Polymeric-based drug and gene delivery is advantageous for a number of reasons. Polymers can increase the circulation time of the cargo, protect the cargo from enzymatic degradation, and improve localization at the target site. The present work focuses on three different polymers for drug and gene delivery: poly-lactic-co-glycolic acid (PLGA), polyethyleneimine (PEI) and polystyrene-co-poly(N-isopropylacrylamide) (PS-co-NIPAM). Each of these polymers has a niche within the drug and gene delivery field, and each has its own advantages and disadvantages. Formulations were prepared using each of the aforementioned polymers with an ultimate goal of translating the formulations to clinical applications.
The drug PD98059, which has been identified as a potential treatment following myocardial infarction or for leukemia, has been loaded into PLGA nanoparticles, microparticles, or pellets. Each of these formulations was characterized based on size and surface morphology, as well as the release rate of the PD98059. Additionally, an HPLC-UV method was developed for detecting PD98059 at low concentrations using the wavelength of maximum absorption. Included with this HPLC-UV method was a method for extracting PD98059 and an internal standard (7-hydroxyflavone) from murine plasma to promote future biodistribution studies.
Formulations were also prepared using PEI and plasmid DNA (pDNA). PEI is a cationic polymer that can improve the circulation time and cellular uptake of pDNA compared with naked pDNA alone. The PEI-pDNA complexes (nanoplexes) were delivered to two endometrial cancer cell lines: Ishikawa H and KLE. The pDNA encoded for mutations to the PLAC1 gene, which is a potential onco-target for endometrial cancers using the CRISPR-Cas9 delivery system. The relative gene expression of PLAC1 was measured after transfection, as well as the relative cell viability. In the Ishikawa cells, there was no improvement in transfection efficiency (decrease in PLAC1 expression) incurred by the PEI, and the cells did not undergo decreases in relative viability. However, in the KLE cells there were large decreases in relative cell viability in response to the nanoplexes, which also made it difficult to determine relative gene expression following transfection.
Transfection was also paired with a genotoxic engineered nanomaterial (ENM) to investigate how co-delivery of nanoplexes and ENMs may influence the efficacy of gene therapy. The relative viability of A549 and Ishikawa H cells indicated that co-delivery was less toxic to cells compared with mono-delivery of either agent. Furthermore, relative expression of the cell cycle regulating p53 gene was increased after 48 hours in Ishikawa cells that received as little as 10 µg/mL CuO ENMs. Further toxicity investigations of CuO ENMs, ZnO ENMs, and SiO2 ENMs were conducted in the two lung epithelial cell lines A549 and Beas-2B, which indicated that the A549 cells are more susceptible to changes in viability as a function of ENM dose than the Beas-2B cells. Furthermore, the A549 cells likely internalized the SiO2 ENMs through the caveolae-mediated endocytosis mechanism.
The uptake and toxicity of PS-co-NIPAM microgels, which are a thermo-responsive polymer, was assessed in HepG2 liver adenocarcinoma cells, which was ~3 µg/mL total (maximum) for a 90 µg/mL dose. As such, the endocytosis mechanism could not be determined for these investigations. The results also indicated that these microgels did not induce any toxicity at 150 µg/mL after 24 hour exposure, which is promising for their development as a biocompatible delivery system.
The overarching goal of this research was to use polymeric formulations, which are known to be both biocompatible and highly tunable, for improved drug and gene delivery. The breadth of applications in this thesis highlights the many potential applications of polymeric formulations, as well as the interdisciplinary advantages to using polymers as carriers for sustained- and/or controlled-delivery.