| Objectives:To prepare influenza vaccine liposomes modified by cationic DOTAP with different charges,and to investigate the physicochemical properties,immunogenicity,stability and safety of them.Methods:1.Using soybean lecithin and cholesterol,cationic DOTAP and neutral phospholipid DOPC(with different DOTAP and DOPC mass ratios of 4:1,3:2,2:3,1:4)as the membrane materials.The freeze-dried powder of influenza vaccine liposomes modified with different charge cations were prepared by freeze-drying combined with freeze-thaw method,and particle size,Zeta potential,and encapsulation efficiency were investigated.2.Kunming mice were divided into PBS group,vaccine stock solution group,varied cationic groups(4:1,3:2,2:3,1:4)and neutral liposome group,the stock solution group was used as the positive control group,and the PBS group was used as the negative control group.After 7,14 and 28 Days of mice intraperitoneal immunization respectively,MTT method was used to detect the proliferation of splenic lymphocytes,and flow cytometry was used to detect the CD4+/CD8+ratio of mouse T lymphocytes to evaluate the cellular immunogenicity.3.Kunming mice were divided into PBS Group,vaccine stock solution group,cationic group(4:1,3:2,2:3,1:4),neutral liposome group,blank group,After 3,7,14,28,42,56 days of mice intraperitoneal immunization,with the blank non-administered group used as the pre-immunization control,the antibody titer ratio of the mouse serum was detected by the micro hemagglutination inhibition method to evaluate the effect of cationic liposomes on the immune function of the mice to evaluate the humoral immunogenicity.4.The cation 4:1 liposome freeze-dried powder and suspension were placed at 4℃,25±2℃(60%±5%)and 40±2℃(75%±5%)for stability test,during the test period,the encapsulation rate,stimulation index,and antibody titer ratio were determined as the indicators.5.Kunming mice were divided into PBS group,vaccine stock solution group,4:1 group and neutral liposome group.The male and the female were equal amount.The mice were immunized intraperitoneally for 14 days.The stock solution group was used as a positive control,and the PBS group was used as a negative control.observing the weight change,organ index and blood index of mice for safety evaluation.Results:1.The average particle size of the four groups of liposomal influenza vaccines with DOTAP:DOPC ratios of 1:4,2:3,3:2,and 4:1 are 437.32nm,475.03nm,485.81nm,and 529.65nm;the zeta potentials are 1.59mV,5.31mV,7.93mV,12.05mVrespectively;the encapsulation efficiency is 89.00%,89.15%,86.07%,81.82%respectively.2.The results of the T lymphocyte proliferation experiment showed that compared with the PBS group,the SI values of the four cationic liposome groups were significantly higher at 7,14,and 28 days of mice immunization(P<0.05)and there was a t difference;The SI values of the four cationic liposome groups were statistically different at 7d and 28d compared with the stock solution group,(P<0.05).Flow cytometry was used to detect the ratio of CD4+/CD8+in mice.The results showed that the CD4+/CD8+values of the four cationic liposome groups were significantly higher than those of the other groups after14 and 28days of immunization(P<0.05).3.The results of hemagglutination inhibition experiment showed that the antibody titer ratio of the stock solution group,and4:1 group were greater than or equal to 4 at 3days of immunization;the antibody titer ratio of the stock solution group,4:1 group,3:2 group and the neutral group were greater than or equal to 4 on the 7th day.The 4:1 group was statistically different from the original solution group and the neutral group(P<0.05);the antibody titer ratio of each group was greater than or equal to 4 at 14d,28d,42d,56d of immunization.and the titer of each group was at.There is a downward trend after56d immunization,but all are greater than or equal to 4,resulting in an effective humoral immune response.4.The results of physical stability showed that when stored at the encapsulation rate of cationic liposome lyophilized powder decreased more slowly than that of the suspension,and the appearance did not change significantly under 4℃,25±2℃(60%±5%),40±2℃(75%±5%)storage condition.After 15 days of storage at4℃,the results of MTT experiment showed that the 4:1 group was significantly different from the PBS group and the neutral group(P<0.05);there was no significant difference in each group at 30 days storage.There was a significant difference between the 4:1 group and the PBS group at 60d and 120d of storage at this temperature(P<0.05);The results of hemagglutination inhibition test showed that the ratio of antibody titers before and after immunization in the 4:1 group was greater than 4 at 15d,30d,60d,and 120d under 4℃ storage.the results of MTT experiment showed that the 4:1 group was significantly different from the PBS group and the stock solution group at 15d of 25±2℃(60%±5%)storage(P<0.05);there was no significant difference between each group after 30days storage;The results of hemagglutination inhibition test showed that the ratio of antibody titers before and after immunization in the 4:1 group was greater than 4 at 15 days,30 days,and 60 days storage.Under 40±2℃(75%±5%),the results of MTT experiment showed that the 4:1 group was significantly different from the PBS group and the neutral group at 15days storage.The results of hemagglutination inhibition test showed that the antibody titer ratio of the 4:1 group was greater than 4 at 15 days of storage.5.Acute toxicity results showed that the mice all survived during the observation period and their body weight increased normally.Compared with the PBS group,there were no significant differences in the number of granulocyte,platelets,and average platelet volume in the cationic group of female and male rats.The white blood cells,lymphocytes and hemoglobin in female mice were significant differences between cation group and PBS group and the stock solution group(P<0.05)in female mice.There was no statistical difference between male rats.There was no statistical difference in the heart,liver,lung,thymus,kidney and brain of the 4:1 group of female and male rats compared with the original solution group.Conclusions:The cationic liposome adjuvants prepared with different mass ratios of DOTAP and DOPC of 4:1,3:2,2:3,1:4 have good stability and can induce cellular and humoral immunity in mice.The mice showed good safety,and the 4:1 group of cations with a charge of more than 10 had the best immune effect. |
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