Bioprocesses Intensification via Microfluidics for the Production of Ionizable Polymeric Nanoparticles for RNA Delivery

The use of therapeutic RNA, propelled by RNA vaccines and oligoribonucleotide gene therapy, is expanding to cancer, autoimmune, and amyloidogenic diseases. Yet instability, off-targeting, Toll-like receptor (TLR) activation, and poor membrane permeability hinder delivery. Hence the need for scalable, efficient, site-specific, non-immunogenic systems. Smart nanoparticles, especially polymeric, enable controlled stimulus-responsive release while improving stability and shelf life. Microfluidic technologies, particularly microreactors, allow continuous nanoparticle production with precise mixing and particle control via laminar flow and tailored channel geometries, and facilitate numbering-up for reproducible lab-to-industrial scale manufacturing. This work focuses on optimizing microfluidic parameters to form complexes between pre-formed ionizable polymeric nanoparticles (IPNs) and a model oligoribonucleotide (<100 bp) to simulate therapeutic sequences. RNA-loaded carriers were produced in 3D-printed microreactors of varying geometries using a central composite design (CCD) to assess how flow dynamics affect nanoparticle properties. IPNs consist of PMMA-co-DEAEMA with DEAEMA 5% w/w, a pH-sensitive comonomer enabling electrostatic RNA binding. Nanoparticles arose by free-radical miniemulsion polymerization at a 9:1 aqueous (water, CTAB, sodium bicarbonate) to oil (hexadecane, MMA, DEAEMA) ratio. After 5-min sonication on ice, polymerization proceeded in an EasyMax-102 reactor using KPS initiator. RNA complexation was evaluated by comparing bulk mixing (Eppendorf) and microfluidic methods employing syringe pumps and Flower Nanotechnology® microreactors. Flow rates ranged from 30 to 3000 µL/min allowing analysis of hydrodynamic impacts. Results show that flow rate, channel geometry, and precursor concentrations significantly influence biophysicochemical properties, such as particle size, surface charge, and polydispersity index (PDI).  Surface responses from CCD experiments identified optimal process variable ranges for nanoparticle production to meet therapeutic requirements. Overall, the study advances reproducible, scalable microfluidic processes for RNA delivery applications and reinforces polymeric nanocarriers and microfluidics in next-generation therapies.

This work was supported by Financiadora de Estudos e Projetos (FINEP) 01.22.0514.00(0503/22) and by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) 260003/015754/2021.