Near infrared enhanced palladium loaded siraitia grosvenorii carbon dots amplify mitophagy for acute lung injury immunotherapy

Abstract

Mitophagy is a self-protection mechanism for cells to eliminate dysfunctional mitochondria, and maintain mitochondrial homeostasis. Thus, precisely inducing mitophagy represents a promising strategy for acute lung injury (ALI) immunotherapy. Here, the mitochondrial targeted palladium loaded siraitia grosvenorii derived carbon dots (CPs@SS31) were engineered designed to integrate PTT, mitophagy induction, and immunoregulation for synergistic enhanced ALI therapy. CPs@SS31 combining with near infrared (NIR) irradiation not only directly scavenged reactive oxygen species to achieve antioxidant and anti-inflammation, but also amplified mitophagy via activating PINK1/Parkin pathway. Furthermore, it specifically targeted mitochondria to increase ATP production and mitochondrial membrane potential, thereby repairing the mitochondrial function of lipopolysaccharide induced cells. Meanwhile, it also demonstrated that CPs@SS31+NIR efficiently induced macrophage M2 polarization, and upregulated CD4⁺ T cells number and CD4⁺/CD8⁺ ratio, thereby activating immunoregulation, and achieving ALI repair therapy. In vitro and in vivo studies both demonstrated the robust alleviated lung inflammation, and accelerated lung tissue repair in ALI rats models. This work proposed an innovative “mitophagy induction-immunoregulation” paradigm, offering a promising strategy for ALI therapy, and being extended to the treatment of other inflammation related diseases.

Graphical abstract

The synergistic enhanced ALI immunotherapy was achieved by the specific mitochondrial targeting of CPs@SS31, and their mediated ROS scavenging, inflammation inhibition, tissue repair, macrophage M2 polarization, T cell immunoactivation as well as mitochondrial function activation to reprogram lung redox homeostasis.

Highlights

• •A mitochondrial targeted palladium loaded carbon dots was prepared for ALI therapy.
• •ROS scavenging and mitophagy activation jointly contributed to anti-inflammation.
• •Macrophage M2 polarization and T cells immunoactivation achieved ALI immunotherapy.

1. Introduction

Acute lung injury (ALI) has become a significant global concern mainly stemming from its high mortality rate and wide etiology [1]. As a common critical illness in intensive care unit (ICU), around 11%∼25% of patients may develop ALI, always accompanied by multiple organ dysfunction [2]. There is no specific drug for ALI treatment, severely limited by the complexity and diversity of its pathogenic mechanism [3]. In clinic, the current ALI treatment is life support techniques such as extracorporeal membrane oxygenation (ECMO). ECMO can only temporarily maintain life, but it can not change the disease progression, and may instead increase pain. Simultaneously, it causes severe irreversible organ damage [4]. Efforts to develop effective ALI therapeutic strategies face challenges associated with the limited bioavailability and biosafety in clinic.

Novel approaches to targeting mitochondria provide promising therapeutic strategies for drug delivery systems in respiratory system diseases [5]. As one of the most essential organelles in cells, mitochondria are the main site for aerobic respiration [6]. They not only participate in the regulation of cellular metabolism, but also provide energy for cellular activities. And they are also engaged in important functions such as cell apoptosis regulation and immunoregulation [7]. In the past few years, there have been many studies employing different mitochondrial targeting strategies in biomedical fields [8,9]. Mitochondria are also the main site for the generation of reactive oxygen species (ROS). Under ALI conditions, mitochondrial function is prone to dysfunction due to the lack of blood and oxygen supply, affecting the balance of cellular energy, and the production and removal of ROS [10]. Excessive ROS can lead to oxidative stress, and further trigger cell death [11]. Additionally, mitochondrial damage can stimulate the release of inflammation related cytokines by recruiting immune cells such as macrophages to the lungs, further exacerbating the inflammatory response [12]. Meanwhile, during the progression of ALI, mitochondrial dysfunction is characterized as the increased oxidative stress, decreased ATP production, and imbalanced mitophagy. They not only affect the integrity of pulmonary cells, but also further strengthen inflammatory reactions, forming a vicious cycle [13].

Significantly, mitophagy, a self-protection mechanism, is an important route for cells to maintain their own function, eliminate dysfunctional mitochondria, and regulate mitochondrial homeostasis [14]. Numerous studies have shown that mitophagy imbalance can damage mitochondrial functions, leading to the occurrence of various diseases [15,16]. Activating the PINK1/Parkin pathway, and inducing mitophagy is beneficial for treating inflammation related diseases [17]. Thus, mitophagy regulation is a key area for ALI therapy. Wan et al. revealed that RUNX1 induced mitophagy by upregulating mitophagy adaptor proteins to alleviate AT2 damage and lung inflammation in ALI [13]. It had also shown that the overexpression of SOCS1 could inhibit lipopolysaccharide (LPS) induced macrophage M1 polarization, reduce the expression of IL-1β, IL-6 and TNF-α, and increase the expression of IL-10. In addition, SOCS1 could promote TFEB nuclear translocation, thereby enhancing autophagy and inhibiting macrophage M1 polarization, ultimately alleviating ALI [18]. Mitochondria play a significant role in the pathological process of ALI, and regulating mitophagy can prominently affect the progression and therapeutic efficacy of ALI. The design of mult-functional nanomaterials offers promising potentials in preventing and treating ALI.

Nanozymes, a type of novel nanomaterials with multiple enzymatic activities, have shown great potentials in the treatment of various diseases [19,20]. Several types of nanozymes, such as metallic element [21,22], metal oxide [23,24], black phosphorus (BP) [25], metal organic framework (MOF) [26,27], covalent organic framework (COF) [28], etc, represent the hopeful candidates in ALI therapy. Among them, carbon dots (CDs) are a type of zero dimensional carbon nanomaterials with significant fluorescent properties, and excellent stability and biocompatibility, which have attracted wide attentions [29]. Natural CDs, with the combination of advantages of natural plants and modern nanotechnology, have shown great potentials in the field of diseases’ treatment [30]. Due to their abundant functional groups such as phenolic hydroxyl, carboxyl, and amino groups on their surface, CDs have multiple enzyme activities, and no toxicity to cells [31]. Research confirmed that CDs from natural plants such as houttuynia cordata, ginkgo biloba leaves, mulberry leaves and goji berries, which were applied to treat diabetic wounds [32], ALI [33] and other diseases [34]. More importantly, by loading metal elements such as selenium (Se) [35], platinum (Pt) [36], ferrum (Fe) [37] and manganese (Mn) [38] on the surface of CDs, the catalytic performance of newly prepared hybrid CDs are significantly enhanced. Liu et al. reported a metal doped CDs for acute kidney injury (AKI) therapy. Compared with CDs alone, Se doped CDs were more effective in scavenging ROS, inhibiting ferroptosis, regulating inflammatory responses, and ultimately efficiently and safely treating AKI [39].

In this study, we reported the mitochondrial targeted ultrasmall Pd loaded CDs (CPs@SS31) through preparing the natural derived CDs from siraitia grosvenorii followed by in situ loading with ultrasmall Pd, and modifying with elamipretide (SS31), which could specifically mediate mitophagy to modulate the levels of oxidative stress, and achieve lung tissue repair. CPs@SS31 exhibited multiple ·OH, ·O₂⁻ and H₂O₂ scavenging ability, responsible for maintaining redox homeostasis. To enable precise mitochondrial targeting, Pd loaded CDs (CPs) were functionalized with SS31. SS31 is a mitochondrial targeting peptide with a unique cationic and aromatic structure, capable of actively crossing the cell membrane, and accurately targeting the inner mitochondrial membrane [40,41]. Hence, the substantial ALI therapy was achieved by an effective mitochondrial accumulation of nanozymes, and their mediated mitochondrial function activation. The CPs@SS31 enabled the specific reprogramming redox homeostasis in the lung microenvironment, resulting in the significant antioxidant, anti-inflammation and tissue repair in LPS induced ALI rats (Fig. 1). It can precisely participate in inducing mitophagy, and activating immunoregulation, to achieve the synergistic ALI therapy. In this study, it showed a rational design of metal loaded CDs that induced mitophagy for ALI therapy, and offered an alternative therapeutic strategy for other inflammation related diseases.

Fig. 1.

Schematic illustration of the preparation of CPs@SS31, and the corresponding in vivo therapy of acute lung injury via NIR enhanced ROS scavenging, inflammation inhibition, macrophage M2 polarization, and T cells immunoactivation, as well as specifically targeting mitochondria, activating mitochondrial function, and inducing mitophagy to reprogram lung redox homeostasis, and promote tissue repair.

2. Experimental methods

Preparation of CPs@SS31: CDs were prepared via the traditional hydrothermal reaction of mechanically crushed siraitia grosvenorii as previously reported [42]. To prepare CPs, 30 mg CDs were dispersed in deionized (DI) water, followed by the addition of 30 mg K₂PdCl₆. The mixture was stirred with magnetic stirring for 1 h before the dropwise addition of 1 mL N₂H₄ solution (Jinshan Reagent, Chengdu, China). The final solution reacted overnight, and then was dialyzed with DI water for 3 days. After freezing drying, CPs were obtained. And the CPs@SS31 were prepared by mixing 30 mg CPs and 10 mg HS-PEG-SS31 (LookChem, China) for 2 h. After high speed centrifugation and re-dispersion for 3 times, CPs@SS31 were collected by freezing drying for further experiments [43,44].

Physicochemical characterization of CPs@SS31: To investigate the molecular structure of CDs, CPs and CPs@SS31, ultraviolet-visible spectrum (UV-vis, Shimadzu, Japan) and Fourier transform infrared spectrometer (FTIR, Shimadzu, Japan) were applied. And the crystallization structure of CDs, CPs and CPs@SS31 was respectively characterized by X-ray diffraction (XRD, MiniFlex 600, Japan). To investigate the morphology of CDs and CPs, transmission electron microscope (TEM, Hitachi, Japan) was used. And their element composition and Pd contents were investigated by X-ray photoelectron spectroscopy (XPS, ESCALAB 250XI+, USA), and inductively coupled plasma-mass spectrometer (ICP-MS, Varian, USA). The zeta potential of CDs, CPs and CPs@SS31 was tested by zeta nanosizer (Nano ZS, Malvern, UK). Finally, to investigate the thermal stability of CDs and CPs, thermal gravity analyzer (TGA, NETZSCH, Germany) was applied.

Dispersion and photothermal effects evaluation: The dispersion of CDs, CPs and CPs@SS31 was implemented as follows. In brief, CDs, CPs and CPs@SS31 were initially dispersed in different solutions: phosphate buffer saline (PBS, pH = 7.6, Gibco, USA), Dulbecco's modified eagle medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Gibco, Billings, USA) and 5 mM H₂O₂ (Aladdin, China) respectively. At predetermined time points (0, 0.5, 1, 2, 4, 8 and 24 h), the images were observed by digital camera. To investigate the photothermal effects, CDs and CPs with different concentrations of 0, 100, 200 and 500 μg/mL were under near infrared (NIR) irradiation (0.5, 1, 1.5 and 2 W/cm², 808 nm, CNI Laser, China) for 15 min. The photothermal images and corresponding temperatures were collected and recorded every minute during irradiation. The photothermal stability was implemented by placing 200 μg/mL CPs under NIR irradiation (1.5 W/cm²) with 5 cycles of “on” and “off”, 15 min for each cycle.

ROS scavenging ability investigation: The ROS scavenging ability was initially evaluated by electron spin resonance (ESR, Bruker, A300, Germany). In details, CDs and CPs were prepared with the concentration of 200 μg/mL, and mixed with the corresponding working solutions, where 5-tert-butoxycarbonyl 5-methyl-1-pyrroline-N-oxide (BMPO, 100 mM), 10 mM xanthione and 1 U/mL xanthione oxidase, and 2, 2, 6, 6-tetramethylpiperidine (TEMPONE, 100 mM) was respectively applied as the working solutions for ·OH, ·O₂⁻ and ¹O₂ scavenging ability testing by ESR. Besides, the ROS scavenging capacities were investigated by ROS testing kits. In brief, 200 μg/mL of CDs or 50, 100 and 200 μg/mL of CPs were incubated with ·OH and ·O₂⁻ testing kits (Solarbio, China). And the solutions were measured at 536 and 530 nm by microplate reader (Thermo Fisher, USA). Specifically, for NIR alone and CPs + NIR, NIR irradiation (0.5 W/cm²) was implemented for 10 min after mixing CPs with the working solutions. To further confirm the effect of SS31 and NIR alone on ROS scavenging capacities, 2, 2-diphenyl-1-picrylhydrazine (DPPH, Nanjing Jiancheng Bio, China) and 2, 2′-diazobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABST, Beyotime, China) testing kits were applied by following the protocols.

Cell viability testing: Mouse monocyte macrophages (RAW264.7, ATCC, USA) were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (Solarbio, China). The fresh culture medium was replaced everyday. When the confluence of cells reached 90%, they were passage for further experiments. At the beginning, the cell viability was evaluated by cell counting kit-8 (CCK-8, Biosharp, China). In brief, the cells were cultured in the 96 well plate with the density of 1 × 10⁴ cells/mL, and incubated with CDs or CPs with the concentration range of 0, 5, 10, 20, 50, 100, 200 and 500 μg/mL for 24 h. The cells were rinsed against with PBS for 3 times, followed by the addition of 10 μL 10% CCK-8 for another 2 h. And the supernatant was finally observed at 450 nm by microplate reader.

Cellular protection ability investigation: the cells were cultured in a 6 well plate with the density of 2 × 10⁶ cells/mL, and stimulated with LPS (Servicebio, China) for 30 min. The treated cells were then incubated with 200 μg/mL of CDs, CPs, CPs@SS31 or CPs@SS31+NIR for 24 h respectively. Finally, the cells were stained with calcein-AM/propidium iodide (PI) solution (Beyotime, China) for 30 min in the darkness before being observed by fluorescent microscope (Olympus, Japan).

Antioxidant and anti-inflammatory abilities in cellular levels: RAW264.7 were cultured, and stimulated with 1 μg/mL LPS for 30 min, followed by incubating with 200 μg/mL samples for another 24 h. Initially, to investigate the intracellular ROS levels, the treated cells were stained with 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, China) for 30 min, and observed by fluorescent microscopy after rinsing with PBS. And the intracellular ROS levels of treated cells were also analyzed by flow cytometry (Beckman, USA). Meanwhile, the inflammatory factors (IL-6 and IL-10) expression levels in the supernatant of treated cells were also analyzed by enzyme-linked immunosorbent assay (ELISA, Fankew, China) following the protocols. Besides, the treated cells were fixed with 4% paraformaldehyde (PFA, Biosharp, China) for 15 min, and treated with blocking buffer (Beyotime, China). And then, the cells were incubated with primary antibodies (anti-IL-6, TNF-α, CD206, CD86, CD31, HSP70 and PINK1, 1 : 200, Proteintech, USA) overnight. After PBS rinsing for 3 times, the cells were then incubated with secondary antibody (Proteintech, USA) for another 2 h. After 4′, 6-diamidino-2-phenylindoledilactate (DAPI, Biosharp, China) staining, the cells were finally observed by fluorescent microscope.

In addition, the related genes expression levels of treated cells were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The total RNA of treated cells was extracted by RNA extraction kit (Thermofisher, China). After cDNA synthesis by reverse transcription kit (Vazyme, China), the qPCR was performed by quantitative PCR instrument (analytikjena, Germany). The relative genes expression levels were calculated by 2^−ΔΔCt, where glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as the reference. The detailed primer sequences for RT-qPCR was illustrated in Table S1. In particular, to evaluate the phagocytic ability of treated cells, neutral red phagocytosis experiment was applied. After washing with PBS for 3 times, the treated cells were incubated with neutral red solution (Solarbio, China) for 1 h. And then, after removing the supernatant, the cells were lysed with lysis buffer (Solarbio, China). The final solution was observed at 540 nm by microplate reader.

Mitochondrial functions investigation: To investigate the mitochondrial targeting ability, RAW264.7 were seeded into a 6-well plate with the density of 2 × 10⁶, and then incubated with Cy5 labeled CPs (Cy5-CPs) or CPs@SS31 (Cy5-CPs@SS31) for 6 h. Later, the cells were stained with mito-tracker green probe assay kit (Beyotime, China) and DAPI. After PBS washing for 3 times, the cells were fixed with 4% PFA for 15 min. The cells were finally observed by confocal laser scanning microscope (ZEISS, Germany). In particular, to prepare Cy5-CPs or Cy5-CPs@SS31, 30 mg CPs or CPs@SS31 was dispersed in PBS, followed by adding 10 mg Cy5-PEG-SH (Lumiprobe, China). The mixture reacted overnight before washing with DI water for 3 times. The final product was obtained after freezing drying.

In mitochondrial levels, to investigate the mitochondrial membrane potential (MMP), the treated cells were rinsed with PBS for 3 times, and added with 5 μg/mL JC-1 working solution (Beyotime, China). The final samples were incubated in the dark for 20 min, and washed with PBS for 3 times before observation by fluorescent microscope. Besides, the MMP of treated cells was also analyzed by flow cytometry. And the ATP content of treated cells was tested by the enhanced ATP kit (Beyotime, China) as the protocols described. Furthermore, to investigate the mitochondrial ROS levels, the treated cells were incubated with mitochondrial superoxide assay kit with MitoSOX red (Yeasen, China) for 30 min. After washing against with PBS for 3 times, the cells were stained with mito-tracker green before observation.

Anti-inflammation mechanisms exploration: The high throughput RNA sequencing was applied to explore anti-inflammation mechanism. In brief, LPS induced RAW264.7 were treated with PBS (control group) or 200 μg/mL CPs@SS31+NIR (CPs@SS31+NIR) for 24 h. The total RNA of cells was then extracted by RNA extraction kit, and the genomic DNA was digested by DNase I (Takara, Japan) before performing RNA sequencing analysis. The differentially expressed genes (DEGs) were identified by using R software (version = 4.42) with the statistical significance of P value < 0.05 and Log₂ Fold change >1. And the kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was conducted to explore the possible pathways and biological functions of CPs@SS31+NIR in ALI therapy.

To confirm the anti-inflammation mechanism, western blotting (WB) was implemented to analyze the relative proteins expression levels of treated cells. In brief, the treated cells were washed with ice-cold PBS, and treated with lysis buffer containing protease inhibitors and phosphatase inhibitors (Solarbio, China). After collecting the supernatant, its protein concentration was adjusted by BCA protein assay kit (Beyotime, China), and the supernatant was separated by protein gel electrophoresis. And then the proteins were transferred to polyvinylidene fluoride (IPVH00010, Millipore, USA) membranes before blocking for 2 h, and the membranes were washed with tris buffered saline with Tween-20 (TBST, Sigma, USA) for 3 times. Furthermore, the membranes were separately incubated with the primary antibody (anti-P62, Parkin, PINK1 and GAPDH, Proteintech, China) overnight at 4 °C. After rinsing with TBST for 3 times, the membranes were incubated with the secondary antibody (Goat Anti-Rabbit, Sangon, China) for another 2 h. At last, the membranes were soaked in 1 mL of UltraSignal ECL WB detection reagent (Beyotime, China), and scanned by automatic chemiluminescence image analysis system (Bio-Rad, USA). In specific, 0.01 mM Mdivi-1 (Yeasen, China) was applied as the inhibitor to further confirm the mitophagy induced anti-inflammation mechanism [45]. The related proteins expression levels of treated cells were also analyzed by WB.

In vivo biodistribution study: Male sprague dawley (SD) rats (180∼220 g, 6-8 weeks) were purchased from the experimental animal center of Guangxi Medical University. All animal experiments were approved by the animal ethics committee of Guangxi Medical University (No. 202411035). To evaluate the biodistribution of CPs@SS31, 0.4 mL Cy5 and Cy5-CPs@SS31 (200 μg/mL) was intratracheal (IT) injected into rats. The fluorescent images of major organs were collected at selected time points (0, 0.5, 1, 2, 4, 8 and 24 h), and observed by in vivo imaging system (IVIS, PerkinElmer, USA) with an excitation and emission wavelength of 646 and 664 nm. Significantly, the feasibility of in vivo photothermal therapy (PTT) was also investigated. After IT administration of CPs@SS31 or saline for 0.5 h, NIR irradiation was implemented in the lung of rats. The photothermal images was collected, and the corresponding temperatures were recorded during NIR irradiation.

In vivo biosafety evaluation: To investigate the hemocompatibility, the fresh blood was collected, and centrifuged at 3000 rpm for 15 min. The red blood cells were dispersed in PBS, and incubated with CPs or CPs@SS31 with the concentrations of 0, 5, 10, 20, 50, 100, 200 and 500 μg/mL for 1 h, with DI water as the positive control, and PBS as the negative control. After centrifuge at 3000 rpm for 10 min, the supernatant was observed by microplate reader, and the hemolysis ratio was calculated as [(OD_s-OD_n)/(OD_p-OD_n)] × 100%, where OD_s, OD_n and OD_p was the optical density (OD) of sample, negative control and positive control. In addition, to evaluate in vivo biosafety, the rats (n = 5) were IT injected with saline (sham group) or CPs@SS31 respectively for 7 days. Their body weight was weighed every day. After 7 days, the rats were sacrificed, and the blood was collected to investigate the blood indicators by fully automatic blood analyzer (Mindray, China). And the major organs were also collected and observed, and cut into sections for hematoxylin and eosin (H&E) staining after 4% PFA fixation.

In vivo ALI therapy evaluation: The ALI animal models were established by IT injection of LPS (5 mg/kg) for 1 h as previously reported [46]. For in vivo ALI therapy, the rats were divided into the following 5 groups (n ≥ 3): rats with IT injection of saline (sham group), and LPS induced rats followed by the IT administration of saline (ALI group), CPs (CPs), CPs@SS31 (CPs@SS31) and CPs@SS31+NIR (CPs@SS31+NIR). NIR irradiation (0.5 W/cm²) was implemented for 3 times within 1 h after IT administration of 0.5 h, 10 min per time. The rats were sacrificed after 24 h’ therapy, and their blood and major organs including heart, liver, spleen, lung and kidney were collected for further evaluation. The blood indicators of treated rats were analyzed by the fully automatic blood analyzer. In specific, after collecting the fresh blood at predetermined time points, the Pd contents in the blood of treated rats were analyzed by ICP-MS.

The macroscopic observation of lung was imaged by digital camera. And the wet/dry ratio of lung tissue was also calculated by weighing the weight of lung before and after oven drying. Besides, the inflammatory factors (IL-6 and IL-10) expression levels of lung tissue were analyzed by ELISA. To investigate the pathological feature, the lung tissue was initially stained by H&E before observation. And the ROS levels of lung tissue were investigated. In details, the tissue section was incubated with DCFH-DA for 30 min. After rinsing with PBS, it was stained with DAPI before observation. For immunohistological staining, the lung tissue was incubated with primary antibodies (anti-IL-6, TNF-α, CD31, HSP70, P62, Parkin and PINK1). After PBS washing for 3 times, the tissue sections were stained with biotin labeled secondary antibody (Proteintech, USA). Markedly, the lung tissue was incubated with primary antibodies (anti-CD86 and CD206) at 4 °C overnight, followed by fluorescent labeled secondary antibody (Proteintech, USA) staining. After DAPI staining, they were observed by fluorescent microscope.

Furthermore, the CD4⁺ and CD8⁺ T cells number in the blood, lung and spleen of treated rats was analyzed by flow cytometry. And the CD4⁺ and CD8⁺ T cells expression levels in the lung tissue of treated rats were also investigated by co-immunofluorescent staining. In details, the lung tissue was incubated with primary antibodies (anti-CD4⁺ and CD8⁺, Biolegend, America) before staining with fluorescent labeled secondary antibody (Proteintech, USA). After DAPI staining, the tissue sections were observed. Finally, the other organs including heart, liver, spleen and kidney were collected, and the corresponding tissue sections were observed after H&E staining.

Statistical analysis: All data were expressed as mean ± standard deviation, and the significance was defined as ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001, and ∗∗∗∗: p < 0.0001, where ∗ indicated the comparison with normal group in cellular levels, or sham group in animal levels.

3. Results and discussion

3.1. Physicochemical properties investigation

To prepare CDs, Siraitia grosvenorii was mechanical crushed followed by hydrothermal reaction at 180 °C for 8 h. And ultrasmall Pd was embedded in CDs to form CPs by in situ reduction. Finally, CPs@SS31 was fabricated by modifying CPs with SS31 via the coordination between HS group of HS-PEG-SS31 and ultrasmall Pd, or Micheal addition between HS group of HS-PEG-SS31 and double bonds of CDs (Fig. 2A). From UV-vis spectrum, no obvious peaks existed in CDs, CPs and CPs@SS31 (Fig. S1), indicating the formation of pure CDs without soluble double bonds chemicals existed. And from FTIR results, the obvious peaks happened at 1630 cm⁻¹ and 3440 cm⁻¹, corresponding to the amino groups and phenolic hydroxyl groups in CDs, CPs and CPs@SS31. The existence of phenolic hydrooxyl groups contributed to the ROS scavenging ability of them [47]. Besides, an extra peak was observed at 2320 cm⁻¹ in CPs@SS31 attributed by SS31 modification (Fig. S2). Meanwhile, by XRD, the obvious peak was observed at 21.6° in CDs, while the extra peaks happened at 40.2° and 46.3° in CPs due to Pd loading (Fig. S3). From TEM image, the obvious spherical dots were shown in CDs and CPs, with the diameter of 2.1 ± 0.1 nm and 4.3 ± 0.4 nm respectively (Fig. 2B and S4). From the above, it indicated the formation of CDs and CPs in a few nm. Specifically, for CPs, its lattice spacing was 0.219 ∼ 0.231 nm (Fig. S5). By zeta sizer, the zeta potential was −9.7 ± 1.1 mV in CDs, slightly decreased to −17.9 ± 1.7 mV in CPs. And it became −17.9 ± 0.4 mV in CPs@SS31 (Fig. 2C). Pd loading could change the zeta potential of CPs, while SS31 modification did not affect the zeta potential of CPs@SS31. Besides, the element composition in CDs and CPs was analyzed by XPS. The obvious C, N and O elements appeared in CDs, while the extra Pd element (6.07%) was shown in CPs compared to CDs (Fig. 2D). From the peak fitting results, no significant differences were observed in C and N elements between CDs and CPs. In specific, from the peak fitting results of Pd element, the obvious Pd⁰ and Pd²⁺ were observed, indicating the multivalent state of Pd existed in CPs (Fig. S6), helpful to the enhanced ROS scavenging ability. By ICP-MS, the Pd content was 10.74 ± 0.11% in CPs (Table S2). It ultimately confirmed the formation of CDs and CPs. After Pd loading, no significant differences existed in chemical structure, molecular structure, morphology and diameter between CDs and CPs, except for the element composition and zeta potential.

Fig. 2.

Preparation and physicochemical characterization of CPs@SS31. A) The synthesis procedure of CPs@SS31. B) TEM image of CPs. (Scale bar = 20 nm) C) Zeta potential of CDs, CPs and CPs@SS31. D) XPS results of CDs and CPs (full spectrum). E) Photothermal images of PBS, CDs and CPs with the same concentration of 200 μg/mL. F) Photothermal effects of PBS, CDs and CPs with the same concentration of 200 μg/mL under NIR irradiation (1.5 W/cm²) (i), different concentrations (0, 100, 200 and 500 μg/mL) of CP under NIR irradiation (1.5 W/cm²) (ii), 200 μg/mL CPs under different NIR irradiation intensity (0.5, 1, 1.5 and 2 W/cm²) (iii) versus time, and the photothermal stability of 200 μg/mL CPs under NIR irradiation (1.5 W/cm²) for 5 “on” and “off” cycles (iv). G) TGA results of CDs and CPs. H) Dispersion of CDs and CPs under different solutions: PBS, FBS, DMEM and 5 mM H₂O₂ (from left to right) versus time. I) ROS scavenging ability of CDs, CPs and CPs + NIR with the same concentration of 200 μg/mL by ESR: ·OH, ·O₂⁻ and ¹O₂. J) ROS scavenging capacity by ·OH and ·O₂⁻ testing kits: CDs, CPs and CPs + NIR with the same concentration of 200 μg/mL.

From photothermal images, for PBS, no changes of temperature happened during NIR irradiation. And the obvious temperature changes existed in CDs and CPs versus irradiation time (Fig. 2E). After statistical analysis, the temperature maintained at 26.8 °C in PBS during NIR irradiation. However, it gradually increased to 61.7 °C in CDs, and 61.8 °C in CPs after 15 min' irradiation (i of Fig. 2F). It displayed the obvious photothermal effects for CDs and CPs. After Pd loading, it did not obviously affect the photothermal effects of CPs. Increasing the concentration of CPs also increased the photothermal effects, which became 51.6, 63.0 and 70.4 °C in CPs of 100, 200 and 500 μg/mL after irradiation for 15 min (ii of Fig. 2F). Similarly, increasing the power intensity of NIR light from 0.5 to 2 W/cm², the corresponding temperature of 200 μg/mL CPs also increased from 44.3 °C to 70.3 °C by 15 min’ irradiation (iii of Fig. 2F). In particular, after 5 cycles of “on” and “off”, the photothermal effects maintained stable in CPs with 200 μg/mL (iv of Fig. 2F). To fully maintain the normal cellular functions, NIR irradiation with the power intensity of 0.5 W/cm² was applied for the following experiments, which was below the critical temperature (45 °C) that causes irreversible damage to normal tissues [48]. Besides, from TGA results, the weight remaining ratio was 10.6% in CDs, and 19.3% in CPs. After comparison, the Pd content was 8.7% in CPs (Fig. 2G). It proved that CDs and CPs possessed the excellent photothermal effects and photothermal stability. Increasing the concentration of CPs, and power intensity of NIR irradiation both contributed to the enhanced photothermal effects. Furthermore, the dispersion of CDs and CPs was also tested. As displayed in Fig. 2H, both CDs and CPs possessed the good dispersion, maintaining stable in 4 different solutions within 24 h.

Finally, the ROS scavenging ability was initially investigated by ESR. As illustrated in Fig. 2I, for ·OH, ·O₂⁻ and ¹O₂ scavenging ability, compared to control group with high intensity, the treatments could decrease their intensity. Among them, CPs@SS31+NIR played the most effective roles. By ROS testing kits, for ·OH scavenging ratio, it was 5.81 ± 0.31% in CDs, 32.14 ± 0.99% in CPs, and 43.56 ± 1.82% in CPs + NIR respectively. Similarly, the ·O₂⁻ scavenging ratio was 50.03 ± 2.19% in CDs, 61.49 ± 0.42% in CPs, and 76.75 ± 0.31% in CPs + NIR (Fig. 2J and Table S3). Prominently, compared to control group (0.71 ± 0.71%), the ·OH scavenging ratio was only 3.56 ± 1.32% in NIR alone, while it was 33.73 ± 5.40% in CPs (Fig. S7 and Table S4). And for CPs at different concentrations (50, 100 and 200 μg/mL), the corresponding ·OH and ·O₂⁻ scavenging ratio were 20.97 ± 0.26% and 5.15 ± 3.06%, 24.39 ± 0.57% and 28.00 ± 2.15%, and 32.27 ± 0.54% and 63.12 ± 1.16% respectively (Fig. S8 and Table S5). Most importantly, the ABST and DPPH scavenging ratio was 62.42 ± 0.62 and 60.55 ± 0.73 in CPs, similar to those in CPs@SS31 (63.09 ± 0.38 and 61.18 ± 0.43). However, the ABST and DPPH scavenging ratio was 2.96 ± 1.48 and 3.72 ± 1.52 in NIR irradiation alone (Fig. S9 and Table S6). As previously reported, NIR irradiation alone could not affect the ROS scavenging ability [49,50]. Compared to CDs, CPs possessed the better ROS scavenging ability due to ultrasmall Pd loading [24]. And SS31 did not contribute to the enhanced ROS scavenging capacities. Thus, the similar ROS scavenging ability happened between CPs and CPs@SS31. Significantly, NIR irradiation could speed up the electron movement, further contributing to the enhanced ROS scavenging ability in CPs + NIR [51,52].

3.2. In vitro biological functions

RAW264.7 cells are murine monocyte leukemia cells, commonly used in inflammation, immunity, and other, and can simulate inflammatory response. From Fig. 3A, the cell viability was above 95% in CDs and CPs with the dose concentration within 200 μg/mL. When their concentration was above 200 μg/mL, the cell viability started to decrease. Herein, 200 μg/mL was applied as the working concentration for further experiments. And from live/dead staining images, compared to normal group, a lot of dead cells (red fluorescence), and the decreased number of live cells (green fluorescence) were observed in control group. The treatments could increase the number of live cells, and CPs@SS31+NIR most effectively increased the live cells number (Fig. 3B and S10). After statistical analysis, the live/dead ratio was 263.57 ± 24.38% in normal group, decreased to 105.64 ± 8.67% in control group. After treatments, it became 146.37 ± 28.61% in CDs (∗∗∗∗), 169.13 ± 16.04% in CPs (∗∗∗), 206.17 ± 9.36% in CPs@SS31 (∗), and 248.47 ± 19.37% in CPs@SS31+NIR (Fig. 3C).

Fig. 3.

In vitro antioxidant and anti-inflammation abilities. A) Cell viability of RAW264.7 incubated with CDs and CPs of different concentrations ranging from 0 to 500 μg/mL. B) Live/dead staining images of treated cells, (Scale bar = 100 μm) and the corresponding live/dead ratio (C). D) Intracellular ROS (DCFH-DA) levels of treated cells by fluorescent microscopy, (Scale bar = 50 μm) and the corresponding quantified results (E). F) Intracellular ROS (DCFH-DA) levels of treated cells by flow cytometry. G) Relative genes expression levels of treated cells by RT-qPCR: IL-6 and IL-10. H) TNF-α expression levels of treated cells by fluorescent microscope. (Scale bar = 50 μm) The corresponding groups were: RAW264.7 without treatments (normal group), and RAW264.7 pre-treated with LPS followed by incubation with PBS (control group), CDs (CDs), CPs (CPs), CPs@SS31 (CPs@SS31) and CPs@SS31 combining with NIR irradiation (CPs@SS31+NIR). (∗ symbol compared with normal group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001).

Next, the IL-6 and IL-10 expression levels in the supernatant of treated cells were analyzed by ELISA. As illustrated in Fig. S11, compared to normal group (51.23 ± 1.69 pg/mL), the IL-6 expression levels were ascended to 368.78 ± 5.96 pg/mL in control group with the significant difference of ∗∗∗∗. After treatments, the IL-6 expression levels were declined, which became 158.47 ± 9.72 pg/mL in CDs, 96.36 ± 3.49 pg/mL in CPs, 78.48 ± 2.35 pg/mL in CPs@SS31, and 68.04 ± 2.23 pg/mL in CPs@SS31+NIR. Conversely, the IL-10 expression was in relatively low levels in normal group (2.64 ± 1.33 pg/mL) and control group (18.25 ± 0.32 pg/mL), which was gradually ascended in CDs (21.76 ± 0.69 pg/mL), CPs (34.44 ± 0.98 pg/mL), CPs@SS31 (45.17 ± 4.71 pg/mL), and CPs@SS31+NIR (64.60 ± 1.97 pg/mL). Next, the intracellular ROS levels of treated cells were also analyzed. As displayed in Fig. 3D, the DCFH-DA was in the highest levels in control group with a lot of green fluorescence observed, which was obviously declined in other groups. After statistical analysis, the mean fluorescent intensity (MFI) was 75.06 ± 8.31 in control group with the significant difference of ∗∗∗∗, subsequently decreased to 50.88 ± 1.23, 25.56 ± 1.80, 12.57 ± 0.86, 6.41 ± 0.39 and 4.94 ± 0.92 in CDs, CPs, CPs@SS31, CPs@SS31+NIR and normal group (Fig. 3E). In specific, compared to control group with a lot of green fluorescence observed, NIR alone had no effects on the intracellular ROS levels of treated cells (Fig. S12A). By statistical analysis, the MFI was 0.87 ± 0.12 in normal group, jumped to 3.64 ± 0.16 and 3.40 ± 0.19 in control group and NIR, both with the significant difference of ∗∗∗∗ (Fig. S12B). Remarkably, it displayed the similar tendency by flow cytometry. The ROS positive cells ratio was in the order of normal group (15.10 ± 0.72%) < CPs@SS31+NIR (12.32 ± 2.60%) < CPs@SS31 (21.97 ± 1.53%) < CPs (34.40 ± 2.79%) < CDs (37.03 ± 0.45%) < control group (46.27 ± 1.64%) (Fig. 3F and S13).

To further investigate the inflammation related genes expression levels, RT-qPCR was applied. As illustrated in Fig. 3G, the IL-6 gene expression was in the relative low levels in normal group, significantly jumped in control group and CDs with the significant differences of ∗∗∗∗ and ∗∗∗∗. Its expression was significantly decreased in CPs, CPs@SS31 and CPs@SS31+NIR compared to control group. Especially in CPs@SS31 and CPs@SS31+NIR, no significant differences existed. Specifically, for NIR alone, it could not affect the IL-6 expression levels of treated cells, displaying the similar levels of IL-6 expression with control group (Fig. S14). On the contrary, for IL-10 gene expression, its expression was in relatively low levels in normal group, control group and CDs with no significant differences. It obviously increased in CPs, CPs@SS31 and CPs@SS31+NIR with the significant difference of ∗∗∗, ∗∗∗∗ and ∗∗∗∗ (Fig. 3G). And for CD31 gene expression levels, they were relatively low in normal group, control group and CDs, gradually enhanced in CPs, CPs@SS31 and CPs@SS31+NIR with the significant difference of ∗, ∗∗∗∗ and ∗∗∗∗ respectively. Similarly, for HSP70 gene expression, it was in relatively low levels in all groups, except for CPs@SS31+NIR, significantly enhanced with the significant difference of ∗∗∗ (Fig. S15).

Meanwhile, the inflammatory factors expression levels of treated cells were also investigated by immunofluorescent staining. As shown in Fig. 3H, the TNF-α expression levels were relatively high in control group, with the obvious green fluorescence observed compared to that of normal group. After statistical analysis, the MFI of TNF-α expression was 9.20 ± 0.70 in normal group, which significantly increased to 70.90 ± 4.68 in control group. After treatments, the TNF-α expression levels were declined with the obviously decreased green fluorescence. The treatments efficiently reduced the MFI of TNF-α to 50.65 ± 4.38 in CDs (∗∗∗∗), 20.83 ± 1.31 in CPs (∗∗), 11.65 ± 1.74 in CPs@SS31, and 7.27 ± 0.25 in CPs@SS31+NIR (Fig. S16). Meanwhile, the IL-6 expression levels in control group were relatively high with the obvious green fluorescence observed compared to normal group. However, the treatments obviously decreased the green fluorescence (Fig. S17A). Quantitatively, the MFI of IL-6 expression was 7.79 ± 0.52 in normal group, which significantly increased to 48.34 ± 5.82 in control group. After treatments, the MFI of IL-6 expression was reduced to 27.54 ± 1.48 in CDs, 19.30 ± 2.29 in CPs, 13.13 ± 0.88 in CPs@SS31, and 8.87 ± 0.53 in CPs@SS31+NIR respectively (Fig. S17B). And as illustrated in Fig. S18, compared to control group, NIR alone could not affect the IL-6 expression levels. After statistical analysis, the MFI of IL-6 expression was 4.06 ± 0.55 in control group, and 4.16 ± 0.88 in NIR with the significant difference of ∗∗∗ and ∗∗∗.

At last, the CD31 and HSP70 expression levels of treated cells were also investigated by immunofluorescent staining. As displayed in Fig. S19A, the CD31 expression was relatively low in normal group, control group, CDs and CPs, ascended in CPs@SS31 and CPs@SS31+NIR with the obviously observed green fluorescence. After statistical analysis, the MFI was relatively low in all groups except for CPs@SS31 (22.33 ± 0.54) and CPs@SS31+NIR (43.68 ± 8.58) with the significant difference of ∗∗ and ∗∗∗∗ respectively (Fig. S19B). Significantly, compared to normal group and control group, NIR alone had no effects on CD31 expression levels, with no significant difference (Fig. S20). In the meantime, the HSP70 expression levels were low in normal group, control group and CDs with little green fluorescence observed. However, the green fluorescence was significantly enhanced in CPs, CPs@SS31 and CPs@SS31+NIR (Fig. S21A). After analysis, the MFI was 34.84 ± 0.79, 42.42 ± 3.83 and 66.21 ± 5.10 in CPs, CPs@SS31 and CPs@SS31+NIR all with the significant difference of ∗∗∗∗ (Fig. S21B).

As previously reported, NIR alone could not affect ROS scavenging ability, and it also could not affect the functions of antioxidant, anti-inflammation and tissue repair [11,23]. CDs and CPs with a certain of ROS scavenging ability, which presented slight antioxidant and anti-inflammation abilities. However, CPs@SS31 could specifically target mitochondria, more efficiently scavenging ROS to achieve antioxidant and anti-inflammation, and slightly promote tissue repair. Specifically, for CPs@SS31+NIR, it displayed the excellent abilities of antioxidant, anti-inflammation and tissue repair, attributed by its enhanced ROS scavenging ability and PTT effects [53].

3.3. In vivo ALI therapy

The in vivo biodistribution of CPs@SS31 was investigated by IVIS. From Fig. 4A and B, by IT injection, Cy5 could retain in the lung for a certain of time, and totally disappear at 4 h, while Cy5-CPs@SS31 would stay in the lung till 8 h. No fluorescence was observed in other organs by IT injection, meaning that there were no enrichment of CPs@SS31 in the major organs except for the lung. Besides, to confirm the feasibility of in vivo PTT, NIR irradiation was implemented in the lung of rats after IT injection for 0.5 h. Compared to sham group with no temperatures changed, the temperature was gradually ascended in CPs@SS31 during NIR irradiation (Fig. 4C). Thus, it maintained the stable temperature in sham group, while it became 44.5 °C in CPs@SS31 after 15 min’ irradiation (Fig. 4D). Remarkably, for in vivo ALI therapy, it required the administration strategy with high biosafety. By the hemolysis testing, it displayed the excellent blood biocompatibility in CDs, CPs and CPs@SS31 with the hemolysis ratio below 5% (Fig. S22). And after IT injection for 7 days, the body weight in sham group and CPs@SS31+NIR both increased gradually, with no obvious changes existed between them (Fig. 4E). Similarly, from the blood indicators, there were no significant differences observed between sham group and CPs@SS31+NIR (Fig. 4F and Table S7). And from the H&E staining images of major organs, no obvious differences existed as well (Fig. 4G), indicating the excellent biosafety by this type of administration strategy.

Fig. 4.

In vivo biosafety evaluation. A) In vivo biodistribution of CPs@SS31 by IVIS, and the corresponding MFI of the lung (B). C) In vivo photothermal images of treated rats versus time under NIR irradiation (0.5 W/cm²), and the corresponding quantified results (D). E) Body weight changes of treated rats versus time. F) Blood indicators of treated rats after 7 days. G) H&E staining images in the major organs of treated rats after 7 days. (Scale bar = 100 μm) The corresponding groups were: rats without treatments (sham group), and rats with IT administration of CPs@SS31 (CPs@SS31).

For in vivo ALI therapy, LPS stimulation was implemented for 1 h before IT injection. After 24 h, the rats were sacrificed for further evaluation. The detailed time schedule of ALI therapy was listed in Fig. 5A. Later, the blood indicators of treated rats were analyzed. As displayed in Fig. S23, among all groups, there were no obvious differences in the blood indicators except for WBC, HGB, PT and APTT, which were in high levels in ALI group compared to sham group, and subsequently decreased after treatments (Table S8). In addition, the Pd contents maintained the relatively low levels in the blood of sham group. However, in CPs and CPs@SS31, it reached a higher levels in the blood at 0.5 h, and gradually decreased versus time. After 6 h, it reached the similar levels as those of sham group, indicating that the Pd elements were thoroughly metabolized from animals (Fig. S24 and Table S9). Compared to that of CPs, the decreased Pd contents were observed in the blood of CPs@SS31 at 0.5 h, indicating the longer retention time of CPs@SS31 in the lung than that of CPs [54]. From the macroscopic observation of lung tissue, compared to sham group with smooth and glossy surface, and no sign of congestion, the significant congestion phenomena existed in the lung tissue of ALI group. After treatments, the congestion phenomena was relived (Fig. 5B). Besides, the wet/dry ratio of lung tissue was also tested. As listed in Fig. 5C, the wet/dry ratio was 5.04 ± 0.03% in sham group, obviously ascended to 10.55 ± 0.53% in ALI group with the significant difference of ∗∗∗∗. After treatments, it was obviously reduced, especially in CPs@SS31+NIR (4.77 ± 0.11%) with no significant difference.

Fig. 5.

In vivo ALI therapy evaluation. A) Time schedule of in vivo animal experiment. B) Macroscopic observation in the lung tissue of treated rats. C) Wet/dry ratio in the lung tissue of treated rats. D) Inflammatory factors expression levels in the blood of treated rats. E) Inflammatory factors expression levels in the lung tissue of treated rats. F) ROS levels in the lung tissue of treated rats. (Scale bar = 50 μm) G) H&E staining images in the lung tissue of treated rats. (Scale bar = 100 μm) H) TNF-α expression levels in the lung tissue of treated rats, (Scale bar = 100 μm) and the corresponding quantified results (I). J) HSP70 expression levels in the lung tissue of treated rats, (Scale bar = 100 μm) and the corresponding quantified results (K). L) CD31 expression levels in the lung tissue of treated rats, (Scale bar = 100 μm) and the corresponding quantified results (M). The corresponding groups were: rats without treatments (sham group), and rats pretreated with LPS followed by IT administration of PBS (ALI group), CPs (CPs), CPs@SS31 (CPs@SS31) and CPs@SS31 combining with NIR irradiation (CPs@SS31+NIR). (”∗” symbol compared with sham group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001).

From ELISA results, the IL-6 expression levels of serum were relatively low in sham group (15.63 ± 0.98 pg/mL), significantly increased to 42.60 ± 2.58 pg/mL in ALI group with the significant difference of ∗∗∗∗. The treatments could decrease its expression levels, especially in CPs@SS31+NIR with no significant difference compared to sham group. Conversely, the IL-10 expression was in relative low levels in sham group (14.22 ± 1.48 pg/mL) and ALI group (11.19 ± 1.48 pg/mL), which obviously increased after treatments. Especially in CPs@SS31+NIR, IL-10 expression was significantly ascended with the significant difference of ∗∗∗∗ (Fig. 5D). Similarly, the IL-6 expression of lung homogenate was the highest in ALI group (250.28 ± 15.77 pg/mL) with the significant difference of ∗∗∗∗. After treatments, they decreased its expression to 142.70 ± 15.77, 104.88 ± 4.62 and 83.22 ± 4.94 pg/mL in CPs, CPs@SS31 and CPs@SS31+NIR. On the contrary, the IL-10 expression of lung homogenate was the highest in CPs@SS31+NIR (65.42 ± 1.24 pg/mL) with the significant difference of ∗∗∗∗, followed by CPs@SS31 (52.98 ± 1.02 pg/mL), CPs (31.36 ± 3.00 pg/mL), ALI group (17.70 ± 2.69 pg/mL), and sham group (25.59 ± 2.54 pg/mL) (Fig. 5E).

Furthermore, the ALI therapy evaluation mainly focused on the pathological characteristics of lung tissue. From Fig. 5F, the ROS levels of lung tissue were relatively high in ALI group with a lot of green fluorescence observed. The treatments obviously decrease the ROS levels of lung tissue, with decreased green fluorescence compared to ALI group. After statistical analysis, the MFI of ROS levels was 13.67 ± 1.59 in sham group, changed to 34.13 ± 2.87 in ALI group (∗∗∗∗), 32.23 ± 1.67 in CPs (∗∗∗∗), 23.95 ± 3.61 in CPs@SS31 (∗∗), and 12.67 ± 2.01 in CPs@SS31+NIR (Fig. S25). And from the H&E staining images of lung tissue, in ALI group, the lung wall became thicken with obvious dense interstices compared to sham group. The treatments obviously relieved the above symptoms (Fig. 5G). In addition, the inflammatory factors expression levels in the lung tissue of treated rats were also investigated. As displayed in Fig. 5H and S26A, the TNF-α and IL-6 expression was in relatively high levels in ALI group compared to sham group. And their expression was significantly declined after treatments compared to ALI group. The average optical density (AOD) of TNF-α and IL-6 expression was 5.47 ± 0.35 and 5.90 ± 0.35 in the lung tissue of sham group, jumped to 9.10 ± 0.96 and 8.70 ± 0.66 in ALI group with the significant difference of ∗∗∗∗ and ∗∗∗. After treatments, it became 8.67 ± 0.38 and 5.83 ± 0.55 in CPs, 7.37 ± 0.31 and 5.03 ± 0.12 in CPs@SS31, and 6.73 ± 0.38 and 4.60 ± 0.75 in CPs@SS31+NIR (Fig. 5I and S26B). Meanwhile, the HSP70 expression of lung tissue was in the relatively low levels in sham group, ALI group, CPs, CPs@SS31 with no significant differences among them, which was significantly ascended in CPs@SS31+NIR with the significant difference of ∗∗∗∗ (Fig. 5J and K). And for CD31 expression, it was in the low levels in sham group, ALI group and CPs, which also increased in CPs@SS31 and CPs@SS31+NIR with the significant difference of ∗∗∗∗ and ∗∗∗∗ respectively (Fig. 5L and M).

From the above, it confirmed that this therapeutic strategy efficiently decreased the ROS levels, downregualted the inflammatory factor expression levels, and promoted the tissue repair factors expression levels to alleviate ALI with high biosafety. Especially in CPs@SS31+NIR, it most efficiently achieved antioxidant and anti-inflammation, and promoted tissue repair, revealed by the unique upregulation of HSP70 and CD31 expression in the lung tissue.

3.4. ALI immunoregulation therapy

Immunotherapy can enhance the function of the immune system, and improve the body's immune capacity, further helpful to anti-inflammation and promoted tissue repair [55]. Macrophages are capable of non-specific phagocytosis of foreign substances, and the strength of phagocytic ability reflects their immune and epidemic prevention functions [56]. As displayed in Fig. S27, the phagocytic ratio was 100 ± 4.62% in normal group, decreased to 19.43 ± 0.67% in control group. The treatments could improve the phagocytic ratio to 93.43 ± 18.21%, 191.22 ± 5.16%, 274.80 ± 7.05% and 313.84 ± 5.44% in CDs, CPs, CPs@SS31 and CPs@SS31+NIR respectively. After LPS stimulation, it obviously decreased the phagocytic ability of macrophage. By treatments, the phagocytic ability had been improved.

Besides, the macrophage polarization levels of treated cells were also investigated. As illustrated in Fig. 6A, the CD86 gene expression was relatively low in normal group (1.00 ± 0.06), significantly increased in control group (23.62 ± 0.79) with the significant differences of ∗∗∗∗. The treatments downregulated its gene expression, which was 21.18 ± 0.63 in CDs, 14.62 ± 0.40 in CPs, 6.69 ± 2.09 in CPs@SS31, and 2.30 ± 0.06 in CPs@SS31+NIR. Conversely, the CD206 gene expression was in the low levels in normal group (1.01 ± 0.17) and control group (1.56 ± 0.06), which gradually increased in CDs (11.50 ± 1.49), CPs (17.25 ± 1.80), CPs@SS31 (22.53 ± 1.04) and CPs@SS31+NIR (34.24 ± 8.25).

Fig. 6.

ALI immunotherapy evaluation. A) CD86 and CD206 genes expression levels of treated cells. B) CD86 expression levels of treated cells by fluorescent microscope. C) The quantified results of CD86 and CD206 expression levels of treated cells by fluorescent microscope. D) CD86 and CD206 expression levels of treated cells by flow cytometry, and the corresponding CD206/CD86 ratio (E). The corresponding groups were: RAW264.7 without treatments (normal group), and RAW264.7 pre-treated with LPS followed by incubation with PBS (control group), CDs (CDs), CPs (CPs), CPs@SS31 (CPs@SS31) and CPs@SS31 combining with NIR irradiation (CPs@SS31+NIR). F) CD86 and CD206 expression levels in the lung tissue of treated rats. G) CD4⁺ and CD8⁺ T cells number in the blood of treated rats by flow cytometry. H) CD4⁺ and CD8⁺ T cells number in the lung of treated rats by flow cytometry. I) CD4⁺ and CD8⁺ T cells number in the spleen of treated rats by flow cytometry. The corresponding groups were: rats without treatments (sham group), and rats pretreated with LPS followed by IT administration of PBS (ALI group), CPs (CPs), CPs@SS31 (CPs@SS31) and CPs@SS31 combining with NIR irradiation (CPs@SS31+NIR). (Scale bar = 50 μm) (”∗” symbol compared with normal group in cellular levels, and sham group in animal levels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001).

In the meantime, from immunofluorescent staining images, the CD86 expression was in high levels in control group with obvious green fluorescence existed, while its expression was reduced in other groups with obviously decreased green fluorescence (Fig. 6B). After statistical analysis, the MFI of CD86 expression was 11.08 ± 0.49 in normal group, significantly increased in control group (57.90 ± 8.47) with the significant differences of ∗∗∗∗. The treatments obviously decrease the MFI to 25.15 ± 2.61 in CDs (∗∗), 17.11 ± 2.26 in CPs, 11.87 ± 2.30 in CPs@SS31, and 11.00 ± 1.28 in CPs@SS31+NIR (Fig. 6C). On the contrary, for CD206 expression, almost no green fluorescence was observed in normal group, control group and CDs. The obvious ascended green fluorescence happened to CPs, CPs@SS31 and CPs@SS31+NIR (Fig. S28). By analysis, the MFI of CD206 expression was 11.80 ± 0.50, 14.73 ± 0.67 and 18.21 ± 3.24 in normal group, control group and CDs. However, it was gradually ascended in CPs (27.72 ± 3.66), CPs@SS31 (35.99 ± 1.88) and CPs@SS31+NIR (66.22 ± 8.20) with the significant difference of ∗∗, ∗∗∗∗ and ∗∗∗∗ respectively (Fig. 6C).

Furthermore, the CD86 and CD206 expression levels were also analyzed by flow cytometry. In normal group, the CD86 number was 58.0%, and CD206 number was 1.9%. And it was 71.1% and 1.7% in the number of CD86 and CD206 in control group. After treatments, the number of CD86 was declined to 15.0% in CDs, 0.3% in CPs, 12.3% in CPs@SS31, and 3.6% in CPs@SS31+NIR. Conversely, the number of CD206 gradually increased to 3.4%, 0.3%, 13.3% and 7.0% in CDs, CPs, CPs@SS31 and CPs@SS31+NIR respectively (Fig. 6D). After calculation, the CD206/CD86 ratio was 0.03 ± 0.00 in normal group, and 0.02 ± 0.00 in control group, gradually increased to 0.22 ± 0.03 in CDs, 0.92 ± 0.03 in CPs, 1.08 ± 0.03 in CPs@SS31, and 1.83 ± 0.21 in CPs@SS31+NIR with the significant difference of ∗∗∗∗, ∗∗∗∗ and ∗∗∗∗ (Fig. 6E).

To further confirm macrophage M2 polarization, the M2 type genes expression levels of treated cells were also analyzed by RT-qPCR. As illustrated in Fig. S29, the IL-4 and IL-13 genes expression levels were relatively high in normal group (1.00 ± 0.08 and 1.00 ± 0.01). Their levels were significantly declined in control group (0.20 ± 0.03 and 0.16 ± 0.03). After treatments, their expression had been promoted, which was 0.39 ± 0.03 and 0.30 ± 0.00 in CDs, 0.43 ± 0.07 and 0.76 ± 0.03 in CPs, 0.61 ± 0.03 and 0.84 ± 0.04 in CPs@SS31, and 0.84 ± 0.09 and 0.95 ± 0.03 in CPs@SS31+NIR. In animal levels, the CD86 and CD206 expression in the lung tissue of treated rats was also analyzed. As shown in Fig. 6F, the CD86 expression (red fluorescence) was obviously ascended in ALI group compared to sham group. The treatments decreased the red fluorescence compared to ALI group. On the contrary, the CD206 expression was relatively low in sham group and ALI group, significantly increased after treatments. By statistical calculation, the MFI of CD86 and CD206 expression was in relatively low in sham group (65.24 ± 0.56 and 67.56 ± 0.57). And in ALI group, the CD86 expression was the highest (233.65 ± 0.53), and the CD206 expression was still in the low levels (65.24 ± 0.56). After treatments, the MFI of CD86 expression was obviously reduced in CDs (109.71 ± 0.39), CPs (82.80 ± 0.67) and CPs@SS31 (61.90 ± 1.49). However, compared to ALI group, the CD206 expression was ascended in CDs (86.45 ± 1.93), CPs (99.64 ± 5.88) and CPs@SS31 (119.16 ± 12.22) (Fig. S30A and S30B). After calculation, the CD206/CD86 ratio was 1.04 ± 0.00% in sham group, and 0.28 ± 0.00% in ALI group, which was significantly ascended in other groups. Especially, the CD206/CD86 ratio jumped to 1.92 ± 0.15% in CPs@SS31+NIR (Fig. S30C).

T cells number activation is beneficial to enhancing cellular immune functions, thereby achieving anti-inflammation and tissue repair [57]. The T cells gene expression levels of treated cells were analyzed by RT-qPCR. As illustrated in Fig. S31, the IL-2 and IFN-γ gene expression was relatively low in normal group (1.00 ± 0.08 and 1.14 ± 0.63), significantly ascended in control group (8.38 ± 1.33 and 11.71 ± 0.59) with the significant difference of ∗∗∗∗. The treatments could decrease their expression, where CPs@SS31+NIR played the most significant roles. To analyze the T cells number in the blood, lung tissue and spleen tissue of treated rats, flow cytometry was applied. As displayed in Fig. 6G, the number of CD8⁺ T cells was 26.1%, and the number of CD4⁺ T cells was 51.4% in the blood of sham group, while it increased to 26.7% in the number of CD8⁺ T cells, and maintained almost the same levels (50.6%) in the number of CD4⁺ T cells in ALI group. After treatments, compared to ALI group, the number of CD8⁺ T cells decreased, and the number of CD4⁺ T cells was ascended. After calculation, the CD4⁺/CD8⁺ ratio was 1.93 ± 0.03% in sham group, and 1.92 ± 0.05% in ALI group, gradually increased to 1.99 ± 0.04% in CPs, 2.22 ± 0.17% in CPs@SS31, and 2.41 ± 0.25% in CPs@SS31+NIR (∗∗) (Fig. S32A). The similar trend was observed in the CD4⁺ and CD8⁺ T cells number of lung tissue and spleen tissue (Fig. 6H and I). And the CD4⁺/CD8⁺ ratio was 2.29 ± 0.11% and 1.94 ± 0.08% in the lung tissue and spleen tissue of sham group, slightly decreased in those of ALI group (1.30 ± 0.04% and 1.56 ± 0.03%) with the significant difference of ∗∗ and ∗∗. The treatments could recover the CD4⁺/CD8⁺ ratio to 2.53 ± 0.17% and 1.95 ± 0.08%, 2.92 ± 0.10% and 2.09 ± 0.07%, and 9.30 ± 0.58% and 2.62 ± 0.13% in the lung tissue and spleen tissue of CPs, CPs@SS31 and CPs@SS31+NIR. However, only in CPs@SS31+NIR, the CD4⁺/CD8⁺ ratio was the highest with the significant difference of ∗∗∗∗ (Fig. S32B and S32C).

From the above, after LPS stimulation, it induced the macrophage M1 polarization with the decreased CD206/CD86 ratio. After treatments, it induced macrophage M2 polarization, achieving anti-inflammation. Among them, CPs@SS31+NIR most efficiently induced macrophage M2 polarization, with the highest M2/M1 ratio in vitro and in vivo. And the treatments could activate T cells number, with the increased CD4⁺ T cells number and CD4⁺/CD8⁺ ratio. CPs@SS31+NIR most effectively increased CD4⁺ T cells number, and decreased CD8⁺ T cells number, significantly promoting the CD4⁺/CD8⁺ ratio. All of these was helpful to achieving anti-inflammation, and promoting tissue repair, therapy reaching ALI immunotherapy.

3.5. Mitochondrial function regulation and its mechanism

To confirm the mitochondrial targeting ability, the immunofluorescent staining was applied. As shown in Fig. 7A, the obvious overlap of green fluorescence and red fluorescence happened to Cy5-CPs@SS31 compared to Cy5-CPs, indicating that SS31 helped CPs@SS31 to specifically target mitochondria. After statistical analysis, the Pearson correlation coefficient was 0.70 ± 0.03 in CPs, increased to 0.81 ± 0.02 in CPs@SS31 with the significant difference of ∗∗∗, indicating the specific mitochondrial targeting of CPs@SS31 (Fig. S33). And the MMP of treated cells was detected by the combination of immunofluorescent staining and flow cytometry. As revealed in Fig. S34A, red fluorescence was equaled to the amount of JC-1 aggregate, while green fluorescence corresponded to JC-1 monomer amount. The red fluorescence was in the high levels, and green fluorescence was relatively low in normal group, while the red fluorescence significantly decreased, and green fluorescence was ascended in control group. The treatments could reduce the green fluorescence, and increase the red fluorescence compared to control group. After statistical analysis, the aggregates/monomers ratio was 2.56 ± 0.81% in normal group, decreased to 0.18 ± 0.07% in control group with the significant difference of ∗∗∗. After treatments, it became 0.73 ± 0.38% in CDs (∗∗), 0.95 ± 0.30% in CPs (∗∗), 1.70 ± 0.55% in CPs@SS31, and 3.07 ± 0.48% in CPs@SS31+NIR (Fig. S34B). Besides, the MMP of treated cells was also analyzed by flow cytometry. After calculation, the aggregate/monomer ratio was 3.25 ± 0.06% in normal group, which changed to 0.42 ± 0.03% in control group, 0.46 ± 0.02% in CDs, 1.49 ± 0.03% in CPs, 2.44 ± 0.11% in CPs@SS31, and 4.06 ± 0.23% in CPs@SS31+NIR (Fig. 7B and S35). In addition, the mitochondrial ROS levels of treated cells were also evaluated. In control group, the mitochondrial ROS levels were the highest with obvious red fluorescence observed. After treatments, it efficiently decreased the red fluorescence (Fig. S36A). By statistical analysis, the MFI of mitochondrial ROS levels was 19.19 ± 0.32 in control group, decreased to 11.18 ± 0.29 in CDs, 8.88 ± 0.77 in CPs, 2.45 ± 0.25 in CPs@SS31, and 0.36 ± 0.02 in CPs@SS31+NIR (Fig. S36B). Finally, the ATP production content was also tested, which was 3.74 ± 0.10 nM in normal group, significantly declined in control group (0.89 ± 0.06 nM). After treatments, it became 2.01 ± 0.08 nM in CDs, 2.55 ± 0.11 nM in CPs, 3.13 ± 0.23 nM in CPs@SS31, and 3.64 ± 0.05 nM in CPs@SS31+NIR (Fig. S37). It confirmed that the treatments could efficiently reduce MMP, decrease mitochondrial ROS levels, and improve ATP production. Especially for CPs@SS31+NIR, the improved recovery of mitochondrial functions was attributed by the synergistic effects of mitochondria targeting and NIR enhanced ROS scavenging.

Fig. 7.

ALI therapeutic mechanism. A) Co-immunofluorescent staining images of cells incubated with Cy5-CPs or Cy5-CPs@SS31 by confocal staining microscope: DAPI (blue), Mito-tracker green (green), and Cy5-CPs or Cy5-CPs@SS31 (red). (Scale bar = 10 μm) B) MMP of treated cells by flow cytometry. C) Heatmap of DEGs between control group and CPs@SS31+NIR: genes with relatively high (red) and low (blue) expression levels. D) Volcano plot of DEGs: 542 upregulated and 499 downregulated genes between control group and CPs@SS31+NIR. E) Anti-inflammation pathways related DEGs by KEGG enrichment analysis (red). F) Anti-inflammation pathways related differential biological functions by GO enrichment analysis (red): molecular function (MF), biological process (BP) and cell component (CC). G) Protein-protein interaction network of mitophagy related proteins. H) P62, Parkin and PINK1 expression levels of treated cells by WB. I) P62, Parkin and PINK1 expression levels in the lung tissue of treated rats. (Scale bar = 100 μm).

Besides, to explore the anti-inflammation mechanism, transcriptome sequencing was applied between control group and CPs@SS31+NIR. As illustrated in Fig. 7C and D, there were 15,090 DEGs observed, where 542 were upregulated, and 499 were downregulated. From KEGG enrichment analysis, PI3K-Akt signaling pathway was the most significant one, together with some others like IL-17 signal pathways, NF-κB signaling pathway, mitophagy-animal and TNF signaling pathway (Fig. 7E). Similarly, from GO enrichment analysis, it was observed that regulation of inflammatory response and mitochondrial outer membrane could be the main activities of ALI therapy during IT administration (Fig. 7F). As displayed in Fig. 7G, the core hub protein SQSTM1 was the central node of the network, directly interacting with RNF26, WDFY3, PINK1 and PRKN, and also serving as a key mediator connecting different functional modules. And PINK1 directly interacted with PRKN and SQSTM1, corresponding to their synergistic effects in ubiquitination dependent mitophagy [58]. In addition, it also presented an indirect correlation between the mitochondrial protein homeostasis and mitophagy, suggesting a synergistic pathway for mitochondrial functions regulation. From the above, it demonstrated that inflammation inhibition and mitophagy induction was helpful to achieving the enhanced ALI therapy by this administration.

Subsequently, the mitophagy pathway related genes of treated cells were quantified analyzed by RT-qPCR. For P62 gene expression, it was relatively low in normal group (1.00 ± 1.05), prominently ascended to 6.62 ± 0.63 in control group with the significant difference of ∗∗∗∗. The treatments could obviously decrease its gene expression, which was 4.11 ± 0.44 in CDs (∗∗∗∗), 2.71 ± 0.11 in CPs (∗∗∗), 0.42 ± 0.01 in CPs@SS31, and 0.18 ± 0.12 (∗) in CPs@SS31+NIR. Conversely, the Parkin genes expression levels were relatively high in normal group (1.00 ± 0.11), obviously reduced in control group (0.02 ± 0.01). After treatments, its gene expression was recovered, especially in CPs@SS31+NIR (2.49 ± 0.15) (Fig. S38). In specific, for NIR alone (0.02 ± 0.00), it could not affect the Parkin gene expression, with the similar levels to that of control group (0.01 ± 0.00) (Fig. S39). In addition, from immunofluorescent staining images, the PINK1 expression was in the relatively low levels in normal group and control group. The treatments promoted its expression levels (Fig. S40A). After statistical analysis, the MFI was 1.13 ± 0.27 in normal group, and 1.57 ± 0.29 in control group, changed to 3.80 ± 0.31, 8.47 ± 4.37, 10.48 ± 1.05 and 30.32 ± 9.48 in control group, CDs, CPs, CPs@SS31 and CPs@SS31+NIR (∗∗∗∗) (Fig. S40B).

To further confirm the mitophagy mediated anti-inflammation mechanism, WB was applied. As shown in Fig. S41 and 7H, the P62, PINK1 and Parkin expression was in the relatively low levels in normal group. After LPS stimulation, it obviously increased P62 expression, and decreased PINK1 and Parkin expression. The treatments could decrease P62 expression, and improve PINK1 and Parkin expression, where CPs@SS31+NIR was the most effective one. After calculation, in control group, it possessed the highest relative P62/GAPDH ratio of 678.18 ± 75.94%, and the lowest relative Parkin/GAPDH (22.84 ± 1.62%) and PINK1/GAPDH ratio (7.10 ± 0.50%), compared to those of normal group (569.81 ± 37.34, 32.36 ± 1.03 and 15.51 ± 1.28%). Significantly, CPs@SS31+NIR most efficiently improved the relative Parkin/GAPDH ratio (78.68 ± 3.15%) and PINK1/GAPDH ratio (68.09 ± 0.44%), and decreased the relative P62/GAPDH ratio (240.11 ± 31.66%) (Fig. S42). Similarly, as illustrated in Fig. S43 and S44, for P62 expression, the relative P62/GAPDH ratio was 68.94 ± 4.66% in control group. And Mdivi-1 significantly promoted the relative P62/GAPDH ratio (89.36 ± 3.40%). However, it was declined in Mdivi-1+CPs@SS31 (67.67 ± 0.66%) with no significant difference, and Mdivi-1+CPs@SS31+NIR (29.38 ± 1.96%) with the significant difference of ∗∗∗∗. For CP@SS31+NIR, the relative P62/GAPDH ratio jumped to 18.80 ± 1.52% with the significant difference of ∗∗∗∗ (Fig. S44A). For Parkin and PINK1 expression, Mdivi-1 could decrease the relative Parkin/GAPDH and PINK1/GAPDH ratio (55.42 ± 0.76% and 42.52 ± 2.16%), especially in the relative Parkin/GAPDH ratio with the significant difference of ∗∗∗∗. However, both of the relative Parkin/GAPDH and PINK1/GAPDH ratio were ascended in other groups, which was 85.57 ± 3.45% and 55.39 ± 0.30% in Mdivi-1+CP@SS31, and 94.62 ± 2.99% and 65.46 ± 6.97% in Mdivi-1+CP@SS31+NIR (∗∗ and ∗∗) (Fig. S44B and S44C).

In animal levels, the P62 expression levels in the lung tissue of treated rats were also analyzed. As shown in Fig. 7I, the P62 expression was in the high levels in ALI group. After treatments, its expression was reduced, especially in CPs@SS31+NIR. By statistical analysis, the AOD of P62 was 14.30 ± 0.10 in sham group, significantly jumped in ALI group (21.10 ± 0.17). The treatments reduced the AOD of P62 to 16.63 ± 0.55, 15.47 ± 0.38 and 14.30 ± 0.10 in CPs (∗∗∗∗), CPs@SS31 (∗∗∗∗) and CPs@SS31+NIR respectively (Fig. S45). Conversely, the Parkin and PINK1 expression in the lung tissue of treated rats was relatively low in sham group and ALI group, obviously promoted after treatments (Fig. 7I). By statistical analysis, the AOD of Parkin and PINK1 was 9.63 ± 0.15 and 7.47 ± 0.15 in sham group, and 10.40 ± 0.10 and 6.87 ± 0.15 in ALI group. The treatments increased the AOD of Parkin and PINK1 to 11.20 ± 0.10 and 8.10 ± 0.10, 11.87 ± 0.15 and 8.40 ± 0.10, and 12.83 ± 0.38 and 8.90 ± 0.10 in CPs, CPs@SS31 and CPs@SS31+NIR (Fig. S45B and S45C).

From the above, it confirmed that CPs@SS31 could specifically target mitochondria. By combining with NIR irradiation, it most efficiently increased the MMP and ATP production, and decreased the mitochondrial ROS levels. By transcriptome sequencing, it also confirmed the mitophagy pathway activation mediated anti-inflammation mechanism. And further experiments also gave the proof that CPs@SS31+NIR achieved the enhanced ALI therapy via inducing mitophagy with the upregulation of PINK1 and Parkin expression levels, and downregulation of P62 expression levels both in vitro and in vivo.

4. Conclusion

In summary, this research proposed an innovative therapeutic strategy by using CPs@SS31+NIR to achieve specifically targeting mitochondria, and regulating lung redox homeostasis for ALI immunotherapy. By ultasmall Pd loading and NIR irradiation, the designed CPs@SS31+NIR possessed the excellent ability of ROS scavenging. In addition, it also efficiently induced macrophage M2 polarization, and activated T cells immunoregulation both in vitro and in vivo. Furthermore, SS31 contributed CPs@SS31 to be enriched in the mitochondria, leading to the increased MMP and ATP content, and decreased mitochondrial ROS levels, and mitophagy activation to modulate mitochondrial homeostasis. Finally, in ALI animal models, CPs@SS31+NIR ultimately achieved alleviating lung inflammation, and promoting tissue repair. This therapeutic strategy offered a solid evidence of inducing mitophagy and activating immunoregulation for ALI therapy with high efficacy and safety, also ensured a promising strategy for precision targeted treatment of inflammation related diseases.

CRediT authorship contribution statement

Jing Zhang: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis, Data curation. Kunpeng Duan: Visualization, Validation, Investigation, Data curation. Qianyue Liu: Visualization, Investigation, Formal analysis, Data curation. Shurong Chen: Writing – original draft, Visualization, Validation, Formal analysis, Data curation. Hongshuai Zheng: Formal analysis. Yan Liu: Formal analysis. Jing Qian: Formal analysis. Mingjing Yin: Formal analysis. Jing Liu: Formal analysis. Jiaxiao Li: Formal analysis. Zhijian Li: Funding acquisition. Min Chen: Formal analysis. Ximei Huang: Formal analysis. Faquan Lin: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Conceptualization. Ming Gao: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Conceptualization. Lin Liao: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Conceptualization.

Data availability statement

The data that support this study are available on request from the corresponding author.

Ethics approval and consent to participate

In vivo biodistribution study: Male sprague dawley (SD) rats (180∼220 g, 6-8 weeks) were purchased from the experimental animal center of Guangxi Medical University. All animal experiments were approved by the animal ethics committee of Guangxi Medical University (No. 202411035).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: