ENGINEERING CELL INSTRUCTUVE MATERIALS
VEGF Presentation and Therapeutic Angiogenesis
Before the studies in our laboratory were conducted, it was known that the implantation of hydrogel scaffolds with bound VEGF (electrostatic or covalently bound) results in vessels that are more highly branched, perfused and mature than when soluble VEGF is encapsulated in the hydrogel. Moreover, although it was (and still is) common practice in bioengineered materials to covalently bind bioactive signals to the matrix, little is done to know the biological ramifications of such immobilization. In these studies we design a system to study cell activation by covalently bound or electrostatically bound ligands. In particular, we studied the role of VEGF- A165 presentation (soluble, electrostatically bound, covalently bound or multivalent) on endothelial cell activation. Our work in this area has introduced technology for the study of signal transduction of bound ligands that could directly compare the signal transduction by soluble ligands. In our approach, we fist grow the cells on PDMS (plastic) sheets and then either expose them to soluble VEGF or flip them on a surface that contained the bound ligand (e.g. VEGF). This way the cells are at the same confluence and “state” before being exposed to VEGF. Using this method we found that covalently bound VEGF can induce the phosphorylation of VEGFR-2 and that release of the VEGF ligand was not necessary. In fact the ligand stays behind on the surface and can induce the phosphorylation of VEGFR-2 on a new set of cells. Further, we found that VEGFR-2 phosphorylation by covalently bound VEGF led to differential signaling than that phosphorylated by soluble VEGF with covalently bound VEGF inducing more Y1214 and p38 activation and soluble VEGF inducing more Y1175 and p42/44 activation. Similar results were found by collagen I bound VEGF-A. Our work with multivalent VEGF also demonstrates differential activation compared to monovalent VEGF, showing that activating endothelial cells with VEGF clusters results in enhanced branching.
1.Anderson SM, Chen TT, Iruela-Arispe ML, Segura T. The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF. Biomaterials. 2009 Sep;30(27):4618-28. PubMed PMID: 19540581; PubMed Central PMCID: PMC2826152.
2.Anderson, S.M., Siegman, S.N., and Segura, T*., "The effect of vascular endothelial growth factor (VEGF) presentation within fibrin matrices on endothelial cell branching" Biomaterials, 2011. 32(30): p. 7432-7443. PMCID: PMC3601829.
3.Anderson, S.M., Shergill, B., Barry, Z.T., Manousiouthakis, E., Chen, T.T., Botvinick, E., Platt, M.O., Iruela-Arispe, M.L., and Segura, T*., "VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF" Integrative biology : quantitative biosciences from nano to macro, 2011. 3(9): p. 887-896. PMCID: PMC3621282.
Controlled Protein Delivery Based on Enzyme Responsive Nanocapsules
The goal of this research is to deliver growth factors in a dormant protected form that could become activated by the protease wound microenvironment in a controlled manner. We have demonstrated that functional proteins including growth factors can be encapsulated within degradable nanocapsules using in situ radical polymerization. The approach is to first surface absorb a monomer using charge interactions (negative surface protein and positive monomer), mix in neutral monomer, mix in a degradable crosslinker and start the polymerization through the addition of radical initiators. The effect of this process is the polymerization of a polymeric network around the protein. By controlling the concentration of the protein, monomers and crosslinker, nanoparticles (nanocapsules) can be generated that are stable at physiological conditions. The choice of the crosslinker is critical for inducing release of the growth factor cargo in the extracellular space such that the growth factor can interact with surface receptors. We chose a plasmin protease degradable crosslinker that can release the cargo at wound sites. Last, to modulate growth factor release rate from the nano capsules, we mixed degradable and non-degradable crosslinker and demonstrated that the ratio of degradable and non-degradable crosslinker can indeed modulate release rate.
1.Yan, M., Du, J., Gu, Z., Liang, M., Hu, Y., Zhang, W., Priceman, S., Wu, L., Zhou, Z.H., Liu, Z*., Segura, T*., Tang, Y*., and Lu, Y*., "A novel intracellular protein delivery platform based on single-protein nanocapsules" Nature nanotechnology, 2010. 5(1): p. 48-53. PubMed PMID: 19935648.
2.Wen, J., Anderson, S.M., Du, J., Yan, M., Wang, J., Shen, M*., Lu, Y*., and Segura, T*., "Controlled Protein Delivery Based on Enzyme-Responsive Nanocapsules" Advanced materials, 2011. Advanced materials, 2011. Oct 18;23(39):4549-53 PubMed PMID: 21910141; PubMed Central PMCID: PMC3263975.
Biomaterials for stem cell transplantation and differentiation
Although stem/progenitor transplantation after stroke leads to improved functional recovery, direct delivery of the cells has met with limited cell survival, and this appears to occur because of cell stress and death of the transplanted cells in the hostile environment of the post-stroke brain. We have begun to study the design of hyaluronic acid injectable hydrogel scaffolds as a vehicle for stem cell transplantation into the stroke cavity to promote their survival and differentiation. After optimizing the process of transplantation (injection speed, needle gauge and final gel stiffness) we transplanted iPS-NPCs into the infarct cavity of stroked immunodeficient NSG mice with or without a hydrogel and found that delivering neural progenitors within our hydrogel promoted increased differentiation of these cells to immature neurons (CDX positive), compared to delivering cells in saline. As mentioned transplantation of iPS-NPCs within a hydrogel matrix resulted in differentiation to immature neurons but no mature neurons were observed. Thus, we are further engineering the HA hydrogel to promote differentiation towards mature neurons. We utilized a multifactorial design of experiments (DOE) approach to systematically modulate three bioactive signal motifs (RGD, YIGSR, IKVAV) to determine their individual and collective effects on encapsulated iPS-NPC survival. After three optimization rounds, the final iteration formulation was further investigated for its ability to promote differentiation of the NPCs. Compared to cells plated in 2D and un-optimized hydrogels, more mature neurons were observed in the optimized hydrogels. Un-optimized hydrogels included RGD only and equimolar amounts of all three peptides (what is commonly done). Overall, our current findings illustrate the benefits of using both the DOE approach in multi-factor systems and culturing NPCs in a three-dimensional hydrogel. We are currently following with in vivo experiments.
1.Zhang, J., Tokatlian, T., Zhong, J., Ng, KT Q., Patterson, M., Lowry, S.W., Carmichael, S.T. Segura, T.* “Physically Associated Synthetic Hydrogels with Long-Term Covalent Stabilization for Cell Culture and Stem Cell Transplantation” Advanced Materials 2011 Nov 16;23(43):5098-103 PubMed PMID:21997799; PubMed Central PMCID: PMC3242734.
2.Lam J, Carmichael ST, Lowry WE, Segura T. Hydrogel Design of Experiments Methodology to Optimize Hydrogel for iPSC-NPC Culture. Adv Healthc Mater. 2014 Nov 5;PubMed PMID: 25378176.
3.Lam J, Lowry WE, Carmichael S, Segura T. Delivery of iPS-NPCs to the Stroke Cavity within a Hyaluronic Acid Matrix Promotes the Differentiation of Transplanted Cells. Adv Funct Mater. 2014 November 26; 24(44):7053.