Co-encapsulation of vitamin D₃ and Folic Acid in Anhydrous Milk Fat Solid Lipid Nanoparticles via a Solvent-Free Double Emulsion Method: Enhanced Stability, Controlled Release, and Gastrointestinal Protection

This study developed and optimised anhydrous milk fat solid lipid nanoparticles (AMF.SLNs) for co-encapsulating and delivering both VD₃ and FA simultaneously. Using a double emulsion method optimised by Response Surface Methodology (RSM) and Central Composite Design, the best formulation contained AMF (320 mg), Tween 80 (3.7% w/v), lecithin (12.5 mg), VD₃ (7.5 mg), and FA (4.2 mg). The optimised AMF.SLNs had a diameter of 126.4 ± 2.8 nm, a zeta potential of −28.6 ± 1.3 mV, spherical morphology, and high encapsulation efficiencies (91.7% VD₃, 87.3% FA). In vitro release studies demonstrated biphasic, sustained release up to 168 hours, with minimal release in simulated gastric fluid (8.4% VD₃, 14.2% FA at 2 h) and accelerated release in intestinal conditions (42.8% VD₃, 64.7% FA at 6 h). Encapsulation significantly improved the photostability and oxidative stability of both micronutrients. Storage at 4 °C for 30 days showed moderate changes in physical stability.