Preparation And Characterization Of Anti-VEGF Monoclonal Antibody Conjugated With Cisplatin Micelles And Targeting Therapy Rabbit Tongue Cancer

Part?Preparation and Characterization of Ab-PEG-PLA-CDDP nano micellesObjective:In this part,anti-vegf monoclonal antibody coupled amphiphilic polymer micelles were prepared and loaded with cisplatin?CDDP?,an insoluble anticancer drug.The amphiphilic polymer carrier was used to encapsulate the drug in the form of self-assembly in water,so as to increase the solubility,extend the half-life,and improve the sustained sustained release and active targeting ability.Methods:In this study,a new carrier material,aminopolyethylene glycol-polylactic acid?NH₂-PEG-PLA?and model drug cisplatin were used to prepare drug carrier micelles by dialysis at a certain ratio.Conjugation of anti-vegf monoclonal antibody:carbon diimine was used as the crosslinking agent to couple anti-vegf monoclonal antibody with the drug carrier micelles to prepare double-targeted micelles Ab-PEG-PLA-CDDP.Ab-PEG-PLA-CDDP micelles were used to calculate the encapsulation rate and drug loading.The drug release of Ab-PEG-PLA-CDDP in vitro was determined.Results:Ab-PEG-PLA-CDDP nano-micelles were successfully prepared by using different solvents,dosing ratio of drugs to carriers and volume ratio of organic phase to water phase,and their particle size and encapsulation efficiency were determined.The optimal preparation method was as follows:acetone was used as solvent,the mass ratio of drug to carrier polymer was 1:15,and the volume ratio of organic phase to water phase was 2:10.The average particle size of the Ab-PEG-PLA-CDDP micelles obtained by the optimal technology was?102 2.08?nm,the encapsulation rate was?64.36 1.18?%,and the drug loading rate was?5.84 0.11?%.Conclusions:In this experiment,the carrier material was peg-pla and the model drug was CDDP,and Ab-PEG-PLA-CDDP nano-micelles were prepared.Single factor test and orthogonal design test were used to optimize the preparation process and select the optimal formulation.The particle size distribution range of polymer micelle measured by laser particle size analyzer meets the requirements.Part?Establishment of methodology for the determination of CDDP content of Anti-VEGF monoclonal antibody-PEG-PLA-CDDP in nano micellesObjective:A method was developed for the determination of the encapsulation rate and drug loading rate of nano-micellar anti-vegf monoclonal antibody-peg-pla-cddp.Methods:The content of CDDP in anti-vegf monoclonal antibody-peg-pla-CDDP micelles was determined by uv spectrophotometry.CDDP standard solution was prepared for spectral scanning to determine the appropriate absorption wavelength.The absorbance under the known wavelength was determined by UV method with the concentration?ug/ml?as the abscissa and the absorbance?A?as the ordinate.The standard curve equation was obtained to determine the precision and recovery.Results:The drug was absorbed at the wavelength of 348nm by uv scanning,and the carrier had no absorption at that wavelength,which did not interfere with the measurement results.The drug presented a linear relationship in the range of 2.0-7.0ug/ml.The standard curve was plotted and the regression equation was obtained:y=0.1069x+0.0046,the intraday and daytime precision was less than 5%,and the average recovery was 99.4%.Conclusion:The encapsulation rate and drug loading of anti-vegf monoclonal antibody-peg-pla-CDDP nano-micelles detected by UV spectrophotometer?UV?are simple and feasible.Part?In vitro inhibition of RSCC-1 cells by anti-VEGF monoclonal antibody-PEG-PLA-CDDPObjective:The inhibitory effect of Ab-PEG-PLA-CDDP nanoparticles on rabbit tongue cancer cells?RSCC-1?was studied in vitro.Methods:Rscc-1 cells were resuscitated and subcultured to the logarithmic growth phase.After co-culture with PBS,NH₂-PEG-PLA blank vector,CDDP of different concentrations and ab-peg-pla-CDDP for different times,MTT was used to detect the cytotoxicity in vitro,and the cell growth inhibition rate was calculated and the inhibition rate curve was drawn.Results:MTT results showed that NH₂-PEG-PLA vector had no cytotoxic effect on rscc-1cells?p<0.05?.The commercially available CDDP dosage form showed obvious cytotoxic effect,and the growth inhibition rate curve showed that it increased gradually with the increase of drug concentration and action time.The growth inhibition rate of Ab-PEG-PLA-CDDP micellar preparation group at 48h was lower than that of the commercial CDDP preparation group?p<0.05?,and there was no statistical difference between the two groups at 96 h?p<0.05?.Conclusions:1)Ab-PEG-PLA-CDDP polymer micelles showed significant inhibitory effects on rscc-1 cells in a time-dependent and dose-dependent manner.2)Ab-PEG-PLA-CDDP polymer micelles have sustained release characteristics compared with the commercially available CDDP formulations.Part?:Establishment of a method for in vivo analysis of anti-vegf monoclonal antibody-peg-pla-cddp nanoparticlesObjective:To establish a method for detecting the distribution of cisplatin in vivoMethods:The content of CDDP in liver and plasma of New Zealand white rabbits was determined by high performance liquid chromatography?HPLC?.The specificity,repeatability,stability,precision and recovery of tissue and plasma were studied and analyzed.Results:The content of anticancer drug CDDP in normal rabbit tissues and plasma was detected successfully.RSD of tissue and plasma samples was less than 5%,RSD of repeatability and stability was less than 10%.The CDDP content in tissues showed a linear relationship within the range of 0.1-10.0ug/ml,and the standard curve equation was drawn based on the data:y=49013x+19141?R²=0.9995?.The content of CDDP in plasma was within the range of 0.1-10.0ug/ml,and the standard curve equation was calculated as y=36268x+9666.7?R²=0.9905?.Conclusions:The content of CDDP in normal rabbit tissues and plasma was determined by high performance liquid chromatography?HPLC?.It provides a basis for the subsequent experiments to verify the determination of CDDP content in tissue and plasma of Ab-PEG-PLA-CDDP polymer micellar targeted treatment of rabbit tongue cancer transplantation animal model.