| The purpose of this study was to design a novel carrier to VB12-Gel-Core-SLN for oral protein delivery. Insulin and thymopentin were selected as the model drug of protein and peptide, respectively. This carrier with gel core, lipid matrix and VB12 surface ligand has higher encapsulation efficiency (EE), lower burst release, and stronger absorption.Firstly, the suitable particulate carried for VB12 covalent bonding was screened from hydrophilic polysaccharides with different active reactive groups, hydrophobic PLGA and lipid. It was found that natural water-soluble polymers with the representatives of Chitosan, hyaluronic acid and sodium alginate were not suitable to prepare the particles with smaller size, lower swelling rate, and stability in gastrointestinal tract; cross-linked hydrogel nanoparticle with the representative of dextran was not suitable for surface bonding with VB12; hydrophobic polymer with the representative of PLGA was not suitable for the covalent binding with VB12 because of its steric hindrance. Therefore, solid lipid nanoparticle (SLN) which did not swelling in water and had no steric hindrance was selected as carrier.Melting-dispersion process was firstly excluded in the SLN preparation process according to the formal studies. SLN prepared by solvent diffusion method or emulsion solvent evaporation method had the particle size of more than 500nm and EE of less than 10%. Obviously, these were methods were not suitable to prepare protein loaded SLN. Double emulsion solvent evaporation method was selected for detail study because SLN prepared by this method had higher EE and smaller particle size.Six types of lipid material were selected and the contact angles were determined to evaluate their hydrophobicity and its order was glyceryl monostearate<glyceryl palmitostearate< glyceryl tripalmitate<glyceryl behenate<stearic acid<octadecyl alcohol< cetyl palmitate. We did the investigation of the relationship between lipid hydrophobicity and the preparation of primary emulsion, double emulsion, solvent evaporation encapsulation of drugs, in vitro release and in vivo absorption. The results shown that lipid hydrophobicity had significant influence on the processes mentioned above. SLN prepared from glyceryl palmitostearate had excellent pharmaceutical properties with the particle size of 216.8±30.9nm and zeta potential of-14.74±1.10mv.The EE and burst release at 4h were 4.23±3.19% and 33.28% respectively for insulin,22.77±4.25% and 47.43% respectively for thymopentin. Compared with the other lipid material, the INS-SLN prepared by glyceryl palmitostearate has suitable hydrophobicity, smaller particle size and lower burst release, which increased its pharmacological bioavailability to 4.53%Sol-gel exchange technique had been utilized to combine with double emulsion and solvent evaporation process to improve the EE of SLN and to decrease its burst release. Ultrasonic was used to prepare primary emulsion to change the inner phase into gel. After the evaporation of solvent, the Gel-Core-SLN was then formed. The EE and burst release for Insulin-Gel-Core-SLN and thymopentin-Gel-Core-SLN were 57.36% and 10.17%, 61.97% and 15.43%. The Gel-Core-SLN shown the higher EE and the lower burst release than the others.VB12 stearate was synthesized by EDC/DMAP method. VB12 stearate distributed in dichlormethane and concentrated near the oil-water interface in dichlormethane-water phase. After loaded with VB12 stearate, the red colloidal suspension of VB12-Gel-Core-SLN was formed with particle size of 314.4±16.8nm and zeta potential of-14.72±1.84mv. The distribution of VB12 on the surface of VB12-Gel-Core-SLN was confirmed by visible spectrophotometry.Insulin and thymopentin were loaded to VB12-Gel-Core-SLN, the particle size, zeta potential, EE and protein stability were investigated to evaluate the stabilities of VB12-Gel-Core-SLN. After 3 months storage, the particle size and zeta potential were increased slightly while there was no significant change in EE, release and protein stability.Insulin-SLN, Insulin-Gel-Core-SLN and Insulin-VB12-Gel-Core-SLN were subjected to in vivo study, their pharmacological bioavailability were 4.53%,6.01% and 9.31% respectively. It had been proved that the higher EE and the lower burst release provide more drugs to Gel-Core-SLN during its absorption. There was the endocytosis mechanism mediated by VB12 during the absorption of VB12-Gel-Core-SLN.The pharmacological bioavailability (PB) of VB12-Gel-Core-SLN was influenced by administration mode, food, amount of VB12 stearate and particle size. The PB value of VB12-Gel-Core-SLN was 5.87% after ileum administration while it was improved to 7.31% if the VB12-Gel-Core-SLN was incubated with homogenate of stomach intestine before ileum administration. If the rats were administrated with VB12 or food containing VB12, and then administrated with VB12-Gel-Core-SLN, the PB value was 6.07% and 6.40% respectively, indicated that the absorption enhancing effect of VB12 was lost. With the addition of 4mg,8mg and 10mg VB12 stearate, the PB value was 7.03%,9.31% and 9.28% respectively and 8mg was the limit. The PB value of VB12-Gel-Core-SLN was decreasing with the increasing of particle size between 361nm and 3662nm. VB12-Gel-Core-SLN with the particle size of 362nm had the highest PB and the value was 9.31%.It was proved that VB12-Gel-Core-SLN is a promising oral protein delivery carrier. |
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