The mRNA (GFP or mouse Il1rn)was synthesized via in vitro transcription (IVT). Briefly, the coding sequence (CDS) of EGFP or mouse Il1rn was inserted into the IVT template plasmid. The CDS was flanked by a T7 promoter and a poly(A) tail sequence. The plasmid was linearized through restriction enzyme digestion, and the mRNA was transcribed. After purification, the size and purity of the synthesized mRNA were analyzed by denaturing agarose gel electrophoresis. The concentration of mRNA was determined by Nanodrop (A260/A280 ratio ∼2.0).

The SM102-LNP formulation was prepared as previously described (Hassett et al., 2019). Briefly, ionizable lipid (SM102), Phospholipid (DSPC) Cholesterol, and PEGylated lipid (DMG-PEG2000) were dissolved in ethanol with a mass ratio of 50:10:38.5:1.5. The LNPs containing mRNA were assembled via the microfluidic device. RNA was dissolved in citrate buffer (pH 4.0, 50 mM) and the aqueous phase and organic phase were combined at a 3:1 volumetric ratio, achieving rapid mixing in a staggered herringbone micromixer. The SM102-LNP/mRNA complexes were diluted in RNase-free PBS buffer and purified using a cutoff membrane (30 kDa). The final concentration of mRNA was 60 μg/mL and the sucrose in PBS was 10%. Efficiency was determined by Quant-iT RiboGreen RNA Assay Kit according to the user’s instructions. Particle Size and Polydispersity Index (PDI, DLS) were measured by Dynamic light scattering. The morphology of SM102 LNPs-Il1rn was observed using transmission electron microscopy (JEOL).

Mouse primary tendon stem cells (purchased from Wuhan Pricella Biotechnology Co., Ltd., CP-M176) and HEK293T (purchased from Bkmam Holdings Co., Ltd., CL0005) were cultured in complete growth medium (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) at 37°C with 5% CO₂. Cells were seeded at an appropriate density. On the 2^nd day, the cells were incubated with SM102 LNPs-GFP mRNA. The GFP signals were checked under fluorescent microscopy at the indicated time point post-transfection. For flow cytometry, at 48 h post-transfection, cells were trypsinized, and the GFP⁺ cell populations were quantified. Untransfected cells served as negative controls.

Mouse primary tendon stem cells or HEK293T cells were seeded cells in 12-well plates (1 × 10⁵ cells/well) and were incubated with SM102 LNPs- l1rn mRNA with indicated dosage. Supernatants were collected at the indicated time point post-transfection and IL-1RA levels were quantified by ELISA (ABclonal, RK04182) following kit instructions. The size of IL-1RA secreted by transfected cells was analyzed by Western blotting, with Recombinant Human IL-1ra Protein Standard (Abcam, ab316382) used as a positive control.

Mouse primary tendon stem cells were seeded at an appropriate density and transfected with SM102-IL-1RA. At 24 h post transfection, the transfected cells were incubated in the presence or absence of 10 ng/mL recombinant mouse IL-1β (SinoBiological, 10139-HNAE). At 24 h post stimulation, the total RNA was extracted using Trizol for qPCR analysis. Primer sequences are listed below:

Primary tendon stem cells were seeded in 24-well plates (2 × 10⁴ cells/well). The cells were incubated with the indicated dose of SM102 LNPs- Il1rn mRNA for 24 h. Then the cells with or without transfection were stimulated with 10 ng/mL recombinant mouse IL-1β (SinoBiological, 10139-HNAE) for 24 h. Then the cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. Subsequently, cells were incubated with anti-COL1A1 antibody (ABclonal, A24112) or anti-COL3A1 (ABclonal, A3795) at 4°C overnight. After washing, ABflo^® 555-conjugated Goat anti-Rabbit IgG (H + L) (ABclonal, AS058) (1:500) was added and incubated at room temperature for 1 h. Actin-Tracker Green-488 (C2201S) was then applied at room temperature for 40 min, followed by two PBS washes. Finally, DAPI (1:1000) was incubated in the dark at room temperature for 10 min. Images were captured using a confocal microscope, and fluorescence intensity is quantified with ImageJ.

The primary tendon stem cells were seeded in 6-well plates, and when the fusion degree reached 90%, a scratch was made using the tip of a 200 μL pipette. Images were taken at fixed positions using an inverted microscope at the indicated time points. The migration rate was calculated as: (initial area - remaining area)/initial area × 100%.

Figure 1 illustrated the group designment, details of treatments, and animal experiments timelines. To establish the mouse tendinitis model, 10-week-old male C57BL/6J mice were chosen to receive two injections of 20 μL of collagenase I (5 mg/mL, Gibco, Cat# 17100017) at the midpoint of the right Achilles tendon 7 days and 4 days before the experiment, respectively. Before experiments, the clinical assessments were performed to confirm the induction of tendinitis of the right Achilles tendon. The mice were randomly divided into the untreat group and SM102 LNPs- Il1rn mRNA group (2 µg of RNA per mouse). Healthy mice were used as the control group. Animals were euthanized at the respective endpoints (week 1 and week 4), and the tissue was collected for follow-up analysis. All animal trials received approval from the Institutional Animal Care and Use Committee of PLA General Hospital (SCXK No. 2019–0018).

For in vivo imaging, C57BL/6 mice (20–25 g) were anesthetized with isoflurane, and 2 μg of SM102 LNPs-luciferase mRNA were injected into the Achilles tendon using a 30G needle. At indicated timepoints post-injection, D-luciferin (150 mg/kg in PBS) was intraperitoneally administered, followed by bioluminescence imaging of with an IVIS Spectrum system (40 s exposure, region-of-interest analysis). Immediately following live IVIS imaging, heart, liver, spleen, lung, and kidney were harvested and imaged ex-vivo. Data were quantified as fluorescent intensity at the region of interest.

Tendon samples were collected and fixed in 4% paraformaldehyde overnight. After dehydration and embedding in paraffin, 5 μm paraffin sections were prepared for Hematoxylin and Eosin (H&E), Masson’s trichrome and Sirius Red staining. For immunofluorescent analysis, sections were blocked at room temperature after antigen retrieval, followed by overnight incubation at 4°C with the indicated primary antibodies [anti-COL1A1 antibody (ABclonal, A24112), anti-COL3A1 antibody (ABclonal, A3795), or anti iNOS antibody (ABclonal, A3774)] and secondary antibodies. The collagen or fluorescent positive area was quantified using ImageJ software.

Tendon tissue was collected and rinsed in cold PBS to remove blood contaminants. Then, the samples were minced into small fragments and incubated into 250 μL RIPA Lysis Buffer (Beyotime, P0038) with protease inhibitors (Beyotime, P1046). Then the mixture was centrifuged for supernatant collection. The ELISA assays for detection of IL-6 (ABclonal, RK04845) and IL-10 (ABclonal, RK04490) were performed according to the user’s instruction.

At week 4 after treatment, gait analysis was performed using the CatWalk XT system (Noldus). In brief, the mice were placed on a glass walking platform and their walking sequences were recorded (step speed range 10–30 cm/s).

At the end of the animal experiment, the tendon samples were collected and snap-frozen in liquid nitrogen. Then the frozen samples were homogenized in TRIzol reagent for total RNA extraction. RNA sequencing was performed using the Illumina sequencing platform. Reads were aligned to the mouse reference genome (mm39). Differential gene expression analysis was performed with DESeq2 (adjusted p-value <0.05, |log2 fold change| >1). Functional enrichment of pathways was analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Gene Set Enrichment Analysis (GSEA) was used to determine the predefined gene sets that were significantly enriched in the treatment group. All computational workflows were executed on Novogene’s online platform (NovoMagic), accessed via https://magic-plus.novogene.com/#/, and default parameters were used. Raw sequencing data were deposited in the NCBI SRA under accession number SUB15328155.

Blood serum samples were collected and immediately analyzed using an automatic biochemical analyzer (BIOBASE, BK280) to assess metabolic parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), creatinine (CREA), and urea. Additionally, the heart, liver, spleen, lung, and kidney were fixed in paraffin, sectioned, and subjected to hematoxylin and eosin (H&E) staining for histological evaluation of tissue morphology.

All data were presented as the mean ± Standard Error of the Mean (SEM). Statistical significance was determined by one-way ANOVA or two way ANOWA with Fisher’s LSD test using GraphPad Prism 9. P < 0.05 was considered significant.

The physicochemical characterization and representative transmission electron microscopy images of SM102-based nano-liposome particles (LNPs)-mRNA was shown in Figures 2A–C. The denaturing agarose gel electrophoresis showed that the size of Il1rn mRNA before and after encapsulation was approximately 900 bp, which was consistent with the expected size (Figure 2D; Supplementary Figure S1). Co-incubation of SM102 LNPs- Il1rn mRNA with HEK293T cells for 48 h and the total protein was extracted to detect the expression of IL-1RA. The result indicated a protein band in the co-incubation group but not in the negative control (Figure 2E). The molecular weight of the band was around 20 kDa, which is comparable with the positive control (Figure 2E; Supplementary Figure S2). In addition, the delivery of GFP mRNA to HEK293T using SM102 LNPs enabled robust protein expression for at least 48 h (Figure 2F). To further evaluate the SM102 LNPs-mediated mRNA transfection efficiency on primary tendon stem cells, SM102 LNPs-GFP mRNA was used for co-incubation. At 48 h post-incubation, flow cytometry analysis showed that the proportion of GFP-positive cells in the treatment group was 74.9% (Figure 2G). Under fluorescence microscopy, significant GFP fluorescence signals were observed in the treatment group from 24 to 72 h post-transfection (Figure 2H). Quantification analysis indicated that the fluorescence intensity of GFP was significantly increased during the 3 days post transfection (Figure 2I). Similarly, ELISA assay showed that the expression of IL-1RA in the supernatant of the SM102 LNPs- Il1rn mRNA treatment group was significantly higher than that of the untreated group from 24 to 72 h (Figure 2J). These results implied the therapeutic time windows of the exogenous protein expression were around 72 h. Additionally, the transfected tendon cells could secrete IL-1RA in a dosage dependent manner (Figure 2K). The above results indicated that SM102 LNPs could efficiently transfect primary tendon stem cells and achieve functional protein expression for at least 72 h.

Inflammatory responses alter the tendon microenvironment (Chisari et al., 2020). Existing evidence indicates that exogenous supplementation of IL-1β will increase the expression of a series of genes such as Interleukin 6 (Il10) in tendon cells (Tsuzaki et al., 2003; Vinhas et al., 2020). To verify the anti-inflammatory effect of IL-1RA, tendon stem cells transfected with or without SM102 LNPs- Il1rn mRNA were stimulated with 10 ng/mL IL-1β for 24 h qPCR results showed that IL-1β significantly upregulated Il6, inducible Nitric Oxide Synthase 2 (Nos2) and downregulated Interleukin 10 (Il10) (Figure 3A). Overexpression of IL-1RA could partially reverse the abnormal changes of the inflammatory-related genes (Figure 3A). Normal tendon ECM is mainly composed of collagen I (Col1), while in tendon lesions, the deposition of disordered collagen III (Col3) significantly increases, and the imbalance of Col3/Col1 ratio becomes a characteristic pathological marker (Tu et al., 2023; Franchi et al., 2007). Immunofluorescence staining confirmed that IL-1β treatment significantly decreased the Col I expression, increased the Col III expression and Col III/Col I in tendon stem cells, while overexpression of IL-1RA in tendon stem cells restored their expression levels comparable to the normal mice (Figures 3B–F). In addition, the effects of IL-1RA on tendon cell migration under IL-1β stimulation were evaluated by cell scratch assays. Scratch assays indicated that IL-1β significantly reduced migration of tendon stem cells at 12 and 24 h, while overexpression of IL-1RA significantly inhibited this effect (Figures 3G,H).

These results proved that IL-1RA can regulate inflammatory levels, improve ECM metabolic imbalance, and inhibit abnormal cell behaviors induced by IL-1β.

Before evaluating the therapeutic efficacy of SM102 LNPs-Il1rn mRNA, we first injected Luciferase mRNA into the tendon region of mice to determine whether SM102 LNPs could effectively deliver mRNA in vivo. Imaging was performed at 1-, 3-, and 7-day post-injection (Supplementary Figure S3A). Fluorescent signals indicated the successful delivery of mRNA to the tendon tissue by SM102 LNPs (Supplementary Figure S3A). Quantification analysis confirmed significantly higher signal levels in injected mice compared to control mice, persisting for 3 days, which is consistent with the in vitro data (Supplementary Figure S3B). To investigate the biodistribution of luciferase protein, major organs were dissected for imaging at 24 h post-injection. Luciferase signals were detectable in typical storage organs such as the liver and spleen (Rosanaly et al., 2025). However, the signal intensity in these organs was significantly lower, accounting for approximately 10% (liver) and 0.5% (spleen) of that observed in tendon tissue (1.7E+05) (Supplementary Figure S3C).

A murine model of tendinopathy was established via type I collagenase injection to evaluate the anti-inflammatory efficacy of SM102 LNPs-IL-1RA in vivo (Figure 4A). Histopathological analysis at 1-week post-treatment revealed that untreated tendons exhibited disorganized extracellular matrix (ECM) architecture, whereas SM102 LNPs- Il1rn mRNA treatment restored regular fiber alignment (Figure 4B). Masson’s trichrome and Sirius red staining demonstrated excessive collagen deposition in the untreated group, which was significantly attenuated by SM102 LNPs-Il1rn mRNA treatment (Figures 4C,D). Immunofluorescence analysis further indicated elevated expression of pro-inflammatory IL-6, matrix-degrading enzymes MMP1/MMP13, and suppressed IL-10 levels in untreated tendons, consistent with sustained inflammation and ECM catabolism. These pathological markers were normalized to near-physiological levels in the treatment group (Figures 4E–I). The ELISA analysis revealed a comparable expression profile for IL-6 and IL-10 in tendon tissues to the immunofluorescence results (Figure 4J). Furthermore, IL-1RA treatment significantly reduced iNOS expression in the tendon tissue of tendinopathy mice (Figures 4K,L).

At 4 weeks post-treatment, untreated tendons persistently displayed disordered collagen architecture and hyperdeposition, while M102 LNPs- Il1rn mRNA- treated mice exhibited mitigated pathological features (Figures 5A–C). Quantitative immunofluorescence analysis of collagen subtypes revealed a marked decrease in Col I and an increase in Col III and the Col III/Col I ratio in tendinopathy mice (Figures 5D–H). In contrast, SM102 LNPs-Il1rn mRNA treatment reversed this imbalance, significantly downregulating Col I, upregulating Col III, and increasing the Col III/Col I ratio compared to the untreated group (Figures 5D–H).

Functional recovery was assessed via 3D gait analysis. The treatment group displayed a footprint intensity map resembling healthy controls (Figure 6A), with quantitative analysis confirming significant improvement in gait patterns and near-restoration to control levels (Figure 6B). For instance, tendinopathy mice exhibited significantly smaller contact areas and lower intensity values than the control group (Figure 6B), indicating pain-induced unloading of the affected limb to minimize mechanical pressure. Conversely, the restoration of these parameters to near-normal levels in the IL-1RA treatment group demonstrates that local IL-1 blockade effectively reverses tendon damage-induced dysfunction. These results demonstrate that SM102 LNPs-IL-1RA alleviates tendinopathy by resolving inflammation, normalizing collagen composition, and restoring motor function.

RNA sequencing analysis of mouse tendon tissues revealed 447 downregulated genes and 317 upregulated genes (|Log2FoldChange| ≥1, Padj ≤0.05) in the SM102 LNPs-Il1rn mRNA group compared to controls (Figure 7A). Differential gene expression analysis (Figure 7B) and Gene Ontology (GO) enrichment (Figure 7E) demonstrated that SM102 LNPs-Il1rn mRNA treatment specifically modulated cellular responses to IL-1 and extracellular matrix (ECM) remodeling (e.g., regulation of collagen metabolic processes and collagen trimer formation), suggesting that the therapy suppresses inflammation by blocking IL-1 signaling while promoting collagen synthesis and ECM repair. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further supported these findings, showing regulation of inflammatory signaling pathways such as NF-κB and IL-17 signaling in the treatment group (Figures 7C, D). The enrichment of the MAPK signaling pathway aligned with Gene Set Enrichment Analysis (GSEA) results (Figure 7E). In addition, GSEA analysis also found enhanced macrophage differentiation and hydrogen peroxide metabolism in treatment groups (Figure 7E).

Collectively, these transcriptomic findings suggest that SM102 LNPs-Il1rn mRNA exerts dual therapeutic effects in tendinitis by targeting IL-1 signaling—simultaneously inhibiting inflammatory cascades and activating collagen-driven ECM restoration. The coordinated modulation of MAPK signaling, macrophage polarization, and H₂O₂ metabolism may further contribute to reestablishing tissue homeostasis.

The safety of SM102 LNPs- Il1rn mRNA was evaluated by H&E staining of major organs and serum biochemical indicators (Figure 8A). Histological analysis showed that the structures of organs such as the heart, liver, spleen, lung, and kidney in the treatment group were intact, and no degenerative changes or inflammatory cell infiltration were observed. Also, there was no significant difference in serum biochemical parameters (such as liver and kidney function indicators) between the treatment group and the control group, confirming the good biological safety of SM102 LNPs- Il1rn mRNA treatment (Figure 8B).