Nanodisc (ND)-forming membrane scaffold proteins or peptides developed from apolipoprotein A-I (apoA-I) have led to considerable promise in structural biology and therapeutic applications. However, the rationale and regularity characteristics in peptide sequence design remain inconclusive. Here, we proposed a consensus-based normalization approach through the reversed engineering of apoA-IΔ1–45 to design reconfigurable apoA-I peptide analogs (APAs) for tunable ND assembly. We present extensive morphological validations and computational simulation analyses on divergent APA-NDs that are generated by our method. Fifteen divergent APAs were generated accordingly to study the assembly machinery of NDs. We show that APA designs exhibit multifactorial influence in terms of varying APA tandem repeats, sequence composition, and lipid-to-APA ratio to form tunable diameters of NDs. There is a strong positive correlation between DMPC-to-APA ratios and ND diameters. Longer APA with more tandem repeats tends to yield higher particle size homogeneity. Our results also suggest proline is a dispensable residue for the APA-ND formation. Interestingly, proline-rich substitution not only provides an inward-bending effect in forming smaller NDs but also induces the cumulative chain flexibility that enables larger ND formation at higher lipid ratios. Additionally, proline-tryptophan residues in APAs play a dominant role in forming larger NDs. Molecular simulation shows that enriched basic and acidic residues in APAs evoke abundant hydrogen-bond and salt bridge networks to reinforce the structural stability of APA-NDs. Together, our findings provide a rational basis for understanding APA design. The proposed model could be extended to other apolipoproteins for desired ND engineering.
Emulsions offer a promising approach for enhancing the bioavailability of lipophilic active compounds when administered orally. Nonetheless, the impact of lipid matrix composition on the efficacy of penetration and bioavailability remains uncertain. This research investigated the effects of solid lipid ratio in emulsions on colloidal stability, mucus permeability, and bioavailability in vivo . To assess colloidal stability in the gastrointestinal tract (GIT), Turbiscan was employed. The results indicated that an elevated solid lipid ratio improved intestinal stability through the formation of aggregations that resisted pancreatic absorption, as confirmed by TEM. The absorption in various intestinal sections was tested using the Ussing Chamber model. Notably, emulsion with 0% solid lipid (G0M10) exhibited the highest cumulative permeation across the duodenum (221.2 ± 21.19 ng), jejunum (713.1 ± 20.93 ng), and ileum (1056.3 ± 392.06 ng) due to its higher in vitro release rate (> 60%) and smaller particle size. The cumulative permeation decreased with increasing solid lipid ratio. CLSM revealed that emulsions with a solid lipid ratio exceeding 50% exhibited poor mucus permeability within 15 minutes due to aggregation during the passage in the GIT. However, over an extended penetration time (30 minutes), higher permeability was observed, reaching approximately 30 μm. In vitro release studies indicated that a higher solid lipid ratio resulted in a reduced release rate of curcumin (<60%) compared to G0M10 (66.9 ± 3.58%). Correlation analysis unveiled a positive link between bioavailability and in vitro release rate, while a negative correlation emerged with the solid lipid ratio. This work underscores the significance of solid lipid ratios in emulsions for optimizing bioavailability through their influence on stability, permeability, and release of lipophilic compounds in the GIT.
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