The DiR-BOA (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide bisoleate) was generously provided by Professor Cao of Shanghai University. R4F (an ApoA1-mimetic peptide, Ac-FAEKFKEAVKDYFAKFWD) was synthesized by Shanghai Apeptide Co., Ltd (Shanghai, China). Celastrol was obtained from MedChemExpress (New Jersey, USA). The ldlA7 (SR-B1−), ldlA (mSR-B1) (SR-B1+) cell lines were gifts from Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). ELISA kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) were supplied by MultiSciences Biotech Co., Ltd (Lianke, China). Murine macrophage colony-stimulating factor (M-CSF) was purchased from PeproTech. Protease Inhibitor Cocktail was purchased from Sigma–Aldrich. Fetal bovine serum (FBS), penicillin/streptomycin and Dulbecco’s modified Eagle’s medium (DMEM) were bought from Gibco (USA). Dimethyl sulfoxide (DMSO) was bought from Sigma (USA). OCT compound was purchased from Sakura Finetek (Torrance, CA, USA). Antibodies β-actin was bought from Boster Company. HRP labeled antibody, Giemsa stainand, EGTA, Nuclear and Cytoplasmic Protein Extraction Kit, Anti-fluorescence quenching sealant and 4′,6-diamidino-2-phenylindole (DAPI) were bought from Beyotime Biotechnology Co., Ltd. LFA-1 was purchased Invitrogen. CXCR2, iNOS, Arg-1, JNK, p-JNK, ERK and p-ERK were purchased from Proteintech (USA). Lamin B, p-p65, IκBα and p-IκBα were purchased from Wanleibio Co., Ltd. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) and anti-NF-kB p65 antibody were were purchased from Abcam. Antibodies to CD16/32, CD11b, F4/80, CD206, CD86 were purchased from Biolegend (USA). RNA isolater Total RNA Extraction Reagent, HiScript® III RT SuperMix for qPCR (+ gDNA wiper), and Taq Pro Universal SYBR qPCR Master Mix were purchased from Vazyme Biotech Co., Ltd. Polycarbonate nuclepore track-etch membranes were purchased from Whatman. Mouse peripheral blood neutrophil isolation Kit, lowry protein concentration determination kit, and Lipopolysaccharide (LPS) were purchased from Beijing Solarbio Science & Technology Co., Ltd. Immunization Grade Bovine type II collagen (CII), Complete Freund’s adjuvant (CFA), and Incomplete Freund’s adjuvant (IFA) were all obtained from Chondrex, Inc. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were purchased from Nanjing Institute of Biological Engineering of China.
Animals and cells
Specific pathogen-free (SPF) kunming (KM) mice (Female, 6–8 weeks old) were purchased from the Laboratory Animal Center of China Three Gorges University (Yichang, Hubei, China). DBA/1 mice (male, 8 weeks old) were obtained from Beijing Huafukang Biotechnology Co., Ltd (Beijing, China). All animal studies were conducted in compliance with protocols that had been approved by the Ethics Committee of China Three Gorges University and in compliance with the experimental guidelines of the National Institutes of Health on the care and use of animals. The RAW264.7 cell line was obtained from Wuhan Procell Life Technology Co., Ltd. The cells were cultured in DMEM containing 10% FBS and 100 U/mL penicillin‒streptomycin at 37 °C in a humidified 5% CO2 atmosphere.
Isolation and identification of peripheral neutrophils
Lipopolysaccharide (LPS, 1.5 mg/kg) was injected intraperitoneally into the mice to activate neutrophils in vivo. After 6 h, peripheral blood from KM mice was collected in tubes containing the anticoagulant EDTA-2K, and neutrophils were isolated with the Mouse Peripheral Blood Neutrophil Isolation Kit. Then, the isolated neutrophils were resuspended in PBS and stored at − 80 °C for subsequent membrane isolation. The cell suspension was stained with Giemsa to further identify neutrophils, and their morphology was examined under an Olympus BX53 (Japan).
Isolation of NMs
To obtain NMs by the homogenization method, frozen neutrophil suspensions were thawed and washed with PBS three times (centrifugation at 800 g). Neutrophils were then suspended in hypotonic lysing buffer containing 225 mM d-mannitol, 30 mM Tris-HCl (pH 7.5), 75 mM sucrose, 0.2 mM EGTA, and a protease inhibitor cocktail. Neutrophils were then disrupted using a Dounce homogenizer with a tight-fitting pestle (40 passes). The homogenized solution was centrifuged at 20,000 g for 25 min at 4 °C. The pellet was discarded, and the supernatant was centrifuged again at 120,000 g and 4 ºC for 60 min. To obtain NMs by hypotonic treatment, neutrophils were first treated with hypotonic lysis buffer on ice for 30 min. The supernatants were pooled and centrifuged at 20,000 g and 4 °C for 20 min. Afterward, the pellet was discarded, and the supernatant was centrifuged again at 120,000 g for 60 min at 4 °C. To obtain NMs by the repeated freeze‒thaw method, 1 mL of precooled double-distilled water was added to resuspend the neutrophils. The mixture was then sonicated at a frequency of 42 kHz for 10 min and subjected to 3 freeze‒thaw cycles. It was first frozen at − 80 °C for 30 min and then thawed at room temperature for another 30 min. After centrifugation at 10,000 g for 10 min at 4 °C, the supernatant was collected and then centrifuged at 120,000 g for another 45 min. Following the centrifugation, NMs isolated by three different methods were collected as the pellet and stored at − 80 ºC for further use. NM protein content was quantified using a Lowry Protein Concentration Determination Kit with a bovine serum albumin standard.
Synthesis and characterization of NM@F127 and R4F-NM@F127
Pluronic F127 polymer loaded with hydrophobic drugs was prepared by thin-film hydration, further mixed with NMs and extruded 20 times through 400 nm and 100 nm polycarbonate membranes with a liposome extruder to prepare NM@F127 nanoparticles. Subsequently, NM@F127 was functionalized with R4F peptide that was capable of binding phospholipids on NMs to prepare R4F-NM@F127. The mean particle size distribution, zeta potential, and PDI of NM@F127 and R4F-NM@F127 were determined with a Malvern Particle Size Analyzer (Malvern Instruments Ltd., Nano-ZS90, Malvern, Worcestershire, UK). The morphology of R4F-NM@F127 was examined using transmission electron microscopy (TEM, JEOL F200, Japan). The absorption wavelength of DiR-BOA-loaded R4F-NM@F127 was measured with a UV‒visible spectrophotometer (AOE Instruments, China).
High-performance liquid chromatography (HPLC)
To calculate the encapsulation efficiency of NM@F127-Cel and R4F-NM@F127-Cel, HPLC was used to investigate the linear relationship between the Cel concentration and the peak area. The chromatographic conditions for celastrol measurement were as follows: mobile phase: methanol/water = 87/13 (V/V), sample size: 40 µL, flow rate: 1.0 mL/min, separation column temperature: 35 ℃, and detection wavelength: 425 nm.
Isolation of BMDMs
The mice were sacrificed and rinsed in 75% alcohol for 5 min. The tibia and femur of the mice were removed in a sterile environment, and the epiphysis at both ends of the bones were cut to facilitate needle insertion. The bone marrow cavity was repeatedly flushed with serum-free DMEM until the bone cavity became white. The bone marrow cells were centrifuged at 1350 rpm for 10 min at room temperature. After discarding the supernatant, ACK lysis buffer was added and incubated for 3 min. Then, DMEM was added to stop the reaction, and the cells were centrifuged and resuspended in the appropriate medium. Filter the suspension using a sterile 70 μm cell strainer. After centrifugation, the cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin‒streptomycin, 50 µM β-mercaptoethanol and 20 ng/mL murine macrophage colony-stimulating factor.
In vitro targeting test
To assess the SR-B1 receptor-targeting ability of R4F-NM@F127, cells were seeded into 96-well plates at a density of 1.5 × 104 cells/mL and cultured overnight at 37 °C in a humidified 5% CO2 atmosphere. After removing the medium, DiR-BOA-labeled NM@F127 and R4F-NM@F127 were added at final concentrations of 2.5 µM, 5 µM, 10 µM and 20 µM and incubated for 3 h. After incubation, the cells were digested with trypsin and washed three times with PBS, and the fluorescence signal intensity of the cells was measured by FCM. For immunofluorescence analysis, cells (3 × 104) were seeded into 8-well chambers to cover the glass bottoms. Then, the cells were incubated with NM@F127 and R4F-NM@F127 at a DiR-BOA concentration of 20 µM for 3 h. The cells were rinsed gently with sterile PBS three times and fixed with 4% paraformaldehyde for 15 min on ice. Cell culture medium containing 5 µg/mL phalloidin was added, and the cells were stained at room temperature for 30 min. Then, the cells were washed three times with PBS. Subsequently, the nuclei were stained with 0.5 µg/mL DAPI for 10 min and rinsed gently with PBS 3 times. Fluorescence images were acquired using a confocal laser scanning microscope with an excitation wavelength of 405 nm for DAPI, 488 nm for FITC and 633 nm for DiR-BOA.
RAW264.7 cells were seeded into 6-well plates at a density of 1 × 106 cells/mL and divided into the control, LPS, free Cel, NM@F127-Cel and R4F-NM@F127-Cel groups. After overnight incubation, the cells were incubated with NM@F127-Cel and R4F-NM@F127-Cel for 30 min, followed by stimulation with LPS (100 ng/mL); 2 mL of medium was added at a concentration of 0.25 µM or 0.5 µM, and the normal group and the LPS group were given the same amount of culture medium for 6 h to analyze the mRNA expression of the M1 macrophage polarization markers iNOS, TNF-α, IL-6 and the M2 macrophage polarization markers IL-10 and Arg-1. Similarly, the cells were also treated with Cel, NM@F127-Cel or R4F-NM@F127-Cel for 12 h to analyze M1/M2 macrophage polarization markers and proteins related to the NF-κB and MAPK signaling pathways. The nuclear and cytoplasmic proteins of RAW264.7 cells were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit. The expression levels of p65 in the nuclear and cytoplasmic of RAW264.7 cells were determined by western blotting.
The cell supernatants of the above groups were collected, and then the levels of cytokines (TNF-α, IL-6, and IL-10) in the cell supernatants of each group were detected with an ELISA kit according to the manufacturer’s instructions. In addition, blood samples were collected from mice on the day of sacrifice. After incubation at room temperature for 2 h, whole blood was centrifuged at 3000 rpm for 10 min. Serum levels of the inflammatory cytokines TNF-α, IL-6 and IL-10 were determined using ELISA kits.
Western blot analysis
Proteins extracted from cell lysates were separated by 10% SDS‒PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder for 1.5 h, the proteins were hybridized with the primary antibodies against β-actin (1:1000), iNOS (1:1000), Arg-1 (1:5000), Lamin B (1:500), p65 (1:5000), p-p65 (1:500), IκBα (1:500), p-IκBα (1:500), JNK (1:6000), p-JNK (1:2000), ERK (1:1000) and p-ERK (1:5000) overnight at 4 °C. The membrane was washed three times with TBST buffer and incubated with a goat anti-rabbit IgG H&L (HRP) secondary antibody (1:5000) for 1 h at room temperature. After washing with TBST, proteins detection was performed using ECL reagent and a ChemiScope 6100 chemiluminescence imaging system (Clinx, Shanghai, China). Quantitative analysis of bands was performed at least three times. In western blotting experiments, Lamin B was used as a control for nuclear proteins, and β-actin was used as a control for other proteins.
Total RNA was extracted from cells using TRIzol reagent and reverse transcribed into cDNA using a reverse transcription kit. Real-time quantitative polymerase chain reaction (RT‒qPCR) was performed on the StepOnePlus RT‒qPCR System using SYBR green. The primer sequences utilized for amplification are shown in Additional file 1: Table S1. Relative expression was calculated by using the 2 −△△Ct method with normalization to β-actin values. Reactions were repeated a minimum of three times in triplicate. Additional file 1: Table S1. shows the primer sequences used for amplification.
Flow cytometry analysis
To assess M1/M2 polarization, RAW264.7 cells were seeded in 96-well plates at 1.5 × 104 cells per well and incubated overnight in complete culture medium. LPS (100 ng/mL) was added for 30 min of incubation, except for the normal group. Then, the cells were treated with fresh complete culture medium containing Cel or NM@F127-Cel or R4F-NM@F127-Cel at a dose of 0.25 µM or 0.5 µM for 24 h, respectively. After incubation, RAW264.7 cells were incubated with CD16/32 for 10 min to block Fc receptors, followed by staining with an APC anti-mouse CD86 antibody and a PerCP/Cyanine5.5 anti-mouse CD206 antibody for 30 min and detection using FCM. For macrophages in the synovial fluid of the joint, first, a syringe was used to inject sterile PBS into the joint cavity, and then the liquid inside was extracted from the mice. The cells were filtered through a 70 μm cell strainer and then washed once with PBS to prepare single-cell suspensions. The single-cell suspensions were incubated with CD16/32 for 10 min to block Fc receptors, followed by staining with an APC/Cyanine7 anti-mouse F4/80 antibody, and a PE/Cyanine7 anti-mouse/human CD11b antibody, APC anti-mouse CD86 antibody, PerCP/Cyanine5.5 anti-mouse CD206 antibody for 30 min and detection using a flow cytometer (Dakewe Biotech Co., Ltd., China). The data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
For immunofluorescence staining, RAW264.7 cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.3% Triton X-100 for 10 min. Then, the cells were washed three times with PBS. Subsequently, the cells were blocked for 30 min at room temperature with 1% bovine serum albumin containing 0.1% Triton X-100. After that, the cells were incubated overnight with an anti-NF-kB p65 antibody (1:100). Following incubation, the cells were rinsed three times with PBS and incubated with a goat anti-rabbit IgG H&L (Alexa Fluor® 594) (1:400) antibody in the dark for 1 h at room temperature. The nuclei were stained with DAPI. Fluorescence images were acquired using a confocal laser scanning microscope (A1R, Nikon, Japan). The data were analyzed using ImageJ software. For immunofluorescence staining of the synovium of the knee joint, freshly harvested knee joints were fixed with 4% paraformaldehyde for 24 h and then decalcified with PBS containing 15% EDTA at 4 ℃ for three days, and the decalcification solution was changed daily. Once decalcification was complete, the samples were washed overnight at 4 ℃. Then, the tissues were cryoprotected in 30% sucrose in PBS at 4 ℃ until they sank and embedded in OCT compound before freezing on dry ice. Tissue Sect. (10 μm thick) were cut on a cryostat (Dakewe Biotech Co., Ltd., China) and mounted on poly-L-lysine-coated slides. After blocking with 1% bovine serum albumin for 1 h, an iNOS polyclonal antibody (1:100) or Arginase-1 polyclonal antibody (1:100) was added and incubated overnight at 4 ℃. Following incubation, the tissue sections were rinsed three times with PBS and incubated with a goat anti-rabbit IgG H&L (Alexa Fluor® 594) (1:400) antibody in the dark for 1 h at room temperature. The nuclei were stained with DAPI. All sections were imaged using a confocal laser scanning microscope with a dry 20×/0.8 NA objective. The data were analyzed using ImageJ software.
Establishing a mouse model of CIA
The collagen-induced arthritis (CIA) animal model was established in DBA/1 mice per the manufacturer’s instructions (Chondrex, USA). Briefly, bovine type II collagen (2 mg/mL) was thoroughly emulsified with an equal volume of complete Freund’s adjuvant (2 mg/mL) by using a T-branch pipe, and 100 µL of the emulsion was administered to mice intradermally at the base of the tail. On day 21 after primary injection, the mice received an intradermal booster injection of type II collagen with an equal volume of incomplete Freund’s adjuvant.
In vivo fluorescence targeting test
A total of 12 CIA mice were randomly divided into 4 groups (3 animals per group), which were intravenously administered DiR-BOA-labeled F127, NM@F127, R4F-NM@F127, or free DiR-BOA (20 nmol). After 1, 3, 6, 12 and 24 h, the mice were anesthetized and imaged with a small animal in vivo imaging system (IVIS Lumina XRMS). After 24 h, the mice were euthanized, and the blood, heart, liver, spleen, lung, kidney, brain and joint were removed. Blood was sampled and centrifuged at 3000 g for 10 min to obtain plasma. Synovial fluid was collected in a tube. The fluorescence of plasma, synovial fluid, and organs was measured using a small animal in vivo imaging system (IVIS Lumina XRMS). To further evaluate the colocalization of nanoparticles with synovial fluid macrophages, the mean fluorescence intensity of DiR-BOA in synovial fluid macrophages was detected by FCM. Confocal microscopy verified that DiR-BOA-labeled R4F-NM@F127 colocalized with synovial macrophages.
Therapeutic effects in CIA mice
Twenty-eight days after the first immunization, mice with CIA were randomly divided into four groups (n = 4): the CIA model group, free Cel-treated group, NM@F127-Cel-treated group and R4F-NM@F127-Cel-treated group. The three treatment groups were then administered via the tail vein at a dose of 5 mg/kg body weight of Cel on days 28 and 38. The model group was injected with an equal volume of PBS by the same method and on the same days. Normal DBA/1 mice were maintained as the control group. Mice were weighed, and the paw thickness of both hind ankle joints was measured with a Vernier caliper and examined for clinical scores every 3 days from day 21. The position of the caliper was the same during each measurement. Paw inflammation was scored visually as follows: 0 = normal paw; 1 = one toe inflamed and swollen; 2 = more than one toe but not the entire paw inflamed and swollen, or mild swelling of entire paw; 3 = erythema and moderate swelling extending to the entire paw; and 4 = severe erythema and swelling of the whole paw and ankle. Each paw was graded with a score from 0 to 4, generating an arthritis score on a scale of 0–16 for each individual mouse. Next, to evaluate joint swelling, the hind paws of the mice were photographed on day 18 (3 days before secondary immunization), day 28 (on treatment day), day 35 (7 days after treatment), day 42 (14 days after treatment) and day 45 with a camera.
Micro-computed tomography (Micro-CT) analyses
Scanning of mouse paws was performed on a Nemo Micro-CT (NMC-200) device from PINGSENG Healthcare Inc. The device uses cone beam CT (cone beam CT) technology, which is an imaging technology that can achieve high resolution. At the beginning of the experiment, the mouse paw was placed vertically into the sample chamber, the scanning tube voltage was set to 60 kV, and the tube current was set to 120 µA. During the scanning process, the detector and the bulb rotated 360° around the central axis of the sample chamber, and in the scanning area, 4000 projections were carried out within the 1200 s scan time. After the image was captured by the detector, it was transferred to a computer, and the image was reverse reconstructed using the FDK method in Avatar software with a pixel size of 8 μm × 8 μm × 9 μm.
Histopathologic analysis of the joints
After the CIA mice were sacrificed on day 45, the hind legs were removed. The knee and ankle joints were fixed and decalcified. Then, the decalcified limbs were dehydrated step by step and embedded in paraffin. Paraffin sections of murine paws were stained with hematoxylin and eosin (H&E) and safranin O (S&O) to evaluate of inflammation and joint destruction. All images were captured and analyzed with an Olympus BX53 microscope (Japan).
The serum toxicological indexes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed using a commercial kit according to the manufacturer’s protocol. For histopathological observation, samples from the heart, liver, spleen, lung, kidney and brain were harvested. The tissues were fixed in 4% paraformaldehyde for paraffin sectioning followed by H&E staining, and all images were captured and analyzed with an Olympus BX53 microscope (Japan).
Statistical analysis was performed using GraphPad Prism (GraphPad Software 8.0.2). Data are presented as the mean ± SEM. If the data conformed to a normal distribution and homogeneity of variance, the experimental results between the two groups were compared by independent sample t test, and the experimental results between multiple groups were compared by one-way analysis of variance (one-way ANOVA). Significant differences between or among the groups are indicated as follows: ns represents no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001; #p < 0.05, ##p < 0.01 and ###p < 0.001.