Synthesize and characterization of M-CAT-NGs
The M-CAT-NGs synthesis route was shown in Fig. 1a. The synthetic route of CS-Arg-Mal was shown in Fig. S2. 1H NMR spectra (Fig. S3) demonstrated that Arg and Mal could be successfully conjugated with CS backbone by EDC/NHS coupling chemistry. The signal peaks at 3.25–3.48 ppm (e’), 1.43–1.85 ppm (d’), and 2.28–2.51 ppm (c’) represented the three methylene groups in Arg, and the signal peak at 6.74–6.87 pm (i’) represented the carbon-carbon double bond of Mal. The FTIR spectra of CS, Arg, Mal and CS-Arg-Mal were shown in Fig. 1b. In the spectra of CS-Arg-Mal, a characteristic peak at 1701 cm− 1 indicating the carbon-oxygen double bond in Mal, an absorption peak at 1630 cm− 1 indicating the presence of guanidinium group, and characteristic peaks at 833 cm− 1 and 697 cm− 1 representing the carbon-carbon double bond in Mal were observed.
(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
The size distribution of CAT was centered at 639.3 ± 9.7 nm, M-NGs was centered at 164.1 nm and that of M-CAT-NGs was centered at 199.3 nm (Fig. S4–5, Fig. 1c). The TEM results showed that the size of M-CAT-NGs was approximately 129.8 nm (Fig. 1d), which was marginally smaller than the values obtained through DLS analysis (Table S1). This discrepancy could be due to the swelling of M-CAT-NGs in solution. The zeta potentials of CAT, M-NGs and M-CAT-NGs were − 9.17 mV, 9.1 mV and 10.3 mV (as shown in Fig. 1e, Table S1). This was a reassuring result, as it has been shown that positively charged nanoparticles typically achieve superior internalization and enhanced cellular uptake efficiency compared to their negatively charged nanoparticles [38]. These results demonstrated that nanoencapsulation of CAT yields uniformly sized particles with reversed surface charge (from negative to positive), both of which were advantageous for subsequent in vivo experiment.
To investigate the stimuli-responsive drug release behavior of the nanogels, the in vitro release profile of CAT from M-CAT-NGs was assessed using a dialysis method in the presence or absence of MMP-9. As demonstrated in Fig. S6, M-CAT-NGs exhibited MMP-9-dependent release kinetics, with approximately 80% of CAT released within 10 h under MMP-9-present conditions. In contrast, the release was significantly slower in the absence of MMP-9, with minimal detectable release during the initial 3 h and only ~ 25% cumulative release by 48 h. The size distribution of the M-CAT-NGs was determined in PBS to evaluate their stability. DLS characterization demonstrated that after 7 days of incubation, there was no significant variation in the particle size of the M-CAT-NGs, indicating that the M-CAT-NGs had good physiological stability in PBS (Fig. S7). The relative enzyme activities of M-CAT-NGs and CAT were shown in Fig. 1f. The results revealed that the enzyme activity of M-CAT-NGs could reach about 80%, which might be caused by the incomplete release of CAT within the nanogels within a brief time frame. In addition, to assess the protective efficacy of the nanogel on catalase activity, CAT and M-CAT-NGs were incubated with trypsin for 4 h, after which their relative enzymatic activities were assessed. As shown in Fig. 1g, after 4 h of trypsin incubation, the enzymatic activity in the M-CAT-NGs group surpassed that of the native CAT group by over 400-fold. The results indicated that the outer layer of nanogels could well protect the enzymatic activity of CAT, which was essential to protect the activity of catalase from the complex environment in vivo.
To evaluate the sensitivity of VPMS to MMP-9 [31], the enzymatic cleavage of VPMS was investigated. As shown in Fig. 1h, the main fragment (Ac-GCRDVPMS = 453.70, Z = 2) cleaved by MMP-9 was identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Moreover, MMP-9-insensitive peptide (VpMS) was used to replace VPMS to prepare MMP-9-insensitive nanogels (CAT-NGs), and detect the effect of MMP-9 on enzyme activity in MMP-9-sensitive nanogels (M-CAT-NGs) and MMP-9-insensitive nanogels (CAT-NGs). When MMP-9 was added, the enzyme activity of M-CAT-NGs was not significantly different from that of native CAT, whereas the enzyme activity of CAT-NGs was significantly decreased compared to that of native CAT (Fig. 1i). The above results reconfirmed that VPMS could be cleaved by MMP-9, thus enabling M-CAT-NGs to sensitively release CAT.
In vitro anti-inflammatory test
Since the potential toxicity of nanomedicines is a point of contention for their biomedical applications [39], multiple cell lines were utilized to evaluate their in vitro biocompatibility. The survival of L929, RAW264.7 and Beas-2b cells was evaluated by CCK-8 assay after treatment with different samples. As shown in Fig. S8, the evaluation of cell viability following 24 h of co-culture revealed that all material concentrations below 480 µg/mL exerted no significant effect on the viability of any of the three cell lines. When the time was extended to 48 h, some of the samples at the concentration of 420 µg /mL and over exhibited reduction in cell viability, probably due to the toxic effects caused by the accumulation of nanogels ingested into the cells with increasing time. Therefore, to ensure the safety of the nanogels, the concentration of 360 µg/mL was used for the following in vitro experiments.
Additionally, CLSM was utilized to observe the uptake of CAT-FITC and M-CAT-FITC-NGs by Beas-2b and RAW264.7 cells. The fluorescence images in Fig. 2a demonstrated that M-CAT-FITC-NGs were more readily taken up by Beas-2b cells compared to CAT-FITC, which could be attributed to the nanogel shell providing favorable solubility and positively-charged surface for endocytosis [40]. A similar trend was observed in RAW264.7 cells, where M-CAT-FITC-NGs exhibited easier uptake by cells than CAT-FITC, further supporting the role of the nanogels formulation in improving drug delivery efficiency (Fig. S9).
ROS are exacerbated during severe asthma and acute exacerbations, which may lead to oxidative damage to tissues, promoting airway inflammation and hyperresponsiveness [28]. H2O2 is a key metabolite of oxidative stress, and high concentrations of H2O2 can induce inflammatory responses that lead to growth arrest and death of cells [41]. Therefore, to preliminarily investigate the anti-inflammatory effect of nanogels, H2O2-stimulated Beas-2b and RAW264.7 cells were used. Based on CCK-8 results, 1 mmol/L H2O2 was used to stimulate both Beas-2b and RAW264.7 cells (Fig. S10–11). The level of intracellular ROS was significantly decreased in the Beas-2b cells of the M-CAT-NGs group and CAT group compared to the H2O2 stimulation alone group (Fig. 2b, Fig. S12). There were higher intracellular ROS levels in the CAT-NGs group compared to the M-CAT-NGs group. This might be due to the lack of the ability of reactive release in CAT-NGs, which resulted in slower kinetics of CAT release from the nanogels, leading to a less efficient ROS scavenging than that of M-CAT-NGs. However, there was no significant change in the M-NGs group compared to the H2O2 group. The result might be due to the fact that CAT was the main component to decompose H2O2. Since CAT was not contained in M-NGs, the ROS level of M-NGs treated cells remained comparable to that of the H2O2 group. Similar results were obtained using microplate reader to measure the relative expression of ROS in the above cells (Fig. S13). Notably, we observed similar trends in RAW264.7 cells (Fig. S14–S15).
(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
Neutrophilic asthma is usually accompanied with bronchial inflammation. Toll like receptors (TLRs) are over-activated, leading to the release of large amounts of pro-inflammatory factors such as IL-6 and IL-1β, and aggravating the level of airway inflammation [42]. Meanwhile, activated neutrophils also secrete inflammatory factors such as TNF-α, further exacerbating airway inflammation and promoting airway remodeling, leading to asthma exacerbation [43, 44]. Thus, regulating inflammation is crucial for the effective management of asthma [45]. To explore the anti-inflammatory effects of M-CAT-NGs, 1 µg/mL LPS was used to pre-stimulate Beas-2b and RAW264.7 cells for 12 h, followed by co-incubation of cells with different samples for 24 h. As shown in Fig. 2c-e, LPS stimulation of cells resulted in the production of high levels of IL-1β, IL-6, and TNF-α. The three inflammatory factors showed similar trends after treatment with different samples. Compared with the LPS group, there was no significant change in the inflammatory factors in the M-NGs and CAT-NGs groups, and the inflammatory factors in the CAT and M-CAT-NGs groups were reduced. Similar to what we observed in the ROS scavenging experiments, the anti-inflammatory capacity of the M-CAT-NGs group was better than that of the CAT-NGs group, which further confirmed the role of MMP-9 responsive release. Similar trends were observed in RAW264.7 cells (Fig. S16). The above results suggested that the M-CAT-NGs might have the ability to modulate inflammation, and therefore have a therapeutic potential for neutrophilic asthma.
Antibacterial test
Microbial infections have been implicated as significant contributors to the onset of neutropenic asthma, and in addition to viral infections, respiratory colonization by NTHi, Staphylococcus aureus, Pseudomonas aeruginosa, and Catamorax can exacerbate symptoms in patients with neutrophilic asthma [46,47,48,49]. Therefore, antimicrobial therapy is also essential in the management of neutrophilic asthma. The results of bacterial live-dead staining experiments showed that the vast majority of NTHi were killed after 24 h of co-incubation with M-CAT-NGs compared to the PBS control (Fig. 3a). The morphology of the bacteria in the different groups was further observed, most of the bacteria in the M-CAT-NGs-treated group lost their original morphology and showed crumpled and fragmented structures, indicating that the nanogels might have exerted their antimicrobial effect by disrupting the bacterial structure (Fig. 3b). In addition, the growth of NTHi at different time points after co-culture of M-CAT-NGs or its different components with NTHi was measured using a microplate reader. As shown in Fig. 3c, except for the CAT group where the optical density (OD) value did not change much, the OD values of all groups decreased with the increase of sample concentration. The excellent bactericidal ability of M-CAT-NGs was further confirmed by the smear plate counting method, in which the bacterial solutions were taken out after co-incubation with PBS or M-CAT-NGs for 24 h and diluted separately (1014-fold dilution for PBS group). The large number of bacteria killed in the M-CAT-NGs group could be confirmed by the counting results in Fig. 3d. In addition, similar results were obtained after incubation of M-CAT-NGs with S. aureus and E. coli (Fig. S17–18). The above results demonstrated that M-CAT-NGs had good antimicrobial capacity by destroying the bacterial structure, which might be attributed to its components with antimicrobial capacity, such as CS-Arg-Mal and ε-PLL. In fact, CS, Arg and ε-PLL have been reported to have good antimicrobial properties due to their positively charged moieties capable of binding to negatively charged compounds on the bacterial surface and disrupting the bacterial morphology [50,51,52]. Based on the experimental results above, it could be concluded that M-CAT-NGs possessed a strong inhibitory effect on different types of bacteria, indicating that M-CAT-NGs was expected to alleviate disease progression of patients with neutrophilic asthma by inhibiting bacterial growth.
(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
Construction of in vivo model and therapeutic effects of M-CAT-NGs in mice
Studies have shown that NTHi was a dominant bacterium in the airway of severe asthma patients and that NTHi colonization in the airway was strongly correlated with the severity of asthma [53]. Therefore, to investigate whether M-CAT-NGs could alleviate the symptoms of neutrophilic asthma in mice, we established an animal model of neutrophilic asthma in mice sensitized by intraperitoneal injection of OVA and infected with NTHi. The method of constructing the animal model was referred to previous studies by our team, as shown in Fig. 4a [44]. Briefly, mice were sensitized by intraperitoneal injection of 50 µg OVA and 25 µL Al(OH)3 per mouse on days 0, 7, and 14. From days 21 to 23, the mice were subjected to daily challenges with a 5% OVA solution for a duration of 45 min. In contrast, mice in the NC group were sham-sensitized with saline and subsequently challenged with an equivalent volume of saline. On day 24, NTHi (106 CFU, 20 µL) was inoculated into the trachea of anesthetized mice and an equal amount of saline was injected into the trachea of mice in the NC group. The mice were then treated with nebulized drug administration with PBS, M-NGs, CAT and M-CAT-NGs on days 25–27. Physiological indices were measured and samples were taken from the mice on day 28. As anticipated, the protein expression level of MMP-9 in lung tissues of the OVA&NTHi group was significantly elevated compared to the NC group (Fig. S19), further validating our material design strategy.
In vivo biodistribution and toxicity test
To explore the ability of M-CAT-NGs to accumulate in the lungs, the distribution and accumulation of CAT and M-CAT-NGs in vivo were evaluated in the neutrophilic asthma mice after nebulized inhalation. As shown in Fig. 4b-c, the M-CAT-FITC-NGs predominantly accumulated in lung and maintained strong fluorescence signals even 24 h after inhalation via nebulization. In contrast, 1 h after nebulization of CAT-FITC, relatively high accumulation of CAT-FITC was observed in the lung, liver, and kidney. By 24 h, the fluorescence signal of CAT-FITC in lung had significantly decreased (Fig. S20). These findings suggested that the nanogel shell substantially enhanced the lung retention of CAT, thereby reducing its distribution to other organs and lowering the risk of systemic side effects.
(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
Airway hyperresponsiveness (AHR) test
AHR is considered a characteristic feature of asthma and the most widely used method to assess AHR is the methacholine inhalation provocation test [54]. Airway resistance (RI) and dynamic compliance (Cdyn) are important indices to assess AHR. Consequently, changes in RI and Cdyn with increasing doses of methacholine were investigated. As shown in Fig. 4d-e, RI in OVA&NTHi group was increased significantly compared to NC group, with the dose of methacholine rising. At a methacholine concentration up to 25 mg/mL, RI reached a maximum, implying that neutrophilic asthma mice showed respiratory distress. The elevated trend of RI was alleviated after M-NGs, M-CAT-NGs, and AM treatments. In addition, compared with mice in NC group, mice in OVA&NTHi group showed a significant decrease in Cdyn under treatments with different concentrations of methacholine, whereas Cdyn was significantly increased in both groups after M-NGs, M-CAT-NGs, and AM treatments (Fig. 4f-g). This indicated that M-NGs, M-CAT-NGs, and AM treatments alleviated dyspnea, enhanced respiratory depth and more effectively increased in expiratory volume in OVA&NTHi-treated mice [55]. The improvement effect of M-CAT-NGs on Cdyn appeared to be superior to that of AM, possibly due to the dual functions of M-CAT-NGs in antibacterial activity and ROS clearance, which contributed to enhanced lung function. Compared with OVA&NTHi group, neither RI nor Cdyn changed significantly in CAT group, probably due to native CAT was unstable and susceptible to decomposition in vivo, while nanogels provided a protective effect to prevent CAT from losing its activity, and improved its solubility so that it could be more easily and efficiently nebulized for inhalation. These results suggested that neutrophilic asthma mice had a severe lung function deficit, as evidenced by high airway resistance and low dynamic lung compliance, which could be alleviated to some extent by M-CAT-NGs.
Modulation of the inflammatory response in vivo assay
To investigate whether M-CAT-NGs could exert a mitigating effect on airway inflammation in neutrophilic asthma mice, the proportion of neutrophils (Ly6G+, CD11b+) in BALF and inflammatory factors in lung tissue were examined. As shown in Fig. 5a-b, the proportion of neutrophils was significantly increased in the OVA&NHTi group compared with the NC group, whereas the proportion of neutrophils was significantly decreased in the M-NGs and M-CAT-NGs groups compared with the OVA&NHTi group, and there was no significant change in the proportion of neutrophils in the CAT group compared with the OVA&NHTi group. M-NGs and M-CAT-NGs could significantly reduce the proportion of neutrophils in BALF of neutrophilic asthma mice probably due to their excellent antibacterial ability and the CAT in M-CAT-NGs could eliminate ROS. There was no significant change in the proportion of neutrophils in the CAT group compared to the OVA&NTHi group might be due to the CAT shows no antimicrobial ability and native CAT is not stable enough and easily broken down in vivo. Besides, IL-1β, IL-6 and TNF-α associated with asthma were tested and obtained similar results (Fig. 5c-e). OVA and NTHi treatment could induce the expression of inflammatory cytokines and produce an inflammatory response. The expressions of IL-1β, IL-6 and TNF-α were not reduced after treatment with CAT but those were significantly decreased after M-NGs, M-CAT-NGs and AM treatments. It was noteworthy that the expressions of IL-1β, IL-6 and TNF-α showed the most significant reduction after treatment with M-CAT-NGs. In addition, ROS aggregation and its mediated oxidative stress damage played an important role in asthma [28]. To investigate whether M-CAT-NGs could mitigate the accumulation of ROS in lung, the expression level of ROS in lung tissues was measured. As shown in Fig. 5f, the level of ROS production in lung tissues was significantly increased after OVA&NTHi treatment compared with NC group. Compared with the OVA&NTHi group, M-NGs, M-CAT-NGs and AM treatment led to a decrease in ROS levels in lung tissue, among which the reduction was most significant after M-CAT-NGs treatment. There was no significant change in the CAT group, indicating that M-NGs and AM might reduce the production of ROS in lung tissue through their antibacterial effects, while M-CAT-NGs could exert therapeutic effects from two perspectives: antibacterial activity and encapsulation of CAT to form nanogels, thereby enhancing CAT solubility and improving its bioavailability. The above results suggested that M-CAT-NGs could regulate inflammatory responses in vivo and potentially treat neutrophilic asthma.
(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
Morphologic changes in lung tissue
Pathologically, the main features of asthma included chronic inflammation, airway tissue remodeling, disruption of epithelial integrity [56], cupped cell hyperplasia [57, 58], and dysregulation of mucus secretion [49]. To investigate whether M-CAT-NGs could ameliorate pathological changes in lung tissue, lung tissues from mice were stained with H&E and observed. As illustrated in Fig. 6a and Fig. S21, the OVA&NHTi group exhibited pronounced thickening of the bronchioles and alveolar septa when compared to the NC group, increased alveolar fusion. No significant improvement in the bronchi and alveoli was observed after CAT treatment. M-NGs and M-CAT-NGs groups showed attenuated bronchiolar thickening in the lung tissues, with the alveolar septa relatively intact, and a significant reduction in the number of inflammatory cell infiltrates. This might be due to both M-NGs and M-CAT-NGs had good antimicrobial capacity, especially M-CAT-NGs also increased the bioavailability of CAT and promoted the uptake of M-CAT-NGs by the epithelial cells, which reduced the effect of bacterial infections on the integrity of the alveolar-capillary barrier and moderated inflammatory responses and reduced the infiltration of inflammatory cells [59].
Studies have shown that infection with NTHi in asthma patients leads to goblet cells proliferation, upregulation of the mucin MUC5AC [60], and airway mucus became more abundant and viscous, resulting in ciliary dysfunction, coupled with mucus embolism and compromised mucosal ciliary clearance [61]. Bronchial stasis impaired pathogen clearance, thereby facilitating the colonization of NTHi. Consequently, NTHi robustly induced the transcription of MUC5AC via the upregulation of the MAPK pathway, while also enhancing the expression of the highly insoluble MUC2 mucin through NF-κB and TGF-β/Smad signaling [62]. Diffuse mucus occlusion of the airways could lead to localized atelectasis, which could worsen symptoms in patients with asthma and greatly increase asthma mortality [63]. Consequently, alleviating mucus secretion within the airways is crucial for the effective management of asthma. The results of AB-PAS staining showed significantly higher levels of airway mucus secretion in the OVA&NTHi group compared with the NC group, indicating that sensitization with OVA, coupled with NTHi infection, markedly elevated mucus secretion levels within the airways of the mice (Fig. 6b and Fig. S22). Compared with the OVA&NHTi group, the M-CAT-NGs group showed a significant decrease in mucus, which might be attributed to the fact that M-CAT-NGs inhibited bacterial colonization within the mucus layer, contributing to an enhanced mucus environment, leading to a decrease in mucus. Similarly, mucus in the airways of mice was also significantly reduced after nebulization with M-NGs compared to the OVA&NTHi group whereas no significant change in mucus was observed in the CAT group, which might be attributed to the excellent antimicrobial capacity of M-NGs and M-CAT-NGs, which inhibited the growth of NTHi, thus alleviating mucus obstruction in the airway. To detect the longer-term therapeutic effect after medication, we conducted lung function tests and pathological section examinations on mice on the 14th and 31st days after the cessation of M-CAT-NGs treatment. As shown in Fig. S23, M-CAT-NGs-treated mice exhibited comparable respiratory parameters to NC controls. H&E and AB-PAS staining revealed resolution of in treated mice at both timepoints, with no rebound pathology (Fig. S24).
(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
H&E staining observations and blood biochemical indicator tests were conducted to investigate whether the structure and function of major organs in mice would be damaged by M-CAT-NGs. As depicted in Fig. S25, treatment with 0.96 mg/mL of M-NGs, CAT, and M-CAT-NGs did not result in any significant alterations to the organ tissue structure when compared to the NC group. Specifically, no pathological alterations were observed in the myocardial striations, and the fundamental structural components of the liver-namely, the central vein, hepatic sinusoids, and bile ducts-exhibited clear structural integrity. The splenic capsule and trabeculae, white and red medulla, and parenchymal marginal regions were also structurally intact. The interstitium and parenchyma of the lungs were well defined. The glomeruli and tubules of the kidneys also did not show any pathologic changes. Similar results were obtained in the blood biochemical indices, and there were no significant changes in all indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), uric acid (UA), blood urea nitrogen (BUN) and creatinine (CRE) in all groups of mice compared with the control group (Fig. S26), which indicated that M-NGs, CAT and M-CAT-NGs had no significant effect on the hepatic and renal functions of mice. In conclusion, M-NGs, CAT, and M-CAT-NGs showed no significant toxicity to mice, either in terms of morphology or liver and kidney functions, providing strong evidence of safety in vivo.
The in vivo results demonstrated that M-CAT-NGs exhibited significant therapeutic effects in OVA&NTHi-induced neutrophilic asthma mice, which were mainly reflected in the ability of M-CAT-NGs to significantly improve the lung function of neutrophilic asthma mice, reduce the proportion of neutrophilic cells, decrease the mucus secretion of the airways, restore the normal morphology of the airways, clean ROS from the lungs, and alleviate inflammation in the lungs. The therapeutic effects of M-NGs were similar to those of M-CAT-NGs, but M-NGs exhibited no significant role in cleaning ROS in vivo. This might be due to M-NGs did not contain the key component, i.e. CAT, for decomposing ROS. Although free-form CAT showed good efficiency in decomposition of H2O2in vitro, it did not show therapeutic effects in neutrophilic asthma mice in vivo. This result might be due to free CAT was unstable and susceptible to destruction and loss of bioactivity.