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: 10702524
• 负责人: Joel Schneider
• 金额: $ 121.91万
• 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/10702524
• 关键词: 3-Dimensional; Adhesives; Adopted; Affect; Affinity; Anti-Bacterial Agents; Appearance; Attenuated; Binding; Biocompatible Materials; Biological; Bovine Serum Albumin; Breast Cancer Cell; Breathing; Buffers; C-terminal; Cancer cell line; Cell Survival; Cells; Characteristics; Charge; Chemicals; Clinical; Combined Modality Therapy; Culture Media; DNA; Data; Disease; Doxorubicin; Drug resistance; Electrostatics; Encapsulated; Engineering; Engraftment; Environment; Epidermal Growth Factor Receptor; Erlotinib; Event; Extracellular Matrix; Face; Family; Feedback; Fluorescence; Fluorescence Polarization; Future; Gel; Genetic; Glass; Glioblastoma; Goals; Gram-Positive Bacteria; Growth; Homo; Hydrogels; Hydrogen Bonding; Hydrophobicity; Immunoglobulin G; Immunomodulators; Implant; In Situ; Intercalating Agents; Investigation; Ionic Strengths; Knowledge; Label; Light; Lymph; Malignant Neoplasms; Malignant neoplasm of brain; Mass Spectrum Analysis; Mechanics; Mesothelioma; Metal Ion Binding; MicroRNAs; Molecular; Molecular Conformation; Morphology; Mussels; N-terminal; NOESY; Nucleic Acids; Oncogenic; Operative Surgical Procedures; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Population; Pre-Clinical Model; Proline; Property; Proteins; Publishing; Recovery; Resistance; Role; Sampling; Serum Proteins; Shapes; Side; Solid; Solvents; Spectrum Analysis; Structure; Surface; Syringes; System; Temperature; Therapeutic; Thinness; Time; Tissue Transplantation; Tissues; Torsion; Toxic effect; Vascularized Composite Allotransplantation; Vertebral column; Vesicle; Vitronectin; Water; Work; alpha-Fetoproteins; base; beta pleated sheet; biological systems; cancer cell; chemotherapy; clinical application; conformer; design; drug discovery; experimental study; foot; hydrophilicity; implant coating; improved; in vivo; kinase inhibitor; monomer; nanoparticle; novel; optical spectra; particle; peptide structure; protein aminoacid sequence; response; restraint; screening; sedimentation velocity; self assembly; small molecule; solid state nuclear magnetic resonance; tumor

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.

AIM 1：掺杂在未标记肽背景的荧光标记的Edans-Max1的时间和浓度依赖性研究被用于遵循组装的早期事件。 CD表明，在添加触发缓冲液后，150 UM Max1保持在水中至少2H展开。然而，缓冲液中肽的荧光发射光谱在同一时间周期内显示出蓝移和强度的增加，这表明尽管没有β-片结构进化，但Max1分区却将其分为疏水环境，例如寡聚物提供的环境。荧光极化表明，Max1在水中是单体（R 1nm），但缓冲液形成了寡聚颗粒（R 4nm）的合奏。接下来，我们证明了低聚物的形成可能是纤维形成的稳定道。我们显示了能够缓慢胶凝的2 mM肽溶液的时间依赖性CD。仅在4H时，系统才开始发展β-片结构，这表明了原纤维形成的起源。 在24小时时，展开的肽和含薄片的结构的混合物相等。 在同一时间周期内进行的荧光相关光谱（FCS）显示了单体的时间依赖性耗竭，而寡聚物的外观则显示出大小的大小，并且纤维纤维仅在以后形成。此外，在以后的时间，TEM显示了直接从低聚物发芽的原纤维。综上所述，我们的数据支持涉及低聚物形成的机制。我们对Proline对自组装作用的作用的调查始于解决Max1在其无序状态下的解决方案结构。当我们准备凝胶时，首先将固体肽溶解在5C的水中，以提供触发自组装的MM库存溶液。在5C（2mm Max1）时，分析沉积速度实验表明肽是单体。同型和异核双重和三谐和NMR实验的结合提供了顺序的主链共振分配。距离限制是从2D Noesy和3d 15n和13c分辨的NOE实验和结构中获得的，该实验和结构使用Xplor-NIH63计算出来。 NMR表明，尽管N和C末端链是无序的，但存在三个不同的未折叠肽种群，每个种群都有定义DPRO-PRO基序的不同扭转角度，即顺式Trans（18％），Trans-Trans（22％）（22％） ）和trans-cis（60％）。预计会在Max1的折叠和组装状态中发现，Di-Pro单元采用典型的II型转弯，预计会构型。在展开的状态下，跨反式构象异构体几乎用I+3（val9-thr12）H键距离为3.3 Angstrom的II'转弯。对于顺式传播和反式CIS构象异构体而言，定义相同电位H键的原子之间的距离要大得多，这些构象异构体不利于发夹形成和纤颤。跨CIS构象是人口最多的构象，因为它投射了高度的N端和C末端链，从而最大程度地降低了系统的能量。鉴于溶液中的大多数肽最终在触发凝胶化后最终聚集成单态原纤维，并且在无序状态下，跨反式构象最小化，这表明脯氨酸异构化是重要的，并且可能是凝胶速率限制步骤机制。目标2：我们不断设计新的肽，以完善我们对肽序列如何影响材料形成，特性和功能的理解。 AIM 2包含两个子Aim：1）探索发夹设计和2）将功能（例如化学弹头和IgG结合域）纳入发夹肽中，以开发亲和力控制的药物释放系统。 AIM3：我们对材料刚性如何影响癌细胞对化学疗法的反应有所了解。众所周知，ECM刚度改变了乳腺癌细胞表型，但是，底物刚度在其化学治疗反应中的作用尚不清楚。常规的培养和癌细胞系对不自然的刚性塑料或玻璃底物的适应会导致其生长，转移潜力的深刻变化，并且正如我们所显示的化学治疗反应。我们证明，当从宿主微环境中去除并在刚性表面培养时，原发性乳腺癌细胞会发生巨大的表型变化，并且对临床批准的化学治疗剂的反应被机械反馈细胞从培养物底物中得到的机械反馈细胞深刻改变。相反，在模仿其宿主肿瘤ECM机械的底物上培养的癌细胞在药物活性和耐药途径方面具有与原位细胞相似的遗传特征。我们的工作突出了一个机会，可以通过将机械刚性作为筛选活动的参数进行整合来改善药物发现工作。在单独的工作中，我们发现我们呈阳性的凝胶只有通过从培养基中吸附的血清蛋白而具有细胞同相。多阶段质谱法表明，至少40种血清蛋白可以通过静电吸引来减轻其毒性吸收凝胶表面。此外，基于细胞的研究表明，单蛋白添加剂，例如牛血清白蛋白，fetuin-A或玻璃纤维素也可以有效。 尽管我们的呈阳性凝胶可用于生物应用，但我们为体内应用开发了固有的细胞相容性负电荷凝胶。例如，肽凝胶Acves3-RGDV保持细胞活力，可用于封装和输送细胞在体内产生长期植入。 AIM 4：我们开发了一种miRNA递送系统来治疗间皮瘤。我们设计了一个表面填充水凝胶（SFH），该水凝胶（SFH）可以在手术期间被注射或喷涂到表面癌症或用作主要疗法。一旦应用，SFH可以响应组织形态的改变并释放miRNA/肽纳米颗粒的变化，从而进入癌细胞，从而减弱其致癌特征。通过单一应用，凝胶在间皮瘤的四个临床前模型中显示出疗效，这表明局部应用肿瘤特异性miRNA的治疗作用。我们还开发了一种多区域水凝胶材料来提供组合疗法。我们开发了一种材料，可有效地将EGFR激酶抑制剂Erlotinib（ERL）和阿霉素（DOX，DNA插源）以ERL/DOX顺序方式提供，以协同杀死胶质母细胞瘤，这是最积极的脑癌。该材料由在水凝胶原纤维网络中交错的球形Dox-vesicles组成，该网络允许时间分辨的小分子独立共递送。我们还开发了一种新颖的植入物涂料。我们证明了衍生自贻贝足蛋白5的肽显示未报告的抗菌特性。这种神秘的功能是设计新型基于肽的抗菌粘合剂水凝胶的灵感，这些蛋白水凝胶对抗药性革兰氏阳性细菌具有活性。最后，我们开发了一种材料，该材料在血管复合材料同育手术后限制组织排斥。我们介绍了可以将免疫调节剂直接传递到移植组织和排水淋巴的材料。