PtCu@BSA nanoparticles, a preparation method and application thereof

Abstract

The application discloses PtCu@BSA nanoparticles and a preparation method and application thereof, and relates to the technical field of nanometer materials.The PtCu@BSA nanoparticles take porous PtCu-based nanoparticles as a framework, and are wrapped with BSA on the surface, which is favorable for enhancing the biocompatibility and stability of the PtCu@BSA nanoparticles; meanwhile, the porous PtCu-based nanoparticles have high targeting capability and electromotive catalytic effect, can well catalyze water decomposition into hydroxyl radicals under the action of an alternating electric field, thereby effectively killing bacteria, and improving the limitation that antibiotics are frequently used in existing drugs for treating osteomyelitis.Meanwhile, in the process of electromotive catalysis, the porous PtCu-based nanoparticles are partially degraded into positively charged ions such as Pt 2+ , Pt 4+ , Cu 2+ , and the ions have strong biofilm penetration, and the ions can synergistically regulate an infected microenvironment, so that local inflammation is subsided.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to PtCu@BSA nanoparticles and a preparation method and application thereof. Background Art

[0002] Osteomyelitis is an infection and destruction of bone caused by aerobic and anaerobic bacteria, mycobacteria, and fungi. It commonly develops in long bones, the feet of patients with diabetes, or at sites of penetrating bone injury due to trauma or surgery. In children, the most common site of infection is the metaphysis of well-vascularized long bones, such as the tibia or femur.

[0003] A literature review indicates that the rate of deep infection after open fractures throughout the body ranges from 2% to 50%. The tibia is the most common site of open fractures and the most common site of bone infection. Research results show that the most common site of osteomyelitis is the lower extremities (accounting for 65%). While the upper extremities are less prone to osteomyelitis than the lower extremities, they are also susceptible to secondary infection following traffic accident injuries. Furthermore, the incidence of spinal osteomyelitis is also high.

[0004] Current treatments for osteomyelitis primarily involve antibiotic therapy and surgical debridement of the osteomyelitis site. Commonly used drugs include glycopeptides such as vancomycin and teicoplanin, which are considered the mainstays of treatment against MRSA in osteomyelitis. Both drugs are available only as parenteral formulations. While vancomycin is the preferred treatment due to its long history of use, it carries an increased risk of adverse events, particularly acute renal failure, particularly in patients with other risk factors, such as concomitant use of nephrotoxic medications, high serum trough concentrations, or prolonged dosing. Furthermore, frequent use of antibiotics can lead to bacterial resistance, compromising therapeutic efficacy. Therefore, improving therapies for osteomyelitis remains an urgent challenge.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a PtCu@BSA nanoparticle and a preparation method and application thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a PtCu@BSA nanoparticle, comprising porous PtCu-based nanoparticles and BSA, wherein the BSA is coated on the surface of the porous PtCu-based nanoparticles.

[0009] In an optional embodiment, the pore size of the porous PtCu-based nanoparticles is 10 to 50 nm;

[0010] Preferably, the porous PtCu-based nanoparticles are synthesized using Pluronic F-127 as a template.

[0011] In an optional embodiment, the loading amount of the BSA is 0.1 to 5 mg of the BSA per 1 mg of the porous PtCu-based nanoparticles;

[0012] Preferably, the encapsulation efficiency of BSA is 130-150%;

[0013] Preferably, the particle size of the PtCu@BSA nanoparticles is 110 to 120 nm;

[0014] Preferably, the potential of the PtCu@BSA nanoparticles is -20 to -10 mV.

[0015] In a second aspect, the present invention provides a method for preparing PtCu@BSA nanoparticles as described in any one of the aforementioned embodiments, wherein the BSA is coated on the surface of the porous PtCu-based nanoparticles.

[0016] In an optional embodiment, the method for preparing the porous PtCu-based nanoparticles comprises: synthesizing using Pluronic F-127 as a template;

[0017] Preferably, the method for preparing the porous PtCu-based nanoparticles comprises dissolving chloroplatinic acid, copper chloride, Pluronic F-127 and acid in a mixed solution, adding an α-ascorbic acid aqueous solution, heating the mixed system to react until the solution turns black, and performing solid-liquid separation to obtain porous PtCu-based nanoparticles;

[0018] Preferably, the reaction temperature of the mixed system is 90-100°C and the reaction time is 3-5h;

[0019] Preferably, the concentration of the α-ascorbic acid aqueous solution is 0.1-0.3 M;

[0020] Preferably, the concentration of chloroplatinic acid is 15-25 mM, more preferably 20 mM.

[0021] Preferably, the concentration of copper chloride is 15-25 mM, more preferably 20 mM.

[0022] Preferably, the concentration of Pluronic F-127 is 7.4-7.6 mg / mL, more preferably 7.4 mg / mL;

[0023] Preferably, the concentration of the acid is 4-8M.

[0024] In an optional embodiment, coating the BSA on the surface of the porous PtCu-based nanoparticles comprises:

[0025] dissolving the porous PtCu-based nanoparticles in an organic solvent and adding lipoic acid to obtain PtCu-COOH nanoparticles;

[0026] The PtCu-COOH nanoparticles are dissolved in water, and the BSA is added and stirred to obtain PtCu@BSA nanoparticles.

[0027] In an optional embodiment, the organic solvent includes C1-C4 low-carbon alcohol;

[0028] Preferably, the mass ratio of the porous PtCu-based nanoparticles to the lipoic acid is 1:8-12; the mass ratio of the porous PtCu-based nanoparticles to the BSA is 1:8-12;

[0029] Preferably, the reaction time of the porous PtCu-based nanoparticles and the lipoic acid is 10 to 15 hours; the reaction time of the PtCu-COOH nanoparticles and the BSA is 10 to 15 hours.

[0030] In an optional embodiment, before adding the BSA, the method further comprises adding EDC and NHS to the aqueous solution containing the PtCu-COOH nanoparticles;

[0031] Preferably, the total mass of the EDC and the NHS added is 5 to 15 times that of the PtCu-COOH nanoparticles;

[0032] Preferably, after obtaining the PtCu-COOH nanoparticles, they are washed and purified using an organic solvent;

[0033] Preferably, after obtaining the PtCu@BSA nanoparticles, they are washed and purified with deionized water.

[0034] In a third aspect, the present invention provides use of the PtCu@BSA nanoparticles according to any one of the aforementioned embodiments or the method for preparing the PtCu@BSA nanoparticles according to any one of the aforementioned embodiments in preparing a medicament for treating osteomyelitis.

[0035] In a fourth aspect, the present invention provides a pharmaceutical composition for treating osteomyelitis, comprising the PtCu@BSA nanoparticles according to any one of the aforementioned embodiments;

[0036] Preferably, the pharmaceutical composition for treating osteomyelitis further comprises a pharmaceutically acceptable excipient;

[0037] Preferably, the pharmaceutically acceptable excipients include one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, osmotic pressure regulators, stabilizers, glidants, flavoring agents, antibacterial agents, suspending agents, coating agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, protective agents, humectants, softeners, absorbents, diluents, empty capsules, matrices, and carrier materials;

[0038] Preferably, the dosage form of the pharmaceutical composition for treating osteomyelitis includes an injection form or an oral dosage form.

[0039] The present invention has the following beneficial effects:

[0040] The PtCu@BSA nanoparticles provided by the present invention use porous PtCu-based nanoparticles as a skeleton, and BSA is coated on the surface of the porous PtCu-based nanoparticles. Modifying BSA on the surface of the porous PtCu-based nanoparticles is beneficial to enhancing the biocompatibility and stability of the PtCu@BSA nanoparticles. At the same time, the porous PtCu-based nanoparticles have a high electrokinetic catalytic effect. Under the action of an AC electric field, they can well catalyze the decomposition of water into hydroxyl radicals, thereby effectively killing bacteria and improving the limitations of the existing osteomyelitis treatment drugs that frequently use antibiotics. At the same time, during the electrokinetic catalytic process, the porous PtCu-based nanoparticles will partially degrade into positively charged ions such as Pt 2+ , Pt 4+ 、Cu 2+ These ions have strong biomembrane penetration and can synergistically modulate the infection microenvironment, thereby reducing local inflammation. Compared with small molecule drugs, PtCu@BSA nanoparticles have better stability in the body, thereby increasing drug circulation time and reducing dosing frequency. PtCu@BSA nanoparticles are distinguished from other drug carriers by their ability to be activated by electric fields, independent of drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 Transmission electron microscopy image (a) of PtCu, transmission electron microscopy image (b) of PtCu@BSA nanoparticles, and particle size distribution diagram (c) prepared in Example 1;

[0043] Figure 2XRD peak pattern of PtCu@BSA prepared for Example 1;

[0044] Figure 3 Infrared absorption peak pattern of PtCu@BSA, PtCu, BSA prepared for Example 1;

[0045] Figure 4 Transmission electron microscope pattern (a) and particle size distribution pattern (b) of porous PdCu-based nanoparticles prepared for Comparative Example 1;

[0046] Figure 5 Transmission electron microscope pattern (a) and particle size distribution pattern (b) of large mesoporous PtNPs nanoparticles prepared for Comparative Example 2;

[0047] Figure 6 Degradation of methylene blue for different examples;

[0048] Figure 7 Effect of PtCu@BSA nanoparticles after electric field treatment on degradation into Pt 2+ , Pt 4+ , and Cu 2+ ;

[0049] Figure 8 Effect of ROS production of Ctrl, E, PtCu@BSA, and PtCu@BSA+E groups;

[0050] Figure 9 Degradation effect of PtCu@BSA on methylene blue under different dosing concentrations;

[0051] Figure 10 Viable and dead bacteria staining pattern after treatment of different example groups Ctrl, E, PtCu, and PtCu+E;

[0052] Figure 11 Bacterial morphology pattern of MRSA after treatment of different example groups;

[0053] Figure 12 X-ray image of tibia of rats in each experimental group;

[0054] Figure 13 Inflammatory thermography image of rats in each experimental group. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not specified in the embodiments, conventional conditions or manufacturer recommended conditions are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0056] The present invention provides a PtCu@BSA nanoparticle, which comprises porous PtCu-based nanoparticles and BSA, wherein the BSA is coated on the surface of the porous PtCu-based nanoparticles.

[0057] In the present invention, the porous PtCu-based nanoparticles are porous PtCu-based nanoparticles having mesopores. Mesopores refer to porous nanomaterials with pore diameters of 10 to 50 nm. The pore diameter of the porous PtCu-based nanoparticles can be, for example, any one of 10, 15, 20, 30, 35, 40, and 50 nm, or a range between any two of these. The porous PtCu-based nanoparticles in the present invention are prepared using a soft film method and are synthesized using Pluronic F-127 as a template to form the porous PtCu-based nanoparticles having mesopores.

[0058] When the pore size of the porous PtCu-based nanoparticles is less than 2nm, it is called small mesopores, and when the pore size is greater than 50nm, it is called large mesopores. In the present invention, by controlling the pore size of the porous PtCu-based nanoparticles to 10-50nm, better electrocatalytic activity and antibacterial performance can be obtained. The porous PtCu-based nanoparticles can produce toxic hydroxyl radicals under electrocatalytic action, causing bacterial death. At the same time, during the electrocatalytic process, the porous PtCu-based nanoparticles will partially degrade into positively charged ions such as Pt 2+ , Pt 4+ 、Cu 2+ , has strong biofilm penetration ability and synergistically regulates the infection microenvironment to relieve local inflammation.

[0059] The present invention utilizes BSA (bovine serum albumin) to coat porous PtCu-based nanoparticles. BSA is a common serum protein that has good biocompatibility in vivo and is not prone to causing immune responses. The effects and advantages of modifying the surface of porous PtCu-based nanoparticles with BSA include increasing biocompatibility and improving stability. Specifically, BSA is a natural protein, and its presence can improve the biocompatibility of porous PtCu nanoparticles, reduce their immunogenicity in vivo, and make the nanoparticles more easily accepted in vivo, thereby improving biocompatibility. BSA molecules are wrapped around the surface of porous PtCu-based nanoparticles, which is conducive to forming a stable protective layer to prevent the nanoparticles from agglomerating and precipitating in the solution, thereby improving stability.

[0060] In some embodiments of the present invention, the loading amount of BSA is 0.1-5 mg BSA per 1 mg of porous PtCu-based nanoparticles; for example, it can be, but is not limited to, any one of 0.1, 0.5, 1, 2, 3, 4 or 5 mg, or a range between any two of them, preferably 2 mg BSA per 1 mg of porous PtCu-based nanoparticles.

[0061] In some embodiments of the present application, the PtCu@BSA nanoparticles have a particle size of 50-500 nm; the particle size may, for example, but not limited to, be 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm, or a range between any two of the above-mentioned values, and is preferably about 120 nm.

[0062] In some embodiments of the present application, the PtCu@BSA nanoparticles have a potential of -20 to -10 mV.

[0063] The method for preparing the PtCu@BSA nanoparticles described above comprises wrapping BSA on the surface of the porous PtCu-based nanoparticles.

[0064] Specifically, the method comprises the following steps:

[0065] S1, synthesizing porous PtCu-based nanoparticles.

[0066] In the present application, the porous PtCu-based nanoparticles are synthesized using Pluronic F-127 as a template. The porous Pt-based nanoparticles synthesized using Pluronic F-127 do not use any toxic reagents, and have high biocompatibility and nanoparticle yield.

[0067] Specifically, chloroplatinic acid, copper chloride, Pluronic F-127, and an acid are mixed and dissolved, an aqueous solution of α-ascorbic acid is added, the mixed system is heated and reacted, the reaction temperature of the mixed system is 90-100°C, the reaction time is 3-5 h, the solution is reacted until the solution becomes black, and solid-liquid separation is performed to obtain the porous PtCu-based nanoparticles.

[0068] The concentration of chloroplatinic acid is 15-25 mM, which may, for example, but not limited to, be 15, 18, 20, 22, or 25 mM, or a concentration range between any two of the above-mentioned values, and is further preferably 20 mM.

[0069] The concentration of copper chloride is 15-25 mM, which may, for example, but not limited to, be 15, 18, 20, 22, or 25 mM, or a concentration range between any two of the above-mentioned values, and is further preferably 20 mM.

[0070] The concentration of Pluronic F-127 is 7.4-7.6 mg / mL, which may, for example, but not limited to, be 7.2, 7.3, 7.4, 7.5, or 7.6 mg / mL, or a concentration range between any two of the above-mentioned values. In an optional embodiment, the concentration of Pluronic F-127 is 7.4 mg / mL.

[0071] The acid includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, nitric acid, and preferably hydrochloric acid. The concentration of hydrochloric acid is 4-8M, for example, but is not limited to, 4, 5, 6, 7, or 8M, or a concentration range between any two of the above. In an optional embodiment, the concentration of hydrochloric acid is 6M.

[0072] The concentration of the aqueous solution of α-ascorbic acid is 0.1-0.3M; for example, but is not limited to, 0.1, 0.2, or 0.3M, or a concentration range between any two of the above. In an optional embodiment, the concentration of hydrochloric acid is 0.1M.

[0073] S2, wrapping BSA on the surface of the porous PtCu-based nanoparticles.

[0074] (1) Dissolving the porous PtCu-based nanoparticles in an organic solvent, adding thioctic acid to obtain PtCu-COOH nanoparticles.

[0075] The organic solvent includes a low-carbon alcohol of C1-C4, and is further preferably ethanol.

[0076] In the present application, by adding thioctic acid, carboxyl groups can be grafted on the porous PtCu-based nanoparticles, and the porous PtCu-based nanoparticles are carboxylated, thereby facilitating subsequent compounding with BSA. The mass ratio of the porous PtCu-based nanoparticles to thioctic acid is 1:8-12; for example, but is not limited to, any one of 1:8, 1:9, 1:10, 1:11, or 1:12, or a range value between any two of the above, and is further preferably 1:10.

[0077] The reaction time of the porous PtCu-based nanoparticles and thioctic acid is 10-15h; for example, but is not limited to, 10, 11, 12, 13, 14, or 15h, or a time range between any two of the above, and is further preferably 12h.

[0078] After obtaining the PtCu-COOH nanoparticles, organic solvent is used for washing and purification to remove the porous PtCu-based nanoparticles and thioctic acid on the surface which are not completely reacted.

[0079] (2) Dissolving the PtCu-COOH nanoparticles in water, adding BSA and stirring to obtain PtCu@BSA nanoparticles, and then using deionized water for washing and purification.

[0080] The mass ratio of the porous PtCu-based nanoparticles to BSA is 1:8 to 12; for example, it can be 1:8, 1:9, 1:10, 1:11, or 1:12, or a range between any two of the foregoing, and is more preferably 1:10. In the present invention, an excess amount of BSA is added during the preparation process to ensure that BSA is fully loaded on the porous PtCu-based nanoparticles, with the loading amount being approximately 0.1 to 5 mg of BSA per 1 mg of porous PtCu-based nanoparticles.

[0081] The reaction time of PtCu-COOH nanoparticles and BSA is 10 to 15 hours, for example, but not limited to, 10, 11, 12, 13, 14 or 15 hours, or a time range between any two of the foregoing, more preferably 12 hours.

[0082] Prior to adding BSA, EDC and NHS are added to the aqueous solution containing the PtCu-COOH nanoparticles. The total weight of the EDC and NHS added is 5 to 15 times the weight of the PtCu-COOH nanoparticles, for example, but not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times, or a mass range between any two of the foregoing, more preferably 10 times. The mass ratio of EDC to NHS can be any ratio, including but not limited to 1:1, 1:4, 3:5, 5:1, and the like.

[0083] The above-mentioned PtCu@BSA nanoparticles can be widely used in the preparation of drugs for treating osteomyelitis.

[0084] Correspondingly, the present invention provides a pharmaceutical composition for treating osteomyelitis, comprising the above-mentioned PtCu@BSA nanoparticles.

[0085] In an optional embodiment, the pharmaceutical composition further comprises optionally pharmaceutically acceptable excipients. In an optional embodiment, the pharmaceutically optional acceptable excipients comprise one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, osmotic pressure regulators, stabilizers, glidants, flavoring agents, antibacterial agents, suspending agents, coating agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, protective agents, humectants, softeners, absorbents, diluents, empty capsules, matrices, and carrier materials.

[0086] In an optional embodiment, the pharmaceutical composition can be in any acceptable dosage form selected in the art, including but not limited to an injection dosage form or an oral dosage form.

[0087] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0088] The main reagents used in the following examples are as follows:

[0089] α-Ascorbic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0090] N-(3-(Dimethylamino)-propyl)-3-ethylcarbodiamine hydrochloride (EDC) was purchased from Beijing Suolai Biotechnology Co., Ltd. (Beijing, China).

[0091] N-Hydroxysuccinimide (NHS) was purchased from J&K Scientific Ltd. (Beijing, China).

[0092] Chloroplatinic acid (H2PtCl6) and copper chloride (CuCl2) were purchased from Chemical Technology Co., Ltd. (Tianjin, China).

[0093] BSA (fetal bovine serum albumin) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0094] All chemicals were used as received without further treatment.

[0095] Example 1

[0096] This embodiment provides a PtCu@BSA nanoparticle, and the preparation method thereof is as follows:

[0097] S1. Synthesis of porous PtCu-based nanoparticles.

[0098] After mixing 1.5 ml of H2PtCl6 (20.0 mM), 1.5 ml of CuCI2 (20 mM), 0.2 ml of HCl (6.0 M) and 50.0 mg of Pluronic-F127, the mixture was dissolved under ultrasonic conditions and 2.0 ml of ascorbic acid (0.1 M) aqueous solution was added to the above solution to obtain the final H2PtCl6 and CuCI2 precursor amounts of 0.03 mmol and 0.03 mmol, respectively. The mixed solution was then kept in an oil bath at 95 ° C for 4 hours. Finally, the sample was collected by centrifugation at 14000 rpm for 10 minutes and the residual Pluronic-F127 was removed by continuous washing / centrifugation cycles with ethanol and water to obtain porous PtCu-based nanoparticles. The prepared porous PtCu-based nanoparticles were stored in ethanol until dried at room temperature before use.

[0099] S2. BSA is coated on the surface of porous PtCu-based nanoparticles.

[0100] The porous PtCu-based nanoparticles were dissolved in ethanol, 10 times mass of thioctic acid was added, and after 12 hours of reaction, the porous PtCu-based nanoparticles were purified by washing with ethanol for multiple times. Then the obtained PtCu-COOH nanoparticles were dissolved in water, 10 times mass of EDC and NHS of the nanoparticles were added to the prepared PtCu-COOH nanoparticle solution, followed by adding 10 times mass of BSA, stirring for 12 hours, finally, the PtCu@BSA nanoparticles were obtained by centrifugation and purified by washing with deionized water for multiple times.

[0101] The transmission electron microscope images and particle size distribution diagrams of the porous PtCu-based nanoparticles and PtCu@BSA nanoparticles prepared in this example are shown in Figure 1 The XRD diagram is shown in Figure 2 The infrared absorption peak diagram is shown in Figure 3

[0102] The particle size of the PtCu@BSA nanoparticles in this example is about 115 nm, the potential of the porous PtCu-based nanoparticles is about -11.2 mV, the potential of the BSA is -12.6 mV, and the potential of the PtCu@BSA is -16.4 mV. The loading amount of the BSA is 2 mg of BSA per 1 mg of porous PtCu-based nanoparticles.

[0103] Example 2

[0104] This example provides a PtCu@BSA nanoparticle, and a preparation method thereof is as follows:

[0105] S1, synthesizing porous PtCu-based nanoparticles.

[0106] After mixing 1 ml of H2PtCl6(25.0 mM), 1 ml of CuCl2(25 mM), 0.1 ml of HCl (8.0 M) and 60.0 mg of Pluronic-F127, the mixture was dissolved under ultrasonic condition, and 1.0 ml of ascorbic acid (0.3 M) aqueous solution was added to the above solution. Then, the mixed solution was kept in an oil bath at 100°C for 3 hours. Finally, the sample was collected by centrifugation at 14000 rpm for 10 minutes, and the residual Pluronic-F127 was removed by multiple continuous washing / centrifugation cycles with ethanol and water, to obtain the porous PtCu-based nanoparticles. The prepared porous PtCu-based nanoparticles were stored in ethanol until use, and dried at room temperature.

[0107] S2, wrapping BSA on the surface of the porous PtCu-based nanoparticles.

[0108] ​The porous PtCu-based nanoparticles were dissolved in ethanol, 12 times mass of thioctic acid was added, and after 15 hours of reaction, the porous PtCu-based nanoparticles were purified by washing with ethanol for several times. Then the obtained PtCu-COOH nanoparticles were dissolved in water, 15 times mass of EDC and NHS of the nanoparticles were added to the prepared PtCu-COOH nanoparticle solution, followed by the addition of 8 times BSA, stirring for 15 hours, finally, the PtCu@BSA nanoparticles were obtained by centrifugation and purified by washing with deionized water for several times.

[0109] Example 3

[0110] This example provides a PtCu@BSA nanoparticle, and the preparation method is as follows:

[0111] S1, synthesis of porous PtCu-based nanoparticles.

[0112] After mixing 2 ml of H2PtCl6(15.0 mM), 2 ml of CuCl2(15 mM), 0.3 ml of HCl(4.0 M) and 40.0 mg of Pluronic-F127, the mixture was dissolved under ultrasonic condition, and 3.0 ml of ascorbic acid(0.2 M) aqueous solution was added to the above solution. Then, the mixed solution was kept in an oil bath at 90°C for 5 hours. Finally, the sample was collected by centrifugation at 14000 rpm for 10 minutes, and the residual Pluronic-F127 was removed by continuous washing / centrifugation cycles with ethanol and water for several times to obtain the porous PtCu-based nanoparticles. The prepared porous PtCu-based nanoparticles were stored in ethanol until use, and dried at room temperature.

[0113] S2, BSA is wrapped on the surface of the porous PtCu-based nanoparticles.

[0114] The porous PtCu-based nanoparticles were dissolved in ethanol, 8 times mass of thioctic acid was added, and after 10 hours of reaction, the porous PtCu-based nanoparticles were purified by washing with ethanol for several times. Then the obtained PtCu-COOH nanoparticles were dissolved in water, 5 times mass of EDC and NHS of the nanoparticles were added to the prepared PtCu-COOH nanoparticle solution, followed by the addition of 12 times BSA, stirring for 10 hours, finally, the PtCu@BSA nanoparticles were obtained by centrifugation and purified by washing with deionized water for several times.

[0115] Comparative Example 1

[0116] This comparative example provides a porous PdCu-based nanoparticle, and the preparation method is different from step S1 of Example 1 in that H2PtCl6(20.0 mM) in the precursor is replaced by Na2PdCl4(20.0 mM). The data are as follows: Figure 4 .

[0117] From Figure 4 It can be seen that the particle size of the nanoparticles is about 110nm. Figure 6 It can be seen that the nanoparticles are platinum, copper, and palladium trimetallic nanoparticles. The experiment on the degradation of methylene blue shows that the degradation effect of the nanoparticles on methylene blue under the action of an alternating electric field is lower than that of PtCu@BSA prepared in Example 1.

[0118] Comparative Example 2

[0119] This comparative example provides a large mesoporous PtNPs nanoparticle, and the preparation method thereof is as follows:

[0120] DPPC and cholesterol were dissolved in chloroform and subjected to rotary evaporation. When the solution was evaporated to a white film, an α-ascorbic acid aqueous solution was added to fully dissolve the white solid. After the dissolution was complete, the solution was ultrasonicated until it became a clear liquid. Subsequently, chloroplatinic acid solution was added to the solution and reacted for 6 hours. The solution eventually turned black. The nanoparticles were washed three times by centrifugation at 10000RPM for 10 minutes to prepare PtNPs nanoparticles. Data as shown Figure 6 .

[0121] from Figure 5 It can be seen that the particle size of the nanoparticles is about 100 nm. Figure 6 It can be seen that the nanoparticles are pure platinum metal nanoparticles. The experiment on the degradation of methylene blue shows that the degradation effect of the nanoparticles on methylene blue under the action of an alternating electric field is lower than that of the PtCu@BSA prepared in Example 1.

[0122] Experimental Example 1: Verifying the ability of PtCu@BSA nanoparticles to generate ROS

[0123] The electrocatalytic activity of the nanoparticles was evaluated by measuring the absorption change of methylene blue solution at 664 nm during the catalytic decomposition of the dye. In a typical process such as photocatalysis, 2 ml of MB dye (3×10 -5 MB) and PtCu@BSA (100 μg / ml) in PBS were placed in cells in a 24-well plate. The cell was equipped with symmetrically arranged platinum electrodes connected to the wells via a salt bridge (f = 3 mm, saturated with KCl). The two electrodes were connected to a DC power supply, and its intensity was monitored by a multimeter. Square wave AC output was achieved by the forward and reverse components of a DC motor. The current duration was controlled to 1 second, 10 seconds, 30 seconds, or 60 seconds. After the power was turned on, 50 μl of the mixture was extracted, diluted 4-fold, and the absorption spectrum of MB was measured.

[0124] from Figure 7 It can be seen that after the electric field is applied, part of the PtCu@BSA nanoparticles is degraded into Pt 2+ , Pt 4+ 、Cu 2+.

[0125] from Figure 8 It can be seen that PtCu@BSA nanoparticles have a strong ability to generate ROS under the action of electric field.

[0126] from Figure 9 It can be seen that PtCu@BSA has a degradation effect on methylene blue at different dosage concentrations (the effect of methylene blue degradation is used to evaluate the ROS generation effect).

[0127] Experimental Example 2: Verification of the antibacterial properties of PtCu@BSA nanoparticles

[0128] Based on the excellent electrocatalytic activity of PtCu@BSA nanoparticles, the present invention studied the antibacterial properties of PtCu@BSA nanoparticles against MRSA, which is the main cause of osteomyelitis. CLSM was used to perform live / dead staining assays to study the effective antibacterial activity of PtCu@BSA nanoparticles. Live and dead bacteria were stained with syringin orange (SYTO, green fluorescence) and propidium iodide (PI, red fluorescence), respectively, and then observed by CLSM. Figure 10 As shown in the figure, the bacteria in the control group (Ctrl) emit green fluorescence, indicating that all bacteria are alive. The bacteria in the EDT electric field group (E) and the PtCu@BSA nanoparticle group (PtCu@BSA) emit green fluorescence, indicating that all bacteria are alive. When the EDT electric field is activated, almost all bacteria in the PtCu@BSA group (PtCu@BSA+E) show red fluorescence, indicating that the electrodynamic effect mediated by PtCu@BSA effectively destroys MRSA. The bacterial morphology of these treated MRSA is observed under a scanning electron microscope, as shown in the figure. Figure 11 As shown in the figure, the bacterial surface of the control group has a complete and smooth membrane structure, while the cell wall of the bacteria in the PtCu@BSA+E group appears wrinkled and broken and due to the positively charged ions (Pt 2+ , Pt 4+ 、Cu 2+ ) release leads to the death of a large number of bacteria.

[0129] Experimental Example 3: Animal Experiment

[0130] Animal experimental procedures: We locally injected PtCu@BSA nanoparticles into rats with osteomyelitis model and applied an alternating electric field (10mHZ / 5mA) to the infected area of ​​the rats' osteomyelitis through an electric field generator for 10 minutes each time.

[0131] After treatment twice a week, X-rays of the tibia of rats infected with osteomyelitis were taken on the 0th and 21st days of treatment to evaluate the relief of the infection site. Figure 12It can be clearly seen that the PtCu@BSA+E group has the best effect, with bone density and bone morphology better than other groups, which also proves that the symptoms have been significantly improved.

[0132] After the treatment period, the rats in different groups were subjected to thermal imaging by the animal thermal imaging verification method. Figure 13 It can be seen that the temperature of PtCu@BSA+E in the rat tibia is lower than that in other groups, which also proves that PtCu@BSA+E has a certain degree of relief of inflammation.

[0133] In summary, the PtCu@BSA nanoparticles provided by the present invention use porous PtCu-based nanoparticles as a skeleton, and BSA is coated on its surface. Modifying BSA on the surface of porous PtCu-based nanoparticles is beneficial to enhancing the biocompatibility and stability of PtCu@BSA nanoparticles. At the same time, porous PtCu-based nanoparticles have high targeting ability and electrokinetic catalytic effect. Under the action of an AC electric field, they can catalyze the decomposition of water into hydroxyl radicals, thereby effectively killing bacteria and improving the limitations of existing osteomyelitis treatment drugs that frequently use antibiotics. At the same time, during the electrokinetic catalytic process, the porous PtCu-based nanoparticles will partially degrade into positively charged ions such as Pt 2+ , Pt 4+ 、Cu 2+ These ions have strong biomembrane penetration and can synergistically regulate the infection microenvironment to resolve local inflammation. Compared with small molecule drugs, PtCu@BSA nanoparticles have better stability in the body, thus increasing drug circulation time and reducing the frequency of drug administration.

[0134] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A PtCu@BSA nanoparticle, characterized in that: The invention comprises porous PtCu-based nanoparticles and BSA, wherein the BSA is coated on the surface of the porous PtCu-based nanoparticles. The preparation method of the porous PtCu-based nanoparticles comprises the following steps: mixing and dissolving chloroplatinic acid, copper chloride, Pluronic F-127 and acid, adding an α-ascorbic acid aqueous solution, heating the mixed system for reaction, reacting the solution until the solution turns black, and performing solid-liquid separation to obtain porous PtCu-based nanoparticles; coating the surface of the porous PtCu-based nanoparticles with the BSA comprises the following steps: dissolving the porous PtCu-based nanoparticles in an organic solvent, adding thioctic acid to obtain PtCu-COOH nanoparticles; and dissolving the PtCu-COOH nanoparticles in water, adding the BSA and stirring to obtain PtCu@BSA nanoparticles.

2. The PtCu@BSA nanoparticles according to claim 1, characterized in that The pore size of the porous PtCu-based nanoparticles is 10-50 nm.

3. The PtCu@BSA nanoparticles according to claim 1, characterized in that The loading amount of the BSA is 0.1-5 mg of the BSA per 1 mg of the porous PtCu-based nanoparticles.

4. The PtCu@BSA nanoparticles according to claim 1, characterized in that The particle size of the PtCu@BSA nanoparticles is 110-120 nm.

5. The PtCu@BSA nanoparticles according to claim 1, characterized in that The potential of the PtCu@BSA nanoparticles is -20~-10 mV.

6. A method for preparing PtCu@BSA nanoparticles according to any one of claims 1 to 5, characterized in that: The BSA is coated on the surface of the porous PtCu-based nanoparticles.

7. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The usage ratio of the chloroplatinic acid, the copper chloride, the Pluronic F-127, the acid and the α-ascorbic acid aqueous solution is 1-2 ml: 1-2 ml: 40-60 mg: 0.1-0.3 ml: 1-3 ml.

8. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The reaction temperature of the mixed system is 90-100° C., and the reaction time is 3-5 hours.

9. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the α-ascorbic acid aqueous solution is 0.1-0.3M.

10. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the chloroplatinic acid is 15-25 mM.

11. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of chloroplatinic acid is 20 mM.

12. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the copper chloride is 15-25 mM.

13. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the copper chloride is 20 mM.

14. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the Pluronic F-127 is 7.4-7.6 mg / mL.

15. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of Pluronic F-127 is 7.4 mg / mL.

16. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The concentration of the acid is 4-8M.

17. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The organic solvent includes C1~C4 low-carbon alcohol.

18. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The mass ratio of the porous PtCu-based nanoparticles to the lipoic acid is 1:8-12; the mass ratio of the porous PtCu-based nanoparticles to the BSA is 1:8-12.

19. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: The reaction time of the porous PtCu-based nanoparticles and the lipoic acid is 10 to 15 hours; the reaction time of the PtCu-COOH nanoparticles and the BSA is 10 to 15 hours.

20. The method for preparing PtCu@BSA nanoparticles according to claim 6, characterized in that: Before adding the BSA, the method further includes adding EDC and NHS to the aqueous solution containing the PtCu-COOH nanoparticles.

21. The method for preparing PtCu@BSA nanoparticles according to claim 20, characterized in that: The total mass of the EDC and the NHS added is 5 to 15 times that of the PtCu-COOH nanoparticles.

22. The method for preparing PtCu@BSA nanoparticles according to claim 20, characterized in that: After obtaining the PtCu-COOH nanoparticles, they are washed and purified using an organic solvent.

23. The method for preparing PtCu@BSA nanoparticles according to claim 20, characterized in that: After obtaining the PtCu@BSA nanoparticles, they were washed and purified using deionized water.

24. Use of the PtCu@BSA nanoparticles according to any one of claims 1 to 5 or the PtCu@BSA nanoparticles prepared by the method for preparing the PtCu@BSA nanoparticles according to any one of claims 6 to 23 in the preparation of a medicament for treating osteomyelitis.

25. A pharmaceutical composition for treating osteomyelitis, characterized in that: It comprises the PtCu@BSA nanoparticles according to any one of claims 1 to 5.

26. The pharmaceutical composition for treating osteomyelitis according to claim 25, characterized in that The pharmaceutical composition for treating osteomyelitis further comprises pharmaceutically acceptable excipients.

27. The pharmaceutical composition for treating osteomyelitis according to claim 26, characterized in that The pharmaceutically acceptable excipients include one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, osmotic pressure regulators, stabilizers, glidants, flavoring agents, antibacterial agents, suspending agents, coating agents, antioxidants, chelating agents, pH regulators, adsorbents, protective agents, humectants, softeners, absorbents, diluents, empty capsules, matrices and carrier materials.

28. The pharmaceutical composition for treating osteomyelitis according to claim 25, characterized in that The dosage form of the pharmaceutical composition for treating osteomyelitis includes an injection form or an oral dosage form.

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