Our lab designs polymeric biomaterials for soft tissue repair and drug delivery with focused applications in ophthalmology. Many chronic or age-related conditions, particularly in the eye, are associated with excess inflammation and oxidative stress. We are designing topical and injectable drug delivery systems for sustained release of therapeutics to mitigate multiple factors, including angiogenesis, reactive oxygen species, and markers associated with fibrosis and inflammation. This talk will focus on nanoparticle and microcapsule systems developed to treat retinal and neurodegenerative conditions. Nanoparticles have been prepared using both polydisulfide and polydopamine chemistries for responsive release of small molecules to protein and antibody therapeutics. These therapeutic delivery systems are also being explored for treatment for other conditions relating to back pain, cancer, women’s health, and wound healing. 

Fibers formed by electrospinning are versatile materials with significant potential for bio/chem/medical applications.  Electrospun fiber membranes display very high surface area and porosity, which greatly enhances interaction with ambient environments and response to external stimuli. Electrospun fibers can be formed in complex shapes, in homogenous and multi-layer (core-sheath) structures, and can be embedded with various functional agents released in a controlled manner [1]. Our group has previously reported electrospun membranes for diverse biomedical applications, including dural repair [2], transdermal drug release [3], controlled drug release for brain tumor therapy [4]. One important consideration is the surrounding media pH, as pH plays a major role in many biological systems. Materials capable of sensing and responding to pH changes can be used to provide various treatment modalities and behaviors. For example, fiber membranes have been developed [5] containing different types of pH sensitive Eudragit polymers that dissolve in specific pH  conditions and release drug payloads in targeted locations of the gastrointestinal system. Carbopol is an exciting excipient in electrospun membranes due to its ability to modulate viscosity as a function of pH [6]. We report the electrospinning of Carbopol (C974P NF) dispersed in a PVP solution. Carbopol/PVP fiber membranes are designed to rapidly hydrate into a hydrogel. Carbopol becomes more responsive at higher pH (>6), increasing uptake volume of buffer solution - 5x of dry mass at pH 4 and 20x at pH 8 in 15 min. Spreadability measurements of the gel-phase indicate that higher pH and Carbopol concentration result in a more viscous network of hydrated fiber membranes. The pH responsive nature of Carbopol-containing electrospun membranes has multiple potential biomedical uses, including wound healing, drug delivery, and contraceptives.

Non-viral vehicles, such as polymer nanoparticles show promise as biomedical tools for the future of gene delivery. Polymer chemistry affords the ability to determine features of the self-assembled nanoparticle by tuning the monomeric sequence of the molecular backbone. Battelle has developed >6,000 novel polymers, and characterized their structure and function as gene delivery vehicles. Unlike lipids, many of these polymers can be directly dispersed in water, without the need for a solvent dilution processing step. However, exploration of formulation for these polymers still provides useful parameters for tuning the characteristics of the PNP. Here, we describe formulation techniques that enable further control over the characteristics and performance of these PNPs as gene delivery vehicles. We selected five polymers from our libraries synthesized via reversible addition-fragmentation chain transfer polymerization. We explored the self-assembly of these polymers into PNP using a direct hydration method as well as nanoprecipitation methods with and without genetic cargo. We measured the particle size via dynamic and static light scattering, exploring the effects of polymer concentration, rate of solvent removal, and payload loading on the nanostructures obtained. We demonstrated the use of nanoprecipitation to produce reproducible, stable PNPs, dispersed in water, that were not possible by directly hydrating the polymers. The mean particle size for the nucleic acid loaded PNPs obtained by nanoprecipition was 280 ± 7 nm for triplicate formulations. We extended this process to four other polymers, demonstrating applicability to a greater collection of our polymer library, and we showed the ability of the process to effectively load nucleic acids into these PNPs. This work demonstrates the capability nanoprecipitation to open the design space of Battelle’s platform to produce and characterize libraries of PNPs with greater diversity and potential as gene delivery vehicles. We are able to use these methods to formulate PNPs loaded with genetic cargos that are not otherwise able to be studied, obtaining in vitro transfection and in vivo biodistribution data. These results lay a foundation to accelerate the development of non-viral gene delivery and new genetic therapies for untreatable and devastating diseases.

A grand challenge in the field of cardiovascular tissue engineering is the production of functional, vascularized myocardial tissue for treating congenital heart defects or injury arising from myocardial infarct.  Central to addressing this challenge is the design of the scaffolding in which cells are organized and delivered, which also critically defines a variety of microenvironmental cues governing cell biology and resulting function.  For engineering cardiac tissue, naturally derived matrices such as hydrogels composed of collagen or fibrin have been the most explored given their ease of use and the ability for cells to physically and enzymatically remodel these materials. However, these natural matrices are difficult to tune (often essential to optimizing the biologic of incorporated parenchymal cells in an engineered tissue graft) and typically rapidly resorb following implantation, rendering their utility in engineering long-term organ replacement therapies limited. In contrast, synthetic or semi-synthetic biomaterials provide a means to highly tunable microenvironmental cues that can be catered to incorporated parenchymal cells and provide control over degradation mechanisms and resorption rates. However, while synthetic hydrogels enable numerous axes of microenvironmental control that can improve the function of long-term tissue grafts, this class of materials typically possess lower potential for cellular remodeling required for tissue assembly, due in large part to their amorphous microstructure and nanoscale porosity. In this talk, I’ll discuss some of the composite strategies developed in my lab to achieve the best of both classes of materials towards the overall goal of designing vascularized myocardial tissue. By integrating synthetic matrix-like fibers into natural hydrogels, we have established a means for engineering organized, contractile myobundles and capillary beds that can integrate with host vasculature upon implantation.

Prepubertal patients undergoing lifesaving yet gonadotoxic treatments have limited options for fertility preservation, especially those with bloodborne or metastatic cancers. Currently, the only available method is cryopreservation and auto-transplantation of ovarian tissue, which carries the risk of reintroducing malignant cells. In vitro culture of ovarian follicles can mitigate this problem; however, traditional materials like alginate prevent large-scale volumetric expansion and have limited functionality. In this report, we utilized a proteolytically degradable polyethylene glycol (PEG)-based hydrogel modified with bioactive, extracellular matrix (ECM) sequestering peptides. This design created a 3D system that can be locally remodeled as a follicle expands and that promotes follicle-follicle and follicle-matrix interactions. Ovaries from 10–12-days old mice were enzymatically digested using Liberase to obtain primary stage follicles that were encapsulated in groups of ten. Hydrogels were formed by pre-reacting 5% wt 8-arm PEG-VS with ECM-sequestering (BMB) peptides followed with crosslinking with plasmin-degradable peptides. Hydrogels were cultured in 150uL of alpha-MEM based media supplemented with fetuin, insulin, transferrin, selenium, bovine serum albumin, and follicle stimulating hormone (FSH) for 12 days, with brightfield images taken and half of the media (75uL) refreshed every two days. At the end of culture, follicles were either fixed in 4% paraformaldehyde for sectioning and staining or incubated in maturation media (alpha-MEM supplemented with fetal bovine serum, epidermal growth factor, human chorionic gonadotropin, and FSH) for in vitro maturation. During culture, murine follicles formed multi-follicle, organoid-like aggregates that exhibited granulosa cell proliferation and antrum formation. In vitro maturation of oocytes collected from follicle aggregates resulted in meiotic resumption to the mature meiosis II stage, and immunofluorescent staining showed intact gap junctions between granulosa cells, suggesting that follicles maintained cell-cell connections critical for successful folliculogenesis and maturation. Conclusion. Co-encapsulation of multiple murine follicles in a biomimetic hydrogel promoted folliculogenesis in vitro. Further development of this platform has the potential to move follicle culture towards a more translatable method for fertility preservation and improved patient quality of life. 

Previously, we have shown that plasmid DNA that encodes transcription factors for β cell patterning (βC) (e.g., Pdx1, Ngn3, Mafa) can drive the direct reprogramming of dermal fibroblasts (DFs) into induced β cells (iβCs) with potential to become an alternative method to treat type I diabetes. Recently, our objective has been to investigate whether plasmid DNA that encodes transcription factors for skin plasticity (SP) (e.g., Tcf3, Sox9, Trp63) can increase DFs multipotency and, therefore, β cell reprogramming efficiency. Combinations of plasmid DNA encoding for 3SP+7βC factors or pCMV6 (control plasmid DNA) were delivered to mouse DFs in vitro and in vivo using electroporation. Gene and protein expressions were analyzed using qRT-PCR or histology as appropriate. The ability of 3SP to open the chromatin landscape was significant 7 days post-electroporation. DFs transfected with 3SP+7βC transcription factors significantly increased gene expression for both insulin 1 and insulin 2 14 days post-electroporation. This increased insulin 1/2 expression correlated with a small population (~1%) of transfected DFs expressing insulin protein 14 days post-electroporation. Our findings suggest that DFs transfected with plasmid DNA encoding for 3SP factors exhibit increased multipotency. Furthermore, DFs transfected with DNA encoding for 3SP+7βC factors have the potential to reprogram into iβCs. Current experimentation includes exploration of βC micro-environmental factors and other βC induction methods. Future development of this alternative cell source to treat and potentially cure type I diabetes could greatly improve quality of life for diabetic patients worldwide.