Design and Utility of Novel Proteinaceous Biomaterials

目的1：将荧光标记的EDANS-MAX1掺杂到未标记的肽背景中，进行时间和浓度依赖性研究，以跟踪组装的早期时间事件。CD显示150 uM的MAX1在水中保持展开，并且在加入触发缓冲液后至少保持2h。然而，缓冲液中肽的荧光发射光谱在同一时间段内显示蓝移和强度增加，这表明尽管没有β -片结构进化，但MAX1分裂成疏水环境，例如低聚物提供的环境。荧光偏振显示MAX1在水中为单体（r为1nm），但在缓冲液中形成低聚粒子集合（r为4nm）。接下来，我们证明了低聚物的形成可能是原纤维形成的途径。我们展示了2毫米肽溶液的时间依赖性CD，该溶液能够缓慢凝胶化。只有在第4小时，系统才开始进化出β -薄片结构，这表明了原纤维形成的起源。24h时，未折叠肽和含片结构的混合物相等。在同一时间段内进行的荧光相关光谱（FCS）显示单体的时间依赖性消耗和伴随大小增长的低聚物的出现，而原纤维仅在较晚的时间形成。此外，在稍后的时间，透射电镜显示原纤维直接从低聚物中萌发。综上所述，我们的数据支持一种涉及低聚物形成的机制。我们从求解无序状态下的MAX1溶液结构开始研究脯氨酸对自组装的影响。当我们制备凝胶时，首先将固体肽溶解在5C的水中，得到触发自组装的mM原液。在5C （2mM MAX1）下，分析沉降速度实验表明肽为单体。同源核和异核双共振和三共振核磁共振实验的组合提供了顺序的骨干共振分配。距离约束分别来自2D NOESY和3D 15N和13c分辨率NOE实验，并使用XPLOR-NIH63计算结构。核磁共振显示，尽管n端和c端链是无序的，但存在三个不同的未折叠肽群，每个群体都有不同的扭转角度，即顺式-反式（18%），反式（22%）和反式（60%）。反式反式构象是预料之中的，因为在MAX1的折叠和组装状态中发现，其中di-Pro单元采用典型的II型转弯。在未展开状态下，反-反式构象接近于形成i '弯，i, i+3 （Val9-Thr12）氢键距离为3.3埃。对于顺-反和反-顺构象，定义相同势能氢键的原子之间的距离要大得多，这不利于发夹形成和纤尖化。反式构象是最密集的，因为它将高电荷的N端和c端链分开最远，使系统的能量最小化。考虑到溶液中的大多数肽在凝胶化触发后最终组装成单形原纤维，并且在无序状态下反式反式构象的填充最少，这表明脯氨酸异构化是凝胶化机制中重要的，并且可能是限速的步骤。目的2：我们不断设计新的肽，以完善我们对肽序列如何影响物质形成，性质和功能的理解。目标2包含两个子目标：1)探索发夹设计；2)将化学弹头和igg结合域等功能整合到发夹肽中，以开发亲和控制药物释放系统。目的3：我们了解了材料硬度如何影响癌细胞对化疗的反应。众所周知，ECM硬度改变乳腺癌细胞表型，然而，底物硬度在其化疗反应中的作用尚不清楚。癌细胞系对非自然刚性塑料或玻璃基质的常规培养和适应导致其生长，转移潜力和化疗反应的深刻变化。我们证明，原发性乳腺癌细胞从宿主微环境中移除并在刚性表面上培养时，会发生显着的表型变化，并且对临床批准的化疗药物的反应被细胞从培养底物接收的机械反馈深刻地改变。相反，在模拟宿主肿瘤ECM机制的基质上培养的癌细胞在药物活性和耐药途径方面与原位细胞具有相似的遗传谱。我们的工作强调了通过将机械刚度作为筛选活动的参数来改善药物发现工作的机会。在单独的工作中，我们发现我们带正电的凝胶仅通过吸附培养基中的血清蛋白而具有细胞相容性。多级质谱分析结果表明，至少40种血清蛋白可以通过静电吸引吸附到凝胶表面，从而改善其毒性。此外，基于细胞的研究表明，单蛋白添加剂，如牛血清白蛋白、胎蛋白a或玻璃体粘连蛋白也可以有效。虽然我们的正电荷凝胶可用于生物应用，但我们已经开发出具有内在细胞相容性的负电荷凝胶用于体内应用。例如，肽凝胶AcVES3-RGDV可维持细胞活力，并可用于在体内包裹和递送细胞，从而实现长期植入。目的4：我们开发了一种用于治疗间皮瘤的miRNA传递系统。我们设计了一种表面填充水凝胶（SFH），可以在手术期间通过注射器或喷雾输送到表面癌症，也可以作为主要治疗方法。一旦应用，SFH可以响应组织形态的改变而改变形状，并释放进入癌细胞的miRNA/肽纳米颗粒，从而减弱其致癌特征。单次应用，凝胶在四种间皮瘤临床前模型中显示出疗效，证明了局部应用肿瘤特异性miRNA的治疗效果。我们还开发了一种多室水凝胶材料来提供联合治疗。我们开发了一种材料，以ERL/DOX顺序方式有效地递送EGFR激酶抑制剂厄洛替尼（ERL）和阿霉素（DOX， DNA插入剂），协同杀死胶质母细胞瘤，这是最具侵袭性的脑癌。这种材料由球形dox -囊泡组成，这些囊泡交织在水凝胶纤维网络中，允许小分子的时间分辨独立共递送。我们还开发了一种新的种植体涂层。我们证明了从贻贝足蛋白5中提取的肽，显示出未报道的抗菌特性。这种隐功能为设计一类新的基于肽的抗菌胶粘剂水凝胶提供了灵感，这种水凝胶对耐药的革兰氏阳性细菌有活性。最后，我们开发了一种材料来限制血管化复合异体移植手术后的组织排斥反应。我们设计的材料可以将免疫调节剂直接输送到移植组织和引流淋巴中。

Aim 1: Time- and concentration-dependent studies of fluorescently labeled EDANS-MAX1 doped into a background of unlabeled peptide were used to follow the early time events of assembly. CD shows that 150 uM MAX1 remains unfolded in water and for at least 2h after the addition of triggering buffer. However, fluorescence emission spectra of peptide in buffer show a blue shift and increase in intensity over the same time-period, indicating that although no beta-sheet structure has evolved, MAX1 partitions into a hydrophobic environment, such as that offered by an oligomer. Fluorescence polarization showed that MAX1 is monomeric in water (r 1nm), but in buffer forms an ensemble of oligomeric particles (r 4nm). Next, we showed that the formation of oligomers is likely on-pathway to fibril formation. We showed time-dependent CD for a 2 mM solution of peptide capable of slowly gelling. Only at 4h does the system begin to evolve beta-sheet structure, suggesting the genesis of fibril formation. At 24h, there is an equal mixture of unfolded peptide and sheet-containing structures. Fluorescence correlation spectroscopy (FCS) performed over the same time-period shows a time-dependent depletion of monomer and concomitant appearance of oligomers that grow in size, with fibrils forming only at later times. Further, at later times, TEM shows fibrils sprouting directly from oligomers. Taken together, our data supports a mechanism involving oligomer formation. Our investigation into the role of proline's influence on self-assembly began with solving the solution structure of MAX1 in its disordered state. When we prepare gels, solid peptide is first dissolved in water at 5C to afford mM stock solutions from which self-assembly is triggered. At 5C (2mM MAX1), analytical sedimentation velocity experiments show that the peptide is monomeric. A combination of homo- and heteronuclear double- and triple-resonance NMR experiments afforded sequential backbone resonance assignments. Distance restraints were obtained from 2D NOESY and 3D 15N- and 13C-resolved NOE experiments and structures calculated using XPLOR-NIH63. NMR shows that although the N-and C-terminal strands are disordered, three distinct populations of unfolded peptide exist, each having distinct torsion angles defining the DPro-Pro motif, namely cis-trans (18%), trans-trans (22%), and trans-cis (60%). The trans-trans conformation was expected, as it is found in MAX1's folded and assembled state where the di-Pro unit adopts a typical type II' turn. In the unfolded state, the trans-trans conformer is close to forming a II' turn with an i, i+3 (Val9-Thr12) H-bond distance of 3.3 angstrom. The distances between atoms defining the same potential H-bond are much greater for the cis-trans and trans-cis conformers, which are not conducive to hairpin formation and fibrillization. The trans-cis conformation is the most populated as it projects the highly charged N- and C-terminal strands farthest apart, minimizing the energy of the system. Given that most of the peptide in solution eventually assembles into monomorphic fibrils after gelation is triggered, and the trans-trans conformation is minimally populated in the disordered state, this suggests that proline isomerization is an important, and possibly rate-limiting step in the gelation mechanism. Aim 2: We continuously design new peptides to refine our understanding of how peptide sequence affects material formation, properties, and function. Aim 2 contains two sub-aims that: 1) explore hairpin designs and 2) incorporate functionality, such as chemical warheads and IgG-binding domains, into hairpin peptides to develop affinity-controlled drug release systems. Aim3: We developed an understanding of how material rigidity influences cancer cell response to chemotherapy. It is known that ECM stiffness alters breast cancer cell phenotype, however, the role of substrate stiffness in their chemotherapeutic response was unclear. Routine culture and adaptation of cancer cell lines to unnaturally rigid plastic or glass substrates leads to profound changes in their growth, metastatic potential, and as we showed, chemotherapeutic response. We demonstrate that primary breast cancer cells undergo dramatic phenotypic changes when removed from the host microenvironment and cultured on rigid surfaces, and that responses to clinically-approved chemotherapeutics are profoundly altered by the mechanical feedback cells receive from the culture substrate. Conversely, cancer cells cultured on substrates mimicking the mechanics of their host tumor ECM have a similar genetic profile to the in situ cells with respect to drug activity and resistance pathways. Our work highlights an opportunity to improve drug discovery efforts by integrating mechanical rigidity as a parameter in screening campaigns. In separate work, we discovered that our positively-charged gels are cytocompatible only by virtue of adsorbing serum proteins from culture media. Multistage mass spectrometry showed that at least 40 serum proteins can absorb to the gel surface through electrostatic attraction ameliorating its toxicity. Further, cell- based studies show that single protein additives such as bovine serum albumin, fetuin-A, or vitronectin can also be effective. Although our positively-charged gels can be used for biological applications, we have developed inherently cytocompatible negatively-charged gels for in vivo applications. For example, peptide gel AcVES3-RGDV maintains cell viability and can be used to encapsulate and deliver cells in vivo enabling long-term engraftment. Aim 4: We developed a miRNA delivery system towards the treatment of mesothelioma. We engineered a surface-fill hydrogel (SFH) that can be syringe- or spray-delivered to surface cancers during surgery or used as a primary therapy. Once applied, SFH can shape-change in response to alterations in tissue morphology and release miRNA/peptide nanoparticles that enter cancer cells attenuating their oncogenic signature. With a single application, the gel shows efficacy in four preclinical models of mesothelioma, demonstrating the therapeutic impact of the local application of tumor-specific miRNA. We also developed a multicompartment hydrogel material to deliver combination therapies. We developed a material that effectively delivers the EGFR kinase inhibitor Erlotinib (ERL) and Doxorubicin (DOX, DNA intercalator) in an ERL/DOX sequential manner to synergistically kill glioblastoma, the most aggressive form of brain cancer. This material is composed of spherical DOX-vesicles interlaced within a hydrogel fibril network that allows time-resolved independent co-delivery of small molecules. We also developed a novel implant coating. We demonstrated that a peptide derived from mussel foot protein-5, displays unreported antibacterial properties. This cryptic function served as inspiration for the design of a new class of peptide-based antibacterial adhesive hydrogel, which are active against drug-resistant Gram-positive bacteria. Lastly, we developed a material that limit tissue rejection after vascularized composite allotransplantation surgery. We enginerred materials that can deliver immune modulators directly to transplanted tissue and to the draining lymph.