Optimization of Tilmicosin-Loaded Nanostructured Lipid Carriers Using Orthogonal Design for Overcoming Oral Administration Obstacle

Tilmicosin (TMS) is widely used to treat bacterial infections in veterinary medicine, but the clinical effect is limited by its poor solubility, bitterness, gastric instability, and intestinal efflux transport. Nanostructured lipid carriers (NLCs) are nowadays considered to be a promising vector of therapeutic drugs for oral administration. In this study, an orthogonal experimental design was applied for optimizing TMS-loaded NLCs (TMS-NLCs). The ratios of emulsifier to mixed lipids, stearic acid to oleic acid, drugs to mixed lipids, and cold water to hot emulsion were selected as the independent variables, while the hydrodynamic diameter (HD), drug loading (DL), and entrapment efficiency (EE) were the chosen responses. The optimized TMS-NLCs had a small HD, high DL, and EE of 276.85 ± 2.62 nm, 9.14 ± 0.04%, and 92.92 ± 0.42%, respectively. In addition, a low polydispersity index (0.231 ± 0.001) and high negative zeta potential (−31.10 ± 0.00 mV) indicated the excellent stability, which was further demonstrated by uniformly dispersed spherical nanoparticles under transmission electron microscopy. TMS-NLCs exhibited a slow and sustained release behavior in both simulated gastric juice and intestinal fluid. Furthermore, MDCK-chAbcg2/Abcb1 cell monolayers were successfully established to evaluate their absorption efficiency and potential mechanism. The results of biodirectional transport showed that TMS-NLCs could enhance the cellular uptake and inhibit the efflux function of drug transporters against TMS in MDCK-chAbcg2/Abcb1 cells. Moreover, the data revealed that TMS-NLCs could enter the cells mainly via the caveolae/lipid raft-mediated endocytosis and partially via macropinocytosis. Furthermore, TMS-NLCs showed the same antibacterial activity as free TMS. Taken together, the optimized NLCs were the promising oral delivery carrier for overcoming oral administration obstacle of TMS.


Effect of emulsifier to lipid ratio on TMS-NLCs
It was firstly evaluated the effect of emulsifier to lipid ratio on the hydrodynamic diameters (HD), polydispersity index (PDI) and zeta potentials (ZP) of TMS-NLCs. Herein, emulsifier to lipid ratio was set in the range from 5% to 60%. Other parameters were fitted to 1:9 of SA to OA ratio, 10% of drug to lipid ratio, 10 mL of water phrase, and 11 mL of cold water dispersion volume. As shown in Table S1, HD of TMS-NLCs gradually decreased with increased amounts of emulsifiers, all PDIs were close to 0.3, and ZPs were ranging from −36 mV to −40 mV. In order to use emulsifiers as little as possible, three levels of 20%, 25% and 30% were used in the later orthogonal test. Table S1. Effect of emulsifier to lipid ratio (ELR) on the hydrodynamic diameters (HD), polydispersity index (PDI) and zeta potentials (ZP) of TMS-NLCs (n=3).

Effect of drug to mixed lipid ratio on TMS-NLCs
It has been reported that the addition amounts of drug would influence the parameters of TMS-NLCs, especially HD. In this study, we investigated that effect of drug to lipid ratio on the parameters of TMS-NLCs. Herein, other parameters were fitted to 1:3 of SA to OA ratio, 25% of emulsifier to lipid ratio, 10% of drug to lipid ratio, 10 mL of water phrase, and 11 mL of cold water dispersion volume. The results showed that HD of TMS-NLCs gradually increased when the drug to lipid ratio was enhanced from 5% to 50% while there was no significant difference in ZP and PDI (Table S3). In order to require the small size of TMS-NLCs, 10%, 20%, and 30% was selected as the three levels of drug to lipid ratio.

Effect of cold water to hot emulsion ratio on TMS-NLCs
Effect of cold water dispersion volume on TMS-NLCs was investigated when other parameters were fitted to 1:3 of SA to OA ratio, 25% of emulsifier to lipid ratio, 10% of drug to lipid ratio and 10 mL of water phrase. As shown in Table S4, the more cold water dispersion volume the bigger HD, while there was no significant changes in ZP and PDI. 2/1, 1/1 and 1/2 was hence chosen as the three levels of cold water to hot emulsion ratio.

Effect of ultrasonic time on TMS-NLCs
Effect of ultrasonic time on TMS-NLCs was evaluated when other parameters were fitted to 1:9 of SA to OA ratio, 30% of emulsifier to lipid ratio, 10% of drug to lipid ratio, 10 mL of water phrase and 11 mL of cold water dispersion volume. As exhibited in Table  S5, HD of TMS-NLCs would gradually decrease along with the increasing ultrasonic time ranging from 5 min to 20 min, while HD would increase when the ultrasonic time was more than 20 min. Generally, TMS-NLCs was relatively superior in all HD, PDI and ZP when the ultrasonic time was 20 min. Therefore, the ultrasoinc time was fitted to 20 min in the orthogonal experiments. Table S5. Effect of ultrasonic time (UT) on the hydrodynamic diameters (HD), polydispersity index (PDI) and zeta potentials (ZP) of TMS-NLCs (n=3).

Effect of cold water dispersion time on TMS-NLCs
Effect of cold water dispersion time on TMS-NLCs was analyzed when other parameters were fitted to 1:9 of SA to OA ratio, 30% of emulsifier to lipid ratio, 10% of drug to lipid ratio, 10 mL of water phrase and 11 mL of cold water dispersion volume. As presented in Table S6, HD of TMS-NLCs would gradually decreased with the heightened cold water dispersion time ranging from 60s to 120s, while HD would increase when the cold water dispersion time was over 60s. On the whole, TMS-NLCs was relatively superior in HD, PDI and ZP when the cold water dispersion time was 60s. Therefore, the cold water dispersion time was set to 60s in the orthogonal experiments. Table S6. Effect of cold water dispersion time (CWDT) on the hydrodynamic diameters (HD), polydispersity index (PDI) and zeta potentials (ZP) of TMS-NLCs (n=3).

Results of Analysis of variance
According to analysis of variance in Table S7, the ratio of drug to mixed lipids had a significant impact on HD (p<0.05). Four factors had no significant impact on EE (p>0.05). The ratio of drug to mixed lipids had a significant impact on DL (p<0.05), which improved with the increasing TMS content. Error 0.08 2 Note: F0.05(2,2)=19.00; p < 0.05 represented significant difference marked by *.