Fabrication Of Self-assembled Chitosan-based Delivery Systems For The Effects Of Sustained And Controlled Release Of Bioactive Compounds

This work was funded by the National Key R&D Program of China?2018YFD0400300?.Recently,food-derived bioactive compounds have received extensive attention for their non-toxic,easily degradable,unique disease prevention,and health regulation effects.However,food-derived bioactive compounds must overcome the extreme p H conditions and various digestive enzyme system of the gastrointestinal tract.Thus,they should also pass through the small intestinal epithelial mucus layers and cells,be intactly transported to the blood circulation,reach the target point and accumulate to a certain amount to exert their unique physiological activity in the body.This phenomenon leads to the fact that most food-derived bioactive compounds have good functional activity in in vitro,but they do not exert their due effects in vivo.The extremely low bioavailability of food-derived bioactive compounds in vivo also limits their more comprehensive development and applications.Consequently,with the rapid development of nano-biotechnology,the formulation of an effective delivery strategy is of great significance for improving the food-derived bioactive compounds'intact absorption and bioavailability.It is also a crucial scientific problem that needs to be solved urgently in food-derived bioactive compounds.Therefore,in the present study,we first designed chitosan-tripolyphosphate?CS-TPP?nanoparticles?NPs?to solve the problem of easy degradation and low permeability of egg-white derived bioactive peptides?EWDP?.The research investigated the entrapment capacity of EWDP with different molecular weights.The results revealed the entrapment mechanism between CS-TPP NPs and EWDP.Thus,we evaluated the protective effect and absorption enhancement of the CS-TPP NPs on the EWDP.Subsequently,to improve the solubility of CS,this study used a chemical modification method to graft small molecules of L-arginine?Arg?,L-lysine?Lys?,N-acetyl-L-cysteine?NAC?,and L-cysteine?CYS?on the main chitosan chain.We obtained the Arg/Lys/NAC/CYS-CS with good water solubility and fabricated the CS-based nano-delivery systems by electrostatic interaction.The newly prepared NPs have a good core-shell structure,p H responsiveness,excellent entrapment,protection,controlled release,sustained release,and absorption enhancement for hydrophilic and hydrophobic bioactive compounds.In the present study,the CS-based delivery systems provide new ideas for improving the bioavailability of food-derived bioactive compounds in vivo and provide strong support for the in-depth research and broad application of food-derived bioactive compounds.The main research contents and conclusions of this article are as follow:?1?This research fabricated the CS-TPP NPs by electrostatic interaction and explored the entrapment capacity of CS-TPP NPs on EWDP with different molecular weights.The FTIR results revealed the entrapment mechanism of CS-TPP NPs on EWDP.Finally,this study evaluated the protection and absorption of CS-TPP NPs on EWDP.It was found that the particle size and the entrapment capacity for EWDP of the CS-TPP NPs increased with the rise of the initial concentration of CS.The entrapment capacity of CS-TPP NPs for EWDP also increased with the rising of environmental p H values.The results of particle size,Zeta potential,and FTIR revealed that the CS is first entangling with EWDP through strong hydrogen bond interactions and electrostatic interactions.After adding TPP,they formed a 3D network structure.When there are small EWDP in the systems,the negatively charged EWDP helps promote the particles'internal shrinkage to create more stable and uniform NPs.However,with the increase in the amount of EWDP,the positively charged-NH3⁺on the surface of the NPs gradually fell,the particle charge decreased,and the system stability dropped sharply.The CS-TPP NPs can significantly prevent the degradation of EWDP by gastrointestinal digestive enzymes in in vitro simulated digestion.The everted rat sac results revealed that the CS-TPP NPs could remarkably improve the intact absorption rate and increase the bioavailability of EWDP.?2?The present study used amphiphilic amino acids Arg and Lys to chemically modify the CS.Additionally,we also used Na OH to change casein?CA?to obtain a water-soluble CA.Subsequently,we got Arg/Lys-CS-CA NPs by electrostatic interaction.We also explored the colloidal properties,entrapment capacity,release,and controlled release properties and permeability enhancement of Arg/Lys-CS-CA NPs.The results showed that the smallest particle size of Arg/Lys-CS-CA NPs is110/82 nm.They are in a uniform and stable spherical core-shell configuration,with good p H response characteristics,28-day storage stability,and mucosal adhesion.Arg/Lys-CS-CA NPs have excellent amphiphilic properties.The entrapment efficiency of the hydrophilic bioactive compounds EWDP is in the field of 81-91%and 76-87%,and the entrapment efficiency of the hydrophobic bioactive compounds curcumin is in the range of 35-74%and 48-87%.Arg/Lys-CS as a hydrophilic shell plays a significant role in the entrapment of hydrophilic bioactive compounds.As an amphiphilic core,CA has a larger hydrophobic area and plays a substantial role in hydrophobic bioactive compounds'entrapment.The main driving force for the entrapment of hydrophilic bioactive compounds by NPs is the strong hydrogen bonding and electrostatic interaction between molecules.The entrapment of hydrophobic bioactive compounds is mainly realized through hydrophobic interaction and hydrogen bonding interaction.In vitro sustained release results showed that Arg/Lys-CS-CA NPs could significantly increase the bioavailability of egg white peptides and curcumin,limit the release of bioactive compounds in the stomach environment,and achieve slow release in the intestinal conditions.The gavage experiments in ICR mice found that Arg/Lys-CS-CA NPs can significantly increase the content of curcumin in the serum of mice and effectively increase the bioavailability of curcumin in vivo.?3?The small molecule substances NAC and CYS containing sulfhydryl groups are grafted to CS?degree of deacetylation?95%?main chain to obtain the water-soluble CS derivative NAC/CYS-CS.We fabricated the NAC/CYS-CS-CA NPs by electrostatic interaction.The present study found that the minimum particle size of NAC/CYS-CS-CA NPs is 89/86 nm.Besides,the NPs have good p H responsiveness,uniform spherical structure in TEM,layer-by-layer self-assembled structure,and 21-day storage stability.The NAC/CYS-CS-CA NPs have excellent amphiphilic properties.The entrapment efficiency of EWDP is 39-67%and 51-63%.For curcumin,the entrapment efficiency is 45-58%and 48-63%.The NPs achieve effective entrapment of EWDP through strong hydrogen bond and electrostatic interaction.For curcumin,the driving force is hydrophobic and hydrogen bond interaction.Both in vitro sustained release results and gastric absorption experiments in ICR mice showed that NAC/CYS-CS-CA NPs have an excellent controlled release,sustained release properties for food-derived bioactive compounds,and can effectively enhance the absorption of curcumin in Caco-2 cells.The intracellular absorption and the content in mouse serum verified the improvement of the bioavailability of the food-derived bioactive compounds in vivo.?4?The present study used NAC/CYS to modify CS?degree of deacetylation75-85%?to obtain water-soluble NAC/CYS-CS.The NAC/CYS-CS was spontaneously cross-linked with self-assembled?-lg?self-aggregated by glutaraldehyde?by electrostatic interaction.The study found that the smallest particle size of the NPs is 118/48 nm,which has good p H responsiveness and system stability.Compared with NAC/CYS-CS-CA NPs,it significantly improves the system stability under p H 6 conditions.NAC/CYS-CS-?-lg NPs are amphiphilic,the entrapment efficiency of EWDP is between 51-89%and 50-81%,and the entrapment efficiency of curcumin is 42-57%and 41-57%.The NPs entrap the EWDP through strong hydrogen bond and electrostatic interaction,and the entrap the curcumin through hydrophobic and hydrogen bond interaction.The NAC/CYS-CS plays a crucial role in the entrapment of EWDP.So the NPs have p H responsiveness to the entrapment of hydrophilic bioactive compounds.Besides,the self-aggregating amphiphilic?-lg core plays a key role in curcumin entrapment,but it can also entrap hydrophilic bioactive compounds to a certain extent.Therefore,the entrapment efficiency of the NPs does not have a p H response to hydrophobic bioactive compounds.The in vitro release results show that NAC/CYS-CS-?-lg NPs can limit the rapid release of hydrophilic and hydrophobic bioactive compounds under gastric conditions,and effectively promote the rapid transport of bioactive compounds on the Caco-2 cell monolayers.The enhancement of absorption can significantly improve the bioavailability of food-derived bioactive compounds in vivo.The permeability of NAC/CYS-CS-?-lg NPs for the egg white-derived bioactive peptide QIGLF can be increased by up to 2.04 times and 4.56 times.Additionally,the apparent permeability of curcumin is 7.45×10^-6 cm/s and 6.94×10^-6 cm/s.