Design and Utility of Novel Proteinaceous Biomaterials

新型蛋白质生物材料的设计与应用

• 批准号: 10486809
• 负责人: Joel Schneider
• 金额: $ 114.85万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486809
• 关键词: Affect; Amyloid; Basic Science; Behavior; Biocompatible Materials; Breathing; Buffers; Caliber; Cells; Characteristics; Charge; Clinic; DNA; Data; Disease; Drug Delivery Systems; Encapsulated; Ensure; Environment; Event; Evolution; Face; Gel; Hydrogels; Hydrophobicity; Implant; Individual; Injectable; Ionic Strengths; Lead; Liquid substance; Literature; Mechanics; Mesothelioma; Micelles; Modeling; Molecular; Morphology; Nucleic Acids; Peptides; Pharmaceutical Preparations; Process; Property; Proteins; RNA; Recovery; Registries; Reporting; Sampling; Sol-Gel Phase Transitions; Solid; Structure; Syringes; Temperature; Therapeutic; Thinness; Time; Tissue Transplantation; Tissues; Water; Work; amphiphilicity; base; clinical application; crosslink; density; design; hydrophilicity; immunoregulation; mechanical properties; nanoscale; novel; parenteral administration; peptide structure; response; self assembly; small molecule; solid state; therapeutic protein

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.

目标1：胶凝的机理。从机械上讲，两个不同的模型可以描述导致凝胶化的事件，这些事件在早期步骤中有所不同。机制1断言，单体肽首先折叠成谨慎的两亲性β-然后将其面部和侧向缔合以形成原纤维。我们最初根据早期数据和描述发夹折叠的文献来偏爱机制1。然而，对淀粉样蛋白和内在无序蛋白质的淀粉样蛋白的机理研究表明，早期步骤可能涉及胶束样寡聚物的形成（机制2）。在这里，展开的肽的快速关联是由疏水性塌陷驱动的，形成低聚物，这可能起到增加肽的局部浓度，并促进其订购以将折叠和组装成富含β的原纤维。两种机制都可以导致定义明确的原纤维簇的演变，我们以后通过冷冻-TEM直接观察到。先前的工作表明，随着网络的发展，各个簇包含悬挂的原纤维末端，它们会生长和互二渗透的相邻簇。聚集的原纤维网络渗透整个样品体积和溶液变为凝胶的确切时间很快（1 wt％肽时1分钟）和浓度取决于。凝胶点之后，网络继续增长，填充空隙，以进一步固化凝胶。 Cryo-TEM建议最终的网络包含缠结并形成支链的原纤维，这两者都是物理交联，有助于定义凝胶的机械性能。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，组装速率更快会导致更多的交联，较小的网格尺寸和更硬的凝胶。关于药物递送，这种网格大小范围与许多治疗蛋白的直径相似，因此影响了它们从凝胶中的释放行为。目标2：肽水凝胶的分子设计。我们不断设计新肽，以完善对序列组成如何影响材料形成和特性的理解。以前，我们发现链数和链登记处会影响局部原纤维形态，并且两链的对称β脱发可重复地聚集成具有一致的形态的原纤维，形成机械定义良好的凝胶最适合递送应用。我们发现，发夹的亲水性面部残留物组成的变化会降低电荷密度可促进折叠，组装和僵硬凝胶的形成。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，发夹的亲水性脸几乎可以容纳任何天然或非天然残留物，而不会影响原纤维形成和凝胶化。 AIM 3：肽原纤维及其网络的分子水平结构。先前的工作使我们了解了局部原纤维结构的理解，这主要基于从TEM，AFM和SANS数据中得出的模型，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告凝胶网络级结构的数据。原纤维如何建立网络？它们是否只是纠缠，它们是否形成分支（正如我们提出的那样），并且在网络中持续存在的凝胶机制早期形成的低聚物的残留物吗？目标4：研究纤维网络和封装治疗之间的身体相互作用。小分子，蛋白质，RNA，DNA和细胞可以通过在触发缓冲液中的治疗溶液中添加到水中的溶液中，直接封装在凝胶网络中。治疗与原纤维网络之间的物理相互作用决定了凝胶封装过程中的每种治疗类型分区的方式，并影响释放的速率。我们的工作表明，根据治疗类型，管理这些过程的规则有所不同。目标5：开发肽材料以临床应用。我们的基础科学实验室探索诊所寻求灵感，导致多个应用项目，包括间皮瘤，组织移植和免疫调节。