Materials

Roburic acid (RBA, CAS: 6812-81-3; catalog B20723) was obtained from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Sodium hyaluronic acid (HA, molecular weight: 11.5 Kd) was purchased from Freda Biopharm Co. Ltd (Shandong, China). Folic acid (FA) was purchased from Aldrich Chemical Co. (St. Louis, MO, USA). NH2-PEG-FA and Fmoc-PEG-OH was purchased from Ruixi Biotech Co. Ltd.

Animals and cells

Healthy male Sprague-Dawley rats (180–220 g) and male BALB/c mice (18–22 g, 6 weeks old) were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). Murine were kept in a room with controlled temperature (24 ± 2 °C) and humidity (55%) with free access to food and water. The animal experiments in this study were conducted in line with China’s national statute regarding the experimental animals and were approved by Sichuan University’s Institutional Animal Care and Ethics Committee. The murine RAW264.7 cell line was purchased from American Type Culture Collection (ATCC, USA). Dulbecco’s modified Eagle Medium (DMEM) with 1% penicillin and streptomycin was used as cell culture medium, which included 10% fetal bovine serum (GIBCO, USA). The cells were incubated at a 37 °C incubator with 5% CO2.

Synthesis of FA-HA-PAE polymer

The synthesis of FA-HA-PAE polymer involved three steps.

Step 1: HA (500 mg), NH2-PEG-FA (2.5 eq.), EDC (4.0 eq.) and NHS (4.0 eq.) were dissolved in 40 °C anhydrous formamide (15 mL) and reacted overnight at 40 °C. The reaction solution was poured into a large amount of acetone to precipitate, and the FA-HA was obtained by filtering.

Step 2: Fmoc-PEG-OH (2 g), acryloyl chloride (2.0 eq.) and triethylamine (2.0 eq.) were dissolved in 20 mL of chloroform and stirred at room temperature for 12 h, followed by washing three times in water. The products were dried with anhydrous sodium sulfate. After decompression concentration, a large amount of ice ether was poured into precipitation, and the products were collected by centrifugation. The Fmoc-PEG-propylene were obtained by vacuum drying. Poly (β-amino ester) (PAE) was synthesized via Michael-addition polymerization. Fmoc-PEG-propylene (1 g), 1,6-bis (acryloyloxy) hexane (10.0 eq.) and 1,3-bis-(4-piperidine) propane (11.0 eq.) were dissolved in 20 mL of chloroform and stirred at 55 °C for 48 h. After decompression and concentration, the reaction solution was poured into a large amount of ice ether to precipitate, and the products were filtered and collected. The Fmoc-PEG-PAE were obtained by vacuum drying. Fmoc-PEG-PAE (1 g) and piperidine (3 mL) were dissolved in 10 mL chloroform and stirred at room temperature for 1 h, followed by washing three times in water. The product was dried with anhydrous sodium sulfate. After decompression concentration, a large amount of ice ether was poured into precipitation, and the products were filtered and collected. The NH2-PEG-PAE products were obtained by vacuum drying.

Step 3: FA-HA (500 mg), NH2-PEG-PAE (6.0 eq.), EDC (7.5 eq.) and DMAP (0.5 eq.) were dissolved in 40 °C anhydrous formamide (15 mL) and reacted overnight at 40 °C. The reaction solution was poured into a large amount of acetone to precipitate. The FA-HA-PAE was obtained by filtering.

Preparation and characterization of FA-HA-PAE nanoparticles (NPs) and RBA-loaded FA-HA-PAE nanoparticles (RBA-NPs)

The micelles could be fabricated through the self-assembly ability of FA-HA-PAE polymers. 5 mg RBA and 15 mg FA-HA-PAE copolymer (RBA:FA-HA-PAE copolymer = 1:3, w-w) were respectively dissolved in 5 mL of DMSO and 30 mL of deionized water and then mixed. The mixture was emulsified by a probe-type ultrasonicator (Scientz, Ningbo, China) at 200 W for 10 min to obtain RBA-NPs. To remove the unloaded drugs and excess DMSO solvent, RBA-NPs were loaded into a dialysis bag (MWCO = 7000 Da) against deionized water for 24 h. The blank NPs were prepared in the same method, except adding RBA. The particle sizes and zeta potentials of blank NPs and RBA-NPs were characterized by dynamic light scattering (DLS) (Malvern ZetaSizer Nano ZS90, UK), and transmission electron microscopy (TEM) (H-600, Hitachi, Japan) was used to monitor their morphologies. The concentration of RBA was determined by UV-vis spectrophotometer (Lambda 365, PerkinElmer, USA) with full wavelength scanning, with the maximum absorption wavelength at 210 nm. In the case of DiD-loaded NPs, the lipophilic dye DiD was used in place of RBA.

The cumulative release of RBA

The cumulative release of RBA was measured using a dynamic dialysis technique in different pH conditions. RBA-NPs (1 mg) were placed into a dialysis bag (MWCO = 7000 Da) and then submerged into 20 mL of PBS buffer within 0.5% Tween 80 at pH 7.4, 6.8 and 5.0 in turns. At certain time intervals (0.5, 1, 2, 4, 6, 12, 24, 36, 48 and 72 h), 1 mL of the release media was withdrawn and replaced with an equivalent volume of fresh media. The concentrations of released RBA were then determined by UV-vis spectrophotometer.

Cellular uptake study

RAW264.7 cells were seeded in 12-well plates at a density of 1×106 cells per well with or without LPS (100 ng/mL) and IFN-γ (20 ng/mL) and cultured at 37 °C for 24 h. The cell culture media were changed with 1 mL of fresh media containing DiD-NPs (1 μg/mL DiD). After 2 h incubation, cells were washed 3 times by PBS. A flow cytometer (BD FACSCelesta, USA) was used to quantitatively analyze the fluorescence intensity of DiD. Cells were fixed and stained with DAPI, and then photographed by laser scanning confocal microscope (LSM 800, Zeiss, Germany).

AIA model

The rats’ base of tails were injected subcutaneously with Complete Freund’s adjuvant (80 μL) containing 10 mg/mL heat-killed mycobacteria (Chondrex, #7027, Washington DC, USA). The development of arthritis progression was tracked on a daily basis and was fully established at 14 days after injection.

Immunofluorescence staining

Tail veins of AIA rats were administered with free DiD or DiD-NPs. Ankle joints were collected 24 h after the last administration to prepare sections. The prepared sections of 10 μm thick slices were stained with CD44 antibody (Affinity Biosciences, DF6392, 1:500), CD68 antibody (Affinity Biosciences, DF7518, 1:500), and FOLR2 antibody (Affinity Biosciences, DF9518, 1:300). Nuclei was stained by DAPI. A laser scanning confocal microscope (LSM 800, Zeiss, Germany) was taken to record the fluorescent distributions in synovial joints.

Biodistribution in AIA rats

Tail veins of AIA rats were injected with free DiD or DiD-NPs. After administration, the biodistribution of DiD in the ankle joints was tested by in vivo imaging measurement of fluorescence intensity using a Caliper IVIS Lumina III In Vivo Imaging System (Perkin Elmer, USA) at specific time points (0.5, 2, 6, 12, 24, and 48 h). After photographing, rats were sacrificed, and hearts, livers, spleens, lungs, kidneys, and paws were collected for in vivo imaging. Image J (National Institutes of Health, USA) was used to quantify the corresponding fluorescence intensity. AIA rats treated with N.S. were set as controls.

Polarization of M1 and M2 phenotype macrophages

M1 macrophage polarization was achieved by treating RAW264.7 with LPS (100 ng/mL) and IFN-γ (20 ng/mL) for 24 h. M2 macrophage polarization was achieved by treating the RAW264.7 with IL-4 (20 ng/mL) + IL-13 (20 ng/mL) for 24 h. THP-1 cells were induced to differentiate into macrophages by adding phorbol myristate acetate (PMA) 100 ng/ml for 48 ~ 72 h. M1 type macrophages were used LPS (100 ng/mL)+IFN- γ (20 ng/mL) treatment for 48 h; M2 type macrophages were used IL-4 (20 ng/mL)+IL-13 (20 ng/mL) for 48 h. THP-1 cells are adherent cells after differentiation. The shift of M1-to-M2 macrophages was induced by RBA-NPs (the equivalent of 20 μM of RBA) for 24 h. Immunofluorescence staining and flow cytometry were used to reveal the frequencies of M1 and M2 macrophages. The levels of M1(iNOS, TNF-α, IL-1β) and M2 (Arg-1, IL-10, TGF-β) markers were determined utilizing ELISA assay. The cells were blocked by blocking buffer (PBS solution with 5% BSA) and fixed by 4% paraformaldehyde, and then incubated with primary antibodies against PE anti-mouse CD68 antibody (Biolegend, 137013, 1:200), PE anti-mouse F4/80 Antibody (Biolegend, 123109, 1:100), PE anti-human CD68 Antibody (Biolegend, 333807, 1:20), FITC anti-mouse CD86 antibody (Biolegend, 105005, 1:100), APC anti-mouse CD206 (MMR) antibody (Biolegend, 141707, 1:100), FITC anti-human CD86 Antibody (Biolegend, 374203, 1:20) and APC anti-human CD206 (MMR) antibody (Biolegend, 321109, 1:50) at 4 °C overnight. After being counterstained with DAPI, the laser scanning confocal microscope (LSM 800, Zeiss, Germany) was used to image the sections.

Flow cytometry

For intracellular staining, the cells were blocked by blocking buffer (PBS solution with 5% BSA) and incubated with FITC anti-mouse CD86 antibody (Biolegend, 105005, 1:100) and APC anti-mouse CD206 (MMR) antibody (Biolegend, 141707, 1:100) at 4 °C for 1 h in a dark place. The cells were then washed in PBS and detected by a flow cytometry (BD FACSCelesta, USA). The results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Therapeutic efficacy evaluation

The forty rats were divided into six groups at random (n = 8): Normal, AIA rats with N.S. (N.S.), AIA rats with dexamethasone (0.5 mg/kg) (Dex), AIA rats with free RBA (5 mg/kg) (RBA), AIA rats with blank NPs (Blank NPs), or AIA rats with RBA-NPs (dose of 5 mg/kg for RBA) (RBA-NPs) with intravenous administration. After arthritis induction, treatment was given on days 17, 20, 23, and 26. In the N.S. group, AIA rats were given with an equal volume of saline. During treatment, the paw thickness of ankle joints was assessed every two days. On day 14, each hind limb was graded on a 0 to 4 scale: 0 means normal; 1 means slight erythema and/or swelling; 2 means moderate redness and swelling; 3 means severe swelling; 4 means ankylosis and inability to bear weight. The limb scores of each mouse were added together to yield a maximal score of 16. On day 28, the rats were sacrificed via anesthesia (pentobarbital sodium, 65 mg/kg, intraperitoneal). The thymus and spleen were taken and weighed right away. The thymus and spleen indices were calculated respectively by dividing the wet weight of the thymus and spleen by body weight (mg/10 g).

Histopathological examination

Twenty-eight days after arthritis induction, all of the rats were sacrificed. The ankle joints were removed and fixed with 4% paraformaldehyde. Ankle joints were decalcified and fixed using 15% tetrasodium ethylenediaminetetraacetic acid for 2 months. Sections were cut at 3 µm thicknesses after processing for paraffin embedding, followed by stained with hematoxylin-eosin (HE) and Safranin-O Fast-Green. A light microscope (Olympus BX53, Tokyo, Japan)was used to observe staining.

Immunohistochemical analysis and TRAP assay

4% paraformaldehyde was applied to fix ankle joints collected before and two days after the last treatment. A 15% tetrasodium ethylenediaminetetraacetic acid solution was then used to decalcify the fixed ankle joints were decalcified with daily changes of a 15% (w/v) tetrasodium ethylenediaminetetraacetic acid solution for 2 months. The decalcified joints were subsequently embedded in paraffin and then sectioned for staining. Commercial streptavidin-biotin complex (SABC) kits (Boster, Wuhan, China) were used to immunolocalize ALP polyclonal antibody (Invitrogen, PA5-106391, 1:200), anti-RANKL (Abcam, ab239607, 1:100), anti-OPG (Abcam, ab203061, 1:200), IL-6 Polyclonal antibody (Proteintech, 23457-1-AP, 1:100), IL-1 beta Polyclonal antibody (Proteintech, 26048-1-AP, 1:100), TNF Alpha Monoclonal antibody(Proteintech, 60291-1-Ig, 1:500) in the joints. The TRAP staining kit (Wako Pure Chemical Industries, Osaka, Japan) was applied to stain these sections following the instructions.

Micro-CT imaging

All rats were sacrificed on day 28 after arthritis induction. After fixed in paraformaldehyde at a concentration of 4%, an ex vivo micro-computed tomography (Micro-CT, SCANCO MEDICAL VivaCT 80, Switzerland) was used to scan the ankle joints were scanned at 70 kV and 113 μA with a 15 μm resolution. The 3D pictures of the distal femur’s joints and trabecular were created by rebuilding the dataset. Furthermore, quantitative analyses were performed for certain morphometric characteristics such as bone mineral density (BMD), bone surface vs. bone volume (BS/BV), trabecular separation (Tb.Sp), and trabecular bone thickness (Tb.Th).

Safety assessment

Healthy rats (200 ± 20 g) were intravenously injected with 5 mg/kg of RBA, blank NPs, or RBA-NPs to investigate the in vivo safety of RBA-NPs. Rats treated with an equivalent volume of saline were set as the N.S. group. The rats were slaughtered two days following the final dose, and key organs including the heart, liver, spleen, lung, and kidney were removed for histological analysis as previously reported. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactic dehydrogenase (LDH), creatinine (CREA), uric acid (UA), white blood cell (WBC), red blood cell (RBC), and platelet (PLT) in the obtained serum from the administered rats were assayed on a Hitachi 7020 automatic biochemical analyzer (Hitachi, Japan). Lung tissue edema was evaluated by the ratio of lung W/D. The lung tissues were divided from the left upper lung lobe. After remove the water, the tissues were weighed firstly and reweighed followed dehydration at 80 °C for 24 h. The results were concluding as the wet weight divided by the dry weight.

Protein extraction

The RAW264.7 cells were washed three times in pre-cooled PBS before being centrifuged at 1000 g for 5 min at 4 °C. After removing supernatant, samples were incubated for 5 min in Lysis buffer (40 mM Tris-HCl, 4% SDS, 2 M Thiourea,7 M Urea, pH 8.5) containing 2 mM EDTA, 1 mM PMSF, and 10 mM DTT. The suspension was then sonicated on ice for 5–15 min before being centrifuged for 20 min at 13,000 rpm, 4 °C. A pre-cooled acetone solution of four volumes was added to the supernatant at −20 °C for 2 h. Centrifugation of the protein pellets and resuspension in a urea/TEAB solution containing 8 M urea and 100 mM TEAB (pH 8.0) were followed by air drying. Protein samples were reduced for 30 min at 56 °C with 10 mM DTT, then alkylated for 30 min at room temperature in the dark with 50 mM iodoacetamide (IAM). 4 volumes of precooled acetone at −20 °C for 2 h were added to the sample for centrifugation. The air-dried protein pellets were resuspended in the above-mentioned urea/TEAB solution. The concentration of total protein was assessed according to the Bradford method. For tryptic digestion, an equivalent amount of protein from each sample (about 100 μg) were employed. The trypsin was introduced at a 1:50 (w/w) enzyme-to-protein ratio. Following digestion at 37 °C for 12–16 h, C18 columns were used to desalt the peptides which were then dried with a vacuum concentration meter. Trypsin was introduced at an enzyme-protein ratio of 1:50 (w/w), and the digestion was carried out at 37 °C for 12–16 h. Peptides were desalted using C18 columns after digestion, and the desalted peptides were dried using a rotary evaporator.

Bioinformatics analysis

Heat maps were drawn using Perseus (1.6.2.2). GeneCodis 3.0 was used for GO and KEGG pathway analysis. FDR (q-value) was used to select proteins of interest. In the quantitative results, the changed protein was considered to be differentially expressed when the fold change of the protein was >1.5 or <0.667 and the p-value was <0.05.

Statistical analysis

The quantitative results were provided as mean ± standard deviation. A Student’s two-sided t-test was used for statistical analysis of a two-group comparison. For multiple comparisons, a two-way analysis of variance (ANOVA) was utilized. A significant difference was considered at P-value < 0.05.