An Experimental Study On Nano-medicine Targeting Glioma And Its Combination With Irradiation Treatment

Glioma is the most aggressive malignancy in the human brain, accounting for 40% of all primary malignant brain tumors. The survival rate is only 6-12 months. Surgery is currently the preferred approach to treating patients with glioma; however, due to the lack of the clear boundaries between cancerous and normal tissues, it is difficult to completely remove the entire tumor lesion. Radiotherapy is another choice for the treatment of glioma, but the tolerance threshold of normal brain cells is lower than those of glioma cells. Chemotherapy is also routinely used to treat glioma.But the chemotherapy of glioma with lower side effectsremains a great challenge because of the existence of the blood-brain barrier(BBB), which prevents nearly all largemolecule and 98% of smallmolecule drugs such as Paclitaxel(PTX) entering the central nervous system, Therefore, brain-tumor-targeted chemotherapy and prolonged survival time of glioma patients have become a hot research topic. Research background:The development of nanotechnology and materials science has greatly promotedthe research of nanoscale drug delivery systems, or simply “nanodrugs”. Compared withtraditional chemotherapy, optimally designed targeting nanodrugs can prolong the blood circulation time of drugs, decrease the systemic toxicity, and increase the accumulation of drugs at the tumor sites by coupling specific ligands, which can recognize their receptors on the target cell membrane, ontothe surface of nanodrugs, and finally increase the transport ability of nanodrugs across the cell membrane, leading to the enhanced cytotoxicity.Currently, there are many brain active targeting delivery systems to be reported, mainly using transferrin(TF), lactoferrin(LF), or desoctonyl ghrelin. By using the specific recognition function of them with the corresponding receptor(TFR, Lf R, GHSR) and the receptor-mediated transport process, drugs can across the BBB to realize brain targeting. But these ligands are endogenous glycoproteins and it isdifficult to avoid the inhibition due to "endogenous/extragenous competition, so people gradually turned to look for" non-endogenous protein or peptide ligands", such as rabies virus polypeptide RVG29. Currently RVG29 and RG29-9Rhave been used with certain cationic polymer vectors for gene delivery to the brain, but the conveyed objects are si RNA, protease, etc.Carriers are cationic polypeptides, liposomes, polymers, dendrimers or pluronicf127 microgel. Subcutaneous glioma modelswere established, but theycannot represent intracranial gliomas. It has not been reported to use RVG29 to carry out brain-targeted delivery of conventional chemotherapeutic agents, especially in the treatment of brain glioma. Research objectives:In the thesis, RVG29-targeted and PTX-loaded micelles M(RVG PTX) were prepared The brain-targeting ability, the inhibitory effects on brain glioma C6 cell proliferation, and the suppression effect on glioma C6 tumor growth of M(RVG PTX) micelles will be examined, and the mechanisms of the brain-targeting and the tumor suppression will be elucidated. The objectives include.1. To investigate the ability of the RVG29-targeted nano-micelles to across the BBB and to clarify its mechanism;2. To examine the effects of RVG29-targeted nano-micelles on C6 cell proliferation and apoptosis, and on glioma tumor volume and survival time of the tumor-bearing animals;3. To observe the contribution of combination of radiotherapy and chemotherapy based on the RVG29-targeted nano-micelles to the inhibition of C6 cell proliferation and apoptosis and to discuss the possibility of combination therapy;.4. To elucidate the mechanisms of C6 cell growth inhibition and cell apoptosis induction by the RVG29-targeted nano-micelles. Research methods:Water-soluble dextran(Dex) is used as carrier polymer. It is firstly carboxylated, and then coupled chemically with paclitaxel(PTX) and RVG29 to prepare PTX-loaded micelles M(PTX)and RVG29-targeted micelles M(RVG,PTX). These micelles are characterized by 1H NMR, fluorescence spectroscopy, DLS, TEM, etc. Orthotopic Wistar rat glioma-models are established by using the stereotactic technique. MTT, FCM, CLSM, MRI, and in vivo imaging system for small animals are used for determination of the biodistribution of the RVG29-targeted micelles in the C6 cells and in the animal body. real-time PCR, Western Blot and immunohistochemistry are used to detect the expression levels of apoptosis-related RNA and proteins. Research Results:1. Starting from Dextran(Dex), conjugate of Dex and PTX(Dex-PTX) and its nano-micelles(M(PTX)), and conjugate of Dex, PTX, and RVG29(RVG-Dex-PTX) and its nano-micelles(M(RVG, PTX)) were prepared. Molecular weight of Dex-PTX is about 16500. Mass of PTX is 20 % of Dex. Mass of RVG29 in RVG-Dex-PTX is 10 % of Dex. Micelle sizes of M(PTX) and M(RVG, PTX) are about 45-60 nm, as determined by DLS.2. The above micelles are labeled with Cy5 dye to prepare fluorescent micelles M(Cy5, PTX) and M(Cy5, RVG, PTX). Cell uptake experiments show that compared with M(Cy5, PTX), more targeted micelles M(Cy5, RVG, PTX) are uptaken by C6 cells. RVG29-shielding tests show that existence of RVG29 on the micelle surface is responsible for the targeting effect of micelles. Ex vivo organ imaging and CLSM tests show that fluorescent signals are visible in the intracranial part of the test rats and the fluorescence intensity in targeted micelle group is stronger than that of non-targeted or RVG29-shielded groups(p<0.05), indicating that the targeted micelles have entered the rat brain. In conclusion, the RVG29 decoration of the micelles is the determining factor for the brain-targeting effect.3. In vitro study shows that the inhibition effect of M(RVG, PTX) micelles against C6 cells is dependent on drug concentration and action time, i.e, the cell inhibition rate increases with action time and drug concentration, and the inhibition rate of M(RVG, PTX) micelles is stronger than M(PTX) micelles. In animal trials, as a result of i.v. injection via tail vein, the average tumor size in targeted-micelle group is obviously smaller than those in control, non-targeted or RVG29-shielded groups(p<0.05). The survival time of the tumor-bearing rats is prolonged and the life quality of the test animals is improved in the targeted group.4. After X-ray irradiation at a dose of 10 Gy, test C6 cells are given M(RVG, PTX) micelles. At a concentration of PTX of 5 ug/ml proliferation inhibition and cell death of C6 cells are observed. The efficiency is obviously higher than the group of single drug or radiation treatment.5. It is found by using FCM, real-time PCR and Western-Blot that use of the RVG29-targeted micelles leads to up-regulation of apoptosis-assistant proteins such as caspase-3/-8/-9 and Bax, and down-regulation of anti-apoptosis proteins like Bcl-xl. The multi-drug-resistance(MDR) proteins such as P-gp is also down-regulated. These results help us understand the mechanisms underneath the C6 cell proliferation and apoptosis. Conclution:In short, RVG29-targeted nano-micelles M(RVG, PTX) have been prepared. In vitro and in vivo studies show that these micelles can across the BBB, initiate C6 cell proliferation, induce C6 cell apoptosis, suppress glioma tumor growth, and prolong survival time of the test rats. It is to say, these RVG29-targeted and PTX-loaded micelles possess anti-tumor efficacy. Moreover, mechanisms of brain-targeting and anti-glioma action of the micelles are discussed. These results will benefit the application of nano-micelles as the carrier of anti-cancer drugs.