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.

AIM 1：设计能够触发水凝胶成就的肽：我们设计和研究了一百多个肽，以了解序列如何调节折叠，组装和结果材料的特性。如下所述，已经付出了很多努力来理解独特的结构扰动对凝胶化的作用。 1。链数和注册表。我们通过制备单链肽和三链β片以与两链发夹进行比较来评估链数对自组装的影响。我们发现，单链肽会自组装形成由具有异质形态的原纤维组成的弱凝胶。 2。转弯类型。在蛋白质中，转弯负责链逆转，有助于定义床单的扭曲，在某些情况下是核折叠的。 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羰基稳定转弯。我们通过用规范的四个残基β-转弯和五个残基[I+G1β-硫硫代序列]替换了转弯，从而研究了转弯类型对折叠，组装和凝胶机械性能的影响。我们发现，每个转弯的固有折叠倾向都会影响发夹折叠和组装的速率，并且每种转弯类型能够逆转预期的链方向，从而导致直径均匀定义的原纤维。重要的是，流变学表明，转弯类型还影响了水凝胶机械刚度。在四个残基转弯中的二酚基序，以及一个含有单个脯氨酸的五分配[型I+G1β-卵子]序列（VPDGT），提供了所研究人员的最僵硬的凝胶。我们目前正在得出一个生物物理模型，以解释转向类型对凝胶网络刚度的依赖性。 3。发夹疏水面的扰动。我们发夹的热诱导的折叠和组装是由疏水效应驱动的，疏水效果取决于温度。我们研究了疏水性和侧链身份如何影响发夹的温度依赖性折叠和组装以及水凝胶流变特性。 4。发夹的亲水脸的扰动。我们已经系统地研究了发夹亲水性面的残留物组成如何影响折叠，组装和材料特性。 5。肽表位和其他功能组的连接。通过将肽表位和其他化学物质共价纳入纤维化网络，可以通过重新组合自组装发夹来增强凝胶的功能。通常，发夹脚手架宽恕了其顺序的改变。可以通过功能化赖氨酸侧链或掺入非天然残基来将部分纳入其N和C末端及其亲水性面部。在这些区域进行修改，最小化折叠，组装和块状材料特性。发夹疏水面的变化不太宽容。迄今为止，我们纳入了各种细胞结合表位（RGD等）和能够指导生物矿化的肽片段。还可以纳入较小的有机功能，例如赖氨酸侧链的索巴胺基团，这些链链允许光层面化。综上所述，我们探索肽序列物质关系的基础研究建立了一个不断发展的设计规则集，使我们能够为目标应用程序合理设计肽，如本报告所示。 AIM 2：表征发夹折叠，自组装机制和由此产生的网络结构。成就1。机械理解。发现了两种凝胶化的机械模型，它们的早期步骤有所不同。最初，我们基于描述发夹折叠的文献以及我们自己的数据而偏爱机制A。但是，最近描述了本质上无序蛋白质的寡聚化的最新出版物表明，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进行调查（vide Infra）。 2。网络特征。最终网络的批量流变学测量表明，我们的水凝胶显示出粘性的行为，让人联想到诸如肌动蛋白等重型交联的，半融的聚合物网络，例如肌动蛋白，其物理特性可以使用Mackintosh理论来预测。 AIM 3：研究小分子，蛋白质和细胞的封装和递送。成就我们为局部治疗剂设计凝胶的努力以蛋白质和细胞为中心。我们最近还开始致力于小分子递送。 1。蛋白质的递送。我们已经表明，通过将展开的肽的溶液添加到水中的蛋白质溶液中并触发凝胶化，大分子可以直接，很容易地封装在凝胶网络中。 2。递送细胞。我们的工作集中在理解凝胶特性如何影响细胞在组织工程和细胞医学治疗中最终使用的封装，传递和行为。 3。小分子输送。我们最近开始探索凝胶提供小分子的潜力。在协作中，我们表明，小分子姜黄素可以在Max8凝胶中与治疗相关的浓度封装，而不会显着影响凝胶流变学特性。这种疏水化合物很少溶于水，但可以分配到原纤维组件的疏水区域中。我们表明姜黄素可以在几天内释放，以实现对模型髓母细胞细胞的作用。这项研究提供了提出下面描述的系统研究的动力，该研究将确定小分子行为并将其从网络中释放出来的规则。 AIM 4：开发用于白介素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，通过负责细胞分化和增殖的细胞因子来维持周围淋巴细胞的一致群体。这是一个新项目，我们最近建立了两项合作，以帮助实现目标。目标5：开发促进血管吻合的水凝胶。这是与整形外科系约翰·霍普金斯（Johns Hopkins）博士博士的新合作项目。他和他的团队是全手移植的领先专家。我们正在开发促进微血管吻合的凝胶，缝合非常小的血管（直径0.2mm）以帮助器官移植。成就Brandacher Lab开发了一种超微型外科手术模型，以研究大鼠后肢同种异体移植中的免疫调节作用。该模型可以适应以研究我们凝胶在有助于吻合方面的功效。