Fig. 2

A Leaking assay performed at different time points: basal, 24 h, 48 h, 72 h, and 7 days after the synthesis. Data represent mean ± SEM (n = 3, independent samples). One-way ANOVA followed by post-hoc Dunnett’s multiple comparison test was used to perform statistical analysis compared to the IF of the corresponding control (basal). B Schematic representation of the rtPA amidolytic activity using a chromogenic substrate, which after hydrolysis by rtPA, the group pNA from the substrate is released. C The proteolytic activity of free rtPA and encapsulated rtPA (CSM@rtPA after storage by lyophilization process or storage at 4 ºC) in presence of the chromogenic substrate is recorded by measuring the absorbance at 405 nm over time. Data represent mean ± SEM (n = 3, independent samples). One-way ANOVA followed by post-hoc Dunnett’s multiple comparison test was used to perform statistical analysis compared with rtPA group (***P < 0.001). D The amidolytic activity is blocked when the rtPA is recognized by PAI-1. The proteolytic activity of free rtPA and encapsulated rtPA (CSM@rtPA) is determined in presence and absence of the PAI-1. Data represent mean ± SEM (n = 3, independent samples). One-way ANOVA followed by post-hoc Dunnett’s multiple comparison test was used to perform statistical analysis compared with the rtPA group (***P < 0.001). E Clots treated with the vehicle, rtPA (1 mg/kg), CSMs (number equivalent to the CSM@rtPA) and CSM@rtPA/L (equivalent to 1 mg/kg rtPA). Data represent mean ± SEM (n = 6, independent measurements per group of treatment). Statistical analysis was assessed by Kruskal–Wallis test followed by Dunn’s multiple test compared with the vehicle treatment group (**P < 0.01). F Thrombolytic effect displayed as fold change of clot mass loss normalized to average clot mass loss with PBS as control treatment. Treatments of either vehicle (PBS), rtPA, CSM and CSM@rtPA/L, or Wortmannin were injected via a microdialysis pump at a speed of 0.3 mL/min. Following treatment, vials were weighed again to determine final clot mass and total clot mass loss for each treatment. Data represents mean fold change ± SEM, (n = 5, independent measurements per group of treatment). One-way ANOVA followed by post-hoc Dunnett’s multiple comparison test was used to perform statistical analysis compared with the vehicle group (***P < 0.001). G Hemolytic effects of CSM and CSM@rtPA/L compared to free rtPA. Hemolytic effect displayed as absorbance fold change from mean PBS (vehicle) treatment at 0.5 h. RBC pellets treated with either vehicle, rtPA, CSM@rtPA/L, CSM, or 0.1% Triton X-100. Three biological replicates performed with three biological replicates each. Data represents mean absorbance fold change ± SEM, (n = 6, independent measurements per group of treatment). One-way ANOVA followed by post-hoc Dunnett’s multiple comparison test was used to perform statistical analysis compared with the vehicle group (***P < 0.001)