Simultaneously Controlling Inflammation and Infection by Smart Nanomedicine Responding to the Inflammatory Microenvironment

Abstract

The overactivated immune cells in the infectious lesion may lead to irreversible organ damages under severe infections. However, clinically used immunosuppressive anti-inflammatory drugs will usually disturb immune homeostasis and conversely increase the risk of infections. Regulating the balance between anti-inflammation and anti-infection is thus critical in treating certain infectious diseases. Herein, considering that hydrogen peroxide (H₂O₂), myeloperoxidase (MPO), and neutrophils are upregulated in the inflammatory microenvironment and closely related to the severity of appendectomy patients, an inflammatory-microenvironment-responsive nanomedicine is designed by using poly(lactic-co-glycolic) acid (PLGA) nanoparticles to load chlorine E6 (Ce6), a photosensitizer, and luminal (Lum), a chemiluminescent agent. The obtained Lum/Ce6@PLGA nanoparticles, being non-toxic within normal physiological environment, can generate cytotoxic single oxygen via bioluminescence resonance energy transfer (BRET) in the inflammatory microenvironment with upregulated H₂O₂ and MPO, simultaneously killing pathogens and excessive inflammatory immune cells in the lesion, without disturbing immune homeostasis. As evidenced in various clinically relevant bacterial infection models and virus-induced pneumonia, Lum/Ce6@PLGA nanoparticles appeared to be rather effective in controlling both infection and inflammation, resulting in significantly improved animal survival. Therefore, the BRET-based nanoparticles by simultaneously controlling infections and inflammation may be promising nano-therapeutics for treatment of severe infectious diseases.

Keywords: anti‐infection and anti‐inflammation treatment; bioluminescence resonance energy transfer; inflammatory microenvironment; myeloperoxidase; neutrophils.

Figure 1

Schematic illustration of the Lum/Ce6@PLGA nanoparticles to respond inflammatory microenvironment for improved anti‐infection and anti‐inflammation therapy.

Figure 2

The characteristics of microenvironment in the patients with infectious acute appendicitis in clinic. A) A scheme for the analysis of effusion in clinical cases. B,C) Representative flow cytometry graphs (B) and statistical data (C) of the proportions of neutrophils in different groups as indicated. D) The correlation between the date of hospital admission and the proportions of neutrophils in effusion of different patients. E) The concentrations of H₂O₂ in the effusion of different patients. (F,G) The activities (F) and concentrations (G) of MPO in different groups as indicated. Data are presented as mean ± SEM. Statistical significance was calculated by one‐way ANOVA with Tukey's post hoc test or Pearson correlation.

Figure 3

Characterization and in vitro functions of Lum/Ce6@PLGA nanoparticles. A) Schematic illustration of the synthesis of Lum/Ce6@PLGA nanoparticles. B) A TEM image of Lum/Ce6@PLGA nanoparticles. C) The luminescence spectra of Lum/Ce6@PLGA nanoparticles in the absence or presence of ClO⁻. “+” means that the nanoparticles incubated with ClO⁻ for evaluating the property of BRET. D,E) Luminescence images (D) and quantitative data (E) of Lum/Ce6@PLGA nanoparticles under different conditions as indicated. F) Representative luminescence images of Lum/Ce6@PLGA nanoparticles incubated with H₂O₂, MPO, and Cl⁻ at different time points. G,H) Representative luminescence images (G) and the luminescence intensities (H) of Lum/Ce6@PLGA nanoparticles after addition with different concentrations of Tempol. J) Representative luminescence images (I) and the luminescence intensities (J) of Lum/Ce6@PLGA nanoparticles after addition with different concentrations of 4‐ABAH. K,L) Representative flow cytometry graphs (K) and statistical data (L) of the percentages of apoptotic neutrophils after treatment with Lum/Ce6@PLGA nanoparticles under different conditions as indicated for 30 min. As the isolated neutrophils were kept in D‐hanks solution containing NaCl, no additional Cl⁻ was added into each group for this experiment. M,N) Representative images (M) and the statistic result (N) of S. aureus colonization at 30 min after incubation with different conditions as indicated. Data are presented as mean ± SEM. Statistical significance was calculated by one‐way ANOVA with Tukey's post hoc test.

Figure 4

In vivo protective effects against LPS‐induced peritonitis by Lum/Ce6@PLGA nanoparticles. A) A schematic illustration of LPS‐induced peritonitis model and treatment with Lum/Ce6@PLGA nanoparticles. B) The concentrations of H₂O₂ in PLF of peritonitis mice at 8 h after challenge (n = 5). C,D) The activities (C) and concentrations (D) of MPO in PLF at 8 h after challenge (n = 4 or 5). E,F) The statistical data of the proportions of neutrophils (E) and monocytes (F) in PLF at 8 hours after LPS challenge (n = 5). G,H) Survival rate (G) and individual body weights (H) of the LPS‐treated mice after different treatments (n = 13). I) The H&E staining images of lung and intestine sections collected from mice at 12 h after different treatments. J,K) The proportions of neutrophils (J) and macrophages (K) in PLF after treatments (n = 4). L) Cytokine levels including TNF‐α, IL‐6, and IL‐1β in PLF collected at 12 h after different treatments (n = 4). Data are presented as mean ± SEM. The statistical significance between two groups was calculated by two‐sided Student's t‐test.

Figure 5

In vivo therapeutic effect against S. aureus‐induced peritonitis by Lum/Ce6@PLGA nanoparticles. A) A schematic illustration of S. aureus‐induced peritonitis model. B<C) The activities (B) and concentrations (C) of MPO in PLF at different time points in the peritonitis mice (n = 3). D) The concentrations of H₂O₂ in PLF at different time points (n = 3). E–H) Representative flow cytometry graphs and statistical data of the proportions of neutrophils (E&F) and monocytes (G,H) in PLF collected from the healthy and peritonitis mice at 8 hours after S. aureus infection (n = 4). I) A schematic illustration of experimental schedule for the treatments of S. aureus‐induced peritonitis. J) Survival rate of the peritonitis mice after different treatments (n = 8). K,L) Representative images (K) and the statistic data (L) of S. aureus colonization in PLF and lung of the mice at 12 h after different treatments as indicated (n = 3). M) The H&E staining images of lung and intestine sections collected from mice at 12 h after different treatments. N,O) Representative flow cytometry graphs (N) and statistical data (O) of the proportion of neutrophils in the PLF at 12 h after different treatments (n = 4). P) The total number of cells in PLF after different treatments. Q) Cytokine levels including TNF‐α, IL‐6, and IL‐1β in the PLF collected from the peritonitis mice at 12 h after different treatments as indicated (n = 4). Data are presented as mean ± SEM. The statistical significance between two groups was calculated by two‐sided Student's t‐test. More than two groups were calculated by one‐way ANOVA with Tukey's post hoc test.

Figure 6

In vivo protective effects against CLP‐induced peritonitis by Lum/Ce6@PLGA nanoparticles. A) A schematic illustration of CLP‐induced peritonitis model. B) The concentration of H₂O₂ in PLF at different time points (n = 3). C,D) The activities (C) and concentrations (D) of MPO in PLF at different time points in the mice with CLP operation (n = 3). E–H) Representative flow cytometry graphs and statistical data of the proportions of neutrophils (E&F), macrophages (G) and monocytes (H) in PLF collected from healthy and peritonitis mice at 8 hours after CLP operation (n = 4). I) A schematic illustration of experimental schedule for the treatments of CLP‐induced peritonitis. J,K) Survival rate (J) and individual body weights (K) of the peritonitis mice after different treatments (n = 7). L) The H&E staining images of lung and cecum sections collected from mice at 12 h after different treatments. M,N) Representative images (M) and the statistic data (N) of bacteria colonization in PLF and major organs of mice at 12 h after different treatments as indicated (n = 3). O) The proportion of neutrophils in the PLF at 12 h after different treatments. P) Cytokine levels including TNF‐α, IL‐6, and IL‐1β in the PLF collected from the peritonitis mice at 12 hours after different treatments as indicated (n = 3). Data are presented as mean ± SEM. The statistical significance between two groups was calculated by two‐sided Student's t‐test. More than two groups were calculated by one‐way ANOVA with Tukey's post hoc test.

Figure 7

In vivo protective effects against pneumonia by Lum/Ce6@PLGA nanoparticles. A) A schematic illustration of S. aureus‐induced pneumonia model. B) Photographs of lung tissues in the mice at different time points. C) The activities and concentrations of MPO in BALF of pneumonia mice at different time points in the pneumonia mice (n = 3). D) The concentrations of H₂O₂ in BALF of pneumonia mice at different time points (n = 3). E–G) Representative flow cytometry graphs and statistical data of the proportions of neutrophils (E), monocytes (F), and macrophages (G) in BALF collected from the healthy and pneumonia mice at 8 hours after infection (n = 4). H) The H&E staining images of lung tissues collected at 8 hours after S. aureus infection. I) A schematic illustration of experimental schedule for the treatments of S. aureus‐induced pneumonia with inhalation administration of Lum/Ce6@PLGA nanoparticles. J) Survival rate of the peritonitis mice after different treatments (n = 7 or 8). K,L) Representative images (K) and the statistic data (L) of S. aureus colonization in BALF and lung tissue of the mice at 12 h after different treatments (n = 3). M) The H&E staining images of the lung tissues collected from mice at 12 h after different treatments. N) The statistical data of the proportions of neutrophils in BALF collected from the pneumonic mice at 12 h after different treatments (n = 4). O) The total number of cells in BALF after different treatments (n = 4). P) Cytokine levels including TNF‐α, IL‐6, and IL‐1β in BALF collected from the pneumonia mice at 12 h after different treatments as indicated (n = 3). Q) A schematic illustration of VSV‐induced pneumonia model. i.n. means intranasal administration, inh. means inhalation administration. R) The statistical data of the proportion of neutrophils in BALF (n = 4). S) The activities and concentrations of MPO in BALF at 8 h post‐infection (n = 4). T) The concentrations of H₂O₂ in BALF of pneumonia mice at 8 h post‐infection. U,V) Survival rate (U) and individual body weights (V) of the pneumonia mice after different treatments (n = 7 or 8). W) The H&E staining images of the lung collected from mice at 12 h after different treatments. X) The statistical data of the proportions of neutrophils at 12 h in BALF after different treatments (n = 3). Data are presented as mean ± SEM. The statistical significance between two groups was calculated by two‐sided Student's t‐test. More than two groups were calculated by one‐way ANOVA with Tukey's post hoc test.