Design and Utility of Novel Proteinaceous Biomaterials

目的1：凝胶化的机理。从机制上讲，两种不同的模型可以描述导致凝胶形成的事件，但它们的早期步骤不同。机制1认为，单体肽首先折叠成离散的两亲性-发夹，然后在表面和侧面结合形成原纤维。我们最初基于早期数据和描述发夹折叠的文献支持机制1。然而，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低聚物的形成（机制2）。在这里，未折叠肽的快速结合是由疏水坍塌驱动的，形成低聚物，这可能会增加肽的局部浓度，并促进它们的排序，从而启动折叠和组装成富含β的原纤维。任何一种机制都可能导致明确定义的原纤维簇的进化，这是我们后来通过冷冻透射电镜直接观察到的。先前的研究表明，单个簇包含悬垂的纤维末端，随着网络的发展，这些末端会生长并穿透相邻的簇。簇状纤维网络渗透整个样品体积并使溶液变成凝胶的确切时间是快速的（在1wt %的肽下1min），并且与浓度有关。凝胶点后，网状物继续生长，填充空隙，使凝胶进一步硬化。低温透射电镜显示，最终的网状结构包含缠绕在一起并形成分支点的原纤维，这两者都是物理交联，有助于确定凝胶的机械性能。通过调整肽浓度或自组装速率，可以改变网络的网状尺寸（20-50 nm）。一般来说，更快的组装速度导致更多的交联，更小的网格尺寸和更硬的凝胶。在药物输送方面，这个网孔大小的范围与许多治疗性蛋白质的直径相似，因此会影响它们从凝胶中的释放行为。目的2：肽水凝胶的分子设计。我们不断设计新的多肽，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会影响局部纤维形态，并且两股对称的β -发夹可重复地组装成具有一致形态的原纤维，形成最适合递送应用的机械定义明确的凝胶。我们发现发夹亲水性面上的残基组成的变化会降低电荷密度，从而促进折叠、组装和形成更硬的凝胶。因此，在较低的溶液pH值、离子强度和温度下，带最少电荷的肽形成凝胶。此外，发夹的亲水表面可以容纳几乎任何天然或非天然的残留物，而不会影响纤维的形成和凝胶化。目的3：肽原纤维及其网络的分子水平结构。先前的工作使我们对局部纤维结构的理解主要基于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一个网络的？它们是简单地纠缠在一起，还是形成分支（正如我们所提出的那样），以及在凝胶化机制早期形成的低聚物残留物是否会在网络中持续存在？目的4：研究纤维网络与包封治疗之间的物理相互作用。小分子、蛋白质、RNA、DNA和细胞可以直接包裹在凝胶网络中，只需将未折叠的肽溶液加入到触发缓冲液中的治疗溶液中。疗法和原纤维网络之间的物理相互作用决定了每种疗法在凝胶包封过程中如何在凝胶内分区，并影响其释放的速度。我们的研究表明，控制这些过程的规则因治疗类型而异。目标5：开发面向临床应用的多肽材料。我们的基础科学实验室从临床中寻找灵感，导致了几个应用项目，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.