| Glioma is the most common brain tumor that threatens the health of people, with high recurrence rate and high mortality. Due to the invasive growth of glioma and blood brain barrier (BBB), glioma is difficult to be cured by surgery, radiotherapy and chemotherapy. Carmustine (BCNU), an important chemotherapeutic alkylating agent, could combine with DNA to form O6-chloroethylguanine and further inhibit its replication. The limited efficacy of BCNU is the tumor resistance, which is associated with the expression of DNA repair proteins in tumor cells, especially O6-methylguanine DNA-methyl transferase (MGMT). MGMT is able to remove the chloroethyl group from O6-chloroethylguanine to protect DNA from alkylating damage. O6-benzylguanine (BG), an inactivator of MGMT, is used to potentiate the efficiency of BCNU by directly depleting MGMT levels in tumor cells. In this study, we intended to construct a core-shell nanoparticle drug delivery system simultaneously loading BCNU and BG simultaneously for targeting treatment of glioma. Poly (lactide-co-glycolides)(PLGA) and Chitosan (CS) were used as carrier materials. PLGA served as the core of NPs carrying BCNU, while CS was used as a coating shell loading BG. BG lying in the external shell could be released from NPs prior to BCNU, to consume the MGMT in tumor cells, which ultimately results in the improved therapeutic effect of BCNU.In the first chapter, the preformulation studies of BCNU and its sensitizer BG were carried out. HPLC analytic method of BCNU and BG was established. The results of BCNU stability in different conditions showed that BCNU was quite stable in organic solvents, whereas it had a rapid degradation in aqueous solutions. The stability of BCNU decreased with the increasing pH value of solution and increasing temperature. C6cells with relatively lower MGMT level, F98cells with relatively higher MGMT level and human primary glioblastoma (HPG) cells were used as the models to evaluate the cytotoxicity using MTT cell proliferation assays. Pretreatment with BG could increase the cytotoxicity of BCNU against C6cells, F98cells and HPG cells. The sensitizing effect of BG depended on the tumor resistance to BCNU, the concentration and administration time of BG. The apoptosis of F98cells induced by BCNU could be increased by the pretreatment of BG.In the second chapter, BCNU+BG loaded PLGA/CS NPs were prepared and characterized. The single factor inspection was investigated, including the molecular weight of PLGA, the concentration of PLGA, the concentration of CS, the kind of surfactant, ultrasonic power, O/W volume ratio and the method to remove organic solvents. Then the optimal prescription was obtained through the orthogonal experiment design. The PLGA/CS NPs in a mean diameter around177.1±3.2nm showed positive zeta potential (19.6±1.8mV). The drug loading efficiency of BCNU and BG in PLGA/CS NPs was (3.05±0.32)%and (8.97±0.81)%, respectively. The transmission electron microscopy (TEM) image and scanning electron microscopy (SEM) image demonstrated that the PLGA/CS NPs had a regular spherical shape with clear core-shell structure.In the third chapter, in vitro evaluations of PLGA/CS NPs were carried out. In plasma, the degration half-life of BCNU in NPs (T50=108.56min) was5.0-fold longer than that of free BCNU (T50=21.55min)(P<0.05). The hemolytic toxicity of non-loaded PLGA NPs was low, as well as the PLGA/CS NPs. C6cells, F98cells and HPG cells, with different MGMT levels, were used as the models to evaluate the cytotoxicity of drug-loaded NPs. After being encapsulated into the NPs, BCNU displayed improved antitumor activity. Furthermore, when BG coexisted with BCNU in the NPs, the IC50values against each cell line were the lowest among four treatment groups. Since not only the BCNU-resistance degrees but also BG sensitization effects in F98cells for all treatment groups were very close to those of HPG cells, F98cell line is more suitable to be the drug-resistance glioma cell model for further in vitro and in vivo experiments. The fluorescence probe (coumarin6) loaded nanoparticles were prepared and characterized. The particle size and zeta potential of coumarin6-loaded NPs were consistent with those of BCNU+BG loaded NPs. In vitro leakage experiment demonstrated that only1.0%free coumarin6released from PLGA/CS NPs at24h in PBS (pH7.4and pH4.0)(Figure S4). Therefore, coumarin6could be used to trace the transport of NPs in vitro and in vivo. The cellular uptake was evaluated by fluorescent microscope and flow cytometry. The cellular uptake of CS surface-modified PLGA NPs is higher than that of non-modified PLGA NPs. Additionally, the cellular uptake of PLGA/CS NPs increased with the decreasing particle size and the prolonged incubation time. Further investigation on the internalization mechanism by F98cells indicated that PLGA/CS NPs mainly be interacted with negative charged cell membranes by electrostatic interactions, and then was internalized by clathrin-mediated endocytosis and caveolae-mediated endocytosis.In the fourth chapter, in vivo transport behaviors of PLGA/CS NPs were investigated. After being encapsulated into NPs, the transport behaviors were significantly changed. The results demonstrated that coumarin6and BG in NPs displayed significant longer retention time, with prolonged AUC, t1/2β and MRT. The biodistribution of coumarin6and BG in PLGA/CS NPs was also changed. Especially, intracerebral accumulations of drug loaded NPs were significantly higher than that of free drugs whether in normal brain or in tumor. For normal brain, the AUCs with an increase2.38-fold for coumarin6and2.44-fold for BG were found, while the multiples for tumor were up to2.21-fold and2.80-fold, respectively.In the last chapter, the antitumor activity and safety of BCNU+BG-PLGA/CS NPs against F98glioma bearing rats was evaluated. The rats treated with the combination therapy of BCNU and BG had a significant improvement in survival. Noteworthily, Rats treated with BCNU+BG-PLGA/CS NPs demonstrated the greatest antitumor activity, showing the longest median survival and the maximal increase in life span among all the treatment groups. Correspondingly, the tumor growth of F98glioma-bearing Fischer rats was detected using magnetic resonance imaging (MRI). The tumor growth was inhibited in each treatment group. Among these, rats treated with BCNU+BG-PLGA/CS NPs showed the smallest tumor size, indicating the greatest inhibitory capacity of BCNU+BG-PLGA/CS NPs. The results of H&E staining and TUNEL revealed that after the treatment of BCNU+BG-PLGA/CS NPs, only a few tumor cells were infiltrated into brain parenchyma and the apoptosis of tumor cells was induced significantly. |
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