NAD(H)-loaded nanoparticles for efficient sepsis therapy via modulating immune and vascular homeostasis

Abstract

Sepsis is a life-threatening organ dysfunction responsible for nearly 270,000 deaths annually in the United States alone. Nicotinamide adenine dinucleotide (NAD⁺), an immunomodulator, can potentially treat sepsis; however, clinical application of NAD⁺ is hindered by its inability to be directly taken up by cells. To address this challenge, a family of nanoparticles (NPs) loaded with either NAD⁺ or the reduced form of NAD⁺ (NADH), hereafter NAD(H)-loaded NPs, were engineered to enable direct cellular transport and replenishment of NAD(H). The NAD(H)-loaded NPs improved cellular energy supply, suppressed inflammation and prevented inflammation-induced cell pyroptosis and apoptosis. Therefore, the NPs can help maintain immune homoeostasis and vascular function, two key factors in the pathogenesis of sepsis. The NAD(H)-loaded NPs demonstrated excellent therapeutic efficacies in treating endotoxemia and multidrug-resistant pathogen-induced bacteremia. In addition, the NAD(H)-loaded NPs prevented caecal ligation and puncture-induced multiorgan injury and improved outcomes of secondary Pseudomonas aeruginosa infections following caecal ligation and puncture, thus potentially leading to a highly innovative and translational approach to treat sepsis efficiently and safely.

Fig. 1 ∣. NAD(H)-loaded NPs replenished cellular NAD(H) pool and prevented inflammation-induced energy depletion.

a, Schematic illustration of NAD⁺ metabolism and cellular uptake. Since NAD⁺ cannot pass through the cell membrane directly, it has to be degraded by extracellular enzymes into several precursors (for example, NAM and NR) which can enter the cells and subsequently enhance the NAD⁺ biosynthesis. Such a conversion process is inefficient as it is regulated and limited by critical enzymes (for example, NAMPT). The NAD(H)-loaded NPs can be taken up by the cells via endocytosis and directly replenish cellular NAD⁺. The CaP or MOF cores can dissolve in acidic endosome, leading to endosome swelling and bursting (due to an increase in osmotic pressure) to release the entrapped payload into cytosol. Created with BioRender.com. NMN, nicotinamide mononucleotide; NMNAT, nicotinamide mononucleotide adenylyltransferase; NRK, nicotinamide riboside kinase. b,c, Size and morphology of NAD⁺-LP-CaP (b) and NADH-LP-MOF (c) characterized by dynamic light scattering and transmission electron microscopy (inset). Representative data of three independent experiments. Scale bars, 200 nm. d,e, The NAD(H) release profiles from NAD⁺-LP-CaP (d) and NADH-LP-MOF (e) under different pH values. Data are presented as mean ± s.d. (n = 3). f,g, Intracellular NAD(H) levels (f) and NAD⁺/NAD(H) ratio (g) in BMDMs incubated with free NAD(H) (10 μM) or an equivalent dose of the NPs. Data are presented as mean ± s.d. (n = 5). Statistical significance was calculated via one-way analysis of variance (ANOVA) with Tukey’s post hoc test. h,i, Quantification of intracellular ATP level (n = 5; h) and cell viability (n = 6; i) in an LPS-mediated energy depletion model. LPS-stimulated BMDMs (LPS, 100 ng ml⁻¹) were treated with free NAD(H) (10 μM) or an equivalent dose of the NPs. Data are presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test.

Fig. 2 ∣. NAD(H)-loaded NPs prevented inflammation-induced cell death.

a,b, Pro-inflammatory cytokine TNF-α (a) and IL-6 (b) levels in BMDM culture supernatant quantified by ELISA. LPS-stimulated BMDMs (LPS, 100 ng ml⁻¹) were treated with free NAD(H) (10 μM) or an equivalent dose of the NPs. Data are presented as mean ± s.d. (n = 3). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. Statistical analyses were done relative to the LPS treatment group. c–f, Analysis of caspase1 activation (demonstrated by fluorochrome-labelled inhibitors of caspases (FLICA) probe 5-carboxyfluorescein-Tyr-Val-Ala-Asp-fluoromethylketone (FAM-YVAD-FMK) staining; n = 5; c and d) and IL-1β release (n = 3; e and f) indicating the activation of canonical (c and e) and non-canonical (d and f) inflammasome pathways. BMDMs were primed with LPS (100 ng ml⁻¹, 3 h) as signal 1, and then either incubated with ATP (2.5 mM, 1 h; for the canonical pathway) or transfected with lipoLPS (100 ng LPS per well, 3 h; for the non-canonical pathway) as signal 2. Free NAD⁺, empty NPs (denoted as eCaP) or the NAD⁺-LP-CaP NPs were added together with signal 1 or signal 2 or both. Data are presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. Statistical analyses were done relative to the positive controls (LPS/ATP or LPS/lipoLPS). g,h, NF-κB p65 nuclear translocation observed by CLSM. BMDMs were pretreated with free NAD(H) or the NPs for 5 h, stimulated with LPS (100 ng ml⁻¹) for 1 h and then immunostained for p65. Representative images of five independent experiments. Scale bars, 20 μm. Data are presented as mean ± s.d. (n = 5). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. Statistical analyses were done relative to the LPS treatment group. i,j, BMDM apoptosis triggered by LPS (100 ng ml⁻¹, 48 h) analysed by annexin V and propidium iodide double staining. FITC, fluorescein isothiocyanate. k, HUVEC apoptosis triggered by TNF-α (80 ng ml⁻¹, 48 h). The cells were treated with free NAD(H) or the NPs at a corresponding dose of 10 μM. l, Fluorescence images of HUVEC monolayer stained for tight junction protein VE-cadherin (green) after incubation with LPS together with free NAD(H) or the NPs for 24 h. Cell nuclei were stained by 4’,6-diamidino-2-phenylindole (DAPI; blue). Representative images of three independent experiments. Scale bar, 50 μm.

Fig. 3 ∣. The therapeutic efficacy of the NPs in a mouse model of endotoxemia.

a, Experimental procedures for the endotoxemia mouse model. Created with BioRender.com. b–d, Survival (b and c) and body weight (d) analysis of the mice receiving phosphate-buffered saline (PBS), free NAD(H) (20 mg kg⁻¹) or an equivalent dose of empty NPs and NAD(H)-loaded NPs, with treatment 1 h after LPS (15 mg kg⁻¹) administration. Data are presented as mean ± s.d. (n = 10). Statistical significance was calculated via log-rank test. e,f, Pro-inflammatory cytokine TNF-α and IL-1β levels in the serum of septic mice receiving different treatments were measured 6 h after LPS challenge. Data are presented as mean ± s.d. (n = 6). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. g, Gene expression in the white blood cells of the mice receiving different treatments over negative controls. The colours in the heatmap present the log₁₀ value of relative gene expression. h, Ex vivo fluorescence images representing the biodistribution of Cy5.5-labelled LP-CaP NPs in healthy and septic mice 4h after NP administration. L, K, S, Lu, H and M represent liver, kidneys, spleen, lungs, heart and thigh muscle, respectively. a.u., arbitrary units. i, Quantitative analysis of the mean fluorescence intensity of the organ or tissue shown in the ex vivo image. Data are presented as mean ± s.d. (n = 3). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. j, The vascular hyperpermeability in LPS-stimulated mice receiving different treatments. Evans blue dye was injected 12 h after LPS challenge, and the amount of the dye retained in the lungs was extracted and quantified. Data are presented as mean ± s.d. (n = 6). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test.

Fig. 4 ∣. Immune cell population variation, apoptosis and caspase1 activation in the blood, lungs and spleen of endotoxemia mice.

a,b, Flow cytometric quantification of monocyte (CD45⁺CD11b⁺Ly6C⁺Ly6G⁻), neutrophil (CD45⁺CD11b⁺Ly6C⁺Ly6G⁺), CD4⁺ T cell (CD45⁺CD11b^lowCD3⁺CD4⁺) and CD8⁺ T cell (CD45⁺CD11b^lowCD3⁺CD8⁺) populations in blood of septic mice 24 h after LPS administration. PBS, free NAD⁺ (20 mg kg⁻¹) or an equivalent dose of LP-CaP and NAD⁺-LP-CaP were i.v. injected 1 h after LPS (7.5 mg kg⁻¹, i.v.) administration. Healthy mice without LPS injection were used as the negative control. Data are presented as mean ± s.d. (n = 5). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. c, Flow cytometric quantification of monocyte and neutrophil populations in the lungs of the septic mice with PBS, free NAD⁺, LP-CaP or NAD⁺-LP-CaP treatment. Data are presented as mean ± s.d. (n = 5). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. d, Representative plots of cell apoptosis for splenic lymphocytes. The grayscale figures on the left show the gating strategy for the CD4⁺ and CD8⁺ T cells. e,f, Quantification of cell apoptosis (e) and caspase1 activation (f) for a variety of immune cells (monocyte and neutrophil in blood, and CD4⁺ and CD8⁺ T cell in spleen) assessed by annexin V/7AAD staining and FLICA assay, respectively. Data are presented as mean ± s.d. (n = 5). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test.

Fig. 5 ∣. Therapeutic performance of the NAD⁺-loaded NPs in bacteria-induced sepsis models.

a, Experimental procedures for the CLP and P. aeruginosa secondary infection model. Mice subjected to CLP received two i.v. injections of PBS, free NAD⁺ (20 mg kg⁻¹) or an equivalent dose of LP-CaP and NAD⁺-LP-CaP at 6 h and 24 h after the surgery and challenged intratracheally with P. aeruginosa (1 × 10⁸CFU in 50 μl PBS) at day 3. A sham group without CLP and a CLP group without the P. aeruginosa challenge were used as control groups. Created with BioRender.com. b,c, Survival (b) and body weight (c) analysis of the mice in the bacteria secondary infection model. Data are presented as mean ± s.d. (n = 14). Statistical significance was calculated via log-rank test. d, Experimental procedures for the model of a polymicrobial blood infection induced by MRSA and P. aeruginosa. Mixed multidrug-resistant bacteria (mixed MRSA and P. aeruginosa with 5 × 10⁷ CFU for each pathogen) were i.v. administrated to induce blood infection and sepsis, and one injection of different treatments, including PBS, NAD⁺-LP-CaP, free Rif (acting as a model antibiotic), Rif-LP-CaP or NAD⁺-Rif-LP-CaP at a dose corresponding to 10 mg Rif per kilogram and 20 mg NAD⁺ per kilogram were i.v. injected 6 h after the infection. Created with BioRender.com. e,f, Survival (e) and body weight (f) analysis of the mice in the blood infection model. Data are presented as mean ± s.d. (n = 10). Statistical significance was calculated via log-rank test. g, Bacterial loads in liver, spleen, lungs, kidneys and blood of the septic mice 12 h after the treatments, determined by serial dilution and plate counting. Data are presented as mean ± s.d. (n = 6). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. Statistical analyses were done relative to the PBS treatment group. h, Blood biochemistry analysis demonstrating the liver (alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)) and kidney (blood urea nitrogen (BUN)) function of the mice with a bacterial blood infection. Data are presented as mean ± s.d. (n = 3). Statistical significance was calculated via one-way ANOVA with Tukey’s post hoc test. Statistical analyses were done relative to the PBS treatment group. i, Representative histological images for tissue sections with haematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, from the healthy mice (control) and the infected mice with PBS or NAD⁺-Rif-LP-CaP treatments. OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. Representative images of three independent experiments. Scale bar, 100 μm.