Nano-material-mediated ankylosing spondylitis treatment preparation capable of specifically inhibiting MSC osteogenesis abnormality and application of nano-material-mediated ankylosing spondylitis treatment preparation

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and discloses a nanomaterial-mediated ankylosing spondylitis treatment preparation for specifically inhibiting MSC (mesenchymal stem cell) osteogenesis abnormality and application of the nanomaterial-mediated ankylosing spondylitis treatment preparation, and the technical scheme is characterized in that the preparation comprises a mannose-modified mesoporous silica nano-drug delivery system and Ikbkb siRNA (small interfering Ribonucleic Acid). The invention discloses a new mechanism that a tmTNF reverse signal drives pathological osteogenesis through a PSMA1-NF-kappa B axis under the stimulation of sTNFR I, and a mannose modified Man-MSN nano delivery system is constructed, so that active targeting on AS focus macrophages is realized; and by specifically knocking down Ikbkb, the key link of immune-osteogenesis transformation can be specifically blocked on the premise of not influencing the overall inflammation level.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, nanomedicine and targeted therapy, and more specifically relates to a therapeutic agent based on mesoporous silica nanoparticles loaded with siRNA, which specifically blocks pathological osteogenic formation in ankylosing spondylitis by targeting the sTNFR-tmTNF-NF-κB signaling axis of macrophages, and its application. Background Technology

[0002] Ankylosing spondylitis (AS) is a bone immune-related disease characterized by chronic inflammation and pathological osteoblastic formation. AS patients often experience pathological osteoblastic formation (new bone formation) after the inflammation subsides, leading to bony ankylosis of the spine. Current clinical drugs (such as NSAIDs and TNF-α inhibitors) primarily target the inflammatory phase and often lack specific blocking ability against already initiated osteogenic signaling.

[0003] Existing research indicates that even after inflammation subsides, mesenchymal stem cells (MSCs) in the local microenvironment can still undergo abnormal osteogenic differentiation. However, the cellular and molecular mechanisms driving this process are not fully understood, and there is a lack of targeted agents that can be precisely delivered to the lesion and specifically inhibit osteogenic differentiation without affecting systemic bone metabolism. Therefore, developing a targeted nanomedicine that targets the core driving mechanism of pathological osteogenic differentiation in AS has significant clinical implications. Summary of the Invention

[0004] This invention addresses the challenge of existing AS treatments failing to specifically inhibit pathological new bone formation. Based on the "tmTNF-macrophage-MSC" interaction mechanism, this invention provides a nanomaterial-mediated therapeutic agent for ankylosing spondylitis that specifically inhibits abnormal MSC osteogenic formation and its application. This agent can target lesion macrophages and block the secretion of osteogenic factors.

[0005] Specifically, the present invention is achieved through the following technical solutions:

[0006] On one hand, the present invention provides a nanomaterial-mediated therapeutic agent for ankylosing spondylitis that specifically inhibits MSC osteogenic abnormalities, the agent comprising a mannose-modified mesoporous silica nanodrug delivery system and 1kbkb siRNA.

[0007] Preferably, the siRNA sequence of the Ikbkb is as follows:

[0008] Chain of Justice: 5'- GUGAACAGAUCGCCAUCAATT-3';

[0009] Antonym chain: 5'-UUGAUGGCGAUCUGUUCACTT-3'.

[0010] Preferably, the particle size of the formulation is 139.16±2.60 nm and the zeta potential is 17.57±3.45 mV.

[0011] On the one hand, the present invention provides a method for preparing the above-mentioned formulation, comprising: first synthesizing aminated mesoporous silica nanoparticles, encapsulating fluorescent probes, adsorbing 1kbkb siRNA, and then preparing the formulation by chemical coupling with mannose receptor targeted modification.

[0012] Preferably, the preparation method includes: dispersing 20 mg of aminated mesoporous silica in DOPC water as an emulsifier, adding 12 OD 1kbkb of siRNA, 2 mg of fluorescent probe and 2 mg of NHS-PEG-mannose under ice bath stirring, stirring in an ice bath for at least 2 h, collecting the aminated mesoporous silica carrying si 1kbkb and fluorescent probe after centrifugation, and modifying the surface to target mannose receptor.

[0013] Preferably, the aminated mesoporous silica is prepared by dispersing 50 mg of mesoporous silica in toluene, adding 0.2 mL of silane coupling agent APTES, reacting overnight under argon protection at 80°C, and washing several times with ethanol and water; the fluorescent probe is Cy5.5, Cy3, Cy5, Cy7 or ICG.

[0014] On the one hand, the present invention provides the use of the above-mentioned formulation in the preparation of a drug for treating ankylosing spondylitis.

[0015] Preferably, the dosage form of the drug is an injection.

[0016] Preferably, the drug is administered via subcutaneous, intramuscular, intravenous, or intra-articular injection.

[0017] Preferably, the target of the drug is 1kbkb.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The formulation provided by this invention has strong targeting and utilizes mannose receptor-mediated endocytosis to significantly improve the accumulation efficiency of drugs in macrophages of diseased joints. Animal experiments have confirmed that this formulation can significantly reduce ectopic ossification and spinal deformities, and prevent and treat osteoarthritis by blocking the secretion of TGFβ3 / BMP2 at the source. Moreover, it has few side effects. Compared with systemic NF-κB inhibitors, this nano-formulation mainly works locally on the lesion and does not affect the function of major organs. Attached Figure Description

[0020] Figure 1 for tmTNF + / + Immunofluorescence staining of mouse ankle tissue sections;

[0021] Figure 2 A schematic diagram of the co-culture and osteogenic induction experiment of macrophages and MSCs;

[0022] Figure 3 Alizarin Red S staining and alkaline phosphatase activity detection and quantitative analysis results (A is the staining result, B is the quantitative analysis result);

[0023] Figure 4 Western blot was used to detect the expression of osteogenic markers COL1 and RUNX2 in co-cultured MSCs (A is the sTNFR I stimulation group, B is the sTNFR II stimulation group).

[0024] Figure 5 Immunofluorescence staining of primary macrophages after stimulation with sTNFRI or sTNFRI II (A represents the effect of TGF-β on TGF-β). (The effect of B on BMP2 expression is denoted by 3).

[0025] Figure 6 Molecular mechanism analysis diagram (A represents tmTNF) + / + The differentially expressed gene intersection between the mouse transcription sequencing and sTNFRI-stimulated rescue experiments (B is the volcano plot of Ikbkb after enrichment analysis of the intersection genes).

[0026] Figure 7 For tmTNF interaction analysis based on dual-label (Flag-intracellular / HA-extracellular) IP-MS;

[0027] Figure 8 Results for NF-κB pathway validation (A: Immunoprecipitation results; B: Western Blot results).

[0028] Figure 9 The results of qPCR detection of TGFβ3 and BMP2 mRNA expression were obtained.

[0029] Figure 10 The flowchart and characterization results of nanomedicine preparation are shown below (A is the preparation flowchart; B is the dispersion characteristics of nanomaterials observed under electron microscopy; C is the comparison of particle size before and after drug loading; D is the potential change before and after drug loading; E is the comparison of ultraviolet spectra before and after drug loading).

[0030] Figure 11 Results of in vivo imaging of mice;

[0031] Figure 12Diagram showing animal experimental treatment grouping;

[0032] Figure 13 For ELISA detection of serum TGF- 3. and BMP2 protein levels;

[0033] Figure 14 Results of Western blot and qPCR assays after nanomedicine treatment (A and B represent IKK cells in macrophages after treatment, respectively). and NF- B pathway activation was detected and quantified by Western blot; C was the detection of macrophage mRNA levels by qPCR.

[0034] Figure 15 Analysis of the in vivo therapeutic effects of nanomedicines (A: gross view of mice; B: Safranin-Fix-Green staining results; C: H&E staining results; D: statistical analysis of newly formed bone area; E: semi-quantitative inflammation score).

[0035] Figure 16 The results are for safety testing (A is the weight statistics of experimental animals, and B is the HE staining results of organs). Detailed Implementation

[0036] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example 1: Validation of the mechanism by which tmTNF-macrophages drive MSC osteogenics

[0041] In this embodiment, C57BL / 6J mice were selected, and membrane-type TNF overexpressing mice (provided by Cyagen (Suzhou) Biotechnology Co., Ltd.) were obtained through transgenic methods, denoted as tmTNF. + / + tmTNF was collected from 10-week-old infants. + / + Immunofluorescence staining was performed on the ankle joints of WT mice (healthy mice), and the results showed that tmTNF... + / + F4 / 80 + TNF- Macrophages (green / purple) and CD90 + Mesenchymal stem cells (MSCs, red) are closely adjacent in their anatomical location, suggesting potential cell-cell interactions (see [link]). Figure 1 ).

[0042] Furthermore, this embodiment constructed a co-culture model of macrophages and MSCs and conducted osteogenic induction experiments, such as... Figure 2 As shown.

[0043] 1) Cell co-culture model and osteogenic induction

[0044] Construction of Transwell co-culture system: tmTNF-overexpressing mice were inoculated in the upper chamber (tmTNF...). + / + ) or primary macrophages from healthy mice, and mouse MSCs were inoculated in the lower chamber.

[0045] Osteogenic induction: sTNFR I or sTNFR II were added to the system for stimulation, while the unstimulated system served as a control.

[0046] 2) Osteogenic capacity testing

[0047] Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) activity detection and quantification were performed on lower chamber MSCs. Results showed that tmTNF activity stimulated by sTNFRI (>10 μg / mL) was significantly increased. + / + Macrophages significantly enhanced the formation of mineralized nodules and the expression of COL1 and RUNX2 in MSCs, and the induction ability of sTNFRI was significantly stronger than that of sTNFRI II (see...). Figure 3 ).

[0048] 3) Factor detection

[0049] Western blotting was used to detect the expression of osteogenic markers COL1 and RUNX2 in co-cultured MSCs. The results showed that sTNFRI treatment significantly upregulated osteogenic proteins, while sTNFRI II treatment also showed upregulation, but the effect was weaker than that of sTNFRI (see [link to article]). Figure 4Immunofluorescence assays revealed that tmTNF levels decreased under stimulation by sTNFRI or sTNFRI II. + / + TGF-β in macrophages (F4 / 80, green) The expression of 3 and BMP2 (red) was significantly increased (see [link]). Figure 5 ).

[0050] The above results confirm the existence of the "tmTNF-macrophage-MSC" mechanism contributing to the bone axis.

[0051] Example 2: Screening and Validation of Molecular Targets PSMA1 and IKBKB

[0052] 1) Multi-omics analysis

[0053] tmTNF + / + With TNFRI - / - Crossbreeding mice (mice with TNFRI knockout) to obtain tmTNF + / + ×TNFR1 - / - Double-positive offspring, resistant to tmTNF + / + and tmTNF + / + × TNFRI - / - Transcriptome sequencing and bioinformatics analysis were performed on mouse macrophages, and a "rescue experiment" under sTNFRI stimulation was conducted to screen for 145 core differentially expressed genes (see [link to study]. Figure 6 Enrichment analysis of the intersecting genes (A in the original text) showed that Ikbkb expression was upregulated, pointing to the NF-κB pathway (see [link to original text]). Figure 6 (B in the middle).

[0054] 2) Identification of interacting proteins

[0055] A dual-tag (Flag / HA) tmTNF vector was constructed, and immunoprecipitation-mass spectrometry (IP-MS) revealed that the intracellular tmTNF domain specifically binds to the proteasome subunit PSMA1 (see [link to original text]). Figure 7 ).

[0056] 3) Pathway validation

[0057] The NF-κB pathway was validated using co-immunoprecipitation (Co-IP). Results showed that the anti-PSMA1 antibody successfully pulled down flag-labeled tmTNF, confirming the endogenous binding of tmTNF to PSMA1 (see [link to relevant documentation]). Figure 8In the diagram, A, Marker, represents the standard reference for protein molecular weight; Input represents whole-cell lysate without immunoprecipitation, serving as a positive control for protein expression; IP: anti-PSMA1 represents the experimental group using anti-PSMA1 antibody for immunoprecipitation, used to detect the binding of PSMA1 to its interacting proteins; IgG represents the negative control group using nonspecific immunoglobulin G, used to exclude nonspecific binding. IB: FLAG and IB: GAPDH represent immunoblotting detection using anti-FLAG antibody and anti-GAPDH antibody, respectively, with GAPDH used as an internal control for sample loading.

[0058] And using Western blotting to detect NF- The activation state of the B pathway indicates that in tmTNF + / + Knockdown of Psma1 (siPsma1) in macrophages significantly inhibited IKK. I B and p65 phosphorylation levels; in tmTNF + / + × TNFRI - / - Stimulation with sTNFRI in macrophages can induce phosphorylation of the above molecules (see...). Figure 8 B, tmTNF + / + +Si NC represents tmTNF transfected with small interfering RNA negative control (si NC). + / + Mouse cell control group, tmTNF + / + +Si Psma1 indicates tmTNF transfected with a small interfering RNA (si Psma1) targeting Psma1 to knock down Psma1. + / + Mouse cell experimental group; tmTNF + / + ×TNFR I - / - This represents the experimental group of progeny cells from crosses between tmTNF+ / + mice and TNFRI gene knockout mice; tmTNF + / + ×TNFR I - / - +sTNFRI indicates the experimental group of progeny cells from crosses between tmTNF+ / + and TNFRI gene knockout mice, which were then supplemented with soluble TNFRI protein.

[0059] Furthermore, qPCR results showed that knocking down Psma1 or Ikbkb significantly inhibited the mRNA expression of downstream TGFβ3 and BMP2 (see [link to qPCR results]). Figure 9 tmTNF + / ++Si Ikbkb indicates that tmTNF was transfected with a small interfering RNA (si Ikbkb) targeting IKBKB and then knocked down by Ikbkb. + / + Mouse cell experimental group).

[0060] In conclusion, Ikbkb is an ideal intervention target.

[0061] Example 3: Preparation and Characterization of Targeted Nanomedicines

[0062] 1) Preparation process

[0063] 50 mg of mesoporous silica was dispersed in toluene, and 0.2 mL of silane coupling agent APTES was added. The reaction was carried out overnight under argon (Ar) protection at 80 °C. The mixture was washed several times with ethanol and water to obtain aminated mesoporous silica (NH2-MSN).

[0064] 20 mg of aminated mesoporous silica was dispersed in water with DOPC emulsifier. 12 ODIkbkb of siRNA, 2 mg of NHS-Cy5.5, and 2 mg of NHS-PEG-mannose were added under ice bath stirring. After stirring under ice bath for two hours, the aminated mesoporous silica-loaded siIkbkb and CY5.5 were collected by centrifugation. The surface was modified with mannose receptor targeting to obtain a targeted nanomedicine, denoted as siIkbkb / Cy5.5@Man-MSN.

[0065] If Ikbkb of siRNA is not added in the above method, the targeting vector is obtained, denoted as Cy5.5@Man-MSN.

[0066] The siRNA sequence of Ikbkb is as follows:

[0067] Chain of Justice: 5'- GUGAACAGAUCGCCAUCAATT-3';

[0068] Antonym chain: 5'-UUGAUGGCGAUCUGUUCACTT-3'.

[0069] 2) Physicochemical characterization

[0070] Transmission electron microscopy (TEM) showed that the particles were uniformly spherical; dynamic light scattering (DLS) showed that the modified particle size increased and the zeta potential decreased, with the particle size of siIkbkb / Cy5.5@Man-MSN being 139.16±2.60 nm and the zeta potential being 17.57±3.45 mV; UV spectroscopy confirmed the successful loading of Cy5.5 (see...). Figure 10 (BE in the middle).

[0071] 3) Targeted validation

[0072] In vivo imaging in mice showed that, 6-72 hours after tail vein injection, the fluorescence enrichment intensity of the mannose-modified group (Man-MSN) at the diseased joint sites was significantly higher than that of the non-targeted control group, confirming its excellent lesion macrophage targeting ability (see...). Figure 11 ).

[0073] Example 4: In vivo treatment efficacy evaluation

[0074] 1) Animal administration

[0075] Select tmTNF + / + In a spontaneous ankylosing spondylitis model mouse, siIkbkb / Cy5.5@Man-MSN was injected weekly via tail vein for 4 weeks (see [link to original text]). Figure 12 ).

[0076] 2) Molecular level assessment

[0077] Western blot and qPCR analysis showed that the level of IKKβ protein in primary macrophages was decreased and p65 phosphorylation was inhibited in the treatment group (see...). Figure 14 Serum and tissue levels of TGF-β3 and BMP2 were significantly decreased (see...) Figure 13 ).

[0078] 3) Gross and histological assessment

[0079] Gross observation showed significant improvement in kyphosis and tail folding in the treatment group mice. Quantitative analysis using safranin-fast green staining revealed a significant reduction in ectopic cartilage formation area in the treatment group (see...). Figure 15 (AB in the middle).

[0080] 4) Specificity description

[0081] Notably, HE staining and inflammation scoring showed no significant difference in inflammation levels between the treatment and control groups, indicating that the therapy specifically blocked the osteogenic process, rather than simply having an anti-inflammatory effect (see...). Figure 15 (CE in the text).

[0082] Example 5: Biosafety Evaluation

[0083] Mouse body weight changes were monitored during treatment, and heart, liver, spleen, lung, and kidney were collected at the endpoint for HE staining. Results showed that mice in the treatment group experienced normal body weight gain, and no significant histopathological damage was observed in major organs, indicating that the nano-formulation has good biocompatibility and safety (see [link to treatment]). Figure 16 ).

[0084] In summary, in AS lesions, membrane-bound TNF (tmTNF), acting as a soluble receptor (sTNFRI) in the receptor-sensing environment, activates the NF-κB pathway by recruiting the intracellular protein PSMA1. This signaling axis leads to macrophage secretion of TGFβ3 and BMP2, thereby inducing pathological osteogenic differentiation in adjacent MSCs. Therefore, blocking the key NF-κB pathway kinase IKKβ (encoded by the Ikbkb gene) can significantly inhibit the expression of these osteogenic factors. Based on this, this invention constructs a nano-formulation loaded with siRNA targeting Ikbkb using a mannose-modified mesoporous silica nanomedicine delivery system. After intravenous injection, this nano-formulation accumulates in macrophages of joint lesions, releasing siRNA to silence IKKβ, blocking PSMA1-mediated NF-κB activation, reducing TGFβ3 / BMP2 secretion, and thus inhibiting osteogenic differentiation of MSCs.

[0085] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A nanomaterial-mediated therapeutic agent for ankylosing spondylitis that specifically inhibits MSC osteogenic abnormalities, characterized in that, The formulation comprises a mannose-modified mesoporous silica nanomedicine delivery system and 1kbkb of siRNA.

2. The formulation according to claim 1, characterized in that, The siRNA sequence of the Ikbkb is shown below: Chain of Justice: 5'- GUGAACAGAUCGCCAUCAATT-3'; Antonym chain: 5'-UUGAUGGCGAUCUGUUCACTT-3'.

3. The formulation according to claim 1, characterized in that, The formulation has a particle size of 139.16±2.60 nm and a zeta potential of 17.57±3.45 mV.

4. The method for preparing the formulation according to any one of claims 1-3, characterized in that, include: Aminated mesoporous silica nanoparticles were synthesized, encapsulated with fluorescent probes and 1kbkb siRNA, and then chemically coupled with mannose.

5. The preparation method according to claim 4, characterized in that, include: 20 mg of aminated mesoporous silica was dispersed in DOPC water emulsion. 12 OD Ikbkb of siRNA, 2 mg of fluorescent probe and 2 mg of NHS-PEG-mannose were added under ice bath stirring. The mixture was stirred under ice bath for at least 2 h. After centrifugation, the aminated mesoporous silica carrying si Ikbkb and fluorescent probe was collected and surface-targeted mannose receptor modified to obtain the product.

6. The preparation method according to claim 5, characterized in that, The aminated mesoporous silica was prepared by dispersing 50 mg of mesoporous silica in toluene, adding 0.2 mL of silane coupling agent APTES, reacting overnight under argon protection at 80 °C, and washing several times with ethanol and water; the fluorescent probe was Cy5.5, Cy3, Cy5, Cy7 or ICG.

7. The use of the formulation according to any one of claims 1-3 in the preparation of a medicament for treating ankylosing spondylitis.

8. The application according to claim 7, characterized in that, The drug is in the form of an injection.

9. The application according to claim 7, characterized in that, The medication can be administered via subcutaneous, intramuscular, intravenous, or intra-articular injection.

10. The application according to claim 7, characterized in that, The target of the drug is 1 kbkb.

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