Fig. 5. DOX-hyd-BSA NPs protect mouse death from sepsis and does not impair the neutrophil generation in the bone marrow.

(A) Mouse survival rates in sepsis after treatments of NPs. Four hours after intraperitoneal LPS (50 mg/kg) challenge to mice, mice were treated with PBS, free DOX, and prodrug DOX-hyd-BSA NPs at 0.2 mg/kg of DOX, respectively. All data expressed are as means ± SD (10 mice per group). Statistical analysis was done using the Kaplan-Meier method. ***P < 0.001 compared to controls (PBS and free DOX). Mouse body weights were measured after treatments of PBS (B), free DOX (C), and DOX-hyd-BSA NPs (D) (equal to 0.2 mg/kg free DOX). Number of neutrophils (E), TNF-α (F), IL-1β (G), and IL-6 (H) in blood, and number of neutrophils (I), TNF-α (J), IL-1β (K), and IL-6 (L) in BALF at 16 and 72 hours after LPS challenge, respectively. N.D. (not detected) represents the mouse death. All data expressed as mean ± SD (five mice per group). (M) Diagram shows the experimental protocol to address whether DOX-conjugated BSA NPs impair neutrophil immune sentinel to the secondary infection. The mice were challenged with LPS (intraperitoneal, 50 mg/kg) or PBS (control). Four hours later, the LPS-challenged mice were intravenously treated with DOX-hyd-BSA NPs at 0.2 mg/kg of DOX. The control mice were not treated with LPS and NPs. Seventy-two hours later, all survival and control (healthy) mice were challenged with LPS [i.t. (intratracheal), 10 mg/kg)]. At 84 hours, BALF was collected to assess neutrophil number (N), TNF-α (O), IL-1β (P), and IL-6 (Q). All data are expressed as means ± SD [seven survival mice for the DOX-hyd-BSA NPs-treatment group (equal to 0.2 mg/kg free DOX), and three healthy mice for the control group]. Statistics were performed by a two-sample Student’s t test. Statistics were performed by a two-sample Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).