| Gliomas account for about 46% of intracranial tumors, and surgical excision is the primary choice in the clinical treatment of gliomas. Because of the specificity of their locations, gliomas usually cannot be removed completely, the treatment always supplemented with radiotherapy and chemotherapy. Glioma in its early stage, the blood-brain barrier (BBB) is relatively complete; with the progression of the disease, it begins to generate a large number of tumor angiogenesis, and the vascular endothelial cell gap goes up to 50-300 nm; Supplemented treatments against the margin of the tumor where usually maintains after tumor resection because of its infiltration with normal brain tissue, and also where BBB still has its function, is significantly important to the prognosis of gliomas treatment.In this study, the anticancer drug Doxorubicin was encapsulated in liposome, the particle size of which was about 100 nm, and the liposome was modified with the target functional group Angiopep-2 that targe to low density lipoprotein receptor-related protein which is highly expressed on BBB and gliomas cells, so that a glioma targeted liposome is achieved to enhance the role of drugs on the lesion site and reduce the systemic side effects.The first part described the preparation and characterization of Doxorubicin (DOX) liposome and carried out the optimization of formulation and preparation process of the liposome. After optimization, drug/lipid ratio in liposome prescription was 1:25, DPPC/PEG-DSPE/DSPE-PEG-MAL was 90:10:2, the encapsulation efficiency was above 99%. The volume based particle size of the liposome was about 100 nm; its Zeta Potential was around-8 mV. The liposome was prepared by film hydration, and the maleimides of DSPE-PEG-MAL on the liposome surface conjugated with the Angiopep-2 covalently. By pH gradient drug loading, Doxorubicin liposome (AL) was obtained at last.Using rat glioma cells (C6) as cell model of in vitro study, the drug delivery characteristics of AL to glioma were investigated, and the results would be compared with DOX and the normal doxorubicin liposome (NL). The qualitative uptake results demonstrated that the uptake of AL was more than that of NL, which showed AL has certain characteristics of glioma targeting.In the second part, the LC-MS/MS method for the quantification of doxorubicin in biological samples was established. Glioma-bearing mouse model was established using C6 cells, and proved by pathological anatomy and histopathology. Pharmacokinetic properties of AL were investigated through drug concentration in plasma and tissues (brain, tumor, heart, spleen, liver, lung, and kidney) of glioma-bearing mice, and the results would be compared with DOX and NL. The pharmacokinetics results showed that DOX, NL, and AL were fast distributed and slowly eliminated, which fitted with three-compartment model. Compared with DOX, doxorubicin liposomes significantly prolonged circulation time of drug in vivo and increased its brain uptake; AUCo-t of AL in the brain and tumor were 2.92 and 7.76 times those of NL, the brain targeting index was 2.8, indicating a certain brain targeting ability of AL. Biodistribution results showed that, AL has longer residence time in brain tissue. These results suggested that the optimized AL can increase brain drug transport, demonstrating that it is an ideal drug delivery system for brain targeting.In the third part, C6 glioma-bearing mouse model was used to evaluate anti-tumor efficacy of AL in vivo, and the results would be compared with the saline group, DOX group and NL group. The tumor inhibitory rate was compared with tumor volume as the main index, and the tumor area were evaluate by histopathologic examination and TUNEL detection of apoptosis. Take the changes of mice body weight as an index, make preliminary evaluation to study the impact of the preparation on the mice survival. And survival analysis was carried out for each group. After treatments, the mice tumor volume of the saline negative control group, DOX group NL group and AL group were 68.63±30.80 mm3,36.47±12.00 mm3,11.93±4.21mm3 and 5.39±4.27 mm, respectively. The tumor volume of AL group was significantly smaller than that of all the other groups, and its histopathologic examination and TUNEL results showed strongest apoptosis of tumor area; its mice gained more weight compared with the other groups administrated with doxorubicin; its median survival time of mice was significantly longer than that of saline group and DOX group, and longer than that of NL group but there was no significant difference. The above results demonstrated that Angiopep-2 modified doxorubicin liposome has certain glioma targeting therapeutic effects. |
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