Carrillo-Conde Brenda R, Roychoudhury Rajarshi, Chavez-Santoscoy Ana V, Narasimhan Balaji, Pohl Nicola L B
Department of Chemical and Biological Engineering, Iowa State University, IA, USA.
J Vis Exp. 2012 Jul 6(65):3967. doi: 10.3791/3967.
Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity. Several biodegradable polymers have been studied as vaccine delivery vehicles(1); in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses. The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices. The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses. C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods. Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).
必须采用涉及材料设计、纳米技术、化学和免疫学等领域的跨学科方法来合理设计有效的疫苗载体。基于纳米颗粒的平台可以延长疫苗抗原的持久性，这可能会提高疫苗的免疫原性。几种可生物降解的聚合物已被研究用作疫苗递送载体(1)；特别是，聚酸酐颗粒已证明能够持续释放稳定的蛋白质抗原，并激活抗原呈递细胞和调节免疫反应。这些疫苗载体的分子设计需要整合聚合物性质的合理选择以及合适靶向剂的掺入。靶向配体和功能化颗粒的高通量自动化制备是一种强大的工具，它将增强研究广泛性质的能力，并将导致可重复的疫苗递送装置的设计。已证明添加能够被免疫细胞上特定受体识别的靶向配体可调节和定制免疫反应。C型凝集素受体(CLRs)是识别病原体表面存在的碳水化合物的模式识别受体(PRRs)。通过CLRs刺激免疫细胞可增强抗原的内化以及随后的呈递，以进一步激活T细胞。因此，碳水化合物分子在免疫反应研究中起着重要作用；然而，这些生物分子的使用常常受到结构明确且纯净的碳水化合物可用性不足的困扰。基于迭代溶液相反应的自动化平台能够使用比传统固相方法显著更少的构建模块量来快速且可控地合成这些合成具有挑战性的分子。在此，我们报告一种用于寡糖自动化溶液相合成的方案，例如基于甘露糖的靶向配体，并采用氟固相萃取进行中间体纯化。在开发出制备基于碳水化合物的靶向剂的自动化方法之后，我们描述了使用如前所述由LabVIEW操作的自动化机器人装置将它们连接到聚酸酐纳米颗粒表面的方法。用碳水化合物进行表面功能化已显示出在靶向CLRs方面的功效，并且提高制造方法的通量以揭示与多参数系统相关的复杂性将具有很大价值(图1a)
