The Study Of The Fabrication Of Combined Biodegradable Implants Delivery Temozolomide And Paclitaxel For Treatment Of Glioma

BackgroundMalignant glioma is the most common type of primary brain tumor in adults. Approximately 5 new cases per 100,000 in population are diagnosed each year. The current standard treatment for malignant glioma consists of surgical resection followed by radiation therapy and aggressive systemic chemotherapy. However, the prognosis for patients with maglignant glioma is relatively poor with a median survival of<2 years. Autopsy studies indicate that 90% of recurrent gliomas occur within 2 centimeters and metastasize rarely, so local chemotherapy provides an alternative drug delivery method for the treatment of brain tumors. Desirable local chemotherapy system could provide sustained and effective drug concentration with well histocompatibility and less neurotoxicity. The present research about local chemotherapy system is focus on biodegradable materials. Gliadel(?) wafer is the first biodegradable chemotherapy system used to treat glioma. The wafer consists of polycarboxyphenoxypropane and sebacic acid in a 20:80 molar ratio and contains 3.85% BCNU. It has showed some success in improving survival of patients with malignant glioma. However, it is only used to treat recurrent glioma by FDA because AGT, a DNA-repair protein found in the majority of brain tumors. So we need to find other effective drugs and safe biodegradable vehicle.Temozolomide(TM) is one of the most effective antineoplastic agents for malignant glial tumors. TM is converted to its active metabolite 3-methyl-(triazen-1-yl) imidazole-4-carboxamide (MTIC) at physiological pH. MTIC induces apoptosis of cells due to alkylation of DNA at the 06 and N7 positions of guanine. It is evident that local delivery of temozolomide by biodegradable polymers is superior to oral administration in a rodent glioma model.Paclitaxel targets microtubules and has been clinically effective against a variety of human cancers, that shows cytotoxicity against glioma in vitro. However, it is prevented from entering brain and penetrating intact blood-brain barrier poorly. Biodegradable polymer implant was developed to deliver paclitaxel against experimental malignant glioma and found to be efficient in a rat model.Most work on electrospinning biodegradable polymers has focused on synthetic materials, notably PLA, PGA, PLGA, and PCL. However, these materials have some disadvantages, such as low hydrophilicity, and aseptic inflammation because of their acid degradation products. Poly-propylene carbonate (PPC) is a degradable material formed from copolymerization of propylene oxide and carbon dioxide. It has low local irritant effect in brain tissue and good histocompatibility with brain. So PPC is the ideal material as release vehicle in brain.Various techniques have been designed to prepare controlled drug delivery systems, including solvent casting/salt leaching, membrane lamination, phase separation, emulsion freezing/drying, and hydrogel freezing/drying. Due to very high interstitial pressure and circulation of cerebrospinal fluid, microspheres have the risk of being expelled out of the target site. The other disadvantage of using microspheres is the high initial burst due to the presence of the drug on the surface which might lead to undesired neurotoxicity. Electrospinning is an attractive approach for the fabrication of fibrous biomaterials. Electrospun mats have larger specific surface areas and smaller pore size for polymer degradation and drug diffusion compared to other controlled drug delivery systems.Therefore, the aim of this study was to designe a high effective local drug delivery system with low loading rate. We combine electrospinning with microspheres delivering temozolomide and paclitaxel, avoiding the possibility of neurotoxicity. The preparation parameters and the safety and effectivity of the system were analyzed.This study includes three parts:PartⅠThe fabrication and study of the biodegradable implantsPartⅡCytotoxic study of the implants against glioma C6 cells in vitroPartⅢThe study of combined implant against glioma in ratsPartⅠThe fabrication and study of the biodegradable implantsObjectiveTo fabricate the drug delivery systems and investigate the preparation parameters, physical characteristics and release propertiesMethods1. Preparation of the drug delivery system:Temozolomide fibers were fabricated by electrospinning, and paclitaxel microspheres were prepared using emulsifying-solvent evaporation method. Then emulsion of microspheres was electrospinned into combined mats. The ratio of temozolomide and paclitaxel was 2:1, 1:1,1:2 respectively, through holding the loading rate of paclitaxel and changing the temozolomide.2. Physical characteristics:The size, distribution and surface morphology of the fibers and microspheres were examined using a scanning electron microscope after gold coating.3. Thermal analysis of drugs in matrices:Thermal analysis was performed using differential scanning calorimetry (DSC) to characterize the physical status of drugs in matrices.4. The drug encapsulation efficiency (EE) and loading rate were calculated according to the following equations: Loading rate=WM/WT×100 Encapsulation efficiency=WM/Dt×100 , where WM is the amount of drug in systems, Dt is the amount of drug used for the preparation, and WT is the weight of systems.5. The drugs releasing study in vitro:the systems were immersed into 20ml PBS buffer in tube incubated at 37℃and 100 rpm in a shaking water bath. At each specified time interval,1h,3h,6h,12h,24h,72h,120h,168h,240h和280h, drugs in solution were detected by UV analysis. Then the time-cumulative drug released plots would be produced.Results1. When PPC was 8%, distance was 15-20cm and voltage was 10-15kV, electrospinning could be performed normally. The TM fibers were smooth and uniform only at 8% by weight. The paclitaxel microspheres have proper size when the stirring rate is 500 rpm and concentration of alginate is 1%. The emulsion of microspheres was electrospinned into the beads-in-string structure at 15cm and a voltage of 12kV. 2. SEM images showed that the diameters of the fibers and microspheres were 3μm and 7.2μm. The surface of fibers and microspheres was smooth and uniform.3. Pure TM and paclitaxel showed an endothermic melting peak at 207.5℃and 223.0℃, but no peak was seen at temperatures of 100-250℃for the samples. It is evident that the drugs were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.4. The loading rate and encapsulation efficiency of paclitaxel in microspheres were 3.9% and 65.3%,2.1% and 94.5% in combined fibers. The loading rate and encapsulation efficiency of TM in combined fibers were 1.2%,2.3%,4.1% and 97.6%, 96.1%,95.7%. The EE of paclitaxel in microspheres is lower, because that it could dissolve in dichloromethane in preparation of microspheres. However, this part of paclitaxel stays in fibers during process of electrospinning.5. The microspheres released paclitaxel for about 8 days with obvious initial burst, and paclitaxel could be released for about 12 days with low initial burst. Because it could prolong the release time and reduce the initial burst when the microspheres were covered in fibers. TM was released for about 12 days from combined fibers. The initial release rate was faster with higher loading rate.Conclusion1. PPC could fabricate the drug delivery system by electrospinning.2. TM and paclitaxel were in an amorphous or disordered-crystalline phase of a molecular or a solid solution state in the matrix.3. Combined delivery systems could reduce initial burst of paclitaxel microspheres and prolong its release time.4. Temozolomide and paclitaxel could be released stably for long time from delivery systems. PartⅡCytotoxic study of the implants against glioma C6 cells in vitroObjectiveTo investigate the effect of implants against glioma C6 cells in vitro and the best ratio of two drugsMethods1. C6 cell culture:Cells were maintained in tissue culture in Dulbecoo's minimum essential medium with 10% fetal bovine serum, in atmosphere of 5% CO2, and 90% relative humidity at 37℃。2. Cytotoxicity test:C6 cells were transferred to a 96-well plate to ensure 1×104 cells per well. The following experimental groups were studied. (1) 2:1 combined fibers; (2) 1:1 combined fibers; (3) 1:2 combined fibers; (4) TM/PPC fibers; (5) paclitaxel microspheres; (6) PPC and alginate; (7) control group, each group in triplicate.3. To detect cell viability:The cytotoxicity assay was conducted by Cell Counting Kit-8(cck-8). The plates were detected through microplate reader. Cell viability was determined according to the following equation:Cell viability (%)=Abs test cells/Abs control cells×100Results1. The cells viability of group (6) and (7) is over 99%. It is evident that the material PPC and alginate has no cytotoxicity to C6 glioma cells.2. The cells viability of group (1) at 1,2,3 days is 52.4±6.3%,27.9±5.3%, 5.5±2.4%, group (2) is 56.3±4.2%,29.3±6.2%,9.1±5.1%,group (3) is 75.7±2.9%,52.1±4.9%,22.9±2.5%, group (4) is 86.7±5.1%,66.4±4.3%,35.4±2.7%, and group (5) is 80.0±4.2%,63.1±4.5,29.5±3.4%. Statistical analysis shows that there are no statistical difference between group (1) and group (2)(P>0.05), but there are obvious difference between group (1) and (3), group (2) and group (3), group (1) and group (4), group (1) and group (5)(P<0.05). All kinds of implants could inhibit the C6 glioma cells continuously.Conclusion1. The material PPC and alginate has no cytotoxicity to C6 glioma cells.2. Combined implants could produce stronger cytotoxicity than single drug implants with low loading rate.3. When the ratio of TM and paclitaxel was about 1:1, the synergistic effect was obvious.PartⅢThe study of combined implants against glioma in ratsObjectiveTo investigate the effect of combined implants (1:1) against glioma in rat model and the security used in brainMethods1. Combined fiber mats were implanted in the surface of hemisphere of four Sprague Dawley rats. The rats were scanned by MRI at 3,7 days.2. Preparation of rat glioma model:31 male (200-250g) Sprague Dawley rats were implanted in C6 cells. All rats should be scanned by MRI after a week. The rats with tumors were divided to two groups:treatment group (n=10) and control group (n=8).3. The sections were subjected to hematoxylin and eosin attaining for histological examination.4. Proliferation cell nuclear antigen (PCNA) was used to evaluate the proliferation of tumor. PCNA-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.5. To evaluate apoptotic activity, the TUNEL technique was used.Results1. There was no brain edema on MRI after the mats were implanted in hemisphere. In microscope, the range of edema is about 0.8mm at 3 day, and 0.5mm at 7day, accompanying with few lymphocyte infiltrating.2. The volume of tumor was 122.1±4.5mm3 at first week. Three weeks after treatment, the volume of treatment group tumor was 184.0±5.4mm3, and the growth rate was 50.8%. The control group was 341.3±11.4mm3 and 180.0%. Significant differences were observed between two groups (P<0.0001).3. The median survival time of treatment group was 42.5 days, and the control group is 28.5 days. Two groups had significantly difference (P<0.05). It is evident that the combined fibers could prolong the survival of rats.4. The percentage of PCNA-positive were 65.4±5.7%,68.6±6.3%,15.3±5.2% with initial tumor, control and treatment group. The PCNA-positive cells decreased obviously after cure.5. The treatment group shows many apoptotic cells, and the control group has no expression of apoptotic cells. Conclusion1. The combined fibers have well histocompatibility and security.2. The combined fibers could inhibit the growth of tumor effectively, decrease proliferation of tumor cells, induce apoptosis of tumor cells, and prolong the rat median survival time.