Design of Experiment (DOE) for Optimization of PLGA Nanoparticles.

Design of Experiments (DOE) is useful tool for formulation optimization. In present study, Design Expert® software (version 13) was used for optimization of Poly(dl-lactide-co-glycolide) (50:50) (PLGA) nanoparticles (NPs). Capecitabine, an approved anticancer drug with short half-life, was used as model drug. Capecitabine NPs can minimize dosing frequency and improve patient compliance. Effect of two numeric (drug and polymer amount) and one categoric factor (pH of W2 phase) was studied on NP Size and Entrapment Efficiency (EE). Drug amount and pH significantly influenced EE. Polymer amount significantly influenced NP size.

METHODS: PLGA NPs were prepared by double emulsion technique. Aqueous drug solution (W1) was emulsified with polymer solution in ethyl acetate using probe sonicator (Misonix) for 2 minutes. Formed primary emulsion was sonicated with second aqueous phase (W2) containing stabilizer Kolliphor®P188 for 1 minute to produce double emulsion. Organic solvent and excess water were evaporated at 600 rpm, 60°C for 1.5 hr and NPs were washed thrice (14,000 rpm, 4°C for 45 min) Lyophilized NPs were sonicated with acetonitrile for 5 min and EE was analyzed using UV/Vis spectrometer at ʎmax 304nm. Particle size was determined using Malvern Nano-ZS.

RESULTS: Experimental data were fitted into models of increasing polynomial complexity. EE data is best described by two-factor interaction model with R² of 0.9145. ANOVA results show model is significant. Predicted R²of 0.8076 is in reasonable agreement with adjusted R² of 0.8824. Drug amount, pH of W2 phase and their interaction are significant (p<0.05). Higher drug amount decreases EE which maybe because polymer amount was not sufficient to encapsulate increased drug amount and more drug migrated to W2 phase. Lower EE at pH 4.5 might be described by drug leaching into W2 phase due to better drug solubility. Diagnostic normal plot, perturbation plots, interaction plots and 3D-surface plot confirm model robustness. Size data was best fitted to linear model showing polymer amount being significant. Higher polymer amount might have decreased dispersion efficiency of first emulsion into W2 phase and led to fusion of semi-formed particles to larger size.

NUMERICAL OPTIMIZATION: The desired goals were set to minimize polymer amount (to increase drug amount per unit weight of NPs), to minimize drug amount and the pH was set at 7.4 to maximize EE. Predicted optimum responses were checked experimentally. Batch prepared with 120mg polymer and 25mg capecitabine has average size of 111.3 nm (predicted value 116.8nm) and EE of 38% (predicted value 40%), both with 5% coefficient of variance.

CONCLUSION: DOE gives an opportunity to look at individual and combined impact of various factors. It can provide valid conclusions with minimal experimental runs, time, and cost. Similar DOE approaches can be used for other PLGA-drug formulations optimization.

CONFLICT OF INTEREST: Authors declare no conflicts of interest.