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Preparation Of Oral Intestinal Stabilization Systems Of Lactoferrin And Their Properties

Posted on:2023-07-31Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y S WeiFull Text:PDF
GTID:1521307103992259Subject:Food Science and Engineering
Abstract/Summary:PDF Full Text Request
Lactoferrin(Lf)is an important whey functional protein with many bioactivities.Lf can exhibit"local effects"in gastrointestinal tract(GIT)or be absorbed into the bloodstream to play"absorption effects",but its oral bioavailability is very low at present.It is essential to overcome the critical problems that hampered the development of oral Lf systems,such as maintaining Lf stability in GIT,achieving specific delivery and being effectively adsorbed into bloodstream.Studies have shown that the composite small intestinal stabilization systems can effectively improve the oral stability of Lf,but there are still problems of low Lf loading efficiency of core and poor controlled release performance of shell structure.Recently,the colon has also been considered a potential absorption site for functional proteins,but there have been no reports on Lf colonic stabilization systems.In view of the above problems,a composite small intestinal stabilization system(i.e.,pH-responsive composite microcapsule)with high loading efficiency of Lf was firstly constructed.The improvement of the small intestinal local effect of the stabilized Lf and its pharmacokinetic behavior were investigated.Then,the efficient colonic stabilization systems were constructed and their performances were discussed.To improve the colonic delivery efficiency of Lf,this study took the improvement of carrier’s hydrophobicity as breakthrough point,and explored the feasibility of preparing highly-hydrophobic core-shell nanofibers using a hydrophilic polymer by one step electrospinning process for the first time.The rule and mechanism of Lf colon-specific delivery and release by a highly-hydrophobic core-shell structured carrier was clarified.Furthermore,combined with the regulation of electrospinning process and parameter conditions,a highly-hydrophobic composite fiber system with different microstructures was constructed,and the influence of the micro-structural changes on the colonic release behavior of Lf was investigated.Meanwhile,the effect of the release rate of Lf on its local effects in the colon and the pharmacokinetic behavior were evaluated.Main research contents and results are as follows:(1)Construction of the pH-responsive composite microcapsule loaded with Lf and its properties.Firstly,an efficient small intestinal delivery system of Lf was constructed.The negatively charged nanoparticle consisting of poly(lactic co-glycolic acid)(PLGA)and sodium polystyrene sulfonate(PSS)were prepared,and cationic Lf was electrostically adsorbed on the surface of PLGA/PSS NPs followed by coating with the pectin to obtain the Lf nanosystem(Lf NS).The loading efficiency of Lf NS under the optimal conditions was73.4%.Then,an anti-solvent process involving non-organic solvents was designed based on the pH-responsive solubility of the enteric-soluble polymer Eudragit S100(ES100),through which Lf NS was successfully encapsulated in the microcapsule system formed by ES100.The optimal concentration of ES100 in the anti-solvent process was 5.0 mg/m L.In vitro release results showed the obtained pH-responsive composite microcapsule(pHCM)could effectively avoid the release of Lf in simulated gastric fluid(SGF,<1%),and realized the controlled release of Lf in simulated intestinal fluid(SIF),the release of Lf in SIF involved a super case II transport mechanism,in which the degradation of the polymer matrix was dominant.In vitro cell experiments showed that pHCM enhanced the proliferation activity of oral Lf to crypt epithelial cells of small intestine.In vivo experiments showed that pHCM significantly increased the oral relative bioavailability of Lf(13.03%vs.1.97%).In addition,the peak concentration Cmax and peak time Tmax of Lf in serum samples were significantly increased(240.83 ng/m L vs.58.91 ng/m L,1.5 h vs.4.0 h).In other words,the constructed pHCM extended the absorption and transport time of Lf through small intestine.(2)Construction of Lf-loaded highly-hydrophobic core-shell nanofiber and its properties.The hydrophilic polymer ES100 was continuously used as the base material to construct an efficient colonic stabilization system of Lf.A suitable biocompatible solvent,acetic acid,was screened as the mono-solvent for ES100 electrospinning,creating highly-hydrophobic ES100nanofiber mat(contact angle 133.8~o).The main mechanism mediating the hydrophilic to highly hydrophobic transition of ES100 is that the surface energy of ES100 electrospun film is significantly lower than that of cast film.Then,in order to solve the incompatibility between shell fluid of ES100 acetic acid solution and aqueous solution,Lf-loaded W/O emulsion was prepared as the core fluid to ensure the successful coaxial electrospinning,and a novel high hydrophobic core-shell nanofiber was successfully prepared by controlling the flow rate of core emulsion at 0.3 m L/h during coaxial electrospinning process.In vitro assay indicated the cumulative release of Lf in the simulated digestion fluids was less than 1%,and92.3%Lf was sustainably released in the simulated colon fermentation fluid(SCF)during 30h.The release of Lf in SCF involved a non-Fickian transport mechanism,wherein the release is controlled by the diffusion release and the polymer matrix erosion.The synergistic action through gut microbiota adhesion and their metabolites,especially short-chain fatty acids,was illustrated for the disintegration of highly-hydrophobic core-shell nanofiber for the first time.MTT results showed that Lf could not exert its anti-proliferation activity effectively due to its slow colonic release rate.(3)Construction of the highly-hydrophobic composite nanofiber system and its regulation on the colonic release behavior of Lf.In order to improve the colonic release behavior of Lf,a highly-hydrophobic composite nanofiber mat(CNM)was constructed by emulsion coaxial electrospinning process,in which the Lf-contained polyvinyl alcohol solution and pectin/ethylcellulose based water-in-oil emulsion were employed as shell and core fluids,respectively.The CNM consists of a continuous highly-hydrophobic EC shell layer,a co-continuous middle layer of EC and pectin particles and a core layer containing Lf.An electrospinning diagram was constructed to screen out suitable viscosity(51-82 c P)and conductivity(960-1300μS/cm)of the dispersed phase for successful electrospinning of shell emulsion.Furthermore,varying mass fraction of the dispersed phase(5%-20%)of shell emulsion during emulsion coaxial electrospinning allowed to obtain CNMs with different microstructures,labeled as 5&95,10&90 CNM,15&85 CNM,20&80 CNMs.The above CNMs all achieved colonic delivery of Lf(>95%),and the time for complete release of Lf in the simulated colon fermentation process is 10 h,7 h,5 h and 3 h,respectively.That is,the higher the content of pectin particles in the middle layer of CNM,the faster the release rate of stabilized Lf in the colon.Lf release in SCF involved complex mechanisms,in which diffusion release of Lf was dominant.(4)The influence of the colonic release rate of Lf on its oral bioavailability.HCT 116cells were used as the colon cancer model,and the effects of the colonic release rate of Lf on the expression levels of cell cycle arrest and apoptosis-related proteins were preliminarily investigated.The results showed that increasing the colonic release rate of Lf could promote the effective inhibition of Lf on the proliferation of HCT116 cell.In vivo assay demonstrated that the changes in the colonic release rate of Lf does not affect its relative oral bioavailability and elimination half life,but the sustainable release of Lf by CNM can prolong its absorption and utilization time in vivo.It was confirmed for the first time that the relative bioavailability of Lf absorbed through the colon was higher than that of Lf absorbed through the small intestine.This study provides a theoretical basis and scientific method for the construction of the efficient oral stabilization delivery system,and is of great significance for promoting the application of Lf in functional foods.
Keywords/Search Tags:Lactoferrin, Small intestinal stabilization system, Colonic stabilization system, Intestinal local effects, Pharmacokinetics
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