Engineering non-viral gene delivery from hydrogel scaffolds

Tissue engineering aims to restore the function of injured or diseased tissue. In tissue engineering, a scaffold is used to provide space for cell growth and supply bioactive factors to guide the transplanted or infiltrating cells to form tissue with normal structure and function. Traditionally, proteins are used as the bioactive factors. However, it is a challenge to incorporate proteins into scaffolds. Physically entrapped proteins diffuse away from the scaffold rapidly, thus are unable to provide sustained biochemical signals to cells. Chemical immobilization, though can retain proteins in the scaffold, requires very mild and well-tailored chemistry in order to prevent altering the elegant 3D structure of the protein and to leave the active domains of the protein intact and oriented toward cells. An alternative approach is to incorporate DNA into scaffolds so that genes encoding for the bioactive factors are transferred to cells to produce proteins in situ. In practice, delivering DNA from scaffolds is inefficient. It is difficult to retain the DNA in the scaffold due to the diffusion and the frequency for DNA to enter cells is low. Though viral particles can transport DNA into cells, they are unsafe to human because virus elicit severe immune response and may lead to cancer. Non-viral gene delivery technology, which uses cationic agents (e.g. polymers, lipids) to condense the negatively charged DNA into nanoparticles that can both effectively retain DNA in scaffolds and efficiently transport DNA into cells, is becoming very attractive for producing the bioactive factors within the scaffold.;There are many technical challenges to be solved prior to successfully delivering condensed DNA from scaffolds. Due to their soft, loose and charged nature, non-viral gene delivery nanoparticles tend to aggregate in salt, protein solutions or at high concentrations. The current technologies used to load these nanoparticles into scaffolds usually lead to severe particle aggregation and inactivation. Controlling DNA release from scaffolds represents another challenge. Natural tissue formation and regeneration are regulated temporally and spatially by a series of protein factors. Thus, tissue engineering scaffolds should temporally and spatially supply these biological cues to regulate cells to form functional tissues, which require the cell-demanded gene transfer. Currently, DNA release is mostly mediated by the hydrolysis of the scaffold, which is determined by the scaffold chemistry, but not the cells in or surrounding the scaffold. As a result, the current scaffold-mediated gene delivery is uncorrelated with the cellular events during tissue regeneration and unable to provide protein factors demanded by the cells temporally and spatially.;In this dissertation research, we engineered novel non-viral gene delivery from hydrogel scaffolds that can bypass the above challenges and deliver genes into cells in a cell-mediated method. Firstly, we developed a universal process for incorporating non-viral gene delivery nanoparticles into hydrogel scaffold, in which concentrated and unaggregated nanoparticles were loaded into poly(ethylene glycol) (PEG), hyaluronic acid (HA) and fibrin hydrogels. The encapsulated nanoparticles were highly active both in vitro and in vivo. Secondly, we developed PEG and HA hydrogels that are degradable by matrix metalloproteinases (MMPs) to deliver DNA. Nanoparticles encapsulated in these hydrogels are not free to diffuse and delivered to cells only when the scaffold surrounding them is degraded by cells through MMPs. Thus, the gene transfer is cell-controlled. We believe the technologies developed in this dissertation will significantly advance the applications of scaffold-mediated gene delivery in tissue engineering.