Dataset Information

Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

ABSTRACT: Gene delivery using injectable hydrogels can serve as a potential method for regulated tissue regeneration in wound healing. Our microporous annealed particle (MAP) hydrogel has been shown to promote cellular infiltration in both skin and brain wounds, while reducing inflammation. Although the scaffold itself can promote healing, it is likely that other signals will be required to promote healing of hard-to-treat wounds. Gene delivery is one approach to introduce desired bioactive signals. In this study, we investigated how the properties of MAP hydrogels influence non-viral gene delivery of polyethylenimine-condensed plasmid to cells seeded within the MAP gel. From past studies, we found that gene transfer to cells seeded in tissue culture plastic differed from gene transfer to cells seeded inside hydrogel scaffolds. Since MAP scaffolds are generated from hydrogel microparticles that are approximately 100 ?m in diameter, they display local characteristics that can be viewed as two-dimensional or three-dimensional to cells. Thus, we sought to study if gene transfer inside MAP scaffolds differed from gene transfer to cells seeded in tissue culture plastic. We sought to understand the roles of the endocytosis pathway, actin and microtubule dynamics, RhoGTPases, and YAP/TAZ on transfection of human fibroblasts.

SUBMITTER: Truong NF

PROVIDER: S-EPMC7290906 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

Truong Norman F NF Lesher-Pérez Sasha Cai SC Kurt Evan E Segura Tatiana T

Bioconjugate chemistry 20181218 2

Gene delivery using injectable hydrogels can serve as a potential method for regulated tissue regeneration in wound healing. Our microporous annealed particle (MAP) hydrogel has been shown to promote cellular infiltration in both skin and brain wounds, while reducing inflammation. Although the scaffold itself can promote healing, it is likely that other signals will be required to promote healing of hard-to-treat wounds. Gene delivery is one approach to introduce desired bioactive signals. In th ...[more]

PMID: 30513197

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Project description:Designing scaffolds for polyplex-mediated therapeutic gene delivery has a number of applications in regenerative medicine, such as for tissue repair after wounding or disease. Microporous annealed particle (MAP) hydrogels are an emerging class of porous biomaterials, formed by annealing microgel particles to one another in situ to form a porous bulk scaffold. MAP gels have previously been shown to support and enhance proliferative and regenerative behaviors both in vitro and in vivo. Therefore, coupling gene delivery with MAP hydrogels presents a promising approach for therapy development. To optimize MAP hydrogels for gene delivery, we studied the effects of particle size and stiffness as well as adhesion potential on cell surface area and proliferation and then correlated this information with the ability of cells to become transfected while seeded in these scaffolds. We find that the void space size as well as the presentation of integrin ligands influence transfection efficiency. This work demonstrates the importance of considering MAP material properties for guiding cell spreading, proliferation, and gene transfer. STATEMENT OF SIGNIFICANCE: Microporous annealed particle (MAP) hydrogels are an emerging class of porous biomaterials, formed by annealing spherical microgels together in situ, creating a porous scaffold from voids between the packed beads. Here we investigated the effects of MAP physical and adhesion properties on cell spreading, proliferation, and gene transfer in fibroblasts. Particle size and void space influenced spreading and proliferation, with larger particles improving transfection. MAP stiffness was also important, with stiffer scaffolds increasing proliferation, spreading, and transfection, contrasting studies in nonporous hydrogels that showed an inverse response. Last, RGD ligand concentration and presentation modulated spreading similar to non-MAP hydrogels. These findings reveal relationships between MAP properties and cell processes, suggesting how MAP can be tuned to improve future design approaches.

Project description:Microporous annealed particle (MAP) hydrogels are promising materials for delivering therapeutic cells. It has previously been shown that spreading and mechanosensing activation of human mesenchymal stem cells (hMSCs) incorporated in these materials can be modulated by tuning the modulus of the microgel particle building blocks. However, the effects of degradability and functionalization with different integrin-binding peptides on cellular responses has not been explored. In this work, RGDS functionalized and enzymatically degradable poly(ethylene glycol) (PEG) microgels were annealed into MAP hydrogels via thiol-ene click chemistry and photopolymerization. During cell-mediated degradation, the microgel surfaces were remodeled to wrinkles or ridges, but the scaffold integrity was maintained. Moreover, cell spreading, proliferation, and secretion of extracellular matrix proteins were significantly enhanced in faster matrix metalloproteinase degrading (KCGPQGIWGQCK) MAP hydrogels compared to non-degradable controls after 8 days of culture. We subsequently evaluated paracrine activity by hMSCs seeded in the MAP hydrogels functionalized with either RGDS or c(RRETAWA), which is specific for ?5?1 integrins, and evaluated the interplay between degradability and integrin-mediated signaling. Importantly, c(RRETAWA) functionalization upregulated secretion of bone morphogenetic protein-2 overall and on a per cell basis, but this effect was critically dependent on microgel degradability. In contrast, RGDS functionalization led to higher overall vascular endothelial growth factor secretion in degradable scaffolds due to the high cell number. These results demonstrate that integrin-binding peptides can modulate hMSC behavior in PEG-based MAP hydrogels, but the results strongly depend on the susceptibility of the microgel building blocks to cell-mediated matrix remodeling. This relationship should be considered in future studies aiming to further develop these materials for stem cell delivery and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Microporous annealed particle (MAP) hydrogels are attracting increasing interest for tissue repair and regeneration and have shown superior results compared to conventional hydrogels in multiple applications. Here, we studied the impact of MAP hydrogel degradability and functionalization with different integrin-binding peptides on human mesenchymal stem cells (hMSCs) that were incorporated during particle annealing. Degradability was found to improve cell growth, spreading, and extracellular matrix production regardless of the integrin-binding peptide. Moreover, in degradable MAP hydrogels the integrin-binding peptide c(RRETAWA) was found to increase osteogenic protein expression by hMSCs compared to RGDS-functionalized MAP hydrogels. These results have important implications for the development of a MAP hydrogel-based hMSC delivery system for bone tissue engineering.

Dataset Information

Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

Publications

Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

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