Formulation design of lyotropic liquid crystalline nanoparticles governs mesophase organization, colloidal stability, and in vitro NAD+ release.

Lyotropic liquid crystalline nanoparticles (LLCNPs) are lipid-based nanosystems with ordered aqueous and lipid domains that can be exploited to accommodate challenging hydrophilic biomolecules. In this study, glyceryl monooleate (GMO)-based LLCNPs were developed for NAD⁺ loading, using Poloxamer 188 (P188) as a steric stabilizer and polyethylenimine (PEI) as a cationic polymer. Empty and NAD⁺-loaded GMO:P188 and GMO:P188:PEI formulations were prepared by a top-down method and characterized in terms of hydrodynamic diameter, polydispersity index, ζ-potential, encapsulation efficiency, storage and stress stability, drug leakage, in vitro release, and internal mesophase organization by SAXS. Increasing Poloxamer 188 content reduced the size of empty GMO:P188 nanoparticles, while PEI incorporation increased particle size and shifted the ζ-potential toward less negative values. NAD⁺ loading increased the hydrodynamic diameter of most formulations without compromising colloidal homogeneity, with encapsulation efficiency ranging from 38.5 ± 4.0% to 44.5 ± 5.0% across the formulation series. PEI-containing LLCNPs showed lower cumulative NAD⁺ release at 24 h ranging from 66 ± 2% to 70 ± 4%, compared with 78 ± 3% to 87 ± 6% for GMO:P188 LLCNPs. Moreover, the highest mean NAD + leakage observed after 90 days of storage was 10.5 ± 2.1%. SAXS analysis confirmed the presence of an inverse bicontinuous cubic mesophase with Pn3̄m symmetry, while formulation composition influenced lattice parameter and long-range ordering. Overall, the results demonstrate that GMO-based LLCNPs can be compositionally tuned to regulate colloidal stability, NAD⁺ retention, mesophase organization, and release behavior, supporting their potential as nanocarriers for hydrophilic charged biomolecules.