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: 9153858
• 负责人: Joel Schneider
• 金额: $ 96.89万
• 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/9153858
• 关键词: Actins; Adopted; Affect; Anastomosis - action; Animals; Behavior; Binding; Biocompatible Materials; Blood Vessels; Buffers; Caliber; Catheters; Cell Differentiation process; Cell Proliferation; Cell Survival; Cells; Characteristics; Chemistry; Collaborations; Complex; Curcumin; Data; Dependence; Disease; Encapsulated; Epitopes; Face; Future; Gel; Hand; Hydrogels; Hydrogen Bonding; Hydrophobicity; Immune; Immune system; Immunologist; Injectable; Interleukin-7; Ligation; Limb structure; Literature; Lymphocyte; Lysine; Mammalian Cell; Measurement; Mechanics; Methods; Modeling; Modification; Molecular; Morphology; Operative Surgical Procedures; Organ Transplantation; Peptide Fragments; Peptides; Performance; Peripheral; Physicians; Plastic Surgical Procedures; Polymers; Population; Positioning Attribute; Proline; Property; Proteins; Publications; RGD (sequence); Rattus; Recovery; Registries; Regulation; Reporting; Research Design; Rheology; Role; Side; Site; Solutions; Structural Biologist; Structure; Surgical Models; Surgical sutures; Syringes; System; T-Cell Development; T-Lymphocyte; Temperature; Therapeutic; Threonine; Tissue Engineering; Tissues; Transplantation; Valine; Water; Work; base; beta pleated sheet; biomineralization; biophysical model; cell behavior; crosslink; cytokine; design; functional group; immunoregulation; macromolecule; medulloblastoma; novel; peptide structure; physical property; protein aminoacid sequence; regenerative therapy; scaffold; self assembly; small molecule; theories

Aim 1: Design peptides that enable triggered hydrogelation Accomplishments: We have designed and studied well over a hundred peptides to gain an understanding of how sequence modulates folding, assembly and resultant material properties. Much effort has been dedicated to understanding the role of distinct structural perturbations on gelation as outlined below. 1. Strand number and registry. We assessed the effect of strand number on self-assembly by preparing single strand peptides and three-stranded beta-sheets for comparison with the two-stranded hairpin. We found that single strand peptides self-assemble to form weak gels composed of fibrils having heterogenous morphologies. 2. Turn type. In proteins, turns are responsible for chain reversal, help define the twist of sheets, and in some cases, nucleate folding. We designed the high propensity type II' (-VDPPT-) turn in MAX1 to include a beta-branched valine at position i to enforce a trans proline bond at the following residue, a -DPLP- unit at the central i+1 and i+2 positions to adopt dihedral angles similar to type II' turn in proteins, and a threonine at the i+3 position to form a side-chain/main chain H-bond to the ith carbonyl to stabilize the turn. We examined the influence of turn type on folding, assembly and gel mechanical properties by replacing this turn with canonical four-residue beta-turns and five residue [type I+G1 beta-buldge] sequences found in the literature. We found that the inherent folding propensity of each turn influenced the rate of hairpin folding and assembly, and that each turn type was capable of reversing the chain direction where intended, resulting in well-defined fibrils of uniform diameter. Importantly, rheology showed that turn type also influenced hydrogel mechanical rigidity. Diproline motifs within the four residue turns, and a five-residue [type I+G1 beta-buldge] sequence (VPDGT) containing a single proline, offered the stiffest gels of those studied. We are currently deriving a biophysical model to explain the dependence of turn type on gel network stiffness. 3. Perturbations to the hydrophobic face of the hairpin. Thermally-induced folding and assembly of our hairpins is driven by the hydrophobic effect, which is temperature dependent. We studied how hydrophobicity and side-chain identity influences the temperature-dependent folding and assembly of the hairpin, as well as hydrogel rheological properties. 4. Perturbations to the hydrophilic face of the hairpin. We have systematically studied how residue composition of the hydrophilic face of the hairpin affects folding, assembly and material properties. 5. Ligation of peptide epitopes and other functional groups. The function of the gels can be enhanced by covalently incorporating peptide epitopes and other chemistries into the fibrillar network via modification of the self-assembling hairpin. In general, the hairpin scaffold is forgiving of alterations to its sequence. Moieties can be incorporated at its N- and C-termini, as well as from its hydrophilic face by functionalizing the lysine side chains or incorporating non-natural residues; modifications at these regions minimally effect folding, assembly and bulk material properties. Changes to the hairpin's hydrophobic face are less forgiving. To date, we have incorporated various cell-binding epitopes (RGD, etc...) and peptide fragments capable of directing biomineralization. Smaller organic functionalities can also be incorporated, such as sorbamide groups from lysine side chains that allow photopolmerization of the fibrillar network. Taken together, our fundamental studies exploring peptide sequence-material relationships establish a continuously evolving basis set of design rules that allow us to rationally design peptides for targeted applications as will be shown throughout this report. Aim 2: Characterize hairpin folding, self-assembly mechanism, and resulting network structure. Accomplishments 1. Mechanistic understanding. Two mechanistic models for gelation were found that differ in their early steps. Initially, we favored mechanism A based on the literature describing hairpin folding, as well as our own data. However, recent publications describing the oligomerization of intrinsically disordered proteins, suggest that these early steps may be more complex as described in mechanism B, which we will investigate in future work (vide infra). 2. Network characteristics. Bulk rheological measurements of the final network indicate that our hydrogels display viscoeleastic behavior reminiscent of heavily crosslinked, semiflexible polymer networks such as actin whose physical properties can be predicted using Mackintosh theory. Aim 3: Study the encapsulation and delivery of small molecules, proteins, and cells. Accomplishments Our efforts to design gels for the local delivery of therapeutics have centered on proteins and cells. We have also recently started working towards small molecule delivery. 1. Delivery of proteins. We have shown that macromolecules can be directly and easily encapsulated in the gel network by adding a solution of unfolded peptide in water to a buffered solution of protein and triggering gelation. 2. Delivery of cells. Our work has focused on understanding how gel characteristics influence the encapsulation, delivery and behavior of cells for eventual use in tissue engineering and cytomedical therapy. 3. Small Molecule Delivery. We have recently begun exploring the potential of our gels to deliver small molecules. In collaboration, we showed that the small molecule, curcumin, could be encapsulated at therapeutically relevant concentrations in MAX8 gels without significantly influencing gel rheological properties. This hydrophobic compound is sparingly soluble in water, but can partition into the hydrophobic regions of the fibril assembly. We showed that curcumin can be released over days to effect action on model medulloblastoma cells. This study provided the impetus to propose the systematic studies described below that will establish the rules by which small molecules behave in, and are released from, the network. Aim 4: Develop hydrogels for Interleukin 7 (IL-7) delivery to modulate T cell survival. In healthy immune systems, consistent populations of peripheral lymphocytes are maintained through cytokines responsible for cell differentiation and proliferation. This is a new project and we have recently established two collaborations to help carry out the aims. Aim 5: Develop hydrogels that facilitate vascular anastomoses. This is a new collaborative project with Dr. Brandacher at Johns Hopkins, Department of Plastic Surgery. He and his team are leading experts in whole hand transplantation. We are developing gels that facilitate micro-vascular anastomoses, the suturing of very small vessels (Diameter 0.2mm) to aid organ transplantation. Accomplishments The Brandacher lab has developed a super-micro surgical model to study immunomodulatory effects in rat hind limb allotransplantation. This model can be adapted to study the efficacy of our gels in aiding anastomosis.

目标 1：设计能够触发水凝胶化的肽 成就：我们设计并研究了一百多种肽，以了解序列如何调节折叠、组装和所得材料特性。人们付出了很多努力来理解不同结构扰动对凝胶化的作用，如下所述。 1. 链号和登记。我们通过制备单链肽和三链β-折叠片与两链发夹进行比较，评估了链数对自组装的影响。我们发现单链肽自组装形成由具有异质形态的原纤维组成的弱凝胶。 2.转弯型。在蛋白质中，转动负责链反转，帮助定义片材的扭曲，在某些情况下，还帮助定义核折叠。我们设计了 MAX1 中的高倾向 II 型 (-VDPPT-) 转角，在位置 i 处包含一个 β 支链缬氨酸，以在随后的残基处强制形成反式脯氨酸键，在中心 i+1 处有一个 -DPLP- 单元，在 i 处有一个 -DPLP- 单元。 +2位置采用类似于蛋白质中II'型转角的二面角，并且i+3位置处的苏氨酸与第i个羰基形成侧链/主链氢键以稳定蛋白质 转动。我们通过用文献中发现的规范的四残基β-转角和五残基[I+G1型β-凸起]序列替换该转角，研究了转角类型对折叠、组装和凝胶机械性能的影响。我们发现，每个转角的固有折叠倾向会影响发夹折叠和组装的速率，并且每种转角类型都能够按预期反转链方向，从而产生直径均匀的明确原纤维。重要的是，流变学表明转弯类型也影响水凝胶的机械刚性。四个残基转角内的二脯氨酸基序和包含单个脯氨酸的五残基[I型+G1 beta-buldge]序列（VPDGT）提供了所研究的最硬的凝胶。我们目前正在推导一个生物物理模型来解释转弯类型对凝胶网络刚度的依赖性。 3.对发夹疏水面的扰动。热诱导的发夹折叠和组装是由疏水效应驱动的，疏水效应与温度有关。我们研究了疏水性和侧链特性如何影响发夹的温度依赖性折叠和组装，以及水凝胶的流变特性。 4. 发夹亲水面的扰动。我们系统地研究了发夹亲水面的残留成分如何影响折叠、组装和材料特性。 5.肽表位和其他官能团的连接。通过修饰自组装发夹，将肽表位和其他化学物质共价结合到纤维网络中，可以增强凝胶的功能。一般来说，发夹支架可以容忍对其序列的改变。通过功能化赖氨酸侧链或掺入非天然残基，可以在其 N 端和 C 端掺入部分，也可以从其亲水面掺入部分；这些区域的修改对折叠、组装和散装材料特性的影响最小。发夹疏水面的变化就不那么宽容了。迄今为止，我们已经整合了各种细胞结合表位（RGD 等）和能够指导生物矿化的肽片段。还可以掺入更小的有机官能团，例如来自赖氨酸侧链的山梨酰胺基团，其允许原纤维网络的光聚合。总而言之，我们探索肽序列与材料关系的基础研究建立了一套不断发展的基础设计规则，使我们能够为目标应用合理设计肽，如本报告中所示。目标 2：表征发夹折叠、自组装机制以及由此产生的网络结构。成就 1. 机理理解。发现两种凝胶化机制模型在早期步骤上有所不同。最初，根据描述发夹折叠的文献以及我们自己的数据，我们倾向于机制 A。然而，最近描述本质无序蛋白质寡聚化的出版物表明，这些早期步骤可能更复杂，如机制 B 中所述，我们将在未来的工作中对其进行研究（见下文）。 2.网络特性。最终网络的整体流变测量表明，我们的水凝胶表现出粘弹性行为，让人想起高度交联的半柔性聚合物网络，例如肌动蛋白，其物理性质可以使用麦金托什理论进行预测。目标 3：研究小分子、蛋白质和细胞的封装和递送。成就 我们设计用于局部治疗的凝胶的努力集中在蛋白质和细胞上。我们最近还开始致力于小分子递送。 1. 蛋白质的输送。我们已经证明，通过将未折叠肽的水溶液添加到蛋白质缓冲溶液中并引发凝胶化，可以将大分子直接且轻松地封装在凝胶网络中。 2. 细胞的递送。我们的工作重点是了解凝胶特性如何影响细胞的封装、递送和行为，以最终用于组织工程和细胞医学治疗。 3.小分子传递。我们最近开始探索凝胶输送小分子的潜力。通过合作，我们发现小分子姜黄素可以以治疗相关浓度封装在 MAX8 凝胶中，而不会显着影响凝胶的流变特性。这种疏水性化合物微溶于水，但可以分配到原纤维组件的疏水性区域中。我们发现姜黄素可以在几天内释放以对模型髓母细胞瘤细胞产生作用。这项研究为提出下面描述的系统研究提供了动力，这些研究将建立小分子在网络中行为和从网络中释放的规则。目标 4：开发用于白细胞介素 7 (IL-7) 递送的水凝胶，以调节 T 细胞存活。在健康的免疫系统中，外周淋巴细胞通过负责细胞分化和增殖的细胞因子维持一致的群体。这是一个新项目，我们最近建立了两项合作来帮助实现这些目标。目标 5：开发促进血管吻合的水凝胶。这是与约翰霍普金斯大学整形外科的 Brandacher 博士的一个新合作项目。他和他的团队是全手移植领域的领先专家。我们正在开发促进微血管吻合的凝胶，即缝合非常小的血管（直径 0.2 毫米）以帮助器官移植。成就 Brandacher 实验室开发了一种超显微手术模型来研究大鼠后肢同种异体移植的免疫调节作用。该模型可用于研究我们的凝胶在辅助吻合方面的功效。