Gene Delivery From Granular Scaffolds for Tunable Biologics Manufacturing.

The understanding of the molecular basis for disease has generated a myriad of therapeutic biologics, including therapeutic proteins, antibodies, and viruses. However, the promise that biologics can resolve currently incurable diseases hinges in their manufacturability. These therapeutics require that their genetic material be introduced to mammalian cells such that the cell machinery can manufacture the biological components. These are then purified, validated, and packaged. Most manufacturing uses batch processes that collect the biologic a few days following genetic modification, due to toxicity or difficulty in separating product from cells in a continuous operation, limiting the amount of biologic that can be produced and resulting in yearlong backlogs. Here, a scaffold-based approach for continuous biologic manufacturing is presented, with sustained production of active antibodies and viruses for 30 days. The use of scaffold-based biologic production enabled perfusion-based bioreactors to be used, which can be incorporated into a fully continuous process.

Nucleic Acid Delivery from Granular Hydrogels.

Nucleic acid delivery has applications ranging from tissue engineering to vaccine development to infectious disease. Cationic polymer condensed nucleic acids are used with surface-coated porous scaffolds and are able to promote long-term gene expression. However, due to surface loading of the scaffold, there is a limit to the amount of nucleic acid that can be loaded, resulting in decreasing expression rate over time. In addition, surface-coated scaffolds are generally non-injectable. Here, it is demonstrated that cationic polymer condensed nucleic acids can be effectively loaded into injectable granular hydrogel scaffolds by stabilizing the condensed nucleic acid into a lyophilized powder, loading the powder into a bulk hydrogel, and then fragmenting the loaded hydrogel. The resulting hydrogel microparticles contain non-aggregated nucleic acid particles, can be annealed post-injection to result in an injectable microporous hydrogel, and can effectively deliver nucleic acids to embedded cells with a constant expression rate. Due to the nature of granular hydrogels, it is demonstrated that mixtures of loaded and unloaded particles and spatially resolved gene expression can be easily achieved. The ability to express genes long term from an injectable porous hydrogel will further open the applications of nucleic acid delivery.

Microporous annealed particle hydrogel stiffness, void space size, and adhesion properties impact cell proliferation, cell spreading, and gene transfer.

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.

Pathways Governing Polyethylenimine Polyplex Transfection in Microporous Annealed Particle Scaffolds.

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 µm 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.