| Objective:The dominant epitope peptide drug E5 used in this study was developed in the early stage of the research group,which can target dendritic cells(DC)and T cells to produce anti-tumor immune response through cytotoxic T lymphocyte(CTL)effect.In this study,polycystic liposomes were prepared by re-emulsification to encapsulate epitope peptides,and their stability was further enhanced by freeze-drying to make up for the high encapsulation rate and low leakage of peptide drugs that were difficult to achieve with existing technologies,so as to facilitate the storage and transportation of peptides.Methods:E5-Multivesicular Liposomes(E5-MVL)suspension was prepared by remulsification,and the encapsulation rate,morphology,particle size and stability were characterized by high performance liquid phase,biological microscope,laser particle size analyzer and other methods.E5 polycystic liposomal lyophilized products were prepared by vacuum freeze-drying technology,and the lyophilization process was further optimized to determine the final lyophilization conditions.The vitro release of E5-MVL suspension and lyophilized products was determined,and the stability of E5MVL after reconstitution was observed.Results:1.220 nm was selected as the wavelength for E5 liquid chromatography analysis,and the E5 methanol solution showed a good linear relationship at a concentration of 10-100 μg/mL,the standard equation was Y=21.20*X-6.478,and the R2 value was 0.9995.The E5 aqueous solution of low-concentration,mediumconcentration and high-concentration group was investigated,and the relative standard deviation RSD was less than 2%,and the precision was good.The detection of E5 by lyophilized protectant and polycystic liposomal drug delivery system does not interfere with each other,and the specificity is good.2.The lyophilization prescription and process of E5-MVL were optimized,and finally 8%mannitol was selected as the lyophilization protective agent,and the lyophilized protective agent was added to the E5-MVL after the preparation was completed by additive,and the pre-freezing temperature was-40℃ and the pre-freezing time was 4 hours of lyophilization process.E5-MVL lyophilized products have good in vitro release and have a sustained release effect.Stability experiments showed that whether at 4℃ or 25℃,the micromorphological edges of liposomal lyophilized products were clear,and the preparation of lyophilized preparations could enhance the preservation time of E5-MVL suspension and effectively improve its stability.The epitope peptide E5-MVL lyophilized powder injection preparation prepared by the best lyophilized prescription and process has a smooth appearance,no collapse,good redispersibility,and rapid reconstitution.The average particle size of E5-MVL suspension was 9.32±0.16 μm,the encapsulation rate was 69.39±0.38%,the average particle size of lyophilized product was 14.33±0.29 μm,the encapsulation rate was 68.55±0.36%,the particle size was good,and the encapsulation rate was qualified.Conclusion:In this study,the E5-MVL drug delivery system(E5-MVL)of polycystic liposomal preparations encapsulated with dominant epitope peptides were constructed by re-emulsification method,and they were prepared into lyophilized powder injection preparations,and the preparation process of polycystic liposomal lyophilized preparations containing dominant epitope peptides was optimized from the perspective of exploring the lyophilization process,indicating that E5-MVL lyophilized powder injection preparations had good appearance morphology,particle size,encapsulation rate,in vitro release rate and stability.In general,an E5-dominated epitope peptide polycystic liposome lyophilized powder injection preparation was prepared,which improved the stability of E5-MVL and extended the storage time. |
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