Petersen Latrisha K, Chavez-Santoscoy Ana V, Narasimhan Balaji
Department of Chemical and Biological Engineering, Iowa State University, Iowa, USA.
J Vis Exp. 2012 Sep 6(67):3882. doi: 10.3791/3882.
Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides(1). This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously(1). This combinatorial platform has been validated with conventional methods(2) and the polyanhydride film and nanoparticle libraries have been characterized with (1)H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion(1-11). The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.
聚酸酐是一类具有出色生物相容性和药物递送能力的生物材料。虽然它们已经通过传统的一次一个样品的合成技术进行了广泛研究，但最近开发了一种高通量方法，能够合成和测试大量聚酸酐文库(1)。这将有助于更高效地优化和设计这些用于药物和疫苗递送应用的生物材料。本工作中的方法描述了可生物降解聚酸酐薄膜和纳米颗粒文库的组合合成以及从这些文库中释放蛋白质的高通量检测。在这种机器人操作方法中（图1），线性致动器和注射泵由LabVIEW控制，这实现了免手动的自动化协议，消除了用户误差。此外，该方法能够快速制造微尺度聚合物文库，减小批量大小，同时产生多变量聚合物系统。这种聚合物合成的组合方法有助于在与传统合成一种聚合物相同的时间内合成多达15种不同的聚合物。此外，在将聚合物文库溶解于溶剂中并沉淀到非溶剂中（用于纳米颗粒）或通过真空干燥（用于薄膜）后，组合聚合物文库可以制成空白或负载蛋白质的几何形状，包括薄膜或纳米颗粒。在将荧光染料偶联的蛋白质加载到聚合物文库中后，可以使用如前所述的基于荧光的检测方法（图2和3）高通量评估蛋白质释放动力学(1)。这个组合平台已经通过传统方法进行了验证(2)，并且聚酸酐薄膜和纳米颗粒文库已经通过(1)H NMR和FTIR进行了表征。这些文库已经针对蛋白质释放动力学、稳定性和抗原性；体外细胞毒性、细胞因子产生、表面标志物表达、粘附、增殖和分化；以及体内生物分布和粘膜粘附进行了筛选(1-11)。本文开发的组合方法能够进行高通量聚合物合成以及负载蛋白质的纳米颗粒和薄膜文库的制造，进而可以在体外和体内进行筛选以优化生物材料性能。
