| 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 carriersThe 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 deliveryGal-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 activityTo 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. |
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