| Glioma is the most common and fatal primary brain tumor. Despite surgical debulking, radiation and chemotherapy, median survival has been changed relatively little. Chemotherapy is the most common method for the treatment of gliomas. However, the great side effects of chemotherapy could not be disregarded. Recently, gene therapy has been followed with interest because of its non-toxicity and high therapy efficiency. But it is commonly believed that cancer is a multi-factor, multi-link, multi-stage complex disease, so that treatment with a single factor often cannot achieve the desired effect of inhibiting tumors. Therefore, it needs to develop an effective drug delivery system for the treatment of glioma.Before tumor cells were killed, drug delivery system would pass through the following steps:drug delivery across the blood-brain barrier (BBB), further into the site of the tumor, then drug release and induce tumor cell apoptosis. Thus, an effective drug delivery system for glioms treatment must has functions of brian-targeting, glioma-targeting, controlled drug release and multi-target treatment.In the present study, we designed the nanoscaled co-delivery system loaded both DOX and pORF-hTRAIL. DOX was loaded on the surface of T7modified Dendrigraft poly-L-lysine (DGL) using Glu as an acid-sensitive linkage, then therapeutic gene pORF-hTRAIL was compacted and encapsulated by the DOX conjugated dendrimers. The co-delivery system has the following characteristics,(1) Brain tumor-targeting. T7peptide, a novel targeting ligand, can specially bind to transferrin receptor (TfR), which is over-expressed on the brain capillaries endothelial cells and many malignant tumor cells [19,20]. Thus, T7peptide targeted to TfR can not only mediate the transport of nanocarriers across the blood-brain barrier (BBB) but also can increase the accumulation of anticancer drugs in brain tumors.(2) pH-sensitivity. The delivery systems were accumulated more effectively in the tumor than normal tissues via T7-mediated endocytosis. Furthermore, the pH-triggered hydrazone bond was designed between DOX and carrier, so that DOX could be released from the carrier after accumulation.(3) Combination therapy. Two drugs target tumor cells at different points, the combination therapy was expected to achieve synergetic anticancer effect at lower drug doses, and result in lower undesirable toxicities.In the first chapter, the physiologic characteristic and treatment of brain glioma was summarized. The most outstanding characteristic of brain glioma was invasive growth. Currently, chemotherapy is the most conventional method for clinical treatment of glioma. However, the treatment efficacy of chemotherapy drugs was limited by their side effects. Gene therapy is a promising new treatment for glioma, but it is difficult to achieve effective outcomes for patients with gene therapy alone. Therefore, new Strategies, such as brain tumor-targeting and combination therapy, were developed for glioma treament.The second chapter focused on the synthesis and in vitro evaluation of T7-PEG-DGL-Glu-NHN-DOX (DGDPT), the molar ratio of each component is1:2:2:2:1(DGL:Glu:DOX:PEG:T7). DGDPT exhibited a pH-dependent drug release characteristic. A MTT assay results showing that DGDPT, which were modified with T7, higher significantly reduced the viability of U87cells. The inhibitory effect of DGDPT co-treated with Tf was superior to that of untreated one.The third chapter focused on the construction and in vitro evaluation of the targeting co-delivery system. The gel retardation assay demonstrated that pDNA could be effectively packed by vectors at a DGL to pDNA weight ratio greater than0.5. The specified ratio of3:1was used in the following experiments.The mean diameter of DGDPT/pORF-hTRAIL was173nm, the zeta potential value was3.2mV. TEM showed that the DGDPT/pORF-hTRAIL NPs was a kind of analogous sphericalshape and compacted structure with a size approximately<200nm. The drug release of DGDPT and DGDPT/pORF-hTRAIL had no significant difference. The drug released from DGDPT/pORF-hTRAIL with a pH-dependent characteristic. T7-modified co-delivery system showed higher efficiency in cellular uptake and gene expression than unmodified co-delivery system in U87MG cells, and Tf facilitated the internalization and transfection. Apoptosis induced by co-delivery system (DGDPT/pORF-hTRAIL) was more serious than that by mono-delivery system (DGPT/pORF-hTRAIL or DGDPT/pGL-3). And the apoptosis signals caused by systems with Tf were stronger than that by systems without Tf. co-delivery system exhibited higher cytotoxicity than mono-delivery systems, combination index (CI) values at30%,50%and70%of growth inhibition effects were0.11,0.13and0.20, respectively. The combination treatment resulted in a synergistic growth inhibition (CI<1) in U87MG cells.The fourth chapter focused on the biodistribution of DGDPT/pORF-hTRAIL nanoparticles in brain glioma-bearing nude mice, as well as the pharmacodynamic evaluation. The T7-modified delivery system exhibited a rapid U87MG tumor targeting as early as1h p.i. An increasing tendency in signal intensity was seen in tumors from1h to4h after the injection of DGDPT/pORF-hTRAIL. Unfortunately, the unmodified delivery system was accumulated mostly in the normal tissues, and cleared up quickly. The induction of apoptosis by co-delivery systems and mono-delivery systems were examined using the TUNEL method, the largest number of apoptotic cells was observed in the tumor tissue which was treated targeting co-delivery system. The change in body weight after treatment was evaluated. After treated with targeting co-delivery system DGDPT/pORF-hTRAIL, the body weight of mice had no obvious change until fiftieth day, while other groups showed a huge decrease in weight after about20days. The median survival time of DGDPT/pORF-hTRAIL group, DGDP/pORF-hTRAIL group, DGPT/pORF-hTRAIL group, DGDPT/pGL-3group, DOX group, and saline group were57,27,33.5,31.5,34,23days, respectively. DGDPT/pORF-hTRAIL effectively prolonged the survival time of tumor-bearing nude mice... |
PDF Full Text Request