Whiteley, Zoe Leigh; (2023) Microfluidic Production of Plasmid DNA Nanogels as Non-Viral Gene Delivery Vectors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years, gene therapy has become recognised as a revolutionary approach for treating genetic conditions. Viruses are commonly employed to deliver the genetic material, owing to their unique ability to enter the cell and deliver their payload. Despite the advantages, drawbacks in terms of safety exist, prompting the development of non-viral alternatives. One such alternative are nanogels, these being hydrogel nanoparticles with cross-linked polymeric structures, with advantages of high encapsulation, biocompatibility and stimuli responsiveness. The main limitations of nanogel production are the lack of reproducibility and homogeneity, which we propose may be addressed using microfluidics. The microfluidic approach involves the reduction of reactions down to the micro-scale, supporting uniform and controlled mixing. The overarching aim of this thesis is therefore to design a microfluidic system for producing plasmid DNA-loaded nanogels, in order to achieve a reproducible, scalable and biologically effective non-viral system. Initially, chitosan nanogels were developed in an acrylic hydrodynamic flow-focussing microchip, which led to fouling of the polymer in the microchannels. Contact angle analysis determined that batches of acrylic used were inconsistent in their surface properties, thus a more reliable glass coaxial flow reactor was designed, which overcame fouling issues and facilitated higher throughput. The chitosan was optimised for gene delivery, whereby the more soluble carboxymethylchitosan was conjugated to branched polyethylenimine. The polymer was cross-linked with sodium tripolyphosphate in the microreactor, producing nanogels of desired size, polydispersity and encapsulation efficiency. The in vitro transfection was assessed in both HEK293T and HT1080 cells which showed higher transfection than the gold-standard reagent. This prompted the use of nanogels as transfection reagents for the manufacture of AAV viral vectors, owing to the high transfection efficiencies in HEK293T cells. Equivalent viral titres to the current commercially available reagents were achieved, albeit at lower cost, greater scalability and higher formulation stability.

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