Valence Engineering Driven Multiple Mechanisms of Superior Antioxidant Nanosheets for Treatment of Pulmonary Fibrosis

doi:10.21203/rs.3.rs-2241692/v1

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Valence Engineering Driven Multiple Mechanisms of Superior Antioxidant Nanosheets for Treatment of Pulmonary Fibrosis

https://doi.org/10.21203/rs.3.rs-2241692/v1

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Idiopathic pulmonary fibrosis is a chronic and highly lethal lung disease that largely associated with oxidative stress. Unfortunately, there is currently no effective antioxidant therapy targeting oxidative stress pathogenesis. One key and great challenge is finding ideal antioxidant materials with superior anti-fibrotic effects. In this study, we report novel antioxidant V₄C₃ nanosheets (V₄C₃ NSs) for treatment of pulmonary fibrosis by scavenging reactive oxygen and nitrogen species. It is found that the subtle auto-oxidation can adjust the valence composition of V₄C₃ NSs and prominently improve their antioxidant behavior. The valence engineering triggers the multiple antioxidant mechanisms of electron transfer, H atom transfer and enzyme like catalysis, thus endowing V₄C₃ NSs with broad-spectrum, high-efficiency and persistent antioxidant capacity. Benefiting from the super antioxidant properties and high biocompatibility, V₄C₃ NSs can significantly prevent myofibroblast proliferation and extracellular matrix abnormality, thus alleviating the progression of pulmonary fibrosis by ROS scavenging, anti-inflammation and rebuilding antioxidant defenses in the bleomycin-induced animal model. This study not only provides an important strategy for designing excellent antioxidant nanomaterials, but also proposes a nanoscheme for the treatment of pulmonary fibrosis and other oxidative stress-related diseases.

Biological sciences/Biochemistry/Biocatalysis

Physical sciences/Nanoscience and technology/Nanoscale materials/Two-dimensional materials

V4C3 nanosheets

valence engineering

antioxidant

reactive oxygen species

pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and highly lethal interstitial disease of the lung, for which there is currently no cure. The average survival time of IPF patients after diagnosis is only 3–5 years.^1,2 Although the pathogenesis has not been defined, it is widely believed that the oxidative stress is closely related to the occurrence of pulmonary fibrosis.^3,4 The overproduction of reactive oxygen species (ROS) causes oxidative damage to alveolar epithelial cells and stimulates the release of fibrogenic mediators, leading to the proliferation of fibroblasts and abnormal deposition of extracellular matrix (ECM), which triggers lung tissue fibrosis.^5,6 By targeting oxidative stress related pathogenesis, antioxidant therapy represents an effective strategy to both prevent and alleviate pulmonary fibrosis. ^7,8 Although there are many small molecules and antioxidant enzymes showing evident activity to reduce ROS, they suffer low antioxidant efficiency and persistence under pathological conditions, which hinders their clinical application. Acetylcysteine has previously been used as prevailed antioxidant substance in treatment of pulmonary fibrosis,⁹ but it often requires combination with other anti-fibrotic drugs defined as “standard of therapy”. Unfortunately, many treatments recommended that the standard therapy has essentially failed,¹⁰ especially the compatible medications (prednisone and azathioprine) are highly toxic and the therapeutic effect of acetylcysteine remained unclear.¹¹ Therefore, it highly necessitates to seek new treatment with ideal antioxidant materials for IPF that liberated from the use of “standard of therapy”.

Nanomaterials have been found that can modulate the production of ROS under specific conditions, providing a new solution for the treatment of ROS-based diseases.¹² Though some nanomaterials (e.g. Ag, Au, Se and Fullerene nanoparticles) have been reported to be useful to alleviate pulmonary fibrosis^13–16, very few studies focus on antioxidant therapy.^13–16 As a class of nanomaterials with enzyme-like catalytic characteristics, nanozymes possess high catalytic efficiency, good stability, and especially the ability to regulate ROS.^17,18 Their antioxidant enzyme-like activity allows them to effectively scavenge ROS (such as H₂O₂ and O₂^•−), thereby showing potential for antioxidant treatment of glioma, Parkinson's disease, Alzheimer's disease and other central nervous system diseases.^19–21 However, nanozymes with single enzyme-like activity are inefficient for scavenging all ROS species, while nanozymes with multi-enzyme like function are often accompanied by multiple catalytic actions.²² For example, many nanozymes mimicking oxidoreductase simultaneously show pro-oxidant and anti-oxidant functions, thus resulting in unfavorable outcomes in complex pathological microenvironments.²³ Currently, it remains a big challenge to extend the antioxidant spectrum of nanozymes due to their single catalytic mechanism. To meet this challenge, we are lighted by integrating multiple mechanisms to develop superior antioxidant nanomaterials with high efficiency and a broad activity spectrum.

As an emerging two-dimensional metal nitride or carbide materials,²⁴ the outstanding electrical conductivity, mixed-valence transition metal and hydrophilic surface allow MXenes not only to show excellent catalytic ability to promote electron transfer but also to exhibit biological activity.^25–27 In our recent investigation on novel nano-antioxidant, we find that MXenes can serve as a model to meet the above requirement for diverse antioxidant mechanisms because of its unique surface and composition structure. Unfortunately, research on the synergistic effects for super antioxidant role and treatment of ROS related diseases is lacking. To this end, V₄C₃ nanosheets (NSs) with 4 + 3 atomic layer structure and abundant valence state of vanadium were selected for antioxidant treatment of IPF (Fig. 1). To balance the valence composition of multi-valence vanadium, a low temperature slow auto-oxidation process was employed to treat V₄C₃ NSs. The valence composition of V species in aged V₄C₃ NSs is beneficial to simultaneously drive the enzyme-like catalytic ability and electron donating behavior of V₄C₃, thus making them exhibit high efficiency, broad spectrum activity, and highly stable antioxidant activity. Benefiting from the super antioxidant capacity, V₄C₃ NSs were demonstrated a significantly therapeutic efficiency to bleomycin-induced pulmonary fibrosis by ROS scavenging, anti-inflammation and rebuilding antioxidant defenses in the lungs. Notably, V₄C₃ NSs show good biocompatibility and renal excretion. This work provides a new strategy and significant insights for the design of high-efficiency antioxidant drugs for the treatment and prevention of ROS-mediated diseases.

Fabrication and characterization of fresh and aged V₄C₃ NSs

V₄C₃ NSs were prepared by typical etching and intercalation processes.²⁸ The detailed procedure is schemed in Fig. 2a and described in supporting information. Hydrofluoric acid (HF) is often-used to etch the aluminum layer in most methods.²⁹ To avoid the high toxicity of HF in operation, we chose the relatively mild mixed-solution method of concentrated HCl and LiF to etch the Al layer. Scanning electron microscope (SEM) images showed the tight “sandwich” structure of V₄AlC₃ MAX phase and the accordion-like structure of multi-layer V₄C₃ (Figure S1 and S2).³⁰ Then, few-layered V₄C₃ NSs were formed by TMAOH intercalation with stirring for 3 days and further ultrasonic treatment under argon atmosphere (Fig. 2a). Due to rich hydrophilic groups, the as-prepared V₄C₃ NSs became well-dispersed in aqueous solution, exhibiting the typical Tyndall effect. Transmission electron microscope (TEM) images show the few-layered V₄C₃ NSs and the well-defined lattice planes, of which the interplanar spacing is calculated to be 0.254 nm, which corresponds to the (-110) plane (Fig. 2b-d).³¹ The average thickness of V₄C₃ NSs was determined to be ≈ 2.3 nm, denoting that the synthesized V₄C₃ is composed of two layers (Figure S4). X-ray diffraction (XRD) results show that the peak at 39.6° disappear, indicating that the Al layer is completely etched. After the etching and stripping process, the characteristic peak (002) appeared and finally shifted to 2θ = 5.3º (Figure S5), suggesting again the formation of few-layered structure of V₄C₃ NSs. The uncontrolled oxidation of few layered MXenes has always been considered one of the limitations for their practical application.³² However, everything has two sides: proper oxidation can equilibrium the valence composition and thus may bring more abundant biological effects.³³ The key is to grasp the oxidative degree. To achieve this, a relatively low temperature (4 ºC) with a long time (~ 8 weeks) (LTLT) aging treatment was employed to allow the slow auto-oxidation. For clarity, fresh V₄C₃ is denoted as V₄C₃-A, and aged V₄C₃ as V₄C₃-B. The structure of V₄C₃-B was characterized systematically in comparison with V₄C₃-A. After the aging treatment, the suspension showed same Tyndall effect and no visible color change to naked eye, but the absorbance decreased considerably in the wavelength range from 200 to 800 nm (Fig. 2h). TEM showed that topographical structure was locally damaged and some edge and surface defects formed after aging, but the NSs showed well-defined lattice planes with distance of 0.254 nm (Fig. 2e-g). The XRD pattern showed the disappearance of diffraction peaks (004), (006) and (008), while the typical peak (002) still stood (Figure S6).

The surface chemical state and element composition were further comparatively analyzed. Fourier transform infrared spectroscopy (FT-IR) was used to characterize the functional groups and chemical bonds on the surface of V₄C₃ NSs. There were abundant hydroxyl groups on the surface for both V₄C₃-A and V₄C₃-B (Fig. 2i). X-ray photoelectron spectroscopy (XPS) was used to analyze the surface chemical bond characteristics and elemental valence distribution of V₄C₃ NSs. The survey spectra confirm the same element existence of V 2p, C 1s, O 1s, and F 1s in V₄C₃-A and V₄C₃-B (Fig. 2j). These results indicate that V₄C₃-B maintains good crystallinity and original few-layered MXenes structure after LTLT aging. Although there is some destruction in surface morphology, both the samples V₄C₃-A and V₄C₃-B have the almost same surface groups, crystal structure, optical property and chemical elements.

Analysis by fitting the high resolution XPS spectra implicated that the valence composition of V, C and O of V₄C₃ readily changed after LTLT aging treatment. In the spectrum of V 2p, there are three groups of deconvolution peaks between 511 and 525 eV, which are V-C/V²⁺ (V 2p 3/2 512.9 eV, V 2p 1/2 520.2 eV), V³⁺ (514.0 eV and 521.1 eV), V⁴⁺ (516.2 eV and 522.4 eV) (Fig. 2k).^33,34 The percentages of V-C/V²⁺, V³⁺ and V⁴⁺ were calculated (Table S3). The V⁴⁺ content increased from 28.4% in V₄C₃-A to 49.3% in V₄C₃-B, while the percentage of V-C/V²⁺ and V³⁺ dropped by ~ 8% and ~ 13%, respectively. This suggests that the valence composition of vanadium goes through a significant change even under gentle auto-oxidation. The O 1s fitting peaks identified the oxygen bonded in V-C-(OH)x, C = O, V-O and V-Ox in both V₄C₃-A and V₄C₃-B. The increase of oxygen content in C = O and V-O and decreased percentage in OH also confirms the oxidation of V₄C₃ and surface absorbed -OH groups (Figure S7a). The C 1s spectra can be fitted into four Gaussian peaks, corresponding to V-C, C-C, C-H, and C-O, respectively (Figure S13b). It is obvious that after aging, the signal intensity of the V-C and C-H peaks decreased, while the signal intensity of the C-C and C-O peaks increased significantly, indicating that V-C bonds were partially broken after oxidation. The histogram clearly shows the composition changes of V with different valence and C in different forms for the two samples (Fig. 2l). To further verify the valence change of V and identify the dominant specie, the ESR spectra of V₄C₃-A and V₄C₃-B NSs were recorded at room temperature (Fig. 2m). No ESR signal was detectable for V₄C₃-A; in contrast, an evident eight-line spectrum appeared in V₄C₃-B. This signal is characteristic for V⁴⁺, because V⁴⁺ contains a 3 d¹ electron coupled to its nuclear spin, I = 7/2 for V⁴⁺.^35,36 These results reveal the significant change in valence composition of V₄C₃ after slow oxidation via LTLT aging treatment. In particular, VO²⁺/VO⁺ ratio changed from 0.8 to 2.2 without generation of V⁵⁺, suggesting VO²⁺ become the dominant V species in V₄C₃-B. This valence composition change results in a positive shift (0.36 eV) of redox potential of V₄C₃ (Figure S8). As expected, the variable valence composition, unique layered crystal structure, and superior water solubility would endow V₄C₃ with unparalleled physicochemical properties and biological activity.

Multiple mechanisms collaborating superior antioxidant activity of V₄C₃ NSs

To comprehensively evaluate the antioxidant capability of V₄C₃ NSs, we tested the scavenging ability of V₄C₃ NSs towards stable free radicals and short-lived radicals including reactive oxygen (H₂O₂, •OH, O₂^−• and PTIO^•) and nitrogen (ABTS^+•, DPPH^•). ABTS^+• and DPPH^• are the nitrogen centered radicals widely used for the determination of total antioxidant capacity of natural antioxidants.³⁷ The highly stable radical cation ABTS^+• is generated by the oxidation of ABTS with potassium sulfite (K₂S₂O₈). The total antioxidant capacity of V₄C₃ NSs was accessed by monitoring its absorption changes in the visible band.³⁸ The results showed that V₄C₃ NSs had high activity to reduce ABTS^+• and DPPH^• in concentration-dependent manner (Fig. 3a-b). It is recognized that the reduction of ABTS^+• is dominated by a single electron transfer (ET) mechanism, while the scavenging of DPPH^• in aqueous solution is mainly conducted by a hydrogen atom transfer (HAT) mechanism.^37,38 The clearance of ABTS^+• and DPPH^• may suggest that the antioxidant activity of V₄C₃ NSs involves both ET and HAT mechanisms.

ROS are highly reactive chemicals containing oxygen, produced either exogenously or endogenously, and are the main source of oxidative stress causing damage to DNA, proteins, and other cellular structures. Because H₂O₂, superoxide and hydroxyl radicals are the dominant ROS in biological systems, they were selected to be the targets of clearance to test the antioxidant activity of V₄C₃ NSs. Considering short life and high activity of •OH and O₂^−•, they were monitored by using ESR spectra coupled with spin trapping technique. •OH was produced by typical Fenton reaction between Fe²⁺ and H₂O₂, while O₂^−• was generated by a classic enzymatic reaction between hypoxanthine and xanthine oxidase. •OH and O₂^−• were rapidly captured by 5, 5-dimethyl-1-pyrroliline nitrogen oxide (DMPO) and 5-tert-butyl-5-methyl-1-pyrroliline nitrogen oxide (BMPO) to form a spin adduct, DMPO/•OH and BMPO/•OOH, respectively. The ESR signal shows the fingerprint spectrum of DMPO/•OH having four lines with 1:2:2:1 intensity and BMPO/•OOH having four-lines with 1:1:1:1 and distinct hyperfine splitting parameters. It was found that the addition of V₄C₃ NSs result in the significant reduction of ESR signal for both •OH and O₂^−•, and the reduction degree is positively correlated to the V₄C₃ NSs concentration (Fig. 3d, e). This confirmed the strong activity of V₄C₃ NSs to scavenge •OH and O₂^−•.

The NSs’ scavenging ability for H₂O₂ was tested by HRP assay. HRP can catalyze the oxidation of 3‘3’5’5-Tetramethylbenzidine hydro-chloride dehydrate (TMB) and color evolution in the presence of H₂O₂. Upon the addition of V₄C₃ NSs, the characteristic absorption peak of oxidized TMB at 650 nm was dramatically reduced, proving that H₂O₂ was reduced by V₄C₃ NSs, thus inhibiting the oxidation of TMB. The reduction of H₂O₂ is proportionally dependent on the concentration of V₄C₃ NSs (Fig. 3c). In addition, we used three free radicals (ABTS^+•, DPPH^• and •OH) to compare the antioxidant activity of V₄C₃ NSs with ascorbic acid (AA) and tannic acid (TA), and the results showed that V₄C₃ NSs have superior antioxidant efficiency, from several times to dozens of times higher than that of the natural antioxidant molecules (AA and TA) (Figure S9 and S10). After 2 months storage, the antioxidant activity of V₄C₃ NSs to reduce ABTS^+•and DPPH^• retained 90% of fresh samples (Figure S11). These results demonstrated that the V₄C₃ NSs exhibit broad-spectrum, high-efficiency and long-stability antioxidant capacity. As discussed above, the fresh V₄C₃ NSs have an -OH bonded surface and multi-valence V with dominant low valence (V²⁺ and V³⁺ account for ~ 70%), which endow V₄C₃ NSs with a strong ability to donate electrons and reductive power. Comparing the reduction potential of V³⁺/V²⁺ and VO²⁺/V³⁺ with that of free radical or ROS, it is evident that φ (V³⁺/V²⁺= -0.255 V) and φ (VO²⁺/V³⁺ = 0.337 V) are sufficiently negative to reduce DPPH^•, ABTS^+•, H₂O₂ and •OH. Therefore, it is thermodynamically favorable that V₄C₃ NSs scavenges them all. Apart from the strong electron donating ability, valent V, especially in the IV state, may act as the catalytic site for the superoxide dismutation reaction, supporting V₄C₃ NSs as SOD mimetics.²⁷ In thermodynamics, the disproportionation of O₂^−• is associated with the oxidation and reduction reactions of O₂^−• with the reduction potential of 0.91 V and − 0.18 V, respectively.³⁹ An ideal SOD or catalyst to mediate the oxidation and reduction reactions has an optimal redox potential of ~ 0.36 V in the middle of 0.91 and − 0.18 V.⁴⁰ The redox potential of either VO²⁺/V³⁺ (φ = 0.337 V) or V₄C₃ NSs (φ = 0.0545 V) falls between that of O₂/O₂^−• and O₂^−•/H₂O₂, theoretically confirming the feasibility of V₄C₃ NSs to catalyze the O₂^−• dismutation like SOD (Fig. 3f). Based on the above analysis, the antioxidant behavior of V₄C₃ NSs may be directed by multiple mechanisms: electron transfer (proton coupled electron transfer), H atom transfer and enzyme-like catalysis for facilitating electron transfer (Fig. 3g). Therefore, V₄C₃ NSs are able to exhibit the high-efficiency and broad-spectrum antioxidant activity.

Next, in order to explore the antioxidant stability, we move on to compare the V₄C₃-A and V₄C₃-B (V₄C₃ NSs before and after LTLT aging treatment) for their antioxidant behavior. Like V₄C₃-A, V₄C₃-B can efficiently scavenge the same free radicals and H₂O₂ (Fig. 3h). After about 2 months of storage, the activity of V₄C₃-B to scavenge ABTS^+•, H₂O₂ and •OH almost the same as that of fresh V₄C₃-A, indicating their high stability of antioxidant activity. V₄C₃-B show decreased activity to scavenge DPPH^•, this may be caused by the reduction of -OH terminals on V₄C₃ surface after slow auto-oxidation because the DPPH^• reduction involves hydrogen atom transfer. More interestingly, it was found that the O₂^−• scavenging activity of V₄C₃-B increased significantly, to ~ 2.7 times higher than that of V₄C₃-A (Fig. 3h and Figure S12). The V(IV) proportion reached approximately 50% in V₄C₃-B and redox potential was measured to be more closer to 0.36 V, which are highly beneficial to enhance SOD-like activity. PTIO^• is an oxygen centered stable free radical and presents a characteristic five-line ESR spectrum. The reduction of PTIO^• is related to electron transfer. V₄C₃-A NSs show considerable ability to reduce PTIO^• (Figure S12). In contrast, V₄C₃-B NSs exhibit significant ability to quench PTIO^• in a concentration dependent manner, suggesting again their superior catalytic activity to facilitate electron transfer (Figure S13). Benefiting from the multiple antioxidant mechanisms driven by surface and valence engineering, V₄C₃ NSs were shown to possess broad-spectrum, high efficiency, good stability and continuously increasing super-antioxidant capacity. In addition, they have high water solubility, thus could define them as an ideal antioxidant material for treatment of idiopathic pulmonary fibrosis.

Protect fibroblast cells from oxidative damage via scavenging ROS

As discussed above, ROS can cause oxidative damage to alveolar epithelial cells so that trigger lung tissue fibrosis. Considering the excellent ROS scavenging capacity, V₄C₃ NSs thus are highly expected to serve as a potential antioxidant for treatment of idiopathic pulmonary fibrosis. The bare V₄C₃ NSs without any modification were found to sharply assemble in physiochemical buffer solution, which limited their further biological application. To address this problem, the V₄C₃-B NSs were modified by tannic acid (TA) to form high dispersive V₄C₃-TA in PBS buffer (Figure S14). We measured that V₄C₃-TA showed the compatible antioxidant activity with bare V₄C₃ NSs toward all the free radicals, which is proposed to afford excellent ROS scavenging capacity in cell and in mice.

To investigate the effect of V₄C₃-TA on redox homeostasis at the cellular level, a series of assays were conducted. Firstly, the toxicity of V₄C₃-TA was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay on normal mouse fibroblast cell L929. The result showed that 80% of L929 cells remained alive even the concentrations of V₄C₃-TA up to 25 µg mL^− 1, indicating low cytotoxicity of V₄C₃-TA towards to normal cell (Figure S15). Myofibroblasts, which are the main effector cells in pulmonary fibrosis diseases, can be transformed from L929 cells after induced by 30 ng mL^− 1 TGF-β1 at 24 h. α-SMA, a marker of fibroblast to myofibroblast transformation, was subsequently evaluated by immunofluorescence. As displayed in Fig. 4a, b, fluorescence intensity of α-SMA was significantly increased by 4.5-fold after stimulated by TGF-β1, implying that fibroblast L929 was successfully activated into myofibroblast. The cell morphology change from spindle shape of fibroblast into long strip of myofibroblast presented more solid evidence for the formation of myofibroblasts (Fig. 4a). After treatment with V₄C₃-TA, the cell morphology returns to normal and α-SMA signal intensity decreases significantly.

Since myofibroblasts have a high oxidative stress state, reducing intracellular ROS level would be necessary for antifibric effect. Following this consideration, we used DCFH-DA fluorescence probe to test the ROS-scavenging capacity of V₄C₃-TA. As imaged in Fig. 4c, d, the intracellular fluorescence intensity of L929 cells increased dramatically after treatment with 30 ng mL^− 1 TGF-β1, suggesting the substantial amount of ROS in myofibroblasts. In comparison, V₄C₃-TA-pretreated myofibroblast cells exhibited a significant decrease in fluorescence intensity, proclaiming the superior ROS scavenging capacity of V₄C₃-TA. Flow analysis further intuitively showed that V₄C₃-TA could efficiently remove intracellular ROS (Fig. 4e, f). Then, oxidative stress damage to intracellular biological macromolecules was evaluated by measuring the level of methylene dioxyamphetamine (MDA), one marker reflecting the level of lipid oxidation. As depicted in Fig. 4g, TGF-β1-treated cells revealed 1.3-fold increase while TGF-β1 + V₄C₃-TA-treated cells afforded similar level of MDA compared to that in PBS group, demonstrating that V₄C₃-TA acted as effective ROS scavengers to restore cellular redox homeostasis into normal level. Finally, the level of α-SMA in TGF-β1 + V₄C₃-TA-treated L929 was significantly decreased by 4-fold compared to that in TGF-β1 group (Fig. 4a), providing credible evidence for the antifibrotic effect of V₄C₃-TA at cellular level.

In vivo therapeutic efficacy on pulmonary fibrosis.

The excellent ability for in vitro ROS scavenging and α-SMA inhibition drives us to further investigate whether or not V₄C₃-TA can protect lung tissue from ROS injury and even alleviate pulmonary fibrosis. The IPF mouse model was firstly established by intratracheally injected bleomycin (BLM) after going through an inflammatory infiltration stage (3–7 days) and fibrosis stage (14–21 days) for a total of 21 days (Fig. 5a). Subsequently, intravenous injection of V₄C₃-TA was performed to verify the potential feasibility of V₄C₃-TA as an antioxidant for treating pulmonary fibrosis diseases. Compared to healthy mice in PBS group, body weight and lung index (that is, the ration of lung weight to body weight) in BLM group respectively decreased by 52% and 59%, while normal body weight and lung index were achieved for BLM + V₄C₃-TA-treated mice (Fig. 5b, c), validating that potential feasibility of V₄C₃-TA for treating pulmonary fibrosis disease. Hematoxylin and eosin (H&E) staining of lung slice from BLM-treated mice showed that collapsed alveolar structure, thick alveolar septum and a large number of in filtratedred blood cells (Fig. 5d), and Masson's trichrome staining also revealed that a large amounts of diffused blue collagen, suggesting the serious pulmonary fibrosis in the BLM-treated mice (Fig. 5d). In contrast, BLM + V₄C₃-TA-treated mice showed integrity alveolar structure and less blue area staining of collagen, presenting visual evidences forV₄C₃-TA-triggered recovery of alveolar structure and efficient inhibition of pulmonary fibrosis (Fig. 5d).

In vivo superior therapeutic effect promoted us to further explore the potential therapeutic mechanism of V₄C₃-TA against pulmonary fibrosis. α-SMA is a marker of epithelial-mesenchymal transition, and collagen I is the major component of the extracellular matrix (ECM). These two proteins are considered as important biomarkers of fibrotic response, and the expressions of which are tested by immunofluorescence (IF), immunohistochemistry (IHC) and western blotting (WB). As shown in Fig. 5e, compared with the PBS group, the red fluorescence intensity of α-SMA in the BLM group was increased by about 14 times, and the green fluorescence intensity of collagen I was increased by about 5 times. Meanwhile, WB revealed that the expression level of α-SMA and collagen I was up-regulated by 70% and 40% in BLM group than that of PBS group (Fig. 5f). IHC results in BLM-interfered lung tissue also showed extremely positive brown particles compared with that in PBS group (Figure S16). The overexpression of these two proteins implies the severe progression of pulmonary fibrosis. Impressively, upon administration of V₄C₃-TA, the level of α-SMA and collagen I restored to be consistent with that of the PBS group. Furthermore, other indicators related to epithelial-mesenchymal transition markers were also detected by IHC, including Fibronectin and E-cadherin. As shown in Fig. 5g, V₄C₃-TA intervention decreased the expression of fibronectin and increased the expression of E-cadherin in lung compared with BLM group. These experimental results explicitly proved that V₄C₃-TA could efficiently inhibit the proliferation of myofibroblasts, reduce ECM abnormal precipitation, and thus alleviate pulmonary fibrosis.

The etiology in IPF suggested that the oxidative stress and the tissue injury could evoke inflammatory response which further accelerates the cycle of oxidative stress and aggravates fibrotic pathology.⁴¹ It is proposed that V₄C₃-TA with ROS scavenging ability can break the vicious cycle, fight inflammation and restore natural oxidative stress defense system to resist fibrosis in mice. To test this assumption, the anti-inflammatory effect of V₄C₃-TA was firstly assessed by histological analysis of TGF-β1 and TNF-α, which are two important inflammatory cytokines potentially participate in the process of pulmonary fibrosis. Enzyme linked immunosorbent assay (ELISA) showed that compared with BLM group, both the expressions of TGF-β1 and TNF-α decreased significantly when treated by V₄C₃-TA (Fig. 5h), suggestive of the anti-inflammatory role of V₄C₃-TA. Then, a series of biochemical detection of lung tissue were performed to reveal the effect of V₄C₃-TA on mouse lung redox homeostasis. Compared to BLM-treated mice, BLM + V₄C₃-TA group showed significantly reduced MDA level, indicating that V₄C₃-TA protected lung tissue from lipid peroxidation damage (Fig. 5i). In addition, V₄C₃-TA can restore the activity of superoxide dismutase (SOD) and catalase (CAT) in the lung tissue, and increase the level of non-enzyme antioxidant GSH (Fig. 5j, l). These results confirmed that V₄C₃-TA not only inhibits the inflammatory outbreaks, but also repairs the antioxidant defense system, reduces pathological damage in the body, and thus blocks the process of pulmonary fibrosis.

We then sought to evaluate the biosafety of V₄C₃-TA. As can be seen in Figure S17, there are no distinguishable inflammatory lesions or histological damage in H&E stains of heart, liver, spleen and kidney of mice in PBS, BLM and BLM + V₄C₃-TA groups. In addition, ICP-MS detection of V element in time-dependent urine revealed that most V₄C₃ could be excreted within 12 h after intravenous injection, suggesting rapid clearance by the kidney and low long-term toxicity of V₄C₃-TA (Figure S18).

By targeting oxidative stress induced pathogenesis, the outstanding antioxidant V₄C₃ nanosheets were developed and applied for the first time to prevent and alleviate pulmonary fibrosis. Benefiting from the unique valence engineer and surface structure, V₄C₃ nanosheets provide multiple collaborative mechanisms involving electron transfer, H atom transfer and enzyme-like catalysis, thus can effectively scavenge ROS and RNS (ABTS^+•, DPPH^•, PTIO^•, •OH, O₂^−•, H₂O₂) and exhibit wide-spectrum, efficient and persistent antioxidant capability. Especially, controlling the gentle auto-oxidation can balance the valence composition of vanadium thus further enhance the antioxidant capacity of V₄C₃. The in vitro results proved that V₄C₃ NSs can significantly scavenge the cellular ROS and prevent the oxidative damage towards fibroblast cells. The in vivo study further demonstrated the anti-inflammatory, antifibrotic effect and remodeling oxidative stress of V₄C₃ to inhibit the myofibroblast proliferation and thus achieve efficient treatment on pulmonary fibrosis. This work provides important implications for designing highly efficient antioxidant nanomaterials for modulating the redox condition and treating oxidative stress-related pulmonary fibrosis disease.

Chemical and Materials

V₄AlC₃ MAX powder (400 mesh) was purchased from 11 Technology (Jilin Province, China). Tannin Acid (TA), Concentrated hydrochloric acid (HCl 12 M), hydrogen peroxide (H₂O₂), horseradish Peroxidase (HRP) and xanthine oxidase (XOD) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), (S)-Nitroso-N-acetylpenicillamine (SNAP), and Ferrous sulfate (FeSO₄) were commercially available from Aladdin Industrial Co. (CA, USA).1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) and Lithium fluoride (LiF) were purified from Shanghai Macklin Biochemical Co., Ltd. Tetramethyl ammonium hydroxide (TMAOH), diethylenetriaminepentaacetic acid (DTPA) and xanthine (HX) were purchased from Sigma-Aldrich (Shanghai, China). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and 5tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) were purchased from Tongren Institute of Chemistry. 2-Phennyl-4,4,5,5-tetramethylimidazoline-q-oxyl 3-Oxide (PTIO) was obtained from Shanghai Maokang Biotechnology Co., LTD. Milli-Q water (18 MΩ cm) was used in the preparation of all solutions. All glassware and autoclave used in the following procedures were cleaned using aqua regia solution (HNO₃/HCl = 1:3 v/v).

Characterization

UV-Vis absorption spectra were obtained using a Cary 5000 UV-Vis Spectrometer (Varian, USA) and a matched quartz cuvette with a path length of 1 cm. The crystal structures of the V₄C₃ NSs were characterized by X-ray diffraction (XRD, D8 Advance diffractometer, Bruker, Germany) using monochromatized Cu Kα radiation (λ = 1.5418 Å). Transmission electron microscope (TEM) and high resolution transmission electron microscope (HRTEM) were captured on a Tecnai G2 F20 U-Twin electron microscope with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) was performed with a Thermo ESCALAB 250XI multifunctional imaging electron spectrometer (Thermo Fisher Scientific, USA) using 150 W Al K_α radiation and a base pressure of approximately 3×10^− 9 mbar. The binding energies were calibrated to the C 1s line at 284.8 eV. AFM images were tested by Dimension Icon produced by Bruker Germany and Zeta potential was tested using Malvern Zetasizer Nano ZS90. Electron spin-resonance (ESR) spectra were obtained using an EMX micro electron resonance spectrometer (Bruker, Germany). The measurements were carried out at room temperature under the following conditions: microwave power 2 mW, modulation amplitude 1.0 G, attenuation 20 dB, and scan range of 100 G. Streaming cell analyzer (BD, FACSCalibur, USA), The fluorescent images were captured by using Confocal and superresolution microscopy (NIKON AIR + STORM, Japan). The absorbance was recorded with a multifunctional enzyme marker (Thermo scientific, Varioskan flash, USA).

Synthesis of multilayer V₄C₃

Typically, 0.4 g V₄AlC₃ was added into a 100 mL Teflon tank and followed by careful addition of 30 ml concentrated hydrochloric acid (HCl, 12 M) and 10 mL 9 M LiF solution (weigh 2.334 g LiF and dissolve in 10 mL deionized water). After stirring for 30 min at room temperature, the mixture was incubated at 140 ºC for five days to obtain a multilayer V₄C₃ solution with the Al layer etched away. Centrifuge it with deionized water (3500 r/min) and wash it to pH > 6. The products were dried under vacuum at 60 ℃ for 12 h.

Synthesis of few layered V₄C₃

Weigh 0.1 g multilayer V₄C₃ powder, place it in 10 mL glass sample bottle, add 5 mL high-concentration tetramethyl ammonium hydroxide solution (TMAOH, 25% in water), stir for 3 days at room temperature (25 ℃). Then, remove the TMAOH solution by centrifuging at 8000 r/min three times. After that, the black precipitate was re-dispersed into 20 mL deionized water, and the 2D V₄C₃ solution was obtained by ultrasound under argon atmosphere for 90 min. Then, a colloidal solution of 2D V₄C₃ nanosheets (NSs) was collected by centrifugation for 20 min at 8000 rpm. The 2D V₄C₃ NSs were dried in a vacuum freeze-dryer for at least 24 h and collected for quantification.

The aged V₄C₃ NSs (V₄C₃-B) were obtained by placing the fresh V₄C₃ NSs with concentration of 1.5 mg/mL at 4 ℃ for 8 weeks.

Synthesis of V₄C₃-TA

In order to obtain specific and effective V₄C₃/TA load, different load ratios were tried, and the results showed that the mass ratio of V₄C₃/TA was 1:0.75, which could remain stable in PBS solution. The specific load steps are as follows: 7.5 mg of TA were dispersed into 1 mL aqueous solution and added to 10 mL after aging V₄C₃ NSs solution (1.1 mg/mL) and left for stirring overnight. The excess TA was removed by centrifugation, and the final concentration was fixed to 1 mg/mL.

ABTS^+• and DPPH• Scavenging Activity

ABTS and DPPH assays were used to measure the total antioxidant capacity of V₄C₃ NSs. The configuration of ABTS^+· solution was carried out according to the literature method, and potassium persulfate was used as the oxidant in order to obtain free radicals. The configuration method is as follows: 0.0374 g ABTS powder was dissolved into 30 mL deionized water, followed by the addition of 0.0066 g K₂S₂O₈ under stirring. The reaction was left to proceed in the dark for 12 h. To test the ABTS^+• reduction, 0.2 mg/mL V₄C₃ with varied volume (3, 6, 9, 12, 15 µL) was added to 3 mL ABTS^+• solution (O.D. = ~0.9). To test the DPPH• scavenging activity, 2.5 mL of 0.1 mg/mL DPPH• ethanol solution was combined with 0.5 mL of deionized water. Then, different volumes (3, 6, 9, 12, 15 µL) of 0.2 mg/mL V₄C₃ were added. Two minutes later, the UV-Vis spectra were recorded to measure the reduction of ABTS^+• and DPPH^•.

•OH Scavenging Activity

The •OH was generated by Fe²⁺ and H₂O₂ through a classical Fenton reaction, and its production and removal process was tested by ESR spectrum. The reaction system solution was 50 µL, consisting of 10 µL 10 mM H₂O₂, 5 µL 50 mM DMPO, and 10 µL 10 mM Fe²⁺ in the absence or presence of different concentrations of V₄C₃ or antioxidants (AA/TA). ESR spectral signals were recorded at different times.

O₂^−• Scavenging Activity

ESR was used to test scavenging activity of V₄C₃ on superoxide (O₂^−•). Superoxide anion was generated in the mixture containing 10 µL 5 mM hypoxanthine (HX) with 10 µL 0.25 mM DTPA, 5 µL 250mM BMPO, and V₄C₃ at various concentrations, and initiated by adding 5 µL 0.4 U/mL XOD. O₂^−• scavenging activity of antioxidants was evaluated from the change of BMPO/•OOH signal intensity at different times.

PTIO· Scavenging Activity

The scavenging activity of PTIO• free radical was measured by ESR spectrometer. For the ESR measurement, 5 µL PTIO• (2 µM) in the absence or presence of different concentrations of V₄C_3, and the ESR spectra was recorded immediately at different interval.

H₂O₂ Scavenging Activity

The H₂O₂ scavenging ability of V₄C₃ was analyzed by HRP-H₂O₂-TMB system. 20 µL H₂O₂ (0.1 M) was firstly mixed with V₄C₃ at desirable concentrations in 3 mL H₂O. 5 min later, 20 µL TMB (20 mM) and 10 µL HRP (1 µg/mL) were added into the above mixture to initiate the TMB oxidation. After 2 min of reaction, the absorption spectra showing characteristic absorption at 652 nm was recorded by a UV-Vis spectrometer.

Cell culture and induction

The L929 mouse fibroblasts are purchased from the Institute of Basic Medicine of the Chinese Academy of Medical Sciences and cultured in MEM supplemented with 10% horse bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO₂ atmosphere. TGF-β1 is purchased from Beijing. Adding a 30 ng/mL TGF-β1 without serum-free culture base will become fibroblast-induced into muscle and fibroblasts.

Vitro cytoprotective test

Cell viability was examined using an MTT assay kit. L929 cells were seeded in a 96-well plate at a density of 8 × 10³ cells/well and incubated for 24 h. The cells were then incubated with different concentrations of V₄C₃ for 24 h, followed by washing with PBS to remove MEM. Then, 10 µL MTT and 90 µL MEM (5 mg/mL 1 × PBS preparation) was added per well at 37°C for 4 h, followed by the addition of 100 µL DMSO, after which absorbance at 490 nm was measured using enzyme standard test.

ROS assessment

Fluorescence analysis ROS: When the L929 cell density of 6-well plate reaches about 60%, except for the PBS group, add TGF-β1 to the remaining groups to induce for 24 h, then replace the medium with new medium, and add different concentrations (0–1) of V₄C₃ for 12 h. After adding the DCF-DA probe to incubate for 30 min at a ratio of 1:1000, wash with PBS to remove any DCFH-DA that had not been taken up, and observe using a microscope. Streaming cell analyzer test ROS: The cells were digested and collected by trypsin after incubation with V₄C₃ for 12 h. After Cell was added to the DCFH-DA probe to incubate for 30 minutes, followed by PBS washing twice and tested by flow cytometry.

Protein extraction and quantification

The cells were induced by 30ng/ mL TGF-β1 for 24 h, and incubated with V₄C₃ for 12 h. The appropriate RIPA lysis solution was added to collect the cells, and the cells were lysed on ice for 30 min. After centrifugation at 12000 g for 20 min, the supernatant was stored in the − 80°C. The extracted proteins were quantitatively determined by SolarBio BCA protein concentration assay.

Lipid oxidation detection

Step refer to biyuntian Lipid oxidation Kit operation: The TBA storage solution and TBA working solution were dissolved at 70°C. 100 µL protein and 200 µL working solution were added and heated under boiling water for 15 min, cooled to room temperature, and centrifuged at 1000 g for 10min. 200 µL supernatant was taken, and the absorbance at 532 nm was measured with a microplate tester.

Mouse lung fibrosis model built by BLM

SPF C57BL/6 mice about 6 weeks old were purchased from Beijing Weitong Lihua Experimental Animal Technology. The pulmonary fibrosis model was constructed by intratracheal instillation after the mice were stabilized for one week, and the mice were randomly divided into three groups (PBS group, BLM group, BLM + V₄C₃ group). Briefly: mice were anaesthetized followed by intratracheal instillation 3 mg/kg of bleomycin. We then waited for the mouse to wake up. Mice received the first dose of V₄C₃ (1 mg·kg^–1) 2 days after bleomycin and were treated every 2 days for 18 days. The mice were killed and lung tissues were collected, part of which were fixed in 4 ℃ formaldehyde, and part of which were washed with PBS and stored at -80 ℃.

Hematoxylin and Eosin (H&E) staining

The lung tissues of mice were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded, and sliced to obtain paraffin sections of about 4 µm. Staining procedure prepared according to Solarbio′s Hematoxylin and Eosin(H&E) staining kit. Simply: Prepared 4 µm tissue samples were deparaffinized using xylene and ethanol. After 5 min of hematoxylin staining and 1 min of eosin staining, the histological morphology was observed under a microscope.

Masson staining

After paraffin sectioning, the slices were pasted at 60°C for 30 min and operated according to Solarbio′s Masson staining kit. In simple terms, Weigert iron hematoxylin stained the nucleus, Ponceaufuchsin stained for 6 min to make the muscle fibers red, and aniline blue stained for 1min to make the collagen fibers blue.

Immunohistochemical staining

The lung slides were heated at 60 ℃ for 2 h, then incubated in 3% H₂O₂ for 30 min to quench endogenous peroxidase activity. Citrate buffer was used for antigen repair。The slides were incubated in 5% BSA for 1h for blocking and in the α-SMA antibody(Collagen Ⅰ antibody)༈α-SMA: 5% BSA = 1:2000 ; Collagen Ⅰ: 5% BSA = 1:1000) overnight at 4 ℃. Then, biotinylated secondary antibody was applied for 1h at room temperature. DAB chromogenic solution for was applied color development and hematoxylin staining for the nucleus performed for 20 s. Staining was observed under the microscope.

Double immunofluorescence staining

Lung tissue was sealed with 3% H₂O₂, then permeabilized using 0.3% triton-100. Tissue was incubated with the primary antibody(α-SMA: 5% BSA = 1:500 ; Collagen Ⅰ: 5% BSA = 1:200) at 4 ℃ overnight, then the secondary antibody (with 488: green light), rabbit antibody (594: red light) and 5% BSA at 1:200 dilution was added for 1 h at room temperature. Tissue was incubated in DAPI stain for the nucleus for 2 min, after which the anti fluorescence quenching agent added. The glass was covered and images taken with laser confocal microscope.

Extraction and quantification of lung tissue protein

The lung tissues placed at -80 ℃ were removed, and 100 µL of RIPA for every 10 mg of tissue was added for lysis. The lung tissues were stirred in a tissue stirrer for 2 min, and then cleaved on ice for 30 min. After centrifugation at 12000 rmp for 20 min, the supernatant was separated and diluted with PBS in Solebo's BCA protein concentration reagent. The previously prepared BCA working solution was added and the solution incubated at 37°C for 20 min, after which the absorbance value at 562 nm was measured with a microplate reader.

Transforming growth factor β1 (TGF-β1)

The serum obtained by centrifugation was quantified with BCA protein, and operated according to Elabscience Transforming Growth factor (TGF-β1) ELISA kit: The samples were activated. Then, biotinylated antibodies were added for incubation, after which the enzyme-binding working solution was added for incubation. The reaction was terminated by adding TMB color, and the absorbance at 405 nm was measured.

TNF-α: BCA protein was quantified after protein was extracted with RIPA lysate. Absorbance at 450 nm was measured according to the kit procedure.

HYP: BCA protein was quantified after protein was extracted with RIPA lysate, and subsequently detected according to HYP ELISA kit.

Catalase detection

Dilute the extracted lung tissue protein with H₂O₂ enzyme buffer solution, then add 10 µL, 250 µM H₂O₂ solution for 3 min, followed by H₂O₂ enzyme to stop the reaction. Then, add color and working solution, and measure the absorbance at 520 nm. It is worth noting that H₂O₂ is more accurate when reduced by 30–50% after the reaction. The catalase activity of each gram sample was calculated according to the catalase detection kit by Biyuntian.

Superoxide enzyme detection

Tissue homogenization was performed by adding 1 g lung tissue to 10ml superoxide (SOD) dismutase activity detection kit from SolarBio. Supernatant was centrifuged at 8000 g for 10 min, and BCA protein was quantified. After that, the unused reagent was added and the absorbance at 560 nm was measured at 37°C for 30 min. Finally, the activity of superoxide enzyme in each mg sample was calculated.

GSH detection

Performed according to the trace reduced glutathione determination kit of Nanjing Jianyue: For 1 g lung tissue, add 9 mL PBS to prepare 10% tissue homogenate in a tissue homogenate machine. Then, centrifuge the supernatant and quantify BCA protein. Add reagents to the supernatant after reaction, centrifuge the supernatant, and the absorbance was measured at 405 nm. Finally, calculate the content of GSH in different mg samples.

Western-blotting test

BCA protein concentration detection kit was used to quantify the extracted protein. Proteins were separated by 8%/12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Non-specific antigens of PVDF membranes were blocked with 5% free-fat milk or BSA in Tris buffer for 4 h. The washed membranes were cut into strips according to the molecular weight of the target protein and incubated with the corresponding primary antibody overnight at 4°C. The primary antibody was removed the next day and the membrane incubated with the secondary antibody for 1 h at room temperature. ECL imaging method was used to display protein bands.

Statistical analysis

Differences between experimental groups were assessed by Student's t-test using GraphPad Prism 8 Software. *p < 0.05 was considered statistically significant; **p < 0.01 and ***p < 0.001 were considered highly significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data needed to support the finding of this study are presented in the article and the Supplementary Information. Any other data related to this work are available from the corresponding authors upon reasonable request. A reporting summary for this article is available as a Supplementary Information file.

Acknowledgements

This work is supported financially by the National Natural Science Foundation of China (62274141), the Program for Zhongyuan Leading Talents of Science and Technology Innovation in Henan Province (204200510016), Postgraduate Education Reform and Quality Improvement Project of Henan Province (YJS2022JD34). Y. Huang gratefully acknowledges National Natural Science Foundation of China (22175055, U1904150), Natural Science Foundation of Henan (212300410003) and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (21IRTSTHN002) for financial support.

Author information

These authors contributed equally: Quan Liu, Yaping Ren.

Contributions

Q.L., Y.P.R., Y.W.H., W.W.H. conceived and designed the experiments; Q.L., Y.P.R. carried out the experiments; H.M.J. conducted the synthesis and characterization of nanoparticles; Q.L., Y.P.R., Y.P.T., Y.W.H., W.W.H. participated in final data analysis and interpretation; Q.L., S.K. X.B.M., W.W.H. wrote and edited the manuscript, Y.W.H, Z.Z. and W.W.H supervised this project and revised the manuscript.

Corresponding authors

Correspondence to Weiwei He or Yongwei Huang.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material available at

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Valence Engineering Driven Multiple Mechanisms of Superior Antioxidant Nanosheets for Treatment of Pulmonary Fibrosis

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Abstract

Figures

Introduction

Results And Discussion

Fabrication and characterization of fresh and aged V4C3 NSs

Multiple mechanisms collaborating superior antioxidant activity of V4C3 NSs

Protect fibroblast cells from oxidative damage via scavenging ROS

Discussion

Methods

Chemical and Materials

Characterization

Synthesis of multilayer V4C3

Synthesis of few layered V4C3

Synthesis of V4C3-TA

ABTS+• and DPPH• Scavenging Activity

•OH Scavenging Activity

PTIO· Scavenging Activity

H2O2 Scavenging Activity

Cell culture and induction

Vitro cytoprotective test

ROS assessment

Protein extraction and quantification

Lipid oxidation detection

Mouse lung fibrosis model built by BLM

Hematoxylin and Eosin (H&E) staining

Masson staining

Immunohistochemical staining

Double immunofluorescence staining

Extraction and quantification of lung tissue protein

Transforming growth factor β1 (TGF-β1)

Catalase detection

Superoxide enzyme detection

GSH detection

Western-blotting test

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Fabrication and characterization of fresh and aged V₄C₃ NSs

Multiple mechanisms collaborating superior antioxidant activity of V₄C₃ NSs

Synthesis of multilayer V₄C₃

Synthesis of few layered V₄C₃

Synthesis of V₄C₃-TA

ABTS^+• and DPPH• Scavenging Activity

H₂O₂ Scavenging Activity