Combination Of 3-Methyladenine Therapy And ASN-Gly-Arg(NGR)-Modified Mesoporous Silica Nanoparticles Loaded With Temozolomide For Glioma Therapy

Part I Preparation and detection of targeted mesoporous silica drug-loading systemObjective1.To prepare a targeted polypeptide Asn-Gly-Arg(NGR)modified mesoporous nano-silica drug delivery system(MSN-TMZ-PDA-NGR).2.To detect the characteristics of the nano drug delivery system.Method1.MSN was mixed with TMZ to allow TMZ to be adsorbed into the mesopores of MSN.The precipitated MSN-TMZ was collected and dried,and stirred in a polydopamine solution to obtain MSN-TMZ-PDA.The purified precipitate was covalently bound in the NGR solution to give MSN-TMZ-PDA-NGR.2.The characteristics of the nano drug-loading system were examined by electron microscopy,infrared spectroscopy and nanoparticle size analyzer.The components in the preparation process were collected to calculate drug loading efficiency and encapsulation efficiency.ResultThe diameters of MSN and MSN-TMZ-PDA-NGR were 62±5.91 and 70±8.26 nm respectively by transmission electron microscopy.The hydrated particle size of the nanoparticles were 93.97±9.43 and 129.2±15.72 nm,the zeta potential were-40.4,21.6 mV,respectively.The infrared spectrum of MSN before and after modification showed characteristic changes.The drug loading rate and encapsulation efficiency of the targeted nano-mesoporous silica drug-loading system are 28.5% and 38%,respectively.ConclusionThe NGR-targeted mesoporous silica nano drug delivery system was successfully prepared.The system has excellent stability,drug loading rate and encapsulation efficiency.Part II Targeting and toxicity study of mesoporous silica nano drug delivery system in vitroObjective1.To verify the prepared MSN-TMZ-PDA-NGR has high uptake of glioma cells and confirm its specific targeting.2.To verify the toxic effects of the prepared MSN-TMZ-PDA-NGR on glioma cells.3.To explore the mechanism of MSN-TMZ-PDA-NGR on cytotoxicity and to verify the induction of autophagy and apoptosis.Method 1.Rat glioma cell line C6,human glioma cell line U87 and rat astrocyte AC were used in this study.Western blot were used to detect the expression of CD13 receptor protein on three cell surfaces under standard conditions.MSN-Rh6G-PDA and MSN-Rh6G-PDA-NGR were prepared by loading Rhodamine 6G in MSN instead of TMZ,and added to the cultured cells,and the uptake of each pharmaceutical preparation by the cells was observed under a fluorescence microscope.2.Glioma cells were cultured in a standardized manner and divided into TMZ,MSN-TMZ-PDA,MSN-TMZ-PDA-NGR and MSN-PDA-NGR groups,and different concentrations of drugs were added to treat the corresponding time.The absorbance values of each group were measured by CCK8 method,and the cell activity and IC50 value were calculated to evaluate the cytotoxicity of the drugs.3.Glioma cells were cultured under standard conditions and MSN-TMZ-PDA-NGR was added at different concentrations and times.Anti-LC3 B,anti-p62,anti-Caspase-3 and other antibodies were selected according to specific autophagy and apoptotic proteins,and the expression of related proteins was detected by Western-blotting method.Autophagy and apoptosis induction of MSN-TMZ-PDA-NGR was verified.4.The cultured glioma cells were treated with TMZ and MSN-TMZ-PDA-NGR at the same dose,respectively,and the cells were collected and fixed at a specific time to prepare an electron microscope observation sample.Autophagy induction of MSN-TMZ-PDA-NGR was visually confirmed under an electron microscope.Result 1.Compared with rat astrocyte AC,CD13 has higher expression on the surface of C6 and U87 cells by Western blotting.The fluorescence spots of MSN-Rh6G-PDA-NGR in the two glioma cells C6 and U87 were significantly higher than those of normal cells by fluorescence microscopy.In both glioma cells,NGR-targeted nanoparticles can be taken up into cells faster and reach a peak in 1 hour.After pretreatment of NGR,the intracellular fluorescent particles were significantly reduced.2.The cytotoxicity of each nano-preparation containing TMZ was evident in the CCK8 cytotoxicity assay.The IC50 values of TMZ,MSN-TMZ-PDA and MSN-TMZ-PDA-NGR were 127.50,422.00 and 21.02 ?g /mL after C6 cells were treated for 24 h,respectively,and 39.71,89.31 and 6.19 ?g /mL after 48 h.After U87 cells were treated for 24 hours,the IC50 values of TMZ,MSN-TMZ-PDA and MSN-TMZ-PDA-NGR were 180.40,719.70 and 17.38 ?g /mL,respectively,and 32.11,124.50 and 7.43 ?g /mL after 48 h,respectively.MSN-PDA-NGR without drug was not significantly cytotoxic.3.By immunoblotting,MSN-TMZ-PDA-NGR induced autophagy and apoptosis in C6 and U87 cells in a time-and concentration-dependent manner.4.Observed by electron microscopy,autophagosomes and autolysosomes appeared in the cells treated with MSN-TMZ-PDA-NGR,and the number was significantly higher than that of TMZ treatment group.Conclusion MSN-TMZ-PDA-NGR has good targeting and cytotoxicity,can induce autophagy and apoptosis in glioma cells,and provide a basis for further experiments.Part III 3-methyladenine combined with NGR-modified mesoporous silica loaded with tromethamine for glioma in vitroObjective To test and verify whether the autophagy inhibitor 3-MA block the autophagy caused by MSN-TMZ-PDA-NGR,while increasing cell apoptosis.Method 1.The optimal MOI of mRFP-GFP-LC3 adenovirus was explored and the glioma U87 cells were transfected with mRFP-GFP-LC3 adenovirus.The induction of autophagy by MSN-TMZ-PDA-NGR and the inhibition of autophagy by 3-MA were observed by laser confocal microscopy.2.Autophagy and apoptosis-specific proteins were detected by Western blot to verify whether MSN-TMZ-PDA-NGR can induce autophagy or apoptosis of glioma.Apoptosis was detected by flow cytometry and Hoechst staining of 3-MA to verify whether autophagy was inhibited and that more apoptosis occurred.Result 1.The glioma cells were successfully transfected with mR FP-GFP-LC3 adenovirus with optimal MOI.MSN-TMZ-PDA-NGR showed more obvious autophagy fluorescence under laser confocal microscopy.After administration of 3-MA,autophagy was inhibited and fluorescence was reduced or disappeared.2.Semi-quantitative analysis by western blot showed that compared with other groups,MSN-TMZ-PDA-NGR resulted in more obvious autophagy,and autophagy was attenuated after combined with 3-MA,resulting in more obvious apoptosis.In flow cytometry and Hoechst staining experiments,MSN-TMZ-PDA-NGR induced stronger apoptosis than other groups,and apoptosis was increased after treatment with 3-MA.ConclusionMSN-TMZ-PDA-NGR-induced autophagy can be blocked by the autophagy inhibitor 3-MA,leading to the occurrence of apoptosis.This provides a theoretical basis for the further combination of the two drugs for in vivo anti-tumor therapy.Part IV 3-methyladenine combined with NGR-modified mesoporous silica loaded with tromethamine for glioma in vivo1.To construct a subcutaneous xenograft tumor model of U87 nude mice.2.To detect the distribution of MSN-Rh6G-PDA-NGR in vivo by IVIS(Animal Imaging System)and evaluate its targeting.3.To observe the effect of targeted nano drug-loading system on tumor weight and growth rate,and evaluate the anti-tumor effect and biosafety of this system.Method 1.Human glioma cell line U87 was cultured in vitro,and 4W old nude mice were selected to subcutaneously under the right anterior armpit to establish a subcutaneous ectopic tumor transplantation model of U87 nude mice.2.Intraperitoneal injection of various Rh6G-containing preparations,the distribution of the drug was observed under the living body fluorescence imaging system,and the in vivo targeting was evaluated.3.Intraperitoneal injection of each TMZ preparation,the tumor growth rate was measured,the tumor volume and mass were measured,and the tumor specimens were stained with Tunel to evaluate the in vivo antitumor activity of each preparation.The biosafety of the formulation was evaluated by monitoring the body weight of nude mice and HE staining of major organs.Result 1.Compared with the control group and MSN-Rh6G-PDA,MSN-Rh6G-PDA-NGR accumulated more prominently in the tumor area in vivo,and the fluorescence intensity showed that the fluorescence intensity in the MSN-Rh6G-PDA-NGR group was 2.35 times that of the non-targeting group.2.After treatment with each TMZ preparation,the tumor growth rate and quality of the MSN-TMZ-PDA-NGR group were significantly lower than those of the TMZ group and the control group.In the combined 3-MA treatment group,the tumor growth rate and quality were significantly lower than the non-combination treatment.Tunel staining showed that the 3-MA combination group resulted in stronger apoptosis.There was no significant difference in HE staining between body weight and vital organs between nude mice.ConclusionMSN-TMZ-PDA-NGR can target tumor models in vivo and exhibit significant anti-tumor activity.Combined with 3-MA,it can enhance anti-tumor effect and has good biosafety.