Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, M.P., India.
Vaccine. 2011 Nov 8;29(48):9026-37. doi: 10.1016/j.vaccine.2011.09.033. Epub 2011 Sep 20.
In this study surface coated poly-(ɛ-caprolactone) (PCL) nanoparticles with chitosan (CS) were developed as a carrier system for nasal immunization using recombinant Influenza A virus (A/California/07/2009) H1N1 hemagglutinin (HA) protein, for the induction of humoral, cellular and mucosal immunity. CS coated PCL (CS-PCL) nanoparticles were characterized in vitro for their percent yield, size, shape, entrapment efficiency, loading capacity and zeta potential. The in vitro release and antigen integrity were also evaluated. Particles were prepared by an emulsion-diffusion-solvent evaporation method. The coated cationic nanoparticles of average size 125.64±6.51 nm with a narrow size distribution (pdi: 0.185±0.032) and a positive surface charge (+22.88 mV) were obtained. HA antigen was efficiently entrapped in CS-PCL nanoparticles (entrapment efficiency 74.84±4.51%, loading capacity 14±2% (w/w)). The molecular weight and antigenicity of the entrapped HA was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. In vitro release study of antigen showed that about 66.47% of entrapped antigen was released within 63 days. The immune-stimulating activity was studied by measuring hemagglutination inhibition (HAI) titer, IgG, IgG1 and IgG2a titer, secretory IgA level in nasal and lung lavage (mucosal secretions) following nasal administration of modified CS-PCL nanoparticles in Balb/c mice and compared with soluble HA antigen administered intramuscular (IM) and with PCL (uncoated) nanoparticles administered intranasal (IN). The numbers of IFN-γ or IL-4 secreting cells in spleen homogenates were also measured 21 day after third immunization. Single IN or IM immunization with antigen-loaded CS-PCL nanoparticles resulted in strong HAI and total IgG responses. These responses were higher than those achieved after booster IM administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The IN administered antigen-CS-PCL nanoparticles induced higher immune responses compared to the other IN antigen formulations, and these responses were enhanced by IN booster vaccinations. Moreover, IM administered soluble HA antigen did not elicit s-IgA in mucosal secretions as it was induced and measured in the case of nasal administration of CS-PCL nanoparticles. In contrast to IM administered antigen CS-PCL nanoparticles induced a balanced Th1 and Th2 response. CS-PCL nanoparticles (cationic nanoparticles) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These findings demonstrate high potential of CS-PCL nanoparticles for their use as a carrier adjuvant for nasal administered influenza antigens.
在这项研究中，使用壳聚糖（CS）对表面涂覆的聚（ε-己内酯）（PCL）纳米颗粒进行了开发，作为使用重组甲型流感病毒（A/加利福尼亚/07/2009）H1N1血凝素（HA）蛋白进行鼻内免疫的载体系统，用于诱导体液、细胞和粘膜免疫。CS 涂覆的 PCL（CS-PCL）纳米颗粒在体外进行了产率、大小、形状、包封效率、载药量和 Zeta 电位的特征分析。还评估了体外释放和抗原完整性。通过乳液扩散溶剂蒸发法制备颗粒。获得了平均大小为 125.64±6.51nm 的带正电荷的阳离子纳米颗粒，具有较窄的粒径分布（pdi：0.185±0.032）和正表面电荷（+22.88mV）。HA 抗原有效地包封在 CS-PCL 纳米颗粒中（包封效率为 74.84±4.51%，载药量为 14±2%（w/w））。如聚丙烯酰胺凝胶电泳和 Western 印迹所示，包封的 HA 的分子量和抗原性得以维持。抗原的体外释放研究表明，约 66.47%的包封抗原在 63 天内释放。通过在 Balb/c 小鼠中鼻内给予修饰的 CS-PCL 纳米颗粒，并与肌肉内（IM）给予可溶性 HA 抗原和鼻内（IN）给予未涂覆的 PCL 纳米颗粒进行比较，研究了免疫刺激活性，并测量了血凝抑制（HAI）滴度、鼻和肺灌洗液（粘膜分泌物）中的 IgG、IgG1 和 IgG2a 滴度。在第三次免疫后 21 天，还测量了脾匀浆中 IFN-γ或 IL-4 分泌细胞的数量。单次 IN 或 IM 免疫接种载抗原的 CS-PCL 纳米颗粒可引起强烈的 HAI 和总 IgG 反应。这些反应高于加强 IM 给予亚单位抗原后的反应，而 IgG1/IgG2a 谱没有发生实质性变化。与其他 IN 抗原制剂相比，IN 给予的抗原-CS-PCL 纳米颗粒诱导了更高的免疫反应，并且通过 IN 加强疫苗接种增强了这些反应。此外，IM 给予的可溶性 HA 抗原不能在粘膜分泌物中诱导 s-IgA，因为在 CS-PCL 纳米颗粒鼻内给药的情况下诱导并测量了 s-IgA。与 IM 给予的抗原 CS-PCL 纳米颗粒相反，诱导了平衡的 Th1 和 Th2 反应。因此，阳离子 CS-PCL 纳米颗粒在鼻内给予时可引起体液（全身和粘膜）和细胞免疫反应。这些发现表明 CS-PCL 纳米颗粒在用作流感抗原鼻内给药的载体佐剂方面具有很高的潜力。
