.
26. Liu T, Li X, Qian Y, Hu X, Liu S. Multifunctional pH-Disintegrable micellar nanoparticles of asymmetrically functionalized β-cyclodextrin-Based star copolymer covalently conjugated with doxorubicin and DOTA-Gd moieties[J]. Biomaterials 2012;33(8):2521-31.
27. Inoue S, Ding H, Portilla-Arias J, Hu J, Konda B, Fujita M, et al. Polymalic acid-based nanobiopolymer provides efficient systemic breast cancer treatment by inhibiting both HER2/neu receptor synthesis and activity[J]. Cancer Res 2011; 71 (4):1454-64.
28. Oishi M, Nagasaki Y. Stimuli-responsive smart nanogels for cancer diagnostics and therapy[J]. Nanomedicine (Lond) 2010;5(3):451-68.
29. Na K, Lee ES, Bae YH. Self-organized nanogels responding to tumor extracellular pH:pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells[J]. Bioconjug Chem 2007; 18(5):1568-74.
30. Wu W, Shen J, Banerjee P, Zhou S. Chitosan-based responsive hybrid nanogels for integration of optical pH-sensing, tumor cell imaging and controlled drug delivery[J].Biomaterials 2010;31(32):8371-81.
31. Wu W, Aiello M, Zhou T, Berliner A, Banerjee P, Zhou S. In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery[J]. Biomaterials 2010;31(11): 3023-31.
32.郝堂娜,乔明曦,李镇,陈大为.pH及温度敏感型生物可降解嵌段共聚物的研究进展[J].药学学报2008(02)：123-27.
33. Zhang H, Mardyani S, Chan WCW, Kumacheva E. Design of biocompatible chitosan microgels for targeted pH-mediated intracellular release of cancer therapeutics[J]. Biomacromolecules 2006; 7(5):1568-72.
34. Ponta A, Bae Y. PEG-poly(amino acid) Block Copolymer Micelles for Tunable Drug Release[J]. Pharm. Res.2010;27(11):2330-42.
35. Murthy N, Xu M, Schuck S, Kunisawa J, Shastri N, Frechet JM. A macromolecular delivery vehicle for protein-based vaccines:acid-degradable protein-loaded microgels[J]. Proc Natl Acad Sci USA 2003;100(9):4995-5000.
36. Gan D, Lyon LA. Tunable swelling kinetics in core--shell hydrogel nanoparticles[J]. J Am Chem Soc 2001;123(31):7511-7.
37. Pelton R. Temperature-sensitive aqueous microgels[J]. Adv Colloid Interface Sci2000；85(1)：1-33.
38. Choi SH, Yoon JJ, Park TG. Galactosylated Poly(N-isopropylacrylamide) Hydrogel Submicrometer Particles for Specific Cellular Uptake within Hepatocytes[J]. Journal of Colloid and Interface Science 2002;251 (1):57-63.
39. Schmid S, Fuchs R, Kielian M, Helenius A, Mellman I. Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants[J]. J Cell Biol 1989; 108(4): 1291-300.
40. Wu W, Shen J, Gai Z, Hong K, Banerjee P, Zhou S. Multi-functional core-shell hybrid nanogels for pH-dependent magnetic manipulation, fluorescent pH-sensing, and drug delivery[J]. Biomaterials 2011;32(36):9876-87.
41. Di Simplicio P, Giannerini F, Giustarini D, Lusini L, Rossi R. The Role of Cysteine in the Regulation of Blood Glutathione-Protein Mixed Disulfides in Rats Treated with Diamide[J]. Toxicology and Applied Pharmacology 1998; 148(1):56-64.
42. Chang L-S, Lin S-R, Huang H-B. Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah[J]. Archives of Biochemistry and Biophysics 2006;454(2):181-88.
43. Circu ML, Stringer S, Rhoads CA, Moyer MP, Aw TY. The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells[J]. Biochem Pharmacol 2009;77(1):76-85.
44. Ou M, Xu R, Kim SH, Bull DA, Kim SW. A family of bioreducible poly(disulfide amine)s for gene delivery[J]. Biomaterials 2009;30(29):5804-14.
45. Saito G, Swanson JA, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfide linkages:role and site of cellular reducing activities[J]. Adv Drug Deliv Rev 2003;55(2):199-215.
46. Ding JX, Shi FH, Xiao CS, Lin L, Chen L, He CL, et al. One-step preparation of reduction-responsive poly(ethylene glycol)-poly (amino acid)s nanogels as efficient intracellular drug delivery platforms[J]. Polym. Chem.2011;2(12): 2857-64.
47. Purushotham S, Chang PE, Rumpel H, Kee IH, Ng RT, Chow PK, et al. Thermoresponsive core-shell magnetic nanoparticles for combined modalities of cancer therapy[J]. Nanotechnology 2009;20(30):305101.
48. Rastogi R, Gulati N, Kotnala RK, Sharma U, Jayasundar R, Koul V. Evaluation of folate conjugated pegylated thermosensitive magnetic nanocomposites for tumor imaging and therapy[J]. Colloids Surf B Biointerfaces 2011;82(1):160-7.
49. Quan C-Y, Wei H, Sun Y-X, Cheng S-X, Shen K, Gu Z-W, et al. Polyethyleneimine modified biocompatible poly(N-isopropylacrylamide)-based nanogels for drug delivery[J]. Journal of Nanoscience and Nanotechnology 2008;8(5):2377-84.
50. Hashida M, Akamatsu K, Nishikawa M, Yamashita F, Yoshikawa H, Takakura Y. Design of polymeric prodrugs of PGE1 for cell-specific hepatic targeting[J]. Pharmazie 2000;55(3):202-5.
51. Duncan R. Development of HPMA copolymer-anticancer conjugates:clinical experience and lessons learnt [J].Adv Drug Deliv Rev 2009;61(13):1131-48.
52. Cheng X, Gao F, Xiang J, Jiang X, Chen J, Zhang J. Galactosylated a,(3-poly[(2-hydroxyethyl)-L-aspartamide]-bound doxorubicin:improved antitumor activity against hepatocellular carcinoma with reduced hepatotoxicity[J]. Anti-Cancer Drugs 2011;22(2):136-47.
53. Di Stefano G, Fiume L, Baglioni M, Bolondi L, Chieco P, Kratz F, et al. Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging[J]. Dig Liver Dis 2008;40(4):278-84.
54.李崇辉,温守明,池木根,孙曼霁.半乳糖多聚赖氨酸的肝靶向特征[J].中国药理学与毒理学杂志1999(2)：110.
55.张灿,丁娅,沈健.新型肝靶向载体n-乳糖酰化壳聚糖的制备与表征[J].JOURNAL OF CHINA PHARMACEUTICAL UNIVERSITY2003;34(5):387-90.
56.徐文殷.去唾液酸糖蛋白受体介导的肝脏靶向性研究进展[J].第二军医大学学报2006(09)：1002-05.
57. Wang S, Xu H, Xu J, Zhang Y, Liu Y, Deng YH, et al. Sustained liver targeting and improved antiproliferative effect of doxorubicin liposomes modified with galactosylated lipid and PEG-lipid[J]. AAPS PharmSciTech 2010;11(2):870-7.
58. Kawakami S, Yamashita F, Nishikawa M, Takakura Y, Hashida M. Asialoglycoprotein receptor-mediated gene transfer using novel galactosylated cationic liposomes[J]. Biochemical and Biophysical Research Communications 1998;252(1):78-83.
59. Jiang JW, Zhang Y. [Targeting delivery effect of galactose receptor-mediated c-myc antisense oligonucleotide on human hepatocellular carcinoma cell line Bel-7402[J].Ai Zheng 2004;23(11):1288-93.
60.王绍宁邓意辉,吴红兵,张玲玲,陈大为.半乳糖化脂质体在小鼠体内肝靶向性评价[J].中国药学杂志2006；(14)：1076-79.
61. Wen SY, Wang XH, Lin L, Guan W, Wang SQ. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome[J]. World J Gastroenterol 2004; 10(2):244-9.
62. Yang R, Meng F, Ma S, Huang F, Liu H, Zhong Z. Galactose-Decorated Cross-Linked Biodegradable Poly(ethylene glycol)-b-poly(ε-caprolactone) Block Copolymer Micelles for Enhanced Hepatoma-Targeting Delivery of Paclitaxel[J]. Biomacromolecules 2011;12(8):3047-55.
63. Suo A, Qian J, Yao Y, Zhang W. Galactosylated poly(ethylene glycol)-b-poly (1-]actide-co-beta-malic acid) block copolymer micelles for targeted drug delivery: preparation and in vitro characterization[J]. Int JNanomedicine 2010;5:1029-38.
64. Wang W, Zhao X, Hu H, Chen D, Gu J, Deng Y, et al. Galactosylated solid lipid nanoparticles with cucurbitacin B improves the liver targetability[J]. Drug Deliv 2010; 17(3):114-22.
65. Lee C-M, Jeong H-J, Kim E-M, Kim DW, Lim ST, Kim HT, et al. Superparamagnetic Iron Oxide Nanoparticles as a Dual Imaging Probe for Targeting Hepatocytes In Vivo[J]. Magnetic Resonance in Medicine 2009;62(6):1440-46.
66. Kabanov AV, Vinogradov SV. Nanogels as Pharmaceutical Carriers:Finite Networks of Infinite Capabilities[J]. Angewandte Chemie International Edition 2009;48(30):5418-29.
67. Na K, Park KH, Kim SW, Bae YH. Self-assembled hydrogel nanoparticles from curdlan derivatives:characterization, anti-cancer drug release and interaction with a hepatoma cell line (HepG2) [J]. J Control Release 2000;69(2):225-36.
68. Liang H-F, Chen S-C, Chen M-C, Lee P-W, Chen C-T, Sung H-W. Paclitaxel-Loaded Poly(y-glutamic acid)-poly(lactide) Nanoparticles as a Targeted Drug Delivery System against Cultured HepG2 Cells[J]. Bioconjugate Chemistry 2006;17(2):291-99.
69. Shen Z, Wei W, Tanaka H, Kohama K, Ma G, Dobashi T, et al. A galactosamine-mediated drug delivery carrier for targeted liver cancer therapy[J]. Pharmacol Res 2011;64(4):410-9.
70. Jeong Y-I, Seo S-J, Park I-K, Lee H-C, Kang I-C, Akaike T, et al. Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly(y-benzyl 1-glutamate) and poly(ethylene glycol) diblock copolymer endcapped with galactose moiety [J]. International Journal of Pharmaceutics 2005;296(1-2):151-61.
71.刘晨江,赵志宏.冬凌草的研究进展[J].中国药学杂志1998(10)：1-5.
72.王瑞林,高增义.冬凌草治疗食管癌、贲门癌101例临床疗效报告[J].河南医学院学报1980(0])：31-37+2.
73.张典瑞,任天池.冬凌草甲素的药学研究进展[J].中国药学杂志2003(11)：1-4.
74.张俊峰,刘加军,陆敏强,李华,陈规划.冬凌草甲素抑制人肝癌bel-7402细胞生长及诱导细胞凋亡的机制研究[J].中草药2006(10)：1517-21.
75.张松王留兴,樊青霞,王瑞林.刺五加叶皂苷联合冬凌草甲素对人食管癌Eca-109细胞的生长抑制作用[J].肿瘤基础与临床2007(03)：226-29.
76.唐新桥,冬凌草甲素对MG-63细胞的增殖抑制、凋亡诱导和转移抑制作用及分子机制[D].中南大学,2008.
77.邓志成,冬凌草甲素抑制人肝癌SMMC-7721细胞增殖及诱导细胞凋亡的机制研究[D].苏州大学,2010.
78.季宇彬,洪宝,高世勇.冬凌草甲素抑制人胃癌sgc-7901细胞生长的G2/M期阻滞机制研究[J].中草药2010(12)：2024-26.
79.江永青,冬凌草甲素诱导MDA-MB-231细胞凋亡机制及抑制肿瘤转移相关酶的实验研究[D].南昌大学,2008.
80.熊向阳,江永青,余乐涵,李华.冬凌草甲素对肿瘤转移相关酶的影响[J].上海中医药杂志2009(03)：73-75.
81.张亚宏,冬凌草甲素及Fas激动型抗体CH11诱导人肿瘤细胞自噬和凋亡调节机制的研究[D].沈阳药科大学,2009.
82. Xu FJ, Kang ET, Neoh KG. pH-and temperature-responsive hydrogels from crosslinked triblock copolymers prepared via consecutive atom transfer radical polymerizations[J]. Biomaterials 2006;27(14):2787-97.
83. Rapoport N. Physical stimuli-responsive polymeric micelles for anti-cancer drug delivery[J]. Progress in Polymer Science 2007;32(8-9):962-90.
84. Shu XZ, Zhu KJ, Song W. Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release[J]. Int J Pharm 2001;212(1):19-28.
85. Jin YH, Hu HY, Qiao MX, Zhu J, Qi JW, Hu CJ, et al. pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation[J]. Colloids Surf B Biointerfaces 2012.
86. Li F, Wu H, Zhang H, Li F, Gu C-h, Yang Q. Antitumor drug Paclitaxel-loaded pH-sensitive nanoparticles targeting tumor extracellular pH[J]. Carbohydrate Polymers 2009;77(4):773-78.
87. Wei W, Yue ZG, Qu JB, Yue H, Su ZG, Ma GH. Galactosylated nanocrystallites of insoluble anticancer drug for liver-targeting therapy:an in vitro evaluation[J]. Nanomedicine 2010;5(4):589-96.
88. Yang Y, Zhang Z, Chen L, Gu W, Li Y. Galactosylated poly(2-(2-aminoethyoxy) ethoxy)phosphazene/DNA complex nanoparticles:in vitro and in vivo evaluation for gene delivery. Biomacromolecules 2010; 11(4):927-33.
89. Mahdavinia GR, Pourjavadi A, Hosseinzadeh H, Zohuriaan MJ. Modified chitosan 4. Superabsorbent hydrogels from poly(acrylic acid-co-acrylamide) grafted chitosan with salt-and pH-responsiveness properties[J]. European Polymer Journal 2004;40(7):1399-407.
90. Chiu YL, Chen SC, Su CJ, Hsiao C W, Chen YM, Chen HL, et al. pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan:in vitro characteristics and in vivo biocompatibility[J]. Biomaterials 2009;30(28): 4877-88.
91. Wei H, Zhang XZ, Cheng H, Chen WQ, Cheng SX, Zhuo RX. Self-assembled thermo- and pH responsive micelles of poly(10-undecenoic acid-b-N-isopropylacrylamide) for drug delivery[J]. J Control Release 2006; 116(3):266-74.
92. Alvarez-Lorenzo C, Concheiro A, Dubovik AS, Grinberg NV, Burova TV, Grinberg VY. Temperature-sensitive chitosan-poly(N-isopropylacrylamide) interpenetrated networks with enhanced loading capacity and controlled release properties[J]. J Control Release 2005;102(3):629-41.
93. Don T-M, Chen H-R. Synthesis and characterization of AB-crosslinked graft copolymers based on maleilated chitosan and N-isopropylacrylamide[J]. Carbohydrate Polymers 2005;61(3):334-47.
94. Zhang J, Wang Q, Wang A. Synthesis and characterization of chitosan-g-poly(acrylic acid)/attapulgite superabsorbent composites[J]. Carbohydrate Polymers 2007;68(2):367-74.
95. Yinsong W, Lingrong L, Jian W, Zhang Q. Preparation and characterization of self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan conjugates[J]. Carbohydrate Polymers 2007;69(3):597-606.
96. Duan C, Zhang D, Wang F, Zheng D, Jia L, Feng F, et al. Chitosan-g-poly(N-isopropylacrylamide) based nanogels for tumor extracellular targeting[J]. In1JPharm 2011;409(1-2):252-9.
97. Xing J, Zhang D, Tan T. Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo[J]. Int JBiol Macromol 2007;40(2):153-8.
98. Stockert RJ. The asialoglycoprotein receptor: relationships between structure, function, and expression[J]. Physiol Rev 1995;75(3):591-609.
99. Yang R, Meng F, Ma S, Huang F, Liu H, Zhong Z. Galactose-decorated cross-linked biodegradable poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer micelles for enhanced hepatoma-targeting delivery of paclitaxel[J]. Biomacromolecules 2011;12(8):3047-55.
100. Park IK, Kim TH, Park YH, Shin BA, Choi ES, Chowdhury EH, et al. Galactosylated chitosan-graft-polyethylene glycol) as hepatocyte-targeting DNA carrier[J]. J Control Release 2001;76(3):349-62.
101. Wang Q, Zhang L, Hu W, Hu ZH, Bei YY, Xu JY, et al. Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery[J]. Nanornedicine 2010;6(2):371-81.
102. Cao X, Li Z, Song X, Cui X, Cao P, Liu H, et al. Core-shell type multiarm star poly(epsilon-caprolactone) with high molecular weight hyperbranched polyethylenimine as core: Synthesis, characterization and encapsulation properties[J]. European Polymer Journal 2008;44(4):1060-70.
103. Bo Q, Zhao Y. Double-hydrophilic block copolymer for encapsulation and two-way pH change-induced release of metal loporphyrins[J]. Journal of Polymer Science Part a-Polymer Chemistry 2006;44(5):1734-44.
104. Wang Q, Zhang L, Hu W, Hu Z-H, Bei Y-Y, Xu J-Y, et al. Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery[J]. Nanomedicine: Nanotechnolog3, Biology and Medicine 2010;6(2):371-81.
105.李琦,孙汉董.冬凌草甲素对麻醉开胸家兔血流动力学的作用[J]. Chinese Pharmacological Bulletin 1992;8(1).
106 陆彬主编.药剂学[M].北京,中国医药科技出版社,2003,254-255.
107毕殿洲主编.药剂学(第四版)[M].北京：人民卫生出版社,2000,450.
108陆彬.药物新剂型与新技术[M].北京,人民卫生出版社,2002：234-235.
109高晓黎,程利勇,孙殿甲等.定量评价去氢骆驼蓬碱注射用乳剂的组织靶向性[J].药学学报2000；35(2)：142.
2.熊微.温度/pH双重敏感纳米凝胶的构建、药物偶联与新型血管栓塞材料的研究[D].华中科技大学,2011.
3. Tamura M, Ichinohe S, Tamura A, Ikeda Y, Nagasaki Y. In vitro and in vivo characteristics of core-shell type nanogel particles:optimization of core cross-linking density and surface poly(ethylene glycol) density in PEGylated nanogels[J]. Acta Biomater 2011;7(9):3354-61.
4. Vinogradov SV, Kohli E, Zeman AD. Comparison of nanogel drug carriers and their formulations with nucleoside 5'-triphosphates[J]. Pharm Res 2006;23(5):920-30.
5.刘永,崔英德,尹国强等.快速响应水凝胶在给药系统中的应用进展[J].高分子材料科学与工程2009(06)：158-62.
6. Galmarini CM, Warren G, Kohli E, Zeman A, Mitin A, Vinogradov SV. Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines[J]. Mol Cancer Ther 2008;7(10):3373-80.
7. Quan CY, Sun YX, Cheng H, Cheng SX, Zhang XZ, Zhuo RX. Thermosensitive P(NIPAAm-co-PAAc-co-HEMA) nanogels conjugated with transferrin for tumor cell targeting delivery[J]. Nanotechnology 2008;19(27):275102.
8. Oishi M, Sumitani S, Nagasaki Y. On-off regulation of 19F magnetic resonance signals based on pH-sensitive PEGylated nanogels for potential tumor-specific smart 19F MRI probes[J]. Bioconjug Chem 2007; 18(5):1379-82.
9. Bae B-c, Na K. Self-quenching polysaccharide-based nanogels of pullulan/folate-photosensitizer conjugates for photodynamic therapy[J]. Biomaterials 2010;31(24):6325-35.
10. Xiong W, Wang W, Wang Y, Zhao Y, Chen H, Xu H, et al. Dual temperature/pH-sensitive drug delivery of poly(N-isopropylacrylamide-co-acrylic acid) nanogels conjugated with doxorubicin for potential application in tumor hyperthermia therapy[J]. Colloids Surf B Biointerfaces 2011;84(2):447-53.
11. Fan T, Li M, Wu X, Li M, Wu Y. Preparation of thermoresponsive and pH-sensitivity polymer magnetic hydrogel nanospheres as anticancer drug carriers[J]. Colloids and Surfaces B-Biointerfaces 2011;88(2):593-600.
12. Stubbs M, McSheehy PM, Griffiths JR, Bashford CL. Causes and consequences of tumour acidity and implications for treatment[J]. Mol Med Today 2000;6(1):15-9.
13. Karve S, Bandekar A, Ali MR, Sofou S. The pH-dependent association with cancer cells of tunable functionalized lipid vesicles with encapsulated doxorubicin for high cell-kill selectivity[J]. Biomaterials 2010;31(15):4409-16.
14. Lee I, Akiyoshi K. Single molecular mechanics of a cholesterol-bearing pullulan nanogel at the hydrophobic interfaces[J]. Biomaterials 2004;25(15):291-18.
15. Yallapu MM, Jaggi M, Chauhan SC. Poly(beta-cyclodextrin)/curcumin self-assembly:a novel approach to improve curcumin delivery and its therapeutic efficacy in prostate cancer cells[J]. Macromol Biosci 2010;10(10):1141-51.
16. Oh NM, Oh KT, Baik HJ, Lee BR, Lee AH, Youn YS, et al. A self-organized 3-diethylaminopropyl-bearing glycol chitosan nanogel for tumor acidic pH targeting:in vitro evaluation[J]. Colloids Surf B Biointerfaces 2010;78(1):120-6.
17. Akiyoshi K, Kobayashi S, Shichibe S, Mix D, Baudys M, Kim SW, et al. Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs:complexation and stabilization of insulin[J]. J Control Release 1998;54(3):313-20.
18. Liu SQ, Tong YW, Yang YY. Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glyco lide) with varying compositions[J]. Biomaterials 2005;26(24):5064-74.
19. Bhuchar N, Sunasee R, Ishihara K, Thundat T, Narain R. Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release[J]. Bioconjugate Chemistry 2011;23(l):75-83.
20. Cai G, Jiang H. pH-sensitive nanoparticles self-assembled from a novel class of biodegradable amphiphilic copolymers based on chitosan[J]. J Mater Sci Mater Med 2009;20(6):1315-20.
21. Vinogradov S, Batrakova E, Kabanov A. Poly(ethylene glycol)-polyethyleneimine NanoGelTM particles:novel drug delivery systems for antisense oligonucleotides[J]. Colloids and Surfaces B:Biointerfaces 1999;16(1-4): 291-304.
22. Choi SH, Lee SH, Park TG. Temperature-sensitive pluronic/poly(ethylenimine) nanocapsules for thermally triggered disruption of intracellular endosomal compartment[J]. Biomacromolecules 2006;7(6):1864-70.
23. Li M, Huang Q, Wu Y. A novel chitosan-poly(lactide) copolymer and its submicron particles as imidacloprid carriers.
24. Zhang C, Qineng P, Zhang H. Self-assembly and characterization of paclitaxel-loaded N-octyl-O-sulfate chitosan micellar system[J]. Colloids and Surfaces B: Biointerfaces 2004;39(1-2):69-75.
25. Duan C, Gao J, Zhang D, Jia L, Liu Y, Zheng D, et al. Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery of oridonin[J]. Biomacromolecules 2011;12(12):4335-43.
26. Liu T, Li X, Qian Y, Hu X, Liu S. Multifunctional pH-Disintegrable micellar nanoparticles of asymmetrically functionalized β-cyclodextrin-Based star copolymer covalently conjugated with doxorubicin and DOTA-Gd moieties[J]. Biomaterials 2012;33(8):2521-31.
27. Inoue S, Ding H, Portilla-Arias J, Hu J, Konda B, Fujita M, et al. Polymalic acid-based nanobiopolymer provides efficient systemic breast cancer treatment by inhibiting both HER2/neu receptor synthesis and activity[J]. Cancer Res 2011; 71 (4):1454-64.
28. Oishi M, Nagasaki Y. Stimuli-responsive smart nanogels for cancer diagnostics and therapy[J]. Nanomedicine (Lond) 2010;5(3):451-68.
29. Na K, Lee ES, Bae YH. Self-organized nanogels responding to tumor extracellular pH:pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells[J]. Bioconjug Chem 2007; 18(5):1568-74.
30. Wu W, Shen J, Banerjee P, Zhou S. Chitosan-based responsive hybrid nanogels for integration of optical pH-sensing, tumor cell imaging and controlled drug delivery[J].Biomaterials 2010;31(32):8371-81.
31. Wu W, Aiello M, Zhou T, Berliner A, Banerjee P, Zhou S. In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery[J]. Biomaterials 2010;31(11): 3023-31.
32.郝堂娜,乔明曦,李镇,陈大为.pH及温度敏感型生物可降解嵌段共聚物的研究进展[J].药学学报2008(02)：123-27.
33. Zhang H, Mardyani S, Chan WCW, Kumacheva E. Design of biocompatible chitosan microgels for targeted pH-mediated intracellular release of cancer therapeutics[J]. Biomacromolecules 2006; 7(5):1568-72.
34. Ponta A, Bae Y. PEG-poly(amino acid) Block Copolymer Micelles for Tunable Drug Release[J]. Pharm. Res.2010;27(11):2330-42.
35. Murthy N, Xu M, Schuck S, Kunisawa J, Shastri N, Frechet JM. A macromolecular delivery vehicle for protein-based vaccines:acid-degradable protein-loaded microgels[J]. Proc Natl Acad Sci USA 2003;100(9):4995-5000.
36. Gan D, Lyon LA. Tunable swelling kinetics in core--shell hydrogel nanoparticles[J]. J Am Chem Soc 2001;123(31):7511-7.
37. Pelton R. Temperature-sensitive aqueous microgels[J]. Adv Colloid Interface Sci2000；85(1)：1-33.
38. Choi SH, Yoon JJ, Park TG. Galactosylated Poly(N-isopropylacrylamide) Hydrogel Submicrometer Particles for Specific Cellular Uptake within Hepatocytes[J]. Journal of Colloid and Interface Science 2002;251 (1):57-63.
39. Schmid S, Fuchs R, Kielian M, Helenius A, Mellman I. Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants[J]. J Cell Biol 1989; 108(4): 1291-300.
40. Wu W, Shen J, Gai Z, Hong K, Banerjee P, Zhou S. Multi-functional core-shell hybrid nanogels for pH-dependent magnetic manipulation, fluorescent pH-sensing, and drug delivery[J]. Biomaterials 2011;32(36):9876-87.
41. Di Simplicio P, Giannerini F, Giustarini D, Lusini L, Rossi R. The Role of Cysteine in the Regulation of Blood Glutathione-Protein Mixed Disulfides in Rats Treated with Diamide[J]. Toxicology and Applied Pharmacology 1998; 148(1):56-64.
42. Chang L-S, Lin S-R, Huang H-B. Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah[J]. Archives of Biochemistry and Biophysics 2006;454(2):181-88.
43. Circu ML, Stringer S, Rhoads CA, Moyer MP, Aw TY. The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells[J]. Biochem Pharmacol 2009;77(1):76-85.
44. Ou M, Xu R, Kim SH, Bull DA, Kim SW. A family of bioreducible poly(disulfide amine)s for gene delivery[J]. Biomaterials 2009;30(29):5804-14.
45. Saito G, Swanson JA, Lee KD. Drug delivery strategy utilizing conjugation via reversible disulfide linkages:role and site of cellular reducing activities[J]. Adv Drug Deliv Rev 2003;55(2):199-215.
46. Ding JX, Shi FH, Xiao CS, Lin L, Chen L, He CL, et al. One-step preparation of reduction-responsive poly(ethylene glycol)-poly (amino acid)s nanogels as efficient intracellular drug delivery platforms[J]. Polym. Chem.2011;2(12): 2857-64.
47. Purushotham S, Chang PE, Rumpel H, Kee IH, Ng RT, Chow PK, et al. Thermoresponsive core-shell magnetic nanoparticles for combined modalities of cancer therapy[J]. Nanotechnology 2009;20(30):305101.
48. Rastogi R, Gulati N, Kotnala RK, Sharma U, Jayasundar R, Koul V. Evaluation of folate conjugated pegylated thermosensitive magnetic nanocomposites for tumor imaging and therapy[J]. Colloids Surf B Biointerfaces 2011;82(1):160-7.
49. Quan C-Y, Wei H, Sun Y-X, Cheng S-X, Shen K, Gu Z-W, et al. Polyethyleneimine modified biocompatible poly(N-isopropylacrylamide)-based nanogels for drug delivery[J]. Journal of Nanoscience and Nanotechnology 2008;8(5):2377-84.
50. Hashida M, Akamatsu K, Nishikawa M, Yamashita F, Yoshikawa H, Takakura Y. Design of polymeric prodrugs of PGE1 for cell-specific hepatic targeting[J]. Pharmazie 2000;55(3):202-5.
51. Duncan R. Development of HPMA copolymer-anticancer conjugates:clinical experience and lessons learnt [J].Adv Drug Deliv Rev 2009;61(13):1131-48.
52. Cheng X, Gao F, Xiang J, Jiang X, Chen J, Zhang J. Galactosylated a,(3-poly[(2-hydroxyethyl)-L-aspartamide]-bound doxorubicin:improved antitumor activity against hepatocellular carcinoma with reduced hepatotoxicity[J]. Anti-Cancer Drugs 2011;22(2):136-47.
53. Di Stefano G, Fiume L, Baglioni M, Bolondi L, Chieco P, Kratz F, et al. Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging[J]. Dig Liver Dis 2008;40(4):278-84.
54.李崇辉,温守明,池木根,孙曼霁.半乳糖多聚赖氨酸的肝靶向特征[J].中国药理学与毒理学杂志1999(2)：110.
55.张灿,丁娅,沈健.新型肝靶向载体n-乳糖酰化壳聚糖的制备与表征[J].JOURNAL OF CHINA PHARMACEUTICAL UNIVERSITY2003;34(5):387-90.
56.徐文殷.去唾液酸糖蛋白受体介导的肝脏靶向性研究进展[J].第二军医大学学报2006(09)：1002-05.
57. Wang S, Xu H, Xu J, Zhang Y, Liu Y, Deng YH, et al. Sustained liver targeting and improved antiproliferative effect of doxorubicin liposomes modified with galactosylated lipid and PEG-lipid[J]. AAPS PharmSciTech 2010;11(2):870-7.
58. Kawakami S, Yamashita F, Nishikawa M, Takakura Y, Hashida M. Asialoglycoprotein receptor-mediated gene transfer using novel galactosylated cationic liposomes[J]. Biochemical and Biophysical Research Communications 1998;252(1):78-83.
59. Jiang JW, Zhang Y. [Targeting delivery effect of galactose receptor-mediated c-myc antisense oligonucleotide on human hepatocellular carcinoma cell line Bel-7402[J].Ai Zheng 2004;23(11):1288-93.
60.王绍宁邓意辉,吴红兵,张玲玲,陈大为.半乳糖化脂质体在小鼠体内肝靶向性评价[J].中国药学杂志2006；(14)：1076-79.
61. Wen SY, Wang XH, Lin L, Guan W, Wang SQ. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome[J]. World J Gastroenterol 2004; 10(2):244-9.
62. Yang R, Meng F, Ma S, Huang F, Liu H, Zhong Z. Galactose-Decorated Cross-Linked Biodegradable Poly(ethylene glycol)-b-poly(ε-caprolactone) Block Copolymer Micelles for Enhanced Hepatoma-Targeting Delivery of Paclitaxel[J]. Biomacromolecules 2011;12(8):3047-55.
63. Suo A, Qian J, Yao Y, Zhang W. Galactosylated poly(ethylene glycol)-b-poly (1-]actide-co-beta-malic acid) block copolymer micelles for targeted drug delivery: preparation and in vitro characterization[J]. Int JNanomedicine 2010;5:1029-38.
64. Wang W, Zhao X, Hu H, Chen D, Gu J, Deng Y, et al. Galactosylated solid lipid nanoparticles with cucurbitacin B improves the liver targetability[J]. Drug Deliv 2010; 17(3):114-22.
65. Lee C-M, Jeong H-J, Kim E-M, Kim DW, Lim ST, Kim HT, et al. Superparamagnetic Iron Oxide Nanoparticles as a Dual Imaging Probe for Targeting Hepatocytes In Vivo[J]. Magnetic Resonance in Medicine 2009;62(6):1440-46.
66. Kabanov AV, Vinogradov SV. Nanogels as Pharmaceutical Carriers:Finite Networks of Infinite Capabilities[J]. Angewandte Chemie International Edition 2009;48(30):5418-29.
67. Na K, Park KH, Kim SW, Bae YH. Self-assembled hydrogel nanoparticles from curdlan derivatives:characterization, anti-cancer drug release and interaction with a hepatoma cell line (HepG2) [J]. J Control Release 2000;69(2):225-36.
68. Liang H-F, Chen S-C, Chen M-C, Lee P-W, Chen C-T, Sung H-W. Paclitaxel-Loaded Poly(y-glutamic acid)-poly(lactide) Nanoparticles as a Targeted Drug Delivery System against Cultured HepG2 Cells[J]. Bioconjugate Chemistry 2006;17(2):291-99.
69. Shen Z, Wei W, Tanaka H, Kohama K, Ma G, Dobashi T, et al. A galactosamine-mediated drug delivery carrier for targeted liver cancer therapy[J]. Pharmacol Res 2011;64(4):410-9.
70. Jeong Y-I, Seo S-J, Park I-K, Lee H-C, Kang I-C, Akaike T, et al. Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly(y-benzyl 1-glutamate) and poly(ethylene glycol) diblock copolymer endcapped with galactose moiety [J]. International Journal of Pharmaceutics 2005;296(1-2):151-61.
71.刘晨江,赵志宏.冬凌草的研究进展[J].中国药学杂志1998(10)：1-5.
72.王瑞林,高增义.冬凌草治疗食管癌、贲门癌101例临床疗效报告[J].河南医学院学报1980(0])：31-37+2.
73.张典瑞,任天池.冬凌草甲素的药学研究进展[J].中国药学杂志2003(11)：1-4.
74.张俊峰,刘加军,陆敏强,李华,陈规划.冬凌草甲素抑制人肝癌bel-7402细胞生长及诱导细胞凋亡的机制研究[J].中草药2006(10)：1517-21.
75.张松王留兴,樊青霞,王瑞林.刺五加叶皂苷联合冬凌草甲素对人食管癌Eca-109细胞的生长抑制作用[J].肿瘤基础与临床2007(03)：226-29.
76.唐新桥,冬凌草甲素对MG-63细胞的增殖抑制、凋亡诱导和转移抑制作用及分子机制[D].中南大学,2008.
77.邓志成,冬凌草甲素抑制人肝癌SMMC-7721细胞增殖及诱导细胞凋亡的机制研究[D].苏州大学,2010.
78.季宇彬,洪宝,高世勇.冬凌草甲素抑制人胃癌sgc-7901细胞生长的G2/M期阻滞机制研究[J].中草药2010(12)：2024-26.
79.江永青,冬凌草甲素诱导MDA-MB-231细胞凋亡机制及抑制肿瘤转移相关酶的实验研究[D].南昌大学,2008.
80.熊向阳,江永青,余乐涵,李华.冬凌草甲素对肿瘤转移相关酶的影响[J].上海中医药杂志2009(03)：73-75.
81.张亚宏,冬凌草甲素及Fas激动型抗体CH11诱导人肿瘤细胞自噬和凋亡调节机制的研究[D].沈阳药科大学,2009.
82. Xu FJ, Kang ET, Neoh KG. pH-and temperature-responsive hydrogels from crosslinked triblock copolymers prepared via consecutive atom transfer radical polymerizations[J]. Biomaterials 2006;27(14):2787-97.
83. Rapoport N. Physical stimuli-responsive polymeric micelles for anti-cancer drug delivery[J]. Progress in Polymer Science 2007;32(8-9):962-90.
84. Shu XZ, Zhu KJ, Song W. Novel pH-sensitive citrate cross-linked chitosan film for drug controlled release[J]. Int J Pharm 2001;212(1):19-28.
85. Jin YH, Hu HY, Qiao MX, Zhu J, Qi JW, Hu CJ, et al. pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation[J]. Colloids Surf B Biointerfaces 2012.
86. Li F, Wu H, Zhang H, Li F, Gu C-h, Yang Q. Antitumor drug Paclitaxel-loaded pH-sensitive nanoparticles targeting tumor extracellular pH[J]. Carbohydrate Polymers 2009;77(4):773-78.
87. Wei W, Yue ZG, Qu JB, Yue H, Su ZG, Ma GH. Galactosylated nanocrystallites of insoluble anticancer drug for liver-targeting therapy:an in vitro evaluation[J]. Nanomedicine 2010;5(4):589-96.
88. Yang Y, Zhang Z, Chen L, Gu W, Li Y. Galactosylated poly(2-(2-aminoethyoxy) ethoxy)phosphazene/DNA complex nanoparticles:in vitro and in vivo evaluation for gene delivery. Biomacromolecules 2010; 11(4):927-33.
89. Mahdavinia GR, Pourjavadi A, Hosseinzadeh H, Zohuriaan MJ. Modified chitosan 4. Superabsorbent hydrogels from poly(acrylic acid-co-acrylamide) grafted chitosan with salt-and pH-responsiveness properties[J]. European Polymer Journal 2004;40(7):1399-407.
90. Chiu YL, Chen SC, Su CJ, Hsiao C W, Chen YM, Chen HL, et al. pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan:in vitro characteristics and in vivo biocompatibility[J]. Biomaterials 2009;30(28): 4877-88.
91. Wei H, Zhang XZ, Cheng H, Chen WQ, Cheng SX, Zhuo RX. Self-assembled thermo- and pH responsive micelles of poly(10-undecenoic acid-b-N-isopropylacrylamide) for drug delivery[J]. J Control Release 2006; 116(3):266-74.
92. Alvarez-Lorenzo C, Concheiro A, Dubovik AS, Grinberg NV, Burova TV, Grinberg VY. Temperature-sensitive chitosan-poly(N-isopropylacrylamide) interpenetrated networks with enhanced loading capacity and controlled release properties[J]. J Control Release 2005;102(3):629-41.
93. Don T-M, Chen H-R. Synthesis and characterization of AB-crosslinked graft copolymers based on maleilated chitosan and N-isopropylacrylamide[J]. Carbohydrate Polymers 2005;61(3):334-47.
94. Zhang J, Wang Q, Wang A. Synthesis and characterization of chitosan-g-poly(acrylic acid)/attapulgite superabsorbent composites[J]. Carbohydrate Polymers 2007;68(2):367-74.
95. Yinsong W, Lingrong L, Jian W, Zhang Q. Preparation and characterization of self-aggregated nanoparticles of cholesterol-modified O-carboxymethyl chitosan conjugates[J]. Carbohydrate Polymers 2007;69(3):597-606.
96. Duan C, Zhang D, Wang F, Zheng D, Jia L, Feng F, et al. Chitosan-g-poly(N-isopropylacrylamide) based nanogels for tumor extracellular targeting[J]. In1JPharm 2011;409(1-2):252-9.
97. Xing J, Zhang D, Tan T. Studies on the oridonin-loaded poly(D,L-lactic acid) nanoparticles in vitro and in vivo[J]. Int JBiol Macromol 2007;40(2):153-8.
98. Stockert RJ. The asialoglycoprotein receptor: relationships between structure, function, and expression[J]. Physiol Rev 1995;75(3):591-609.
99. Yang R, Meng F, Ma S, Huang F, Liu H, Zhong Z. Galactose-decorated cross-linked biodegradable poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer micelles for enhanced hepatoma-targeting delivery of paclitaxel[J]. Biomacromolecules 2011;12(8):3047-55.
100. Park IK, Kim TH, Park YH, Shin BA, Choi ES, Chowdhury EH, et al. Galactosylated chitosan-graft-polyethylene glycol) as hepatocyte-targeting DNA carrier[J]. J Control Release 2001;76(3):349-62.
101. Wang Q, Zhang L, Hu W, Hu ZH, Bei YY, Xu JY, et al. Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery[J]. Nanornedicine 2010;6(2):371-81.
102. Cao X, Li Z, Song X, Cui X, Cao P, Liu H, et al. Core-shell type multiarm star poly(epsilon-caprolactone) with high molecular weight hyperbranched polyethylenimine as core: Synthesis, characterization and encapsulation properties[J]. European Polymer Journal 2008;44(4):1060-70.
103. Bo Q, Zhao Y. Double-hydrophilic block copolymer for encapsulation and two-way pH change-induced release of metal loporphyrins[J]. Journal of Polymer Science Part a-Polymer Chemistry 2006;44(5):1734-44.
104. Wang Q, Zhang L, Hu W, Hu Z-H, Bei Y-Y, Xu J-Y, et al. Norcantharidin-associated galactosylated chitosan nanoparticles for hepatocyte-targeted delivery[J]. Nanomedicine: Nanotechnolog3, Biology and Medicine 2010;6(2):371-81.
105.李琦,孙汉董.冬凌草甲素对麻醉开胸家兔血流动力学的作用[J]. Chinese Pharmacological Bulletin 1992;8(1).
106 陆彬主编.药剂学[M].北京,中国医药科技出版社,2003,254-255.
107毕殿洲主编.药剂学(第四版)[M].北京：人民卫生出版社,2000,450.
108陆彬.药物新剂型与新技术[M].北京,人民卫生出版社,2002：234-235.
109高晓黎,程利勇,孙殿甲等.定量评价去氢骆驼蓬碱注射用乳剂的组织靶向性[J].药学学报2000；35(2)：142.