Phenolic acid-strontium nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a phenolic acid-strontium nano-particle and a preparation method and application thereof, the phenolic acid-strontium nano-particle is a nano-structure obtained by self-assembly by using a method of modifying phenolic acid by metal strontium coordination, according to the prepared caffeic acid-strontium nanoparticles, coordination modification of phenolic acid molecules is achieved through coordination of strontium, carboxyl of phenolic acid and catechol groups, and the caffeic acid-strontium nanoparticles are of a spherical structure, small and uniform in particle size, good in dispersity, simple in preparation process, low in cost and easy to produce in batches; after the caffeic acid is subjected to coordination modification by strontium, the capability of targeted inhibition of the caffeic acid on the TRPV1 is remarkably enhanced, so that the problem of calcium overload of a focus is solved, and the problems of inflammatory polarization of macrophages and sensitization of damaged sensory neurons, which are caused by abnormal calcium influx due to high expression of the TRPV1 at the arthritis focus part, are effectively and synchronously solved; and the compound has excellent bone repair performance and extremely high biological safety, and has wide application prospects in preparation of drugs for preventing and / or treating inflammatory bone related diseases and preparation of bone repair drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, particularly to the field of nanomedicine, specifically to a phenolic acid-strontium nanoparticle, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a major form of joint inflammation, typically affecting joints in the hands and feet. RA causes progressive joint pain, swelling, and stiffness. In recent years, the prevalence of RA in China has been approximately 0.4%, but this figure is rising annually due to population aging, with pain being the primary reason for 70% of patients seeking medical attention. More importantly, due to the increasing chronic pain, functional impairment, and risk of complications, RA severely impacts patients' quality of life and work, imposing a significant economic burden on individuals and society.

[0003] The pathological microenvironment of rheumatoid arthritis (RA) is mainly characterized by oxidative stress in the joints and high levels of inflammatory mediators (IL-1β, TNF-α, and IL-6, etc.), erosion of articular cartilage and subchondral bone, and sensitization of nociceptive neurons. Currently, clinical treatment for RA generally employs anti-inflammatory therapy, primarily using glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), classic antirheumatic drugs such as methotrexate, and monoclonal antibody drugs. However, these drugs inevitably have serious toxic side effects and lack direct treatment for pain caused by bone erosion and nociceptive neuron sensitization in arthritis. Therefore, designing safe and effective novel drugs to inhibit and alleviate the inflammatory environment, pain, and bone erosion of RA, targeting the pathogenesis and disease microenvironment of RA, is a crucial issue for public health.

[0004] Intracellular calcium ions influx into nerves and TRPV1 (Transient Receptor Potential Vanilloid 1) is a ligand-gated cation channel that induces calcium ion influx upon activation. On the one hand, excessive calcium ion influx is associated with abnormal nerve excitation; on the other hand, excessive calcium ion influx can also cause inflammatory polarization of macrophages. Therefore, targeting and antagonizing TRPV1 can simultaneously achieve anti-inflammatory pain and anti-neuralgia.

[0005] Natural phenolic acids are a class of organic compounds widely found in plant-based foods. They are unique metabolites of plants. They are not only common polyphenolic antioxidants in people's diets, but also important pharmacologically active small molecules in traditional Chinese medicine and a "gold mine" for modern drug development. They possess various biological activities such as antioxidation, anti-inflammation, and anticancer activity. Due to their rich and diverse chemical structures, they have become a source of many receptor protein modulators, providing templates for drug development. However, they face certain limitations in the development of finished drugs or functional foods, such as poor water solubility, low bioavailability, and weak targeted binding ability. These shortcomings severely limit their application.

[0006] Given the current situation where natural phenolic acids (caffeic acid) have poor water solubility and low utilization, rheumatoid arthritis drugs have serious toxic side effects, and there is a lack of drugs that directly treat pain caused by bone erosion and nociceptive neuron sensitization in arthritis, no effective solutions have yet been proposed. Summary of the Invention

[0007] The purpose of this invention is to address at least one deficiency in the prior art by providing phenolic acid-strontium nanoparticles, their preparation method, and applications. It innovatively discovers that natural phenolic acid molecules (caffeic acid, ferulic acid, gallic acid, chlorogenic acid) are an excellent ligand library for TRPV1, possessing both anti-inflammatory and antioxidant properties. By modifying the molecular structure of natural phenolic acids with metal coordination, this invention uses caffeic acid as the target and employs metal coordination self-assembly nanotechnology to prepare caffeic acid-strontium nanoparticles. These nanoparticles can enhance the targeting and antagonism of TRPV1 receptors, endowing them with bone repair activity. This innovatively achieves direct regulation of arthritis neurosensitization and tissue repair, solving the problems of poor water solubility and low utilization of natural phenolic acids (caffeic acid), the serious toxic side effects of drugs for rheumatoid arthritis, and the lack of drugs that directly treat pain caused by bone erosion and nociceptive neuron sensitization in arthritis.

[0008] The caffeic acid-strontium nanoparticles of this invention are simple to prepare, exhibit good nanoparticle dispersibility, and high biosafety. They can target TRPV1 receptors to inhibit joint inflammation and pain sensitization, effectively reduce downstream oxidative stress induced by immunogens, and repair damaged tissues. These caffeic acid-strontium nanoparticles possess high biocompatibility, dispersion stability, and in vivo safety. Based on the natural antioxidant and anti-inflammatory properties of phenolic acid ligands, they simultaneously target and inhibit TRPV1 receptors, promoting bone tissue repair, thus providing a safe and efficient treatment option for the prevention and treatment of rheumatoid arthritis.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention is to provide phenolic acid-strontium nanoparticles, wherein the phenolic acid-strontium nanoparticles are nanostructures obtained by self-assembly using a method of coordinating and modifying phenolic acid with metallic strontium; wherein the phenolic acid is at least one of caffeic acid, ferulic acid, gallic acid, and chlorogenic acid.

[0010] Furthermore, the phenolic acid-strontium nanoparticles are caffeic acid-strontium nanoparticles, which achieve coordination modification of phenolic acid molecules through coordination of strontium with the carboxyl and catechol groups of phenolic acid.

[0011] Furthermore, the preparation method of the caffeic acid-strontium nanoparticles includes the following steps: S1. Strontium precursor powder is dissolved in ultrapure water to prepare strontium precursor aqueous solution; S2. Mix the alkaline aqueous solution with caffeic acid powder, and after the caffeic acid powder dissolves, dilute to volume with ultrapure water to prepare a caffeic acid aqueous solution. S3. Add the strontium precursor aqueous solution obtained in step S1 to the caffeic acid aqueous solution obtained in step S2, stir to react, collect the precipitate by centrifugation, wash and vacuum dry to obtain the caffeic acid-strontium nanoparticles.

[0012] Further, in step S1, the concentration of the strontium precursor in the aqueous solution is 1~50 mg / mL; preferably, the concentration of the strontium precursor is 7.4 mg / mL.

[0013] Further, in step S1, the strontium precursor includes at least one of strontium chloride, strontium nitrate, strontium acetylacetone, and strontium sulfate; preferably, the strontium precursor is strontium chloride.

[0014] Further, in step S2, the alkaline aqueous solution includes at least one of sodium hydroxide, potassium hydroxide, and ammonia; preferably potassium hydroxide.

[0015] Further, in step S2, the concentration of the alkaline aqueous solution is 0.5~5 M; preferably 0.25 M.

[0016] Further, in step S2, the concentration of caffeic acid in the aqueous caffeic acid solution is 2~10 mg / mL, and the volume ratio of alkaline aqueous solution to ultrapure water is 1:5~1:15; preferably, the final concentration of caffeic acid in the aqueous caffeic acid solution is 41 mM (about 7.4 mg / mL), and the final concentration of potassium hydroxide is 250 mM.

[0017] Further, in step S3, the mass ratio of the strontium precursor to caffeic acid is 1:1 to 20:1; preferably 10:1.

[0018] In one specific embodiment, in step S1, the strontium precursor powder is strontium chloride, the amount of which is 0.37 g, and the volume of ultrapure water is 50 mL; in step S2, the alkaline aqueous solution is potassium hydroxide, the concentration of which is 2.5 M, and the volume of which is added is 500 μL; the amount of caffeic acid is 0.037 g, which is dissolved and then diluted with water to a final volume of 5 mL.

[0019] Further, in step S3, the strontium precursor aqueous solution is added dropwise to the caffeic acid aqueous solution at a rate of 0.5~1.5 mL / min; preferably, it is added dropwise at a rate of 1 mL / min.

[0020] Further, in step S3, the stirring reaction is carried out at a speed of 300~800 rpm for 3~5 h; further, the stirring reaction is carried out at a speed of 550 rpm for 4 h.

[0021] Further, in step S3, the centrifugation parameters are 10000~14000 r speed for 8~12 min; preferably, 12000 r speed for 10 min.

[0022] Further, in step S3, the cleaning involves one wash with ultrapure water and two washes with a 1:1 mixture of ethanol and ultrapure water. Preferably, the ethanol is analytical grade anhydrous ethanol.

[0023] Further, in step S3, the operating conditions for vacuum freeze drying are: vacuum degree -0.8 ~ -1 MPa, cold trap temperature -80℃, and drying time of 5~7 h; preferably, drying for 6 h.

[0024] Furthermore, the caffeic acid-strontium nanoparticles obtained in step S3 have a spherical structure and an average particle size of 100~150 nm.

[0025] Furthermore, the average particle size of the caffeic acid-strontium nanoparticles is 136.4 ± 4.3 nm.

[0026] The caffeic acid-strontium nanoparticles prepared in this invention are the first of their kind obtained by the inventors. It is also the first time that caffeic acid has been found to achieve anti-inflammatory and analgesic effects by antagonizing TRPV1 to inhibit calcium ion influx. Furthermore, the coordination modification of caffeic acid with metallic strontium not only solves the problems of poor water solubility, low bioavailability, and easy metabolism of caffeic acid, but also endows it with bone repair activity, which can be used for anti-inflammatory and analgesic effects and bone repair in arthritis.

[0027] A second aspect of the present invention is to provide a phenolic acid-strontium nanoparticle formulation, wherein the active ingredient of the formulation comprises phenolic acid-strontium nanoparticles as described in the first aspect.

[0028] Furthermore, the phenolic acid-strontium nanoparticle formulation is a caffeic acid-strontium nanoparticle formulation, which is an injection solution prepared by dissolving caffeic acid-strontium nanoparticles in a biocompatible solution, wherein the biocompatible solution is physiological saline and / or phosphate buffer.

[0029] Furthermore, in the injection solution, the concentration of the caffeic acid-strontium nanoparticles is 1~10 mg / mL; preferably 4~6 mg / mL, more preferably 5 mg / mL.

[0030] Furthermore, the concentration of the phosphate buffer solution is 0.01 mM, and the pH is 6.5~8.5, preferably pH 7.4.

[0031] Furthermore, the physiological saline contains NaCl at a concentration of 0.9 wt%.

[0032] Furthermore, the preparation of the injection solution by dissolving the caffeic acid-strontium nanoparticles in a biocompatible solution specifically involves placing the caffeic acid-strontium nanoparticles in a vacuum freeze-drying oven, drying them at a cold trap temperature of -80°C for 6 hours, and then resuspending them in a certain volume of biocompatible solution to obtain the injection solution.

[0033] Furthermore, the injection method of the injection solution includes joint injection or intravenous injection.

[0034] Furthermore, the effects of the caffeic acid-strontium nanomedicine include at least one of the following: enhancing targeted inhibition of TRPV1 thereby inhibiting calcium overload, promoting bone tissue repair, and scavenging reactive oxygen species.

[0035] In a specific embodiment of the present invention, mice are used as the subjects, and the concentration of caffeic acid-strontium nanoparticles in the injection solution is 2~7 mg / kg, preferably 5 mg / kg.

[0036] Understandably, the use in humans is adaptable, and as a preferred option, the concentration of caffeic acid-strontium nanoparticles in the injection solution suitable for intra-articular or intravenous administration in humans is 20~80 mg / kg, preferably 45.15 mg / kg (±10%).

[0037] A third aspect of the present invention is to provide an application of phenolic acid-strontium nanoparticles as described in the first aspect or phenolic acid-strontium nanoformulations as described in the second aspect, the application including at least one of the following applications: application in the preparation of medicaments for the prevention and / or treatment of inflammatory bone-related diseases, application in the preparation of bone repair medicaments, and application in the preparation of medicaments that target and antagonize TRPV1.

[0038] Furthermore, the inflammatory bone-related diseases include inflammatory arthritis characterized in vivo by inflammatory neuropathic hyperalgesia, bone erosion, and excessive ROS, more preferably rheumatoid arthritis.

[0039] Furthermore, drugs that target and antagonize TRPV1 can be used to prevent or treat diseases related to calcium overload caused by high TRPV1 expression.

[0040] Furthermore, the drug is at least one of the following drugs having the functions of: enhancing the normalization of nerve sensitization by targeting and inhibiting TRPV1, restoring weight loss induced by rheumatoid arthritis in mice, clearing LPS / IFN-γ-induced intracellular reactive oxygen species, inducing pro-inflammatory M1 macrophages to differentiate into anti-inflammatory M2 macrophages, reducing the expression of inflammatory factors in synovial tissue, increasing the expression of anti-inflammatory factors in synovial tissue, promoting bone function, inhibiting and reducing joint swelling, inhibiting bone erosion, increasing bone mineral density, and reducing ROS levels.

[0041] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention discovers that four natural phenolic acids—caffeic acid, ferulic acid, gallic acid, and chlorogenic acid—all possess the potential to target TRPV1. Taking caffeic acid as an example, by modifying the molecular structure of the natural phenolic acid—caffeic acid—with metal coordination, caffeic acid-strontium nanoparticles were prepared. These nanoparticles exhibit a spherical structure, small and uniform particle size, and good dispersibility. They significantly enhance the ability of caffeic acid to target and inhibit TRPV1, thereby resolving calcium overload in lesions. Simultaneously, they effectively address macrophage inflammatory polarization and nociceptive neuron sensitization caused by abnormal calcium influx due to high TRPV1 expression at arthritis lesions. Furthermore, through metal... The coordination modification of caffeic acid molecules with strontium endows caffeic acid-strontium nanoparticles with excellent bone repair properties. They exhibit excellent dispersion stability in physiological solutions, can be diluted with physiological saline or phosphate buffer, and can be used directly in clinical settings without the need for solubilizers, thus solving the clinical application problem of poor water solubility and low utilization of natural phenolic acid (caffeic acid). Moreover, the preparation process is simple, low-cost, easy to mass-produce, and has good therapeutic effects and extremely high biosafety. It has broad application prospects in the preparation of drugs for the prevention and / or treatment of inflammatory bone-related diseases and in the preparation of bone repair drugs. Attached Figure Description

[0042] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a diagram showing the molecular docking results of different phenolic acids with TRPV1 protein in one embodiment of the present invention; Figure 2This is a TEM image (a) and elemental analysis diagram (b) of caffeic acid-strontium nanoparticles in one embodiment of the present invention; Figure 3 This is a diagram showing the particle size distribution of caffeic acid-strontium nanoparticles in one embodiment of the present invention. Figure 4 This is an isothermal titration calorimetric result diagram of the affinity of caffeic acid (CA) and caffeic acid-strontium nanoparticles (CA-Sr NPs) for TRPV1 in one embodiment of the present invention; Figure 5 In one embodiment of the present invention, caffeic acid-strontium nanoparticles are used to remove ABTS. + The experimental results of antioxidant capacity are shown in the figure; Figure 6 This is a diagram showing the experimental results of caffeic acid-strontium nanoparticles (CA-Sr NPs) clearing LPS-induced oxidative stress in macrophages in one embodiment of the present invention. Figure 7 This is a confocal representation of an embodiment of the present invention showing how caffeic acid-strontium nanoparticles (CA-Sr NPs) inhibit LPS / IFN-γ-induced calcium influx into macrophages (a) and a statistical graph of calcium ion fluorescence signals (b). Figure 8 This is a flow cytometry image of the repolarization assay of LPS / IFN-γ-induced macrophages by caffeic acid-strontium nanoparticles (CA-Sr NPs) in one embodiment of the present invention; wherein, the Q1 partition (CD86) - CD206 + M2 macrophages; Q3 partition (CD86) + CD206 - ) are M1 macrophages; Figure 9 This is a confocal representation of an embodiment of the present invention showing how caffeic acid-strontium nanoparticles (CA-Sr NPs) inhibit capsaicin-CAP-induced calcium influx in primary dorsal root ganglion cells. Figure 10 This is a patch-clamp detection result of caffeic acid-strontium nanoparticles (CA-Sr NPs) inhibiting capsaicin-CAP-induced abnormal electrical signals in primary dorsal root ganglion cells in one embodiment of the present invention. Figure 11 This is a statistical graph showing the action potential frequency of caffeic acid-strontium nanoparticles (CA-Sr NPs) inhibiting capsaicin-CAP-induced abnormal electrical signals in primary dorsal root ganglion cells in one embodiment of the present invention. Figure 12 This is an optical microscope image (a) and a statistical graph (b) of Alizarin Red ASR staining of MC3T3 after different treatments in one embodiment of the present invention; wherein, OBIM is osteoblast positive induction solution; Figure 13This is an H&E staining result of pathological sections of major organs in mice treated with caffeic acid-strontium nanoparticles (CA-Sr NPs) according to an embodiment of the present invention. Figure 14 This is a curve showing the measurement of ankle swelling in mice with rheumatoid arthritis during treatment with caffeic acid-strontium nanoparticles (CA-Sr NPs) in one embodiment of the present invention. Figure 15 This is a graph showing the measurement of mechanical pain (a) and thermal pain (b) in the soles of mice treated with caffeic acid-strontium nanoparticles (CA-Sr NPs) during an embodiment of the present invention. Figure 16 This is a representative immunofluorescence image of dorsal root ganglion tissue in mice treated with caffeic acid-strontium nanoparticles (CA-Sr NPs) according to an embodiment of the present invention; wherein, in part a, green: c-Fos expression; red: TRPV1 expression; blue: cell nucleus; part b represents TRPV1. + c-Fos + Percentage of double-positive cells; Figure 17 This is a diagram showing the results of ankle bone structure analysis in mice with rheumatoid arthritis after treatment with caffeic acid-strontium nanoparticles (CA-Sr NPs) according to an embodiment of the present invention. Part a is a representative Micro-CT image of the ankle joint; part b is an analysis of the bone volume fraction of the ankle joint after different treatments; part c is a bone area density analysis; part d is an analysis of the trabecular spacing; and part e is an analysis of the average bone mineral density. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0045] The caffeic acid used in this invention was purchased from MCE, catalog number: HY-B1829A.

[0046] Unless otherwise specified, all manufacturers of other products in this invention embodiment can be obtained through commercial purchases.

[0047] In some embodiments of the present invention, a method for preparing caffeic acid-strontium nanoparticles is provided, specifically including the following steps: Strontium precursor powder was dissolved in ultrapure water to prepare a strontium precursor aqueous solution with a concentration of 1-50 mg / mL. Caffeic acid powder was added to an alkaline aqueous solution with a concentration of 0.5-5 M. After the caffeic acid powder dissolved, the solution was diluted with ultrapure water to prepare a caffeic acid aqueous solution with a concentration of 2-10 mg / mL. Then, the strontium precursor aqueous solution was added dropwise to the caffeic acid aqueous solution at a mass ratio of 1:1 to 20:1 (strontium precursor to caffeic acid 1:1 to 20:1). The mixture was stirred for 3-5 h, and the precipitate was collected by centrifugation at 10000-14000 r for 8-12 min. The precipitate was washed 1-3 times and vacuum dried for 5-7 h under vacuum conditions of -0.8 to -1 MPa and a cold trap temperature of -80℃ to obtain caffeic acid-strontium nanoparticles.

[0048] The present invention will be described by way of example below.

[0049] Example 1 - Screening of natural phenolic acid molecules targeting TRPV1 In this embodiment, the docking binding energies of common phenolic acids (caffeic acid, ferulic acid, gallic acid, and chlorogenic acid) with TRPV1 were detected, and the results are as follows: Figure 1 As shown.

[0050] from Figure 1 The results show that the binding energy of caffeic acid to TRPV1 is -6.438 kcal / mol; the binding energy of ferulic acid to TRPV1 is -5.964 kcal / mol; the binding energy of gallic acid to TRPV1 is -6.032 kcal / mol; and the binding energy of chlorogenic acid to TRPV1 is -7.617 kcal / mol. Among these, the binding energies of the four commonly used natural phenolic acids—caffeic acid, ferulic acid, gallic acid, and chlorogenic acid—to TRPV1 are all less than -4 kcal / mol, indicating that these four natural phenolic acid molecules all have the potential to target TRPV1.

[0051] In the following examples, only caffeic acid will be used as the experimental subject for illustration.

[0052] Example 2 - Preparation of Caffeic Acid-Strontium Nanoparticles and Their Injection Solution This embodiment relates to a preferred method for preparing caffeic acid-strontium nanoparticles and their injection solution.

[0053] (1) Preparation of caffeic acid-strontium nanoparticles: Strontium chloride (0.37 g) was dissolved in 50 mL of ultrapure water to obtain a strontium chloride aqueous solution. 500 μL of potassium hydroxide aqueous solution (2.5 M) was added to 0.037 g of caffeic acid powder, and the solution was dissolved and then diluted to 5 mL with ultrapure water to obtain a caffeic acid aqueous solution. The strontium chloride aqueous solution was then added dropwise to the caffeic acid aqueous solution at a rate of 1 mL / min, and the mixture was stirred for 4 h. The reaction solution was centrifuged at 12000 r for 10 min, and the precipitate was collected. The precipitate was washed once with ultrapure water and twice with a 1:1 mixture of ethanol and ultrapure water. The precipitate was then freeze-dried under vacuum to obtain caffeic acid-strontium nanoparticles (CA-Sr NPs). The freeze-drying conditions were: vacuum degree -0.8 ~ -1 MPa, cold trap temperature -80℃, and drying time 6 h.

[0054] (2) The characterization of the above-mentioned caffeic acid-strontium nanoparticles (CA-Sr NPs) was analyzed. The TEM image, elemental mapping image, and particle size distribution image of the caffeic acid-strontium nanoparticles (CA-Sr NPs) are shown below. Figure 2 and Figure 3 As shown.

[0055] from Figure 2 It can be seen from the analysis that CA-Sr NPs have a spherical structure, small and uniform particle size, and good dispersibility. Elemental analysis shows that caffeic acid and strontium are successfully coordinated. Figure 3 From this, we can see that the average particle size of CA-Sr NPs is about 136.4 nm.

[0056] (3) Preparation of injection solution for caffeic acid-strontium nanoparticles CA-Sr NPs: Caffeic acid-strontium nanoparticles were placed in a vacuum freeze-drying oven and dried at a cold trap temperature of -80°C for 6 hours. They were then resuspended with a small volume of sterile phosphate physiological buffer solution (0.01 mM, pH = 7.4) to obtain a stock solution of caffeic acid-strontium nanoparticles with a concentration range of 1~10 mg / mL, the concentration of which is adjustable.

[0057] In subsequent in vivo injection experiments, a certain volume of biocompatible solution (physiological saline or phosphate buffer) was added to the stock solution sample vial according to the calculated ratio based on the actual concentration and volume used. The vials were then resuspended by sonication in a water bath for 2 minutes to prepare injection solutions of caffeic acid-strontium nanoparticles at different concentrations. The optimal concentration of caffeic acid-strontium nanoparticles in the above injection solutions was 4–6 mg / mL, with 5 mg / mL used for mice.

[0058] In animal experiments, the injection dose for mice is 5 mg / kg, with an intra-articular injection volume typically of 20 μL. Since the weight of mice is usually between 18 and 25 g, this concentration of injection solution conforms to general principles of use in animal experiments. Furthermore, in cell experiments, the dosage is typically between 10 μg / mL and 50 μg / mL; therefore, a 5 mg / mL stock solution also meets the requirements for administration in cell experiments.

[0059] Example 3 - Experiment on the enhanced targeting of TRPV1 and scavenging of reactive oxygen species by caffeic acid-strontium nanoparticles This embodiment verifies the performance of the caffeic acid-strontium nanoparticles prepared in Example 1, including isothermal titration calorimetry determination of the affinity of caffeic acid for TRPV1 before and after coordination with strontium, and verification of the ability of caffeic acid-strontium nanoparticles to scavenge reactive oxygen species.

[0060] (1) Isothermal titration calorimetry for determining affinity: Because caffeic acid has extremely low water solubility, a 2% DMSO aqueous solution was used to dissolve it. The TRPV1 protein was dissolved using the same solvent, and affinity was determined using an isothermal titration calorimeter. The results are as follows: Figure 4 As shown.

[0061] from Figure 4 The results show that the dissociation constant Kd between caffeic acid-strontium nanoparticles (CA-Sr NPs) and TRPV1 is 249 nM, while the dissociation constant Kd between caffeic acid (CA) and TRPV1 is 1.5 μM. This indicates that strontium coordination modification of caffeic acid enhances the affinity of caffeic acid for TRPV1 and strengthens its ability to target TRPV1. Since TRPV1 is usually highly expressed at the lesion site of arthritis pain, and high expression of TRPV1 is related to macrophage inflammatory polarization and sensory neuron sensitization, this property of caffeic acid-strontium nanoparticles is beneficial for their therapeutic application in arthritis pain.

[0062] (2) Experiment on the ability to scavenge reactive oxygen species: A certain amount of ABTS solution was mixed with potassium persulfate solution and stored in the dark for 12 hours to allow ABTS to be completely oxidized to ABTS. +This was used as the working stock solution for ABTS. ABTS was prepared using phosphate buffer (pH=7.4, 0.01 mM). + The solution is diluted to an appropriate concentration so that its absorbance at a specified wavelength is approximately 0.7 ± 0.05, which is ABTS. + Working solution. Then, sample solutions of caffeic acid-strontium nanoparticles (CA-Sr NPs) at different concentrations (1.25, 2.5, 5, 10, 15, 20, 40 μg / mL) were mixed with a certain volume of ABTS+ working solution, incubated at room temperature in the dark for 5 minutes, and the absorbance (A405 nm) was measured. The results are as follows. Figure 5 As shown.

[0063] from Figure 5 It can be concluded that with the increase of the concentration of caffeic acid-strontium nanoparticles (CA-Sr NPs), ABTS... + The content of caffeic acid-strontium nanoparticles (CA-Sr NPs) gradually decreased, indicating that CA-Sr NPs have a dose-dependent reactive oxygen species scavenging ability, which is beneficial for their therapeutic application in inflammatory bone-related diseases.

[0064] Example 4 - In vitro experimental verification of caffeic acid-strontium nanoparticles This embodiment uses caffeic acid-strontium nanoparticles prepared in Example 1 to study their in vitro efficacy.

[0065] (1) Experiment on the scavenging of reactive oxygen species by primary macrophages: Primary macrophages (extracted from the bone marrow of 6-8 week old C57 mice) were first co-incubated with 100 ng / mL LPS for 8 hours. LPS / IFN-γ-treated cells were then treated with different doses of caffeic acid-strontium nanoparticles (CA-SrNPs) (12 and 25 μg / mL, prepared using complete cell culture medium). PBS treatment served as a negative control. After 12 hours, intracellular ROS levels were detected using the DCFH-DA reactive oxygen species probe. The results are as follows: Figure 6 As shown.

[0066] from Figure 6 As can be seen, CA-Sr NPs can effectively scavenge LPS / IFN-γ-induced intracellular reactive oxygen species at a concentration of 12 μg / mL, which has certain guiding significance for the actual dosage of the injection solution used for in vivo injection in animals.

[0067] (2) Experiment on calcium ion content in primary macrophages: Primary macrophages were first co-incubated with LPS / IFN-γ (100 ng / mL, 20 ng / mL) and IL-4 (20 ng / mL) for 12 hours to induce differentiation into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype, respectively, designated as LPS / IFN-γ group (M1 positive control group) and IL-4 group (M2 positive control group). Cells treated with LPS / IFN-γ were then treated with CA-Sr NPs (12 μg / mL) for 12 h, designated as the treatment group (LPS / IFN-γ + CSNs group). After Fluo-4 AM staining, green fluorescence signals were observed by confocal microscopy. The results are as follows: Figure 7 As shown.

[0068] from Figure 7 As can be seen from the data, compared with the LPS / IFN-γ group, the treatment group LPS / IFN-γ + CSNs group and IL-4 group had lower intracellular calcium ions. It is known that a large influx of calcium ions can stimulate macrophages to inflammatory polarization towards M1. This suggests that CA-Sr NPs can inhibit the calcium influx of macrophages under LPS / IFN-γ, and may thus inhibit the M1 polarization of macrophages under LPS stimulation.

[0069] (3) Repolarization experiment of primary macrophages: Primary macrophages were first co-incubated with LPS / IFN-γ (100 ng / mL, 20 ng / mL) and IL-4 (20 ng / mL) for 12 hours to induce differentiation into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype, respectively, designated as the LPS / IFN-γ group (M1 positive control group) and the IL-4 group (M2 positive control group). Cells treated with LPS / IFN-γ were then treated with CA-Sr NPs (12 μg / mL) for 12 h, designated as the treatment group (LPS / IFN-γ + CSNs group). Flow cytometry was then used to analyze the expression of CD86 and CD206 in each group. The results are as follows: Figure 8 As shown.

[0070] from Figure 8 It can be concluded that, compared with the LPS / IFN-γ group, the Q1 partition (CD86) of the treatment group LPS / IFN-γ + CA-Sr NPs group is higher. - CD206 + The number of cells increased significantly, and the Q3 partition (CD86) showed a significant increase. + CD206 - The number of cells decreased significantly, showing the same trend as the IL-4 group (M2 positive control group), indicating that CA-SrNPs can effectively induce pro-inflammatory M1 macrophages to differentiate into anti-inflammatory M2.

[0071] (4) Experiment on calcium ion content in primary dorsal root ganglion cells: Primary dorsal root ganglion cells (extracted from 2-week-old SD rats) were first loaded with the calcium ion fluorescent probe Fluo-4AM, and then incubated with the TRPV1 standard agonist (capsaicin, CAP). Specifically, the cells were incubated for 1 h in a medium containing capsaicin (final concentration 10 μM); then, caffeic acid-strontium nanoparticle (CA-Sr NPs) stock solution was added to the incubation system for 1 h to achieve a final concentration of 20 μg / mL. Fluorescence was observed using a confocal microscope with a live-cell workstation module. The results are as follows: Figure 9 As shown.

[0072] from Figure 9 The results show that after the addition of the standard TRPV1 agonist (capsaicin, CAP), dorsal root ganglion cells showed increased intracellular calcium ion signaling. However, after further incubation with CA-Sr NPs, the increased calcium ion influx was inhibited. This indicates that CA-Sr NPs can inhibit abnormal nerve excitation by antagonizing TRPV1 receptors, blocking their activation, and inhibiting abnormal calcium ion influx.

[0073] (5) Patch-clamp experiment on primary dorsal root ganglion cells: Primary dorsal root ganglion cells were first incubated on poly-L-lysine-coated cell slides. Standardized patch-clamp techniques were then used to record different drug treatments. Specifically, capsaicin stock solution (10 mM) was first added to achieve a final capsaicin concentration of 10 μM. After recording action potentials with patch-clamp, the aforementioned caffeic acid-strontium nanoparticle stock solution was added to achieve a final caffeic acid nanoparticle drug concentration of 20 μg / mL. The action potential results of the dorsal root ganglion cells are as follows: Figure 10 , Figure 11 As shown.

[0074] from Figure 10 and Figure 11 The results show that, compared with the untreated group, the frequency of action potentials was significantly increased after capsaicin CAP treatment under current stimulation of 20, 40 and 60 pA. However, after further treatment with CA-Sr NPs, the frequency of action potentials was significantly lower than that of the CAP group and also lower than that of the untreated group under the same current stimulation, indicating that CA-Sr NPs can effectively inhibit the abnormal action potentials induced by capsaicin CAP.

[0075] (6) Osteogenic induction experiment of MC3T3 cells: MC3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were subjected to standard osteoblast-induced medium (OBIM) and Sr... 2+Caffeic acid (CA) and CA-Sr NPs (10 μg / mL) were co-cultured under the following conditions: 5% CO2, 37℃, using commercially available MEM-α medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The medium was replaced with fresh medium every 3 days. At day 21, Alizarin Red (ASR) staining was performed, and the results are as follows: Figure 12 As shown.

[0076] from Figure 12 As can be seen, compared with the untreated group, the ASR positive signal of each treatment group was significantly improved. In addition, the CA-Sr NPs group showed the most ASR positive signal, which indicates that CA-Sr NPs can effectively promote the formation of calcium mineralization nodules by osteoblast progenitor cells, indicating that CA-Sr NPs have good osteogenic properties.

[0077] Example 5 - In vivo experimental verification of the effects of caffeic acid-strontium nanoparticles This embodiment studies the in vivo efficacy of the caffeic acid-strontium nanoparticles prepared in Example 1, using the treatment of rheumatoid arthritis in mice as an example.

[0078] (1) Constructing a mouse model of rheumatoid arthritis: Healthy male C57 mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used to induce rheumatoid arthritis by intra-articular injection (single injection near the ankle joint, volume 25 μL) of Complete Freund's Adjuvant (purchased from Sigma, catalog number: F5881-10ML) one week after acclimatization to the new environment. Different treatments were administered on the fifth day after Freund's adjuvant induction. The treatment evaluation period lasted for 4 weeks. During this period, the body weight and joint circumference of all mice were recorded every other day as one of the evaluation criteria for rheumatoid arthritis.

[0079] (2) Treatment experiment for rheumatoid arthritis in mice: Following the method in Example 1, the prepared caffeic acid-strontium nanoparticles (CA-Sr NPs) were dispersed in phosphate buffer (0.01 mmol / L, pH = 7.4) to obtain a CA-Sr NPs injection solution with a concentration of 5 mg / mL. Caffeic acid was dissolved in a small amount of alkaline aqueous solution with pH = 9 and diluted with phosphate buffer. The pH was then adjusted to neutral with dilute hydrochloric acid to prepare a caffeic acid (CA) injection solution with a concentration of 5 mg / mL.

[0080] The above-mentioned injection solutions were administered via joint injection to treat rheumatoid arthritis mice in the corresponding experimental groups (CA dose: 5 mg / kg, CA-Sr injection dose: 5 mg / kg), and experimental observations were recorded. Specifically, mice were divided into four groups: control group (healthy group), positive control group (adjuvant-induced rheumatoid arthritis), CA treatment group (for adjuvant-induced rheumatoid arthritis mice), and CA-Sr NPs treatment group (for adjuvant-induced rheumatoid arthritis mice). All mice received one injection per day for 28 days. In this embodiment, the dosage of CA-Sr NPs used in live mouse treatment was 5 mg / kg, and the injection solution was prepared at 5 mg / mL. The injection volume was determined according to the actual weight of the mouse; for example, a 20 g mouse injected with 5 mg / mL CA-Sr NPs should receive 20 μL of the injection solution.

[0081] After the experimental evaluation period, tissue samples were taken from the heart, liver, spleen, lungs, kidneys, and joints of all mice for pathological section analysis. Joint tissue and dorsal root ganglion tissue were also taken for pathological examination. The results are as follows: Figure 13 As shown, from Figure 13 Pathological sections of the major organs of mice showed no pathological features in the major organs of the CA-Sr NPs treatment group, indicating that CA-Sr NPs have good biocompatibility. In this embodiment, pathological examination of the major organs of rheumatoid arthritis mice used in the in vivo treatment experiment was performed. No adverse reactions were found with the caffeic acid-strontium nanoparticle injection at the therapeutic concentration in this embodiment, demonstrating its good safety profile.

[0082] according to Figure 14 The results of joint swelling measurement showed that the CA and CA-Sr NPs treatment groups could effectively relieve swelling of inflamed joints, and CA-Sr NPs showed the best swelling reduction ability.

[0083] according to Figure 15 It can be seen that both CA and caffeic acid-strontium nanoparticles CA-Sr NPs inhibited and reduced joint thermal pain and mechanical pain in mice with rheumatoid arthritis, and CA-Sr NPs had better efficacy.

[0084] Twenty-eight days into treatment, tissue samples from the dorsal root ganglia of mice were taken for pathological analysis, and the results were as follows: Figure 16 As shown. From Figure 16 The results showed that activated TRPV1+ positive cells were significantly increased in the rheumatoid arthritis mouse group, while they were significantly downregulated in the CA and CA-Sr NPs treatment groups, especially the CA-Sr NPs group, which showed the most significant downregulation. This indicates that CA-Sr NPs can effectively inhibit the transduction of pain signals in arthritis.

[0085] On day 28 of treatment, mouse ankle joints were harvested for Micro-CT imaging analysis. Figure 17 As can be seen, both CA and caffeic acid-strontium nanoparticles CA-Sr NPs inhibited bone erosion in mice with rheumatoid arthritis, but CA-Sr NPs showed better bone repair effects and increased bone mineral density in the ankle joints of mice with rheumatoid arthritis.

[0086] Furthermore, based on the difference between mouse experimental doses and human doses, and according to the relevant empirical formula: human drug dose (mg / kg) = 9.01 × mouse drug dose (mg / kg), the concentration of caffeic acid-strontium nanoparticles in the injection solution suitable for intra-articular injection in humans can be calculated to be around 45.05 mg / kg.

[0087] In summary, this invention innovatively discovers that four natural phenolic acids—caffeic acid, ferulic acid, gallic acid, and chlorogenic acid—all possess the potential to target TRPV1. Taking caffeic acid as an example, caffeic acid-strontium nanoparticles (CA-Sr NPs) were prepared using a simple method, and the prepared CA-Sr... NPs exhibit excellent dispersion stability. Through coordination modification of caffeic acid molecules via the carboxyl and catechol groups of strontium, the ability to target TRPV1 is significantly enhanced. This effectively inhibits TRPV1-mediated abnormal calcium influx from macrophages and dorsal root ganglion cells; suppresses inflammatory polarization of macrophages and neurosensitization of dorsal root ganglion cells; effectively reduces ROS levels at rheumatoid arthritis lesions; and promotes osteogenic repair using the bioactivity of metallic strontium. They demonstrate significant effects in the prevention and treatment of rheumatoid arthritis, not only alleviating joint swelling induced by rheumatoid arthritis in mice and effectively reducing the expression of inflammatory factors in synovial tissue, but also increasing the expression of anti-inflammatory factors. An injection prepared from caffeic acid-strontium nanoparticles (CA-Sr NPs) effectively inhibits bone erosion and promotes bone repair in mice after treatment, exhibiting good safety. They have important application value in the preparation of drugs for the prevention and / or treatment of inflammatory bone-related diseases, in the preparation of bone repair drugs, and in the preparation of drugs targeting and antagonizing TRPV1.

[0088] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A phenolic acid-strontium nanoparticle, characterized in that, The phenolic acid-strontium nanoparticle is a nanostructure obtained by self-assembly of a phenolic acid modified by coordination of a metal strontium; wherein the phenolic acid is at least one of caffeic acid, ferulic acid, gallic acid, and chlorogenic acid.

2. The phenolic acid-strontium nanoparticle of claim 1, wherein, The phenolic acid-strontium nanoparticle is a caffeic acid-strontium nanoparticle, which is modified by coordination of strontium with the carboxyl and catechol groups of the phenolic acid.

3. The phenolic acid-strontium nanoparticle of claim 2, wherein, The preparation method of the caffeic acid-strontium nanoparticle comprises the following steps: S1, dissolving a strontium element precursor powder in ultrapure water to prepare a strontium precursor aqueous solution; S2, mixing an alkaline aqueous solution with a caffeic acid powder, and after the caffeic acid powder is dissolved, adding ultrapure water to constant volume to prepare a caffeic acid aqueous solution; S3, adding the strontium precursor aqueous solution obtained in step S1 to the caffeic acid aqueous solution obtained in step S2, stirring and reacting, collecting the precipitate by centrifugation, washing, and vacuum drying to obtain the caffeic acid-strontium nanoparticle.

4. The phenolic acid-strontium nanoparticle of claim 3, wherein, In step S1, the concentration of the strontium precursor in the strontium precursor aqueous solution is 1-50 mg / mL; and / or In step S1, the strontium element precursor includes at least one of strontium chloride, strontium nitrate, strontium acetylacetone, and strontium sulfate.

5. The phenolic acid-strontium nanoparticle of claim 3, wherein, In step S2, the alkaline aqueous solution includes at least one of sodium hydroxide, potassium hydroxide, and ammonia water; and / or In step S2, the concentration of the alkaline aqueous solution is 0.5-5 M; and / or In step S2, in the caffeic acid aqueous solution, the concentration of the caffeic acid is 2-10 mg / mL, and the volume ratio of the alkaline aqueous solution to ultrapure water is 1:5-1:

15.

6. The phenolic acid-strontium nanoparticle of claim 3, wherein, In step S3, the mass ratio of the strontium precursor to the caffeic acid is 1:1-20:1; and / or In step S3, the strontium precursor aqueous solution is added dropwise to the caffeic acid aqueous solution at a speed of 0.5-1.5 mL / min; and / or In step S3, the stirring and reaction is performed at a speed of 300-800 rpm for 3-5 h; and / or In step S3, the centrifugation parameters are 10000-14000 r for 8-12 min; and / or In step S3, the washing is performed once with ultrapure water and twice with a 1:1 mixture of ethanol and ultrapure water; and / or In step S3, the operating conditions of the vacuum freeze-drying are: vacuum degree -0.8 to -1 MPa, cold trap temperature -80℃, and drying time 5-7 h; and / or The caffeic acid-strontium nanoparticle prepared in step S3 has a spherical structure, and the average particle size is 100-150 nm.

7. A phenolic acid-strontium nanoformulation, characterized in that, The effective component of the preparation includes the phenolic acid-strontium nanoparticle according to any one of claims 1-6.

8. The phenolic acid-strontium nanoformulation according to claim 7, characterized in that, The phenolic acid-strontium nanoparticle preparation is a caffeic acid-strontium nanoparticle preparation, which is an injection prepared by dissolving the caffeic acid-strontium nanoparticle in a biocompatible solution; wherein the biocompatible solution is physiological saline and / or phosphate buffer.

9. The phenolic acid-strontium nanoformulation according to claim 8, characterized in that, The coffee acid-strontium nanoparticle is dissolved in a biocompatible solution to prepare an injection solution, specifically: the coffee acid-strontium nanoparticle is placed in a vacuum freeze-drying box, dried at a cold trap temperature of-80 DEG C for 6 hours, and then resuspended in a certain volume of a biocompatible solution to obtain an injection solution; wherein the concentration of the coffee acid-strontium nanoparticle in the injection solution is 1-10 mg / mL.

10. Use of a phenolate-strontium nanoparticle as defined in any one of claims 1 to 6 or a phenolate-strontium nanoparticle formulation as defined in any one of claims 7 to 9, characterized in that, The application includes at least one of the following applications: application in preparing a drug for preventing and / or treating an inflammatory bone-related disease, application in preparing a bone repair drug, and application in preparing a drug for targeting antagonizing TRPV1.

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