Abstract Background The imbalance of macrophage polarization plays a pivotal role in the progression of rheumatoid arthritis (RA). Reprogramming macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype is considered a promising therapeutic strategy. Methods To address this challenge, Panax notoginseng polysaccharides (PNP) with varying molecular weights were chemically conjugated with deoxycholic acid (DC) to obtain amphiphilic conjugates (PNP-DC), which self-assembled into micelles (PNP-Ms). After screening for optimal molecular weight, folic acid (FA) was introduced onto the micelle surface, and Polyphyllin I (PPI) was encapsulated to form FA-modified, PPI-loaded micelles (FA-PPI-Ms) with macrophage-targeting capability. Results FA-PPI-Ms showed enhanced cellular uptake via FA receptor–mediated endocytosis and effectively eliminated reactive oxygen species (ROS), reduced inflammatory cytokine production, and exhibited good biosafety. In vivo, FA-PPI-Ms significantly alleviated joint swelling and inflammation in RA rat models. Mechanistic studies based on RNA sequencing and experimental validation revealed that FA-PPI-Ms suppressed the JAK2/STAT3 signaling pathway, thereby promoting M2 macrophage polarization and restoring the M1/M2 balance. Conclusion This study presents a novel FA-PPI-Ms delivery system for targeted macrophages. By modulating polarization through inhibition of JAK2/STAT3 signaling, the system offers a promising therapeutic strategy for RA and potentially other inflammatory diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03576-8. Keywords: Rheumatoid arthritis, Panax Notoginseng polysaccharide, Micelles, Macrophage polarization, JAK2-STAT3 Introduction Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation, cartilage destruction, and pannus formation as its primary pathological manifestations [[42]1]. The morbidity, disability rate, and mortality associated with RA have been increasing annually, posing a significant threat to human health and resulting in substantial economic burdens [[43]2]. Although the pathogenesis of RA remains incompletely understood, macrophages—crucial immune cells involved in both innate and adaptive immunity—have long been recognized as playing a pivotal role in its development [[44]3]. These cells can be polarized into distinct phenotypes in response to various signals from the microenvironment, primarily including pro-inflammatory M1 type and anti-inflammatory M2 type macrophages [[45]4]. M1 macrophages are central to promoting inflammation and bone destruction within the cytokine network characteristic of RA, while M2 macrophages contribute to anti-inflammatory processes and tissue repair in the later stages of inflammation [[46]5]. Given the significant role of macrophage polarization in the pathogenesis of RA, strategies aimed at inhibiting M1 macrophage polarization or inducing M2 macrophage polarization through pharmacological interventions may represent a promising new approach for RA treatment [[47]6, [48]7]. Despite recent progress in pharmacologic therapies—including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and biologic disease-modifying antirheumatic drugs (bDMARDs)—these agents primarily alleviate symptoms rather than address the underlying immune dysregulation. Moreover, their long-term use is often associated with systemic side effects and a lack of specificity toward immune cells. Therefore, there remains a pressing need to develop targeted strategies capable of modulating immune cell behavior within the RA microenvironment [[49]8]. Polyphyllin I (PPI) is an active compound extracted from the rhizome of Paris polyphylla, known for its selective inhibitory effects on various cancer cell types, including osteosarcoma, liver, ovarian, gastric, and myeloma cells, and is frequently employed to impede tumor progression [[50]9, [51]10]. Recent studies have indicated that PPI serves as an effective immune adjuvant, possessing anti-inflammatory and immunoregulatory properties, which can significantly ameliorate synovial inflammation in the ankle joint of collagen-induced arthritis mice [[52]11, [53]12]. The underlying mechanism is likely linked to the inhibition of pro-inflammatory effector production mediated by NF-κB, which in turn modulates macrophage polarization. Consequently, PPI is regarded as a representative agent for regulating macrophage polarization balance in the treatment of RA [[54]13]. Nevertheless, the clinical application of PPI is considerably limited by its poor solubility, low oral bioavailability, and, more critically, its high toxicity, which can result in hemolysis following intravenous administration and lead to liver and kidney toxicity with prolonged use. Nanodrug delivery systems present a promising strategy to address the limitations of current treatments for PPI [[55]14, [56]15]. These nanocarriers can mitigate drug toxicity, enhance drug solubility, prolong drug circulation time, decrease drug clearance, and facilitate controlled delivery to the target disease site. Consequently, the integration of PPI with effective delivery strategies represents a novel approach in the treatment of RA. Polymer nanomicelles serve as ideal carriers to improve the solubility, bioavailability, half-life, and targeting of hydrophobic drugs [[57]16, [58]17]. Over the past decade, polymer-based micellar therapies have emerged as an appealing new strategy for RA treatment [[59]18, [60]19]. Polysaccharides, which are the primary macromolecular constituents in traditional Chinese medicine, are derived from a diverse array of sources. Nanocarriers formulated from traditional Chinese medicine polysaccharides typically exhibit excellent biocompatibility, biodegradability, and low toxicity [[61]20, [62]21]. Thus, polysaccharide-based polymer nano-micelles have garnered increasing attention in the field. Panax notoginseng polysaccharides (PNP) are active components derived from Panax notoginseng (Burk.) F.H. Chen, recognized for their antioxidant, anti-inflammatory, and immunomodulatory properties, as well as their ability to ameliorate inflammatory diseases by regulating macrophage polarization [[63]22]. PNP contains various functional groups, including hydroxyl, carboxyl, and other groups, which can be chemically modified with deoxycholic acid to synthesize amphiphilic polymers [[64]23]. The hydrophobic interactions of these polymers in aqueous solutions lead to self-aggregation into micelles, facilitating the encapsulation of PPI. Folic acid (FA) exhibits a strong affinity for FA receptors, which are overexpressed on activated macrophages [[65]24]. Consequently, the surface of FA-modified polymer micelles can actively bind to these receptors, promoting endocytosis and enabling targeted drug release within the cells [[66]25]. However, the polysaccharide of Panax Notoginseng is a complex polysaccharide composed of various components, and the diversity in molecular weight results in significant differences in the properties and activities of these components [[67]26]. Based on this understanding, we hypothesize that constructing self-assembled nanocarriers with varying average molecular weights of PNP may lead to notable differences in the physical and chemical properties of the carriers. These properties include structural appearance, particle size distribution, drug loading capacity, and stability. In summary, this study focused on PNP with varying molecular weight intervals, which were conjugated with deoxycholic acid to create PNP nanocarriers, and to identify the molecular weight interval of PNP that is most suitable for constructing these nanocarriers. Additionally, we incorporated FA modifications on the surface of the carriers to endow them with active targeting capabilities, achieving the specific delivery of PPI and PNP, thereby establishing a ‘medicine-based medicine’ model that promotes the effective accumulation of host-guest complexes with pronounced anti-inflammatory effects in diseased areas (Scheme [68]1). This research not only provides a scientific foundation for the comprehensive study of self-assembled nanocarriers derived from PNP but also establishes a robust basis for their broader application in anti-RA therapy. Scheme 1. [69]Scheme 1 [70]Open in a new tab Schematic representation of engineered Panax notoginseng polysaccharide micelles inhibit macrophage polarization and delay the progression of rheumatoid arthritis via JAK2-STAT3 signaling pathway Results and discussion Composition and MW distribution of purified polysaccharide fractions The detailed composition and MW distribution of the purified polysaccharides (PNP) were determined via HPLC and HPSEC (Table [71]1). The results revealed that PNP fractions (PNP-1 to PNP-4) primarily consist of Glucose, Galactose, Arabinose, Mannose, Rhamnose and Glucuronic Acid. The average MW of the fractions were 256.45 Da (PNP-1), 3589.22 Da (PNP-2), 4187.94 Da (PNP-3), and 9549.93 Da (PNP-4). The variations in MW and sugar composition suggest distinct chemical structures and glycosylation patterns, which could influence their performance in drug delivery systems. The MW of polysaccharides is crucial for their physicochemical properties and biological activities. Higher MW polysaccharides with abundant free hydroxyl groups may enhance interactions with drug molecules, while lower MW polysaccharides could form micelles with a low polydispersity index (PDI), thereby improving uniformity in drug distribution [[72]27–[73]29]. Additionally, mannose and rhamnose residues in PNP-2 may contribute to specific immune-targeting pathways [[74]30–[75]32]. These compositional and structural differences likely affect the self-assembly behavior of polysaccharides in solution and their interaction with PPI. Therefore, selecting an appropriate polysaccharide matrix requires a comprehensive evaluation of MW, sugar composition, and solution behavior, guiding the subsequent optimization of polysaccharide micelles. Table 1. Molecular weight and monosaccharide composition of polysaccharides PNP1-4 Polysaccharide Molecular Weight (Da) Sugar composition PNP-1 256.45 Glucose, Galactose, Arabinose PNP-2 3589.22 Glucose, Mannose, Rhamnose PNP-3 4187.94 Glucose, Mannose, Galactose PNP-4 9549.93 Glucose, Galactose, Glucuronic Acid [76]Open in a new tab Synthesis and characterization of modified polysaccharides DC was conjugated to the polysaccharides through ester bond formation between the carboxyl group of DC and the hydroxyl groups of the polysaccharides, as illustrated in Fig. [77]1A. The ^1H NMR spectra revealed characteristic shifts in PNP-2 between δ 2.9–5.5 ppm, attributable to -CH[2]- and -CH- of the polysaccharide backbone. New peaks at δ 0.5–1.3 ppm in the PNP-2-DC spectrum were identified as -CH[3] and -CH[2]- from DC, indicating successful conjugation (Fig. [78]1B, Fig. [79]S1-[80]S4). Furthermore, FTIR analysis showed the disappearance of -COOH signal (1700–1750 cm⁻¹) and the emergence of ester carbonyl stretching vibrations (1735–1750 cm⁻¹) post-DC modification, confirming ester bond formation (Fig. [81]1C, Fig. [82]S5-[83]S8). Fig. 1. [84]Fig. 1 [85]Open in a new tab Synthesis and Characterization of Modified Polysaccharide Micelles. (A) Schematic representation of the esterification process for conjugating DC to polysaccharides. (B) ^1H NMR spectra of PNP-2-DC. (C) FTIR spectra of PNP-2-DC. (D) Representative images of particle sizes and appearance of PNP-1-DC micelles. (E) Representative images of particle sizes and appearance of PNP-2-DC micelles. (F) Representative images of particle sizes and appearance of PNP-3-DC micelles. (G) Representative images of particle sizes and appearance of PNP-4-DC micelles. (H) Schematic illustration of the conjugation of FA to PNP-2-DC. (I) ^1H NMR spectra of FA-PNP-2-DC. (J) FTIR spectra of FA-PNP-2-DC The DC-modified polysaccharides exhibited amphiphilic properties, enabling self-assembly into core-shell structured micelles in aqueous media. These DC-modified polysaccharides (PNP-1 ~ 4-DC) successfully formed micelles with distinct particle sizes and PDI values, as summarized in Table [86]2 and visualized in Fig. [87]1D-G. The laser irradiation results show a significant Tyndall effect, indicative of good dispersion stability. Notably, PNP-2-DC formed micelles with an average size of 99.77 nm and a low PDI of 0.1123, suggesting superior uniformity. Micelles of this size (~ 100 nm) are optimal for passive targeting of inflamed arthritic tissue via the enhanced permeability and retention (EPR) effect, with a PDI of less than 0.2 indicating high colloidal stability suitable for drug delivery applications [[88]33]. To further evaluate the self-assembly behavior of these micelles, the critical micelle concentration (CMC) of each formulation was determined. As shown in Supporting Information Table [89]S1, PNP-1-DC, PNP-2-DC, PNP-3-DC, and PNP-4-DC exhibited CMC values of 0.0542, 0.0294, 0.0313, and 0.0352 mg/mL, respectively. These results indicate that all four types of micelles could spontaneously form in aqueous solution at low concentrations, with PNP-2-DC showing the lowest CMC. This suggests enhanced thermodynamic stability and strong self-assembling ability, further supporting its selection as the optimal carrier. Using DC-modified polysaccharides as carriers, PNP micelles encapsulated PPI with encapsulation efficiencies as follows: PNP-1-DC (71.02 ± 7.16)%, PNP-2-DC (93.65 ± 3.81)%, PNP-3-DC (87.67 ± 3.11)%, and PNP-4-DC (57.08 ± 1.48)%. PNP-2-DC micelles showed the highest encapsulation efficiency and optimal size, highlighting their potential as drug delivery carriers. Table 2. Particle size and polydispersity index (PDI) of modified polysaccharide micelles (mean ± SD) (n = 3) Micelle Diameter (nm) PDI PNP-1-DC 77.72 ± 1.51 0.1877 ± 0.0085 PNP-2-DC 99.77 ± 7.99 0.1123 ± 0.0265 PNP-3-DC 185.83 ± 8.56 0.1147 ± 0.0588 PNP-4-DC 265.60 ± 5.22 0.2283 ± 0.0153 [90]Open in a new tab Synthesis and characterization of FA-PNP-Ms To further improve targeting efficiency, FA was conjugated to the PNP-2-DC micelles, leveraging the folate receptor-mediated active targeting mechanism [[91]34]. The synthetic pathway is illustrated in Fig. [92]1H. The ^1H NMR spectra of FA-PNP-DC micelles revealed aromatic peaks at δ 6.5–8.5 ppm, characteristic of the FA moiety, while FTIR spectra showed revealed characteristic stretching vibrations of the -COOH from FA in the range of (1700–1750 cm⁻¹), which were significantly absent in the FA-PNP-DC spectrum. Additionally, compared to PNP-DC, the FA-PNP-DC exhibited new absorption peaks between (1450–1600 cm⁻¹), corresponding to the aromatic ring (C = C) stretching vibrations of FA. The carbonyl stretching vibrations associated with ester bonds also showed noticeable shifts and changes in intensity around (1735–1750 cm⁻¹). These spectral changes confirm the successful conjugation of FA onto the polysaccharide backbone via esterification, indicating the successful modification of the polymer (Fig. [93]1I and J). The dual-functionalized FA-PNP-DC micelles exhibited amphiphilic properties and improved targeting potential towards inflamed tissues. Structural characterization of micelles FA-PNP-2-DC micelles encapsulating PPI exhibited distinct solubility characteristics in aqueous solutions, as shown in Fig. [94]2A. Free PPI appeared as a suspension with visible white particulates due to its poor water solubility. In contrast, the PPI-Ms formed a pale-yellow solution, while the FA-modified FA-PPI-Ms resulted in a clear, stable yellow solution, indicating that micellar encapsulation significantly improved the solubility of PPI in water. SEM analyses revealed that PNP-2 existed in an amorphous form (Fig. [95]2B), while FA-PNP-2-DC micelles adopted a spherical morphology post-self-assembly (Fig. [96]2C-D). FTIR spectra of the formulation (Fig. [97]2E) showed significant alterations in the characteristic peaks of PPI. The peaks associated with PPI, particularly in the regions of (1250–1500 cm⁻¹) and (2800–3000 cm⁻¹), exhibited a marked decrease in intensity or disappeared entirely. These changes in both peak intensity and shape suggest that PPI was successfully encapsulated within the micelles. The encapsulation likely restricted the molecular vibrations of PPI, preventing the expression of its original infrared characteristics. DLS measurements confirmed an increase in micelle size from (107.67 ± 2.83) nm (PPI-Ms) to (115.93 ± 9.46) nm (FA-PPI-Ms) upon FA modification, with PDI of 0.0950 ± 0.0832 and 0.1580 ± 0.0827, respectively (Fig. [98]2F-I). Additionally, Zeta potential measurements indicated a surface charge shift from (-3.40 ± 0.70) mV to (-11.23 ± 1.83) mV after FA modification (Fig. [99]2J-L; Table [100]3), promoting stability and reduced plasma protein interaction, essential for minimizing aggregation and enhancing targeted delivery [[101]35]. Fig. 2. [102]Fig. 2 [103]Open in a new tab Characterization and Evaluation of Micelles. (A) Visual appearance of free PPI, PPI-Ms, and FA-PPI-Ms in aqueous solutions. (B) SEM image of PNP-2, scale bar = 2 μm. (C) SEM image of FA-PPI-Ms, scale bar = 2 μm. (D) TEM image of FA-PPI-Ms, scale bar = 50 nm. (E) FTIR spectra of FA-PPI-Ms. (F) Quantitative analysis of particle size of the varying micelles. (G) Quantitative analysis of PDI value of the varying micelles. (H) Representative images of particle sizes of PPI-Ms. (I) Representative images of particle sizes of FA-PPI-Ms. (J) Quantitative analysis of zeta potential of the varying micelles. (K) Representative images of zeta potential of PPI-Ms. (L) Representative images of zeta potential of FA-PPI-Ms. (M-N) Changes in particle size (M) and PDI (N) of FA-PPI-Ms were quantitatively analyzed after 30 d of placement at 4 °C. (O) Critical micelle concentration (CMC) of FA-PPI-Ms. (P) In vitro release profile of PPI from FA-PPI-Ms, PPI-Ms and free PPI. All data are presented as mean ± SD (n = 3) Table 3. Particle size, PDI, and zeta potential of micelles (mean ± SD) (n = 3) Micelles Diameter (nm) PDI Zeta potential (mV) PPI-Ms 107.67 ± 2.83 0.0950 ± 0.0832 -3.40 ± 0.70 FA-PPI-Ms 115.93 ± 9.46 0.1580 ± 0.0827 -11.23 ± 1.83 [104]Open in a new tab EE%, stability, and drug release profile The PPI-Ms demonstrated an EE of (93.09 ± 5.76)%, while the FA-PPI-Ms showed a slightly lower EE of (92.70 ± 5.40)%, indicating a high drug-loading capacity for both formulations. The stability of FA-PPI-Ms was further assessed over 30 days. The EE of FA-PPI-Ms decreased gradually to (87.39 ± 0.83)% at day 10, (85.78 ± 0.74)% at day 20, and (82.95 ± 4.28)% at day 30, suggesting good long-term stability (Table [105]4). Throughout the storage period, the EE remained above 80%, with the micelle size consistently ranging between 100 and 200 nm (Fig. [106]2M) and a PDI maintained around 0.2 (Fig. [107]2N). These results highlight the robust stability and efficient drug-loading capability of FA-PPI-Ms, even under extended storage conditions. Moreover, FA-PPI-Ms demonstrated favorable colloidal stability in PBS containing 10% FBS at 37 °C, with particle size increasing moderately from (115.93 ± 9.46) nm at 0 h to (143.81 ± 4.26) nm at 48 h, suggesting good serum stability for systemic administration (Supporting Information Fig. [108]S14). The low critical micelle concentration (CMC) of 0.0263 mg/mL for FA-PNP-2-DC, determined using pyrene as a fluorescence probe, underscores its stability against dilution in physiological environments (Fig. [109]2O). Table 4. Stability of micelles (mean encapsulation efficiency % ± SD) (n = 3) Micelles 0 d 10 d 20 d 30 d PPI-Ms 93.09 ± 5.76 89.77 ± 2.02 85.99 ± 0.10 83.60 ± 3.41 FA-PPI-Ms 92.70 ± 5.40 87.39 ± 0.83 85.78 ± 0.74 82.95 ± 4.28 [110]Open in a new tab In vitro release studies revealed a rapid burst release for free PPI, with (86.79 ± 2.45)% of the drug released within the first 4 h (Fig. [111]2P). In contrast, FA-PPI-Ms exhibited a much more controlled release pattern, with only (33.98 ± 2.31)% of PPI released in the initial 2 h and achieving a cumulative release of (78.22 ± 12.77)% over a 48-hour period. This delayed release profile of FA-PPI-Ms suggests their potential for sustained drug delivery, making them a promising option for the prolonged therapeutic management of chronic inflammatory diseases such as arthritis. Cell targeting and cellular uptake of Ms In the synovial fluid of patients with RA, a significant accumulation of inflammatory M1 macrophages is observed [[112]36]. These M1 macrophages not only release pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), thereby exacerbating the inflammatory response, but also upregulate the expression of FRβ on their cell surfaces [[113]25]. Consequently, FRβ has emerged as an attractive target for the development of targeted drug delivery systems for RA therapy [[114]24, [115]37]. In this study, we utilized LPS to stimulate RAW 264.7 cells to differentiate into M1 macrophages, thereby establishing an in vitro model that mimics the in vivo inflammatory microenvironment. Given that FA can specifically bind to FRβ, we selected FA as the targeting ligand for FA-PPI-Ms to enhance the delivery efficiency of drugs to M1 macrophages in inflamed joints. Coumarin (Cou), a fluorescent probe, was used to label the different micelle formulations, and their uptake capacity was evaluated using fluorescence microscopy. In unstimulated and LPS-stimulated macrophages, no green fluorescence was detected in the blank control group, whereas pronounced green fluorescence was observed in the culture medium containing the drug-loaded micelles. As illustrated in Fig. [116]3, there was no significant difference in fluorescence intensity between Cou-Ms and FA-Cou-Ms in unactivated RAW264.7 macrophages. This is attributed to the low expression of FRβ on the cell membrane of resting macrophages, which limits the cellular uptake of FA-PPI-Ms. In contrast, in LPS-activated RAW264.7 macrophages, FA-Cou-Ms exhibited the strongest green fluorescence. This phenomenon is due to the upregulation of FRβ in activated macrophages, coupled with the high affinity of FA for FRβ, leading to increased cellular uptake of the micelles. Moreover, when free FA was added to the culture medium, the cellular uptake of FA-Cou-Ms was significantly reduced. This reduction is likely because free FA competes with FA-Cou-Ms for binding to FRβ on the cell surface, thereby decreasing the internalization of FA-Cou-Ms by the cells. Quantitative analysis via flow cytometry confirmed the cellular uptake efficiency of various micelle formulations, with results consistent with those observed under fluorescence microscopy. Collectively, these findings demonstrate that FA-Cou-Ms exhibit excellent targeting ability toward inflammatory macrophages. Fig. 3. [117]Fig. 3 [118]Open in a new tab Cellular uptake of different formulations on activated/non-activated RAW264.7 cells. (A) Representative fluorescence microscope images of RAW264.7 cells treated with different formulations on activated/non-activated RAW264.7 cells, Scale = 50 μm; (B) Cellular uptake of activated/non-activated RAW264.7 cells incubated with different formulations. (C) Quantitative analysis of fluorescence intensity treated with different formulations of micelles to activated/non-activated macrophages. (D) Quantitative analysis of cellular uptake of activated/non-activated RAW264.7 cells treated with different formulations. Data are presented as mean ± SD (n = 3). One-way ANOVA with Bonferroni’s test was used for statistical comparison. ^*P < 0.05 In vitro cytotoxicity against RAW264.7 cells To ensure the safety of subsequent administration, CCK-8 method was used to detect the survival rate of activated RAW264.7 cells treated with free PPI and different micelles under the same concentration gradient of PPI (Fig. [119]S9). The cytotoxicity of free PPI was significantly higher than that of the other groups. The PNP-MS without PPI had the lowest cytotoxicity, indicating that when delivering drugs, it was mainly PPI that exerted the therapeutic effect on inflammatory macrophages, while PNP as a membrane material did not add additional toxicity. Among the micelles with different formulations, FA-PPI-MS showed the strongest toxicity to inflammatory macrophages, which was since the FA ligand in FA-PPI-MS can bind to the overexpressed FRβ on inflammatory macrophages. Therefore, FA-PPI-MS directly targets inflammatory macrophages and increases the drug delivery efficiency of PPI. Apoptosis is an orderly programmed cell death mode, which is crucial for biological evolution, body homeostasis maintenance and phylogeny. The process includes cell morphological changes and chromosomal DNA damage. We investigated the effects of different formulations of micelles on the apoptosis of activated RAW264.7 macrophages. As shown in Fig. [120]S10, the apoptosis rate of RAW264.7 cells treated with PBS was 6.33%±0.68%, the apoptosis rate of RAW264.7 cells treated with free PPI was 28.99%±1.46%, and the apoptosis rate of RAW264.7 cells treated with PNP-Ms was 11.19%±0.58%. The apoptosis rate was 16.52%±2.15% in PPI-Ms group and 18.17%±2.65% in FA-PPI-Ms group. These results were consistent with the cytotoxicity assay, and once again confirmed that FA-PPI-Ms is of excellent targeting properties. We performed live/dead cell assays using calcein-AM and PI staining to visually assess cell death induced by the different formulations. Live cells labeled with calcein-AM emit green fluorescence, whereas the nuclei of PI-labeled dead cells emit red fluorescence. As shown in Fig. [121]S11, the cell death rate was increased in the different micelle treatment groups compared with the PBS group, and the FA-PPI-Ms group had the highest cell death rate. This result is consistent with previous cytotoxicity assay and apoptosis results. Effects of FA-PPI-Ms on ROS, inflammatory factors and polarization of RAW264.7 cells in vitro The development of RA is often associated with an imbalance in oxidative stress, leading to a significant accumulation of ROS [[122]38]. This increase in ROS contributes to the secretion of pro-inflammatory cytokines, thereby amplifying the inflammatory response and ultimately resulting in cartilage destruction and bone erosion [[123]39–[124]41]. Consequently, the reduction of ROS is essential for suppressing the inflammatory response and providing joint protection. Our results indicate that ROS levels, as well as the expression levels of TNF-α and IL-1β, were significantly elevated in RAW264.7 cells following LPS stimulation, while the expression levels of IL-10 and transforming growth factor-beta (TGF-β) were significantly diminished. Following a series of micelle incubations, ROS levels were notably reduced, along with a simultaneous decrease in the expression levels of pro-inflammatory factors TNF-α and IL-1β. Among the tested micelles, FA-PPI-Ms demonstrated the most effective reduction in both ROS and pro-inflammatory factor expression in LPS-induced RAW264.7 cells. Additionally, FA-PPI-Ms significantly enhanced the expression level of the anti-inflammatory factor TGF-β (Fig. [125]4A-F). These findings suggest that FA-PPI-Ms effectively clears intracellular ROS and improves the inflammatory microenvironment, which is critical for mitigating the progression of RA. Fig. 4. [126]Fig. 4 [127]Open in a new tab Anti-inflammatory and macrophage polarization activity in vitro. (A) Scavenging effects of varying formulations on ROS, scale bar = 50 μm; (B) Statistical analysis of relative expression of ROS treated with different formulations. (C-F) Levels of IL-1β, TNF-α, IL-10 and TGF-β in cell culture supernatant after treating with different formulations. (G) Representative fluorescence images of CD86 staining of values with varying formulations, scale bar = 50 μm; (H) Representative fluorescence images of CD206 staining of values with varying formulations, scale bar = 50 μm; (I) Analysis of relative fluorescence intensity in (G); (J) Analysis of relative fluorescence intensity in (H); (K) Flow cytometric histograms of proportion of M1 macrophages in different formulations; (L) Flow cytometric histograms of proportion of M2 macrophages in different formulations; (M) Quantitative analysis of proportion of M1 macrophages in different formulations; (N) Quantitative analysis of proportion of M2 macrophages in different formulations. Data are presented as mean ± SD (n = 6). One-way ANOVA with Bonferroni’s test was used for statistical comparison. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 Macrophages are well known for their high heterogeneity as immune cells, which can be polarized into M1 and M2 phenotypes depending on the tissue microenvironment and pathological conditions [[128]42]. The imbalance between M1 and M2 macrophages plays a crucial role in the onset and progression of RA [[129]43]. M1 macrophages express proteins such as CD86 and iNOS, secrete pro-inflammatory factors like TNF-α and IL-1β, adversely affecting chondrocyte synthesis and catabolism, inhibiting chondrogenesis, and promoting the development or exacerbation of RA [[130]44]. In contrast, M2 macrophages express insulin-like growth factor-1 (IGF-1) and CD206, producing anti-inflammatory factors, including IL-10 and TGF-β, which are involved in the anti-inflammatory response, promote tissue repair, and inhibit or delay the progression of RA [[131]45]. Therefore, promoting the transformation of macrophages from the M1 to the M2 phenotype represents a promising treatment strategy for RA. Our results confirm that FA-PPI-Ms can inhibit the secretion of TNF-α and IL-1β in RAW264.7 cells while promoting the secretion of TGF-β. However, it remains unclear whether FA-PPI-Ms exert these effects by regulating macrophage polarization. To investigate this, we induced M1 polarization in RAW264.7 cells using LPS and assessed the ratio of the M1 marker CD86 to the M2 marker CD206 through immunofluorescence staining, thereby evaluating the effects of various micelles on reversing LPS-induced M1 polarization. The results indicated a significant increase in the proportion of CD86-positive RAW264.7 cells treated with LPS. Although the proportion of CD206-positive cells also increased slightly due to stress, this change was not statistically significant, highlighting a notable imbalance between M1 and M2 macrophages. Interestingly, PNP-Ms, PPI-Ms, and FA-PPI-Ms all significantly reversed these alterations, leading to a substantial reduction in the proportion of CD86-positive cells and a significant increase in CD206-positive cells, with the FA-PPI-Ms group demonstrating the most pronounced effect on these indicators (Fig. [132]4G-J). Flow cytometry results further corroborated the impact of FA-PPI-Ms on the repolarization of LPS-treated macrophages, with quantitative analysis revealing that FA-PPI-Ms significantly reduced the proportion of CD86 and increased the proportion of CD206 (Fig. [133]4K-N). Furthermore, to strengthen these observations, we additionally measured the expression of two key polarization-related genes, inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1). The results showed that LPS stimulation markedly upregulated iNOS expression and downregulated Arg-1, confirming M1 polarization. Treatment with FA-PPI-Ms reversed this trend by significantly reducing iNOS and elevating Arg-1 levels, thereby further validating their role in promoting M2 polarization (Supporting Information Fig. [134]S15). These findings suggest that FA-PPI-Ms may exert their anti-inflammatory effects by facilitating the polarization of macrophages from M1 to M2. Biodistribution of different micelles in CIA rats In this study, DiR fluorescent probes were used instead of PPI to be encapsulated into micelles, and the accumulation and distribution of different micelles in CIA rats were detected by intravenous administration and observation under the real-time fluorescence imaging system. In the saline group, no fluorescent signal was detected throughout imaging. Free DiR rapidly undergoes drug metabolism and clearance from the blood circulation after injection, and its fluorescent signal is barely detectable at the site of arthritis. Due to the passive targeting of micelles, both DiR-Ms and FA-DiR-Ms showed clear fluorescence signals at the site of joint inflammation. However, the intensity and duration of the fluorescence signal of DiR-Ms were less significant than that of FA-DiR-Ms (Fig. [135]5B). This is due to the presence of a large number of inflammatory M1 macrophages at the site of joint inflammation, the overexpression of FRβ on M1 macrophages, and the ability of the FA ligand of FA-DiR-Ms to specifically bind to FRβ and actively target M1 macrophages, which results in a more efficient enrichment of FA-DiR-Ms in inflamed joints. Fig. 5. [136]Fig. 5 [137]Open in a new tab Targeting behavior and anti-arthritic efficacy in CIA rats. (A) Schematic illustration of experimental timeline progression; (B) In vivo fluorescence real-time imaging observation of CIA rats treated with varying formulations; (C) Effect of varying formulations on inflammatory cytokines in the serum of CIA rats; (D) Thermographic map depicting footprint pressure in CIA rats; (E) 2D representation of plantar pressure in rats; (F) 3D visualization of plantar pressure in rats. Data are presented as mean ± SD (n = 6). Statistical significance was determined using one-way ANOVA with Dunnett’s test. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 Effect of different micelles on gait parameters We evaluated the effects of different formulations in terms of pain relief and the return to normal walking patterns of rats. The greater the weight-bearing capacity of the rats, the less severe the joint pain. As shown in Fig. [138]5D-F, CIA rats showed a significant decrease in paw pressure compared with normal rats, indicating reduced weight-bearing capacity and greater joint pain. Paw pressure gradually recovered after treatment in different drug groups, among which FA-PPI-MS had the best recovery, indicating that FA-PPI-MS can more effectively deliver drugs to the inflammatory joints due to the modified FA ligand, which can reduce joint pain and improve the walking pattern of rats. Therapeutic effects on CIA rats The collagen-induced arthritis (CIA) model exhibits the same immunological and pathological characteristics as RA, including chronic, symmetrical, and polyarticular lesions [[139]46, [140]47]. These features make the CIA model an ideal choice for studying the pathogenesis of RA and screening potential therapeutic agents. Successful CIA induction in rats is indicated by symptoms such as joint swelling, weight loss, reduced fur glossiness, slight hair loss, joint redness and deformity, and impaired mobility. As seen in Fig. [141]6A, the model group exhibited slow weight gain due to reduced activity and food intake caused by joint swelling and pain. In the treatment groups, the free PPI group showed better weight gain than the model group, but due to the adverse effects of PPI, the weight remained at a relatively low level, with slower weight gain compared to the different micelle treatment groups. By day 21 of treatment, the FA-PPI-Ms group showed a significant increase in body weight. Additionally, the FA-PPI-Ms group showed a significant reduction in paw volume and arthritis index (Fig. [142]6B-C). Fig. 6. [143]Fig. 6 [144]Open in a new tab The degree of joint swelling and changes in bone structure in CIA rats. (A) Body weight changes in arthritic rats monitored over time for each group; (B) Measurement of paw thickness in arthritic rats over time for each group; (C) Temporal assessment of arthritis scores in arthritic rats over time for each group; (D) Illustrative images of hindlimbs corresponding to each experimental group; (E) Representative X-ray images of CIA rats in the different groups; (F) Representative micro-CT images of ankle joints and knee joints corresponding to each experimental group. Data are presented as mean ± SD (n = 6) The degree of joint swelling is a key indicator for assessing the severity of arthritis in CIA rats and can be used as a preliminary basis for judging the effectiveness of treatment. We measured joint swelling by observing the degree of joint redness and swelling at the end of treatment and by statistically analyzing changes in paw volume and arthritis index. Representative images of the hind paws of CIA rats after treatment showed that the FA-PPI-Ms group had significantly reduced hind paw swelling and skin color close to normal, with no obvious redness compared to the model group (Fig. [145]6D). Clearly, the excellent therapeutic effect of FA-PPI-Ms is related to its FA ligand’s precise targeting ability towards M1 macrophages at the site of joint inflammation. To better evaluate the anti-inflammatory effect of FA-PPI-Ms, we measured cytokine levels in the serum of CIA rats (Fig. [146]5C) and found that FA-PPI-Ms significantly downregulated inflammatory cytokines TNF-α and IL-1β, and upregulated anti-inflammatory cytokines TGF-β and IL-10. This result further confirmed that FA-PPI-Ms can significantly reduce immune response and effectively alleviate arthritis symptoms. Bone destruction is a major contributor to RA disability and loss of joint function, and can be an important clinical indicator for assessing the severity of arthritis [[147]48, [148]49]. Subsequently, X-ray and micro-CT were used to conduct an in-depth evaluation of CIA rats receiving different treatments. As shown in Fig. [149]6E-F, the model group rats exhibited significant bone damage in the knee and ankle joints, characterized by rough joint surfaces and unclear bone boundaries. After treatment with free PPI, bone boundaries improved, but joint surfaces remained uneven, and bone damage persisted. Treatment with PNP-Ms and PPI-Ms resulted in clearer bone boundaries and partially restored smooth joint surfaces, with significant reduction in bone damage. However, in CIA rats treated with FA-PPI-Ms, there was a notable restoration of bone structural integrity, characterized by clear bone boundaries and relatively smooth joint surfaces. These results indicate that FA-PPI-Ms can effectively repair bone erosion caused by RA. Furthermore, quantitative evaluation of bone damage in each treatment group was performed through histomorphometric analysis of micro-CT images (Fig. [150]S12). The results showed that, compared to the model group, FA-PPI-Ms significantly increased BMD, BV/TV, BS/TV, Tb.N, and Tb.Th, while reducing Tb.Sp. These findings reveal the strong potential of FA-PPI-Ms in inhibiting the progression of RA and improving bone deterioration and cartilage erosion. Histological analysis In the macroscopic aspect, FA-PPI-Ms reduced the degree of swelling in CIA rats, which was further analyzed by staining and microscopic analysis of ankle and knee joints. HE staining showed that joints in the model group were destroyed by hyperplastic synovial tissue without obvious joint boundaries (Fig. [151]7A, D). The joint tissue was infiltrated by a large number of inflammatory cells. After free PPI treatment, the joint damage was slightly improved, but synovial hyperplasia and inflammatory cell infiltration were still serious. After PNP-Ms and PPI-Ms treatment, the joints showed a moderate degree of immune cell infiltration and synovial hyperplasia, and the joint boundaries gradually became clear. Notably, after FA-PPI-Ms treatment, the pathological state of the joint cavity in CIA rats was significantly changed and tended to the normal joint cavity state, which was characterized by clear bone boundary, minimal immune cell infiltration and disappearance of synovial hyperplasia. To further assess the extent of cartilage tissue degradation and destruction, Safranin O-fast green staining was performed on joint sections (Fig. [152]7B, E). In the knee and ankle joints of the normal control rats, the cartilage tissue showed clear staining after safranin O staining, and the cells were very closely arranged. In contrast, articular cartilage of RA rats showed marked degeneration, with a large reduction of glycosaminoglycan components in the cartilage matrix. Cartilage degradation was significantly attenuated by FA-PPI-Ms in different treatment groups, suggesting that FA-PPI-Ms partially reversed the degenerative changes in RA. Finally, toluidine blue staining was performed on the knee and ankle joints (Fig. [153]7C, F). The cartilage tissue appeared dark blue, and the chondrocytes appeared blue-purple due to proteoglycan secretion. Compared with the blank control group, the cartilage tissue of the model group showed cartilage surface exfoliation, significantly reduced staining depth, and looser cell arrangement. After treatment with different nano-formulations, the preservation of cartilage was significantly improved. In particular, the treatment effect of FA-PPI-Ms group was significant, the cartilage surface remained almost intact and smooth, the proteoglycan distribution in the bone cavity also became uniform, and the blue-purple color of the cartilage area after staining was more distinct. The results of histological analysis were consistent with the results of arthritis score and micro-CT examination, which indicated that FA-PPI-Ms had a significant therapeutic effect on RA. Fig. 7. [154]Fig. 7 [155]Open in a new tab Histological analysis of the knee and ankle joints of rats. (A) HE staining of knee cartilage; (B) Safranin O staining of knee cartilage; (C) Toluidine blue staining of knee cartilage; (D) HE staining of ankle cartilage; (E) Safranin O staining of ankle cartilage; (F) Toluidine blue staining of ankle cartilage, scale bar = 100 μm (n = 6) The regulatory effect of FA-PPI-Ms on macrophage polarization in vivo Given the remarkable ability of FA-PPI-Ms to restore the M1/M2 macrophage balance in vitro, we visually examined the polarization of macrophages in the joints of rats using immunofluorescence. As illustrated in Fig. [156]8A-D, compared to the rats of control group, the expression level of the M1 macrophage-specific biomarker (CD86, green) in the inflammatory joints of the model group was significantly elevated (Fig. [157]8A, C), while the expression levels of PPI and PNP were slightly decreased. Conversely, the expression level of the M2 macrophage-specific biomarker (CD206, green) was slightly increased (Fig. [158]8B, D). Notably, treatments with PPI-Ms and FA-PPI-Ms exhibited a more pronounced phenotypic shift from M1 to M2, which contributed to their strong anti-inflammatory effects. Additionally, we employed qRT-PCR to assess the expression of CD86 and CD206 mRNA in the synovium of each group of rats. The results were consistent with those of immunofluorescence staining, confirming that FA-PPI-Ms had the ability to restore the balance of M1/M2 macrophages. Fig. 8. [159]Fig. 8 [160]Open in a new tab Effect on macrophage polarization in CIA rats with varying formulations. (A-B) Images of immunofluorescence results of CD86 and CD206 in knee synovial tissues after treatment; (C-D) Images of immunofluorescence results of CD86 and CD206 in ankle synovial tissues after treatment, scale bar = 50 μm; (E-F) qRT-PCR analysis of CD86 and CD206 mRNA expression in knee synovial tissues after treatment. Data are presented as mean ± SD (n = 6). One-way ANOVA followed by Bonferroni’s test was used. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 Safety evaluation Although micellar carriers are expected to enhance the targeting ability of drugs and reduce toxic side effects, it is still necessary to conduct comprehensive safety evaluation in rats after administration. After treatment, the safety of different micelles was investigated by pathological examination of various organs, blood routine examination and liver and kidney function indexes. HE images showed that no obvious tissue damage and pathological abnormalities were observed in the micellar groups with different formulations (Fig. [161]S13A). Moreover, the levels of BUN, Crea, ALT and AST in serum of rats showed no statistically significant differences among different preparation groups (Fig. [162]S13B), indicating that FA-PPI-Ms had good biocompatibility in vivo. Transcriptomic analysis of FA-PPI-Ms regulates macrophage polarization To further understand the potential regulatory mechanism of FA-PPI-Ms on macrophage polarization, we conducted transcriptomic sequencing analysis of RAW264.7 cells stimulated by LPS in PBS and FA-PPI-Ms treatment groups. The results are illustrated in Fig. [163]9A present a volcanic map highlighting the significantly different genes across each sample group. In comparison to the model group with the FA-PPI-Ms group, 54 differential genes were identified, including 33 up-regulated and 21 down-regulated genes. The expression pattern cluster (heat map) of these significantly different genes is depicted in Fig. [164]9B. To elucidate the underlying biological processes, gene ontology (GO) analysis was conducted for the differentially expressed genes in the Model and FA-PPI-Ms treatment groups. The GO analysis revealed that biological processes are primarily associated with biological regulation, cellular processes, reproductive processes, and responses to stimuli. Cellular components predominantly include cell parts, cell membranes, and organelles, while molecular functions mainly encompass catalytic activity, molecular function regulation, and molecular transducer activity (Fig. [165]9C-D). Furthermore, through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the cell signaling pathways regulated by these significantly different genes were examined (Fig. [166]9E-F), including the Ribosome, Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, Nitrogen metabolism, and Th1 and Th2 cell differentiation, among others. Notably, the JAK-STAT pathway is crucial in regulating macrophage polarization by FA-PPI-Ms, as it specifically targets the mechanisms underlying macrophage polarization. Studies have demonstrated that the JAK2-STAT3 signaling pathway, a critical component of the JAK-STAT pathway, plays a significant role in regulating macrophage polarization [[167]50, [168]51]. Activation of the JAK2-STAT3 pathway promotes the polarization of macrophages toward the M1 phenotype, resulting in an imbalance between M1 and M2 macrophages, which exacerbates synovial inflammation and tissue damage in RA model [[169]52, [170]53]. Fig. 9. [171]Fig. 9 [172]Open in a new tab Transcriptomic analysis elucidated the mechanism of action of FA-PPI-Ms in the treatment of RA. (A) Volcano plot of significantly differentially expressed genes between the model group and the FA-PPI-Ms group, where blue circles represent downregulated genes and red circles represent upregulated genes; (B) Heatmap of significantly differentially expressed genes in cells of the model group and the FA-PPI-Ms group, where deeper blue indicates lower expression levels and deeper red indicates higher expression levels; (C) Histogram of GO functional annotation of significantly differentially expressed genes between the model group and the FA-PPI-Ms group; (D) Histogram of GO functional enrichment of significantly different genes between the model group and the FA-PPI-Ms group; (E) Bubble plot of GO functional enrichment of significantly differentially expressed genes between the model group and the FA-PPI-Ms group; (F) Histogram of KEGG pathway enrichment of genes with significant differences between the model group and the FA-PPI-Ms group; (G-H) To validate the transcriptomics results, qRT-PCR was employed to measure the mRNA expression levels of JAK (G) and STAT3 (H) in RAW264.7 cells; (I) Western blot was utilized to assess the expression levels of proteins associated with the JAK2-STAT3 signaling pathway; (J-K) Statistical analysis of the ratios of p-JAK2/JAK2 (J) and p-STAT3/STAT3 (K) was conducted. Data are presented as mean ± SD (n = 3), ^*P < 0.05, ^**P < 0.01 We further validated the transcriptomic findings using qRT-PCR, which demonstrated a consistent trend. The mRNA expression levels of JAK2 and STAT3 were significantly elevated in the model group. Furthermore, mRNA expression levels of JAK2 and STAT3 were markedly reduced following treatment with FA-PPI-Ms (Fig. [173]9G-H). Additionally, we examined whether similar differential trends were observable at the protein level by Western blot. Compared to the control group, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 in the model group cells were significantly increased. Following incubation with FA-PPI-Ms, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were significantly decreased (Fig. [174]9I-K). These results suggest that FA-PPI-Ms may regulate macrophage polarization and mitigate the progression of RA by inhibiting the activation of the JAK2-STAT3 signaling pathway. FA-PPI-Ms restrained the activation of JAK2-STAT3 signaling pathway in RAW264.7 cells to reverse M1/M2 imbalance JAK2 plays a crucial role in cytokine receptor signaling by phosphorylating STAT3, a significant member of the STAT protein family, which is commonly involved in regulating macrophage polarization in RA [[175]54, [176]55]. The overactivation of STAT3 can lead to the disruption of downstream genes, promote the release of pro-inflammatory cytokines such as TNF-α and IL-6, and enhance the differentiation of macrophages into the M1 phenotype [[177]56]. This exacerbates the levels of pro-inflammatory factors in the joint, intensifying the inflammatory response and creating a vicious cycle that hinders the recovery of bone tissue [[178]1]. Inhibiting the JAK2/STAT3 pathway can suppress the polarization of M1 macrophages, promote the polarization of M2 macrophages, and slow the progression of RA [[179]57]. Based on the above results, we hypothesize that FA-PPI-Ms may modulate macrophage polarization, at least in part, through the JAK2-STAT3 signaling pathway to mitigate the progression of RA. To further validate this hypothesis, we introduced the JAK2 selective inhibitor AG490 to LPS-activated RAW264.7 cells with/without FA-PPI-Ms (Fig. [180]10). Our results indicated that FA-PPI-Ms exhibited effects comparable to those of AG490 in regulating macrophage polarization and inflammatory cytokine levels. Notably, when the JAK2-STAT3 signaling pathway in RAW264.7 cells was partially inhibited by AG490, the regulatory effects of FA-PPI-Ms on macrophage polarization were nearly abolished, demonstrating that AG490 effectively blocked the actions of FA-PPI-Ms (Fig. [181]10A-O). Thus, it can be reasonably concluded that FA-PPI-Ms may regulate macrophage polarization, at least partially, through the JAK2-STAT3 signaling pathway, thereby improving the inflammatory microenvironment and delaying the progression of RA. Fig. 10. [182]Fig. 10 [183]Open in a new tab Validation of the mechanism by which FA-PPI-Ms reverse M1/M2 imbalance. (A-B) qRT-PCR analysis of CD86 and CD206 mRNA expression on activated RAW264.7 cells treated with different formulations; (C-D) Flow cytometric histograms of proportion of M1and M2 macrophages in different formulations; (E-F) Quantitative analysis of proportion of M1 and M2 macrophages in different formulations; (G-H) Representative fluorescence images of CD86 and CD206 staining of values with varying formulations, scale bar = 50 μm; (I-J) Analysis of relative fluorescence intensity in (G-H); (K-L) qRT-PCR analysis of JAK2 and STAT3 mRNA expression on activated RAW264.7 cells treated with different formulations; (M) Western blot analysis of JAK2-STAT3 signalling axis-related protein expression. (N-O) Semi-quantitative analysis of p-JAK2/ JAK2 and p-STAT3/ STAT3 levels by Image J, Data are presented as mean ± SD (n = 3). One-way ANOVA followed by Bonferroni’s test was used. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 Conclusion In summary, we successfully synthesized PNP nanocarriers by coupling a series of PNP with deoxycholic acid across various molecular weight intervals. We determined the optimal molecular weight range of PNP that facilitates the formation of PNP-Ms with the best physical and chemical properties. This discovery not only establishes a stable foundation for further research but also positions PNP as a promising material for the development of nanodrug carriers. Subsequently, we developed FA-PPI-Ms with macrophage-targeting properties aimed at slowing the progression of RA through macrophage reprogramming strategies. Our results demonstrate that FA-PPI-Ms effectively clear ROS and inhibit the inflammatory response, exhibiting good biosafety. Notably, FA-PPI-Ms exhibited the most significant therapeutic effects in the knee and ankle joints of RA rats, as evidenced by marked reductions in joint swelling, arthritis scores, bone erosion areas, and joint pathological injury. Mechanistically, FA-PPI-Ms inhibited the JAK2/STAT3 signaling pathway and facilitated the reprogramming of M1 macrophages into M2 macrophages, thereby restoring the M1/M2 macrophage ratio and inhibiting the inflammatory response. However, several limitations should be acknowledged. First, the CIA rat model used in this study may not fully replicate the immunopathological features of human RA, potentially affecting the translatability of our findings. Second, the long-term biosafety, pharmacokinetic profile, large-scale manufacturing feasibility, and immunogenicity of FA-PPI-Ms in humans require further investigation prior to clinical translation. Nevertheless, our results strongly support the therapeutic potential of FA-PPI-Ms for RA and establish a promising platform for exploring macrophage-reprogramming-based strategies in inflammatory diseases. Experimental section Materials and reagents Polyphyllin I (PPI) Purchased from Chengdu Pufei De Biotech Co.,Ltd. with a purity of ≥ 98%. Water-Soluble Polysaccharides of Panax notoginseng (PNP) acquired from Shaanxi Yuzhou Biotechnology Co., Ltd. Deoxycholic Acid (DC), Folic Acid (FA), Galactose, Mannose, Glucuronic Acid, Galacturonic Acid, Ribose, Arabinose, 4-Dimethylaminopyridine (DMAP), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride (EDC·HCl), Anhydrous Dimethyl Sulfoxide (DMSO), These reagents were sourced from Shanghai Aladdin Biochemical Technology Co., Ltd. Rhamnose Purchased from Myrell Biotechnology Co., Ltd. All chemicals and reagents used were of analytical grade. JAK2, STAT3 and β-actin primers were purchased from Shanghai Shenggong Biological Engineering Co., LTD. SteadyPure universal RNA Extraction kit, Evo M-MLV reverse transcription Premix kit and SYBR Green Pro Taq HS Premix qPCR Kit were order to Hunan Accurate Biology Co., LTD. AG490 was purchased from MedChemExpress. Anti-JAK2 (phospho-Tyr1007/1008) antibody were purchased from MedChemExpress (New Jersey, USA). Anti-STAT3 (Phospho-Y705), anti-STAT3, anti-JAK2, anti-β-actin antibodies and HRP-labelled secondary antibody was obtained from Wuhan Boster Biotechnology Co., Ltd (Wuhan, China). RAW 264.7 cells were obtained from iCell Bioscience Inc. (Shanghai, China). SD rats (SPF-grade, male, aged: 6 weeks, weight: 140–160 g) were purchased from Liaoning Changsheng Biological Co., LTD. (Certificate No.: SCXK (Liao): 2023-0001). All procedures were conducted in accordance with the guidelines of the Liaoning University of Traditional Chinese Medicine Institutional Animal Care and Use Committee. This research was approved by the Liaoning University of Traditional Chinese Medicine Institutional Animal Care and Use Committee (NO. 210000420240204). Fractionation of polysaccharides from Panax notoginseng Dissolve PNP in deionized water at a concentration of 10 mg/mL. To remove proteins, perform the Sevag method by adding a mixture of chloroform and n-butanol (4:1, v/v) to the solution, then vortex and let it stand to allow phase separation. Repeat the extraction until no protein precipitate is visible between the phases. Retain the aqueous phase for subsequent ultrafiltration. Pass the pretreated PNP solution (after protein removal) through a 10 kDa ultrafiltration membrane. Collect the filtrate, adjust the ethanol concentration to 40% (v/v), and allow the solution to stand until precipitation occurs. Collect the precipitate and lyophilize to obtain PNP-4. Filter the previous step’s filtrate through a 5 kDa membrane. Collect the resulting filtrate, adjust the ethanol concentration to 60% (v/v), and let it stand at 4 °C. Collect the precipitate and lyophilize to obtain PNP-3. Pass the filtrate from the previous step through a 5 kDa membrane. Collect the filtrate, adjust the ethanol concentration to 80% (v/v), and allow it to stand at 4 °C. Collect the precipitate and lyophilize to obtain PNP-2. Filter the previous step’s filtrate through a 1 kDa ultrafiltration membrane. Collect the filtrate, concentrate, and lyophilize to obtain PNP-1. Determine the polysaccharide molecular weight (MW) using high-performance size exclusion chromatography (HPSEC). Monosaccharide compositions of PNP1-4 were analyzed by HPLC following hydrolysis with 3 M TFA and PMP derivatization [[184]58]. Preparation of deoxycholic acid (DC)-modified PNP (PNP-1 to PNP-4), folic acid and DC dual-modified polysaccharides (FA-PNP-DC), and corresponding micelles DC modification of PNP-1 ~ 4 Weigh 196.29 mg of deoxycholic acid (DC), 61.09 mg of 4-dimethylaminopyridine (DMAP), and 95.85 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·Cl). Place a 200 mL three-neck flask onto an iron stand over a magnetic stirrer and add the reagents sequentially, and 10 mL of anhydrous dimethyl sulfoxide (DMSO). Under N[2] protection, stir at room temperature at 500 rpm for 0.5 to 1 h to activate the carboxyl groups. Dissolve 400.00 mg of Panax notoginseng polysaccharide (PNP-1 ~ 4) in 20 mL anhydrous DMSO with sonication to ensure complete dissolution. Slowly add the PNP solution dropwise to the activated DC reaction mixture and continue stirring at room temperature for 48 h. After completion, add 10 times the volume of CH[3]CH[2]OH and allow the mixture to stand overnight to precipitate the product. Collect the precipitate, transfer it to a dialysis bag (selected based on the MW of PNP fractions), and dialyze against distilled water for 48 h. Freeze-dry the dialyzed product to obtain DC-modified PNP-1 ~ 4 samples. Preparation of FA-PNP-DC Place a 200 mL three-neck flask onto an iron stand over a magnetic stirrer and add 220.70 mg of FA, 196.29 mg of DC, 61.09 mg of DMAP, 95.85 mg of EDC·Cl, a magnetic stir bar, and 10 mL of anhydrous DMSO. Under N[2] protection, stir at 500 rpm for 0.5 to 1 h at room temperature to activate the carboxyl groups. Dissolve 400.00 mg of DC-modified PNP in 20 mL of anhydrous DMSO with sonication for complete dissolution. Slowly add the DC-modified PNP solution dropwise to the reaction mixture and continue stirring at room temperature for 48 h. After reaction completion, add ten times the volume of CH[3]CH[2]OH and let the mixture stand overnight to precipitate the product. Collect the precipitate, transfer to a dialysis bag (selected based on MW), and dialyze against distilled water for 48 h. Freeze-dry the dialyzed product to obtain FA-DC dual-modified PNP (FA-PNP-DC). Preparation of FA-PNP-DC micelles encapsulating polyphyllin I (FA-PPI-Ms) To prepare FA-PPI-Ms, dissolve FA-PNP-DC and DSPE-PEG[2000] in water and add an ethyl acetate solution of PPI. Shake the mixture gently, then sonicate the resulting hydration solution at 200 W for 10 min using a probe sonicator. Remove the ethyl acetate by rotary evaporation. To obtain uniform FA-PNP-DC micelles, extrude the solution twice through a polycarbonate membrane with a pore size of 0.22 μm. Following the same protocol, DC-modified PNP-1 ~ 4 micelles encapsulating PPI were prepared. Additionally, fluorescent probe micelles were synthesized by replacing PPI with DiR or Coumarin for imaging applications. Characterization of PNP-1 to PNP-4, FA-PNP-DC, and corresponding micelles Samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d[6]) and analyzed using a Bruker 500 MHz NMR spectrometer (Bruker, Germany). ^1H NMR spectra were recorded to investigate the molecular structure and chemical environment of the samples. Fourier transform infrared (FTIR) spectra were obtained using a Bruker Equinox 55 FTIR spectrometer (Bruker, Germany) in the range of 400–4000 cm^− 1. Samples were prepared by spreading them on KBr discs and drying them for 2 min under infrared drying lamps. Each spectrum was recorded by averaging 32 scans at a resolution of 4 cm^− 1 to improve signal-to-noise ratio and accuracy. The particle size distribution, polydispersity index (PDI), and zeta potential (ζ) of the micelles were measured using a Malvern Zetasizer Nano ZS90 (Malvern, UK) employing dynamic light scattering (DLS) techniques. Micelle morphology was examined using a JEOL JEM-1200EX transmission electron microscope (JEOL, Japan). A 5 mL micelle solution was placed onto a formvar-carbon-coated copper grid and allowed to air dry. For negative staining, a 2% phosphotungstic acid solution was added to the grid, air-dried, and then observed under TEM. High-resolution images were acquired to confirm the self-assembled nanostructure of the micelles. The morphological changes of the polysaccharides before and after self-assembly were analyzed using a scanning electron microscope (SEM). This method provides detailed surface structure images. The concentration of PPI in the micelles was determined using a high-performance liquid chromatography system (HPLC, D1100, Dalian Elite, China). The mobile phase consisted of acetonitrile and water (70:30, v/v), with a detection wavelength of 203 nm. Calibration curves constructed using standard solutions were employed to quantify the saponin content in micelle formulations, ensuring precise and accurate measurements. The encapsulation efficiency (EE, %) of PPI in the micelles was determined by first separating the unencapsulated drug using Sephadex G-50 column chromatography. After the free drug was removed, the micelle suspension was analyzed by HPLC to quantify the amount of encapsulated drug. The encapsulation efficiency was calculated using the following formula: graphic file with name d33e1260.gif The stability of the PPI-Ms and FA-PPI-Ms was evaluated by storing the samples at 4 °C for 30 days. The particle size, PDI, and EE were measured on days 10, 20, and 30 to monitor any changes over time. To further assess colloidal stability under physiological conditions, the formulations were incubated in PBS containing 10% fetal bovine serum (FBS) at 37 °C. The in vitro release of PPI from free drug, PPI-Ms, and FA-PPI-Ms was investigated using dialysis. A 1 mL solution of each formulation was placed in a dialysis bag with a MW cut-off of 3000 Da and immersed in 50 mL of release medium (PBS, pH 7.4, containing 0.5% Tween 80) at 37 °C on a shaking incubator. At predetermined time points (2, 4, 8, 12, 24, and 48 h), 0.5 mL of the release medium was withdrawn and replaced with an equal volume of fresh medium. The amount of PPI in the release medium was quantified using HPLC. The cumulative release (CR) was calculated using the formula: graphic file with name d33e1270.gif Where (C[i]) is the drug concentration at the i-th time point (mg/mL), (V[i]) is the volume of release medium at the i-th time point (mL), and (m[drug]) is the initial amount of drug loaded (mg). The critical micelle concentration (CMC) of the micelles was determined using pyrene as a fluorescence probe. The fluorescence intensity of pyrene was measured at various micelle concentrations, and the CMC was determined from the inflection point in the fluorescence intensity-concentration curve. Intracellular uptake RAW 264.7 cells were seeded in 6-well plates at 1.5 × 10^5 cells per well, and M1-type macrophage model was established with LPS. After the model was established, the original medium was removed, and fresh medium containing micelles (Cou-Ms, FA-Cou-Ms, FA-Cou-Ms + FA) with the same coumarin concentration (3 µM) was added and incubated for 2 h. At the end of the incubation period, the cells were fixed with 4% paraformaldehyde for 30 min and stained with Hoechst 33,342 in the dark for 15 min. After staining, the images were imaged under fluorescence microscope. Cellular uptake was further assessed using flow cytometry. RAW 264.7 cells at 3 × 10^5 cells/well were seeded in 6-well plates, and the M1-type macrophage model was established by LPS. After treatment, cells were collected, washed by centrifugation, resuspended in 300 µL PBS, and entered for flow cytometry analysis. Cell viability The effects of free PPI, PNP-Ms, PPI-Ms and FA-PPI-Ms on the viability of RAW 264.7 cells induced by LPS were detected by CCK-8 method. A total of 1 × 10^4 cells in logarithmic growth phase were seeded in a 96-well plate, and the cells were treated with the indicated concentrations of free PPI, PNP-Ms, PPI-Ms, and FA-PPI-Ms, respectively. After 48 h of incubation, a certain amount of CCK-8 solution was added to each well and incubated for 2 h. The cell viability was assessed by measuring the absorbance of the samples at a wavelength of 450 nm, and the cell viability and half maximal inhibitory concentration (IC[50]) were recalculated. Cell viability was calculated using the following formula: graphic file with name d33e1313.gif Cell apoptosis Annexin V/FITC/ propidium iodide (PI) double staining was used to evaluate the effect of different formulations on the apoptosis of RAW 264.7 cells after activation. RAW 264.7 cells in the logarithmic growth phase were seeded into 6-well culture plates at a density of 3 × 10^5 cells per well. After cell adherence, the cells were treated with LPS solution for 12 h. At the end of induction, media containing free PPI, PNP-Ms, PPI-Ms, and FA-PPI-Ms were added to each culture well. After 24 h of culture, cell samples were collected for staining, and finally the apoptosis rate was quantified using flow cytometry (BD Biosciences, New Jersey, USA). Calcein-AM/PI staining RAW 264.7 cells were seeded in 48-well plates at a density of 2 × 10^4 cells/well. After cell adherence, cells were induced by LPS solution for 12 h, and then incubated with free PPI, PNP-Ms, PPI-Ms, and FA-PPI-Ms for 24 h. After incubation, 200 µL of PBS containing Calcein-AM (0.2 µM) and PI (2.5 µM) was added to each well, which was treated at 37 ℃ in the dark for 30 min. After washing with PBS, unbound dye was removed, and observed under a fluorescence microscope (Nikon EclipseE800, Nikon, Tokyo). Intracellular reactive oxygen (ROS) levels 2’,7’ -dichlorodihydrofluorescein diacetate (DCFH-DA) staining was used to assess the intracellular ROS scavenging ability of the different preparations. RAW 264.7 cells were seeded in 48-well plates at a density of 2 × 10^4 cells/well. After cell adherence, except for the blank control group, the cells in the other groups were induced by LPS solution for 12 h, and then incubated with PBS (blank control and model group), PNP-Ms, PPI-Ms, FA-PPI-Ms for 24 h. The original medium was replaced by DCFH-DA reagent prepared by DEME medium without FBS. The cells were then incubated at 37 ℃ for 30 min. The stained cells were imaged under a fluorescence microscope. Macrophage polarization in vitro RAW 264.7 cells were seeded in 6-well plates at a cell density of 3 × 10^5 cells/well, induced by LPS and incubated for 24 h in various treatment groups. Thereafter, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100, and non-specific binding was blocked with PBS solution containing 5% BSA. After CD86 (M1 macrophage markers) and CD206 (M2 macrophage markers) antibodies were incubated at 4℃ for 12 h, secondary antibodies were added and incubated at room temperature in the dark for 1 h. The nuclei were stained with Hoechst 33,342 for 15 min, and the images were observed by fluorescence microscope. Meanwhile, the mean fluorescence intensity was quantified by flow cytometry. Anti-inflammatory effect in vitro To explore the effect of the different formulations on the expression of proinflammatory cytokines in vitro, 2 × 10^5 RAW 264.7 cells were seeded in each well of a 6-well plate and induced by the addition of LPS for 12 h, followed by incubation with the different formulations for 24 h. The supernatants were collected at the end of the incubation, and the concentrations of TNF-α, IL-1β, IL-10, Arg-1, iNOS and TGF-β in the supernatants were determined according to the instructions of the ELISA kits. After lysing macrophages, an ELISA kit was used to detect the effects of different administration groups on the expression of Arg-1 and iNOS. Establishment and administration of the CIA model In the present study, the establishment of the CIA rat model followed the method described previously in the reference [[185]59]. Schematic diagram of experimental methods was shown in Fig. [186]5A. During the primary immunization phase, rats were injected with a mixture of fully emulsified chicken type II collagen and complete Freund’s adjuvant at a 1:1 volume ratio through the caudal base position. After the primary immunization, the rats received a second injection, this time with a mixture of fully emulsified chicken type II collagen and incomplete Freund’s adjuvant, again in a 1:1 volume ratio. In the subsequent treatment study, rats were randomly assigned to receive saline, free PPI, PNP-Ms, PPI-MS, FA-PPI-Ms (PPI: 2 mg·kg^− 1), respectively. Body weight, arthritis score and paw thickness were measured and recorded every 3 days during treatment on a scale of 0–4 (0, baseline; 1, mild inflammation with slight discoloration of one limb; 2, moderate involvement affecting two limbs; 3, advanced inflammation involving three limbs; 4, extensive arthritis affecting the whole limb and digits). On the 27th day of the experiment, the swelling of hind feet and X-ray pictures of the rats in each group were taken and recorded. Biodistribution of blood in the body To explore the distribution characteristics of different micelle preparations, the CIA rats were injected with normal saline, free DiR, DiR-Ms and FA-DiR-Ms through the tail vein, respectively. The in vivo fluorescence imaging system (IVScope8200, Clinx, China) was used to capture and record the fluorescence signals of rats in each group at the time points of 1, 3, 6, 12, 24, 48, and 72 h after injection. Giat analysis CatWalk XT system (Noldus Information Technology, Netherlands) was performed using a high-speed camera system in which rats walked on a transparent running strip and gait was recorded from an abdominal perspective, and the camera system captured each gait cycle at 100 frames per second and 1920 × 1080 pixels resolution. Paw movements were automatically tracked and analysed to extract parameters such as time to first landing, step length, stride length, braking time, support time and gait frequency. Each animal was required to run on the walkway three times. Micro-CT imaging At the end of the treatment, the knee and ankle joints were fixed in 4% formaldehyde. The fixed samples were scanned in a high-resolution micro-CT scanner to obtain 3D images of the joint. The scan length was 2.5 cm, and the parameters were as follows: scanning voltage 90 kV, current 80 µA, high resolution, and scanning time 4 min. Data were analyzed using the manufacturer’s software, calibrated for analysis. The bone mineral density (BMD, g/cm^3), bone volume fraction (BV/TV, %), bone surface to bone volume (BS/TV, 1/mm), trabecular separation (Tb.Sp, mm), trabecular thickness (Tb.Th, mm), and number of trabeculae (Tb.N, 1/mm) were quantitative analyzed. Detection of serum related factors Serum samples were centrifuged at 3000 rpm for 5 min and stored at -20 ℃. The concentrations of TNF-α, IL-1β, IL-10 and TGF-β in serum were detected by ELISA kits according to the instructions of the manufacturer. Histological and immunofluorescence analysis Histological sections were stained with hematoxylin-eosin (HE), Safranin O fast green (SO) and toluidine blue (TB). Immunofluorescence staining with anti-CD86 and anti-CD206 antibodies was performed, and the stained sections were examined by a fluorescence microscope. Safety evaluation At the end of the treatment cycle, to ensure the safety of the treatment, we implemented a series of assessments in the rats. This involves collecting rat blood samples and extracting organs such as heart, liver, spleen, lungs and kidneys from them. Subsequently, the organ samples were stained with HE for histopathological examination to evaluate the possible effects of micelle treatment with different formulations. Serum toxicological parameters such as blood urea nitrogen (BUN), creatinine (CRE), serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. RNA sequencing (RNA-Seq) analysis Total RNA was extracted from the cells of both the Model group and the FA-PPI-Ms group using a column purification method. The concentration and purity of the RNA were assessed through ultraviolet spectrophotometry. RNA integrity was evaluated utilizing the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Messenger RNA (mRNA) was isolated via Oligo (dT) magnetic bead enrichment, followed by random fragmentation, and subsequently, complementary DNA (cDNA) was synthesized using reverse transcriptase. A sequencing library was then generated on the Illumina NovaseqTM 6000 platform (LC-Bio Technology CO, Ltd, Hangzhou, China), incorporating an index code into the attribute sequence of each sample. For transcriptome data analysis, differential expression analysis was conducted using the DESeq function with duplication to estimate size factors and the nbinomTest, or the edgeR libraries without duplication. The resulting p-values were adjusted using Benjamini and Hochberg’s method to control the false discovery rate. After conducting the DESeq2 test, P-values were adjusted, with a threshold of 0.05 established for identifying differentially expressed genes. The clusterProfiler R software package was employed to assess the statistical enrichment of differentially expressed genes within the KEGG pathway, considering significant enrichment to be indicated by a corrected P < 0.05. qRT-PCR Total RNA was extracted from rat synovial tissue and RAW264.7 cells using an RNA extraction kit, and the concentration and purity of the RNA were assessed through ultraviolet spectrophotometry. cDNA was synthesized with the Evo M-MLV reverse transcription Premix kit, utilizing the extracted RNA as a template. Subsequently, in vitro amplification was conducted using the SYBR Green Pro Taq HS Premix qPCR Kit. The expression of β-actin was normalized and calculated using the 2^−ΔΔCt method. The sequences of the primers employed are presented in Table [187]S1. Western blot Total proteins were extracted from synovial tissue and RAW264.7 cells in each group and subsequently normalized. Protein Sample Loading Buffer was added, and the proteins were heated to induce denaturation. The protein samples (20 µg) were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF, Bio-Rad, Anaheim, CA, USA), and washed three times with TBST. 5% skim milk powder solution was used to block the membranes, which were then incubated overnight at 4 °C with anti-JAK2, anti-JAK2 (phospho-Tyr1007/1008), anti-STAT3, anti-STAT3 (Phospho-Y705), and anti-β-Actin antibodies (1:1000, v/v). After washing three times with TBST, the secondary antibody, labeled with HRP (1:1000, v/v), was incubated for 1 h at RT. The PVDF membranes were developed using BeyoECL Plus solution and imaged with a gel imager. Using β-actin as the internal control, ImageJ software was utilized to analyze the gray values of the target proteins semi-quantitatively and to calculate their relative expression. Statistical analysis Data were statistically analyzed using GraphPad Prism 9.0 software and presented as mean ± SD. One-way ANOVA, accompanied by Bonferroni’s or Dunnett’s multiple comparison tests, was employed to evaluate differences between groups. Additionally, Student’s t-test was utilized to compare two groups. P < 0.05 was considered statistically significant. Electronic supplementary material Below is the link to the electronic supplementary material. [188]Supplementary Material 1^ (27.7MB, doc) Acknowledgements