Electrostatically triggered hydrophobic self-assembly of protein hydrogels

描述（由申请人提供）：3D组织工程支架深层缺氧限制了包埋细胞的活力，导致局部坏死。新型生物材料的开发能够以生理方式将氧气局部输送到这些细胞中，将减轻这些细胞群中的氧气消耗和坏死。我们的实验室最近一直在研究位置形成的蛋白质水凝胶和球状蛋白质自组装的新机制，利用静电荷和疏水相互作用的变化在生理温度下形成水凝胶。在适当控制的pH条件下，部分变性的蛋白质将保持必要的二级结构，这将是功能所需的。本研究将利用对诱导蛋白自组装的新认识，开发一种新的基于白蛋白/肌红蛋白的支架，该支架可以根据邻近细胞的需要输送氧气。白蛋白已被广泛表征，可通过从血液中分离或通过重组DNA技术获得，具有生物相容性，并已用于诸如血管移植等医疗器械，而肌红蛋白已被充分表征，且易于获得携带氧气的球状蛋白。为了合理地设计用于此目的的水凝胶，将采用理论和实验相结合的方法来评估这些新材料，同时为理解和设计更好的蛋白质水凝胶材料提供基本框架。基于初步结果，我们怀疑可以用作ph诱导水凝胶形成构建块的蛋白质的类型和/或大小存在根本限制。为了确定这些边界，我们将a)执行一系列系统的原子分子动力学模拟来确定pH变性白蛋白和肌红蛋白的结构，b)从原子模拟中建立粗粒度模型，并执行布朗动力学模拟来研究白蛋白和肌红蛋白的凝胶形成及其力学特性，c)实验研究蛋白质凝胶的形成。测量它们的机械性能并测试模拟的预测，使用这些结果对模型进行微调，d)预测和制造具有功能性肌红蛋白的变性白蛋白凝胶的最佳条件，e)表征合并肌红蛋白的稳定性，f)评估水凝胶的氧结合和释放动力学。这项研究是解决问题的理论和实验方法的真正整合：理论原子和粗粒度蛋白质表示与卓越的计算能力相结合，目前允许模拟生物相关尺寸和时间尺度的系统，而先进的成像和光谱技术可以直接比较模拟和实验。可靠和预测模型的发展将使模拟能够预测和指导改进生物材料的设计。本提案中开发的技术为设计用于伤口愈合和透析等领域的其他功能蛋白质水凝胶支架奠定了基础。

DESCRIPTION (provided by applicant): Oxygen depletion deep within 3D tissue engineered scaffolds limits the viability of embedded cells, causing localized necrosis. The development of new biomaterials that can deliver oxygen locally to these cells in a physiological manner will mitigate oxygen depletion and necrosis in these cell populations. Our laboratories have recently been investigating in situ-forming protein hydrogels and novel mechanisms for globular protein self-assembly that utilize changes in electrostatic charges and hydrophobic interactions to form a hydrogel at physiological temperatures. Under the proper controlled pH conditions, the partially denatured proteins will maintain the necessary secondary structure that will be needed for function. This proposal will utilize this new understanding of induced protein self-assembly to develop a new albumin/myoglobin-based scaffold that can deliver oxygen on demand by neighboring cells. Albumin has been extensively characterized, is readily available via isolation from blood or via recombinant DNA technology, biocompatible, and has been used in medical devices such as vascular grafts while myoglobin is well characterized and readily available oxygen carrying globular protein. In order to rationally design the hydrogel for this purpose a combination of theoretical and experimental approaches will be used to evaluate these new materials while providing fundamental framework for understanding and designing better protein hydrogel materials. Based on preliminary results, we suspect there are fundamental limitations to the types and/or size of proteins that can be used as pH-induced hydrogel forming building blocks. To delineate those boundaries, we will a) perform a series of systematic atomistic molecular dynamics simulations to determine the structure of the pH denatured albumin and myoglobin, b) build coarse-grained models from the atomistic simulations, and perform Brownian Dynamics simulations to study gel formation and their mechanical properties for albumin and myoglobin, c) experimentally study the formation of the protein gels, measure their mechanical properties and test the predictions from the simulations, use these results for fine tuning of the model, d) predict and fabricate the optimal conditions for formation of denatured albumin based gels with functional myoglobin proteins, e) characterize the stability of incorporated myoglobin, and f) assess oxygen binding and release kinetics from the hydrogel. This research is a true integration of theoretical and experimental approaches to solve a problem: theoretical atomistic and coarse-grained protein representations in combination with superior computational power currently allow simulation of systems of biologically relevant size and timescale while advanced imaging and spectroscopic techniques enable direct comparison between simulations and experiments. The development of reliable and predictive models will enable the simulations to predict and guide the design of improved biomaterials. The techniques developed in this proposal lay the foundation for the design of additional functional protein hydrogel scaffolds for applications in areas such as wound healing and dialysis.