Design and Utility of Novel Proteinaceous Biomaterials

目的1：凝胶化机理。从机理上讲，两种不同的模型可以描述导致凝胶化的事件，其早期步骤不同。机制1认为，单体肽首先折叠成离散的两亲性β-发夹，然后表面和侧面缔合形成原纤维。我们最初倾向于基于早期数据和描述发夹折叠的文献的机制1。然而，对淀粉样蛋白形成肽和内在无序蛋白的机制研究表明，早期步骤可能涉及胶束样寡聚体的形成（机制2）。在这里，未折叠肽的快速缔合由疏水性塌缩驱动以形成寡聚体，这可以起到增加肽的局部浓度并促进它们的排序以引发折叠和组装成富含β的原纤维的作用。任何一种机制都可能导致明确定义的原纤维簇的演变，我们在稍后的时间直接通过冷冻TEM观察到。先前的工作表明，单个簇包含悬垂的纤维末端，随着网络的发展，这些纤维末端生长并渗透到相邻的簇中。成簇的原纤维网络使整个样品体积膨胀并且溶液变成凝胶的确切时间是快速的（在1重量%肽下1分钟）并且是浓度依赖性的。在凝胶点之后，网络继续生长，填充空隙，以进一步硬化凝胶。低温透射电镜表明，最终的网络包含缠结并形成分支点的原纤维，两者都是有助于定义凝胶机械性能的物理交联。通过调节肽浓度或自组装速率，可以改变网络的网格尺寸（20-50 nm）。通常，更快的组装速率导致更多的交联、更小的网格尺寸和更硬的凝胶。关于药物递送，该范围的筛目尺寸类似于许多治疗性蛋白质的直径，因此影响它们从凝胶中的释放行为。目的2：多肽水凝胶的分子设计。我们不断设计新的肽，以完善我们对序列组成如何影响材料形成和性质的理解。先前，我们发现链数和链注册影响局部原纤维形态，并且双链对称β-发夹可再现地组装成具有一致形态的原纤维，其形成最适合于递送应用的机械上明确限定的凝胶。我们发现，发夹的亲水性表面上的残基组成的变化，降低电荷密度，促进折叠，组装和形成更硬的凝胶。因此，最小电荷的肽在较低的溶液pH值、离子强度和温度下形成凝胶。此外，发夹的亲水面可以容纳几乎任何天然或非天然的残基，而不影响原纤维形成和凝胶化。目的3：肽纤维及其网络的分子水平结构。以前的工作给了我们一个局部原纤维结构的理解，主要是基于来自TEM，AFM和SANS数据的模型，但没有关于组装中肽的确切分子排列的细节。此外，我们几乎没有直接报道凝胶的网络级结构的数据;原纤维是如何结合形成网络的？它们是否只是简单地缠结在一起，是否形成分支（正如我们所提出的），以及在凝胶化机制早期形成的低聚物的残余物是否持续存在于网络中？目的4：研究微纤维网络与微囊化药物之间的物理相互作用。小分子、蛋白质、RNA、DNA和细胞可以通过将未折叠肽的水溶液加入到触发缓冲液中的治疗溶液中而直接包封在凝胶网络中。治疗和原纤维网络之间的物理相互作用决定了每种治疗类型在其封装期间如何在凝胶内分配，并影响其释放的速率。我们的工作表明，这些过程的规则根据治疗类型而不同。目的5：开发具有临床应用前景的多肽材料。我们的基础科学实验室期待对灵感的临床，导致几个应用项目，包括间皮瘤，组织移植和免疫调节。

Aim 1: The mechanism of gelation. Mechanistically, two distinct models can describe the events leading to gelation that differ in their early steps. Mechanism 1 asserts that monomeric peptides first fold into discreet amphiphilic beta-hairpins that then associate facially and laterally to form fibrils. We initially favored mechanism 1 based on early data and literature describing hairpin folding. However, mechanistic studies of amyloid forming peptides and intrinsically disordered proteins suggest that the early steps may involve the formation of micelle-like oligomers (mechanism 2). Here, the rapid association of unfolded peptides is driven by hydrophobic collapse to form oligomers, which may act to increase the local concentration of peptide and facilitate their ordering to initiate folding and assembly into beta-rich fibrils. Either mechanism could lead to the evolution of clusters of well-defined fibrils, which we directly observe by cryo-TEM at later times. Prior work showed that individual clusters contain dangling fibril ends that grow and interpenetrate neighboring clusters as the network evolves. The exact time at which the clustered fibril network percolates the entire sample volume and the solution becomes a gel is fast (1min at 1 wt% peptide) and concentration dependent. After the gel point, the network continues to grow, filling the voids, to further rigidify the gel. Cryo-TEM suggests that the final network contains fibrils that entangle and form branch-points, both are physical crosslinks that help define the gel's mechanical properties. The mesh size of the network can be varied (20-50 nm) by adjusting the peptide concentration or the rate of self-assembly. In general, faster rates of assembly lead to more crosslinks, smaller mesh sizes, and stiffer gels. With respect to drug delivery, this range of mesh sizes is similar to the diameters of many therapeutic proteins and thus, influences their release behavior from the gel. Aim 2: Molecular design of peptide hydrogels. We continuously design new peptides to refine our understanding of how sequence composition affects material formation and properties. Previously, we found that strand number and strand registry influence local fibril morphology, and that two-stranded symmetrical beta-hairpins reproducibly assemble into fibrils having consistent morphology that form mechanically well-defined gels best suited for delivery applications. We found that changes in residue composition on the hairpin's hydrophilic face that reduce charge density promotes folding, assembly, and the formation of stiffer gels. Thus, minimally charged peptides form gels at lower values of solution pH, ionic strength and temperature. Further, the hairpin's hydrophilic face can accommodate nearly any natural or non-natural residue without affecting fibril formation and gelation. Aim 3: Molecular-level structure of peptide fibrils and their networks. Previous work gave us an understanding of the local fibril structure largely based on models derived from TEM, AFM, and SANS data, but no detail with respect to the exact molecular arrangement of peptides in the assembly. Further, we have little data directly reporting on the network-level structure of the gel; how do the fibrils associate to form a network? Do they simply entangle, do they form branches (as we have proposed), and do remnants of oligomers formed early in the gelation mechanism persist in the network? Aim 4: Study the physical interactions between fibrillar network and encapsulated therapy. Small molecules, proteins, RNA, DNA and cells can be directly encapsulated in the gel network by adding a solution of unfolded peptide in water to a solution of therapy in triggering buffer. Physical interactions between the therapy and the fibril network determine how each therapy type partitions within the gel during its encapsulation, and influences the rate at which it is released. Our work suggests that the rules governing these processes differ according to therapy type. Aim 5: Develop peptide materials towards clinical applications. Our basic science lab looks towards the clinic for inspiration, leading to several applied projects, including mesothelioma, tissue transplantation and immune modulation.