Silk-engineered bioactive nanoparticles for targeted alleviation of acute inflammatory disease via macrophage reprogramming

Abstract

Significant progress has been made in the development of potential therapies for diseases associated with inflammation and oxidative stress. Nevertheless, the availability of effective clinical treatments remains limited. Herein, we introduce a novel silk-based bioactive material, TPSF, developed by sequentially conjugating Tempol and phenylboronic acid pinacol ester to silk fibroin. This innovative reactive oxygen species (ROS) scavenging material not only effectively eliminates free radicals and hydrogen peroxide but also readily self-assembles into nanoparticle forms (TPSN). In vitro experiments have demonstrated that TPSN exhibits significant anti-inflammatory activities and cytoprotective effects against ROS-mediated damage. Consistently, in murine models of acute lung and kidney injury, TPSN outperforms the small-molecule antioxidant NAC, exhibiting superior therapeutic efficacy. Mechanistically, TPSN has the capability to reprogram M1-like macrophages toward an M2-like state. Importantly, biocompatibility assays confirm that TPSN has good safety profiles. Consequently, TPSN, characterized by its favorable protective effects and excellent biocompatibility, exhibits considerable promise as a therapeutic intervention for inflammation-related diseases. This innovative strategy, which incorporates multifunctional antioxidant components into the silk fibroin matrix, effectively addresses oxidative stress and acute inflammation. Furthermore, it highlights the potential of modified silk fibroin materials in the management and mitigation of inflammation-led tissue damage.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-024-03055-6.

Introduction

Inflammation, a physiological response to diverse harmful stimuli, is integral to the pathogenesis of numerous diseases. It serves as a defense mechanism against infections, hormonal imbalances, injuries, pathogenic microorganisms, and other physiological disruptions [1, 2]. However, when dysregulated, inflammation can contribute to the onset and progression of various conditions, such as rheumatoid arthritis, neurodegenerative disorders, chronic obstructive pulmonary disease, cancer, and other autoinflammatory diseases [3–6]. To treat these diseases effectively, numerous strategies have been devised to attenuate inflammation and impede disease progression. Traditionally, various anti-inflammatory agents, including nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, have been employed in the management of inflammatory diseases [7, 8]. However, these synthetic drugs often come with numerous side effects, such as an increased risk of cardiovascular and nephrotoxic events, as well as systemic adverse effects attributable to their non-specific distribution [9–11]. Hence, biotherapies have emerged as a viable alternative for the treatment of inflammation [12]. These biotherapies are specifically designed to target distinct inflammatory pathways. For instance, certain agents are capable of reducing the activity of specific cytokines or their receptors, such as canakinumab and anakinra [13, 14]. Other agents inhibit the migration of lymphocytes into tissues, exemplified by efalizumab [15, 16]. Additionally, some agents, like abatacept, prevent the binding of monocyte-lymphocyte costimulatory molecules [17]. Notably, anti-cytokine therapies have demonstrated promising outcomes in clinical applications, particularly in the treatment of autoimmune diseases such as inflammatory bowel disease, psoriasis, and rheumatoid arthritis [12, 18]. Nevertheless, it is crucial to acknowledge that these biotherapies may compromise the host’s immune defense against infection and potentially contribute to oncogenesis [19, 20].

It is well-established that inflammation is closely linked to oxidative stress. The imbalance between the generation of reactive oxygen species (ROS) and the availability of antioxidants or radical scavengers results in oxidative stress [21–23]. During the initial phase of inflammation, activated inflammatory and immune cells produce elevated levels of ROS to eradicate pathogens and activate pathways that further propagate the inflammatory response [24, 25]. However, the overproduction of ROS during the inflammatory response can, in turn, exacerbate oxidative stress, leading to tissue damage and chronic inflammation-associated disorders [26–29]. Given the strong link between oxidative stress and various inflammation-related diseases, the efficacy of combined antioxidant and anti-inflammatory therapy is being explored as a strategy to combat multiple pathologies. Small molecule antioxidants, such as superoxide dismutase (SOD, the only enzyme capable of eliminatingO₂^•− in mammalian cells) [30], SOD-catalase and glutathione peroxidase (GPXs) mimetics [31, 32], as well as N-acetylcysteine (NAC, a precursor of glutathione) [33, 34], have been investigated for their potential in treating sepsis, diabetes, coronary artery disease, and other inflammatory conditions [26]. Although some of these agents have demonstrated promise in preclinical studies, clinical trials have frequently produced mixed results [34, 35]. For example, NAC is already used clinically for the treatment of paracetamol toxicity [33]. However, its limited efficacy in scavenging free radicals and the inconsistent outcomes observed in clinical trial have been largely disappointing [34].

Recent advancements in nanotechnology have enabled the creation of functional nanomaterials derived from both natural and synthetic sources. These nanomaterials possess inherent anti-oxidative and anti-inflammatory properties, which can mitigate inflammation-related diseases. Their unique characteristics offer significant potential for overcoming the inherent limitations of small-molecule antioxidants, thereby obviating the necessity for incorporating additional therapeutic compounds or biologics [36–39]. For instance, a combined SOD/Catalase (CAT) mimetic nanoplatform, which included the SOD-mimetic Tempol (Tpl) and the CAT-mimetic β-cyclodextrin, was utilized for the treatment of inflammatory bowel disease (IBD) [40]. While β-cyclodextrin conjugated with phenylboronic acid pinacol ester (PBAP) was efficaciously alleviated the symptoms of peritonitis in mice [41]. Also, polydopamine-based nanoparticles served as ROS scavengers for periodontal disease therapy [42]. In addition to organic anti-oxidative nanosystems, inorganic nanozymes with intrinsic catalytic ROS-scavenging capabilities have also been explored for their potential applications in inflammation-related diseases [37]. These composite nanozymes, fabricated from materials like ceria-zirconia, show enhanced catalytic activity for ROS depletion and have been evaluated for their efficacy in the treatment of sepsis [43]. Despite substantial progress in the application of biomaterials for the treatment of inflammatory disorders over recent decades, a limited number of nanomaterials have received approval for clinical therapy [44, 45]. This limitation is primarily attributed to concerns related to biosafety, the complexity of synthesis process, and high costs [46]. In light of these challenges, it is anticipated that the development of safer and more effective therapeutic nanomaterials, capable of ROS-scavenging, will enhance the management of inflammation-related diseases.

Naturally sourced silk, a protein-based biopolymer derived from silkworm (Bombyx mori), has received FDA approval for medical applications [47]. Owing to its excellent biocompatibility, robust mechanical properties, non-cytotoxicity, and inherent biodegradability, silk has a longstanding use as suture materials [48]. Moreover, its applications extend across various biomedical and biotechnological domains, including vascular prostheses [49, 50], structural implants [51], wound dressings [52, 53], and tissue engineering [54, 55]. Silk fibroin (SF), a primary component of silk protein, is easily acquired and consists of a diverse array of amino acids [56]. These amino acids possess functional groups, including amines, alcohols, and carboxyl groups, which enable facile chemical modifications to incorporate drug molecules, peptides/enzymes, and other reactive groups [57, 58]. For example, SF modified with glycidyl methacrylate (GMA) has been effectively employed as a bioink for digital light processing (DLP) printing [59]. Additionally, Poly-L-lysine (PLL) conjugated SF has been applied as a coating on drug-loaded poly(lactic-co-glycolic acid) microparticles to regulate drug release [60]. Beyond its role as an auxiliary material, it is a pleasant surprise that SF inherently possesses antioxidant properties [61, 62]. However, there is a scarcity of research exploring the potential enhanced antioxidant effects that could be achieved through chemical modifications.

In light of the detrimental effects of ROS and persistent inflammation on tissue health, we propose a novel pathogenesis-guided modification of SF material for enhanced therapeutic efficacy. Herein, we engineered bioactive SF by incorporating PBAP and Tpl units, hypothesizing that this modified material would exhibit superior antioxidant and anti-inflammatory properties. As a proof of concept, we sequentially conjugated Tpl and PBAP onto SF scaffold (abbreviated as TPSF). Such a material featured multiple ROS-scavenging capabilities (toward H₂O₂, •OH, O₂^•−, DPPH•, and HClO), presenting therapeutic effects efficaciously in inflammation-associated diseases (Scheme 1). In vitro experiments were conducted to assess the anti-oxidative, anti-inflammatory, and macrophage reprogramming abilities of TPSF in its nanoparticulate form (TPSN). Additionally, the therapeutic efficacy of TPSN was validated using two murine models of inflammatory diseases.

Scheme 1.

Schematic illustration depicting silk-engineered bioactive nanoparticles for the therapy of acute inflammatory disease by inhibiting the excessive inflammatory responses and oxidative stress. AEC represents the alveolar epithelial cell

Experimental section

Materials

4-dimethylaminopyridine (DMAP), 4-(Hydroxymethyl) phenylboronic acid pinacol ester (PBAP), 1,1-carbonyldiimidazole (CDI), phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharides (LPS, O26: B6) were purchased from Sigma-Aldrich (USA). Silkworm cocoons were supplied by our research group. 4-Glycidyloxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical (Tempol) and lecithin (from soybean) were bought from TCI (Shanghai, China). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000 (DSPE-PEG) and Cyanine5 NHS ester (Cy5) were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). Penicillin-Streptomycin solution, RPMI 1640, DMEM-F12 and Dulbecco’s modified eagle medium (DMEM) were provided by Gibco (USA). Hydrogen peroxide solution, anhydrous dimethyl sulfoxide (DMSO), and dichloromethane (DCM) were purchased from Chron Chemicals (Chengdu, China). N-acetyl-L-cysteine (NAC), cisplatin, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and annexin V-FITC apoptosis detection kit were obtained by Beyotime Biotechnology (Shanghai, China). PE-F4/80 antibody, APC-Ly6G antibody, FITC-CD11b antibody and FITC-annexin V apoptosis detection kit with 7-AAD were purchased from Biolegend (USA). IL-1β ELISA kit, MCP-1 ELISA kit, IL-6 ELISA kit and TNF-α ELISA kit were acquired from Fine Test (Wuhan, China).

Synthesis and characterization of TPSF material

Shredded cocoons (2.5 g) were added into boiling Na₂CO₃ solution (1.0 L, 0.02 mol/L) for at least 30 min and then washed several times with distilled water to remove excess Na₂CO₃ and silk sericin. After air-drying at room temperature, the resulting SF (200 mg) was dissolved in LiBr solution (4.0 L, 9.3 mol/L) and stirred magnetically at 60 °C for 2 h. Subsequently, Tempol (257.5 mg) was introduced into the mixture and stirred at 60 °C for 3 h. The mixture was then centrifuged at 12,000 rpm for 10 min to remove any undissolved substances, and the supernatant was dialyzed (molecular weight cutoff, 8000 Da) against deionized water for 48 h. Finally, the Tempol modified SF material (TSF) was obtained through freeze-drying.

To synthesize a reactive oxygen species (ROS)-scavenging silk fibroin material (defined as TPSF), 1.11 g PBAP and 1.53 g CDI were added into 10 mL anhydrous DCM in a round-bottom flask and reacted under oxygen- and water-free conditions for 30 min. Then the mixture was washed three times with deionized water, followed by further washing with saturated NaCl solution. The organic phase was concentrated by rotary evaporation and dried under vacuum to yield CDI-activated PBAP (CDI-PBAP). Subsequently, TSF (100 mg), CDI-PBAP (800 mg) and DMAP (440 mg) were dissolved in 15 mL anhydrous DMSO and reacted at 30 °C for 72 h under a nitrogen atmosphere. After filtration, the solution was added dropwise into 150 mL deionized water, followed by centrifugation at 8,000 rpm for 10 min. The precipitate was then washed and centrifuged thrice. Finally, the TPSF was obtained by drying under vacuum. TPSF analogs were synthesized by adding different mass ratios of TSF and CDI-PBAP (ranging from 1:1–1:8) as described above.

¹H NMR spectra were conducted on a nuclear magnetic resonance (NMR) spectrometer operating at 600 MHz (DD2, Agilent, USA). Fourier transform infrared (FT-IR) spectra were recorded on a 100 S FT-IR spectrometer (PerkinElmer, USA). Peak fitting was performed using the PeakFit 4.0 software. Electron paramagnetic resonance (EPR) spectra were acquired on an electron paramagnetic resonance spectrometer (EMXplus-9.5/12, Bruker, USA).

Fabrication of various nanoparticles

A modified self-assembly method was employed to prepare TPSF nanoparticles (TPSN). Briefly, DSPE-PEG (9 mg) and lecithin (6 mg) were dissolved in 0.6 mL of ethanol, into which 20 mL of deionized water was added. The mixture was heated to 65 °C for 1 h. After the mixture had been restored to room temperature, TPSF (40 mg) dissolved in DMSO (3 mL) was added dropwise. Then, the mixture was immediately self-assembled by ultrasonication at 80 W for 3 min, followed by centrifugation at 15,000 rpm for 10 min to remove the organic solvent. The resultant TPSN was resuspended in deionized water and stored at 4 °C. Additionally, a similar method was employed to prepare Cy5-loaded TPSN (Cy5-TPSN). DSPE-PEG (6 mg), DSPE-PEG-Cy5 (3 mg) and lecithin (6 mg) were dissolved in 0.6 mL of ethanol, followed by the same procedure to fabricate Cy5-TPSN.

The TSF nanoparticles (TSN) were prepared based on a previously reported method [63]. In brief, 1 mL TSF solution (10 mg/mL) was added dropwise into 10 mL acetonitrile under magnetic stirring at room temperature. Then, the mixture was immediately self-assembled by ultrasonication at 80 W for 3 min. Finally, TSN were obtained after centrifugation and resuspension. On the other hand, SF nanoparticles (SN) were prepared in a similar manner.

Characterization of nanoparticles

The mean size, size distribution profiles and zeta potential of the nanoparticles were determined using a Zetasizer NanoZS (Malvern Instruments, UK). The morphology of the nanoparticles was observed by transmission electron microscopy (TEM) using a JEM-1400 microscope (JEOL, Tokyo, Japan). And scanning electron microscopy (SEM) was performed on a FIB-SEM microscope (Crossbeam 340, Zeiss, Germany). In addition, the mean size and zeta potential of TPSN were determined to assess its stability in H₂O, saline, and PBS after storage at 4 °C for various periods. Meanwhile, the mean size and zeta potential were measured at different pH levels and temperatures. Moreover, the DPPH• scavenging capacity of TPSN was assessed using the method described below, with a 2 h incubation period.

Elimination of H2O2 and superoxide anion by TPSF

Different concentrations of TPSF (varying from 0.125, 0.25, 0.5, 0.75, 1.25, to 2.5 mg/mL) were incubated in 2 mL of PBS containing 100 mmol/L H₂O₂ at 37 °C for 24 h. The Hydrogen Peroxide Detection Kit (Nanjing Jiancheng Bioengineering Institute, China) was used to detect the concentration of remaining H₂O₂. Following the instructions, the absorbance at 405 nm was measured, and the H₂O₂-scavenging capacity was calculated.

In a separate experiment, the superoxide anion scavenging capacity of TPSF was assessed using a Superoxide Anion Free Radical Detection Kit (Nanjing Jiancheng Bioengineering Institute, China). Various concentrations of TPSF (varying from 0.1, 0.25, 0.5, 1, 2, to 4 mg/mL) were incubated with mixture solutions according to the instructions, followed by measuring the absorbance at 550 nm. Of note, the superoxide anion was generated by the reaction between xanthine and xanthine oxidase. When the electron transfer substance and Gress color developer were added, the reaction system turned purplish-red. The superoxide anion and H₂O₂ scavenging capabilities of SF and TSF were examined using the same method as described above.

Elimination of hydroxyl radical by TPSF

To evaluate the •OH-scavenging ability of TPSF, methylene blue (16 µg/mL) was employed as an indicator of •OH, which was generated by mixing isometric H₂O₂ (100 mmol/L) and FeCl₂ (0.5 mg/mL). Various concentrations of TPSF (varying from 0.03, 0.07, 0.13, 0.21, 0.26, 0.39, 0.53 to 0.66 mg/mL) were incubated with aforementioned mixture for 90 min at room temperature. Then, the absorbance of the mixture was recorded using a microplate reader at 666 nm, and the •OH-scavenging rate was calculated. In addition, photographs were also collected after incubating SF (1 mg/mL) and TPSF (1 mg/mL) solution with •OH-containing mixture.

Scavenging of DPPH radical by TPSF

A DPPH• methanol solution (100 µg/mL) was incubated with isometrical different concentrations of TPSF solution, varying from 0.05, 0.1, 0.3, 0.5, 0.8, 1.0, to 1.5 mg/mL at room temperature. The absorbance of the mixture was measured by microplate reader at 520 nm after incubation for different periods in the dark. Similarly, DPPH• solutions incubated with various concentrations of TPSF or SF solution (0.5 mg/mL) were photographed.

Scavenging of hypochlorite by TPSF

To examine the hypochlorite scavenging capability of TPSF, a luminescent nanoprobe (Lu-bCD NPs) was prepared according to our previous research [64]. Specifically, the luminescence is generated by the reaction of Lu-bCD NPs with hypochlorite. Lu-bCD NPs (10 mg/mL) were rapidly mixed with different concentrations of hypochlorite, varying from 1.25, 2.5, 5.0, 10, 25, 50, to 100 mmol/L in a black 96-well plate. Then, the luminescence imaging of the mixture was immediately recorded by an Imaging Spectrum System (Newton 7.0 FT-400 plus, France). The imaging parameters were set as follows: exposure time = 5 min, f/stop = 1, and binning = 8. The luminescence intensity was linearly fitted to the concentration of hypochlorite to establish the standard curve. Subsequently, various concentrations of TPSF (25 µL, varying from 0, 0.5, 1.0, 2.5, 5.0, to 10 mg/mL) were incubated with NaClO solution (475 µL, 100 mmol/L) at room temperature for 15 min. Then the mixed solution (150 µL) was rapidly reacted with Lu-bCD NPs (150 µL, 10 mg/mL) and imaged immediately. The hypochlorite scavenging rate was calculated based on the standard curve. Meanwhile, imaging of hypochlorite scavenging assay of SF solution (1 mg/mL) and TPSF solution (1 mg/mL) was also recorded.

In vitro anti-inflammatory activities of TPSN

RAW 264.7 cells were seeded into 12-well plates at 5 × 10⁵ cells per well and cultured overnight. After preincubation with 25 and 50 µg/mL of TPSN for 2 h, the cells were then stimulated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h. Specifically, the cells in normal group were cultured in fresh medium alone, while those in model group were treated without TPSN. The levels of IL-1β and TNF-α in the cell culture supernatants were measured by ELISA. In another experiment, RAW264.7 cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. After similar treatments, the cells were collected for RT-qPCR analysis.

Regulation of the macrophage phenotype by TPSN in vitro

RAW264.7 cells were seeded into 12-well plates at a cell density of 5 × 10⁵ per well with 1 mL medium. After overnight incubation, the medium was replaced with fresh medium containing LPS (100 ng/mL) and IFNγ (5 ng/mL). Then the cells were incubated for 33 h. Subsequently, TPSN (50 µg/mL) was added to co-incubate for 8 h. In the normal group, cells were treated with growth medium alone, while cells in the model group were treated with LPS and IFN-γ. PE anti-mouse F4/80 antibody, BV650 anti-mouse CD206 antibody and PE-Cy7 anti-mouse CD86 antibody were used to mark different phenotype macrophages. Finally, cells were collected and resuspended in 0.1 mL PBS, mixed thoroughly with the antibodies, and incubated at room temperature for 30 min in the dark. After washing, samples were tested by a flow cytometer and analyzed using Flowjo V10 software. F4/80⁺ and CD86⁺ were identified as M1 phenotype macrophages, while F4/80⁺ and CD206⁺ were considered as M2 phenotype macrophages.

Anti-apoptosis activity of TPSN in vitro

HK-2 (human renal tubular epithelial) cells were inoculated into 12-well plates at a cell density of 2 × 10⁵ per well with 1 mL culture medium and incubated overnight. Cells were then co-incubated with TPSN (varying from 12.5, 25, to 50 µg/mL) and cisplatin (10 µmol/L) for 24 h. Subsequently, both the culture supernatant and cells were collected. After centrifugation, the cells were resuspended in binding buffer containing Annexin V-FITC (Annexin V) and 7-AAD. Gently vortexed and incubated in the dark at room temperature for 15 min. Finally, flow cytometry analysis was performed immediately.

In another experiment, HK-2 cells were seeded into 96-well plates at 1 × 10⁴ cells per well and incubated with 1 mL culture medium overnight. The culture medium was replaced with fresh medium containing different nanoparticles (50 µg/mL) and cisplatin (15 µmol/L). After incubation for 24 h, 10 µL of CCK-8 solution was added to the culture medium, and the optical density (OD) value was measured by microplate reader at 450 nm.

Anti-migration evaluation of TPSN in vitro

RAW264.7 cell migration was assessed using 24-well transwell chambers with 8 μm pore size membranes. In brief, the primary intraperitoneal neutrophil cells were extracted as described above. Except for the control group, 0.5 mL primary intraperitoneal neutrophil cells (1 × 10⁵ cells/mL) was added to the lower chamber, and 0.1 mL RAW264.7 cells (2 × 10⁵ cells/mL) was added to the upper chamber. Only RAW264.7 cells were added in the control group. Then, 0.1 mL fresh medium containing 50 µg/mL TPSN was added to the upper chamber in the TPSN group. The cells in model group were treated with fresh medium alone. After 6 h, all non-migrated cells were gently removed from the upper chamber with a cotton swab. Finally, the migrated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet for 15 min. The migrated cells were photographed by an Inverted Epifluorescence Microscope (Nikon, Japan), and five randomly selected fields were counted to determine the number of migrated cells.

Animals

Male C57BL/6 mice (20–22 g), BALB/c mice (20–22 g) and Sprague Dawley rats (180–200 g) were obtained from Chongqing Byrness Weil biotech Ltd, China. Mice and rats were acclimatized to laboratory conditions for at least one week prior to the experiment. All animals were received food and water ad libitum and housed in standard conditions, including appropriate temperature, humidity and light.

In vivo lung targeting of TPSN in LPS-induced ALI mice

Female BALB/c mice were administered 50 µL of LPS (0.5 mg/mL) intranasally to induce acute lung injury (ALI). After stimulation with LPS for 0.5 h, the mice were intravenous (i.v.) administered with Cy5-TPSN at 10 mg/kg. Meanwhile, the healthy mice received the same dose of Cy5-TPSN or PBS as control groups. At 12 h after treatment, all mice were euthanized, and lungs were excised for ex vivo imaging using the 3D In Vivo Optical Imaging System (Newton 7.0 FT-400 plus). Additionally, lung cryosections were stained with CD68 (Servicebio, Catalog No. GB113109, China) or Ly6G (Servicebio, Catalog No. GB11229, China) antibodies. The fluorescence images were acquired by confocal microscopy (Carl-Zeiss LSM900, Germany).

In vivo therapeutic effects of TPSN in ALI mice

The ALI mice model was established as mentioned above. Firstly, mice were randomly divided into four groups: (1) the control group, healthy mice were i.v. injected with saline through tail vein; (2) the model group, ALI mice were treated with saline; (3) the TPSN group, ALI mice were i.v. administered with TPSN at 0.5 or 2 mg/kg. At 12 h post injection, the mice were euthanized and bronchoalveolar lavage fluid (BALF) was collected according to a previously reported method [64]. The levels of IL-1β, TNF-α, and MCP-1 in BALF were assayed by ELISA. To compare the therapeutic effects of different agents, mice were randomly divided into six groups: (1) healthy mice in control group were i.v. injected with saline via tail vein; (2) ALI mice in model group were similarly treated with saline; 3–6) ALI mice were i.v. administered with NAC, SN, TSN, and TPSN at a dose of 2 mg/kg, respectively. After 12 h, all mice were euthanized, and the lungs were collected. The levels of inflammatory factors in the serum and lung tissues homogenate was determined by ELISA. The left upper lodes of the lung were excised and weighed immediately to obtain the wet weight. Then, the dry weight of the lung tissues was obtained after drying at 60 °C for 48 h. The wet/dry weight ratio of the lungs was calculated. H&E staining of lung paraffin sections was also performed.

In vivo therapeutic effects of TPSN in AKI mice

Male C57BL/6 mice were single intraperitoneal (i.p.) injection with 15 mg/kg cisplatin to induce acute kidney injury (AKI). Mice were randomly divided into different groups as described above and weighed. After stimulation with cisplatin, various formulations were i.v. injected immediately and administered again at 48 h post-injection. All mice were weighed, euthanized after 72 h, and the kidneys were collected. Meanwhile the kidneys were also weighed to calculate the organ index and the serum was isolated for testing the concentrations of UREA and CREA. The levels of H₂O₂ in renal tissue homogenates were determined following the instruction of hydrogen peroxide detection kit (Beyotime). Paraffin sections of the kidneys were stained with H&E and PAS.

Analysis of inflammatory cells by flow cytometry

Lung and kidney single-cell suspensions were prepared according to the methods detailed in the supplementary material. Subsequently, cells from the lungs and kidneys were stained with FITC-conjugated anti-mouse CD11b antibody, PE-conjugated anti-mouse F4/80 antibody, and APC-conjugated anti-mouse Ly6G antibody, respectively. After incubation for 20 min in the dark, cells were washed with cold PBS and resuspended in 100 µL PBS for flow cytometry analysis. Data analysis was performed using FlowJo V10 software.

Total RNA extraction and RT-qPCR

Total RNA was extracted from the kidney and lung tissues using Trizol reagent (Invitrogen, USA), and from RAW264.7 cells using Steady Pure Quick RNA Extraction Kit (Accurate Biotechnology Co.,Ltd, China) according to the manufacturer’s instruction. cDNA was synthesized using PrimeScript™ RT Master Mix (Takara Bio, China) and quantified by a Nano Drop spectrophotometry (Thermo Scientific™ Nano Drop one, USA). qPCR was carried out on the Applied Biosystems QuantStudio 1 Plus (Thermo Scientific, USA) using the SYBR Prime qPCR Set (Baoguang Biotechnology Co., Ltd, China) following the manufacturer’s recommendations. The primers are listed in Supplementary Table S1. Relative mRNA expression levels were calculated, and the data were normalized to GAPDH.

Immunofluorescence analysis

Paraffin-embedded sections of the lungs or kidneys were obtained following a routine procedure. The sections were deparaffinized, hydrated, and subjected to antigen repair, followed by blocking with goat serum. The primary antibodies for CD68 (Servicebio, Catalog No. GB113109, China), Ly6G (AiFangbio, Catalog No. SAF013, China), Kim-1 (Novus, Catalog No. NBP176701SS, USA), iNOS (Proteintech, Catalog No.18985-1-AP, China), AQP1 (Servicebio, Catalog No. GB11310-1, China), and Arg1 (AiFangbio, Catalog No. AF01565, China) were used, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using TSA-conjugated fluorescent dyes. All fluorescence images were acquired by CLSM.

Immunohistochemical staining

Paraffin-embedded sections were subjected to a procedure similar to that used for immunofluorescence staining. The primary antibodies for IL-6 (AiFangbio, Catalog No. AF06790, China), HO-1 (Beyotime, Catalog No.AF1333, China), and Nitrotyrosine were used. A DAB chromogen solution was used to react with HRP-conjugated secondary antibodies. The images were acquired by a fluorescence microscope (Axio Imager M2, Zeiss, Germany).

TUNEL staining assay

To analyze tissue apoptosis, paraffin-embedded sections of the lungs and kidneys were treated as described above with minimal modifications. The sections were repaired with protease K, permeabilized with 0.1% Triton, and stained with TUNEL detection kit (AiFangbio, Catalog No. AFIHC032) according to the manufacturer’s protocol. Nuclei were counterstained with DAPI, sealed with anti-quench sealing medium, and then photographed by fluorescence microscopy. TUNEL positive cells were quantified using Image J software.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD) unless otherwise specified. Statistical analysis was performed with SPSS22 software. One-way analysis of variance (ANOVA) with post-hoc LSD tests was performed for multiple group comparisons. A value of p < 0.05 was considered statistically significant (denoted as *p < 0.05, **p < 0.01, ***p < 0.001).

Results and discussion

Synthesis and characterization of bioactive TPSF material with ROS-scavenging ability

Tpl is a SOD-mimetic agent that can effectively neutralize superoxide anions and oxygen radicals [65]. Meanwhile the PBAP unit is able to stoichiometrically inactivate H₂O₂ [41]. Thus, we designed a silk-based ROS scavenger, defined as TPSF, which was simultaneously chemically modified with both Tpl and PBAP units. We hypothesize that nanomaterials based on TPSF could serve as candidates for targeted therapy of acute inflammatory diseases by restoring the redox balance in the pathological microenvironment. The synthetic process of TPSF is illustrated in Fig. 1A and B. Specifically, TPSF was synthesized via Tpl substitution of the primary amines of SF in an alkaline environment, followed by modification with CDI-activated PBAP on the hydroxyl groups of SF. According to a previously reported study [59], Tpl-conjugated SF (TSF) materials were fabricated with a final Tpl concentration of 282 mmol/L. For PBAP modification, the mass ratios of TSF to CDI-PBAP and the reaction time were determined based on H₂O₂ scavenging efficiency and ¹H NMR spectra (Fig. S1A, B), resulting in a 1:8 ratio for TPSF synthesis over a 72 h reaction period. The ¹H NMR spectra of TPSF indicated the characteristic signals at about 7.4 and 7.7 ppm, which were assigned to the phenyl groups of PBAP (Fig. 1C). Of note, the proton signals at approximately 4.0, 4.4, 6.7, and 7.0 ppm were significantly reduced or even disappeared after modification with the active units, suggesting that the active units were covalently grafted onto the serine and tyrosine residues of SF. Besides, the TPSF showed severely attenuated absorption at around 3300 cm^− 1 in the Fourier-transform infrared (FT-IR) spectrum, which could be attributed to the substitution of hydroxyl groups on SF (Fig. 1D). Also, new signals observed at approximately 1750 cm^− 1 and 1600 –1450 cm^− 1 were identified as carbonyl and phenyl groups, respectively. Compared with SF materials, modified SF products exhibited changes in the amide I, II, and III vibration modes at 1647, 1518, and 1235 cm^− 1, respectively. The deconvolution of the peak fitting results indicated an increase in the proportion of random coil and α-helix structures within TPSF, facilitating the self-assembly into well-defined and morphologically uniform nanoparticles (Fig. S1C-F). Furthermore, the electron paramagnetic resonance (EPR) spectrum revealed that the characteristic triplet signal attributed to Tpl emerged in TSF and TPSF (Fig. 1E). These findings collectively indicate that Tpl and PBAP units were successfully modified on SF.

Fig. 1.

Design, preparation, and characterization of functionalized SF material (TPSF). (A) Schematics showing the preparation process of TPSF. (B) The synthetic route for the functionalization of SF to form TPSF. (C-E) ¹H NMR (C), FT-IR (D) and EPR (E) spectra of different materials including SF, TSF and TPSF

Next, we evaluated the ROS-scavenging capacity of TPSF in vitro. Initially, we investigated the H₂O₂ elimination capability by incubating TPSF with H₂O₂ solution for 24 h. Upon detecting the residual H₂O₂ concentration, a dose-dependent elimination pattern was observed (Fig. 2A). Additionally, the superoxide anion (O₂^•−) scavenging capacity was assessed. Our results indicated that TPSF was able to scavenge O₂^•− in a dose-response manner after reacting with the hybrid system for 40 min (Fig. 2C). Furthermore, to quantify the free radical and hypochlorite (ClO⁻) eliminating capabilities of TPSF, established methods derived from published literature were employed [65, 66]. Briefly, OH radical (•OH) was produced by the Fenton reaction, and methylene blue (MB) served as the •OH indicator probe. When •OH was generated, the color of mixture solution containing MB rapidly changed from dark blue to pale blue. Notably, the color of mixed system showed minimal changes after the addition of 1 mg/mL TPSF and the •OH scavenging rate correlated with the TPSF concentration (Fig. 2B; Fig. S2C). Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), a nitrogen-free radical, was also eliminated by TPSF in a dose and time-associated pattern (Fig. 2D). The color of DPPH• solution shifted from modena to lavender upon the addition of TPSF (Fig. S2A, D). Additionally, a luminescent nanoprobe was employed to detect the capacity of TPSF to scavenge hypochlorite (ClO⁻). As expected, TPSF was capable of elimination ClO⁻ (Fig. 2E; Fig. S2B). Compared with SF and TSF alone, TPSF can efficiently scavenge both H₂O₂ and free radical (Fig. 2F, G). Furthermore, the results of ¹H NMR and HPLC analyses demonstrated that TPSF hydrolyzed to produce HMP (4-hydroxymethylphenol) in a 10 mmol/L H₂O₂ solution (Fig. S3). Collectively, all these results demonstrate that TPSF possesses a robust ROS-scavenging abilities.

Fig. 2.

Assessment of the ROS-scavenging capability of TPSF and characterization of TPSN. (A-C) Elimination of H₂O₂ (A), OH radical (B) and superoxide anion (C) by TPSF after incubation for 24 h, 90 min and 40 min, respectively. (D) Concentration-dependent and time-dependent scavenging of the DPPH radical by TPSF. (E) Hypochlorite-scavenging efficiency of TPSF at different doses after 15 min incubation. The left panel shows typical fluorescent images at different doses of sodium hypochlorite, while the right panel presents a quantitative analysis of the hypochlorite-scavenging efficiency of TPSF. (F-G) Comparative analysis of scavenging capabilities of different materials for H₂O₂ and DPPH radical. (H) Schematic diagram illustrating the preparation of TPSN. (I-K) Size distribution profiles (I), representative TEM (J) and SEM (K) image of TPSN. The mean size of TPSN was 291 ± 4 nm and the mean zeta potential was − 33 ± 2 mV. (L-N) The mean size distribution of TPSN in deionized water (L), saline (M), PBS (N) during 10 days

Preparation and characterization of TPSN

TPSN was fabricated using a modified self-assembly method, as illustrated in Fig. 2H. In brief, a small amount of DSPE-PEG and lecithin solution formed the aqueous phase, into which the TPSF solution was incrementally added. After ultrasound-assisted self-assembly, spherical-like TPSN was obtained, as presented in transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images (Fig. 2J, K). The crystalline structure of TPSN was evaluated using XRD, which indicated a substantial presence of amorphous regions (Fig. S6C). This low crystallinity of TPSN may enhance the susceptibility to degradation in vivo. Dynamic light scattering (DLS) analysis revealed a narrow size distribution profile with a mean diameter of 291 ± 4 nm (Fig. 2I). Also, the zeta potential value was − 33 ± 2 mV. Notably, the average size of TPSN could be altered by adjusting the TPSF mass and the oil-to-water volume ratio. For example, when TPSF mass was 40 mg and 30 mg at an oil-to-water volume ratio of 1:10, the diameters of the negatively charged TPSN were 324 ± 0.3 nm and 325 ± 8 nm, respectively, with corresponding polydispersity index (PDI) values of 0.19 ± 0.01 and 0.16 ± 0.04 (Fig. S4A, B, E, F). While the volume ratio was changed to 3:20, the mean sizes were 291 ± 4 nm and 334 ± 5 nm, with respective PDI values of 0.15 ± 0.04 and 0.17 ± 0.03 (Fig. S4C, D, E, F). Overall, for the following experiments, TPSN was synthesized using 40 mg of TPSF at a volume ratio of 3:20.

Subsequently, the stability of TPSN was assessed by monitoring the size and zeta potential changes of TPSN in distilled water, saline, and PBS at predetermined time points (Fig. 2L-N, Fig. S6A). The size of TPSN exhibited minimal changes over a 10-day period in saline and PBS, while it performed hardly any changes in distilled water. Additionally, the zeta potential and PDI values remained negatively charged and below 0.3, respectively. Furthermore, TPSN exhibited stability under weakly acidic and basic conditions, and within a temperature range from 4 °C to 37 °C at pH = 7.4 (Fig. S6E, F).

Additionally, the ROS-scavenging capability and hydrolysis profiles of TPSN was also examined. Typically, the DPPH• elimination ability of TPSN was similar to that of the TPSF material and still correlated with the concentration of TPSN (Fig. S6B). Subsequently, it was found that the hydrolysis rate and degree of TPSN increased significantly when incubated with H₂O₂ at 1 × 10^− 2 mol/L. In contrast, when incubated in PBS, a much lower rate of hydrolysis was observed (Fig. S6D), confirming that TPSN could be ROS responsive hydrolysis.

In vitro anti-oxidative stress and anti-inflammation activity of TPSN

The pivotal feature of inflammatory cells is intracellular ROS elevation [67]. Recovery of intracellular redox homeostasis is an important strategy for attenuating inflammation-associated diseases. Macrophages and neutrophils play critical roles in the pathogenesis of numerous inflammatory diseases. Therefore, we first examined the cellular uptake of TPSN in macrophages and neutrophils. To quantify and visualize the in vitro phagoptosis of TPSN, similar Cy5-TPSN was obtained by labeled Cy5 fluorescent dye (Fig. S5A). After incubating Cy5-TPSN with RAW264.7 macrophage cells, cellular internalization was detected by flow cytometry and confocal laser scanning microscopy (CLSM). It was shown that the internalization of Cy5-TPSN exhibited a time- and dose-dependent pattern in RAW264.7 and primary intraperitoneal neutrophil cells (Fig. S8, 9). Particularly, significant red fluorescence was observed in RAW264.7 cells after incubation with 25 µg/mL of Cy5-TPSN for 1 h. The intensity of red fluorescence signal increased with the incubation time and dose of Cy5-TPSN. Based on these findings, the fluorescence intensity of Cy5-TPSN was further quantified by flow cytometry. Likewise, a similar phenomenon of internalization in primary intraperitoneal neutrophil cells was discovered (Fig. S9). These findings collectively demonstrate that TPSN can be rapidly and effectively phagocytosed by macrophages and neutrophils.

Due to the significant role of ROS in inflammation-related diseases, we proceeded to assess the antioxidant stress capacity of TPSN in vitro. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) probe, a fluorescent indicator that signals the presence of ROS upon oxidation, was used to evaluate the cellular ROS levels. Compared to model group, RAW264.7 cells preincubated with TPSN exhibited significantly lower fluorescence intensity after PMA stimulation (Fig. S12A, B). It was demonstrated that TPSN can effectively reduce the intracellular ROS level in inflammatory cells. To further verify the anti-ROS capability of TPSN, NAC and nanoparticles prepared with different modifying materials were selected as concurrent control treatments. NAC was included due to its previously established antioxidant properties, which are attributed to its ability to elevate intracellular GSH levels [68]. The nanoparticles SN and TSN were fabricated based on SF and TSF respectively, and then characterized by DLS and TEM (Fig. S5B, C). The mean sizes of SN and TSN were 223 ± 2.6 nm and 289 ± 2.6 nm, respectively. Simultaneously, the zeta-potential values were evidently negative, measured at -19.2 mV and − 14.37 mV. As expected, TPSN exhibited a superior ability to inhibit the generation of intracellular ROS compared to both SN and TSN. Furthermore, the efficacy of TPSN was comparable to, or slightly exceeded, that of the NAC treatment. (Fig. S12C, D). Consistent with the findings based on flow cytometric analysis, fluorescent images showed weaker fluorescence in cells treated with TPSN (Fig. S12 F, upper row).

Subsequently, we investigated whether TPSN could protect macrophages from ROS-induced cell damage. As shown by CCK8 assay, exposing RAW264.7 cells to 2 × 10^− 4 mol/L H₂O₂ and different concentrations of TPSN simultaneously resulted in a significant cellular protective effect (Fig. S12E). Notably, cells treated with high concentration of TPSN (50 µg/mL) revealed considerably higher viability compared to those treated with fresh medium alone. In addition, as demonstrated by flow cytometry analysis, incubation with 2 × 10^− 4 mol/L H₂O₂ led to marked apoptosis, particularly when compared to cells treated with culture medium alone. By contrast, preincubation with TPSN (50 µg/mL) significantly mitigated H₂O₂-induced cell apoptosis (Fig. S12G, F, lower row). Meanwhile, cells in other groups, including those treated with TSN and NAC, also exhibited lower apoptosis rates but still retained a higher degree than those treated with TPSN. Collectively, TPSN revealed obvious cell protective effects by inhibiting cellular ROS generation and oxidative stress-mediated cell apoptosis. These findings correspond to the characterization results of TPSF, further demonstrating its ability to scavenge ROS.

Based on the above results, we subsequently investigated the anti-inflammatory capacity of TPSN in vitro. As demonstrated in Fig. 3A-B, the secretion of inflammatory cytokines (IL-1β and TNF-α) increased dramatically in LPS-stimulated RAW264.7 cells. Conversely, treatment with TPSN resulted in a reduction of inflammatory factors. In a separate assay, we also found that the relative mRNA expression levels of IL-6, IFN-γ, and TGF-β were decreased in cells treated with TPSN (Fig. S11A-C). As well know, macrophage migration plays a critical role in various inflammatory diseases due to the accumulation of pro-inflammatory cytokines [69]. Therefore, we evaluated the anti-migratory effect of TPSN on cells. Results of transwell assay confirmed that TPSN can inhibit macrophage migration (Fig. 3C, D). However, cells in the model group, induced by neutrophils alone, showed a large number of migrated cells. At the same time, TPSN can inhibit the migration of neutrophils themselves (Fig. S11D, E). Taken together, it indicates that TPSN possesses significant anti-inflammatory and anti-oxidative stress abilities.

Fig. 3.

Assessment of anti-inflammatory and anti-oxidative stress capabilities and modulation of macrophage phenotype by TPSN treatment. (A-B) The expression levels of IL-1β (A) and TNF-α in the RAW264.7 cell culture medium following treatment with various doses of TPSN. (C) Quantitative analysis of migrated cells across five fields. (D) Typical optical microscopy images of primary neutrophils-induced migration in RAW264.7 cells, positively stained with crystal violet. (E, F) Representative flow cytometric profiles illustrating the proportion of M1 (F4/80⁺CD86⁺, E) and M2 (F4/80⁺CD206⁺, F) macrophages after TPSN treatment. RAW264.7 cells were stimulated with 100 ng/mL LPS and 5 ng/mL IFN-γ for 33 h, followed by treatment with 50 µg/mL TPSN for 8 h. (G, H) Quantitative analysis of M1 and M2 phenotype macrophage, respectively. (I) Immunofluorescence staining of iNOS and Arg1 in lung sections of ALI mice treated with TPSN. The right panel indicates zoomed-in images of the boxed fields regions corresponding to fluorescence signals. Blue, DAPI-nuclear staining; green, iNOS; red, Arg1. (J) The intracellular ROS level in lung tissues of ALI mice treated with TPSN. Single-cell suspensions of lung tissues were stained with DCFH-DA and analyzed by flow cytometry. (K, L) The mRNA expression levels of pro-oxidant genes (NOX2) and antioxidant genes (HO-1) in lung tissues. (M) Immunohistochemical staining of HO-1 in lung sections. Data are presented as mean ± SD (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001

Promotion of macrophage phenotype transformation and regulation of redox homeostasis by TPSN

Given the significant role of macrophages in the progression of inflammation, we further investigated the potential of TPSN to modulate macrophage phenotypes. Initially, we employed LPS and IFN-γ as stimulating factors to induce the transformation of RAW264.7 macrophages into the M1 phenotype. After co-incubation with 50 µg/mL TPSN, cells were collected for staining and flow cytometry analysis. The results presented in Fig. 3E-H indicated that cells in the TPSN group exhibited a lower M1 phenotype (F4/80⁺CD86⁺) and a higher M2 phenotype (F4/80⁺CD206⁺) compared to the cells stimulated by LPS and IFN-Fγ alone. These results suggest that TPSN can regulate the transformation of pro-inflammatory M1-type macrophages into anti-inflammatory M2-type macrophages in vitro. Furthermore, we conducted verification of the results in mice with acute lung injury (ALI). The mice model was established according to a previously described method, briefly induced with LPS via intranasal delivery [65]. Upon comparison of ALI mice treated with TPSN, analysis of the fluorescence image demonstrated that ALI mice showed stronger iNOS protein (green fluorescence) and weaker Arg-1 protein signals (red fluorescence signal), which respectively represented the M1 and M2 type macrophages (Fig. 3I). Totally, TPSN can effectively induce the macrophage phenotype transformation from M1 to M2 forms both in vitro and in vivo. Additionally, after incubating a single-cell suspension of lung tissue with DCFH-DA probe, flow cytometry results showed a reduced ROS level in lung tissue cells from TPSN-treated ALI mice (Fig. 3J). Meanwhile, the RT-qPCR analyses demonstrated the downregulation of pro-oxidant genes (NOX2, NADPH oxidase 2) and antioxidant genes (HO-1, heme oxygenase-1) after TPSN treatment (Fig. 3K, L). This was also consistent with the immunohistochemistry results of HO-1 (Fig. 3M). These results illustrated that TPSN was able to inhibit oxidative stress and restore the balance between prooxidants and antioxidants in the lesions. Consequently, TPSN can not only facilitate the shift of macrophages toward an anti-inflammatory state but also contribute to the restoration of redox balance in injured sites.

Targeted therapy of ALI in mice with TPSN

Building upon the aforementioned promising results, in vivo studies were performed to investigate the therapeutic efficacy of TPSN in experimental animal models of inflammatory diseases. ALI represents a common life-threatening pulmonary condition characterized by oxidative stress and severe inflammation [70]. Prior to therapeutic evaluation, we examined the biodistribution of TPSN initially. After i.v. administration of Cy5-TPSN in normal BALB/c mice, we collected blood samples and major organs for fluorescence imaging. As shown in Fig. S13A, a correspondingly rapid clearance pattern from blood was observed. The fluorescence intensity decreased by half after approximately 1.5 h post-injection. By contrast, the fluorescent signals rapidly increased in the major organs, including lungs and kidneys (Fig. S14).

Subsequently, we investigated whether TPSN can be effectively accumulated at injured lung tissues. All mice were treated as illustrated in Fig. 4A. We compared the fluorescence intensity of Cy5 in the lungs of normal and ALI mice administrated with Cy5-TPSN. Results from ex vivo imaging exhibited the accumulation of Cy5-TPSN in normal lungs, accompanied by significantly higher fluorescence signals in injured lung tissues (Fig. 4B, C). It can be attributed to the increased permeability caused by the disruption of vascular endothelial and epithelial barriers in injured lungs. Furthermore, immunofluorescence staining was used to evaluate the cellular distribution of Cy5-TPSN. CD68 and Ly6G were used to mark the macrophages and neutrophils, respectively. In ALI mice treated with Cy5-TPSN, Cy5 fluorescence was observed in both neutrophils and macrophages (Fig. 4D). In contrast, no fluorescence signals were found in both cells of normal mice. Additionally, a comparatively higher level of Cy5 fluorescence was observed in lung cryosections of ALI mice. Moreover, TPSN was confirmed to be engulfed by neutrophils and macrophages in cell experiments. Together, these results indicated that phagocytosis of inflammatory cells can enhance the injured site targeting capacity of TPSN. Additionally, cellular uptake of Cy5-TPSN by pulmonary microvascular endothelial cells (PMVEC) occurred in a dose- and time-dependent manner (Fig. S10). Therefore, TPSN can effectively accumulated in damaged lungs, thereby providing a foundation for subsequent therapeutic studies.

Fig. 4.

In vivo targeting performance and therapeutic efficacy of TPSN in ALI mice after i.v. administration. (A) Schematic illustration of the establishment of ALI mice model and treatment regimens. (B, C) Typical ex vivo images (B) and quantitative analysis (C) indicating the accumulation of Cy5-TPSN in the lungs at 12 h after i.v. administration of Cy5-TPSN. (D) Confocal microscopy images of lung cryosections showing the co-localization analysis between Cy5-TPSN and CD68⁺ macrophages or Ly6G⁺ neutrophils. (E) The lung wet-to-dry weight ratios after different treatment. (F-I) Flow cytometric quantitative analysis and typical dot plots of neutrophil (F, H) and macrophage (G, I) proportions in pulmonary tissues of ALI mice. (J) Confocal microscopy images of CD68⁺ macrophages and Ly6G⁺ neutrophils in lung cryosections of ALI mice. The ALI mice model was established with 50 µL LPS at 0.5 mg/mL via intranasal delivery. After 0.5 h, mice received different formulations by i.v. injection. In the therapeutic efficacy assay, mice in the model group received saline, whereas the other groups were treated with NAC, SN, TSN, or TPSN at a dosage of 2 mg/kg, respectively. At 12 h after various treatments, mice lungs were collected for quantitative and histological analyses. Data are presented as mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Encouraged by the efficient anti-inflammatory and targeting capability of TPSN, the therapeutic effect of TPSN was investigated in ALI mice. The levels of pro-inflammatory cytokines IL-1β, TNF-α, and MCP-1 in bronchoalveolar lavage fluid were obviously decreased after treatment with TPSN at a dosage of 2 mg/kg (Fig. S15). In contrast, administration of TPSN at 0.5 mg/kg did not yield a therapeutic outcome, except for a reduction in IL-1β levels.

Furthermore, the therapeutic efficacy of TPSN was also compared in more detail with those of NAC, SN, and TSN. The lung wet/dry weight ratio, an index of pulmonary edema caused by acute inflammation, was remarkably decreased by therapy with 2 mg/kg TPSN and NAC, compared to ALI mice treated with saline (Fig. 4E). Similarly, TSN therapy could also improve the pulmonary edema of ALI mice, except for SN treatment. These results were consistent with the narrow-spectrum ROS-scavenging capacity of TSN. As mentioned above, ALI is mechanistically associated with pulmonary infiltration of immune cells, such as neutrophils and macrophages [70]. Excessive infiltration usually causes damage to lung tissues because of the large production of pro-inflammatory cytokines and chemokines [71]. Thus, the neutrophils and macrophages in lung tissues were measured by flow cytometry analysis and immunofluorescence staining. Compared to normal mice, a significant increase in neutrophils and macrophages was observed in ALI mice treated with saline (Fig. 4F-I). However, TPSN treatment effectively decreased the proportion of neutrophils and macrophages to 10.45 ± 3.86% and 8.64 ± 2.12%, respectively. Likewise, TSN, with limited ROS-scavenging capability, displayed relatively weaker inhibition of inflammatory cell infiltration. Of note, both of them were superior to NAC treatment. However, treatment with SN did not demonstrate any therapeutic effect. Consistent with the flow cytometric analysis results, the therapeutic effect of TPSN was also confirmed in immunofluorescence staining (Fig. 4J). Therapy with TPSN resulted in the weaker expression of CD68 (green fluorescence) and Ly6G (red fluorescence) in the sections of lung tissues. These results were in accordance with the decreased levels of inflammatory cytokine (Fig. 5A-H; Fig. S16). The gene expression of pro-inflammatory factors, including IL-1β, IL-6, TNF-α, and MPO, was significantly downregulated after TPSN therapy. Meanwhile, similar results were found through measuring the level of inflammatory factors in serum and lung tissue homogenates by ELISA. Further hematoxylin and eosin (H&E)-stained sections were used to evaluate the pathological changes in lung tissues. As illustrated in Fig. 5I (upper panel), the lung tissues of ALI mice exhibited notable pathological changes, including significant infiltration of neutrophils (indicated by green arrow), thickened alveolar walls, collapsed alveolar, and parenchymal lesions (indicated by black arrow), along with a large number of necrotic epithelial cells and infiltrated neutrophils filling the bronchial lumen (indicated by red arrow). Whereas only a few histological abnormalities were observed after TPSN treatment. Additionally, immunohistochemical staining of nitrotyrosine was performed to evaluate the anti-oxidative stress capability of TPSN (Fig. 5I, lower panel). Nitrotyrosine, considered a sensitive biomarker of oxidative stress, is formed by peroxynitrite-mediated nitration on protein tyrosine residues. Compared to NAC, SN, and TSN treatments, TPSN therapy resulted in a lower expression of nitrotyrosine, almost equal to that of normal mice. Moreover, therapeutic effects of TPSN were also confirmed by detecting apoptotic cells in lung tissues. Notably, flow analysis revealed that TPSN therapy mitigates cell apoptosis in the lung tissue of ALI mice (Fig. 6A, B). TdT mediated dUTP nick-end labeling (TUNEL) staining of the lung tissue sections exhibited similar results (Fig. 6C). Compared to normal mice or TPSN-treated ALI mice, much stronger TUNEL positive signals were found in ALI mice. Likewise, SN showed no anti-apoptotic effects, and TSN could partly protect cells from apoptosis. Collectively, TPSN demonstrated superior tissue protection capabilities compared to NAC and TSN therapy, as evidenced by lower lung wet/dry weight ratios, reduced pulmonary infiltration of inflammatory cells, decreased secretion of pro-inflammatory factors, inhibited formation of oxidation products, and mitigated cell apoptosis in lung tissues. Consequently, these results demonstrate that TPSN, with effectively lung targeting capacity and anti-inflammatory properties, holds promise as a potential therapeutic strategy for ALI.

Fig. 5.

Evaluation of the anti-inflammatory effect of TPSN and histological analysis in ALI mice. (A-D) Quantification of mRNA levels for IL-1β (A), IL-6 (B), TNF-α (C) and MPO (D) in lung tissues of ALI mice following different treatments. (E-H) Serum expression levels of IL-1β (E), IL-6 (F), TNF-α (G) and IFN-γ (H) in ALI mice as measured by ELISA. (I) H&E staining (upper panel) and nitrotyrosine immunohistochemically staining (lower panel) of lung tissue sections. Black arrows indicate alveolar wall thickening and substantial lung tissues. Green arrows indicate areas of neutrophils infiltration. Red arrows point to accumulative necrotic epithelial cells and extravasated neutrophils in the bronchial lumen. Data in (A-H) are mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6.

Analysis of anti-apoptotic capability of TPSN in lung tissues from ALI mice subjected to various formulations. (A-B) Flow cytometric profiles (A) and quantitative data (B) of apoptotic lung tissue cells after different treatments. (C) TUNEL-stained analysis of lung sections. Data are mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001

Therapeutic efficiency of TPSN in Cisplatin-Induced AKI mice

To validate the universality of the aforementioned silk fibroin-based nanomedicine, we examined the therapeutic efficacy of TPSN in a murine model of cisplatin-induced acute kidney injury (AKI). It is well-documented that oxidative stress and inflammation are critical factors in the pathogenesis of cisplatin nephrotoxicity [72]. This nephrotoxicity represents a significant adverse effect on normal organs in patients undergoing cisplatin-based chemotherapy, substantially impacting their clinical outcomes [72]. Therefore, the cytoprotective effect of TPSN was evaluated in cisplatin-induced cell damage. Considering that the renal epithelial cells are susceptible to oxidative stress in AKI, we used HK-2 cells to study cytoprotective properties of TPSN against ROS damage. As shown in Fig. 7A-F, HK-2 cells can endocytose TPSN in a dose- and time-dependent manner, as evidenced by flow cytometry and confocal microscopy analysis. Next, we examined the inhibitory effect of TPSN on intracellular ROS production (Fig. 7G-I). After treatment with 10 µmol/L cisplatin, HK-2 cells showed a dramatically increased in intracellular ROS. In comparison, when cells were co-incubated with TPSN, the intracellular ROS significantly decreased. Qualitative analysis of intracellular ROS was confirmed by fluorescence imaging. Consistent with our expectations, the ROS signals (green fluorescent) were markedly diminished following TPSN treatment. Moreover, flow cytometry analysis was employed to assess the inhibition capability of TPSN against cisplatin-induced cell apoptosis and necrosis (Fig. 7J, K). It clearly demonstrated that cisplatin can promote HK-2 cells apoptosis and necrosis. By contrast, the apoptotic ratio of HK-2 cells was significantly decreased after treatment with TPSN. Notably, the high-dose TPSN (50 µg/mL) showed a better cytoprotective effect than other doses. Meanwhile, we additionally compared cytoprotective effect of TPSN with NAC, SN, and TSN in CCK8 assay. As illustrated in Fig. 7L, cisplatin markedly reduced the cell viability of HK-2 cells, and this decline was noticeably alleviated by TPSN. For other treatments, NAC and TSN exhibited a cytoprotective effect, consistent with their inhibition of H₂O₂-induced apoptosis in RAW264.7 cells. Taken together, these results demonstrate that TPSN can protect cells against cisplatin-induced cell damage by reducing intracellular ROS levels, inhibiting apoptosis, and preventing necrosis.

Fig. 7.

Evaluation of cytoprotective effects of TPSN in cisplatin-induced HK-2 cells damage. (A, B) Flow cytometric profiles (A) and quantification counts (B) indicating dose-dependent cellular uptake of Cy5-TPSN in HK-2 cells after 2 h incubation. (C) Fluorescence images showing dose-dependent internalization of Cy5-TPSN in HK-2 cells. (D, E) Typical flow cytometric curves (D) and quantitative analysis (E) of time-dependent cellular uptake of Cy5-TPSN at 25 µg/mL. (F) Confocal microscopy images indicating time-dependent internalization of Cy5-TPSN at 25 µg/mL in HK-2 cells. (G, H) Flow cytometry profiles (G) and quantitative data (H) of intracellular ROS generation in HK-2 cells after treatment with different dose of TPSN. (I) Fluorescence images showing a representative reduction in intracellular ROS generation after TPSN treatment. The HK-2 cells were stimulated by 10 µmol/L cisplatin, followed by co-incubation with TPSN for 24 h. (J, K) Typical flow cytometric dot plots (J) and quantitative analysis (K) of apoptotic HK-2 cells after TPSN treatment. The HK-2 cells were induced with 15 µmol/L cisplatin, and then co-incubated with TPSN for 24 h. (L) Cell viability of HK-2 cells exposed to 15 µmol/L cisplatin and different formulations for 24 h. Data are mean ± SD (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001

According to a reported established method, AKI was induced in mice by intraperitoneal injection of a single high dose of cisplatin (15 mg/kg, Fig. 8A). Based on the results of pharmacokinetics and biodistribution of TPSN, we observed a stronger accumulation of Cy5-TPSN in injured kidneys compared to healthy kidneys (Fig. S13B, C). These results were confirmed by immunofluorescence analysis (Fig. S13D). All of these findings suggested that TPSN can be efficiently targeted to injured kidneys. Next, we assessed the therapeutic effect of TPSN in AKI mice. After treatment with TPSN at 2 mg/kg, the levels of serum urea (UREA), serum creatinine (CREA), and IL-1β were significantly decreased (Fig. S17). Well-documented clinical indicators of renal dysfunction include high levels of UREA and CREA [73]. By contrast, TPSN at a dose of 0.5 mg/kg only showed a reduction in CREA and IL-1β expression.

Fig. 8.

Therapeutic effects of i.v. delivered TPSN in AKI mice. (A) Schematic illustration of the establishment and treatment regimens for cisplatin-induced AKI in mice. (B) Relative changes in body weight of AKI mice after different treatments. (C, D) Serum expression levels of UREA (C) and CREA (D) in AKI mice after various treatments. (E-I) Relative mRNA expression levels of KIM-1 (E), TNF-α (F), IL-6(G), MCP-1 (H) and IL-1β (I) in kidneys of AKI mice treated with distinct formulations. (J) TUNEL staining of the kidney cryosections from AKI mice. (K)Staining images of kidney sections marked with KIM-1 antibody (upper panel), H&E (middle panel) and PAS (lower panel). Aquaporin-1 (AQP1) is a biomarker of proximal tubule cells. The yellow arrows represented tubules with necrosis, epithelial anoikis cavitation or loss of brush border. Triangles indicated casts formation in tubes. Mice in model group were received with saline, while other groups were respectively treated with NAC, SN, TSN or TPSN at 2 mg/kg. Data are mean ± SD (n = 4–5). *p < 0.05, **p < 0.01, ***p < 0.001

Furthermore, in a separate experiment, we compared the therapeutic effects of TPSN with NAC, SN, and TSN at a dose of 2 mg/kg. As expected, AKI mice treated with TPSN or TSN showed relief from weight loss, whereas AKI mice (saline treated) exhibited dramatic bodyweight loss within 48 h (Fig. 8B). As indicated in Fig. 8C-D, treatment by TPSN significantly decreased the levels of UREA and CREA in AKI mice, demonstrating that TPSN could effectively restore renal function. By comparison, TSN only partially reduced the serum UREA level, indicating its limited capacity for ROS-scavenging. Similarly, NAC and SN did not show any therapeutic effect. Furthermore, the gene expression of pro-inflammatory factors, including TNF-α, IL-6, MCP-1, and IL-1β, was markedly inhibited by TPSN (Fig. 8F-I). This result was verified by immunohistochemistry staining for IL-6 (Fig. S19). Moreover, flow cytometry was used to analyze the infiltration of neutrophils and macrophages. Consistent with the RT-qPCR results, TPSN demonstrated the capability to inhibit neutrophil and macrophage infiltration (Fig. S18A-C). Kidney injury molecule-1 (KIM-1) is a novel biomarker that is closely associated with the renal tissue damage [74]. Indeed, the gene expression of KIM-1 in AKI mice was dramatically elevated compared to that in mice treated by TPSN (Fig. 8E). Meanwhile, immunofluorescence analysis showed similar expression patterns of KIM in AKI mice after TPSN therapy (Fig. 8K). Besides, histological analyses were conducted using H&E and PAS staining (Fig. 8K). Kidneys of AKI mice represented abnormal architecture with remarkable necrosis, epithelial anoikis cavitation, or loss of brush border in tubules (shown as yellow arrow). And the formation of cast structures in tubules (indicated by black triangles) was also noted, a feature that is regarded as a significant pathological indicator of renal injury. Similarly, PAS staining also revealed a large amount of glycogen deposition in the kidney tubules of AKI mice. Of note, all these histological abnormalities were effectively reversed by TPSN treatment.

The role of oxidative stress in cisplatin-induced nephrotoxicity is well established. Excessive accumulation of ROS within the proximal tubules triggers tubular cell apoptosis [72]. As illustrated by the TUNEL assay, apoptotic cells in TPSN group were obviously decreased compared to AKI mice treated with saline (Fig. 8J; Fig.S18E). Meanwhile, the H₂O₂ level in the homogenate of renal tissue was also measured. As expected, the H₂O₂ level in AKI mice was significantly reduced after TPSN treatment (Fig. S18D). Overall, AKI mice treated with other formulations only showed a significant decrease in some of the parameters assessed. Similar to the findings in ALI models, treatment of cisplatin-challenged mice with TPSN achieved desirable protective effects, as evidenced by notable reductions in the levels of UREA, CREA, KIM-1, H₂O₂, pro-inflammatory cytokines, and kidney tissue injury. Taken together, these results demonstrate that TPSN could effectively restore renal function in AKI mice.

Biocompatibility of TPSN

In light of its desirable anti-oxidative stress and anti-inflammatory activities in vitro and vivo, both animal experiments and in vitro assays were performed to assess the safety of TPSN. Firstly, the cytotoxicity of TPSN was evaluated in RAW264.7 and HK-2 cells. After incubation for 6 h and 12 h in RAW264.7 cells, relatively high cell viability was observed regardless of different doses of TPSN (Fig. S7A, B). Even at a dose of 250 µg/mL, 5-fold relative to the cellular administrated dose, the proportion of viable cells remained higher than 90% after incubation with TPSN for 12 h. Similarly, HK-2 cells incubatied with TPSN for 8 h showed negligible cytotoxicity (Fig. S7C). Furthermore, the hemolysis assay in vitro showed no hemolysis even at dose as high as 2 mg/mL of TPSN (Fig. S7D).

Moreover, acute toxicity tests were conducted in healthy mice following i.v. injection of TPSN at 100 and 200 mg/kg, respectively. Mice in all groups maintained their typical daily diet and water intake without experiencing weight loss or any general behavioral disorders (Fig. S20A). On day 14 after administration, the mice were euthanized. The blood routine test analysis revealed no significant alternations in typical hematological parameters, such as platelet (PLT), red blood cell (RBC), and hemoglobin (HGB) levels (Fig. S20B-D). Moreover, the biochemical parameters relevant to liver and kidney functions also showed no distinct variations between groups (Fig. S20E-H). Meanwhile, examination of H&E-stained sections of the brain, heart, liver, spleen, lungs, and kidneys revealed negligible injuries and pathological patterns (Fig. S21). In addition, monitoring the biochemical parameters related to liver and kidney function on days 1, 3, 5, and 7 post-administration revealed no significant differences between the groups (Table S2). Furthermore, TPSN can degrade into small molecule HMP in the homogenates of lung, kidney, and liver tissues (Fig. S22). Overall, these preliminary data indicate that TPSN has a good safety profile after i.v. administration, with no observed cytotoxicity or in vivo systemic toxicity, even at doses 50–100 times higher than the therapeutic dose. This additional evidence substantiates the safety and efficacy of our proposed strategy for constructing bioactive nanoparticles utilizing silk fibroin.

Conclusions

In summary, we have engineered a bioactive material exhibiting robust ROS-scavenging capacities by facilely conjugating two functional moieties onto a SF scaffold. This material demonstrates effective elimination of H₂O₂, O₂^•−, and free radicals. Moreover, the bioactive material can be readily processed into nanoparticles, exhibiting significant antioxidant, anti-inflammatory, and macrophage reprogramming capabilities. Cellularly, TPSN demonstrated significant efficacy in inhibiting intracellular ROS generation, inflammatory cell migration, and the secretion of pro-inflammatory factors. It also conferred protective effects against oxidative stress and cisplatin-induced apoptosis. In murine models of ALI and AKI, TPSN exhibited significant targeting capabilities and a remarkable ability to suppress excessive inflammation and restore redox homeostasis in the affected organs. Importantly, TPSN therapy yielded more favorable outcomes compared to the clinical small molecule drug NAC. Of note, both in vitro and in vivo experiments have demonstrated that TPSN possesses the ability to reprogram macrophage phenotypes from a pro-inflammatory to an anti-inflammatory state. Furthermore, these studies have illustrated that TPSN exhibits excellent biocompatibility and protective effects against ROS-mediated damage. Consequently, TPSN holds potential as an effective and safe nanotherapeutic agent for treating various diseases associated with oxidative stress and inflammation. The pharmacological efficacy of TPSN could be significantly enhanced through surface modification with targeting or therapeutic functional groups. Additionally, it is expected to serve as a drug delivery system, enabling the exploration of potential synergistic interactions between its inherent anti-inflammatory properties and therapeutic agents.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

R. Liu and H. He conceived the experiments; R. Liu carried out the experiments; R. Liu, L. Li, Y, Wang and H. He analyzed the data, composed the figures and wrote the draft; H. Zuo, Y. Wang, L. Li, Q. Xia and H. He analyzed the data, and figures; Y. Wang, L. Li, and H. He supervised the project, analyzed the data and revised the manuscript and figures.

Funding

This work was supported by the National Key Research and Development Program of China (2022YFD1201600), the State Key Program of National Natural Science of China (32030103), the Fundamental Research Funds for the Central Universities (XDJK2020TJ001), the Natural Science Foundation of Chongqing, China (CSTB2022NSCQ-LZX0302, CSTB2022NSCQ-MSX0761, CSTC2020JCYJ-CXTTX0001, CSTB2022NSCQ-MSX1177), the Key Project of Science and Technology Research Program of Chongqing Municipal Education Commission, China (KJZD-K202200205) and the Chongqing innovation supporting program for oversea returned talents, China (CX2023069).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals recommended by the National Institutes of Health. All procedures and protocols were approved by the Animal Ethics Committee at Third Military Medical University (Chongqing, China), approval No. AMUWEC20230257.

Consent for publication

All authors agree for publication.

Competing interests

The authors declare no competing interests.