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Date of Award
Olivia M. Merkel
Stephan M. Patrick
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that occurs naturally within the cell in a sequence-specific manner to break down double stranded RNA (dsRNA) and regulate RNA expression. Small interfering RNA (siRNA) is an intermediate in the RNAi process and synthetic siRNA can be used to down-regulate overexpressed genes as a potential therapeutic. However, the application of siRNA therapy in the clinic is limited due to an absence of biocompatible and efficient delivery systems. To address these issues, this dissertation focuses on the preparation of various polymeric nanoparticles for the delivery of siRNA as cancer therapy.
First, modifications of the endogenous polyamine spermine were employed to synthesize various cationic oligospermine conjugates that electrostatically condense the negatively charged siRNA molecules. Through the addition of hydrophobic oleic acid, our synthesized bis-spermine molecules spontaneously assembled into uniform micellular nanoparticles that are smaller than 80 nm in diameter and are capable of encapsulating siRNA. Furthermore, flow cytometry revealed that these particles had better intercellular uptake when compared to the un-modified bis-spermine particles.
Traditionally, polymeric nanoparticles are formed through standard batch reactor mixing processes. Unfortunately, the use of batch reactor mixing is unfeasible for larger scale production that is needed for clinical translation of these nanoparticle therapeutics. Microfluidic assembly of polymeric nanoparticles offers a suitable solution to these challenges and was investigated for the assembly of polyethyleneimine-graft-polycaprolactone-block-poly (ethylene glycol) (PEI-g-PCL-b-PEG) tri-block copolymer micelleplexes. Microfluidic mixing of these particles resulted in a more uniform particle distribution and smaller overall size. While these microfluidic particles did not show any benefit in our in vitro experiments, intratracheal administration of the microfluidic particles resulted in a significant increase of reporter gene knockdown in vivo.
Excision repair cross-complementation group 1 enzyme (ERCC1) is a structure specific endonuclease that plays a crucial role in the nucleotide excision repair (NER) pathway. Numerous clinical trials have reported that over-expression of ERCC1 is significantly correlated with low cisplatin chemosensitivity due to its central role in repairing the DNA damage caused by the drug. Here, the delivery of microfluidic PEI-PCL-PEG micelleplexes containing siRNA for the ERCC1 complex was optimized. These microfluidic siRNA / micelleplexes elicited robust ERCC1-XPF protein and mRNA knockdown within lung cancer cells Subsequently, the knockdown of ERCC1-XPF with this polymeric system resulted in a 1.8-fold change in cisplatin IC50 after colony formation assays were conducted. These combined results demonstrate the effectiveness of various polymeric nanoparticles for the delivery of siRNA therapies for the treatment of cancer.
Feldmann, Daniel Paul, "Preparation And Utility Of Polymeric Nanoparticles For The Delivery Of Sirna As Cancer Therapy" (2018). Wayne State University Dissertations. 1924.