| Objectives:To explore the optimal preparation process,stability,physicochemical properties and immunogenicity of influenza vaccines liposomes modified by PEG3000 and PEG6000.Methods:PEG3000,PEG6000 modified influenza vaccine liposome was prepared by freeze-thaw-lyophilization method,and the stimulation index(SI)was taken as the main evaluation index by MTT method,then the optimal preparation process was determined by one-way analysis of variance.The resulted product made from the optimal process was intraperitoneally administered into mice.After 7,14,and 28 days of immunization,the cell immunogenicity was explored by MTT method taking Sl into consideration,and surface-labeled method indexing with CD4+/CD8+、CD3+CD8+、CD3+CD4+、CD3+ratio.Humoral immunogenicity was conducted by micro-hemagglutination inhibition method taking antibody titer ratio as an evaluation index,and surface-labeled method indexing with CD3-CD45R+ratio.Carbon clearance method and surface-labeled method were employed to investigate the non-specific immunogenicity by taking phagocytic index(PI)and CD3-CD49b+as an evaluation parameter.PEG3000 modified liposome lyophilized powder,PEG300 modified liposome suspension,PEG6000 modified liposome lyophilized powder,PEG600 modified liposome will be prepared by the above optimal process Suspension and neutral liposome lyophilized powder were placed at 4 ℃,25±2 ℃and 37 ± 2℃ to conduct stability test.The encapsulation rate was used to evaluate the physical stability,and the antibody titer ratio and viscera index were used as indicators to evaluate biological stability.Results:the optimal preparation method was to add PEG3000,PEG6000 with a molar ratio of 4%to lecithin after liposome formation.After intraperitoneal injection of PEG3000(or PEG6000)modified liposome lyophilized powder,unmodified neutral liposome lyophilized powder,non-liposome vaccine stock solution and PBS negative control group,immunized mice were immunized with one immunization cycle. The cell immunogenicity,spleen lymphocytes results showed that there is a significant difference between PEG3000 modified liposome lyophilized powder and the others(P<0.05)in terms of SI value,CD4+/CD8+,CD3+ CD4+,and CD3+,and higher than the unmodified neutral liposome group,indicating that PEG3000 modified influenza vaccine liposomes enhance cell immunogenicity;There is no significant difference between CD3+CD8+in PEG3000 modified liposome group and other groups,indicating PEG3000 modified influenza vaccine liposomes did not stimulate the increase of cytotoxic T-cell subsets;There is a significant difference between PEG6000 modified liposome lyophilized powder and the others(P<0.05)in terms of SI value,CD4+/CD8+,CD3+ CD4+,CD3+ CD8+,CD3+,and higher than the unmodified neutral liposome group,indicating that PEG6000 modified influenza vaccine liposomes enhance cell immunogenicity;The CD3+CD4+ in the PEG3000 and PEG6000 modified liposome lyophilized powder group in thymic T lymphocytes was significantly different from other groups(P<0.05),and higher than that in the unmodified neutral liposome group,indicating PEG3000 and PEG6000 modified influenza vaccine liposomes stimulated T helper cell an increase in cell subsets;there is no significant difference between CD3+CD8+,CD3+ in the PEG3000 and PEG6000 modified influenza vaccine liposomes and other groups,indicating that PEG3000 and PEG6000 modified influenza vaccine liposomes do not stimulate the T cell subsets to increase.In the humoral immunogenicity,the PEG3000 and PEG6000 modified liposomes lyophilized powder group had HI≥ 4,and the antibody titer ratio were superior to other groups;there is a significant difference between PEG3000 and PEG6000 modified liposome lyophilized powder and the others in terms of CD3+CD45R+ratio,and higher than neutral liposome group,indicating that PEG3000 and PEG6000 modified influenza vaccine liposomes enhance humoral immunogenicity.The non-specific immunoimmunogenicity,PEG3000 and PEG6000 modified liposomes lyophilized powder group had no significant difference in PI value from the PBS negative control,indicating that the liposomes of PEG3000 and PEG6000 modified influenza vaccine liposomes were uneasily engulfed by MPS;The CD3-CD49b＋of EG3000 and PEG6000 modified influenza vaccine liposomes was significantly different from other groups(P<0.05),and higher than the unmodified neutral liposome group,indicating that the EG3000 and PEG6000 modified influenza vaccine liposomes stimulated the increase of NK cell subsetsImmunized mice were immunized by intraperitoneal injection of PEG3000 and PEG6000 modified liposome lyophilized powderprepared with the best prescription,unmodified neutral liposome lyophilized powder,non-liposome vaccine stock solution and PBS negative control group.During the 7d,14d and 28d,the SI value of PEG6000 modified influenza vaccine liposomes in cellular immunogenicity was significantly different from other groups(P<0.05),and it was better than PEG3000 modified influenza vaccine liposomes,indicating that as the molecular weight increases,cell immunogenicity increases;PEG6000 modified influenza vaccine liposomes and PEG3000 modified influenza vaccine liposomes in humoral immunogenicity are not significantly different;PEG6000 modified influenza vaccine liposomes and PEG3000 modified were no significant difference in non-specific immunogenicity,indicating that non-specific immunogenicity does not increase with increasing molecular weight.Physical stability of PEG3000 modified influenza vaccine liposomes lyophilized powder,PEG6000 modified influenza vaccine liposomes lyophilized powder,PEG3000 modified influenza vaccine liposomes suspension and PEG6000 modified influenza vaccine liposomes suspension with 3 months under the storage conditions of 4℃,25±2 ℃ and 37± 2℃.The encapsulation rate of PEG3000 modified influenza vaccine liposome lyophilized powder and PEG6000 modified influenza vaccine liposome lyophilized powder dropped is lower than that of its suspension,and the encapsulation efficiency under 4℃ storage conditions is higher than 25±2℃ and 37±2 ℃ storage conditions,indicating that PEG3000 modified influenza vaccine liposomes yophilized powder and PEG6000 modified influenza vaccine liposones yophilized powder.In the investigation of the biological stability of the PEG3000 modified influenza vaccine liposome lyophilized powder and the PEG6000 modified influenza vaccine liposome lyophilized powder,when stored at 4℃,PEG3000 modified influenza vaccine liposomes and PEG6000 modified influenza vaccine liposomes have a HI≥4 within 3 months;when stored at 25±2℃,PEG3000 modified influenza vaccine liposomes and PEG6000 modified influenza vaccines liposomes had HI≥4 within 3 months,and the antibody titer ratio under 4℃ storage conditions was higher than 25±2℃ storage conditions,indicating that the PEG3000 modified influenza vaccine liposome lyophilized powder and PEG6000 modified influenza vaccine liposome lyophilized powder has better biological stability,and 4℃ is the best storage conditionConclusion the optimal preparation method was to add PEG3000,PEG6000 with a molar ratio of 4%to lecithin after liposome formation.he above two target products can Produce better immunogenicity;PEG6000 modified influenza vaccine liposomes have better cell immunogenicity than PEG3000 modified influenza vaccine liposomes;The humoral immunogenicity and non-specific immunogenicity,PEG3000 and PEG6000 modified influenza vaccine liposomes were no significant difference.It is indicated that it has humoral immunogenicity but is not easily swallow by MPS,which prolongs the residence time in the blood.PEG6000 modified influenza vaccine liposomes and PEG3000 modified influenza vaccine liposomes have better stability. |
PDF Full Text Request