A fibroblast-like synoviocyte-targeted nanoparticle and preparation method and application thereof

Abstract

This invention discloses a nanoparticle targeting fibroblast synovial cells, its preparation method, and its application, which is loaded with a BET inhibitor. Specifically, the preparation method of this invention includes the following steps: 1) modifying the surface of gold nanoparticles with PEG; 2) modifying the terminal amino group of PEG with FAPI; 3) loading I-BET151 onto the surface of the gold nanoparticles using host-guest interactions. This invention applies nanomedicine to the treatment of rheumatoid arthritis, combined with low-dose radiotherapy, using a BET inhibitor to precisely target fibroblast synovial cells and diseased joints, effectively inhibiting the proliferation of synovial fibroblasts, reducing the body's immune response, and showing a significant inhibitory effect on rheumatoid arthritis.

Description

Technical Field

[0001] This invention belongs to the technical field of nanomedicine preparation, specifically relating to a nanoparticle loaded with a BET inhibitor that precisely targets fibroblast synovial cells and its preparation method, the nanoparticle prepared by this method, and the application of the nanoparticle in combined radiotherapy for rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic, persistent synovitis. Rheumatoid arthritis synovial fibroblasts (RASFs) play a crucial role in the pathogenesis and progression of RA. Their abnormal overproliferation is not only a key marker of synovitis but also exhibits tumor-like invasive biological behavior. RAFLS secrete large amounts of pro-inflammatory cytokines and matrix metalloproteinases (MMPs), thereby exacerbating local inflammatory responses in the joint and promoting the degradation and destruction of cartilage and bone tissue. Furthermore, RAFLS can recruit immune cells to the site of inflammation through various mechanisms and cause these cells to remain in the affected joint tissues, thus forming and maintaining a local immune-inflammatory microenvironment, further aggravating tissue damage.

[0003] Traditional treatment for rheumatoid arthritis (RA) primarily relies on pharmacological therapies, including nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and glucocorticoids (GCs) to relieve symptoms and inhibit disease progression. Traditional immunosuppressants and biologics mainly work by modulating immune cell function; however, their tissue specificity is limited and their bioavailability is poor, often leading to systemic toxicity and inadequate disease control, especially in advanced RA.

[0004] Therefore, it is necessary to develop new nanomedicines that combine nanotechnology with radiotherapy to effectively improve the symptoms of rheumatoid arthritis, minimize adverse reactions, and ultimately achieve significant synergistic anti-rheumatoid arthritis efficacy. Summary of the Invention

[0005] This invention discloses small molecule-modified nanoparticles and their preparation method. This method is applicable not only to the modification of gold nanoparticles but also to the modification of other inorganic or organic nanoparticles, providing an innovative and universal strategy and means for the preparation of multifunctional nanocomposites. This invention directly targets RA-FLS cells (fibroblast-like synovial cells in rheumatoid arthritis) with its therapeutic strategy, potentially inhibiting the release of pro-inflammatory factors at the source and slowing down or even blocking degenerative changes in cartilage and bone tissue.

[0006] The present invention adopts the following technical solution: A nanoparticle targeting fibroblast synovial cells includes the nanoparticle and modifications to its surface, said modifications including methoxy polyethylene glycol, polyethylene glycol-(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate complex, mercapto-β-cyclodextrin, and BET inhibitors (e.g., I-BET151).

[0007] The fibroblast-targeting synovial cell nanoparticles disclosed in this invention are BET inhibitor-loaded nanoparticles that precisely target fibroblast-synovial cells. The nanoparticles are metal nanoparticles. The polyethylene glycol-(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate complex is a reaction product of carboxylated polyethylene glycol and (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate. The metal nanoparticles of this invention precisely target diseased small joints, effectively releasing the drug (BET inhibitor) in the diseased joint. Local radiotherapy to the diseased joint may fundamentally reverse the progression of rheumatoid arthritis (RA).

[0008] In this invention, the preparation method of the above-mentioned BET inhibitor-loaded nanoparticles precisely targeting fibroblast synovial cells includes the following steps: forming a modifier on the surface of the nanoparticles to obtain nanoparticles targeting fibroblast synovial cells. Further, the nanoparticles modified with carboxylated polyethylene glycol and mercapto-β-cyclodextrin are reacted with activated (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate, and then reacted with the BET inhibitor to obtain nanoparticles targeting fibroblast synovial cells, which are BET inhibitor-loaded nanoparticles precisely targeting fibroblast synovial cells.

[0009] In the above technical solution, the mass ratio of nanoparticles, methoxy polyethylene glycol, carboxy polyethylene glycol and mercapto-β-cyclodextrin is 1:(15-25):(30-50):(5-10); the mass ratio of modified nanoparticles to (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate and BET inhibitor is 1:(1-3):(0.5-1).

[0010] Preferably, the mass ratio of nanoparticles, methoxy polyethylene glycol, carboxylated polyethylene glycol, and mercapto-β-cyclodextrin is 1:(18-22):(35-45):(8-9); the mass ratio of modified nanoparticles to (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate and BET inhibitor is 1:(1.5-2.5):(0.7-0.9).

[0011] For example, the mass ratio of nanoparticles, methoxy polyethylene glycol, carboxylated polyethylene glycol, and mercapto-β-cyclodextrin is 1:20:40:8.8; the mass ratio of modified nanoparticles to (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate and BET inhibitor is 1:2:0.8.

[0012] A nanomedicine comprising the aforementioned nanoparticles targeting fibroblast synovial cells; which can effectively inhibit the proliferation of synovial fibroblasts.

[0013] This invention discloses the application of the above-mentioned nanoparticles or nanomedicines loaded with BET inhibitors that precisely target fibroblast synovial cells in the preparation of nanomedicines, especially in the preparation of drugs for the treatment of rheumatoid arthritis.

[0014] This invention utilizes (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate and I-BET151 (a BET inhibitor) to bind together on the surface of nanoparticles, developing a simple, environmentally friendly, and stable nanoparticle as a nanomedicine for precisely targeting diseased joints. This method not only effectively alleviates the symptoms of rheumatoid arthritis but also effectively reduces the side effects of drug treatment, and is of great significance for combining with low-dose radiotherapy.

[0015] This invention modifies the surface of nanoparticles with methoxy polyethylene glycol, polyethylene glycol-(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate complex, mercapto-β-cyclodextrin, and I-BET151 (a BET inhibitor) to obtain nanoparticles loaded with BET inhibitors that precisely target fibroblast synovial cells. The specific preparation steps are as follows: (1) Modifying PEG on the surface of nanoparticles: according to the mass ratio of nanoparticles: methoxy polyethylene glycol thiol: carboxy polyethylene glycol thiol: mercapto-β-cyclodextrin = 1~2: 20: 40: 1, add methoxy polyethylene glycol thiol and carboxy polyethylene glycol thiol to the nanoparticle stock solution, stir at room temperature for 12~24 hours, and then obtain PEG-modified carboxy functionalized nanoparticles by ultrafiltration centrifugation and resuspension with water. (2) Modify (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate on the terminal amino group of PEG: activate in DMSO for 6 hours according to the molar ratio of this substance: carbodiamine: N-hydroxysuccinimide = 1:1.2~1.5:1.2~1.5; add the activated product to the mother liquor of the PEG-modified carboxyl functionalized nanoparticles obtained in step (1), stir at room temperature for 12~24 hours, and centrifuge to obtain nanoparticles that specifically and precisely target diseased joints; (3) Dissolve 1 mg of I-BET151 (BET inhibitor) in DMSO, add the aqueous solution of nanoparticles obtained in step (2), and react at room temperature in the dark for 24 h to obtain nanoparticles loaded with BET inhibitor that precisely target fibroblast synovial cells.

[0016] In this invention, the chemical structural formula of (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate (FAPI) is as follows:

[0017] The chemical structural formula of I-BET151 (BET inhibitor) is as follows:

[0018] Most nanoparticles in the prior art are suitable for the preparation method of the present invention, which can ultimately realize the preparation of nanomedicines loaded with BET inhibitors that precisely target fibroblast synovial cells; preferably, the nanoparticles are selected from metal nanoparticles.

[0019] Since synovial cells are located within the joint cavity, targeted intra-articular drug delivery is crucial for achieving therapeutically effective concentrations while minimizing systemic distribution. This invention addresses these challenges by employing a strategy that uses nanoparticles to precisely target diseased small joints and effectively inhibit fibroblast proliferation. Combined with nanotechnology and radiotherapy, this approach effectively improves rheumatoid arthritis symptoms while minimizing adverse reactions, ultimately achieving a significant synergistic therapeutic effect against rheumatoid arthritis.

[0020] In this invention, the methoxy polyethylene glycol thiol is a polyethylene glycol with two ends modified by methoxy and mercapto groups, respectively, and is selected from M-PEG. 2000 -SH, M-PEG 5000 -SH, M-PEG 10000 -SH, M-PEG 20000 Any one of -SH or a mixture thereof in any proportion; more preferably, the methoxy polyethylene glycol thiol is M-PEG. 5000 -SH; plays a stabilizing role, preventing nanoparticles from precipitating out of the original solution.

[0021] In this invention, the carboxyl polyethylene glycol thiol is a polyethylene glycol with two ends modified by carboxyl and thiol groups, respectively, and is selected from NH2-PEG. 2000 -SH, NH2-PEG 5000 -SH, NH2-PEG 10000 -SH, NH2-PEG 20000 Any one of -SH or a mixture thereof in any proportion; more preferably, the aminopolyethylene glycol thiol is NH2-PEG. 5000 -SH; serves as a functional modifier.

[0022] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: (1) This nanomedicine can be precisely targeted to the diseased joints of rheumatoid arthritis by tail vein injection, achieving specific drug delivery and effectively inhibiting the progression of the disease.

[0023] (2) The nanosystem loaded with BET inhibitor can significantly inhibit the activity of synovial fibroblasts, reduce the immune response in vivo, and reduce systemic side effects.

[0024] (3) Radiotherapy combined with this nanomedicine showed excellent inhibitory effects on rheumatoid arthritis and has good potential for clinical translation.

[0025] (4) The preparation method of the present invention is simple, fast, stable and controllable, green and environmentally friendly, and greatly shortens the preparation cycle. It provides a universal new strategy for the rapid construction of multifunctional nanomaterials and has broad application prospects. Attached Figure Description

[0026] Figure 1 A schematic diagram illustrating the preparation of nanomedicines loaded with BET inhibitors for precise targeting of fibroblast synovial cells.

[0027] Figure 2(a) Zeta potential, (b) hydration particle size, TEM images and diameter statistics (n = 100 nm) of FAPI-Au-CD nanoparticles (c) and FAPI-Au-I-BET151 nanoparticles (d), respectively, (e) stability of FAPI-Au-I-BET151 nanoparticles, and (f) I-BET151 release curves of FAPI-Au-I-BET151 over time in solutions with different pH values.

[0028] Figure 3 Quantitative analysis of FAPI-Au-I-BET151 loaded with FAPI (a) and I-BET151 (b).

[0029] Figure 4 To evaluate the inhibitory effect on RAFLS proliferation in vitro; (a) images of RAFLS colony formation under different treatment conditions, (b) proliferation analysis by Click-iTEdU detection, (c) cell cycle distribution of RAFLS cells after different treatments, (d) quantitative analysis of cell cycle, and (e) expression levels of Brd4 and CCXD1 proteins in RAFLS cells.

[0030] Figure 5 To monitor the biodistribution of nanoparticles in CIA model mice at different time points using IVIS imaging technology.

[0031] Figure 6 The in vivo therapeutic effects of FAPI-Au-I-BET151 nanoparticles were observed; (a) a schematic timeline of the FAPI-Au-I-BET151 and RT treatment regimens, (b) clinical scores, (c) changes in claw thickness, (d) body weight. Data are expressed as mean ± standard deviation (n=5), (e) histopathological evaluation of the affected joints by hematoxylin-eosin (HE) and safranin O staining, and (f) representative micro CT images of the claw at the end of each group's experiment. p < 0.05, ** p < 0.01, *** p <0.001.

[0032] Figure 7 The inhibitory effect of FAPI-Au-I-BET151 nanoparticles on the immune response under in vitro RT conditions; representative flow cytometry results of Trge cells (a), Th17 cells (b), and M1 / M2 macrophages (c); changes in the levels of IL-6, IL-1A, TNF-α, and IL-10 in each treatment group;*p < 0.05, ** p < 0.01, *** p <0.001.

[0033] Figure 8 For the Figure 6 (a) HE staining of major organs (heart, liver, spleen, lung and kidney) collected from CIA mice on day 37 after different treatments.

[0034] Figure 9 (a) Complete blood count and (b) Blood biochemistry of mice at different time points after injection of FAPI-Au-I-BET151, and (c) Hemolysis rate and (d) UV absorption spectrum of mouse erythrocyte supernatant at different incubation concentrations.

[0035] Figure 10 The study included: (a) single-cell sequencing analysis of immune cell content in diseased joints; (b) synodic microenvironment cellular landscape identified by single-cell RNA sequencing technology; (c) quantitative analysis of changes in cell composition under different conditions; (d) high-resolution subpopulation clustering and annotation analysis of B cell lineages; (e) quantitative assessment of dynamic changes in B cell lineages among different sample groups; (f) expression profile analysis of lineage-defining marker genes in monocyte and macrophage subpopulations; and (g) quantitative analysis of dynamic changes in monocyte and macrophage lineages among different sample groups.

[0036] Figure 11 It is the core transcriptional regulatory network of the RAFLS phenotype. Detailed Implementation

[0037] This invention successfully developed nanoparticles loaded with BET inhibitors and possessing radiosensitizing properties for targeted therapy of rheumatoid arthritis (RA). Under X-ray irradiation assistance, this nanosystem achieves synergistic and precise treatment of severe RA by loading the novel RA therapeutic drug I-BET151 onto biocompatible gold nanoparticles with significant radiosensitizing properties.

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to explain and illustrate the technical solutions in the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise stated, the materials, reagents, instruments, etc. used in the following embodiments can be obtained by commercial means, the specific preparation operations and testing methods of the present invention are conventional techniques, the animal experiments meet the relevant requirements, and conventional statistical analysis is used.

[0039] Example 1: Preparation of gold nanoparticles and PEG modification of their surface Gold nanoparticles were prepared using the classic sodium citrate-reduction method for chloroauric acid: 200 mL of ultrapure water and 12 mg of chloroauric acid tetrahydrate were added to a two-necked flask. The mixture was stirred with a magnetic stirrer at 600 rpm for 5 min at room temperature, and then incubated at 160 °C. o C was added to 6 mL of 1% trisodium citrate dihydrate solution and the reaction was kept at a warm temperature for 50 min to obtain wine-red gold nanoparticles (AuNPs solution). 20 mg M-PEG 5000 -SH, 40 mg HOOC-PEG 5000 -SH and 8.8 mg of mercapto-β-cyclodextrin were dissolved in 1 mL of ultrapure water and added to the above AuNPs solution (1 mg of gold nanoparticles) under sonication. The mixture was stirred overnight at room temperature (500 rpm) and then centrifuged (12000 rpm, 15 min) for purification. The supernatant was removed. The gold nanoparticles precipitated at the bottom were resuspended in 1 mL of ultrapure water to remove free amino groups. The mixture was centrifuged (14000 rpm, 15 min) again, and the supernatant was discarded. The precipitate was retained and was identified as PEG-modified carboxyl-functionalized gold nanoparticles (COOH-Au).

[0040] Example 2: PEG-terminated FAPI on the surface of gold nanoparticles Following a molar ratio of carboxyl group: carbodiamine: N-hydroxysuccinimide on gold nanoparticles = 1:1.2:1.5, the PEG-modified carboxyl-functionalized gold nanoparticle solution (200 mL, containing 2 mg of PEG-modified carboxyl-functionalized gold nanoparticles, which were concentrated by centrifugation) prepared in Example 1 was activated in 1 mL DMSO for 6 hours, and then 2 mg of FAPI was added. The reaction was stirred at room temperature for 12 h. After centrifugation (11000 rpm, 10 min) 3 times, the supernatant was removed to obtain nanoparticles (FAPI-Au) that specifically target synovial fibroblasts.

[0041] Example 3: Cyclodextrin encapsulation of I-BET151 in gold nanoparticles 0.8 mg of I-BET151 was dissolved in DMSO and added to the FAPI-Au prepared above. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the mixture was centrifuged (12000 rpm, 15 min) to remove free drug. After washing three times with ultrapure water, the nanomedicine loaded with BET inhibitor and precisely targeting fibroblast synovial cells (FAPI-Au-I-BET151) was obtained. The above preparation process is illustrated as follows: Figure 1 As shown.

[0042] Example 4: Chemical characterization of AuNPs, COOH-Au, FAPI-Au, and FAPI-Au-I-BET151 Figure 2 (a) Zeta potential, (b) hydration particle size, (c) FAPI-Au-CD nanoparticles, (d) FAPI-Au-I-BET151 nanoparticles, (e) stability of FAPI-Au-I-BET151 nanoparticles, and (f) I-BET151 release curves of FAPI-Au-I-BET151 over time in solutions with different pH values.

[0043] Figure 3 Quantitative analysis of FAPI-Au-I-BET151 loaded with FAPI (a) and I-BET151 (b).

[0044] The measured surface Zeta potentials of the synthesized AuNPs, HOOC-Au, FAPI-Au, and FAPI-Au-I-BET151 were -17.87±2.78 mV, -25.6±0.24 mV, -0.5±0.05757 mV, and -12.83±0.08 mV, respectively. They were clearly visible in the 1500-1700 cm⁻¹ infrared spectrum. -1 The characteristic absorption band within the range is caused by the C=O stretching vibration of FAPI. High-performance liquid chromatography (HPLC) analysis showed that the coupling yield of FAPI was 66%. The encapsulation efficiency (EE%) of the nanoparticles for I-BET151 was determined to be 75%.

[0045] These results confirm that both FAPI and I-BET151 were successfully modified onto the surface of the nanoparticles. The hydrodynamic diameter of the synthesized AuNPs in aqueous solution ranged from 50 to 80 nm. Meanwhile, transmission electron microscopy (TEM) analysis showed that the synthesized nanoparticles possessed a uniform and well-defined spherical morphology. To evaluate the colloidal stability of the FAPI-Au-I-BET151 nanoparticles, their hydrodynamic diameter in different aqueous solutions, including water, PBS, 10% fetal bovine serum, and cell culture medium, was monitored using dynamic light scattering (DLS). The results showed that the particle size did not change significantly during the 6-day observation period. Subsequently, the pH-dependent drug release characteristics of FAPI-Au-I-BET151 in buffer solutions at pH 6.5, 7.4, and 8.0 were examined. Under all tested pH conditions, I-BET151 was gradually released from the nanoparticles in a time-dependent manner, with the highest release rate at pH 6.5, indicating that a weakly acidic environment promotes drug release.

[0046] Example 5: Nanomedicine loaded with BET inhibitor precisely targets fibroblast synovial cells to inhibit synovial fibroblast proliferation. To verify the antiproliferative effect of nanoparticles, a colony formation assay was used to evaluate the inhibitory effect of FAPI-Au-I-BET151 nanoparticles on cell proliferation. Cells were seeded at low density and cultured for a period of time (50 μg / mL nanoparticles) to allow single cells with sustained proliferative capacity to form colonies, which were then fixed, stained, and counted. This assay quantitatively analyzed the colony formation capacity of RAFLS cells after exposure to different treatments. DNA synthesis levels were further verified using a 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay. Radiotherapy also effectively inhibited RAFLS cell proliferation, and FAPI-Au-I-BET151 nanoparticles exhibited a significantly enhanced radiosensitizing effect. To verify whether the inhibitory effect of I-BET151 on RAFLS cell proliferation affected cell cycle progression, cell cycle analysis was performed. Flow cytometry analysis showed that, compared with other treatment groups, RAFLS cells treated with FAPI-Au-I-BET151 mainly underwent G1 phase arrest, and the proportion of cells in S and G2 / M phases was significantly reduced. These results indicate that FAPI-Au-I-BET151 nanoparticles mainly inhibit fibroblast proliferation and growth by promoting G1 phase cell cycle arrest, thereby regulating the overall cell cycle process. Subsequently, Western blotting and immunofluorescence experiments were used to detect the effects of these gold nanoparticles on the expression of Brd4 and CCND1 proteins. The dual inhibition of Brd4 and CCND1 further indicates that FAPI-Au-I-BET151 disrupted the G1 phase arrest observed in RAFLS cells by interfering with cell cycle progression. These results suggest that radiotherapy has application value as a synovial ablation strategy to inhibit fibroblast proliferation.

[0047] See results Figure 4 To evaluate the inhibitory effect on RAFLS proliferation in vitro; (a) images of RAFLS colony formation under different treatment conditions, (b) proliferation analysis by Click-iTEdU detection, (c) cell cycle distribution of RAFLS cells after different treatments, (d) quantitative analysis of cell cycle, and (e) expression levels of Brd4 and CCXD1 proteins in RAFLS cells.

[0048] Example 6: In vivo imaging study of nanomedicines The synergistic therapeutic effect of low-dose radiotherapy was further investigated in a collagen-induced arthritis (CIA) model. Nanomedicines were pre-modified in vitro with NH2-Cy5.5. Subsequently, multi-timepoint IVIS imaging was performed on CIA model mice receiving different intravenous treatments. The distribution of Cy5.5 nanoparticles (10 mg / kg) in vivo was observed using an IVIS imaging system after tail vein injection. Compared with the unmodified gold nanoparticle control group, mice receiving FAPI-modified gold nanoparticles showed significantly enhanced fluorescence signals at the joints of their limbs. The signal intensity gradually increased over time, peaked at 6 hours, and then continuously decreased. The results are shown in [Figure number missing]. Figure 5 .

[0049] Example 7: In vivo treatment study of nanomedicines The synergistic therapeutic effect of low-dose radiotherapy in a conventional collagen-induced arthritis (CIA) model was further investigated. Figure 6 As shown in Figure a, starting on day 18 after the initial collagen-induced immunization, mice were randomly divided into five groups: (1) saline group, (2) radiotherapy (RT, 2 Gy) group, (3) I-BET151 (5 mg / kg) group, (4) FAPI-Au-I-BET151 (5 mg / kg) group, and (5) FAPI-Au-I-BET151 (5 mg / kg) combined with RT group. CIA mice were intravenously injected with FAPI-Au-I-BET151 nanoparticles via the tail vein every two days and irradiated with X-rays at five-day intervals. Figure 6 As shown in b and 6c, the saline control group exhibited significant erythema and edema of the paws and maintained a consistently high arthritis score throughout the study. Some symptoms were relieved in the RT and I-BET151 groups, but the FAPI-Au-I-BET151 and FAPI-Au-I-BET151+RT groups showed the most significant anti-inflammatory effects. In contrast, the FAPI-Au-I-BET151+RT group had the lowest clinical score. Figure 6 As shown in d, the body weight of all mice remained relatively stable throughout the study. Furthermore, hematoxylin-eosin (HE) stained histopathological sections showed that, compared to the control group, RA mice treated with RT or I-BET151 alone exhibited reduced bone erosion, decreased vascular nodule formation, and reduced synovial hyperplasia. The FAPI-Au-I-BET151 combined with radiotherapy (RT) regimen showed only minor pathological changes, a result further confirmed by safranin O staining, revealing the presence of glycosaminoglycans (GGAs) in the cartilage matrix. Figure 6e). However, significant proteoglycan depletion was observed in the saline treatment group, suggesting substantial damage to the articular cartilage. Crucially, the cartilage tissue of mice receiving the combined treatment remained largely intact. MicroCT was subsequently used to assess bone destruction in the inflamed joints of each group. Mice in the saline group showed the most severe bone erosion, while the combined treatment group showed almost complete remission of bone damage by microCT in the late stage of arthritis. Figure 6 f). These findings strongly suggest that the combination of targeted nanomedicines and low-dose radiotherapy offers a promising treatment strategy for rheumatoid arthritis (RA).

[0050] Example 8: Study on the activation of in vivo immune effects of nanomedicines After treatment, flow cytometry was used to assess splenic T cell subsets and macrophage polarization. The proportion of Foxp3+CD25+ regulatory T cells (Tregs, immunosuppressive subsets of CD3+CD4+ T cells) in the spleen of mice treated with different methods was significantly increased, with the most significant increase observed in the FAPI-Au-I-BET151+RT group. In the CIA model, Th1 cells primarily activate macrophages and B cells via IFN-γ to inhibit bone formation, while Th17 cells directly drive osteoclastogenesis and synovial inflammation via IL-17A. These two cytokines synergistically amplify the inflammatory response: IFN-γ (Th1) and IL-17A (Th17) jointly activate synovial macrophages and fibroblasts. Flow cytometry analysis of the two main cytokines secreted by T cells showed that the levels of inflammatory cytokines in the FAPI-Au-I-BET151+RT group were significantly lower than those in the FAPI-Au group. Cytokines can amplify the inflammatory response by regulating macrophage polarization, thereby disrupting the dynamic balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages. The ratio of M1 to M2 macrophages was further examined.

[0051] Figure 7 The inhibitory effect of FAPI-Au-I-BET151 nanoparticles on immune response under in vitro RT conditions; representative flow cytometry results of Treg cells (a), Th17 cells (b), and M1 / M2 macrophages (c); changes in the levels of IL-6, IL-1A, TNF-α, and IL-10 in each treatment group (d).

[0052] like Figure 7As shown, without X-ray irradiation, the M1 / M2 ratio of the FAPI-Au-I-BET151 group was slightly lower than that of other groups, which may be attributed to the inhibitory effect of I-BET151 on RAFLS proliferation. However, under X-ray irradiation conditions, the macrophage polarization deviation in the FAPI-Au-I-BET151+RT group was the smallest, accompanied by a significant decrease in the expression of pro-inflammatory cytokines (including interleukin-6 (IL-6), interleukin-1α (IL-1α), and tumor necrosis factor-α (TNF-α)), while the levels of anti-inflammatory cytokines such as interleukin-10 (IL-10) were significantly increased. FAPI-Au-I-BET151 effectively inhibited immune activation in CIA mice by inhibiting RAFLS proliferation.

[0053] Example 9: Major organ analysis at the end of treatment Hematoxylin and eosin (H&E) were applied to major organs such as the heart, liver, spleen, and kidneys. Figure 8 The staining results showed no obvious inflammation or tissue damage, further confirming the biocompatibility of the therapeutic agent. Overall, these results strongly demonstrate the excellent biocompatibility of FAPI-Au-I-BET151 nanoparticles.

[0054] Example 10: Analysis of immune infiltration in diseased joints using single-cell sequencing results To investigate the potential mechanism of FAPI-Au-I-BET151 combined with radiotherapy in the treatment of rheumatoid arthritis (RA), single-cell RNA sequencing analysis was performed on joint tissues of three groups of mice: (1) saline control group (RA model group), (2) FAPI-Au-I-BET151 monotherapy group, and (3) FAPI-Au-I-BET151 combined with radiotherapy group (RT group). The RA group showed significant immune infiltration, with B cells accounting for 20.1% and monocytes / macrophages accounting for 35.2%. FAPI-Au-I-BET151 monotherapy significantly reduced the B cell burden to 13.5%, a level that was maintained (13.6%) in the combined treatment group FAPI-Au-I-BET151+RT, indicating that I-BET151 is the main factor leading to B cell exhaustion. FAPI-Au-I-BET151 monotherapy had a limited impact on monocytes / macrophages (decreasing from 35.2% to 32.8%), while the introduction of radiotherapy in the combined radiotherapy group significantly reduced this proportion to 26.6%. These data suggest a synergistic effect, meaning that radiotherapy can specifically target the myeloid cell compartments remaining after monotherapy. Based on these groundbreaking findings, the differential responses of activated B cells and classical monocytes were further investigated. High-resolution subset analysis was used to precisely pinpoint the targets of each treatment regimen.

[0055] Figure 9 (a) Complete blood count and (b) Blood biochemistry of mice at different time points after injection of FAPI-Au-I-BET151, and (c) Hemolysis rate and (d) UV absorption spectrum of mouse erythrocyte supernatant at different incubation concentrations.

[0056] In the rheumatoid arthritis (RA) group, the B-cell lineage was dominated by activated B cells (51.9%), indicating a strong local inflammatory response. Both FAPI-Au-I-BET151 combination therapy and FAPI-Au-I-BET151 combined with radiotherapy (RT) effectively suppressed this cell population, demonstrating its significant efficacy in inhibiting adaptive immune activation. Within the myeloid lineage, classical monocytes were the most abundant subset in the RA group (49.4%), continuously driving the recruitment of inflammatory progenitor cells. Consistent with the overall lineage analysis, FAPI-Au-I-BET151 monotherapy only had a limited effect in reducing the number of this subset.

[0057] Example 11: Evaluation of the effect of FAPI-Au-I-BET151 on fibroblast synovial cells using single-cell sequencing results By comparing the RA group and the FAPI-Au-I-BET151 group, it was found that 65 genes were upregulated in the RA group, but these genes could be inhibited by FAPI-Au-I-BET151. These genes showed significant enrichment during wound healing and leukocyte migration, indicating that FAPI-Au-I-BET151 monotherapy can effectively inhibit the invasive profibrotic and migratory phenotype of fibroblast-like synovial cells (FLS).

[0058] Figure 10 The study included: (a) single-cell sequencing analysis of immune cell content in diseased joints; (b) synodic microenvironment cellular landscape identified by single-cell RNA sequencing technology; (c) quantitative analysis of changes in cell composition under different conditions; (d) high-resolution subpopulation clustering and annotation analysis of B cell lineages; (e) quantitative assessment of dynamic changes in B cell lineages among different sample groups; (f) expression profile analysis of lineage-defining marker genes in monocyte and macrophage subpopulations; and (g) quantitative analysis of dynamic changes in monocyte and macrophage lineages among different sample groups.

[0059] DoRothEA network analysis revealed that the RA phenotype is driven by a dense regulatory network centered on Hif1a (hypoxia-inducible factor 1-α), which activates downstream fibrotic / metabolic targets such as Ccn2 (CTGF), Lox, Mif, and Pkm. FAPI-Au-I-BET151 treatment significantly disrupted this pathological module, as evidenced by a significant downregulation of these target genes in the FAPI-Au-I-BET151 group compared to the RA group. To investigate the additive effect of radiotherapy, the transcriptional profiles of tumor tissues were compared between the FAPI-Au-I-BET151+RT group and the FAPI-Au-I-BET151 group. The transcriptional differences between the two groups were weak (only 7 genes were upregulated in the FAPI-Au-I-BET151+RT group), suggesting that FLS reprogramming is primarily achieved through FAPI-Au-I-BET151 monotherapy. However, regulatory network analysis of the FAPI-Au-I-BET151+RT group showed that the Hif1a regulatory network had a sparse regulatory effect on Hsp90b1. Enriched pathways included antigen processing and presentation, as well as the upregulation of heat shock protein (Hsp90b1), indicating that radiotherapy can induce a mild stress response in FLS cells and has potential immunogenicity. This mechanism differs significantly from the antifibrotic mechanism of FAPI-Au-I-BET151.

[0060] Example 12: Biosafety Evaluation of Nanomedicines To verify the biosafety of the prepared FAPI-Au-I-BET151, 15 male DBA1 mice aged 7-8 weeks and weighing approximately 22g were used. FAPI-Au-I-BET151 at a concentration of 5 mg / kg was injected via the tail vein using an insulin injection at different time points (d0, d20, and d40), with 3 mice in each group. The uninjected group on day 0 served as a blank control. Changes in blood routine (MCV, HCT, MCHC, MCH, HGB, RBC, WBC) and blood biochemical (AST, UA, ALT, ALP, CREA, UREA) indicators were compared at different time points. Results are shown below. Figure 11 .

[0061] This invention innovatively develops nanoparticles loaded with a BET inhibitor and possessing radiosensitizing properties for targeted therapy of rheumatoid arthritis (RA). With the assistance of radiotherapy, this nanosystem achieves synergistic and precise treatment of severe RA by loading the novel RA therapeutic drug I-BET151 onto biocompatible gold nanoparticles with significant radiosensitizing properties. Both in vitro and in vivo studies have confirmed that FAPI-Au-I-BET151 nanoparticles can specifically target diseased joints, inhibiting synovial fibroblast proliferation and alleviating inflammatory responses through the stepwise release of I-BET151. In vivo experiments showed that this nanomedicine effectively inhibited the abnormal proliferation of synovial fibroblasts. Single-cell analysis showed that FAPI-Au-I-BET151 significantly reduced immune activation levels in CIA mice. Simultaneously, related experiments confirmed that FAPI-Au-I-BET151 possesses excellent biocompatibility characteristics. Therefore, this nanomedicine of the present invention can provide a robust and diverse solution for the precise diagnosis and efficient treatment of rheumatoid arthritis.

Claims

1. A nanoparticle targeting fibroblast synovial cells, comprising the nanoparticle and modifications thereof on its surface, characterized in that, The modifiers include methoxy polyethylene glycol, polyethylene glycol-(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate complex, mercapto-β-cyclodextrin, and BET inhibitors.

2. The nanoparticles targeting fibroblast synovial cells according to claim 1, characterized in that, Nanoparticles can be one or more of metallic nanoparticles and non-metallic nanoparticles.

3. The nanoparticles targeting fibroblast synovial cells according to claim 1, characterized in that, The polyethylene glycol-(S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate complex is a reaction product of carboxylated polyethylene glycol and (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate.

4. The method for preparing the targeted fibroblast synovial cell nanoparticles according to claim 1, characterized in that, The process includes the following steps: forming a modifier on the surface of nanoparticles to obtain nanoparticles that target fibroblast synovial cells.

5. The method for preparing the targeted fibroblast synovial cell nanoparticles according to claim 4, characterized in that, Nanoparticles modified with methoxy polyethylene glycol, carboxy polyethylene glycol, and mercapto-β-cyclodextrin were reacted with (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate, and then reacted with a BET inhibitor to obtain nanoparticles targeting fibroblast synovial cells.

6. The method for preparing nanoparticles loaded with BET inhibitors for precise targeting of fibroblast synovial cells according to claim 5, characterized in that, The reaction is carried out at room temperature for 5 to 20 hours.

7. The method for preparing nanoparticles loaded with BET inhibitors for precise targeting of fibroblast synovial cells according to claim 4, characterized in that, The mass ratio of nanoparticles, methoxy polyethylene glycol, carboxylated polyethylene glycol, and mercapto-β-cyclodextrin is 1:(15-25):(30-50):(5-10); the mass ratio of modified nanoparticles to (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide trifluoroacetate and BET inhibitor is 1:(1-3):(0.5-1).

8. A nanomedicine, characterized in that, Its active ingredients include the fibroblast-targeting synovial cell nanoparticles as described in claim 1.

9. The application of the fibroblast-targeting synovial cell nanoparticles of claim 1 in the preparation of nanomedicines.

10. The use of the fibroblast-targeting synovial cell nanoparticles of claim 1 in the preparation of a drug for treating rheumatoid arthritis.

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