Design and Utility of Novel Proteinaceous Biomaterials

目标1：设计能够触发氢化成就的肽：我们已经设计和研究了一百多种肽，以了解序列如何调节折叠，组装和所得材料特性。许多努力已经致力于理解不同的结构扰动对凝胶的作用，如下所述。1. 链数和注册表。我们通过制备单链肽和三链β片来评估链数对自组装的影响，并与双链发夹进行比较。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. 把类型。在蛋白质中，旋转负责链反转，帮助定义片的扭曲，在某些情况下，细胞核折叠。我们设计了MAX1的高倾向型II‘ （- vdppt -）转折，在i位置包含一个β支化的valine，以在接下来的残基上形成反式脯氨酸键，在i+1和i+2位置包含一个- dplp -单元，以形成类似于蛋白质II’转折的二面角，在i+3位置包含一个苏氨酸，以形成与第i羰基的侧链/主链氢键，以稳定转折。我们通过用文献中发现的典型的四残基β -匝和五残基[I型+G1型β -凸]序列代替匝型对折叠、组装和凝胶力学性能的影响进行了研究。我们发现，每个回合的固有折叠倾向影响了发夹折叠和组装的速度，并且每个回合类型都能够在预期的地方逆转链的方向，从而产生均匀直径的明确定义的原纤维。重要的是，流变学表明，转弯类型也影响水凝胶的机械刚度。在这些研究中，四个残基圈内的双脯氨酸基序和含有单个脯氨酸的五个残基[I型+G1 β -构筑]序列（VPDGT）提供了最坚硬的凝胶。我们目前正在推导一个生物物理模型来解释旋转类型对凝胶网络刚度的依赖。3. 对发夹疏水性面的扰动。发夹的热诱导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同一性如何影响发夹的温度依赖性折叠和组装，以及水凝胶流变性能。4. 对发夹亲水性面的扰动。我们系统地研究了发夹亲水性面的残留物组成对折叠、组装和材料性能的影响。5. 肽表位和其他功能基团的连接。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，发夹支架对其序列的改变是可以容忍的。通过功能化赖氨酸侧链或加入非天然残基，可以在其N端和c端以及亲水性表面结合部分；这些区域的修改对折叠、组装和散装材料性能的影响最小。改变发夹的疏水面就不那么容易了。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。更小的有机功能也可以加入，例如赖氨酸侧链上的sorbamide基团，允许纤维网络的光聚合。综上所述，我们探索肽序列-材料关系的基础研究建立了一套不断发展的设计规则，使我们能够合理地为目标应用设计肽，这将在本报告中展示。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。机械的理解。发现凝胶形成的两种机制模型在其早期阶段有所不同。最初，基于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，最近发表的描述内在无序蛋白寡聚化的文章表明，这些早期步骤可能比机制B中描述的更复杂，我们将在未来的工作中对此进行研究（见下文）。2. 网络的特点。最终网络的体积流变学测量表明，我们的水凝胶表现出粘弹性行为，让人想起重交联的半柔性聚合物网络，如肌动蛋白，其物理性质可以使用Mackintosh理论预测。目标3：研究小分子、蛋白质和细胞的包封和递送。我们设计用于局部递送治疗药物的凝胶的努力主要集中在蛋白质和细胞上。我们最近也开始研究小分子递送。1. 传递蛋白质。我们已经证明，通过在蛋白质缓冲溶液中加入未折叠肽溶液并触发凝胶化，大分子可以直接和容易地封装在凝胶网络中。2. 传递细胞。我们的工作重点是了解凝胶特性如何影响细胞的包封，传递和行为，最终用于组织工程和细胞医学治疗。3. 小分子递送。我们最近开始探索我们的凝胶输送小分子的潜力。在合作研究中，我们发现小分子姜黄素可以以治疗相关的浓度包被在MAX8凝胶中，而不会显著影响凝胶流变学特性。这种疏水化合物很少溶于水，但可以分割成纤维组装的疏水区域。我们发现姜黄素可以在数天内释放，对成神经管细胞瘤模型细胞产生作用。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。目的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，通过细胞因子负责细胞分化和增殖来维持外周淋巴细胞的一致种群。这是一个新项目，我们最近建立了两个合作来帮助实现目标。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布兰达切尔博士合作的一个新项目。他和他的团队是全手移植领域的顶尖专家。我们正在开发促进微血管吻合的凝胶，用于缝合非常小的血管（直径0.2mm），以帮助器官移植。Brandacher实验室开发了一种超显微手术模型来研究大鼠后肢异体移植的免疫调节作用。该模型可用于研究我们的凝胶辅助吻合的效果。

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.