Prominent Platforms for the Synthesis of Size-Controllable Polymeric Nanoparticles for Gene Delivery

Abstract

Gene therapy, a promising and emerging medical approach, involves delivering exoge- nous DNA or RNA into host cells to manipulate their genetic expression profiles. In cancer treatment, targeting gene therapy offers significant advantages over traditional methods, such as precise control of targeted genes, high efficiency at low dosage, and minimal side effects. However, protecting the integrity of genetic materials during delivery poses a critical challenge. The polymeric nanoparticles (PNPs), as an appealing approach for safeguard- ing genetic material, present benefits such as a high payload, stable colloidal and storage stability, and versabtile functionalization.This study focuses on the development of novel PNP gene delivery platforms. The PNPs consist of perfluoroheptanoic acid (PFHA), polyethylenimine (PEI), and low molecular weight heparin (LHP). These components can condense nucleic acids into compact nanopar- ticles (NPs) while maintaining robust stability and minimal cytotoxicity. To minimize thebatch-to-batch heterogeneity in NPs’ properties, we introduced two distinct platforms in- cluding the rotor syringe mixing (RSM) system and a microfluidic chip-based system to streamline and ensure the consistency of the NP synthesis process. These platforms offer precise and rigorous control of the parameters of NP synthesis and in turn result in monodis- persed NPs as indicated by the significantly reduced polydispersity index (PDI). In this study, RSM system was first employed as it utilized a syringe pump to control the flow rate and a rotor to control the stirring rate for controllable synthesis and homoge- neous mixing. The synthesis via RSM is highly tunable as it has been found that parameters including the reagent concentrations, injection flow rates, and stirring velocities all affect the outcomes of NPs. However, the RSM synthesis method can only process limited volume at one time, which limit the scalability of such process. The microfluidic chip-based system was then utilized to address the scale-up challenge. After evaluating multiple microfluidic channel configurations, we chose a design with a broader central channel bordered by two narrower side channels. The central channel introduced air and serve as the pneumatic control channel. The reagent solutions were infused into side channels by syringe pumps. This channel configuration helped balance the pressure within different channels on the chip, which effectively resolve issues like leakage and diminished controllability. With optimized air speed and solution infusion rates, NPs can be produced in a highly efficient and consistent manner. New customized microfluidic chip designs are also being pursued for better out- comes. Furthermore, this microfluidic method was able to produce ultra-small NPs around 20 nm in diameter. The NPs produced from both synthesis methods exhibited a well-balanced performance in terms of desired physicochemical properties, robust stability, efficient cellular uptake and endosomal escape profiles leading to successful gene transfection. This research offers an in-depth exploration of gene delivery and help paves the way for enhanced gene therapies, signaling a transformative phase in future medical treatments.