Study On Surface Modification And Enhanced Oral Absorption Of Tilmicosin-loaded Nanostructured Lipid Carriers

Tilmicosin is a macrolide antibiotics for animals,which has strong antibacterial effect and good pharmacokinetic characteristics.However,its clinical application is limited by its poor palatability and low solubility.As a new drug carrier,nanostructured lipid carriers（NLCs）can improve the solubility,permeability and oral bioavailability of drugs,and have the advantages of good biocompatibility,high drug loading and entrapment efficiency,and good stability.Surface modified NLCs can increase the retention time of drugs in intestinal tract,promote intestinal cell uptake and drug lymphatic transport pathway,and further improve the oral efficacy of drugs.In this paper,three hydrophilic compounds and three positively charged compounds were selected to modify the surface of the optimized tilmicosin nanostructured lipid carriers（TMS-NLCs）.The drug loading,entrapment efficiency,stability and in vitro release characteristics were used as indicators to evaluate the quality of the optimized tilmicosin nanostructured lipid carriers.Then,one kind of surface modified TMS-NLCs was selected from each group.IPEC-J2 and MDCK-ch Abcg2/Abcb1 cell lines were used to investigate the intracellular accumulation of TMS-NLCs with different surface properties.The transport mechanism and permeability of TMS-NLCs were studied by cell monolayer model and in situ unidirectional perfusion model of chicken small intestine.The preparation was expanded by high pressure homogenization method,and the pharmacokinetic parameters of TMS-NLCs with different surface properties were compared in chickens.Finally,the mice model of Salmonella typhimurium infection was established to evaluate the therapeutic effect of commercial TMS solution and TMS-NLCs with different surface properties.The results showed that surface modified NLCs can be successfully prepared by high shear ultrasonic homogenization method.The particle size of the six modified NLCs was less than 200 nm,PDI was less than 0.3,and the absolute value of zeta potential was higher than 20 m V.The appearance of the modified NLCs were well dispersed milky white liquid.The addition of different compounds influenced the properties of TMS-NLCs.The entrapment efficiency of hydrophilic modified NLCs was higher than 93%,and the entrapment efficiency of positive modified NLCs was significantly decreased,but it had strong mucosal adhesion.Chitosan（CS）and polyvinyl alcohol（PVA）modified NLCs have good stability,so these two kinds of surface modified TMS-NLCs were selected for subsequent experiments.In the simulated gastrointestinal fluid,the cumulative release rate was API>CS-TMS-NLCs>TMS-NLCs>PVA-TMS-NLCs,indicating that the NLCs showed slow release characteristics.The cytotoxicity results showed the survival rate was higher than 85%when the concentration of NLCs was lower than 50μg/m L,which could be used for subsequent cell test.The uptake of CS and PVA modified NLCs by IPEC-J2 and MDCK-ch Abcg2/Abcb1 cells was significantly increased.The intracellular accumulation of TMS in NLCs group was significantly higher than that in API Group.The results of two-way transport test showed that NLCs could significantly reduce the efflux rate of TMS and improve the permeability of TMS.Further study on its transport mechanism showed that NLCs mainly transported across cell monolayer through energy dependent active transport process.Finally,in situ unidirectional perfusion of chicken jejunum showed that the effective permeability of CS and PVA modified NLCs was significantly higher than that of TMS solution.In order to expand the production of the preparation,the preparation process of high pressure homogenization was optimized with homogenization pressure and circulation times as the factors.The pharmacokinetic results showed the C_maxof CS-TMS-NLCs and PVA-TMS-NLCs were higher than that of TMS-NLCs,and the area under the concentration time curve（AUC）of CS-TMS-NLCs and PVA-TMS-NLCs were1.13 and 1.29 times higher than that of unmodified NLCs,respectively.The mouse model of Salmonella typhimurium infection was established.The mortality rate of positive control group was 50%.The infected mice showed clinical symptoms such as depression,rough hair,shivering and slow reaction.The survival rate of mice in the treatment group was higher than 87.5%,and the weight loss of mice in the oral NLCs group was less than that in the TMS solution group.The number of viable bacteria in lung,liver and spleen in CS and PVA modified TMS-NLCs groups decreased significantly compared with the positive control group.The number of viable bacteria in jejunum and ileum in PVA-TMS-NLC group was significantly less than that in other treatment groups.In conclusion,the results showed that the surface modification TMS-NLCs could reduce the particle size of the carrier and change the surface characters,thus enhancing the permeability in cell monolayer and in vivo intestinal perfusion model.Further,the oral bioavailability of TMS was improved through the chicken pharmacokinetic test.Finally,it showed a better therapeutic effect in the mouse model of Salmonella typhimurium infection.