Let's get physical [electronic resource] : physical entrapment of cells and drugs for localized delivery within physically cross-linked, injectable hydrogels

Delivery of stem cells and drugs using hydrogels is a promising strategy in regenerative medicine for repairing tissue damage and dysfunction. Hydrogels have a high water content, supporting high payloads of encapsulated cells and drugs with good cell viability and drug bioactivity. Injectable hydrogels are desirable for clinical applications, because they can localize the payload at the site of injury in a minimally invasive manner. Physically cross-linked, injectable hydrogels are particularly useful because they do not require chemical cross-links, which are often cytotoxic. However, physically cross-linked networks may still require extreme shifts in the environmental conditions, including pH, temperature, and ionic strengths. To address this limitation, a Mixing-Induced Two- Component Hydrogel (MITCH) was designed to hetero-assemble at physiological conditions. MITCH is shear-thinning, injectable, and self-healing and can be used for physical entrapment of a wide range of target drugs for prolonged delivery. This thesis presents a biophysics and biomaterials approach for improved control over the stability and timing of drug delivery to stem cells within a novel protein-engineered, injectable hydrogel. A broad review of injectable hydrogels, from naturally-derived and protein-engineered materials, for drug delivery is presented in Chapter 1, providing a foundation for the experimental designs employed in subsequent chapters. Chapter 2 describes the characterization of MITCH for encapsulation of multiple drug targets, cells, and co- encapsulation of cells and drugs. MITCH cross-linking occurs by molecular-based recognition hydrogen bonding between complementary peptide domains at physiological conditions, enabling physical entrapment of cells and drugs during gelation. First, the loading capacity and release kinetics of drugs from MITCH is described for drugs with a wide range of molecular weights. Next, the cytocompatability and encapsulation of cells within MITCH is demonstrated. Lastly, co-encapsulation of cells and a therapeutically relevant drug is used to demonstrate that drugs remain localized and/or are consumed by cells within MITCH. In all cases drugs and cells are readily encapsulated within MITCH by simple mixing and physical entrapment of these components within the cross-linked polymer network. The remainder of the thesis focuses on the utility of MITCH for sustained, localized delivery of drugs to co-encapsulated cells, with an emphasis on design strategies for improved control over the stability and timing of delivery of the target drug. In Chapter 3, I characterize the binding affinities between cationic polymers and plasmid DNA and characterize the biophysical stability of the polyplexes and lipoplexes using classical transfection reagents branched polyethylenimine and Lipofectamine. I show that polyplexes formed by stoichiometric binding at non-buffered and buffered conditions have similar biophysical properties and DNA protection at physiological conditions. I then demonstrate that high binding affinity may be required for prolonged transgene expression of cells with slow proliferation, such as mouse adipose derived stromal cells (mADSCs) in 2D and 3D culture platforms. Transgene expression of mADSCs was observed for at least 19 days in 3D culture and could be improved in a dose-dependent manner. In Chapter 4, I discuss the development of a microfluidic platform for high loading, high encapsulation efficiency, and prolonged release of growth factors from alginate microgels. In this proof-of-concept study, I demonstrate the successful formation of alginate microgels by off-chip cross-linking. I show that the shape and size distribution of alginate microgels alters with varying divalent ion identity, but that this does not impact the effective encapsulation efficiency or significantly impact release. However, I show that release kinetics are less variable at high cross-linking densities and therefore are more desirable for controlled delivery of growth factors. In Chapter 5, I present a strategy for dual-stage delivery of multiple growth factors from alginate microgels embedded within MITCH. We explored the impact of dual-stage delivery in 3D cultures on regulation of adipogenesis of human adipose derived stromal cells (hADSCs). I show that the shear-thinning properties of MITCH are not disrupted by incorporation of alginate microgels and that the bulk material properties are suitable for promoting differentiation toward cell types from soft tissues, such as adipocytes. In fact, enhanced lipid accumulation was observed in the absence of adipogenic media and growth factors for hADSCs cultured with MITCH containing empty alginate microgels. Moreover, lipid accumulation on a per cell basis increased above baseline or spontaneous differentiation as a result of dual-stage delivery of fibroblast growth factor 1 (FGF-1) and bone morphogenetic factor 4 (BMP-4). Lastly, characteristic gene expression of the terminally differentiated cells as a function of 3D culture and dual-stage delivery suggests that the predominant cell type present are brown-like adipocytes. This is the first study to demonstrate the efficacy of FGF-1 and BMP-4 in differentiation of hADSCs toward brown adipocytes. Overall, these results demonstrate the importance of improved stability and bioactivity of a diverse range of drugs in promoting the desired cell behavior of a clinical relevant and abundance cell source, the adipose derived stromal cells. This work specifically demonstrates the efficacy of localized delivery within an injectable hydrogel network, suggesting that co-encapsulation of cells and drugs will improve the efficacy of cell delivery for many applications in regenerative medicine.

Keywords: Bioengineering.
Advisor: Heilshorn Sarah primary advisor. Thesis advisor
Melosh Nicholas A. advisor. Thesis advisor
Yang Fan (Bioengineering researcher and teacher) advisor. Thesis advisor