An Investigation of Gene Delivery Barriers for Poly(beta-amino ester)s and Hybrid Gold-Polymeric Nanoparticles

Gene therapy is promising because nearly all inheritable diseases and cancer has an underlying genetic component. However, the development of a gene vector capable of delivering nucleic acids efficiently while remaining safe has been a challenge. Generally speaking, viral vectors are highly adept at delivering nucleic acids intracellularly. Viral vectors, however, have been known to be associated with insertional mutagenesis and immunogenicity and in severe cases, death. Furthermore, viral vectors are difficult to chemically modify for optimization and to mass produce. To date, despite that there have been more than 2000 gene therapy clinical trials worldwide, the U.S. FDA has yet to approve a gene therapy application. The lack of gene therapy approval by regulatory bodies delineates the need for safer and more effective vectors. A safer alternative to viral-based vectors are cationic, amine-containing polymeric vectors. Cationic polymers are capable of ionically complexing nucleic acid and forming nanoparticles on the order of 100-250 nm. This particle diameter is capable of extravasating through mal-formed tumor vasculature. The polymers can be engineered to have a buffering ability that allows the nucleic acid cargo to escape the lysosomal degradation pathway post-endocytosis. Ester-containing backbone polymers are capable of hydrolytic degradation, which helps mitigate toxicity. However, much is unknown regarding how polymer structure affects gene delivery. To date, the polymer vectors which work well have been discovered through empirical methods. Understanding how polymer structure affects gene delivery function would allow for a more rational approach to designing new vectors for gene delivery. The objective of this work has been two-fold: the first objective has been to elucidate non-viral gene delivery barriers – in particular investigating polymer structure-function relationships for polymeric vectors; the second objective has been to develop a polymeric/inorganic hybrid, theranostic-enabling nanoparticle platform technology capable of co-delivering DNA and siRNA and modulating temporal release. More specifically, this work details how polymer structure of poly(β-amino ester)s (PBAEs) affects polymer-DNA binding and how binding affects transfection levels, viability, and nanoparticle physical properties (zeta potential and diameter). We found transfection levels are biphasic with respect to binding in two human cancer cell lines and that binding constants in the range of (1-6)x104 M-1 were necessary but not sufficient for optimal transfection. We also investigated the comparative binding strengths of branched and linear polyethyleneimine, poly(L-lysine) and PBAEs with plasmid DNA and found PBAEs have the weakest binding. This work also details new bioassays including a more high throughput method for assessing cellular and nuclear uptake rates using flow cytometry. This method may be used for elucidating structure-function relationships in various cell types. An auto-fitting, first order mass-action kinetic model was developed in MatLab to quantify the intracellular delivery rate constants for comparing delivery bottlenecks of various polymer structures in various cell lines. This model was used to assess rate differences between polymers which do not transfect well, tansfect mediocre, and transfect well in primary human glioblastoma in vitro. The model recapitulated the experimental data with good agreement without needing to extrapolate data from literature. Principal component analysis is a method to look at large data sets with unknown variable correlations and to quantify how each variable is correlated with another, as well as which and to what degree each variable may drive another. Principal component analysis was utilized to look at 27 physico-chemical properties and cell gene delivery outcomes (i.e., uptake, transfection levels, and viability). We found that certain key parameters, such as hydrophobicity, drove uptake and transfection. The development of a theranostic-enabling platform technology involved gold nanoparticles and a layer-by-layer coating process to create polymer-inorganic hybrids. Gold nanoparticles are relatively biocompatible, are monodisperse, have interesting optical properties, have photothermal capabilities, and are easily chemically modified via thiol bonds. Gold nanoparticles can be synthesized to absorb a desired wavelength of electromagnetic radiation and can be imaged through various modalities such as photoacoustics or X-rays for diagnostic purposes. The theranostic-enabling technology was capable of co-delivering DNA and siRNA as well as delivering two layers of DNA with two different expression time profiles. Co-delivering DNA and siRNA could allow for the knockdown of a dysfunctional aberrant protein while replacing it with a functional protein. The ability to express proteins with different time profiles could have multiple applications such as controlling stem cell differentiation