Design And Fabrication Of M Cells-targeting Starch-based LBL Self-assembled Microcapsules And Their Targeting Controlled Release Mechanism

With the improvement of living standards,bioactive molecules have surging great interests in modulating human health.However,bioactive moleculars suffer multiple barriers in the digestive tract during oral administration,which yields a low bioavailability.Using controlled release delivery systems is a feasible pathway to improve the bioaccessibility and bioavailability.M cells,located in the gut-associated lymphoid tissue of the Peyer's patches,have high-transcytotic capacity for bioactive molecules due to their unique morphology and specific properties in lack of hydrolytic enzymes and glycocalyx on the cell surface,which in turn increasing the bioavailability.Herein,rational designation of M cells-targeting delivery systems is essencial to improve the bioavailability of bioactive molecules.This study summarized the physiological features of the digestive tract and properties of M cells,analyzed the relationships between structures of starch-based materials,digestive tract environment,multi-scale structure and M cells-targeting delivery behaviors of the starch-based Lb L self-assembly microcapsules.And finally,oral M cells-targeting starch-based carrier materials and M cells-targeting starch-based Lb L self-assembly microcapsules delivery systems were obtained via structuring starch with M cell-targeting ligand,controlling the intermolecular interactions during the assembly process,storage,and the transition in the digestive tract.The underlying mechanism would provide significant guidance to improve the bioavailability of functional bioactive molecules.On the basis of the characteristics of M cell receptor and in the interactions between M cell-targeting ligand and specific receptor,molecular docking and molecular dynamic simulation along with MM?GBSA method were used to understand the effects of the structure and grafting amount of RGD containing peptides,and molecular weight of starch-based carrier materials on binding energy,conformation,and binding sites of the M cell-targeting ligand and receptor,aimed to unravel the roles of the grafting amount of RGD containing peptides and molecular weight of starch-based materials on M cells targeting properties.Results showed that GRGDS peptides have better targeting binding ability to M cell specific receptor,and play a decisive role during the targeting binding of starch-based carrier materials to M cell specific receptor,whilst,starch-based carrier materials increased their interaction with M cell specific receptor via reducing the molecular weight.M cells-targeting starch-based carrier materials with GRGDS grafting content from 0.50%to 1.12%and molar mass from 7.04×10⁴ g/mol to 2.06×10⁶ g/mol,and and substitute degree(DS)of carboxymethyl group from 0.228 to 0.252 were respectively obtained via chemical and enzymatic modification.In vitro studies were then conducted to unravel the M cells-targeting properties and their underlying mechanism of the materials,for better understanding the effects of GRGDS peptides grafting amount and molecular weight of the starch-based carrier materials on their M cells-targeting properties.Results indicated that the increase in GRGDS grafting amount along with the reduction in molecular weight significantly enhanced M cells-targeting ability and the cell transport efficiency of the carrier materials.It could be concluded that GRGDS peptides is the key factor affecting the M cells-targeting properties of the starch-based carrier materials,whilst,M cells-targeting starch-based carrier materials can be structured via controlling the GRGDS peptides grafting amount and molecular weight.For better understand the intermolecular self-assembly behaviors and the molecular mechanism of controlled release behaviors for bioactive moleculars of the M cells-targeting starch-based Lb L self-assembly microcapsules delivery systems,molecular dynamic simulation along with MM?PBSA and GBSA method was combined with multiple analytical methods,to analyse the effect of starch-based carrier materials structure,self-assembly conditions and digestive tract environment on the laws of the structure development and controlled release properties of the M cells-targeting starch-based Lb L self-assembly microcapsules.Results showed that GRGDS peptides would reduce the interaction between different starch-based carrier materials,and the reducing of molecular weight of starch-based carrier materials was benefit to optimize the multi-scale structure and M cells-targeting recognition of the microcapsules.Based on these findings,M cells-targeting starch-based Lb L assembly microcapsules with high encapsulation for immuneactive protein and compact fractal structure,Z-average size was 130-250 nm were obtained through fabrication with starch-based carrier materials with GRGDS peptides grafting amount from 0.50%to 0.55%,molecular weight from 7.01×10⁴ to 2.06×10⁶ g/mol and DS of carboxymethyl group from0.228 to 0.243.The storage stability of the microcapsules were analysised,and the effects of p H,structure of the starch-based carrier materials on the interaction between starch-based carrier materials,microcapsules multi-scale structures,targeting controlled release behaviors for immunoactive proteins were systematically investigated.Results showed that microcapsules fabricated with starch-based carrier materials with higher molecular weight and more layers showed better controlled release and M cells-targeting and higher transport efficiency by M cells.Overall consider the impact of molecular weight and GRGDS peptides grafting amount of the starch-based materials and the assembly layer quantity,microcapsules with three layers and Z-average particle size was 219.4-238.9 nm,which fabricated by starch-based carrier materials(GRGDS grafting amount=0.55%,Mw=4.61×10⁵ g/mol,carboxymethyl group DS=0.240)possess better controlled release(deliver 80.08%-81.55%of immunoactive protein to the M cells),M cells-targeting ability and higher transport efficiency(8.76×10^-5-9.38×10^-5 cm/sec).In this study,the relationship among the structure of starch-based carrier material,the environmental conditions of the digestive tract,the multi-scale structure of microcapsules and the M cells-targeting controlled-release characteristics of microcapsules were established from the molecular to molecular interaction level,through the methods of target modification and structural optimization of starch molecules.The results indicated that the M cells-targeting of starch-based carrier material and layer-by-layer self-assembly behavior between starch molecules and immunoactive proteins,oral controlled release delivery behavior and M cells-targeting properties of the fabricated microcapsules could be regulated.The M cells-targeting starch-based carrier materials and their layer-by-layer self-assembled microcapsules delivery system with excellent loading ability,stability,controlled release behaviors and M cells-targeting properties and the related design methods were obtained.This study provides theoretical guidance and technical support for the fabrication of oral M cells-targeting delivery system for bioactive molecules and improves their biological effectiveness.