| Glioma is the most common brain tumor, and primary brain tumors are one of the 10 main causes of death by cancer. Malignant brain gliomas can rarely be cured by surgery, radiotherapy or chemotherapy due to (1) the invasive growth of brain gliomas and (2) the blood-brain barrier (BBB). Gene therapy offers a promising cure of brain glioma, but therapeutic genes can not target to glioma spontaneously. Thus, it needs to develop a brain glioma-targeting gene delivery system to make exogenous genes express throughout the focus.In this study, two strategies were designed according to the physiological characteristic of the BBB during the process of brain glioma:(1) In the late phase of brain glioma, the integrity of the BBB is disrupted, and the brain glioma possesses EPR effect as other tumors, therefore the drug delivery system modified with brain glioma-targeting ligands could concentrate to the glioma; (2) In the early phase of brain glioma, the integrity of BBB is still retained, peptides with high affinity to both BBB and glioma may mediate the drug delivery system crossing the BBB then targeting to the glioma.According to the above two strategies, two novel non-viral nano-scaled gene delivery systems were constructed with means of pharmaceutics, macromolecular chemistry and biology. Polyamidoamine (PAMAM) was applied as the main macromolecular gene vector. CTX, a glioma-targeting peptide, or Angiopep, a BBB-glioma-bitargeting peptide, was modified to the gene vector, with polyethyleneglycol (PEG) as a spacer. There are three important attributes of the drug delivery system:(1) employing novel cationic macromolecular material, PAMAM, as the main gene vector, with high gene encapsulation ability; (2) modified with CTX, a peptide with high affinity and specificity to glioma; (3) modified with Angiopep, a peptide with high affinity to both BBB and glioma.In the first part, the physiologic characteristic and current treatment of brain glioma was summarized. The most outstanding characteristic of brain glioma was invasive growth. A growing tumour mass may disturb this interaction of astrocytes with endothelial cells, which is important in maintaining a functional BBB, particularly when it depends on contact between astrocytes and endothelial cells. Malignant brain gliomas can rarely be cured by surgery, radiotherapy or chemotherapy, and gene therapy offers a promising cure of brain glioma. Tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) is a signaling molecule with characteristics of TNF. TRAIL is able to induce cell apoptosis of gliomas without affecting the normal cells. In this study, plasmid DNA expressing TRAIL-pORF-TRAIL-was used as the therapeutic genes. Moreover, PAMAM was applied as the main macromolecular gene vector, CTX-a glioma-targeting peptide with high affinity to the glioma mediated by the matrix metalloproteinase-2 (MMP-2) , and Angiopep-a BBB-glioma-bitargeting peptide with high affinity to the gliomas and BBB through the low density lipoprotein receptor related protein (LRP) on the surface of glioma cells and the brain capillary endothelial cells (BCECs), were used for modifying the gene delivery system.In the first section of the second part, PAMAM-PEG-CTX and the DNA-loaded nanoparticles were synthesized and the in vitro characteristics were evaluated. After modified with CTX, the cellular uptake of the vector as well as the DNA-loading nanoparticles in C6 glioma cells was significantly increased, while the uptake level was maintained in normal cells (293 cells). The cellular uptake of PAMAM-PEG-CTX in C6 cells was inhibited in 4℃, showing that the vector may be internalized into C6 cells through endocytosis.In the second section, biodistribution and brain expression of PAMAM-PEG-CTX/DNA nanoparticles in brain glioma-bearing mouse were evaluated, as well as the pharmacodynamic evaluation. Compared to PAMAM/DNA, the distribution of PAMAM-PEG-CTX/DNA in the brain glioma was more concentrative. The results of frozen sections showed that PAMAM-PEG-CTX/DNA could increase the expression of DNA inside the glioma while PAMAM/DNA could induce DNA expressing on the edge out of the glioma, and it may relate to the new vessels of the glioma and the BBB. According to the results of pharmacodynamics, PAMAM-PEG-CTX/pORF-TRAIL could induce apoptosis inside the brain gliomas in mouse, showing comparative effectiveness to the commercially available oral drug Temozolomide (TMZ), while PAMAM/DNA could induce apoptosis on the edge of the brain gliomas; the median survival time of PAMAM-PEG-CTX/pORF-TRAIL and TMZ was 59.5 days and 49 days respectively, and 50% of the mouse treated with PAMAM-PEG-CTX/pORF-TRAIL lived longer than 70 days while all of the mouse treated with TMZ died within 70 days. Results above showed that PAMAM-PEG-CTX/pORF-TRAIL could treated with brain glioma effectively.In the first section of the third part, PAMAM-PEG-Angiopep and the DNA-loaded nanoparticles were synthesized, and the in vitro and in vivo characteristics were evaluated. After modified with Angiopep, the cellular uptake of the vector in BCECs increased and exhibited concentration-dependent mode. PAMAM-PEG-Angiopep and its DNA-loaded nanoparticles may be internalized into BCECs through LRP-mediated endocytosis. Angiopep could increase the trans-BBB efficiency of the DNA-loaded nanoparticles in vitro. The brain uptake of angiopep-modified nanoparticles increased with the ratio of angiopep modified, and the gene expression of the angiopep-modified nanoparticles was also increased. PAMAM-PEG-Angiopep/DNA nanoparticles showed excellent brain-targeting efficiency.In the second section, biodistribution of PAMAM-PEG-Angiopep/DNA nanoparticles in brain glioma-bearing mouse were evaluated, as well as the pharmacodynamic evaluation. Compared to PAMAM/DNA, the distribution of PAMAM-PEG-Angiopep/DNA in the brain glioma was more concentrative. The nanoparticles should cross the BBB then further concentrate to the glioma with the interaction of Angiopep and LRP. According to the results of pharmacodynamics, PAMAM-PEG-Angiopep/pORF-TRAIL could induce apoptosis inside the brain gliomas in mouse; the median survival time of PAMAM-PEG-Angiopep/pORF-TRAIL and TMZ was 56.5 days and 49 days respectively. Results above showed that PAMAM-PEG-Angiopep/pORF-TRAIL could treated with brain glioma effectively.
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