Spatiotemporally programmed co-delivery via double emulsions enables absorption synergy of 5-MTHF-ca and vitamin D3

This study introduces an innovative double emulsion (DE) system for the co-delivery of water-soluble 5-methyltetrahydrofolate calcium (5-MTHF-Ca) and lipid-soluble vitamin D 3 (VD 3 ), addressing the challenge of synchronized delivery for nutrients with divergent solubility. Through a two-step emulsification strategy, co-loaded DEs (C-5-MTHF-Ca & VD 3 ) exhibited superior colloidal stability compared to single-loaded counterparts (S-5-MTHF-Ca and S-VD 3 ), retaining 88.17 ± 2.64 % of 5-MTHF-Ca and 81.99 ± 2.45 % of VD 3 after 15-day storage. During in vitro digestion, co-loaded DEs demonstrated delayed lipid hydrolysis (38.01 ± 5.55 % FFA release at 30 min vs. 43.88 ± 13.79 % for S-VD 3 ) and preserved structural integrity through gastric phases, while synchronized intestinal micellar incorporation increased VD 3 bioaccessibility by 19.82 %. Ex vivo fluorescence tracking in mice revealed spatiotemporal co-localization of both actives, with jejunal accumulation peaking at 2–4 h post-administration, aligning with Ussing chamber results showing 2.13-fold enhanced VD 3 permeation and 1.80-fold enhanced 5-MTHF-Ca transport in co-loaded systems. Pharmacokinetic studies in rats confirmed bioavailability enhancement, with co-loaded DEs achieving 1.37-fold higher VD 3 AUC (6707.08 ± 1123.82 vs. 4898.36 ± 685.16 ng·h/mL) and 1.19-fold increased 5-MTHF-Ca C max compared to single-loaded formulations. The extended T max (by twofold) and prolonged t 1/2 K a (1.81-fold for 5-MTHF-Ca) indicated optimized intestinal retention, attributed to the phase-separated W/O/W architecture protecting actives from premature degradation. These findings establish a platform for nutrient co-delivery, where rational DE design enables spatiotemporally coordinated gastrointestinal transit, and synergistic absorption.