Entrapment of recombinant staphylokinase by liposomes: formulations, preparation, characterization and behavior in vivo

Recombinant staphylokinase (r-Sak), which was first marketed in 2005, has proven to be a promising thrombolytic agent with a higher specificity for fibrin and a lower risk of hemorrhage. However, due to its rapid biodegradation and clearance, its efficacy is greatly reduced. Liposomes might prolong the residence time of the protein and peptide, but no efforts have been made to encapsulate r-Sak in liposomes. The aim of the present study was to prepare and characterize a liposomal formulation of r-Sak and investigate its behavior in vivo. Also, the effects of the liposomal preparation conditions and formulation factors on the bioactivity of r-Sak were evaluated. In this paper, using a modified thin-film dispersion method, r-Sak was successfully formulated in mostly small unilamellar liposomes. The entrapping efficiency associated with interior-entrapped and surface-adsorbed r-Sak was 71.2% and, of this, 15.5% was adsorbed on the surface of the vesicles. Also, the results showed that 0.3% w/v Triton X-100 had no significant effect on r-Sak bioactivity, determined by the "lytic-circle" method. However, the processing parameters, including higher temperatures, organic solvents, homogenization and sonication, can significantly reduce the bioactivity of r-Sak, which can be markedly restricted by human serum albumin (HSA). In vivo, after intravenous administration of the r-Sak liposome formulation to rats at doses of 0.41, 0.82,1.24 mg/kg, the estimated plasma half-life was about 52,102 and 87 min, respectively, which was, respectively, 6.1-, 9.4- and 6.3-fold longer than that observed in the control groups after administration of similar doses of the free r-Sak formulation (p < 0.001), and the AUC values were 384, 534 and 582% greater than those of r-Sak solution, respectively, resulting in clearances (CL) that were significantly lower than those seen in the control groups (p < 0.001). In conclusion, liposomes are an effective carrier for r-Sak, prolonging its plasma half-life, leading to a potential enhancement of its therapeutic effect and a reduction in its toxicity.