Vila Ana, Sánchez Alejandro, Janes Kevin, Behrens Isabel, Kissel Thomas, Vila Jato José Luis, Alonso María José
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, Santiago de Compostela, Spain.
Eur J Pharm Biopharm. 2004 Jan;57(1):123-31. doi: 10.1016/j.ejpb.2003.09.006.
High molecular weight (Mw) chitosan (CS) solutions have already been proposed as vehicles for nasal immunization. The aim of the present work was to investigate the potential utility of low Mw CS in the form of nanoparticles as new long-term nasal vaccine delivery vehicles. For this purpose, CS of low Mws (23 and 38 kDa) was obtained previously by a depolymerization process of the commercially available CS (70 kDa). Tetanus toxoid (TT), used as a model antigen, was entrapped within CS nanoparticles by an ionic cross-linking technique. TT-loaded nanoparticles were first characterized for their size, electrical charge, loading efficiency and in vitro release of antigenically active toxoid. The nanoparticles were then administered intranasally to conscious mice in order to study their feasibility as vaccine carriers. CS nanoparticles were also labeled with FITC-BSA and their interaction with the rat nasal mucosa examined by confocal laser scanning microcopy (CLSM). Irrespective of the CS Mw, the nanoparticles were in the 350 nm size range, and exhibited a positive electrical charge (+40 mV) and associated TT quite efficiently (loading efficiency: 50-60%). In vitro release studies showed an initial burst followed by an extended release of antigenically active toxoid. Following intranasal administration, TT-loaded nanoparticles elicited an increasing and long-lasting humoral immune response (IgG concentrations) as compared to the fluid vaccine. Similarly, the mucosal response (IgA levels) at 6 months post-administration of TT-loaded CS nanoparticles was significantly higher than that obtained for the fluid vaccine. The CLSM images indicated that CS nanoparticles can cross the nasal epithelia and, hence, transport the associated antigen. Interestingly, the ability of these nanoparticles to provide improved access to the associated antigen to the immune system was not significantly affected by the CS Mw. Indeed, high and long-lasting responses could be obtained using low Mw CS molecules. Furthermore, the response was not influenced by the CS dose (70-200 microg), achieving a significant response for a very low CS dose. In conclusion, nanoparticles made of low Mw CS are promising carriers for nasal vaccine delivery.
高分子量（Mw）壳聚糖（CS）溶液已被提议作为鼻腔免疫的载体。本研究的目的是研究低Mw的CS以纳米颗粒形式作为新型长期鼻腔疫苗递送载体的潜在效用。为此，先前通过对市售CS（70 kDa）进行解聚过程获得了低Mw（23和38 kDa）的CS。破伤风类毒素（TT）用作模型抗原，通过离子交联技术包裹在CS纳米颗粒中。首先对负载TT的纳米颗粒进行尺寸、电荷、负载效率和抗原活性类毒素的体外释放表征。然后将纳米颗粒经鼻内给予清醒小鼠，以研究其作为疫苗载体的可行性。CS纳米颗粒还用FITC-BSA标记，并通过共聚焦激光扫描显微镜（CLSM）检查其与大鼠鼻黏膜的相互作用。无论CS的Mw如何，纳米颗粒的尺寸都在350 nm范围内，带正电荷（+40 mV），并且相当有效地结合了TT（负载效率：50-60%）。体外释放研究表明，最初有一个突释，随后是抗原活性类毒素的持续释放。经鼻内给药后，与液体疫苗相比，负载TT的纳米颗粒引发了增强且持久的体液免疫反应（IgG浓度）。同样，在给予负载TT的CS纳米颗粒6个月后，黏膜反应（IgA水平）明显高于液体疫苗。CLSM图像表明CS纳米颗粒可以穿过鼻上皮，从而运输相关抗原。有趣的是，这些纳米颗粒为免疫系统提供更好的相关抗原接触机会的能力并未受到CS Mw的显著影响。事实上，使用低Mw的CS分子可以获得高且持久的反应。此外，反应不受CS剂量（70-200μg）的影响，在非常低的CS剂量下也能产生显著反应。总之，低Mw CS制成的纳米颗粒是鼻腔疫苗递送的有前途的载体。
