Novel protein-lipid composite nanoparticles as delivery systems of vitamin B12

• Author / Creator

  Liu, Guangyu

• Many protein and lipid based nanoparticles have been developed to encapsulate hydrophilic nature health products (NHPs) with the purpose to improve their oral bioavailability. However, the efficacy of these nanoparticles was usually compromised due to low encapsulation efficiency, low physical stability or poor release behaviour in the gastrointestinal tract. This work aims to combine protein and lipid to develop novel protein-lipid composite nanoparticles for hydrophilic NHP delivery. Specifically, vitamin B12 was focused which is essential for human life. The absorption of vitamin B12 is an intrinsic factor mediated and cubilin receptor dependent process. Gastrointestinal problems can impair the absorption pathway and lead to vitamin B12 deficiency. Thus, in this work the developed protein-lipid composite nanoparticles were applied as vehicles to load vitamin B12 and improve its absorption through alternative pathways in vivo.The first part of the thesis introduces the design, preparation and characterization of the protein-lipid composite nanoparticles. The nanoparticles were prepared using a modified double emulsion solvent evaporation method and the transmission electron microscope (TEM) demonstrated their unique three-layer structure (phospholipid layer, α-tocopherol layer and barley protein shell) and inner water compartments for loading of vitamin B12. In addition, the nanoparticles had homogeneous size distribution (size 243 nm, polydispersity index 0.17) and high encapsulation efficiency of vitamin B12 (69%). Such nanoparticles could also resist the gastric digestion, with only 10.4% of vitamin B12 released into the simulated gastric fluid after 2 h incubation. This is because the mechanical pressure treatment enhanced the formation of intermolecular β-sheets in barley protein, leading to solid interfacial films that could resist the harsh stomach environment. Also, protein hydrophobic groups were “locked” in the compact interfacial network. This greatly limited the gastric degradation of nanoparticles by pepsin which preferentially attacked the hydrophobic sites of proteins. Whereas in the simulated intestinal fluid, quicker degradation of nanoparticles was observed, with 65.8% vitamin B12 found in the release medium after 6 h.In the second study, the performance of these nanoparticles was improved by modifying the protein shell through succinylation, which significantly increased the particle surface charge, hydrodynamic diameter and decreased the surface hydrophobicity. The modified nanoparticles still maintained the favorable three-layer structure and high vitamin B12 encapsulation efficiency. The increased surface charge and spatial extension of succinate chain on nanoparticle surface improved the nanoparticle stability in both physiological buffer and water. The crosslinking by succinate minimized the leakage of vitamin B12 to 4.5% during one month of storage. Moreover, succinylation slowed down the pancreatic digestion of protein shell because the succinyl-lysyl-peptide bond was resistant to the tryptic hydrolysis. As a result, the modified nanoparticles demonstrated more sustainable release behavior in the simulated intestinal fluid, with around 62.1% vitamin B12 released in 10 h. In the last study, the biological responses of original and modified nanoparticles were evaluated. Both nanoparticles could enter the Caco-2 cells through non-specific endocytosis (clathrin mediated endocytosis and macropinocytosis) and increased the uptake efficiency of vitamin B12 over 20 folds. The original and modified nanoparticles also showed good mucoadhesive capacities (over 29.1%) which potentially prolonged the residence time of vitamin B12 in the intestine. A 14-day in vivo toxicity study showed no evidence of toxicity in rats. In vivo efficacy study showed that the developed vitamin B12 loaded nanoparticles could increase the serum vitamin B12 level and decrease the methylmalonic acid level more efficiently than the free form in a vitamin B12 deficiency rat model. Overall, this research represented the development of novel protein-lipid composite nanoparticles with elaborated design. Nanoparticles had many desired physicochemical properties (e.g. high encapsulation efficiency, good stability, controlled release behavior) and good biological responses (enhanced mucoadhesive property, increased vitamin B12 absorption), which overcame the limitations of sole protein and lipid based nanoparticles. Therefore, such nanoparticles have significant potential to improve the absorption of vitamin B12 in humans, especially those suffering from vitamin B12 malabsorption. This system could also be applied as a platform to deliver many other hydrophilic NHPs.

• Subjects / Keywords

  • succinylation
  • vitamin B12
  • oral nano-delivery
  • protein-lipid composite nanoparticle

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-59r3-x029

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.