Design and Utility of Novel Proteinaceous Biomaterials

Division Of Basic Sciences - Nci

Investigators

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Abstract

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.