Preparation and Stability of Influenza Vaccine Liposomes Modified by PEG6000
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摘要:
目的 筛选出PEG6000流感疫苗脂质体的最佳制备工艺并且评价其稳定性。 方法 将小鼠免疫7 d，通过MTT法确定加入PEG6000的最佳摩尔百分比和最佳制备工艺。采用冻融冻干法制备PEG修饰的流感疫苗脂质体，将样品分别储存于不同温度[4 ℃、（25±2） ℃、（37±2） ℃]下，在设定时间取样测定包封率、胸腺指数和抗体滴度，考察其物理和生物稳定性。 结果 MTT实验结果表明，PEG6000占磷脂摩尔百分比为4%时为最佳用量（P < 0.01），最佳工艺为后修饰法。PEG6000修饰的流感疫苗脂质体冻干粉在（37±2） ℃条件下放置6个月后，包封率仍在80%以上；在（25±2） ℃放置3个月后抗体滴度比 > 4，表明仍具有较好的免疫效应。 结论 PEG6000占磷脂摩尔百分比为4%时为最佳用量，采用后修饰法制备为最佳工艺；PEG6000脂质体具有良好的物理稳定性及生物稳定性。 Abstract:Objective To screen out the best method of PEG6000 modified liposomes for influenza vaccine and evaluate its stability. Methods After 7 d of immunization, the optimal molar percentage of PEG6000 and the optimal preparation method were assumed by MTT method. The PEG-modified influenza vaccine liposomes were prepared by freeze-thaw freeze-drying method. The samples were stored at different temperatures （4 ℃, 25±2 ℃, 37±2 ℃）, and the encapsulation rate and thymus index and antibody titer were measured at a fixed time. So the physical and biological stability of the samples were investigated. Results The MTT experiment showed that PEG6000 was the best dosage when the molar percentage of phospholipid was 4% （P < 0.01）, and the best process was post-modification method.The entrapment rate of PEG6000 modified liposome for influenza vaccine was still higher than 80% when stocked at 37±2 ℃ for 6 months.At 25±2 ℃ for 3 months, the antibody titer was more than 4, indicating that the antibody still had nice immune effection. Conclusions PEG6000 best dosage of phospholipid when the molar percentage of PEG6000 is 4%, and post-modification method is the best preparation process. PEG6000 liposome has nice physical stability and biological stability. -
Key words:
- Liposomes /
- PEG6000 /
- Influenza vaccine /
- Stability
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表 1 7 d 时的刺激指数[n = 5，（
$ \bar x \pm s $ ）]Table 1. SI on 7 d [n = 5，（
$ \bar x \pm s $ ）]分组 刺激指数SI值 F P PEG6000占磷脂的百分摩尔比 2% 1.3317 ± 0.0364△# 119.404 < 0.001* PEG6000占磷脂的百分摩尔比4% 1.5943 ± 0.0184△ PEG6000占磷脂的百分摩尔比 6% 1.4141 ± 0.0257△## PBS 阴性对照组 1.0458 ± 0.8870## 中性脂质体 1.3307 ± 0.0946△## 疫苗原液组 1.2421 ± 0.0459△## *P < 0.05。与PBS阴性对照组比较，△P < 0.05；与PEG6000占磷脂的百分摩尔比4%比较，##P < 0.05。 
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表 2 7 d 时的刺激指数[n = 5，（
$ \bar x \pm s $ ）]Table 2. SI on 7 d [n = 5，（
$ \bar x \pm s $ ）]分组 刺激指数SI值 F P PEG6000共成膜组 1.2629 ± 0.0350△## 7.643 < 0.001* PEG6000后修饰组 1.3407 ± 0.0210△ 疫苗原液组 1.0808 ± 0.0410△## 中性脂质体组 1.1647 ± 0.0450△## PBS阴性对照组 1.0335 ± 0.0070## *P < 0.05。与PBS阴性对照组比较，△P < 0.05；与PEG6000后修饰组比较，##P < 0.05。
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表 3 PEG6000修饰的流感疫苗脂质体的包封率（%）
Table 3. The encapsulation rate of PEG-modifed influenza vaccine liposome
温度（℃） 时间（月） PEG6000冻干粉r PEG6000混悬液 4 0 95.51 62.40 1 91.17 67.02 2 86.54 60.35 6 82.20 28.18 25 ± 2 0 95.69 68.08 1 95.38 55.16 2 95.27 42.27 6 86.54 35.02 37 ± 2 0 97.43 70.86 1 95.65 65.81 2 93.12 65.69 6 83.83 0.14
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表 4 PEG修饰的流感疫苗脂质体的胸腺指数（%）
Table 4. The thymus index of PEG-modified influenza vaccine liposome （%）
温度（℃） 时间（月） PBS 中性脂质体组 PEG6000脂质体组 疫苗原液组 4 1 4.98 6.04 6.14 4.77 2 4.49 5.69 5.99 5.13 3 4.22 5.29 5.32 5.07 6 3.89 4.62 4.90 5.16 25 ± 2 1 4.98 6.04 - 4.77 2 4.49 5.69 - 5.13 3 4.22 5.29 5.33 5.06 6 3.89 4.62 4.85 5.16
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表 5 PEG 修饰的流感疫苗脂质体的抗体滴度比
Table 5. The antibody titer ratio of PEG-modified influenza vaccine liposome
温度
（℃）时间
（月）原液 中性脂质
体组PEG6000脂
质体组4 1 5.78 4.44 4.44 2 5.54 3.98 4.19 3 5.71 4.14 4.46 25 ± 2 1 5.78 4.17 4.37 2 5.54 4.06 4.34 3 5.71 3.98 4.05 免后免前抗体滴度 > 4表明具有免疫效应。
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