| Malignant gliomas represent the largest group of brain tumors in humans and with few exceptions are highly invasive tumors. Resection besides with external beam radiation and systemic chemotherapy are effectiveness, but invariably recur because of the inflltrative nature of the disease. The median survival of patients with high-grade glioblastomas is approximately no more than 1 year. Carmustine (l,3-bis(2-chloroethyl)-l-nitrosourea, BCNU) has been used most frequently for several decades because of its demonstrated in vitro activity against glioma cell lines and relative ability to cross the blood-brain barrier. Its clinical effectiveness , however, has been limited because of the short half-life, the small fraction of the systemically administered dose reaching the tumor at an effective concentration ,and serious systemic toxicities including delayed hematopoietic depression, cytotoxic effects on liver and pulmonary fibrosis. Most of glioma recurrences are local, occurring within 2cm of the original lesion. Based on the special biological behaviors, researchers focused on local drug delivery system. Direct introduction of chemotherapeutic agent with high concentration and long tumor cell exposured by sustained release biodegradable polymer placed in the tumor resection cavity, bypassing the limitation of blood-brain barrier and decreasing systemic toxicity, explored a new way for the treatment of malignant glioma.Poly-Lactide-co-Glycolide (PLGA) is a copolymer formed by condensation polymerization using lactide acid (LA) and glycolide acid (GA), connected by ester linkage. The primary metabolism products in vivo are lactide acid and glycolide acid, successivly completely degrade to CO2 and H2O, and biocompatible to human tissues. The degradation of PLGA is a hydrolysis action, and the degradation rate depends on LA/GA ratio, molecular weight, crystallinityand glass transition temperature. In vitro and in vivo, a bulk erosion mechanism has been considered as the main degradation pathway for PLGA: random chain scission on the linkage of ester bonds in the polymer backbone proceeds homogeneously through the device accompanied by formation of carboxylic acid end groups which accelerate the degradation by autocatalytic action in the centre.We fabricated and prepared BCNU-loaded PLGA microspheres by solvent evaporation and extraction using PLGA with four different molecular weights and LA/GA ratio. We selected appropriate PLGA material by investigating morphous and diameters. Determination of the optimal experimental conditions based on orthogonal design. Explore possible influential factors including poly(vinyl alcohol) with different degree of polymerization and its concentration, PLGA concentration, agitation rate and methyl cellulose concentration, We found that the main influential factor is PVA concentration, then PLGA concentration, agitation time, the last is methyl cellulose concentration. We got the most suitable condition is: PVA concentration in dispersing phase is 1.5%, PLGA concentration in organic phase is 2%, agitated 60 minutes, methyl cellulose concentration is 0.05%. Observation of the surface and cross-section of the microspheres shows us that BCNU was well incorporated inside the microsphere. The drug maintained original activity by high performance liquid chromatography. Determination of residual methylene chloride content in microspheres by vapor phase chromatography, and the result demonstrated that is lower than other researchers reported. Sustained release microparticles were compression molded into wafers using self-made devices. The wafers were 3mm diameter, lmm height and 10.0± 0.24mg in weight.BCNU release profiles determined by high performance liquid chromatography, and the in vitro release study shows that the microspheres can release drug prolongly for 3 weeks. 4.34 % of the drug were released in first 24 hours, 50% of the drugs can be released in 8.34 days. Comparing to in vitro release, in vivo release test shows that it can be released in shorter time (about 2weeks), and with 16.06% released in first 24 hours, and 50% canbe released in 2.14 days. Preparing microspheres with 3H labeled BCNU and implanted in rats brain tissue, then determined the concentrations in brain and serum in different time by liquid scintillation. We found that the concentrations in implanted side are 6-70times higher than that of the contralateral. The drug diffused for 8 millimeters and maintained stable local effective concentrations. The microspheres provided high local drug concentrations, meanwhile without any remarkable side-effects. To further characterize the biocompatibility of PLGA in the brain, we examined the gliotic response following implants of blank wafer into the brain of rats using hematoxylin and eosin stain, and only minimal gliotic reaction were found at different time points. These results demonstrate that PLGA is well tolerated by implantation into the brain and the astrocytic response to PLGA is largely a consequence of mechanical trauma of the surgery. The biocompatibility of PLGA to the CNS provides further support for its protended wide use as sustained and localized drug delivery carrier to the brain.We established a method for the determination of BCNU in phosphate-buffered solution to study the released kinetics of BCNU-loaded poly (lactic-co-glycolic acid) PLGA microspheres in vitro and in vivo. The calibration curve was linear within the range of 0.005~0.35umol/ml and the measurable lowest limit was 0.005umol/ml. The results showed that BCNU can be released from the microspheres for over 3 weeks. This HPLC method is simple, sensitive and accurate. It is suitable for released kinetics study of sustained release microspheres in vitro.The cytotoxicity assay of BCNU-loaded PLGA wafer against C6 tumor cell was conducted by putting the wafer in cell culture medium and then measured by flow cytometry. We found the wafers can induce tumor cells necrosis and apoptosis. Mortality rate increased from 5.35± 1.99% to 20.15±2.96%, advanced stage apoptosis rate elevated from 4.25 +1.1 l%to 32.33 + 2.62% and early apoptosis rate from 6.44+0.57% to 21.82+3.04%. Cell cycle also changed after...
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