Nicolynn E. Davis

Modular enzymatically crosslinked protein polymer hydrogels for in situ gelation

Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, complete crosslinking occurred within 2 min, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.

Harnessing the Immunomodulatory and Tissue Repair Properties of Mesenchymal Stem Cells to Restore β Cell Function

Islet cell transplantation has therapeutic potential to cure type 1 diabetes (T1D), which is characterized by autoimmune-mediated destruction of insulin-producing β cells. However, current success rates are limited by long-term decline in islet graft function resulting partially from poor revascularization and immune destruction. Mesenchymal stem cells (MSCs) have the potential to enhance islet transplantation and prevent disease progression by a multifaceted approach. MSCs have been shown to be effective at inhibiting inflammatory-mediated immune responses and at promoting tissue regeneration. The immunomodulatory and tissue repairing properties of MSCs may benefit β cell regeneration in the context of T1D. This review will elucidate how MSCs can minimize β cell damage by providing survival signals and simultaneously modulate the immune response by inhibiting activation, and proliferation of several immune cell types. In addition, MSCs can enhance islet graft revascularization, maintaining long-term β cell viability and function.

Multivalent Protein Polymer MRI Contrast Agents: Controlling Relaxivity via Modulation of Amino Acid Sequence

Magnetic resonance imaging is a noninvasive imaging modality with high spatial and temporal resolution. Contrast agents (CAs) are frequently used to increase the contrast between tissues of interest. To increase the effectiveness of MR agents, small molecule CAs have been attached to macromolecules. We have created a family of biodegradable, macromolecular CAs based on protein polymers, allowing control over the CA properties. The protein polymers are monodisperse, random coil, and contain evenly spaced lysines that serve as reactive sites for Gd(III) chelates. The exact sequence and length of the protein can be specified, enabling controlled variation in lysine spacing and molecular weight. Relaxivity could be modulated by changing protein polymer length and lysine spacing. Relaxivities of up to approximately 14 mM(-1) s(-1) per Gd(III) and approximately 461 mM(-1) s(-1) per conjugate were observed. These CAs are biodegradable by incubation with plasmin, such that they can be easily excreted after use. They do not reduce cell viability, a prerequisite for future in vivo studies. The protein polymer CAs can be customized for different clinical diagnostic applications, including biomaterial tracking, as a balanced agent with high relaxivity and appropriate molar mass.

Synthesis and characterization of a new class of cationic protein polymers for multivalent display and biomaterial applications

Monodisperse protein polymers engineered by biosynthetic techniques are well suited to serve as a basis for creating comb-like polymer architectures for biomaterial applications. We have developed a new class of linear, cationic, random-coil protein polymers designed to act as scaffolds for multivalent display. These polymers contain evenly spaced lysine residues that allow for chemical or enzymatic conjugation of pendant functional groups. Circular dichroism spectroscopy and turbidity experiments have confirmed that these proteins have a random coil structure and are soluble up to at least 65 degrees C. Cell viability assays suggest these constructs are nontoxic in solution up to a concentration of 100 microM. We have successfully attached a small bioactive peptide, a peptoid-peptide hybrid, a poly(ethylene glycol) polymer, and a fluorophore to the protein polymers by chemical or enzymatic coupling, demonstrating their suitability to serve as multivalent scaffolds in solutions or as gels.

Engineering Surfaces for Substrate-Mediated Gene Delivery Using Recombinant Proteins

Immobilized fibronectin and other natural proteins have been utilized to enhance substrate-mediated gene delivery, with apparent contributions from the intrinsic bioactivity and also physical properties of the immobilized proteins. In this report, we investigated the use of recombinant proteins, compared to the full-length fibronectin protein, as surface coatings for gene delivery to investigate the mechanisms by which fibronectin enhances gene transfer. The recombinant fibronectin fragment FNIII(7-10) (FNIII) contains the alpha(5)beta(1) binding domain of fibronectin and supports cell adhesion, whereas the recombinant protein polymer PP-12 is also negatively charged and has a molecular weight similar to FNIII, but lacks cell binding domains. Transfection was compared on surfaces modified with FNIII, full-length fibronectin, or PP-12. The full-length fibronectin provided the greatest extent of transgene expression relative to FNIII or PP-12, which was consistent with the amount of DNA that associated with cells. FNIII had 4.2-fold or 4.7-fold lower expression levels relative to fibronectin for polyplexes and lipoplexes, respectively. PP-12 produced expression levels that were 317-fold and 12.0-fold less than fibronectin for polyplexes and lipoplexes, respectively. Although expression was greater on FNIII relative to PP-12, the levels of DNA associated per cell with FNIII were similar to or less than those with PP-12, suggesting that the bioactive sequences may contribute to an enhanced intracellular trafficking. For lipoplexes delivered on FNIII, the efficiency of intracellular trafficking and levels of caveolar DNA were greater than that observed with either the full-length fibronectin or PP-12. For polyplexes, fibronectin fragment resulted in greater intracellular trafficking efficiency compared to PP-12 protein polymer. Recombinant proteins can be employed in place of full-length extracellular matrix proteins for substrate-mediated gene delivery, and bioactive sequences can influence one or more steps in the gene delivery process to maximize transfection.

A preconditioning regimen with a PKCɛ activator improves islet graft function in a mouse transplant model.

Transplantation of islets isolated from deceased donor pancreata is an attractive method of β-cell replacement therapy for patients with type 1 diabetes (T1D). However, the loss of islet cell viability and function during the peritransplant period is a limiting factor to long-term islet engraftment. Activation of the isoenzyme PKCe may improve islet survival and function. The current study assesses the effects of PKCe activation on islet graft function in a syngeneic streptozotocin-induced diabetic mouse model. Islets were isolated from wild-type BALB/c mice preconditioned with either a PKCe activator (ψεRACK) or a TAT carrier control peptide. Islets were further treated with the same agents during isolation, purification, and incubation prior to transplantation. Two hundred seventy-five islet equivalents were transplanted under the kidney capsule of streptozotocin-induced diabetic BALB/c mice. Islet function was assessed by measurement of blood glucose levels every 3 days for 42 days after transplant and through an intraperitoneal glucose tolerance test (IPGTT). The time for return to euglycemia in mice transplanted with islets treated with ψεRACK was improved at 14 ± 6 days versus 21 ± 6 days with TAT-treated islets. The IPGTT showed a 50% reduction in the area under the curve associated with an improved insulin response in mice transplanted with ψεRACK-treated islets compared to TAT-treated islets. A preconditioning regimen using PKCe agonist before pancreatic recovery and during islet isolation improves islet graft function and resistance to high glucose stress after transplantation.