Reducing the Side Effects of Thrombolytic Agent by Regulating its Release Rate from Microcapsules

Scientific Research Question: Streptokinase (SK) is the most effective and accessible thrombolytic agent. However SK therapy is accompanied by bleedings or rethrombosis as a result of the exhaustion of plasminogen (Pg) and fibrinogen (Fg) in plasma. The rapid clearance from bloodstream and significant side effects restrict the use of SK in thrombolytic therapy. The aim of this study was to reduce the side effects of SK by including it in microcapsules with a polyethylene glycol (PEG) shell, which temporarily isolates the SK (before its release) from contact with blood proteins.

Methods: PEG microcapsules containing SK were prepared by the method of double emulsification, using an ultrasonic device and polyvinyl alcohol as emulsifying agent. To increase the efficiency of encapsulation, SK was included in microcapsules together with albumin or polyethylenimine. PEG 20 and 40 kDa were used as a polymer shell of microcapsules. The releasing rate from microcapsules and percent of SK inclusion were studied by measuring the SK fibrinolytic activity. The kinetics of plasma clots lysis by native or capsulated SK was studied by decrease in the height of clots. Drop of Fg and Pg levels in plasma surrounding clot were measured.

Findings: For variations of SK releasing rate from microcapsules the water-soluble high molecular PEG 20 and 40 kDa and different PEG/SK ratio were used. According to our optimized method, microcapsule samples containing 8 or 12 mg PEG (20 or 40 kDa) per 1000 IU SK were obtained: 8-PEG₂₀-SK, 8-PEG₄₀-SK, 12-PEG₂₀-SK и 12-PEG₄₀-SK, respectively. The SK inclusion percent was found to be 90-95% (by activity) for all obtained capsulated samples. The times of complete release of the thrombolytic agent from the capsulated samples of 8-PEG₂₀-SK and 12-PEG₂₀-SK were 45 and 60 min, and from the samples of 8-PEG₄₀-SK and 12-PEG₄₀-SK were 75 and 90 minutes, respectively. Initial lysis rates of plasma clots immersed in human plasma by capsulated SK samples decreased with increase in the PEG/SK ratio and molecular weight of PEG. However, all capsular forms of SK, like native SK, produced 100% lysis of plasma clots in 4 h. The capsulated SK samples induced the lower side effects as compared to free SK. The half-times of Pg exhaustion were found to be 6, 8.5, 13, 15 and 16.5 min, and half-times of Fg exhaustion were 4, 34, 38, 40 and 41 min for free SK, 8-PEG₂₀-SK, 12-PEG₂₀-SK, 8-PEG₄₀-SK and 12-PEG₄₀-SK capsulated samples, respectively.

Conclusions: By variation of conditions it was obtained the PEG-SK-microcapsules of various compositions, which contained 90-95% of the thrombolytic agent (by activity) and released it with different rates. Capsulated SK samples, like native SK, produce 100% lysis of plasma clots in 4 h. All capsulated SK samples cause the lower exhaustion of important plasma proteins compared to free SK. The 12-PEG₄₀-SK capsulared sample containing the higher molecular weight PEG and PEG/SK ratio causes the lowest side effects.