Intelligent response nanoprobe for targeted diagnosis and treatment of osteoarthritis and preparation method thereof

Abstract

The invention provides an intelligent response nanoprobe for targeted diagnosis and treatment of osteoarthritis and a preparation method of the intelligent response nanoprobe, and belongs to the field of nanomedicine. According to the method, amino cerium oxide is innovatively connected to the surface of mesoporous gadolinium-doped Prussian blue (Gd-HMPB) coated with hyaluronic acid, and an anti-inflammatory drug is packaged into pores of the Gd-HMPB in an SMT (Surface Mount Technology) manner, so that the targeted intelligent response nano probe for diagnosis and treatment of osteoarthritis (OA) is prepared. Under the targeting action of hyaluronic acid, the intelligent response nanoprobe accurately reaches the OA cartilage part, and the inflammation position is accurately judged by virtue of a magnetic resonance imaging technology. In the acidic microenvironment of inflammation, SMT released by the nanoprobe can cooperate with HMPB and CeO2 nanoenzyme to remove ROS and inflammatory factors and promote polarization of macrophages to anti-inflammatory macrophages, so that the effect of remodeling the OA microenvironment is achieved, and a new thought is expected to be provided for clinical precise diagnosis and treatment of OA.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine, specifically relating to a smart responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method. Background Technology

[0002] Osteoarthritis (OA), a common degenerative joint disease, not only imposes significant long-term health costs on patients but also on society as a whole. The pathological features of OA primarily include progressive loss of articular cartilage, osteophyte formation, and changes in the periarticular and subchondral bone, leading to joint stiffness, chronic pain, and functional impairment. Studies have shown that three major biological factors—proteolytic enzymes, pro-inflammatory cytokines (TNF-α, IL-17A, IL-1), and reactive oxygen species (ROS)—cause and exacerbate cartilage degeneration in OA. Currently, effective treatments for OA include intra-articular drug delivery, including nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and corticosteroids. However, widely used NSAIDs only provide short-term pain control in OA, requiring repeated administration, which severely limits their effectiveness. Long-term systemic use, on the other hand, can cause serious side effects, such as gastrointestinal complications and osteoporosis. This is because these exogenous synthetic drugs not only possess a certain degree of cytotoxicity but also require increased dosages due to their inability to target OA, further increasing cytotoxicity. Therefore, it is necessary to develop a new drug that can both treat inflammation and maximize the protection of patients and drug delivery systems in order to improve the treatment effect of OA.

[0003] Studies have shown that the main characteristic of osteoarthritis (OA) is cartilage damage, primarily caused by an unfavorable OA microenvironment. As a crucial factor affecting joint homeostasis, the OA microenvironment, through both biological and mechanical processes, disrupts the balance between chondrocyte and ECM synthesis and degradation, leading to articular cartilage degeneration and fibrosis. The OA microenvironment mainly includes excessive inflammatory mediators, overexpressed matrix metalloproteinases (MMPs), and excessive reactive oxygen species (ROS). As key pro-inflammatory cytokines in OA pathology, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) can induce proteolytic enzymes and stimulate the production of other inflammatory factors [IL-6, IL-8, and nitric oxide (NO)] and ROS, thereby accelerating cartilage destruction. Furthermore, ROS plays a vital role in regulating cartilage metabolism, chondrocyte apoptosis, ECM synthesis and degradation, and cytokine production, contributing significantly to the occurrence and development of osteoarthritis. In addition, excessive ROS can also increase inflammation and accelerate chondrocyte catabolism and apoptosis by participating in multiple signaling pathways that damage or activate cytokines. Therefore, reducing the levels of ROS and inflammatory mediators in the OA microenvironment and reshaping the joint microenvironment may be a potential treatment strategy for OA.

[0004] Nanoparticles (NPs) have been reported to passively target inflammatory sites and accumulate within inflamed tissues via extravasation through the endovascular system and subsequent inflammatory cell-mediated isolation (ELVIS) effect. Currently, mesoporous nanomaterials are attracting widespread attention due to their unique properties, such as high loading capacity, chemical and physical stability, low toxicity, and ease and inexpensive production in the laboratory. Prussian blue (PB), approved by the US Food and Drug Administration (FDA) as a clinical antidote, has garnered significant attention due to its excellent biocompatibility and safety. Furthermore, PB nanozymes can scavenge reactive oxygen species (ROS) that are excessively produced during inflammation and cause oxidative damage to DNA, proteins, and lipids. Recent studies in animal models have found that this system can deliver antioxidants to diseased tissues, thereby improving symptoms of inflammation-related diseases. These findings suggest that we can utilize mesoporous Prussian blue to prepare nano-drug delivery systems for the precise diagnosis and treatment of osteoarthritis (OA).

[0005] The human body has three main antioxidant enzymes: peroxidase, catalase, and superoxide dismutase (SOD), which prevent damage caused by oxidative stress by scavenging reactive oxygen species (ROS). However, the activity of these natural enzymes is easily lost due to the influence of the inflammatory microenvironment, such as pH, temperature, and proteases. Therefore, much research has focused on the development of nanozymes, i.e., nanoparticles with natural enzyme activity. Among the many nanozymes, CeO2 NPs have attracted great attention in the biomedical field due to their good biocompatibility and multi-enzyme activity. Studies have shown that CeO2 NPs possess superoxide dismutase and catalase activities, as well as hydroxyl radical scavenging activity, making them more effective than non-catalytic antioxidants in scavenging reactive oxygen species (ROS), while being more stable than free enzymes. Therefore, combining CeO2 nanozymes with novel drug delivery carriers to achieve personalized drug delivery at the molecular level, reducing unnecessary costs and toxicity from ineffective treatments for OA patients, represents a promising treatment method for ROS-related osteoarthritis.

[0006] Cytotoxicity is the biggest obstacle to chemotherapy, and endogenous products with anti-inflammatory effects may be a good alternative. Hyaluronic acid (HA), the most widely distributed viscous polysaccharide in the human body, is a natural unbranched polymer composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. It is a major component of the extracellular matrix (ECM) (belonging to GAGs) and possesses good hydrophilicity and biocompatibility. Recent studies have shown that self-assembled HA nanoparticles (HANPs) can not only actively target the pathological site (CD44) expressing the HA receptor in OA, but also show therapeutic potential by interfering with the interaction between the LMW-HA-CD44 fragment and the pathogenesis of OA. In short, nano-sized HA can precisely target OA tissue and has significant anti-inflammatory effects, making it a potential natural polymer for the treatment of OA.

[0007] Magnetic resonance imaging (MRI) is an advanced medical imaging diagnostic technique that allows for non-invasive and rapid detection of biological soft tissues and internal organs. It utilizes the different resonance signals generated by different tissues under the influence of an external magnetic field to create images. MRI can effectively detect tissue necrosis, local ischemia, and various malignant lesions (such as tumors), enabling early diagnosis and monitoring of organ transplantation. In clinical MRI, approximately 30-40% of diagnoses require the use of MRI contrast agents to improve image contrast and clarity. Currently, the most widely used clinical MRI contrast agent is gadolinium chelate—gadopylene dihydropyridine (Gd-DTPA), due to the presence of gadolinium (Gd... 3+ The seven unpaired electrons in Gd-containing protons effectively reduce the T1 and T2 relaxation times of hydrogen protons in water, thereby increasing the signal intensity in MRI (showing positive enhancement). Therefore, Gd-containing protons... 3+ MRI contrast agents play an important role in the diagnosis of clinical MRI diseases. Summary of the Invention

[0008] The purpose of the embodiments in this specification is to provide a smart responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method, so as to ensure sufficient drug penetration into cartilage tissue and achieve the purpose of effective diagnosis and treatment of osteoarthritis.

[0009] This invention innovatively connects cerium aminooxide to the surface of hyaluronic acid-coated mesoporous gadolinium-doped Prussian blue (Gd-HMPB) and encapsulates the anti-inflammatory drug SMT within the pores of the Gd-HMPB, thus preparing a targeted intelligent responsive nanoprobe. Under the targeting effect of hyaluronic acid, the intelligent responsive nanoprobe precisely reaches the cartilage site of osteoarthritis (OA), and the location of inflammation can be accurately determined using magnetic resonance imaging (MRI). In the acidic microenvironment of inflammation, the SMT released by the nanoprobe can synergistically work with HMPB and CeO2 nanozymes to scavenge ROS and inflammatory factors, and promote the polarization of macrophages into anti-inflammatory macrophages, thereby reshaping the OA microenvironment and potentially providing new insights for the precise clinical diagnosis and treatment of OA.

[0010] A method for preparing a targeted smart responsive nanoprobe for the diagnosis and treatment of osteoarthritis includes the following steps:

[0011] (1) Mesoporous Gd-HMPB loaded with anti-inflammatory drugs SMT: First, 1–100 g of polyvinylpyrrolidone (PVP) and 0.1–3.0 g of potassium ferricyanide were dissolved in 10–100 mL of hydrochloric acid aqueous solution with a concentration of 0.001–0.1 mol / L, named solution A. Then, 10–100 g of PVP and 0.1–3.0 g of gadolinium hexahydrate were dissolved in 10–100 mL of hydrochloric acid aqueous solution with a concentration of 0.001–0.1 mol / L, named solution B. Next, solutions A and B were mixed at a volume ratio of 1:0.5–1:5 and reacted in an oven at 50–100 °C for 12–36 h to obtain Gd-containing Prussian blue nanoblocks (Gd-PB). To prepare porous Prussian blue, 0.1–1.0 g of Gd-PB and 0.1–2.5 g of PVP were sequentially dissolved in 200–1000 mL of 0.1–10 mol / L hydrochloric acid solution, and then etched at high temperature in a stainless steel autoclave at 70–200 °C for 1–5 h to obtain Gd-containing porous Prussian blue nanobulbs (Gd-HMPB). Then, 0.1–1.0 g of Gd-HMPB was stirred with 1–10 g of S-methylisothiourea sulfate (SMT) at room temperature for 12–36 h to obtain the drug-loaded nanoprobe Gd-HMPB@SMT (GHPS).

[0012] (2) HA-coated Gd-HMPB@SMT: Mix 5-70 mL of sodium nitrite solution (1-10 g / L) and 25-125 mL of acetic acid solution (5-15 g / L), and add 1-10 g of low molecular weight hyaluronic acid to the mixture. Stir at 25-80°C for 2-10 h. After the reaction is complete, neutralize the reaction solution with 0.01-1 mol / L sodium hydroxide and freeze-dry. Next, dissolve 0.1-1.0 g of ultra-low molecular weight sodium hyaluronate in 1-10 mL of water and permeate the solution through a 50-200 mL acidified gel column. Finally, freeze-dry the collected solution to obtain ultra-low molecular weight hyaluronic acid. Subsequently, add 10-40 mg of ultra-low molecular weight hyaluronic acid to 0.5-5 mL of 1 mg / mL GHPS solution and react at room temperature for 1-12 h to obtain Gd-HMPB@SMT@HA (GHPSH).

[0013] (3) Preparation of NH2-CeO2: 0.1–1 g of cerium nitrate hexahydrate and 0.4–4.5 g of polyallylamine hydrochloride were dissolved sequentially in 5–50 mL of ethylene glycol and stirred vigorously at 40–80 °C for 1–30 min. Then, 0.1–10 mL of ammonia water was rapidly injected into the mixed solution using a syringe, and stirring was continued at 40–80 °C for 2–6 h. After the reaction was complete, the solution was centrifuged at high speed and washed with deionized water to obtain cerium nitrate oxide (NH2-CeO2).

[0014] (4) Preparation of Gd-HMPB@SMT@HA@CeO2: 1-100 μL of NH2-CeO2 aqueous solution with a concentration of 5-30 g / L was slowly added dropwise to 0.5-2 mL of GHPSH aqueous solution with a concentration of 0.1-1.0 g / L, and the solution was shaken in a shaker at 25-50 °C for 6-24 h to obtain the targeted intelligent response nanoprobe Gd-HMPB@SMT@HA@CeO2 (GHPSHCe).

[0015] Furthermore, in step (1), the particle size of GHPS is 50-200 nm and the surface potential is -5-5 mV.

[0016] Furthermore, in step (2), the mass ratio of GHPS to HA is 1:0.2 to 1:80.

[0017] Furthermore, in step (2), the particle size of GHPSH is 50-200 nm and the surface potential is -30-0 mV.

[0018] Furthermore, in step (3), the mass ratio of cerium nitrate hexahydrate to polyallylamine hydrochloride is 1:0.02 to 1:2.5.

[0019] Furthermore, in step (3), the particle size of NH2-CeO2 is 1-30 nm and the surface potential is 20-40 mV.

[0020] Furthermore, in step (4), the mass ratio of GHPSH to NH2-CeO2 is 1:0.02 to 1:100.

[0021] Furthermore, the targeted smart response nanoprobe GHPSHCe has a particle size of 20–200 nm and a surface charge of -10–10 mV.

[0022] Furthermore, the targeted intelligent response nanoprobe GHPSHCe achieved a drug release rate of 47.40% in a simulated OA microenvironment at pH 6.0, which is 2.06 times higher than the release rate (22.98%) in the normal physiological environment of human cartilage at pH 7.4.

[0023] Furthermore, the targeted intelligent response nanoprobe GHPSHCe is a novel OA therapeutic agent with a mesoporous Gd-HMPB framework, internally loaded with the anti-inflammatory drug SMT, surface coated with HA, and encapsulated with CeO2.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) Gd-HMPB not only has good biocompatibility and biosafety, but also can inhibit inflammation by scavenging reactive oxygen species and inflammatory factors. Moreover, it can perform magnetic resonance imaging while loading anti-inflammatory drugs, thereby achieving effective diagnosis and treatment of osteoarthritis.

[0026] (2) As a potential natural polymer for treating OA, hyaluronic acid can precisely target OA sites, has a significant anti-inflammatory effect, and can act as a lubricant as a filler in joints. This allows the smart nanoprobe GHPSHCe to precisely target cartilage tissue while reducing friction between bone ends.

[0027] (3) The enzyme-like activity of CeO2 NPs enables them to scavenge reactive oxygen species (ROS) and promoting factors, while promoting macrophage polarization to anti-inflammatory type, and can decompose and release anti-inflammatory SMT in OA acidic environment, thereby achieving precise regulation and treatment of osteoarthritis.

[0028] (4) The preparation method of the present invention is stable and highly reproducible. The prepared targeted intelligent responsive nanoprobe has good biosafety and high targeting. It can accurately locate the inflammatory site through magnetic resonance imaging technology and achieve targeted drug release at the inflammatory site, thereby realizing the accurate diagnosis and treatment of osteoarthritis. It is expected to provide new ideas for the clinical precision diagnosis and treatment of OA. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments or prior art of this specification, the drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Transmission electron microscopy (TEM) images of different nanoparticles of the present invention: (A) TEM image of Gd-HMPB (scale bar 100 nm), (B) TEM image of GHPSHCe (scale bar 100 nm).

[0031] Figure 2 The hydrated particle size diagrams of Gd-HMPB, GHPS, GHPSH and GHPSHCe prepared in this invention are shown.

[0032] Figure 3 Surface potential diagrams of Gd-HMPB, SMT, GHPS, GHPSH, CeO2, and GHPSHCe prepared in this invention;

[0033] Figure 4 The ultraviolet spectra of Gd-HMPB, SMT, HA, CeO2 and GHPSHCe prepared in this invention are shown.

[0034] Figure 5 This is a schematic diagram related to magnetic resonance imaging of the GHPSHCe nanoprobe of the present invention; (A) T1 magnetic resonance imaging signal image,

[0035] (B) T1 magnetic resonance imaging relaxation rate curve;

[0036] Figure 6 This is the standard curve of SMT in the GHPSHCe nanoprobe of this invention;

[0037] Figure 7 The drug loading rate and encapsulation efficiency of the GHPSHCe nanoprobe of this invention;

[0038] Figure 8 This invention provides a method for determining the drug release curves of the GHPSHCe nanoprobe under different conditions.

[0039] Figure 9 The superoxide dismutase (SOD)-like activity of the GHPSHCe nanoprobe of this invention;

[0040] Figure 10The catalase-like (CAT) activity of the GHPSHCe nanoprobe of this invention is shown in the following figures: (A) absorbance of H2O2 at 240 nm after mixing with different concentrations of GHPSHCe, and (B) decomposition rate of H2O2 under the catalysis of GHPSHCe.

[0041] Figure 11 The peroxidase-like (POD-like) activity of the GHPSHCe nanoprobe of this invention is shown in the graphs: (A) absorbance values ​​at 650 nm of TMB oxide reacting with different concentrations of GHPSHCe, and (B) changes in absorbance values ​​of TMB oxide at different times under the catalysis of GHPSHCe.

[0042] Figure 12 The present invention uses the CCK-8 assay to evaluate the cytotoxicity of GHPSHCe nanoprobes against ATDC5 chondrocytes, RAW264.7 macrophages and HUVEC endothelial cells;

[0043] Figure 13 To assess the blood compatibility of the GHPSHCe nanoprobe of this invention;

[0044] Figure 14 MRI imaging was used to evaluate the cartilage targeting and retention time of GHPSHCe in the control group and early OA model;

[0045] Figure 15 This is a flowchart illustrating the steps of a method for preparing a targeted intelligent response nanoprobe for the diagnosis and treatment of osteoarthritis, according to an embodiment of the present invention. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0047] Example 1:

[0048] (1) Mesoporous Gd-HMPB loaded with anti-inflammatory drugs (SMT): First, 4g of polyvinylpyrrolidone (PVP) and 0.128g of ferricyanide were dissolved in 40mL of 0.01mol / L hydrochloric acid aqueous solution, named solution A. Then, 4g of PVP and 0.18g of gadolinium hexahydrate (Gd(NO3)3·6H2O) were dissolved in 40mL of 0.01mol / L hydrochloric acid aqueous solution, named solution B. Next, solutions A and B were mixed at a volume ratio of 1:0.5 to 1:5 and reacted in an oven at 80℃ for 24h. Gd-containing Prussian blue nanoblocks (Gd-PB) were obtained. To prepare porous Prussian blue, 0.111 g of Gd-PB nanoparticles and 0.555 g of PVP were sequentially dissolved in 111 mL of 1.0 mol / L hydrochloric acid solution, and then etched at high temperature for 3 h in a stainless steel autoclave at 140 °C to obtain Gd-containing porous Prussian blue nanobulbs (Gd-HMPB). Then, 0.5 mg of Gd-HMPB nanoparticles were stirred with 5 mg of SMT at room temperature for 12 h to obtain the drug-loaded nanoprobe Gd-HMPB@SMT (GHPS).

[0049] (2) HA coating of Gd-HMPB@SMT: 12 mL of 58 mmol / L sodium nitrite solution and 75 mL of 166 mmol / L acetic acid solution were mixed. 2.2 g of low molecular weight hyaluronic acid was added to the mixture, and the mixture was stirred at 25 °C for 6 h. After the reaction was complete, the reaction solution was neutralized with 0.1 mol / L sodium hydroxide and freeze-dried. Next, 1.0 g of ultra-low molecular weight sodium hyaluronate was dissolved in 10 mL of water, and the solution was permeated through a 100 mL acidified gel column. The collected solution was freeze-dried to obtain ultra-low molecular weight hyaluronic acid (HA). 20 mg of HA was dissolved in 1 mL of 1.0 mg / mL GHPS and reacted at room temperature for 3 h to obtain Gd-HMPB@SMT@HA (GHPSH).

[0050] (3) Preparation of NH2-CeO2: 126 mg of cerium nitrate hexahydrate and 450 mg of polyallylamine hydrochloride were dissolved sequentially in 10 mL of ethylene glycol and stirred vigorously at 40–80 °C for 5 min. Then, 1.6 mL of 30% ammonia solution was rapidly injected into the mixed solution using a syringe, and stirring was continued at 60 °C for 3 h. After the reaction was complete, the solution was centrifuged at high speed and washed with deionized water to obtain cerium nitrate oxide (NH2-CeO2).

[0051] (4) Preparation of Gd-HMPB@SMT@HA@CeO2: 10 μL of NH2-CeO2 aqueous solution with a concentration of 10 g / L was added dropwise to 1 mL of GHPSH aqueous solution with a concentration of 1.0 g / L, and the solution was shaken at 180 rpm for 12 h in a shaker at 37 °C to obtain the targeted intelligent response nanoprobe Gd-HMPB@SMT@HA@CeO2 (GHPSHCe).

[0052] Transmission electron microscopy (TEM) images show porous Gd-HMPB nanoparticles ( Figure 1 A) and the targeted intelligent response nanoprobe GHPSHCe have highly uniform morphology. Figure 1 B). From intraporous drug loading in Gd-HMPB to surface modification to construct GHPSHCe, the hydrated particle size of the nanoparticles gradually increased slightly, with the hydrated particle size of GHPSHCe being approximately 164 nm. Figure 2 The Zeta potential test results showed that the surface potentials of Gd-HMPB, SMT, GHPS, GHPSH, CeO2, and GHPSHCe were -2.48mV, -2.93mV, -4.92mV, -9.71mV, 23.67mV, and -0.29mV, respectively. Figure 3 This indicates the successful encapsulation of GHPSHCe. Furthermore, the UV absorption spectrum of GHPSHCe also shows characteristic absorption peaks for Gd-HMPB (706 nm), SMT (227 nm), HA (243 nm), and CeO2 (206 nm). Figure 4 This further proves the successful preparation of the nanoprobe GHPSHCe.

[0053] To investigate the T1 magnetic resonance imaging capability of the targeted intelligent responsive nanoprobe GHPSHCe, we prepared a series of GHPSHCe solutions with different concentrations and performed magnetic resonance imaging on them. Figure 5 As shown, with the increase of GHPSHCe nanoparticle concentration, Gd 3+ As the concentration increases, the aqueous solution of the nanoprobe becomes brighter. Figure 5 The higher the T1 WI signal, the stronger the T1 magnetic resonance imaging capability. Calculations show that the T1 relaxation rate of GHPSHCe is 8.312 mM. -1 s -1 .

[0054] To calculate the loading capacity of the targeted smart response nanoprobe onto SMT, we first performed UV spectroscopy tests on SMT solutions of different concentrations and plotted a standard curve. Figure 6 Next, based on its standard curve, the coating efficiency and drug loading rate of the smart response nanoprobe to SMT were calculated for different SMT feed amounts, and the results are as follows: Figure 7As shown, the drug loading rate of Gd-HMPB on SMT changed with the SMT concentration, especially when the SMT concentration was 10 mg / mL, the drug loading rate (47.46%) and encapsulation efficiency (52.20%) of the smart responsive nanoprobe to SMT were the highest. Notably, the drug release rate of the responsive nanoprobe in the simulated OA microenvironment pH=6.0 was as high as 47.40%, which is 2.06 times higher than the release rate (22.98%) in the normal physiological environment of human cartilage pH=7.4. Figure 8 This may be due to the fact that the degradation of CeO2 in a weakly acidic environment promotes the effective and rapid release of SMT.

[0055] Superoxide dismutase (SOD) plays a crucial role in maintaining the body's oxidative and antioxidant balance, scavenging superoxide anion free radicals and protecting cells from damage. Detection of GGHPSHCe solution using an SOD kit showed that GGHPSHCe effectively inhibits O2· - The inhibitory effect increases with increasing concentration. Figure 9 This indicates that the prepared targeted intelligent response nanoprobe has good SOD activity. Subsequently, after testing the catalase (CAT) activity of GHPSHCe using a UV spectrophotometer, it was found that the scavenging rate of H2O2 by GHPSHCe was concentration-dependent. Figure 10 A). Moreover, the scavenging rate of H2O2 gradually increases over time. Figure 10 B). After the reaction is complete, a large number of visible bubbles are produced, and the number of bubbles increases with the duration of the reaction. This indicates that GHPSHCe has CAT activity, enabling the decomposition of H2O2 into H2O and O2. Next, we used TMB, the natural substrate for POD, to verify the POD activity of GHPSHCe. Figure 11 As shown in Figure A, the color of the solution gradually deepens with increasing GHPSHCe concentration, indicating an increase in the production of TMB oxidation products. Furthermore, the absorbance of the reaction solution at 650 nm increases linearly with time. Figure 11 B) indicates that GHPSHCe has good POD-like activity, and its POD activity is specific to concentration and time dependence.

[0056] The cytotoxicity of different concentrations of nanoprobes was assessed using the CCK-8 assay, which showed that... Figure 12 With increasing GHP-PSHCe concentration, the survival rates of ATDC5 chondrocytes, RAW264.7 macrophages, and HUVEC cells did not show a significant decrease (>90%), indicating that the intelligent nanoprobe has good biosafety. Furthermore, the hemolysis rate of rat erythrocytes treated with the intelligent nanoprobe (0–500 μg / mL) was less than 5%. Figure 13This further confirms the good biocompatibility of the GHPSHCe nanoprobe.

[0057] To further verify the targeting ability of the prepared GHPSHCe nanoprobes for osteoarthritis and their T1 magnetic resonance imaging (T1) capability, we induced an osteoarthritis model in the legs of SD rats and injected the GHPSHCe nanoprobes into the joint cavity. Magnetic resonance imaging results showed ( Figure 14 The smart nanoprobes showed higher signal intensity in the articular cartilage of the OA group than in the control group, indicating that the targeted smart response nanoprobes can effectively target OA cartilage tissue and perform T1 magnetic resonance imaging.

[0058] For those skilled in the art, the present invention is not limited to the details of the exemplary embodiments described above. The embodiments are illustrative and not restrictive, and the scope of protection of the present invention is defined by the appended claims rather than the foregoing description. Therefore, no reference numerals in the claims should be construed as limiting the scope of the claims.

[0059] The above description is merely an embodiment of one or more embodiments of this specification and is not intended to limit the scope of these embodiments. Various modifications and variations can be made to these embodiments by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this specification should be included within the scope of the claims.

Claims

1. A smart responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method, characterized in that, The method includes the following steps: (1) Mesoporous Gd-HMPB loaded with anti-inflammatory drugs SMT: First, 1–100 g of polyvinylpyrrolidone (PVP) and 0.1–3.0 g of potassium ferricyanide were dissolved in 10–100 mL of hydrochloric acid aqueous solution with a concentration of 0.001–0.1 mol / L, named solution A. Then, 10–100 g of PVP and 0.1–3.0 g of gadolinium hexahydrate were dissolved in 10–100 mL of hydrochloric acid aqueous solution with a concentration of 0.001–0.1 mol / L, named solution B. Next, solutions A and B were mixed at a volume ratio of 1:0.5–1:5 and reacted in an oven at 50–100 °C for 12–36 h to obtain Gd-containing Prussian blue nanoblocks (Gd-PB). To prepare porous Prussian blue, 0.1–1.0 g of Gd-PB and 0.1–2.5 g of PVP were sequentially dissolved in 200–1000 mL of 0.1–10 mol / L hydrochloric acid solution, and then etched at high temperature in a stainless steel autoclave at 70–200 °C for 1–5 h to obtain Gd-containing porous Prussian blue nanobulbs (Gd-HMPB). Then, 0.1–1.0 g of Gd-HMPB was stirred with 1–10 g of S-methylisothiourea sulfate (SMT) at room temperature for 12–36 h to obtain the drug-loaded nanoprobe Gd-HMPB@SMT (GHPS). (2) HA-coated Gd-HMPB@SMT: Mix 5-70 mL of sodium nitrite solution (1-10 g / L) and 25-125 mL of acetic acid solution (5-15 g / L), and add 1-10 g of low molecular weight hyaluronic acid to the mixture. Stir at 25-80°C for 2-10 h. After the reaction is complete, neutralize the reaction solution with 0.01-1 mol / L sodium hydroxide and freeze-dry. Next, dissolve 0.1-1.0 g of ultra-low molecular weight sodium hyaluronate in 1-10 mL of water, and permeate the solution through a 50-200 mL acidified gel column. Finally, freeze-dry the collected solution to obtain ultra-low molecular weight hyaluronic acid. Subsequently, add 10-40 mg of ultra-low molecular weight hyaluronic acid to 0.5-5 mL of 1 mg / mL GHPS solution and react at room temperature for 1-12 h to obtain Gd-HMPB@SMT@HA (GHPSH). (3) Preparation of NH2-CeO2: 0.1–1 g of cerium nitrate hexahydrate and 0.4–4.5 g of polyallylamine hydrochloride were dissolved sequentially in 5–50 mL of ethylene glycol and stirred vigorously at 40–80 °C for 1–30 min. Then, 0.1–10 mL of ammonia water was rapidly injected into the mixed solution using a syringe, and stirring was continued at 40–80 °C for 2–6 h. After the reaction was complete, the solution was centrifuged at high speed and washed with deionized water to obtain cerium nitrate oxide (NH2-CeO2). (4) Preparation of Gd-HMPB@SMT@HA@CeO2: 1-100 μL of NH2-CeO2 aqueous solution with a concentration of 5-30 g / L was slowly added dropwise to 0.5-2 mL of GHPSH aqueous solution with a concentration of 0.1-1.0 g / L, and the solution was shaken in a shaker at 25-50 °C for 6-24 h to obtain the targeted intelligent response nanoprobe Gd-HMPB@SMT@HA@CeO2 (GHPSHCe).

2. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (1), the particle size of GHPS is 50-200 nm and the surface potential is -5-5 mV.

3. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (2), the mass ratio of GHPS to HA is 1:0.2 to 1:

80.

4. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (2), the particle size of GHPSH is 50-200 nm and the surface potential is -30-0 mV.

5. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (3), the mass ratio of cerium nitrate hexahydrate to polyallylamine hydrochloride is 1:0.02 to 1:2.

5.

6. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (3), the particle size of NH2-CeO2 is 1-30 nm and the surface potential is 20-40 mV.

7. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: In step (4), the mass ratio of GHPSH to NH2-CeO2 is 1:0.02 to 1:

100.

8. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: The targeted smart response nanoprobe GHPSHCe has a particle size of 20–200 nm and a surface charge of -10–10 mV.

9. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: The targeted intelligent response nanoprobe GHPSHCe achieved a drug release rate of 47.40% in a simulated OA microenvironment at pH 6.0, which is 2.06 times higher than the release rate (22.98%) in the normal physiological environment of human cartilage at pH 7.

4.

10. The intelligent responsive nanoprobe for targeted diagnosis and treatment of osteoarthritis and its preparation method as described in claim 1, characterized in that: The targeted intelligent response nanoprobe GHPSHCe is a novel OA therapeutic agent with a mesoporous Gd-HMPB framework, internal loading of the anti-inflammatory drug SMT, surface coating of HA, and encapsulation with CeO2.

Images