壳聚糖纳米载体材料性能对抗乙肝免疫核糖核酸免疫活性的影响(英文)

Influence of properties of Chitosan Nanocarrier material on immune activity of anti-hepatitis B immune ribonucleic acid

背景:近年来抗乙肝免疫核糖核酸作为一种免疫治疗剂,对慢性肝炎的治疗作用备受重视,但临床应用效果不稳定,因此构建一个能有效保护免疫核糖核酸并能被组织更好吸收的免疫核糖核酸运载体系具有重要意义。壳聚糖作为一种新辅料取得了良好的效果。目的:制备抗乙肝免疫核糖核酸的壳聚糖纳米载体,观察其免疫活性。方法:实验于2003-12/04-12在华中科技大学同济医学院环境医学研究所完成。①制备壳聚糖-免疫核糖核酸纳米粒采用复凝聚方法。②壳聚糖-免疫核糖核酸纳米粒形态、粒径和表面电位测定采用扫描电镜、原子力显微镜、Zeta电位/粒度分析仪观察。③壳聚糖对包裹的免疫核糖核酸的保护作用观察采用核糖核酸酶保护试验。④壳聚糖-免疫核糖核酸纳米粒免疫活性采用白细胞黏附抑制试验测定。体内白细胞黏附抑制指数测定:取小白鼠30只,随机分4组,生理盐水组5只、壳聚糖组5只、壳聚糖-免疫核糖核酸纳米粒组10只和免疫核糖核酸组10只。腋下和腹股沟皮下分别多点注射生理盐水、壳聚糖、壳聚糖-免疫核糖核酸纳米粒和免疫核糖核酸溶液后,无菌取出小鼠脾脏,制成单细胞悬液,加入乙肝疫苗和小牛血清1640液及实验试剂,单纯脾细胞悬液作为对照,根据公式[(对照孔A值-实验孔A值)/对照孔]×100%计算黏附抑制指数。体外白细胞黏附抑制指数测定:取健康小鼠1只,制备脾细胞悬液,于培养孔板中加入健康小鼠脾细胞悬液、乙肝疫苗、壳聚糖-免疫核糖核酸纳米粒或免疫核糖核酸溶液或壳聚糖,对照孔不加乙肝疫苗,测定方法同上。结果:①壳聚糖-免疫核糖核酸纳米粒的物理特性:新鲜制备的壳聚糖-免疫核糖核酸纳米粒多呈球形,放置时间在48h以内形态较稳定,但粒径略有增大,放置时间超过72h以后肉眼可见液体中有絮状物析出。Zeta电位/粒度分析仪测定放置3,24,48,72h的壳聚糖-免疫核糖核酸纳米粒的平均粒径分别为132.60,138.46,167.28,486.24nm,平均表面电位为+12.8,+12.5,+10.59,+3.86mV。②壳聚糖-免疫核糖核酸纳米粒的酶保护试验结果:由于壳聚糖与免疫核糖核酸形成了复合物,免疫核糖核酸未被核糖核酸酶降解。当减少壳聚糖-免疫核糖核酸纳米粒量而加大核糖核酸酶量,免疫核糖核酸部分得到保护,部分被核糖核酸酶降解。③壳聚糖-免疫核糖核酸纳米粒免疫活性:体内白细胞黏附抑制试验结果纳米粒组显著高于其他3组(34.51±13.25,-5.50±8.78,5.87±2.06,12.39±6.51,P<0.01),免疫核糖核酸组显著高于壳聚糖和生理盐水组(P<0.05)。体外白细胞黏附抑制试验壳聚糖-免疫核糖核酸纳米粒白细胞黏附抑制率高于免疫核糖核酸和壳聚糖的黏附抑制指数(31.00%,6.55%,7.12%)结论:壳聚糖纳米粒可以有效的保护免疫核糖核酸,提高抗乙肝免疫核糖核酸的利用率和免疫活性,其保护作用与核糖核酸酶呈剂量效应关系。

BACKGROUND： In recent years, being a kind of immune therapeutic form, hepatitis B immune ribonucleic acid （iRNA） has drawn a great atten- tion in treatment of chronic hepatitis B. But its clinical effects are not stable, for which, it is significant to construct iRNA earrier system that can not only protect RNA effectively, but also be well absorbed by tissues. Being a kind of new supplementary material, chitosan has provided good results. OBJECTIVE： To observe the immune activity of anti-hepatitis B iRNA by preparing its chitosan nanoearrier. METHODS： The experiment was performed in Institute of Environmental Medicine of Tongji Medical College of Huazhong University of Science and Technology. ① Chitosan-iRNA nanoparticlcs were prepared by complex coacervation method. ② Particle shape, diameter and surface potential were measured and observed with scan eleetromicroscope, atomie power microscope and Zeta potential/particle analyzer. ③RNA enzymatic protection test was used to observe the protection of chitosan to encapsulated iRNA. ④ Leukocytic adherence inhibition （LAI） test was used to determine the immune activity of chitoson-iRNA nanoparticlcs. Determination of inhibition index of leukocytie adherence in vivo： 30 white mice were employed and randomized into 4 groups, named physiological saline group （5 mice）, chitosan group （5 mice）, chitosan-iRNA nanoparticle group （10 mice） and iRNA group （10 mice）. After subcutaneous injection of physiological saline, chitosan, chitosan-iRNA nanoparticales and iRNA solution in subaxillary region and inguinal groove, the spleen of mouse was collected aseptically to prepare unicellular suspension and hepatitis B vaccine; calf serum 1640 solution and experimental reagent were added. Simple spleen cell suspension was taken as the control. The adherence inhibition index was calculat ed aecording to the formula [（A value of control pore-A value of experimental pore）/control pore] × 100%. Determination of inhibition index of leukoeytic adherence in vitro： 1 healthy mouse was employed to prepare spleen cell suspension. In culture pore-plate, spleen cell suspension of healthy mouse, hepatitis B vaecine and chitosan-iRNA nanoparticles or iRNA solution or ehitosan were added. In the control group, hepatitis B vaccine was not added. The determination method was the same as the above. RESULTS： ① Physical properties of chitosan-iRNA nanoparticles： the fresh prepared ehitosan-iRNA nanoparticle was of sphericity, stable in morphology within 48-hour placement, but its diameter was enlarged slightly and floecule was separated out from solution and visible with naked eyes af- ter placement over 72 hours. It was determined with Zeta potential/particle analyzer that average particle diameters in 3 hours, 24 hours, 48 hours and 72 hours were 132.6, 138.46, 167.28 and 486.24 nm successively and the average surface potentials were ＋12.8, ＋12.5, ＋10.59 and ＋3.86 mV. ② Result of enzymatic protection test of chitosan-iRNA nanoparticle： since chitosan interacts with iRNA to form the complex, iRNA has not been decomposed by RNase. When the amount of chitosan-iRNA nanoparticle is reduced and RNase is increased, a part of iRNA will be protected and another part will be decomposed by RNase. ③ Immune activity of chitosan-iRNA nanoparticle： it was indicated in LAI test in vivo that the result in nanopartitie group was higher than the other three groups （34.51±13.25,-5.50±8.78, 5.87±2.06,12.39±6.51 ,P 〈 0.01）, and that in iRNA group was higher significantly in chitosan and physiological saline groups （P 〈 0.05）. It was indicated in LAI test in vitro that LAI rate of chitoson-iRNA nanoparticle was higher than that of iRNA and chitosan （31.00%,6.55% ,7.12%）. CONCLUSION： Chitosan nanopartiele can protect effectively iRNA andimprove utilization rate and immune activity of hepatitis B iRNA and its protection presents dose-effect relationship with RNase.