(609f) Microfluidic Engineering of pDNA Nanogels Using a Co-Axial Flow Reactor

Gene therapy requires vectors that enable the safe transport of genetic material across cell membranes. Nanogels (NGs) posses multifunctional characteristics viz. sizes <200 nm which enable them to enter the cell via endocytosis and high drug loading and encapsulation capability for a wide range of bioactive molecules such as pDNA, mRNA, oligonucleotides. Their biocompatibility, safety as carriers, and diverse drug release properties (stimuli-responsiveness, controlled response at the target site), render them an excellent model to be used as carriers for gene therapy. In this work, a coaxial flow (CFR) reactor is used to synthesize Carboxymethyl chitosan grafted Polyethylenimine nanogels (CMC-bPEI-pDNA NGs) as carriers for pDNA delivery, due to its laminar flow mixing, suitable for shear sensitive pDNA, ease of operation, and a simple and economic reactor assembly. The CFR is made up of an inner capillary (Inner diameter 0.14 mm) inserted into a larger capillary (Inner diameter 1.12 mm) to form a core flow surrounded by a sheath flow. Mixing occurs at the point where the core and sheath streams meet (outlet of the inner capillary), and the reaction proceeds across the length of the reactor. For polymeric NGs, the cationic polyplexes formed by the polymer complex and pDNA, not only condense the pDNA into smaller particles but are also expected to adhere to the negatively charged cell membrane via electrostatic interactions, thus overall facilitating cellular uptake.

We used the CFR to engineer cross-linked cationic nanogels of diameter <200 nm, with a polydispersity index (PDI) <0.3, encapsulation efficiency (EE) >90% and a high transfection efficiency (>80%). We performed a systematic parametric optimisation including variation of residence time, flow ratio, NP ratio (number of moles of nitrogen in polymer to moles of phosphate in pDNA and cross-linker), cross-linker concentration and pDNA loading. The reagents, polymer solution was added through the core capillary, while the pDNA and cross-linker through the sheath capillary of the CFR, at a particular flow ratio - FR (ratio of core flowrate to the sheath flowrate). The CFR decreased the reaction time for complexation of nanogels to as low as 7 s. All NGs prepared with NP ratios from 0.2 to 48 had sizes below 200 nm (measured using Dynamic Light Scattering) and in the range for endocytic uptakes. However, these NGs were negatively charged for NP < 5. It is interesting to note that the surface charge on NGs can be manipulated in a CFR from cationic to anionic at higher flow ratios (core/sheath) FR>2 or when the location of the reagent streams were interchanged. Hence, cationic monodisperse nanogels with diameter <200 nm and %EE>85% were produced. This tuneability of the overall charge is advantageous as it can avoid the requirement of an additional coating on the nanogels performed to render them cationic for systemic administration. We also found that the encapsulation efficiency can be increased to >99.5% by increasing the cross-linker concentration. Furthermore, we could load pDNA as high as 75 ng/μL and still attain highly monodisperse NG sizes: ~165 nm, PDI < 0.1, EE>99%. The final optimized formulation of nanogels was then scaled-up using a larger CFR and increasing the throughput tenfold. Overall, these NGs successfully deliver the payload to HEK293T cells, similar to the commercially available non-viral vector Lipofectamine 3k after 72 hours.