PEG3000修饰的脂质体流感疫苗冻干粉稳定性及安全性

Stability and safety of PEG3000 modified liposome influenza vaccine in a form of freeze-dried powder

目的考察冻融冻干法制备的PEG3000修饰的脂质体流感疫苗冻干粉稳定性及安全性。方法将冻融冻干法制备的PEG3000修饰的脂质体流感疫苗混悬液和冻干粉分别储存于不同温度[4、(25±2)、(37±2)℃]下,第0、1、2、6月取样,Lowry法检测包封率,考察物理稳定性;将冻干粉分别置4、(25±2)℃条件下,原液始终储存于4℃,第1、2、3月取样,腹腔免疫小鼠7 d后,采血分离血清,微量血凝抑制法检测免疫后与免疫前抗体滴度比,考察生物稳定性。将小鼠随机分为PEG3000修饰的脂质体流感疫苗冻干粉组(PEG组)、原液组、脂质体组(佐剂组)、PBS组(阴性对照组),经小鼠腹腔给药(15μg血凝素),进行急性毒性试验;分别于0、14和28 d经小鼠腹腔给药(6μg血凝素),进行长期毒性试验。结果PEG3000修饰的脂质体流感疫苗冻干粉(37±2)℃保存6个月,包封率达80%以上;(25±2)℃保存3个月,小鼠免疫后与免疫前抗体滴度比≥4。急性毒性试验期间,小鼠未见任何明显急性毒性反应。长期毒性试验期间,各实验组小鼠一般状况良好;体重和摄食量与阴性对照组比较未见明显变化;血液学指标均在正常范围内波动,未发现与给药相关的毒性反应;各组织脏器系统解剖观察及脏器重量均未见明显与给药相关的毒性病理学改变。结论PEG3000修饰的脂质体流感疫苗冻干粉具有良好的稳定性;经小鼠急性和长期毒性试验未见明显毒性反应,具有良好的安全性。

Objective To investigate the stability and safety of PEG3000 modified liposome influenza vaccine in a form of freeze-dried powder prepared by freeze-thaw freeze-drying method.Methods The suspension and freeze-dried powder of PEG3000 modified liposome influenza vaccine were stored at various temperatures[4,(25±2)and(37±2)℃]separately,from which samples were taken 0,1,2 and 6 months after storage,determined for the encapsulation rate by Lowry method,and evaluated for physical stability.The freeze-dried powder was stored at 4 and(25±2)℃,while the bulk at 4℃,from which samples were taken and inoculated i.p.to mice.Serum samples were collected 7 d after immunization and determined for the ratio of antibody after immunization to that before immunization by micro-hemagglutination inhibition test to evaluate the biological stability.Eighty mice were randomly divided into four groups,twenty for each,and immunized i.p.with PEG3000 modified liposome influenza vaccine in a form of freeze-dried powder(PEG group),bulk of the vaccine(bulk group),liposome(adjuvant group)and PBS(negative control group)respectively,while those in PEG and bulk groups were injected i.p.with 15μg hemagglutinin for acute toxicity test.Meanwhile,another eighty mice were divided into four groups by the above-mentioned method,and injected i.p.with 6μg hemagglutinin on days 0,14 and 28 after immunization for long-term toxicity test.Results The encapsulation rate of freeze-dried powder of PEG3000 modified liposome influenza vaccine was more than 80%after storage at(37±2)℃for 6 months,while the antibody titer ratio after storage at(25±2)℃for 3 months was not less than 4.No any obvious acute toxic reactions of mice were observed in acute toxicity test.However,in long-term toxicity test,the general state of mice in test groups were normal,while the body weight and food intake showed no significant change as compared with those in control group;the hematological indexes were fluctuated within the normal range,and no toxic reactions associated with administration were found;no obvious toxic pathological changes associated with administration were observed in various tissues and organs in anatomy.Conclusion The PEG3000 modified liposome influenza vaccine in a form of freeze-dried powder showed good stability,while showed no obvious toxic reactions in acute and long-term toxicity tests,indicating a good safety.