Polymeric Nanoparticle-Based DNA AND siRNA Delivery for Cancer Treatment and Stem Cell Engineering
Tzeng, Stephany Yi
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The fields of biomaterials, nanobiotechnology, and gene and drug delivery have all progressed over the past decades and have rapidly become a focus of research for many applications. In particular, gene delivery, with cargoes including DNA, small interfering RNA (siRNA), and short hairpin RNA (shRNA), is a very attractive tool for research purposes as well as clinical application. The ability to change a cell's expression at the genetic level affords researchers great flexibility in studying a cell's behavior in relation to its gene expression in a laboratory setting. More translational applications for which gene therapy can be a useful tool include cancer therapy and regenerative medicine. For the latter, cells must be directed to grow or behave in strictly defined mann;ers, an issue that is often addressed via administration of soluble factors or spatial or mechanical cues during the cell culture period. While these are by no means strategies to be disregarded, cells can be guided more directly using gene therapy. For example, in some cases, stem cell differentiation is controlled primarily by or inhibited by known factors. While designing drugs to target the specific proteins of interest is dependent on protein structure as well as the ability to deliver the drug, knowing the gene sequence could allow us to deliver or suppress the gene directly, bypassing undruggable protein targets. In the case of disease treatment, many diseases, including inherited and some acquired diseases like cancer, are genetic in origin or are affected by the patient's genetic background. The biodegradable polymer nanoparticles we have designed are able to combat such diseases by changing the gene expression of cancer cells, such as by decreasing their expression of survival factors or causing them to overexpress apoptotic factors that cause cell death. Unlike traditionally studied viral methods, our synthetic nanoparticle system avoids many of the safety concerns surrounding viruses, including toxicity, severe inflammatory or immune response, and the potential for insertional mutagenesis. Non-viral gene delivery is an exceedingly versatile tool; as will be shown below, many types of therapeutic nucleic acids can be delivered using our polymers, and once delivery to a given cell type is optimized, the sequence of the genes being delivered do not affect the properties of the nanoparticles, therefore allowing for essentially any gene to be delivered using our system. Poly(β-amino ester)s (PBAEs), a newer class of biomaterials effective in gene delivery, have been developed for DNA and siRNA delivery to human GBM cells. Importantly, specific chemical structures that have a strong effect on delivery of one nucleic acid type over the other have been identified, and overall trends in polymer structures have been correlated with transfection efficacy. PBAEs that can transfect 2-D and 3-D brain cancer cultures while having minimal effect on fetal brain cells have also been found. This phenomenon was verified in a different species and tissue type, namely rat liver cancer and healthy hepatocytes transfected in a co-culture system. Tumor cell death was caused in vitro after transfection with functional DNA coding for suicide genes or other methods of causing cancer cell apoptosis. DNA and/or siRNA delivery of functional genes was also used for stem cell engineering and regenerative medicine by causing overexpression or knockdown of transcription factors. In this series of applications, siRNA delivery to bone marrow-derived mesenchymal stem cells caused enhanced osteogenic differentiation; DNA delivery to embryonic stem cell-derived neural stem cells caused enhanced neuronal differentiation and maturation; and DNA delivery to adipose-derived mesenchymal stem cells induced secretion of growth factors to enhance vascularization of these cultures for ischemia treatment. Finally, with an eye toward eventual translation of this technology to the clinic, we assessed the ability of PBAE/DNA nanoparticles to overcome in vivo barriers. Procedures to make this technology more translatable are detailed in this work, and we have also shown in a proof-of-concept experiment the ability of our nanoparticles to transfect cancer cells in vivo. The work presented in this thesis can serve as a starting point for future transfections and for rationally designing PBAEs with the most effective structures. In addition, the PBAE conditions discovered can be used for eventual in vivo studies of cancer. These methods may be used on their own for regenerative medicine and cancer therapy applications or could serve as a complementary tool along with conventional strategies and treatments in the future.
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