An Experimental Study On Copolymer-Paclitexal Conjugate Micelles For Inhibitory Effect Of C₆ Glioma

Gliomas takes nearly 44%of all human intracranial malignant tumors. The treatment of glioma challenges neurosurgeons.At present,the conventional treatments of glioma are surgery,radiotherapy and chemotherapy. Though surgery is preferred,operations can not totally remove this kind of invasive malignant tumors.Radiotherapy is usually recommended for gliomas while its surgery is unsatisfing.Traditional radiotherapy can't advoid brain edema because of cytonecrosis.Chemotherapy play an significant role in clinical if the tumor is localled in deeply,perhaps the tumor infringes upon important functional area,and thus the risk of surgery is so big that total removal of the tumor tissue is impossible.If these tumors are medicine sensitive, chemotherapy should be considered as the first choice.Paclitaxel(PTX)is a newly discovered broad-spectrum anticancer medicine PTX isolated from Taxus brevifolia,and has been widely applied in recent years.Being a microtubule stabilizer,paclitaxel disturbs the dynamic balance between microtubule protein andmicrotubule protein polymer, promotes micrubule assembly,inhibits their disassembly and eventually arrests cells in the mitosisAnd the cell cycle progression in late G₂/M phase is blocked via abnormal spindle formation.Then the cellular apoptosis is induced.At present,in the clinical treatment,none-water-solubility,serious allergic reaction and bad permeability of the blood brain barrier of this compound seriously limit its role in the clinical treatment.Therefore,it is necessary to take measures to reduce its side effect.Changing PTX medicine dosage-form,we can enhance its medicine density in the brain,and elevate its curative effect.In 1906,Pnrililich proposed a concept of drug-targeting,ie.medicine could be selectively distributed in the pathological change spots.Up to now,therapy of C₆ glioma by paclitaxel-nanometer micelle has not been reported.Using PEG-PLA as a carrier and modern nanometer micellar target technology,we synthesized the PEG-PLA/PTX micell.In vitro and in vivo study,we discussed its anti-tumor function and the target distribution characteristic,as well as its possible mechanism of anti-tumor function.Our study will provided the experimental basis for new target treatment of brain tumors.The anticancer function and the function mechanism of PEG-PLA/PTX micells against C₆ glioma of the Wistar rat established with sterotactic technique was observed by MTT assay,cell growth curve,morphological study, flow cytometry,RT-PCR,and fluorescence microscopy,HE staining method and the technique of SABC immunohistochemistry in vitro and in vivo.We have studied the effect and mechanism of PEG-PLA/PTX micells against C₆ cells in order to furnish the theoretical basis for developing and exploiting new drug carrier for anticancer medicine.This study contains following work:In this study we first used biodegradable PPG-PLA as the Paclitaxel carrier and synthesized the PEG-PLA/PTX by the chemical linkage.The PEG-PLA/PTX has following chemical characteristic:â‘ The PEG-PLA/PTX has a good water-solubility,its average diameter is 55-80 nm,can form a smooth spheres of narrow diameler.Compared with the blank carrier,PTX elles showed no in shape and size.We can adjust the nanometer granule particle size to realize the passive target treatment goal.â‘¡The PEG-PLA/PTX has a relatilly rapid release of PTX at early stages 30%in first2 day.and then has steady release 80%after 22 days.Because PTX was combined the high polymer PEG-PLA by covalent bonds,only when the chemical bonds broke down,the medicine only can be released.Therefore,by PEG-PLA/PTX we can realize long-term release of drugs and can reduce the medicine dosage in the clinical treatments.In vitro experiments showed:â‘ The experimental result indicated that the PEG-PLA/PTX have the stronger cytotoxicity to the C₆ cells than PTX and its suppression rate has the density and the time dependence;The PEG-PLA/PTX also showed the good medicine slow release effect.â‘¡Useing class type cell technique,the RT-PCR and the indirect immunity fluorescence method,we observed the effect of the PEG-PLA/PTX and the PTX on inhibiting the C6 cell line multiplication.This action was realized by C₆ cell apoptosis.During this process the cell of G2-M phase increased,the cell S phase droped.With increasing medicine dosage of the PEG-PLA/PTX and taxus,the apoptosis effect of C6 cell became more and more evident,and the effect of PEG-PLA/PTX was more than it of the dissociation taxus.â‘¢The experimental results indicated that in the anti-tumor process of the PEG-PLA/PTX,the Bcl-2 gene expression cut down,the Caspase-3 and the AIF gene expression increased.The PEG-PLA/PTX may reduce the medicine pump(the P-g protein) to affect and reduces the P-g protein expression and to suppress MDR gene duplication.The PEG-PLA/PTX can increase the taxus response time in vitro and s medicine density in vitro.The result of laser altogether focused the microscope test further confirm this kind of function.The experimental result indicated that the empty micells don have cytotoxicity to the brain tumorsIn vivo experimentâ‘ The C₆ brain glioma models in Wistar rats were all established successfully by the MRI.The average survival spans were 2 weeks. The survival time of control group was13.5±2.7day The survival time of PEG-PLA/PTX micells group was 44.8±7.8 day.The survival time of experiment group was significantly prolonged.The PEG-PLA micells have good biocompatibility.â‘¢It showed that micelles,it could increase the expression of AIF and caspase-3,;It also could inhibit the expression of MDRI,Bcl-2,and Survivin in cells,and this effect was concentration dependant.â‘£PEG-PLA/PTX micells may targeted aggregate tumor sites,the density of PEG-PLA/PTX micells in tumor site was associated the number of injection.The study of biodistribution in mice indicatedthat the micelles could signify increase the drug accumulation in Brain compared to PTX solution.The mechanisms of targeted aggregation were associated with component of micells,also associated with the local breakage of blood brain barrier by brain tumor.PEG-PLA micelles could enhance the uptake of PTX in C₆ cell lines and this effect was concentration dependent of carriers.In the study of the reversing mechanism,the influence of micelles to cell expression of MDR1mRNA and P-g protein in cells were investigated.It showed that micelles could decrease the the expression of MDR1mRNA..The mechanisms of targeted aggregation were associated with component of micells,also associated with the local breakage of blood brain barrier by brain tumor.Creative of this research:1.For the first time,biodegradable PEG-PLA conjugated with paclitaxel by covalent bond and formed "shell-core" structure aqueous solublenan -oparticle;2.It the first time at home and abroad that we take study of the biodegradable PEG-PLA conjugated with paclitaxel by covalent bond and formed "shell-core" structure aqueous soluble nanoparticle;used for treatment of glioma in according to the special neurotoxic effect of PTX.used for treatment of intracranial malignant tumor as an anti-tumor drug. 3.For the first time at home and abroad to explore the mechanism of glioma cells apoptosis induced by PEG-PLA/PTX micells.Our research showed that PEG-PLA/PTX micells can increase casepase-3 and AIF mRNA level;decerease MMPmRNA level;enhancing the expression of casepase-3.4.We proved the anti-glioma effect of PEG-PLA/PTX micells by in vitro and in vivo.Results Potent cytotoxic study,and the results of this study will provide us a new idea for finding target anti-glioma drugs and experimental basis for clinical application of PEG-PLA/PTX micells...