Biophysical guided development of peptide-protein gels for 3D cell culture

生物工程三维（3D）基质在再生医学和细胞生物学，特别是肿瘤生长研究方面具有相当大的前景。在后一个领域，有必要改进三维（3D）癌症模型，以在生物相关的受控环境中测试对新疗法的反应。目前使用的2D体外细胞模型无法复制肿瘤的许多关键特征，包括细胞-基质相互作用和基质刚度的变化，并且需要更能代表肿瘤微环境（TME）的模型来更快地确定治疗策略。基于自组装肽基材料的基质最近显示出相当大的前景，因为它们可以被编程组装成代表细胞外基质（ECM）的定义良好的纳米结构，并且还可以优化以实现关键特性的可调性，例如机械性能。在这方面，Mata小组最近的研究表明，由肽两亲体（PAs）与蛋白质共同组装形成的凝胶具有开发生物学相关的3D细胞培养物作为卵巢癌和胰腺癌模型的潜力。尽管这些材料具有广泛的应用潜力，但为确定的疾病模型优化/调整这些材料是一个漫长的过程；有必要制定更迅速和合理的分析办法来支持这一进程。虽然测试生物功能相对简单，但分析其他关键特征（如基质纳米结构和机械性能）却面临着更大的挑战，特别是在水合环境中。生物物理技术，如原子力显微镜（AFM），在这方面显示出相当大的前景，因为它们具有独特的能力，以纳米尺度的分辨率探测基质材料的结构和机械性能3。该项目旨在开发一个生物物理“工具箱”，以指导开发和更快速地识别/预测理想应用的最佳肽蛋白凝胶特性。该项目将以Allen教授和Williams教授在AFM和相关生物物理方法应用于生物材料分析和开发方面的专业知识为基础，特别是与肽/蛋白质自组装和聚集相关的技术。此外，它还将建立在Allen和Mata最近开展的工作的基础上，展示基于原子力显微镜的杨氏模量测量的概念验证。“工具箱”最终将包括一个或多个生物物理分析，这将有助于快速识别最佳条件，以提供特定肽-蛋白水凝胶系统所需的特性。为了实现这一目标，初始研究（1-18个月）将侧重于现有的凝胶系统，并将建立初步的基于AFM的分析，例如成像研究，以探索水凝胶结构和材料特性（刚度、粘弹性）的测量/变化。将分析数据与现有数据以及通过组织培养中材料特性和性能的大量分析获得的数据进行比较，将允许对AFM方法进行初步评估，以提供所需的信息（12-24个月）。最初的重点将放在表征良好的肽蛋白水凝胶和简单实验变量（例如介质和缓冲液）的影响上，然后转移到更复杂的不太明确的基质，包括其他生物分子和/或细胞。在最初的“评估”阶段之后，将进行实验以迭代测试和完善生物物理方法（18-36个月），并最终能够评估其优化和预测选定应用程序的肽水凝胶性能的能力（36-42个月）。

Bioengineered three-dimensional (3D) matrices hold considerable promise for regenerative medicine and cellular biology, and in particular for investigations of tumour growth. In this latter area, there is a need for improved three-dimensional (3D) cancer models to test responses to new treatments in a biologicallyrelevant, controlled environment. The 2D in-vitro cell models currently used fail to replicate many key features of tumours, including cell-matrix interactions and changes in matrix stiffness, and models that are more representative of the tumour microenvironment (TME) are needed to identify therapeutic strategiesmore quickly.Matrices based on self-assembling peptide-based materials have shown considerable recent promise, as they can be programmed to assemble into well-defined nanostructures representative of the extracellular matrix (ECM), and also optimized to enable tunability of key characteristics, such as mechanical properties. In this regard, recent research from the Mata group has demonstrated the potential of gels formed from peptide amphiphiles (PAs) co-assembled with proteins, to develop biologically relevant 3D cell cultures as models for ovarian1 and pancreatic cancer2.Despite their potential for a wide range of applications, the optimization/adaptation of such materials for defined disease models is however a lengthy process; there is a need to develop more rapid and rational analytical approaches to support this process. While it is relatively straightforward to test for biofunctionality, it is the analysis of other key characteristics such as matrix nanostructure and mechanical properties that present a significantly greater challenge, particularly in a hydrated environment. Biophysical techniques, such as atomic force microscopy (AFM), have shown considerable promise in this regard due theirunique ability to probe the structure and mechanical properties of matrix materials, with nanometre scale resolution3. This project will aim to develop a biophysical 'tool-box' to guide the development and more rapid identification/prediction of optimal peptide-protein gel properties for desired applications. The project will build on the expertise of Prof. Allen and Williams in the application of AFM and related biophysical approachesfor biomaterials analysis and development, in particular that related to peptide/protein self-assembly and aggregation. It will in addition build on very recent work initiated between Allen and Mata, demonstrating proof-of-concept AFM based measurements of Young's Modulus of his hydrogel materials.The 'tool box' will ultimately comprise one or more biophysical assays which will facilitate rapid identification of optimal conditions to provide the characteristics desired in a particular peptide-protein hydrogel system. To achieve this, initial studies (months 1-18) will focus on existing gel systems and will establish preliminary AFM based assays e.g. imaging studies to explore hydrogel structure and measurement/variation in material properties(stiffness, viscoelasticity). Comparison of data from the assays, with existing data and that obtained through bulk assays of material properties and performance in tissue culture, will allow initial evaluation of the AFM approaches to provide the required information (months 12-24). Initially the focus will be on well characterized peptide-protein hydrogels and the impact of simple experimental variables (e.g. media and buffers), before moving to more complex less well defined matricesincluding other biomolecules and/or cells. After the initial 'evaluation' phase, experiments will be performed to iteratively test and refine the biophysical approach(es) (months 18-36) and ultimately to enable evaluation of their ability to optimize and predict the performance of peptide-hydrogels for chosen applications (months 36-42).