Double-coated PLGA nanoparticles with hierarchical surface architecture for CD44-targeted siRNA delivery

Efficient delivery of small interfering RNA (siRNA) remains a materials challenge because it requires nanocarriers that stabilize polyanionic cargo, support cellular interactions, and enable cytosolic delivery. Although poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are used due to biocompatibility, biodegradability, and regulatory acceptance, siRNA delivery with PLGA requires interfacial engineering to meet these constraints. Here, a modular double-coated PLGA NP platform (dcNPs2.0) is developed and optimized for siRNA complexation, surface functionalization, and scalable manufacturing. The system comprises a PLGA core coated with a polyethyleneimine (PEI) interlayer to mediate siRNA binding, followed by a hyaluronic acid (HA) outer layer, which improves colloidal stability and promotes CD44-mediated uptake. Process optimization, including transition from batch nanoprecipitation to microfluidic fabrication, provides high yield, excellent reproducibility, narrow size distributions, and increased siRNA loading. X-ray photoelectron spectroscopy confirms hierarchical multilayer assembly. The optimized dcNPs2.0 formulation exhibited robust physicochemical stability during storage, in serum-containing media, and following lyophilization with appropriate cryoprotection. Functional evaluation of dcNPs2.0 demonstrated efficient HA-mediated cellular uptake and effective silencing following siRNA delivery in both two-dimensional monolayers and three-dimensional spheroids of MDA-MB-231 cells. Overall, this work establishes a scalable, rationally engineered PLGA nanoplatform that integrates extracellular targeting with intracellular delivery requirements for siRNA therapeutic applications. Graphical Abstract Schematic representation of the development and evaluation of double-coated nanoparticles (dcNPs) for siRNA delivery. dcNPs were designed with a poly(lactide-co-glycolic acid) (PLGA) core, polyethyleneimine (PEI), siRNA, and a hyaluronan (HA) outer coating. The formulation process was optimized from conventional nanoprecipitation, which showed batch-to-batch variability, through process refinement to improve concentration conditions, siRNA loading, and reproducibility, and finally translated to a microfluidic platform (dcNPs 2.0), enabling highly monodisperse, reproducible, and scalable NP production. The optimized dcNPs2.0 were then biologically evaluated for CD44-mediated cellular uptake and intracellular siRNA release, leading to gene silencing in 2D and 3D cancer cell models. Created with BioRender.