Cell cultures
The Raw264.7 cell line is immortalized cells bought from Shanghai Fuheng Biological Company (cat. No. FH0328). Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was used for the culturing of the Raw264.7 cells.
Primary bone marrow‑derived macrophages (BMMs) cells were extracted from the whole bone marrow of the tibia and femur bone in male 6-week-old C57BL/6 mice (GemPharmatech, Nanjing, China). Then, cells were cultured in minimal essential medium alpha (α-MEM) supplied with 30 ng/mL macrophage colony-stimulating factor (M-CSF; cat. no. 416-ML; R&D Systems, Minneapolis, MN, USA), 10% FBS, and 1% penicillin–streptomycin. Both cells were cultured in an incubator at 37 °C and 5% CO2.
Intracellular ROS detection
Raw264.7 cells (30-40% confluence) seeded onto a confocal dish were stimulated with lipopolysaccharide (LPS; 100 ng/mL; cat. no. S1732; Beyotime Institute of Biotechnology, China) and treated via different concentrations of spirulina (SP, cells/mL, 1.2 × 106, 2.5 × 106, 5.0 × 106, 1 × 107) for 24 h. Then, these cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; 10 μM; 30 min; Beyotime Institute of Biotechnology, China), and the ROS level was analyzed per the manufacturer s protocol. Finally, the pictures were captured using a Leica DM4000 B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany), and the relative fluorescence intensity was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Quantitative real-time PCR analysis
Raw264.7 were cultured in six-well plates at the density of 3 × 105 cells/well. Then these cells were stimulated to M1-like (LPS; 100 ng/mL) or M2-like (Interleukin-4; IL-4; 20 ng/mL; Beyotime Institute of Biotechnology, China) polarization and treated via different concentrations of SP (cells/mL, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) for 24 h.
For Keap1, Nrf2, and Homx1 detection, Raw264.7 were cultured in six-well plates at a density of 3 × 105 cells/well. Then, these cells were treated with different concentrations of SP (cells/mL, 6.2 × 105, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) for 24 h.
Primary BMMs were seeded onto six-well plates at a density of 3 × 105 cells/well. Then, these cells were stimulated to the osteoclast phenotype with 30 ng/mL M-CSF and 50 ng/mL receptor activator of nuclear factor-κB ligand (RANKL; R&D Systems, Minneapolis, MN, USA) and treated by SP (2.5 × 106 cells/mL) or MTX (4 μM) for 0, 1, 3, and 5 days.
For RNA extraction, TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to isolate total RNA from both cells. The complementary DNA (cDNA) was reverse transcribed from the obtained total RNA using the cDNA Synthesis Kit. RT-qPCR was performed using the TB Green Premix Ex Taq Kit on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) per the following conditions: Denaturation at 95 °C for 30 s; 40 cycles of 95 °C for 3 s and 60 °C for 34 s; and then 95 °C for 15 s, 60 °C for 60 s and finally, 95 °C for 15 s. Specific primer pairs were designed using NCBI blast, and the sequences are provided in Supplementary Table 1. β actin gene expression was used as the internal control. Target gene expression levels were determined using the 2−ΔΔCq method.
Flow cytometry analysis
Raw264.7 were cultured in six-well plates at the density of 3 × 105 cells/well. Then these cells were stimulated to M1-like or M2-like polarization and treated via different concentrations of SP (cells/mL, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) for 24 h. Next, cell suspensions were prepared and blocked in a 0.3% bovine serum albumin (BSA) solution, incubated with primary antibodies (including PE-CD86, APC-CD206, and FITC-CD11b; BD Biosciences, USA) for 30 min, washed with fresh 0.3% BSA, and applied to flow cytometry analysis on an LSRFortessa flow cytometer (BD Biosciences, USA) to count at least 10 000 events. Finally, the FlowJo software (Version 10.4, BD Biosciences, USA) was used to analyze the result with a gating strategy shown in Supplementary Fig. 21.
TRAP staining assay
Primary BMMs were seeded onto 96-well plates at the density of 8 × 103 cells/well (or onto a 24-well plate at the density of 2 × 104 cells/well). Then these cells were stimulated to osteoclast phenotype with 30 ng/mL M-CSF and 50 ng/mL RANKL and treated via different concentrations of SP (cells/mL, 6.2 × 105, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) or different concentrations of MTX (μM; 0.25, 0.5, 1, 2, 4) for 5–7 days until the matured osteoclasts could be observed in the plates. Then, plates were washed with phosphate-buffered saline (PBS) 3 times and fixed with 4% paraformaldehyde for 15 min. Then, tartrate-resistant acid phosphatase (TRAP) staining was applied immediately at 37 °C for 30 min at dark. Images were captured using an optical microscope (Olympus, Tokyo, Japan). The osteoclasts were TRAP-positive cells with more than three nuclei, and the area and number of these osteoclasts were quantified using the ImageJ software (NIH, Bethesda, MD, USA).
Immunofluorescence of podosome actin belt
Primary BMMs were seeded onto 24-well plates at the density of 2 × 104 cells/well. Then these cells were stimulated to osteoclast phenotype with 30 ng/mL M-CSF and 50 ng/mL RANKL and treated via different concentrations of SP (cells/mL, 6.2 × 105, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) or different concentrations of MTX (μM; 0.25, 0.5, 1, 2, 4) for 5–7 days until the matured osteoclasts could be observed in the plates. Then, plates were washed with PBS, fixed with 4% paraformaldehyde, permeabilized by 0.2% Triton X-PBS solution, repeatedly washed by PBS, and stained with FITC-labeled phalloidin to bounds with the F-actin ring in cells for 1 h in darkness. The nuclei were stained at RT for 10 min without light by 4′, 6-diamidino-2-phenylindole (DAPI). Images were captured using a Leica DM4000 B epifluorescence microscope.
Protein extraction and western blot (WB) analyses
Primary BMMs were seeded onto six-well plates at a density of 3 × 105 cells/well. Then, these cells were stimulated to the osteoclast phenotype with 30 ng/mL M-CSF and 50 ng/mL RANKL and treated by SP (2.5 × 106 cells/mL) or MTX (4 μM) for 0, 1, 3, 5 days; or 0, 10, 20, 40 min, or 24 h.
Raw264.7 were cultured in six-well plates at a density of 3 × 105 cells/well. Then these cells were treated via different concentrations of SP (cells/mL, 6.2 × 105, 1.2 × 106, 2.5 × 106, 5 × 106, 1 × 107) for 24 h.
Total cellular proteins were extracted from cultured cells using a RIPA lysis buffer supplemented with phosphatase and protease inhibitors (Roche, Basel, Switzerland). and the content of proteins was quantified with BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). The exact amount of proteins was resolved on 4–20% SDS–PAGE gel for separation and was electroblotted onto 0.22-μm PVDF membranes (Merck-Millipore, CA, USA). Membranes were blocked with 5% skimmed milk-PBS at room temperature for 1 h and then incubated overnight at 4 °C with primary antibodies (diluted 1:1000 in 5% BSA-TBST). Primary antibodies included NFATc1 (cat. no. ab2796; mouse mAb), c-Fos (cat. no. 9F6; rabbit mAb), CTSK (cat. no. E7U5N; rabbit mAb), phospho-P38 (cat. no. Thr180/Tyr182, D13.14.4E; rabbit mAb), P38 (cat. no. D13E1; rabbit mAb), phospho-ERK (cat. no. Thr202/Tyr204, and D3F9; rabbit mAb), ERK (cat. no. 137F5; rabbit mAb), IκBα (cat. no. L35A5; mouse mAb), phospho-P65 (cat. no. Ser536, 93H1; rabbit mAb), P65 (cat. No. D14E12; rabbit mAb), SOD2 (cat. no. 10701-1-AP; rabbit mAb), HO-1 (cat. no. 10701-1-AP; rabbit mAb), NRF2 (cat. no. 80593-1-RR; rabbit mAb), KEAP1(cat. no. 80744-1-RR; rabbit mAb), GPX1 (cat. no. ab22604; rabbit mAb), GPX4 (cat. no. ab125066; rabbit mAb), HA-tag (cat. no. C29F4; rabbit mAb), Ubiquitin (cat. no. 10201-2-AP; rabbit mAb), LC3 (cat. no. #4108; rabbit mAb), and β-actin (cat. no. D6A8; rabbit mAb). Membranes were washed three times using Tris-buffered saline-Tween20 (TBST) and subsequently incubated with anti-rabbit or anti-mouse IgG (H + L; DyLight™ 800 4 × PEG conjugate; Cell Signaling Technology, Danvers, MA, United States) secondary antibody (1:5000 dilution) for 1 h at RT in the dark. Membranes were washed extensively in TBST, and protein immunoreactivity was detected on a LI-COR Odyssey fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, USA). The gray values of each protein were measured using Image J software (National Institutes of Health, United States). The original scans of blots and gels were displayed in the Source data file.
Confocal fluorescence microscopy
Primary BMMs (30–40% confluence) seeded onto a confocal dish were stimulated to osteoclast phenotype with 30 ng/mL M-CSF and 50 ng/mL RANKL and treated with 2.5 × 106 cells/mL SP for 30 min.
Raw264.7 cells (30–40% confluence) seeded onto a confocal dish were treated via different concentrations of SP (cells/mL, 1.2 × 106, 2.5 × 106, 5 × 106) for 24 h.
Then, the dishes were washed by PBS, fixed with 4% paraformaldehyde, permeabilized by 0.2% Triton X-PBS solution, and then incubated with primary antibodies (p-P38, p-P65, KEAP1, Ubiquitin, and LC3) at 4 °C overnight. The next day the dishes were repeatedly washed by PBS and stained with Alexa Fluor 488 and 555 Conjugate secondary antibodies (anti‐rabbit, anti-mouse, 1:500; Cell Signaling Technology) for 1 h in darkness. The nuclei were stained at RT for 10 min without light by DAPI. Images were captured using a Leica DM4000 B epifluorescence microscope. The IOD/DAPI and the relative fluorescence intensity were calculated using Image-Pro Plus 6.0 software.
Protein degradation assay
Raw264.7 cells were cultured in six-well plates at the density of 3 × 105 cells/well. Then these cells were pretreated with cycloheximide (CHX; 50 nM; cat. no. S7418; Selleck Chemicals, China) for 2 h to inhibit the synthesis of proteins, and the next step the lower three well cells were treated with MG132 (10 μM; cat. no. S2619; Selleck Chemicals, China) or chloroquine (Chlq; 25 nM; cat. no. S6999; Selleck Chemicals, China) for 10 h. Concurrently, cells were treated via different concentrations of SP (cells/mL, 2.5 × 106 and 5 × 106) for 12 h. Finally, the proteins were extracted and applied to WB analysis.
Ubiquitylation modification assay
293 T cells were transfected with HA-PLVC, HA-KEAP1, and Myc-Ubiquitin or transfected with Flag-PLVC, Flag-NRF2, HA-KEAP1, and Myc-Ubiquitin (synthesized, purchased from Shanghai Ai Bosi Biological Technology Co., Ltd.) using lipofectamine 3000 (cat. no. L3000015; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The next day, cells were treated via different concentrations of SP (cells/mL, 2.5 × 106 and 5 × 106). After 24 h, cells were stimulated with MG132 (10 μM) for 8 h at 37 °C and then suspended and washed three times with PBS. The proteins were extracted with 1.2 ml NETT solution (1% Triton X-100, 2.5 M NaCl, 0.9 M Tris–HCl [pH 7.5], 0.25 M EDTA, 100 mM PMSF), 200 μL lysate was used as input whilst the remaining 1000 μL samples were incubated with 30 μL HA-tagged or Flag-tagged magnetic beads at 4 °C overnight. The samples were then boiled at 99 °C for 10 min and finally subjected to WB analysis.
RFP-GFP-LC3B assay
RFP-GFP-LC3B (Component A, Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit, Invitrogen Waltham, MA, USA; #P36239) were added to Raw264.7 cells (30–40% confluence) seeded onto a confocal dish for 16 h, then these cells were treated via different concentrations of SP (cells/mL, 1.2 × 106, 2.5 × 106, 5 × 106) for 24 h. Finally, cells were processed using confocal laser scanning microscopy with Hoechst stain to nuclei.
Isolation of cell membrane from macrophage
RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Beyotime). The macrophage cell membranes (MCMs) were prepared using previously reported methods with some modifications. Briefly, RAW 264.7 cells were washed three times with phosphate buffer saline (PBS, Corning) and then incubated in ice-cold hypotonic lysing buffer (50 mM Tris–HCl, 10 mM magnesium sulfate at pH 7.5) for 4 h at 4 °C. Then, sucrose was added to the cell suspension and eventually reached a sucrose concentration of 0.25 M. RAW264.7 cells were repeatedly disrupted using a dounce homogenizer with a tight-fitting pestle. After that, the cell suspension was centrifugated at 300 × g for 20 min (4 °C). The supernatant was collected while the precipitate was resuspended in the hypotonic lysing buffer and subjected to the dounce homogenizer treatment again. The supernatants were saved and centrifuged at 18,000×g for 40 min (4 °C) to collect the cell membrane. The supernatant was washed twice with cold PBS and stored at −80 °C for further use. The protein quantity of the purified MCMs was determined by a bicinchoninic acid protein (BCA, Beyotime) assay.
Synthesis of macrophage cell membranes (MCMs)-engineered SP
Spirulina platensis (SP) and Zarrouk’s media were obtained from Guangyu Biological Technology (Shanghai, China). SP was maintained in Zarrouk’s medium at 25 °C under light irradiation (3000–6000 lx). SP powder was purchased from the Macklin Biochemical Technology (Shanghai, China). The macrophage cell membranes (MCMs)-cloaked SP were synthesized by a reported extrusion method28,29,30. Briefly, SP was cracked into small fragments by continuous magnetic stirring at room temperature for 12 h. Afterward, the fractured SP was filtrated through a 5 μm cell strainer and concentrated to a concentration of 5 × 108 cells/mL. To prepare MCMs-cloaked SP, MCMs were physically extruded through a 5 μm porous polycarbonate membrane (Membrane Space). Then, MCMs were cloaked onto the SP by co-extruding the two components through a 5 μm porous polycarbonate membrane. The extrusion process was repeated for at least 20 times. The SP@M was then collected and redispersed in DI water at 4 °C for further use. Fresh SP@M was recommended to be prepared before use.
Dead/alive assay of SP
Fragmented SP was obtained by continuously stirring SP for 12 h. Boiled SP was obtained by boiling the SP for 10 min. Then, SP, fragmented SP, and boiled SP were gathered and stained with fluorescein diacetate (FDA) for 30 min at 37 °C. Then, the CLSM images were captured using a Leica DM4000 B epifluorescence microscope.
Protein identification of MCMs and SP@M
Then, SP, MCMs, and SP@M protein were subjected to SDS–PAGE gel electrophoresis for separation and following the electroblotting process for further WB assay. The protein pattern was identified with Coomassie blue staining. The primary antibodies used were TNFR2 (cat. no. 19272-1-AP), CCR2α (cat. no. 16153-1-AP), and CD36 (cat. no. 18836-1-AP) purchased from Proteintech Inc, Wuhan, China.
Isolation and characterization of SP-derived exosomes
100 mL of SP cell culture media (1 × 107 cells/mL, free of FBS, cultured for 24 h) was harvested and centrifuged at 2000×g for 30 min to remove cells and debris. Then, the supernatant containing the cell-free culture media was transferred to a new tube, and 50 mL of the Total Exosome Isolation reagent (Cat. no. 478359; Thermo Fisher Scientific, Waltham, MA, United States) was added, mixed, vortexed, and incubated at 4 °C overnight. We centrifuged this mixture at 10,000×g for 1 h at 4 °C, and gathered and resuspended the extracted exosomes in 400 μL saline. Then, the TEM test was carried out and images were captured using a Hitachi TEM system (HC/HR select = HC-1, accelerating voltage = 80,000, emission = 10.2, vacuum = 6.9E−05 Pa). The nanoparticle size distribution and concentration were measured using a ZetaView instrument (Particle Metrix).
Establishment of CIA model
All animal experiments were authorized by the Institutional Animal Ethics Review Board of the Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (Approve No. SH9H-2024-T246-1). Bovine type-II collagen solution (2 mg/mL) and complete Freund’s adjuvant (CFA) (4 mg/mL) were shaken to emulsion. For the first immunization, male mice (n = 36; DBA/1 J; divided into six groups: Sham group without immunization; saline group; Macrophage membrane [MCMs] group; macrophage membrane-cloaked spirulina [SP@M] group; Spirulina [SP] group; Methotrexate [MTX] group, GemPharmatech, Nanjing, China) were treated by intradermal injection of the complete emulsion at the end of the tail. For the boost immunization, the mice were injected with the emulsion of bovine type-II collagen solution (2 mg/mL) with incomplete Freund’s adjuvant (IFA) on day 21.
Biodistribution in the CIA mouse model
SP was initially labeled with Cy5-PEI to obtain Cy5-labeled SP. Then, Cy5-labeled SP@M was prepared using the previously described macrophage cell membrane modification process. CIA mice were intravenously injected with Cy5-labeled SP or Cy5-labeled SP@M (100 μL, at an SP concentration of 5 × 107 cells/mL). The biodistribution of Cy5-labeled SP and Cy5-labeled SP@M was then 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, 6, and 24 h post-injection) (n = 3 in each group). Then, the major organs and the four limbs were dissected to evaluate the fluorescence intensity.
Assessment of paw swelling and clinical score
The CIA mice were administered 100 µL of PBS, MCMs, SP@M (4 × 107 cells/mL), SP (4 × 107 cells/mL), and MTX (5 mg/kg) intravenously every three days for a total of 10 times. Non-immunized mice in the sham group were administered PBS intravenously. And mice were evaluated every 3 days for paw swelling scores and clinical scores, and the hind paw diameters were measured by a digital caliper. A blinded researcher gave paw scorings (score ~ 0-4) based on the following criteria: 0, normal; 1, mild redness of ankle or tarsal joints; 2, mild redness and swelling extending from the ankle to the tarsals; 3, moderate redness and swelling from ankle to metatarsal joints; 4, severe redness and swelling encompassing the ankle, foot, and digits67. Clinical severity was graded as 0 (normal), 0.5 (erythema and edema in only one digit), 1 (erythema and mild edema of the footpad, or ankle or two to five digits), 2 (erythema and moderate edema of two joints [footpad or ankle, two to five digits]), 3 (erythema and severe edema of the entire paw), or 4 (reduced swelling and deformation leading to incapacitated limb)68. At the end, images of the left hind paws of mice were captured.
Behavioral assessment
On day 60, the behaviors of the mice were analyzed via the Von Frey test, hot plate test, and gait analysis. In this study, the von Frey test was performed on each animal to measure mechanical allodynia in mice. We used flexible nylon von Frey filaments in logarithmic increments of force (0.04–2 g), and the “up-down method” was used. Every result was recorded to assess the 50% mechanical paw withdrawal threshold response (g).
To determine behavioral response to noxious heat, the surface temperature of the hot plate was maintained at 55 °C. We placed the mice on the hot plate and started timing until a quick withdrawal or flick of the paw was observed, which is defined as latency (s). Twenty seconds was set as a cutoff time to prevent mouse paw injury. For the accuracy of the results, we measured three times at 10-min intervals, and the results were averaged.
All our gait analyses were based on a CatWalk runway; animals were trained daily on a CatWalk runway until they were able to make consecutive uninterrupted runs for at least one week. Valid data were recorded at least four times for each animal, with an average of six animals per group; all trials marked by the software as compliant were reviewed manually; if the animal stopped or turned back during the experiment, the recording was discarded. Print area and max contact area parameters were collected for each paw, respectively, and blindly evaluated.
μCT
In the end, the right ankles of mice were collected and applied to μCT analysis. A high-resolution Skyscan 1275 micro-CT scanner (Bruker micro-CT, Kontich, Belgium) was used to perform the micro-computed tomography (CT) scanning with the following parameter settings: source voltage, 46 kV; source current, 75 μA; AI, no filter A; A pixel A size A, 9.000546 μm; rotation step, 0.4°. The bone volume (BV)/total volume (TV) of talus bone was calculated with software (Version: 6.5-3, SCANCO Medical AG, Switzerland) by evaluating and analyzing the three-dimensional region of interest (ROI). For the erosion index, six sites in the ankle joint were scored: the talus, navicular bone, medial cuneiform bone, and the bases of the first, second, and third metatarsals. Each site was scored on a scale of 0–3 (0 = normal; 1 = pitting; 2 = full thickness holes in small-medium areas; and 3 = full thickness holes in medium-large areas) with a maximum score of 18. The scores of the arthritic hind paw determined by the two observers were averaged to obtain the final erosion score69.
Histology and immunofluorescence staining
Ankles from mice were fixed in 4% paraformaldehyde for 48 h and embedded into paraffin. Then, they were cut into 8 µm sections and stained with Safranin O/Fast Green (SO/FG), TRAP, and Hematoxylin and eosin (H&E). The SMASH score, including synovitis, cartilage destruction, and bone erosion, was analyzed according to a previous article48. The number of osteoclasts and TRAP-positive multinucleated osteoclasts per field (Oc.S/BS) was calculated.
These tissue sections were deparaffinized in graded xylene, rehydrated in graded alcohol solutions, and then incubated in antigen retrieval buffer (Roche, Basel, Switzerland) at 37 °C for 30 min. After natural cooling, the slides were placed in PBS (pH 7.4) and washed thrice for 5 min each. An autofluorescence quencher was added to the tissue sections for 5 min, rinsed with running water for 10 min, and then blocked with BSA for 30 min. The sections were subsequently incubated with primary antibodies in a wet box at 4 °C overnight. Primary antibodies were used at 1:100 dilution and included Anti-NRF2, Anti-KEAP1, anti-ubiquitin, anti‐LC3, anti‐CD86, and anti-CD206. The next day, the slides were immersed in PBS and washed three times, then incubated with Alexa Fluor 488 and 555 Conjugate secondary antibody (anti‐rabbit, anti‐mouse, 1:500; Cell Signaling Technology) for 50 min at room temperature in the dark. The slides were again immersed in PBS, washed three times in PBS, and then counterstained in the dark with DAPI solution for 10 min at room temperature. Finally, sections were observed using a Leica DM4000 B epifluorescence microscope. The IOD/DAPI was calculated using Image-Pro Plus 6.0 software.
Immunohistochemistry staining
The paraffin sections were processed using an immunohistochemistry kit (cat. no. G1215-200T; Servicebio Technology) according to the manufacturer’s instructions. The primary antibodies used were anti-TNF-α and anti-CTSK. Images were captured using a Leica DM4000 B microscope. The IOD value and positive cells (%) were calculated using Image-Pro Plus 6.0 software.
RNA seq analysis
Total RNA of ankles from the saline group (n = 3) and SP@M group (n = 3) were extracted as per the manufacturer’s protocol. Then, they were analyzed via RNA (transcriptome) sequencing as Wuhan Huada Gene Technology Co., Ltd. (China): Volcano Plot (|log2FC| ≥ 1, FDR ≤ 0.001), Gene Oncology (GO) of biological pathway and cellular component analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and gene set enrichment analysis (GSEA) was used by mRNA relative expression as Transcripts PerKilobase Million (TPM) to review further pathways involved on the Mybgi platform (WuhanHuada Gene Technology, https://mybgi.bgi.com/tech/login).
Hemolysis assay
The hemolytic toxicity of SP and SP@M on mice red blood cells (RBCs) was preliminarily assessed using a hemolysis assay. Briefly, 500 μL of mice with 5% erythrocytes (Shanghai Lab, Animal Research Center Co., Ltd) was centrifuged at 200×g for 15 min, after which the supernatant was removed. The erythrocytes were resuspended to 10 mL with saline following three washes of the precipitate. The SP and SP@M were added to the erythrocyte suspension at a final concentrations range of 1.25 × 106, 2.5 × 106, 5.0 × 106, 1.0 × 107 cells/mL, whereas the RBCs treated with saline as negative control and Triton X-100, 2% (v/v) as positive control. The RBCs of each group were incubated at 37 °C for 1 h and then centrifuged at 200×g for 15 min. Subsequently, 100 µL of the supernatant was taken, and the absorbance at 540 nm was measured via Infinite M200 Pro microplate reader (Nanoquant, Tecan, Swiss). The percentage of hemolysis was calculated using the following formula:
$${{\rm{Hemolysis}}} {{\rm{rate}}} \, (\%)\, =\,({{\rm{OD}}} {{\rm{samples}}}-{{\rm{OD}}} {{\rm{background}}})\\ /({{\rm{OD}}} {{\rm{positive}}} {{\rm{control}}}-{{\rm{OD}}} {{\rm{negative}}} {{\rm{control}}}) \times 100\%$$
(1)
Biocompatibility assay
The major organs, including the heart, liver, spleen, lungs, and kidneys of the previous CIA mouse models, were harvested from the euthanized mice for further H&E staining and histology assay to evaluate the toxicity of MCMs, SP, and SP@M.
The supplementary biochemical analysis was applied to the DBA/1J mice (n = 12; divided into 2 groups: saline group and macrophage cell membranes-cloaked spirulina [SP@M] group). 100 μL saline and SP@M (4 × 107 cells/mL) were administrated intravenously in the tail once every three days (10 times). When injections finished, all mice were euthanized, and the serum was harvested. Then the levels of albumin (ALB), total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (γ-GT), triglyceride (TG), cholesterol (CHOL), glucose (GLU), urea nitrogen (BUN), and creatinine (CRE) ion Calcium (Ca), and ion Phosphote (P) were measured using the Pointcare V3 automatic biochemical analyzer (Tianjin MNCHIP Technologies Ltd., Tianjin, China) with commercial diagnostic kits.
Statistics and reproducibility
Data in this study were obtained from three to six independent experiments or repeated measurements. Significant differences among groups were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc test or the Kruskal–Wallis test with Dunn’s post hoc test. All analyses were calculated with GraphPad software (version 8.0; NY, USA). Differences were defined as significant with a p-value of <0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
