Mechanistic correlation of mixing kinetics and PLGA nanoparticles size in a hydrodynamic flow focusing microfluidic device

Microfluidics has emerged as an innovative approach for tuneable and controlled synthesis of nanoparticles, offering distinct advantages over conventional methods. Precise control of the physicochemical properties of nanoparticles is essential for optimising their therapeutic performance. In this study, we address the analytical challenge of predicting Poly (lactic- co -glycolic acid) (PLGA) nanoparticle size during synthesis by investigating whether mixing time (τ mix ), measured in-line and in real time within a hydrodynamic flow focusing (HFF) microfluidic platform, can serve as a predictor of nanoparticle size. Because nanoparticle size is conventionally assessed only offline after synthesis is complete, there is currently no methodology for real-time size prediction during microfluidic manufacturing. To evaluate mixing time as an analytical predictor, flow rate ratio (FRR) and total flow rate (TFR) were systematically varied to generate a broad range of mixing conditions. While FRR is widely regarded as the dominant parameter controlling nanoprecipitation, the influence of TFR on mixing dynamics and nanoparticle formation remains less understood. Mixing time was quantified in-line using a novel flow-visualisation method, and the observed trends were subsequently validated using computational fluid dynamics (CFD). Results from flow visualisation and CFD showed that τ mix is strongly dependent on FRR but that, despite being neglected from most theoretical models, TFR also has a measurable effect on τ mix . This effect was further demonstrated through PLGA NPs synthesis under 16 different flow conditions. As expected, decreasing FRR reduced mixing time and led to the formation of smaller nanoparticles. However, increasing TFR also reduced mixing time and consequently decreased nanoparticle size. For example, at a constant FRR of 0.05, increasing TFR from 250 μL/min to 1000 μL/min reduced PLGA NP size from 156.6 ± 2.56 nm to 128.9 ± 2.53 nm. Together, these results establish τ mix as a real-time, in-line measurable parameter that correlates strongly with offline-determined PLGA nanoparticle size. In this analytical framework, nanoparticle size is treated as the target quality attribute, while mixing time, derived from in-line flow visualisation, is evaluated as a measurable surrogate parameter for predicting that attribute during microfluidic nanoprecipitation.