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，特别是在肿瘤生长的研究中具有相当大的前景。在后一个领域，需要改进的三维(3D)癌症模型，以在与生物相关的受控环境中测试对新治疗的反应。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硬度的变化，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。基于自组装多肽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)的明确纳米结构，并且还可以优化以实现关键特性的可调，例如机械性能。在这方面，MATA小组最近的研究表明，由多肽两亲分子(PA)与蛋白质共同组装形成的凝胶有潜力开发具有生物学意义的3D细胞培养，作为卵巢癌和胰腺癌的模型2。尽管它们具有广泛的应用潜力，但这种材料的优化/适应定义的疾病模型是一个漫长的过程；需要开发更快速和合理的分析方法来支持这一过程。虽然生物功能的测试相对简单，但对基质纳米结构和机械性能等其他关键特征的分析提出了更大的挑战，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，因为它们具有以纳米级分辨率探测基质材料的结构和机械性能的独特能力3。该项目的目标是开发一种生物物理‘工具箱’，以指导开发和更快速地识别/预测预期应用的最佳多肽-蛋白质凝胶特性。该项目将建立在艾伦教授和威廉姆斯教授在应用原子力显微镜和相关生物物理方法进行生物材料分析和开发方面的专业知识的基础上，特别是与多肽/蛋白质自组装和聚集有关的领域。此外，它还将建立在Allen和Mata之间最新开始的工作基础上，展示基于概念验证AFM的水凝胶材料杨氏模数的测量。“工具箱”最终将包括一种或多种生物物理分析，这将有助于快速识别最佳条件，以提供特定多肽-蛋白质水凝胶体系所需的特性。为了实现这一目标，初步研究(1-18个月)将重点放在现有的凝胶系统上，并将建立基于AFM的初步分析，例如探索水凝胶结构和材料特性(硬度、粘弹性)测量/变化的成像研究。将化验数据与现有数据和通过组织培养中材料特性和性能的批量分析获得的数据进行比较，将能够对AFM方法进行初步评估，以提供所需的信息(12-24个月)。最初，重点将放在具有良好特性的多肽-蛋白质水凝胶和简单的实验变量(例如，介质和缓冲液)的影响上，然后转移到更复杂、定义较少的基质，包括其他生物分子和/或细胞。在最初的“评估”阶段之后，将进行实验以反复测试和改进生物物理方法(18-36个月)，并最终能够评估它们针对选定应用优化和预测多肽-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).