Rapidly Inhibiting the Inflammatory Cytokine Storms and Restoring Cellular Homeostasis to Alleviate Sepsis by Blocking Pyroptosis and Mitochondrial Apoptosis Pathways

Abstract

Pyroptosis, systemic inflammation, and mitochondrial apoptosis are the three primary contributors to sepsis's multiple organ failure, the ultimate cause of high clinical mortality. Currently, the drugs under development only target a single pathogenesis, which is obviously insufficient. In this study, an acid-responsive hollow mesoporous polydopamine (HMPDA) nanocarrier that is highly capable of carrying both the hydrophilic drug NAD⁺ and the hydrophobic drug BAPTA-AM, with its outer layer being sealed by the inflammatory targeting peptide PEG-LSA, is developed. Once targeted to the region of inflammation, HMPDA begins depolymerization, releasing the drugs NAD⁺ and BAPTA-AM. Depletion of polydopamine on excessive reactive oxygen species production, promotion of ATP production and anti-inflammation by NAD⁺ replenishment, and chelation of BAPTA (generated by BA-AM hydrolysis) on overloaded Ca²⁺ can comprehensively block the three stages of sepsis, i.e., precisely inhibit the activation of pyroptosis pathway (NF-κB-NLRP3-ASC-Casp-1), inflammation pathway (IL-1β, IL-6, and TNF-α), and mitochondrial apoptosis pathway (Bcl-2/Bax-Cyt-C-Casp-9-Casp-3), thereby restoring intracellular homeostasis, saving the cells in a state of "critical survival," further reducing LPS-induced systemic inflammation, finally restoring the organ functions. In conclusion, the synthesis of this agent provides a simple and effective synergistic drug delivery nanosystem, which demonstrates significant therapeutic potential in a model of LPS-induced sepsis.

Keywords: BAPTA-AM; HMPDA; NAD+; pyroptosis; sepsis.

Scheme 1

Schematic illustration of the preparation route, the sepsis animal model establishment process, and the treatment mechanism of NPs. A) Schematic diagram of preparation of HMPDA@BA/NAD⁺@LSA NPs. B) Therapeutic mechanism of BA‐AM and NAD⁺ co‐loaded NPs in an LPS‐induced sepsis mouse model.

Figure 1

Synthesis and characterization of HMPDA@BA/NAD⁺@LSA NPs. A) Representation schematic of HMPDA@BA/NAD⁺@LSA NPs. B) Size distributions and C) ζ‐potential of HMPDA NPs, HMPDA@BA/NAD⁺ NPs, and HMPDA@BA/NAD⁺@LSA NPs. TEM image of D) HMPDA@BA/NAD⁺ NPs and E) HMPDA@BA/NAD⁺@LSA NPs. F) N₂ adsorption–desorption isotherm and pore‐size distribution of HMPDA@BA/NAD⁺ NPs. G) Influence of incubation time on BA‐AM and NAD⁺ loading content (calculated by determination of free drug content) in HMPDA NPs. H) Size distributions and a photograph of the “Tyndall effect” after the response of HMPDA NPs in simulated media (PBS) at 37 °C. I) TEM image of HMPDA NPs’ responsiveness in simulated media (PBS: pH 6.5) for 1 h. J) Evaluation of ROS consumption by HMPDA (30 µg mL⁻¹). Data are means ± SD, n = 3.

Figure 2

Restoration of cellular energy homeostasis. Cell viability (CCK‐8) of A) AML‐12 cells and B) HK‐2 cells was incubated with different doses (based on BA‐AM) of HMPDA@LSA NPs and HMPDA@BA/NAD⁺@LSA NPs for 24 h. C) Relative hemolysis ratios of various concentrations of HMPDA@BA/NAD⁺@LSA NPs. The pretreatment process of the following experiments is consistent, and the specific operation is as follows: H₂O₂‐stimulated AML‐12 cells were incubated with PBS, HMPDA@LSA (HMPDA: 320 ng mL⁻¹), free NAD⁺ (320 ng mL⁻¹), free BA‐AM (200 ng mL⁻¹), HMPDA@NAD⁺@LSA (NAD⁺: 320 ng mL⁻¹), HMPDA@BA@LSA (BA‐AM: 200 ng mL⁻¹), HMPDA@BA/NAD⁺@LSA (BA‐AM: 200 ng mL⁻¹, NAD⁺: 320 ng mL⁻¹) for 12 h, respectively. D) NAD⁺, E) NADH, and F) ATP levels in H₂O₂‐stimulated AML‐12 cells with different treatment. G) Semiquantitative fluorescence results and H) fluorescence images of the JC‐1 assay to measure mitochondrial membrane depolarization in H₂O₂‐stimulated AML‐12 cells after different treatments. JC‐1 is a probe for detecting mitochondrial membrane potential. JC‐1 “aggregates” at a higher mitochondrial membrane potential (red fluorescence, normal state). At a lower mitochondrial membrane potential, JC‐1 become “monomers” (green fluorescence, apoptotic state). Data are mean ± SD, n = 6.

Figure 3

Cellular protective effect of HMPDA@BA/NAD⁺@LSA NPs. The pretreatment process for the following experiments is consistent, and the specific steps are as follows: H₂O₂‐stimulated HK‐2 cells were incubated with PBS, HMPDA@LSA (HMPDA: 320 ng mL⁻¹), free NAD⁺ (320 ng mL⁻¹), free BA‐AM (200 ng mL⁻¹), HMPDA@NAD⁺@LSA (NAD⁺: 320 ng mL⁻¹), HMPDA@BA@LSA (BA‐AM: 200 ng mL⁻¹), HMPDA@BA/NAD⁺@LSA (BA‐AM: 200 ng mL⁻¹, NAD⁺: 320 ng mL⁻¹) for 12 h, respectively. A) The alteration of Ca²⁺ level in the H₂O₂‐stimulated HK‐2 cell line with different treatments was determined by flow cytometry. B) Quantitative flow cytometry results for the relative Ca²⁺ level. C) Fluorescence images, and D) semiquantitative results of intracellular ROS. E) The cell viability of H₂O₂‐induced acutely injured HK‐2 cells after 12 h of treatment. F) Fluorescence images and G) semiquantitative results of AM/PI‐stained injured HK‐2 cells after various treatments. Data are mean ± SD, n = 6.

Figure 4

In vivo therapeutic effect in mice sepsis model. A) Experimental flowchart of the treatment process. B) Ex vivo NIR imaging of major organs (heart, liver, spleen, lungs, and kidneys) in the sepsis mice. Data are mean ± SD, n = 3. C) Survival rate of sepsis mice within 1 d after different treatments. Data are mean ± SD, n = 16. D) Photos of major organs of each group after dissection. The wet/dry ratio of E) liver, F) kidney, and G) lung in sepsis mice from each group. H,I) Blood serum ALT and AST levels from each group, respectively. J) AKP levels of liver tissue in different groups. K,L) Blood serum CRE and BUN levels from each group, respectively. Data are mean ± SD, n = 6.

Figure 5

In vivo therapeutic effect in the sepsis mouse model. A) Liver tissue SOD, and B) MDA concentrations for different groups in the sepsis animal model. Dihydroethidium (DHE) staining of C) liver, D) kidney, and E) lung tissue in different groups (red fluorescence: DHE; blue fluorescence: cell nucleus). F) DHE fluorescence semiquantitative results in different groups. Data are mean ± SD, n = 6.

Figure 6

In vivo therapeutic effect in the sepsis mice model. A) H&E staining of liver, kidney, lung, and spleen tissue. Green arrows indicate central hepatic vein ischemia; yellow arrows indicate liver necrosis cell; white arrows indicate inflammatory cell infiltration; blue arrows indicate renal tubular epithelial cell exfoliation site; black arrows indicate necrotic shedding of renal tubular epithelial cells to form casts; and red arrows indicate accumulation of neutrophils. B) TUNEL staining of liver, kidney, and lung tissue (red fluorescence: TUNEL positive cells; blue fluorescence: cell nucleus). Data are mean ± SD, n = 6.

Figure 7

In vivo therapeutic mechanisms. A) Western blotting analysis of NF‐κB expression and semiquantitative results. Data are mean ± SD, n = 3. B) Immunofluorescent staining images of NLRP3 in the liver, kidney, and lung (red fluorescence: NLRP3; blue fluorescence: cell nucleus). Data are mean ± SD, n = 6. C) Western blotting analysis of NLRP3 expression and semiquantitative results. Data are mean ± SD, n = 3. D) Immunofluorescent staining images of the ASC of the liver, kidney, and lung (green fluorescence: the ASC; blue fluorescence: the cell nucleus). Data are mean ± SD, n = 6. Western blotting analysis of E) Casp‐1, F) TNF‐α, G) IL‐6, and H) IL‐1β expression and semiquantitative results. 1–5 represent the control group, the sepsis (PBS) group, the HMPDA@LSA group, the HMPDA@NAD⁺@LSA group, and the HMPDA@BA/NAD⁺@LSA group, respectively. Data are mean ± SD, n = 3.

Figure 8

In vivo therapeutic mechanisms. Western blotting analysis of A) Bcl‐2, B) Bax, C) Bcl‐2/Bax, D) Cyt0‐C, E) Casp‐9, and F) Casp‐3 expression, and semiquantitative results. 1–5 represent the control group, the sepsis (PBS) group, the HMPDA@LSA group, the HMPDA@NAD⁺@LSA group, and the HMPDA@BA/NAD⁺@LSA group, respectively. Data are mean ± SD, n = 3.

Figure 9

Schematic illustration of cellular regulatory mechanism of HMPDA@BA/NAD⁺@LSA NPs.