Asymmetric Flow Field-Flow Fractionation (AF4) with Fluorescence Detection for Direct, Real-Time, Size-Resolved Measurements of Drug Release from Polymeric Nanoparticles
Polymeric nanoparticles (NPs) are typically designed to enhance the efficiency of drug delivery by controlling the drug release rate. Hence, it is critical to obtain an accurate drug release profile. This study presents the first application of asymmetric flow field-flow fractionation (AF4) with fluorescence detection (FLD) to characterize release profiles of fluorescent drugs from polymeric NPs, specifically poly lactic-co-glycolic acid NPs loaded with enrofloxacin (PLGA-Enro NPs). In contrast to traditional release measurements requiring separation of entrapped and dissolved drugs (typically by dialysis) prior to quantification, AF4-FLD provides in situ purification of the NPs from unincorporated drugs, along with direct measurement of the entrapped drug. Size distributions and shape factors are simultaneously obtained by online dynamic and multi-angle light scattering detectors. The AF4-FLD and dialysis approaches were compared to evaluate drug release from PLGA-Enro NPs containing a high proportion (≈ 88%) of unincorporated (burst release) drug at three different temperatures spanning the glass transition temperature (30 °C for PLGA-Enro NPs). The AF4-FLD analysis was able to identify size-dependent release rates across the entire continuous NP size distribution, with smaller NPs showing faster release. The AF4-FLD method also clearly captured the expected temperature dependence of the drug release (from almost no release at 20 °C to rapid release at 37 °C). In contrast, dialysis was not able to distinguish these differences in the extent or rate of release of the entrapped drug because of interferences from the burst release background, as well as the dialysis lag time. A mechanistic diffusion model that integrates data from both AF4-FLD and dialysis further supported the advantages of AF4-FLD to capture the true release rate of entrapped drug and avoid artifacts observed in dialysis. Overall, this study demonstrates the novel application and unique advantages of AF4-FLD methods to obtain direct, size-resolved release profiles of fluorescent drugs from polymeric NPs.