| Glioma is the most common and aggressive type of primary intracranial neoplasm, accounting for more than40%of all primary central nervous system tumors. Chemotherapy is essential to surgical treatment because traditional surgical resection could not prevent the recurrence of glioma.Therefore, an effective drug delivery system for the treatment of glioma should be developed.In the first chapter, transferrin (Tf) was used to modify cisplatin (Cis) liposome (lipo)(Tf-lipo) as Tf receptor was overexpressed on both brain capillary endothelial cells and glioma cells. Then its potential sequential targeting to glioma was characterized. In bEnd3/C6co-culture blood-brain barrier(BBB) models, higher transport efficiency across the BBB and cytotoxicity in basal C6cells induced by Cis-lipo(Tf) than both Cis-lipo and Cis-solution was showed, suggesting its sequential targeting effect. Interestingly, in BBB models, similar liposomal morphology as that in donor solution was first demonstrated in the receptor solution. Meanwhile, a greater acquisition in the lysosome of bEnd3, distribution sequentially into the nucleus of C6cells could be observed for the Cis-lipo(Tf), and pre-incubation of chlorpromazine and Tf inhibited this process, indicating a clathrin-dependent endocytosis is involved in the transport of Cis-lipo(Tf) across the BBB.In the second part, Tf, togeter with another ligand folate (F) that was overexpressed on the C6cells, were used to modify doxorubicin(DOX) liposome (Tf(F)-dox-lipo) in order to achieve a more ideal sequential targeting. The liposome was prepared by thin-film evaporation method and DOX was loaded by ammonium sulfate gradient method.Folate was conjugated to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000to get the DSPE-PEG2000-Folate, and it was confirmed by NMR analysis. Furthermore, the kinds of phospholipids, ratio of drug to lipid, hydration medium concentration and drug loading time were optimized. And parameters, such as particle size, encapsulation efficiency and stability, were determined to evaluate the liposomes. Then, DSPC was used as the main components of phospholipids,120mM ammonium sulfate was applied for the hydration medium, the drug to lipid ratio was1:15, drug loading time was determined to be60mins, and stable dual-ligands modified liposomes with high encapsulation efficiency was successfully developed. Meanwhile, rats brain blood capillary endothelial cells (bEnd3) and astrocytes were used to construct in vitro blood-brain barrier(BBB), which was further combined with rats glioma C6cells to construct the composite BBB model that could be applied in dual-targeting studies in vitro. The uptake mechanism of doxorubicin liposomes in bEnd3cells, transport ratios through the BBB and toxicity in C6glioma cells were investigated. The Tf(F)-dox-lipo released the DOX in a sustained manner, and it showed a higher uptake in bEnd3cells than conventional liposomes (p<0.05). In additrion, the uptake for Tf(F)-dox-lipo was reduced after pretreatment of transferrin and folic acid, indicating both clathrin-dependent and caveolae-dependent manner were involved in endocytosis way. Meanwhile, in the composite BBB model, Tf(F)-dox-lipo showed a higher transport ratio across BBB and a stronger subsequent toxicity in basal C6cells than other liposome treatments.In the third chapter, in vivo effectiveness of both dual targeting and chemotherapy for Tf(F) liposome were evaluated. Glioma rat model was established by injection of C6cells into the striatum.The model was verified at both tissues level and cells level. After Tf(F)-dox-lipo was administrated intravenously, it could transport across BBB and further distribute into the brain glioma as shown in-vitro imaging technology and brain frozen section. The C6glioma volume was significantly decreased and the survival time of the tumor-bearing mice were greatly increased after intravenous administration of the Tf(F)-dox-lipo as compared with the control group. The anti-tumor effect of the Tf(F)-dox-lipo was also demonstrated by MRI (magnetic resonance imaging), H&E (hematoxylin and eosin) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis. Meanwhile, the P-gp expression of BBB for the Tf(F)-dox-lipo treatment was also lower than the Dox solution and Dox-Lipo treatments. Therefore, the dual-targeting doxorubicin liposomes (Tf(F)-dox-Lipo) were successfully prepared and were demonstrated to improve the therapeutic efficacy of brain glioma as compared with the control group, together with less toxicity than Dox solution, showing a dual-targeting effect. These results indicated that this dual-targeted liposome could be a promising carrier for glioma chemotherapy in the future. |
PDF Full Text Request