The Effect Of Size And Surface Charge On The Property Of Tilmicosin-loaded Hydrogenated Castor Oil Nanoparticle Suspensions

Backgroud:Traditional formulations of tilmicosin have drawbacks of high toxic effect, fast metabolism, and low bioavailability. Frequent injection or high doses over the therapeutic period will increase the cost. High doses could also result in drug resistance which poses a threat to human health. Solid lipid nanoparticles (SLN) have lots of advantages, such as enhancing therapeutic effects, reducing the residual of drug, prolonging the circulation time in vivo and decreasing the toxic effects. SLN could be a potential drug carrier system for veterinary drug. Particle size and surface charge are important characteristics of nanoparticles, and have great effect on the property of nanoparticles drug carriers.Methods:The tilmicosin-loaded hydrogenated castor oil nanoparticle (TMS-HCO-NP) suspensions were prepared with hydrogenated castor oil as lipid material, polyvinyl alcohol as surfactant and quaternary ammonium salt as surface modifier by a hot homogenization and ultrasonic technique. The size of the nanoparticles was controlled by varying the PVA concentration, and the surface charge was modified by adding quaternary ammonium salt. The effects of the size and surface charge on the property of the suspensions were studied. The physicochemical characteristics of TMS-HCO-NP were investigated by optical microscope, scanning electron microscopy (SEM) and photon correlation spectroscopy (PCS). The drug loading of the TMS-HCO-NP was measured by ultraviolet spectrophotometer. The release profiles of TMS-HCO-NP with different particle sizes and zeta potentials were studied. The effects of particle size and surface charge on cytotoxicity of TMS-HCO-NP suspensions were evaluated with BHK, Vero and RAW264.7cell lines, and the macrophage phagocytosis of TMS-HCO-NP was studied through confocal laser system. In vitro antibacterial activity of TMS-HCO-NP suspensions with different particle size and surface charge were investigated using three different methods. The acute toxicity of TMS-HCO-NP suspensions with different particle sizes was evaluated in ICR mice. The stability of TMS-HCO-NP suspensions was studied after stored at room temperature and at4℃for6months.Results:The surfactant significantly affected the size of nanoparticles. When prepared with PVA concentrations of0.2%,1%and5%, the mean diameters of the nanoparticles in the three suspensions were920±35nm,452±10nm and151±4nm, respectively. The three suspensions displayed similar biphasic release profiles, but the suspension of smaller-sized particle showed faster initial release. None of the three suspensions were cytotoxic at clinical dosage levels on BHK and Vero cell lines. At high drug concentrations, TMS-HCO-NP suspensions with different sizes exhibited a concentration-dependent cytoxicity on the three cell lines. Phagocytosis experiments showed that the larger particles could be uptaken more easily. Time-kill curves showed that within12hours the suspension with150nm particles had the most potent bactericidal activity, but later the suspensions with larger-sized particles showed increased antibacterial activity. The results of the novel method also suggested that smaller-sized particles had better antibacterial effect. The suspension with the smallest-sized particle showed significantly more acute toxicity in mice, due to faster drug release. All three suspensions exhibited good stability at4℃and at room temperature for at least6months. The proportion of quaternary ammonium salt affected the surface charge of nanoparticles. When TMS-HCO-NP suspensions were prepared with2%dioctadecyl dimethyl ammonium chloride (DDAC) and difeeerent concentration of PVA (0.2%,1%and5%), the zeta potential of the nanoparticles were43.1±1.2mv,38.9±1.5mv and26.6±1.2mv; the particle sizes were885±16nm,499±6nm and169±4nm; the polydispersity indexes were0.344±0.041,0.309±0.012and0.478±0.005. While the TMS-HCO-NP suspensions were prepared with2%didodecyldimethylammonium bromide (DDAB), the zeta potential were48.2±1.5mv,38.6±1.3mv and27.3±1.2mv; the particle sizes were822±4nm,481±6nm and175±2nm; the polydispersity indexes were0.427±0.036,0.456±0.076and0.532±0.031. With the increment of the proportion of quaternary ammonium from2%to4%, the zeta potential was increased, but the size distribution became wider. The drug loading of positive charged nanoparticles was slight higher than that of the negatively charged ones. Compared with negatively charged TMS-HCO-NP suspensions, the positively ones showed similar release patterns but slower initial release. Cytotoxicity study showed that low concentration of TMS-HCO-NP suspensions with positive surface charge did not affect proliferation of BHK and Vero but RAW264.7, which could be due to different uptake mechanisms of nanoparticles. The antibacterial activity of TMS-HCO-NP suspension with positive surface charge was slightly higher than that with the negative surface charge.These results demonstrate that the size and surface charge could be controlled by varying the PVA concentration and adding quaternary ammonium salt. Particle size and surface charge significantly affected the property of TMS-HCO-NP suspensions. Particle size and surface charge are important considerations for the development of drug-SLN formulations.