Catalase-encapsulated matrix metalloproteinase-9 responsive nanogels for modulation of inflammatory response and treatment of neutrophilic asthma

Abstract

Asthma is a chronic disease with typical pathological features such as airflow limitation, airway inflammation and remodeling. Of these, neutrophilic asthma is considered to be the more severe and corticosteroid-resistant subtype of asthma. Increasing evidence suggests that patients with neutrophilic asthma often accompany with dysbiosis of the internal microbiota, where the increased abundance of non-typeable Haemophilus influenzae (NTHi) is closely related to the neutrophilic asthma phenotype. Furthermore, emerging evidence suggests that reactive oxygen species (ROS) are pivotal in the pathogenesis of neutrophilic asthma. In this study, matrix metalloproteinase-9 (MMP-9)-responsive, catalase-loaded nanogels (M-CAT-NGs) were synthesized, which was composed of MMP-9-sensitive peptide (VPMS), arginine-grafted chitosan and maleimide (CS-Arg-Mal), catalase (CAT), sodium citrate (SC) and ε-poly(L-lysine) (ε-PLL). The M-CAT-NGs showed potent antimicrobial effects and exerted excellent therapeutic effects in the presence of MMP-9 by causing VPMS rupture and responsive release of CAT. In vitro experiments revealed that M-CAT-NGs effectively inhibited the proliferation of NTHi, Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli), while also demonstrating the capacity to modulate the inflammatory response induced by lipopolysaccharide (LPS) and hydrogen peroxide (H₂O₂) stimulation. In vivo experiments demonstrated that nebulized inhalation of M-CAT-NGs was effective in reducing airway hyperresponsiveness (AHR), alleviating inflammation, downregulating the expression level of ROS in the lung tissues, thus enabling the effective management of neutrophilic asthma. Thus, the development of M-CAT-NGs has shown strong potential for the clinical management of neutrophilic asthma by modulating the inflammatory response.

Keywords: Antibacterial nanogels; Inflammatory modulation; MMP-9 responsive; Neutrophilic asthma.

Scheme 1

Schematic illustration of MMP-9-responsive, catalase-loaded nanogels (M-CAT-NGs) for the management of neutrophilic asthma

Fig. 1

(a) Synthesis route of M-CAT-NGs. (b) FTIR spectra of CS, Arg, Mal-NHS and CS-Arg-Mal. (c) Size distribution analysis of M-CAT-NGs suspended in PBS, as determined using a Malvern Nano-ZS90 (n = 3). (d) TEM image of M-CAT-NGs. (e) Zeta potentials of CAT, M-NGs and M-CAT-NGs (n = 3). (f) Relative enzyme activities of M-CAT-NGs and CAT (n = 3). (g) Relative enzyme activities of CAT and M-CAT-NGs after 4 h of trypsin incubation. (h) The VPMS fragment cleaved by MMP-9, determined by UPLC-MS. (i) Relative enzyme activities of M-CAT-NGs and CAT-NGs relative to CAT in the presence of MMP-9 (n = 3), *p < 0.05, ***p < 0.001

Fig. 2

(a) Cellular uptake of CAT-FITC and M-CAT-FITC-NGs on Beas-2b cells for 12 h, measured by CLSM. The scale bar in the image corresponding to each sample indicate a length of 20 μm. (b) Representative images of intracellular ROS in Beas-2b cells detected by the peroxide-sensitive probe DCFH-DA. The scale bar in the image corresponding to each sample indicate a length of 50 μm. (c-e) Expression levels of IL-1β, IL-6, and TNF-α in Beas-2b cells after different sample treatments (n = 3), **p < 0.01, ***p < 0.001

Fig. 3

(a) Live-Dead staining images of NTHi following incubation with either PBS or a 0.36 mg/mL M-CAT-NGs solution. The scale bars indicated a length of 40 μm. (b) SEM images of NTHi following a 24 h incubation with either PBS or a 0.36 mg/mL M-CAT-NGs solution were presented. The scale bars represented 2 μm and 1 μm, respectively. (c) Growth curves of NTHi incubated with different samples (n = 3). (d) Counts of colonies on agar plates coated with NTHi solution with different samples (n = 3), ***p < 0.001

Fig. 4

(a) Schematic diagram of the establishment of the in vivo model along with the corresponding treatment protocol. (b) Fluorescence image of major tissues at 1, 12, and 24 h after nebulized M-CAT-FITC-NGs. (c) The statistics of (b). The insets of (c) represented local magnification. ROI: region of interest. (d) The variations in respiratory system resistance in response to various treatments. (e) The alterations in respiratory system resistance following treatment with 25 mg/mL methacholine (n = 6). (f) The modifications in lung dynamic compliance in response to various treatments. (g) The variations in lung dynamic compliance following administration of 25 mg/mL methacholine (n = 6), *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

(a) Flow cytometric analysis of neutrophils (CD11b⁺/Ly6G⁺) present in BALF. (b) The count of the percentage of neutrophils in (a) (n = 6). (c-e) Expressions of IL-1β, IL-6 and TNF-α in lung after different sample treatments (n = 6), (f) ROS expression in lung tissues after different treatments. The scale bar represented 200 μm, ***p < 0.001

Fig. 6

(a) Histological examination of the lungs of mice in each group by H&E staining. (b) Bronchial AB-PAS staining images of lung tissues in each group of mice. The scale bars indicated 100 μm and 50 μm, respectively