| The abnormal stree caused by space-occupying lesions of glioma which is invasive growth in the central nervous system(CNS)and microenviroment(hypoxia,peripheral tissue ischemia)would induce nerve tissue injury.What's more,the infiltrative nature of glioma makes comolete surgical resection difficult,thus chemotherapy or postoperative chemotherapy is essential.However,the non-selective toxicity of antineoplastic agents,clinical chemotherapy and radiotherapy would further induce acute or chronic nerve injury.As mentioned above,neural damage in CNS is one of the critical factors for impairment of body function,consciousness,language and cognitive.Meantime,approximately all of macromolecular drugs and most of small-molecule compounds cannot reach the brain tissue because of blood-brain barrier.As a result,nerve repair and the method of permeating BBB combined to cure malignant glioma or other brain tumor will be a nove perspective for improving clinic treatment.Based on the the functions of neural repair and anti-tumor,we constructed the self-assembled GM1 micelles loading antineoplasyic agents for the treatment of glioblastoma,which has the capabilities of overcoming BBB and eliminating tumor cells.Meanwhile,as a widely used drug for clinical treatment of neurodegenerative diseases,GM1 may accelerate the functional recovery of the damaged nerves without any other therapeutic agents.The main research work and corresponding results were done in this thesis:(1)The DOX/GM1 micelles were prepared through dialysis method.Orthogonal experiment design(OED)with three factors and three levels was proposed.The drug loading efficiency of these nine combinations were evaluated to identify the best combination and the optimum micelles was prepared by GM1 concentration: 50mg/m L,DOX concentration: 3mg/mL,GM1/DOX: 5/1.The drug loading of DOX/GM1 micelles was 9.33%,and the micelles showed high encapsulation efficiency(97.05%).Nanoparticle size analysis revealed that the DOX-loaded GM1 aggregated to particle with average size of 226 nm.The zeta potential measurement showed that the surface of micelles is negatively charge with – 45.7 mV.The DOX /GM1 micelles were spherical with average diameter of 5~21 nm in the dehydrated state.Moreover,the in vitro drug release experiments demonstrated that cumulative release of DOX was 66.53% within 16 days,the release of DOX from micelles was slow and sustainable without a burst release effect.(2)It is obvious that DOX loaded by GM1 micelles could be endocytosed by HUVECs and C6 cells,which would avoid the drug resistance protein being excreted.The in-vivo animal imaging results showed fluorescence signal from DiR/DOX micelles was accumulated in the mice brain compared to DiR.Furthermore,DOX/GM1 micelles and DOX solution were injected into the circulation of transgenic fluorescent zebrafish(flk1:GFP)through the heart and then the in-vivo imaging of the brain was monitored.Mixed micelles extravasated extensively from vessels(green),which evidenced by fluorescence of DOX(red)within the brain parenchyma.In contrast,doxorubicin was not observed in the brain but confined with the blood vessels.The results indicated that GM1 micelles could effectively deliver the drugs into brain tissue by overcoming the BBB.(3)The study on pharmacodynamics of DOX/GM1.C6-bearing Wistar rats models were established with the success rate of 88%.The brain tumor-bearing rats were randomly divided into five groups and were treated with physiological saline(blank control group),free-DOX solution,free-GM1 solution,DOX+GM1 mixed solution and DOX/GM1 micelles respectively.Each group was valued by Magnetic Resonance Imaging,hematoxylin and eosin staining and Kaplan-Meier survival curves to monitor the tumor size,invasiveness of tumor cells and survival outcome.The group treated with DOX/GM1 showed the greatest prolonging rats survival among all the treatment groups.Meanwhile,the assessment of the Garcia neurological scores was recoded.The neuroscore of rats in GM1,GM1+DOX and GM1/DOX groups remained at least 10 points,which showed fine quality of life in rats with glioblastoma.Indicating the nerve repair and neurotrophic function of GM1.This result also comfirmed GM1 micelle is capable of passing the BBB.Considering to the property of permeating BBB of GM1 micelles,it is implementable to achieve the multiple aims of neuron repairment and tumor treatment at the same time without using other carries. |
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