In vitro evaluation of drug release characteristics and clotting under static and dynamic conditions. (A) tPA loading efficacy comparing FSN and CS-IgG particles (n = 3 nanogel samples). (B) tPA release studies comparing FSN and CS-IgG particles (n = 3 nanogel samples). (C) Absorbance-based polymerization-degradation assay with tPA loaded particles (n = 3 clots per condition). (D) In vitro clotting under dynamic conditions in a fluidic device that contained a stationary fibrin clot located in the clot reservoir and that used flow solutions with fibrinogen and thrombin (control) (n = 7 replicates), fibrinogen and thrombin with unloaded FSNs (n = 4 replicates), fibrinogen and thrombin with nonbinding core-shell (CS) nanogels (n = 3 replicates), fibrinogen with unloaded FSNs without thrombin and thus no active coagulation (n = 3 replicates), fibrinogen and thrombin with tPA-FSNs (n = 4 replicates), fibrinogen and thrombin with tPA alone (n = 3 replicates), and fibrinogen and thrombin with tPA loaded into nonbinding CS nanogels (n = 3 replicates) and tPA-C/S-IgG (n = 3 replicates). Alexa-Fluor 488–labeled fibrinogen was used in all fibrinogen solutions and the stationary clot. All nanogels used are fluorescently labeled (rhodamine B), and arrows point to nanogels present in the fluidic device above. Images were taken on an EVOS FL Auto Imaging System at 4× magnification, and the remaining conditions are shown in supplemental Figure 1. The solid line represents the initial clot boundary, and the dotted line represents the clot boundary after 20 minutes of flow at a wall shear rate of 1 s⁻¹, which was used to quantify clot growth. (E) Quantification was performed using ImageJ software. Data are presented as average ± standard deviation. Clot dynamics were analyzed via a 1-way analysis of variance (ANOVA) with a Tukey’s post hoc test using a 95% confidence interval. Fib, fibrinogen; Th, thrombin. *P < .05; ***P < .001; ****P < .0001.