In the formulation of nanoparticles, poly(lactic-co-glycolic acid) (PLGA) is commonly employed due to its Food and Drug Administration and European Medicines Agency approval for human use, its ability to encapsulate a variety of moieties, its biocompatibility and biodegradability, and its ability to offer a range of controlled release profiles. Common methods for the production of PLGA particles often adopts harsh solvents, surfactants/stabilisers and in general are multi-step and time consuming processes. This limits the translation of these drug delivery systems from bench to bedside. To address this, we have applied microfluidic processes to develop a scale-independent platform for the manufacture, purification and monitoring of nanoparticles. Thereby the influence of various microfluidic parameters on the physicochemical characteristics of the empty and the protein loaded PLGA particles was evaluated in combination with the copolymer employed (PLGA 85:15, 75:25 or 50:50) and the type of protein loaded. Using this rapid production process, emulsifying/stabilising agents (such as polyvinyl alcohol) are not required. We also incorporate in-line purification systems and at-line particle size monitoring. Our results demonstrate the microfluidic control parameters that can be adopted to control particle size and the impact of PLGA copolymer type on the characteristics of the produced particles. With these nanoparticles, protein encapsulation efficiency varies from 8 to 50% and is controlled by the production parameters employed; higher flow rates, combined with lower flow rate ratios should be adopted to promote higher protein loading. In conclusion, herein we outline the process controls for the fabrication of PLGA polymeric nanoparticles incorporating proteins in a rapid and scalable manufacturing process.
Figure 1. Optimisation of the process parameters for the production of polymeric nanoparticles using microfluidics. Polymeric nanoparticle were prepared by microfluidics and their physicochemical characteristics (particle size (bars) and PDI (dots)) at (A) FRR 1:1, (B) FRR 3:1 and (C) FRR 5:1 were measured. The effect of FRR on (D) ZP was also measured. Results represent the mean ± SD of at least three independent batches. SEM micrographs of (E) PLGA 75:25 nanoparticles FRR 1:1, (F) PLGA 75:25 nanoparticles FRR 3:1 and (G) PLGA 75:25 nanoparticles FRR 5:1 at 10 mL/min TFR.
Figure 2. The role of PVA in polymeric nanoparticles prepared by microfluidics. The effect of incorporating different concentrations of PVA (0.5%, 1% and 2%) as stabiliser in the H56 loaded PLGA 75:25 nanoparticles formulated using microfluidics TFR 10 mL/min and FRR 1:1 was investigated in terms of (A) particle size (grey bars) and PDI (white dots) and (B) ZP. A stability study of the particles at 4◦C in Tris 10 mM (pH 7.4) was carried out for a month: (C) particle size and (D) size distribution of H56 loaded PLGA 75:25. Results represent mean ± SD of triplicate measurements.
Figure 3. An overview of the process parameters measured during the production process of PLGA nanoparticles: A) Manufacture - polymer recovery after manufacture of the PLGA 50:50, 75:25 and 85:15 nanoparticles formulated using microfluidics (MF) at TFR 10 mL/min and FRR 1:1, 3:1 and 5:1, B) Purification via TFF - physicochemical characteristics before (MF) and after purification (TFF) and recovery of the polymeric nanoparticles (FRR 1:1, TFR 10 mL/min) and C) Monitoring - the particle size was monitored with Malvern OFF-line and AT-line in order to demonstrate the capability of the microfluidics method for continuous manufacturing of PLGA nanoparticles (particle sizes and intensity graphs for PLGA nanoparticles produced at FRR 1:1 and TFR 10 mL/min). For this study nanoparticles without loaded protein were studied.
Figure 4. Incorporation of proteins within the PLGA nanoparticles. The effect of the FRR on the physicochemical characteristics of the PLGA nanoparticles incorporating OVA (TFR 10 mL/min): (A) particle size and particle size distribution, (B) ZP and (C) encapsulation efficiency (% of initial amount added (* dilution factor); 0.2 mg/mL OVA) vs loading (wt/wt %). Results represent mean ± SD of at least triplicate measurements. SEM micrographs of OVA-loaded (D) PLGA 50:50, (E) PLGA 75:25 and (F) PLGA 85:15 nanoparticles formulated at FRR 1:1 and TFR 10 mL/min after purification.
Figure 5. The effect of the TFR on the physicochemical characteristics of OVA loaded PLGA nanoparticles (FRR 1:1): (A) particle size (bars) and particle size distributions (dots) and (B) ZP. Encapsulation efficiency (EE%) (solid line) vs loading (wt/wt %) (dotted line) for (C) PLGA 50:50, (D) PLGA 75:25 and (E) PLGA 85:15. Results represent mean ± SD of triplicate measurements.
Figure 6. The effect of the initial protein concentration loaded on the physicochemical characteristics of the PLGA nanoparticles produced using microfluidics at FRR 1:1 and TFR 10 mL/min: (A) particle size (bars) and PDI (dots), (B) encapsulation efficiency (EE%) and (C) loading (wt/wt%) for PLGA 50:50, PLGA 75:25 and PLGA 85:15. Results represent mean ± SD of triplicate measurements.