| BackgroundThe glioma which is the the most common primary brain tumor with thehigh incidence is one of the top frequently occur malignant cancers in Chinese.70%gliomas are glioblastoma and anaplastic astrocytoma, and the treatment hasnot important progression. The main treatment of glioma is surgery supported byradiotherapy，chemotherapy，targeted therapy.etc.The main reasons that gliomasare very difficult to resect entirely include the infiltrative growth and the closenessto important functional areas or nerve tracts. More than90%recurrence of gliomanear the tumor cavity within2cm provides an alternative drug delivery method forthe treatment of brain tumors. Desirable local chemotherapy system could providesustained and effective drug concentration to make the tumor stop grow ordisappear and prevent to recur.Local sustained-release chemotherapy has two important points：sustained-release and controlled release， that needs carrier material more strictly.The mature intracranial malignant glioma chemotherapy formulation ofbiodegradable polymer is BCNU sustained release dosage forms forpharmaceutical carrier in PCPP: SA vector, it has a good biocompatibility andbiodegradability, non-cytotoxic. Some defects are found in the application of thesustained release carrier with release agent polyanhydride (PCPP-SA)process,such as cell-attached weak, the poor hydrophilicity, aseptic inflammation causedby acidic degradation products, inflammatory cell infiltration in the local braintissue and brain edema in experimental and clinical applications. In addition, inthe process of some experiments experimenters find that the histocompatibility ofdegradation material is poor, the scar tissue appears clearly, that is easy to affectthe diffusion of slow-release drug, degradation fragments are failed to absorb in time, swim in the tumor cavity, that is dangerous to cause ventricle systemblocked.PLGA slow-release microspheres system which can control the size ofmicrospheres, delay the drug degradation, prolong drug release, targeted release,reduce drug toxicity and irritation, is a good slow-release drug carrier. Nanometercarrier has its slow-release effect, at the same time, open the blood-brain barrierfurther more, also have targeted role, and nanometer carrier surface can carrytargeting ligand which can increase the rate of tumor cells to absorb at the sametime.Nanometer carrier cross the blood-brain barrier into the brain, and drugsplay a role in brain tissue by means of diffusion or carrier degradation. Changingthe nanometer carrier proportion of material can control the degradation ofnanometer carrier，control the speed of drug release，and have the effect of slowrelease and controlled release. Nanometer carrier has a passive targeting afterentering the human body, is easy to be swallowed by the mononuclearmacrophage system and gathered in the liver, spleen, but not gathered in the brain.Hydrophilic surfactant modified nanometer carrier can extend its half-life in thebody, and increase the brain targeting.Temozolomide (TMZ) is a new type of imidazole tetrazines cell cyclenon-specific drugs in recent years in the domestic and international clinical usedfor the treatment of glioma as a breakthrough. Alternate bioavailability of thetemozolomide, after all, the oral temozolomide systemic administration inevitablydamaging other organs of the body damage is very obvious, such as bone marrowsuppression. In clinical MGMT-positive patients use temozolomide more thannegative with the poorer effect of chemotherapy, and to increase the dose of drugswill inevitably lead to more toxicity. The topical administration has the followingadvantages:（1）Directly effecting on the tumor site can avoid the blood-brainbarrier，and increase the choice of brain tumor chemotherapy drugs.(2) Improvethe local drug concentration, reduce systemic toxic effects.(3) Gliomas mainly recur locally,90%of gliomas recur in the range of2cm around the primary tumorsite, which make it ideal for local chemotherapy, especially for MGMT-positivepatients. Temozolomide cann，t control the growth of glioma entirely combiningthe radiotherapy.This experiment plans to prepare Temozolomide-PLGA sustained-releasemicrosphere, with PLGA as the carrier, Temozolomide as sustained-release drug,at the same time study TMZ-PLGA sustained-release microsphere preparationprocess, release rule in vivo and the inhibitory effect of glioma cells in vitro，provides a new train of thought and method for interstitial chemotherapy ofglioma, and, provides a feasible treatment measures for the survival of gliomapatients.This study includes two parts:Part I The fabrication and study of the biodegradable implantsPart II Cytotoxic study of the implants against glioma C6eells in vitroPart I The fabrication and study of the biodegradable implantsObjeetive：To prepare Temozolomide-PLGA sustained-release microsphere,analyse production parameters, the morphological characteristics and releasecurve in vitro.Methods：Prepare Temozolomide-PLGA sustained-release microspheres byultrasonic emulsification-solvent evaporation method. And observe the fourdifferent molecular weight slow-release microspheres,surface morphology with amicroscope, and measure the microsphere diameter. Detect drug loadings andencapsulation efficiency with high performance liquid chromatograph. Then usehigh performance liquid chromatography method to detect the rest of the amountof TMZ in microspheres, calculate the release quantity of drugs in the differentperiods, draw the release curve.Results: Control the production condition of the Temozolomide-PLGAsustained-release microsphere strictly, especially the temperature requirements.The ultrasonic emulsification-solvent evaporation method can get stable structure, uniform slow-release microspheres. The bigger the molecular weight,the greater the microsphere diameter. Light microscope and electron microscope,the microsphere,s surface is smooth, has no cracks, gathering is not obvious, themicrospheres are uniform. In experiments the largest drug loadings of differentmolecular weight TMZ-PLGA slow-release microspheres was10.2%. All theslow-release microspheres coating rate is over90%, that ensures drug loss rarely.Accurately controlling the TMZ-PLGA slow-release microspheres drug loadingscan solve this slow-release system problem that the high drug loading can lead toserious phenomenon of sudden release. Results of methylene chloride residuedetermination: Samples,residual methylene chloride was5.70‰, less than3%which the foreign literature reports, has reached the requirement of intracranialapplication. From the release curve of TMZ-PLGA slow-release microspheres,TMZ-PLGA slow-release microspheres system can continue to release more than2weeks, and meet the requirements of interstitial chemotherapy in the cycle.Conclusion:PLGA is very suitable for preparation of slow-releasemicrospheres as carrier. Temozolomide-PLGA Sustained-release Microsphereconforms to the biomechanical rule, can reduce sudden release effect, and extenddrug slow-release time. The preparation slow-release microspheres of differentmolecular weight and different drug loadings can release Temozolomide longduration.Part II Cytotoxic study of the implants against glioma C6eells in vitroObjective:Analyse that whether the TMZ-PLGA slow-release microspheresin vitro has the ability to continue to inhibition of C6glioma cells. Observe theinfluence of slow-release microspheres on glioma cell apoptosis, necrosis and cellactivity.Methods: Culture cells and passage the cells when growth area wasaccounted for over90%of the culture bottle bottom. Transfer C6cells to the96-hole culture plate. Samples can be divided into two groups: experimental groupand blank control group. Experimental group: Temozolomide-PLGA Sustained-release Microsphere, divide into2.5%,5%,7.5%and10%groupsaccording to the drug loading. Blank control group: Don’t join any drugs. All thesamples culture in the cell incubator. Half of the samples are analysing bymultiparameter flow cytometry instrument. Another half of the samples aredetected the absorption photometric value (OD) of each hole at450nm withautomatic enzyme standard instrument.Results:Flow cytometry instrument tests that2.5%,5%,7.5%and10%TMZ-PLGA slow-release microspheres are confirmed to kill C6cells, and the effectincreases with the drug loading increasing. Detect the activity of glioma C6cellswith MTT method after1,2,3d, the experimental group and the control groupwere statistically significant differences on the analysis of variance.7.5%and10%group are no significant statistical difference. Groups2.5%and7.5%,5%and7.5%,2.5%and10%,5%and10%respectively have significant difference.Confirm that the higher the drug loading, the bigger interactions of inhibiting thegrowth of cells in a certain extent.Conclusion: The TMZ-PLGA slow-release microsphere of different drugloadings all have the inhibitory effect of killing the glioma C6cells. Slow-releasemicrospheres with high drug loadings is higher effect than the sustained releasemicrospheres of low on the activity of inhibiting tumor. The higher drug loadings,the more serious sudden release phenomenon of slow-release microspheres. Thedrug loading about7.5%can not only keep drug concentration in effectiveconcentration levels, and can reduce the release effect, is a more appropriate scopeof the drug. |
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