From Screening to Optimization: Strategic Implementation of Design of Experiments (DOE) for Robust Nanoparticle Formulation.

Design of experiments (DOE) offers a powerful, systematic framework for optimizing nanoparticle (NP) formulations by replacing inefficient one-factor-at-a-time (OFAT) methods. By enabling the simultaneous evaluation of multiple variables, DOE uncovers critical factor interactions and identifies true global optima-critical for quality-by-design approaches. Despite its potential for systematic innovation, DOE remains underutilized in nanomedicine due to its perceived complexity; this review provides a practical roadmap to bridge the gap between statistical theory and robust NP optimization. It provides a practical overview of DOE concepts, including factor selection, design choice, graphical interpretation of results (perturbation/contour plots), model validation (regression analysis and ANOVA), and numerical optimization via desirability function (D). Common pitfalls and best-practice strategies are discussed to support reliable model building and decision-making. A practical case study on poly(lactic-co-glycolic acid) (PLGA) NPs illustrates a multistage workflow: utilizing Taguchi screening to isolate key factors, followed by central composite design (CCD), for precise surface mapping. Numerical optimization using Design-Expert® software maximized EE% (highest importance) within size/zeta ranges, yielding optimal conditions (5 mg drug amount, 4 mL aqueous volume; D = 0.961). Confirmation runs (EE 41.2%, NP size 124 nm, zeta potential -15 mV) validated predictions (EE 47.6%, NP size 133 nm, zeta potential -17.2 mV), confirming model reliability. Ultimately, by bridging conceptual foundations with practical implementation, this review aims to encourage broader adoption of DOE, particularly among emerging formulation scientists, and serves as a roadmap to accelerate scalable NP development, fostering data-driven innovation and improving efficiency in nanomedicine research. Moreover, future integration of artificial intelligence (AI) and artificial neural networks (ANNs) with DOE will drive a predictive, data-driven approach to NP optimization-accelerating robust, scalable, and regulatory-ready nanomedicine development with fewer experiments.