Multivariate Analysis for the Optimization of Microfluidics-Assisted Nanoprecipitation Method Intended for the Loading of Small Hydrophilic Drugs into PLGA Nanoparticles

Authors: E. Chiesa, R. Dorati, T. Modena, B. Conti, and I. Genta

Journal: International Journal of Pharmaceutics

DOI: 10.1016/j.ijpharm.2017.11.044.

Publication - Summary

November 24, 2017


Polymeric nanoparticles are efficient drug delivery vehicles that can deliver a wide range of bioactive agents. Specifically, poly(lactic-co-glycolic acid) copolymer (PLGA) based nanoparticles are biocompatible and biodegradable and approved by the US Food and Drug Administration (FDA) for human use. PLGA nanoparticles have been used for delivery of small molecule drugs, peptides, and nucleic acids; however, their bench-to-clinic translation has been challenging due to lack of a reproducible and scalable manufacturing method. PLGA nanoparticles have been previously generated using emulsification, evaporation, or bulk mixing-nanoprecipitation methods. Additionally, particle size uniformity and control has been challenging in PLGA nanoparticle manufacturing, further limiting their clinical application. In their 2017 publication in the International Journal of Pharmaceutics, the Genta lab from University of Pavia, Italy, have studied and optimized a method for generation of PLGA nanoparticles using the NanoAssembrTM platform for reproducible and scalable microfluidic nanoparticle formulation.

In order to optimize PLGA nanoparticle synthesis, the Genta lab varied several formulation parameters. In brief, the method applied in this article rapidly and reproducibly mixes an organic and an aqueous solvent in a microfluidic mixer. This allows fine control over the solvent/antisolvent precipitation of the nanoparticle components. Total flow rate (TFR) and Flow rate ratio (FRR) are two parameters affecting particle size that can be easily modified when using the NanoAssemblr Benchtop instrument. TFR is the speed at which the two streams of organic and aqueous solvents are pumped through the micromixer; and FRR is the volumetric ratio of aqueous/organic solvent streams. Past studies have indicated the variations in TFR and/or FRR can lead to size and polydispersity index (PDI) change in nanoparticles. The authors applied a randomized full factorial design to further study the effects of formulation variables. Combinations of different TFRs, FRRs, PLGA concentrations, and drug-to-polymer ratios were tested. N-acetylcysteine (N-Ac), a model hydrophilic small molecule drug, was used to assess encapsulation efficiency in the worst-case scenario since its entrapment with the highly hydrophobic polymer is typically challenging. It was found that for PLGA nanoparticles increasing the TFR from 5 to 15 mL/min and increasing of aq/organic FRR from 1:1 to 5:1 led to an overall size reduction with all the particles exhibiting reasonably small polydispersity indices (< 0.2). However, polymer concentration and drug-to-polymer ratio did not significantly affect the particle size. Three different organic solvents were tested as the initial solvent for PLGA, in addition to acetonitrile (results mentioned above), acetone and DMSO were chosen. The same nanoparticle composition with acetone led to smaller particles with higher particle surface charge compared to when acetonitrile or DMSO was used as the solvent. In the next step, polyvinyl alcohol (PVA), a surfactant, was used to see if it can improve particle characteristics. PVA increased particle size while reducing the surface charge. Hence, it was decided to move forward without surfactant and acetonitrile as the optimal organic solvent.  Next the effect of polymer molecular weight (MW) (25, 57 and 90 kDa) on particle size was investigated and it was found that MW directly affects PLGA nanoparticle size. This phenomenon can be used to alter and customize nanoparticle size by merely modifying molecular characteristics of the polymer. The PLGA nanoparticles were then used to encapsulate N-Ac. The influence of TFR and FRR were tested on encapsulation efficiency it was found that a TFR of 15 mL/min and an aqueous:organic FRR of 5:1 lead to N-Ac encapsulation of 65% or higher. Overall, higher TFR and FRR led to higher encapsulation efficiencies. In comparison to the NanoAssemblr microfluidic mixing, the bulk mixing underperformed and generated particles with large sizes and wide size distributions and thus required at least one additional step of filtration or centrifugation to achieve reasonable size and PDI. Moreover, at its best, bulk mixing only achieved about 21% encapsulation efficiency, which is significantly lower than the 65% achieved through microfluidic mixing. Interestingly, the N-Ac release rate of bulk-mixing nanoprecipitation was significantly higher than microfluidic formulated nanoparticles (90% vs 20% for 1 hour at 37˚C).

The findings of the Genta team confirm the advantages of the NanoAssemblr platform for development of polymeric nanoparticles. Specifically, PLGA polymers, which are well-known for their biocompatibility and biodegradability can be remarkably utilized using this novel microfluidic mixing technique to overcome hurdles such as particle size and polydispersity. Moreover, these finely size-tuned nanoparticles can entrap significant amounts of model drugs and are thus promising delivery vehicle for future clinical applications.


Design of Experiment-assisted evaluation of critical process (total flow rate, TFR, flow rate ratio, FRR) and formulation (polymer concentration and structure, drug:polymer ratio) variables in a novel microfluidics-based device, a staggered herringbone micromixer (SHM), for poly(lactic-co-glycolic acid) copolymer (PLGA) nanoparticles (NPs) manufacturing was performed in order to systematically evaluate and mathematically describe their effects on NPs sizes and drug encapsulation; a small hydrophilic moiety, N-acetylcysteine, was chosen as challenging model drug. SHM-assisted nanoprecipitation method consistently yielded NPs with tailor made sizes (in the range of 100-900 nm) and polydispersity index range from 0.061 to 0.286. Significant effects on NPs sizes were highlighted for TFR and FRR: increasing TFR (from 5 to 15 mL/min) and decreasing FRR (from 1:1 to 1:5 v/v, acetonitrile: buffer) NPs with mean diameter <200 nm were obtained. SHM technique allowed for flexible, application-specific tuning of PLGA NPs size using organic solvents with relatively low toxicity (acetone, acetonitrile), varying aqueous phase composition (Tris buffer vs PVA aqueous solution) and PLGA characteristics (Mw ranging from 25-90 kDa, capped or un-capped PLGA, different lactide:glycolide molar ratio). A very satisfactory N-Ac encapsulation efficiency (more than 67%) and a prolonged release (by 168 h) were achieved.

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