The intracellular fate and transport mechanism of shape, size and rigidity varied nanocarriers for understanding their oral delivery efficiency.

Nanocarriers have become an effective strategy to overcome epithelial absorption barriers. During the absorption process, the endocytosis mechanisms, cell internalization pathways, and transport efficiency of nanocarriers are greatly impacted by their physical properties. To understand the relationship between physical properties of nanocarriers and their abilities overcoming multiple absorption barriers, nanocarriers with variable physical properties were prepared via self-assembly of hydrolyzed α-lactalbumin peptide fragments. The impacts of size, shape, and rigidity of nanocarriers on epithelial cells endocytosis mechanisms, internalization pathways, transport efficiency, and bioavailability were studied systematically. The results showed that nanospheres were mainly internalized via clathrin-mediated endocytosis, which was then locked in lysosomes and degraded enzymatically in cytoplasm. While macropinocytosis was the primary pathway of nanotubes and transported to the endoplasmic reticulum and Golgi apparatus, resulting in a high drug concentration and sustained release in cytoplasm. Besides, nanotubes can overcome the multi-drug resistance by inhibiting the P-glycoprotein efflux. Furthermore, nanotubes can open intercellular tight-junctions instantaneously and reversibly, which promotes transport into blood circulation. The aqueous solubility of hydrophobic bioactive mangiferin (Mgf) was improved by nanocarriers. Most importantly, the bioavailability of Mgf was the highest for cross-linked short nanotube (CSNT) which outperformed free Mgf and other formulations by in vivo pharmacokinetic studies. Finally, Mgf-loaded CSNT showed an excellent therapeutic efficiency in vivo for the intervention of streptozotocin-induced diabetes. These results indicate that cross-linked α-lactalbumin nanotubes could be an effective nanocarrier delivery system for improving the epithelium cellular absorption and bioavailability of hydrophobic bioactive compounds.

The kinetic mechanism of cations induced protein nanotubes self-assembly and their application as delivery system.

The amphiphilic proteins can be used as building blocks (BBs) forming various self-assemblies. Understanding their self-assembly mechanism is important for designing novel nanomaterials. Herein, the BBs dimers were first prepared from carboxyl-abundant enzymolyzed α-lactalbumin (α-lac) at 50 °C. Then the unidentate coordination of Ca2+ between the BBs caused a ß-sheet stacking to further self-assemble into nanotubes (NTs). Compared with the traditional "one-pot" method, a step-wise new method was applied to study hydrolysis, aggregation and self-assembly processes separately. The α-lac was hydrolyzed into 11 kDa amphiphilic peptides independent of temperature while a BBs dimer was formed at 50 °C by hydrophobic interaction. Ca2+ induced a conformational change of BBs and promoted these BBs gradually aggregate into 10 strands of filaments, which twisted into helical ribbons by electrostatic repulsion. Ca2+ further induced the twisted helical ribbons closed into NTs driven by the reduction of line tension energy. Besides, the carboxyl-Ca2+ coordination dominated NTs elongation in the longitudinal direction and filaments aggregation in the lateral direction with the same binding stoichiometry of 1:1 respectively. Finally, NTs successfully encapsulated curcumin and improved the viscosity of liquid food. α-Lac NTs show a high potential as a delivery system for food applications.