Nanoparticle compound for inhibiting bone and soft tissue infection as well as preparation method and application thereof

Abstract

The invention provides a nanoparticle compound for inhibiting bone and soft tissue infection as well as a preparation method and application thereof, and belongs to the technical field of medical biological materials. The nanoparticle compound is a gentiopicroside, sericin, hyaluronic acid and endolysin liposome nanoparticle, and a gentiopicroside, sericin, hyaluronic acid and endolysin co-delivery nano drug delivery system targeting the S100A4 site is designed based on the target S100A4 closely related to bone infection through Ai molecular docking and nano drug delivery technologies. The nanoparticle compound provided by the invention has better stability and bioavailability, and can be used for improving and relieving inflammatory response and increasing wound healing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biomaterials, and in particular relates to a nanoparticle complex for inhibiting bone and soft tissue infection, and a preparation method and application thereof. Background Art

[0002] At present, the research on intractable orthopedic diseases such as bone nonunion and bone infection is relatively limited, and there is a lack of in-depth research on the interactions between cells and molecules, resulting in a lack of clinical diagnosis and treatment methods. The application of bioinformatics multi-omics technology helps to clarify the cell, signal and molecular interaction network in the bone microenvironment under disease conditions, and screens out key targets with potential for early diagnosis and targeted treatment through big data analysis technology, providing potential means for the clinical diagnosis and treatment of intractable orthopedic diseases.

[0003] Gentianopicroside is an iridoid glycoside component of Gentianaceae plants. Gentianopicroside has anti-inflammatory effects. Gentianopicroside is derived from the dried roots and rhizomes of Gentiana triflora Pa11, Gentiana rho-dantha Franch, Gentiana manshurica Kitag, or Gentiana rigescens Franch. Gentianopicroside (Scabrans G5) is also known as gentiopicroside, and its molecular formula is C 16 H 20 O9, with a molecular weight of 356.32 and a chemical name of 5-Ethenyl-6-(β-D-glucopyranosyloxy)-5,6-Dihydro-1H,3H-pyrano[3,4-c]pyran-1-one, belongs to the class of cyclopentadiene ether terpene glycosides and is easily soluble in solvents such as methanol, water and ethanol. Existing studies have shown that gentiopicroside has anti-inflammatory and analgesic effects. Gentiopicroside has a certain inhibitory effect on the acute inflammation model of xylene-induced mouse ear swelling and egg white-induced rat toe swelling, as well as the chronic inflammation model of rat granuloma; gentiopicroside can increase the pain threshold for pain caused by heat and chemical stimulation.

[0004] Mannose is an organic compound with the molecular formula C6H 12O6, molecular weight 180.156, colorless or white crystalline powder. It is a carbohydrate that plays an important role in human metabolism, especially in the glycosylation of specific proteins. Mannose, the only carbohydrate nutrient used clinically, is widely distributed in body fluids and tissues, especially in nerves, skin, testicles, retina, liver and intestines. It is directly used to synthesize glycoproteins and participate in immune regulation. Many diseases are caused by the lack of enzymes in mannose saccharification. Regulate the immune system. There are 4 receptors on the surface of macrophages that can capture antigens, all of which contain mannose. Increase wound healing. Anti-inflammatory effect. Inhibit tumor growth and metastasis, and increase cancer survival rate.

[0005] Hyaluronic acid is an acidic mucopolysaccharide. D-glucuronic acid and N-acetylglucosamine are linked by β-1,3-glycosidic bonds, and disaccharide units are linked by β-1,4-glycosidic bonds. The number of disaccharide units can reach as many as 25,000. The molecular weight of hyaluronic acid in the body ranges from 1,000 to 20 million Daltons. With its unique molecular structure and physical and chemical properties, hyaluronic acid exhibits a variety of important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and movement of proteins, water and electrolytes, and promoting wound healing.

[0006] Sericin is a natural macromolecular protein wrapped in the surface of silk fiber. It is mainly secreted by the silk glands of silkworms and is one of the main proteins that make up the cocoon. Sericin is widely used in fabric finishing coatings, cosmetic additives, as well as food, medicine, and functional biomaterials due to its good water solubility, cell adhesion and proliferation activity, lower immunogenicity, and unique in situ fluorescence, antioxidant activity, and tyrosinase inhibition. Sericin has a skin anti-inflammatory effect, and water-soluble sericin and its hydrolyzate have obvious therapeutic effects on skin diseases. Sericin is also a good anti-inflammatory agent for the treatment of skin diseases. When sericin is mixed with yeast extract obtained by enzyme or acid hydrolysis, it can also become a new skin topical medicine, which can also promote collagen production in dermal fibroblasts and effectively prevent or alleviate various skin diseases.

[0007] Endolysin is a peptidoglycan hydrolase encoded by bacteriophages. It is synthesized at the end of the gene expression phase of the phage proliferation and lytic cycle and regulates the release of progeny virions from infected cells by degrading bacterial peptidoglycan. Endolysin proteins are usually composed of two functional domains: a nitrogen-terminal catalytic domain and a carbon-terminal cell wall binding domain. Based on the specificity of the enzyme's action site, endolysin proteins can be divided into four categories: (1) N-acetylglucosaminidase, which cleaves the glycosyl group in peptidoglycan; (2) N-acetylmuramidase (lysozyme or muramidase), which acts on the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetyl-D-glucosamine; (3) N-acetylmuramoyl-L-alanine amidase (NAM-amidase), which cleaves the amide bond between N-acetylmuramic acid and L-alanine; (4) endopeptidase, which cleaves the connection between peptide chains. Endolysin can be used as a biological antibacterial agent with a very low incidence of bacterial resistance; Endolysin has species-specific lysis activity and will not affect other bacteria around the host; At the same time, Endolysin also has high enzymatic activity, which allows bacteria to be lysed within minutes or even seconds. However, there is no report on the combined use of gentiopicroside targeting the S100A4 site with mannose and hyaluronic acid and endolysin, and the use of liposome nano-drug delivery system to inhibit bone and soft tissue infection. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a nanoparticle complex for inhibiting bone and soft tissue infection, and a preparation method and application thereof. The present invention uses Ai molecular docking technology, based on the target S100A4 closely related to bone infection, to design a drug gentiopicroside targeting the S100A4 site. Through the self-assembly method of drug delivery technology, gentiopicroside is loaded into the sericin + HA hyaluronic acid + liposome nano drug delivery system. Gentiopicroside can improve the reduction of inflammatory response and increase wound healing. The use of the nano delivery system makes the nanoparticles loaded with gentiopicroside have better stability and bioavailability, and gentiopicroside can improve the effect of reducing inflammatory response and increasing wound healing.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a nanoparticle complex for inhibiting bone and soft tissue infection, wherein the nanoparticle complex is gentiopicroside+sericin+hyaluronic acid+endolysin liposome nanoparticles.

[0010] In a second aspect, the present invention provides a method for preparing a nanoparticle complex for inhibiting bone and soft tissue infection, comprising the following steps: Step 1: prepare a mixed solution I by mixing 1-1000 mg / ml hyaluronic acid-ethanol solution and 1-2000 mg / ml liposome-ethanol solution at a volume ratio of 1:(1-60); Step 2: adding 1-10 mg / ml sericin-ethanol solution to mixed solution I to prepare mixed solution II, wherein the volume ratio of the sericin-ethanol solution to the mixed solution I is 1:(1-25); Step 3: Add 1-5 mg / ml gentiopicroside-ethanol solution to mixed solution II to prepare mixed solution III, wherein the volume ratio of the mixed solution II to the gentiopicroside-ethanol solution is 5:(1-20); Step 4: During the stirring process, the mixed solution III is added dropwise into the pure water system through a microfluidic chip to prepare a mixed solution IV; Step 5: Continuously dialyze the mixed solution IV in pure water; centrifuge the solution after the dialysis to remove the precipitate; then pass it through a liposome propeller, and the resulting supernatant is a solution of gentiopicroside + sericin + hyaluronic acid liposome nanoparticles; Step 6: dissolving endolysin in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution.

[0011] Preferably, the liposome in step 1 is any one or more of soybean lecithin (L-α-phosphatidylcholine, PC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylcholine (DSPC), bletilla striata polysaccharide, glycyrrhizic acid, hyaluronic acid, and sericin.

[0012] Preferably, the mixing volume ratio of the hyaluronic acid-ethanol solution to the liposome-ethanol solution is 1:5.

[0013] Preferably, in step 2, the volume ratio of the sericin-ethanol solution to the mixed solution I is 1:1.

[0014] Preferably, in step 3, the volume ratio of the mixed solution II to the gentiopicroside-ethanol solution is 1:1.

[0015] Preferably, in step 4, the speed of the mixed solution III passing through the microfluidic chip is 10 s / drop; after the mixed solution IV is prepared, it also includes the process of magnetic stirring dispersion, continued stirring and ultrasonic treatment; the magnetic stirring dispersion is carried out at room temperature; the continued stirring time is 0.5-1.0 hour; the ultrasonic treatment is 150w-300w probe type ultrasound in a 36-38°C water bath, and the ultrasonic treatment time is 5-20min.

[0016] Preferably, in step 5, the mixed solution IV is dialyzed continuously in pure water for 8-12 hours, during which the solution is replaced several times; after the dialysis is completed, the solution is centrifuged at 2500-3500r / min for 5-10 min to remove the precipitate; then it is passed through a 100kDa liposome propeller, and the continuous dialysis uses a dialysis bag with a molecular cutoff of 10-14kDa, and the continuous dialysis time is 8-12 hours.

[0017] Preferably, after step 6, the step of adjusting the pH to 6.5-7.5 and adding 1,2-hexanediol is further included. The pH is adjusted using 3% hydrochloric acid, and the final concentration of 1,2-hexanediol after addition is 0.05-0.10 mg / ml.

[0018] In a third aspect, the present invention also provides the use of the nanoparticle complex in the preparation of drugs or other medical materials for inhibiting bone and soft tissue infection.

[0019] Compared with the prior art, the present invention has the following technical effects: The present invention uses Ai molecular docking and nano drug delivery technology, based on the target S100A4 closely related to bone infection, to design a gentiopicroside + endolysin co-delivery nano drug delivery system targeting the S100A4 site. Gentiopicroside has an inhibitory effect on acute inflammation or chronic inflammation, and reduces inflammatory reactions. Gentiopicroside can promote muscle cell proliferation, relieve pain, promote wound healing, and has the effects of enhancing the antioxidant stress system and scavenging oxygen free radicals. Endolysin has a strong antibacterial effect. Moreover, the preparation method of the targeted nanoparticle complex in the present invention and the operating steps during application are relatively simple, and it is also very suitable for the development, promotion and application of nano drugs for reducing bone and soft tissue infection and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the structural formula of gentiopicroside.

[0021] Figure 2 This is the two-dimensional interaction diagram between gentiopicroside and 7PSQ.

[0022] Figure 3 This is the three-dimensional interaction diagram between gentiopicroside and 7PSQ.

[0023] Figure 4 is the average particle size of the nanoparticle composite prepared in Example 1 of the present invention.

[0024] Figure 5 is the zeta potential of the nanoparticle complex prepared in Example 1 of the present invention.

[0025] Figure 6 This is the inhibitory effect of the nanoparticle complex obtained in Example 1 of the present invention on Staphylococcus aureus.

[0026] Figure 7 This is the inhibitory effect of the nanoparticle complex obtained in Example 1 of the present invention on Pseudomonas aeruginosa.

[0027] Figure 8 This is a negative staining TEM scanning image of the nanoparticle complex obtained in Example 1 of the present invention.

[0028] Fig. 9 This is a graph showing the antibacterial effects of the nanoparticle complex obtained in Example 1 of the present invention on different bacterial species at different concentrations. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The reagents, kits and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0030] The present invention uses computer Ai molecular docking technology, based on the target S100A4 closely related to bone infection, to design a drug targeting the S100A4 site. Target name: S100A4, PDBID: 7PSQ (https: / / www.rcsb.org / structure / 7PSQ), Organism: Homo sapiens, Resolution: 1.91 Å. A spherical docking pocket is set with the position of the original ligand (NU074381b) in the protein crystal data as the center, with the specific position of x = -15.8874, y = -28.780, z = 15.1981, and radius = 10.051 Å. Molecular docking and binding free energy calculation are performed, and the binding free energy calculation results screen out the Chinese medicine monomer gentiopicroside. The free binding energy of gentiopicroside is 179.646, and the original ligand (NU074381b) is 107.952. Gentiopicroside free binding energy is better than the original ligand. Therefore, gentiopicroside was selected as a drug targeting the S100A4 site and used as the main core component of the nanoparticle complex for inhibiting bone and soft tissue infection. Figure 1-Figure 3 .

[0031] A method for preparing a nanoparticle complex for inhibiting bone and soft tissue infection comprises the following steps: Step 1: prepare a mixed solution I by mixing 1-1000 mg / ml hyaluronic acid-ethanol solution and 1-2000 mg / ml liposome-ethanol solution at a volume ratio of 1:(1-60); the liposome is any one or more of soybean lecithin (L-α-phosphatidylcholine, PC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylcholine (DSPC), bletilla striata polysaccharide, and glycyrrhizic acid; Step 2: adding 1-10 mg / ml sericin-ethanol solution to mixed solution I to prepare mixed solution II, wherein the volume ratio of the sericin-ethanol solution to the mixed solution I is 1:(1-25); Step 3: Add 1-5 mg / ml gentiopicroside-ethanol solution to mixed solution II to prepare mixed solution III, wherein the volume ratio of the mixed solution II to the gentiopicroside-ethanol solution is 5:(1-20); Step 4: During the stirring process, the mixed solution III is added dropwise to the pure water system through the microfluidic chip to prepare the mixed solution IV; the speed of the mixed solution III passing through the microfluidic chip is 10 s / drop; after the mixed solution IV is prepared, it also includes the process of magnetic stirring dispersion, continued stirring and ultrasonic treatment; the magnetic stirring dispersion is carried out at room temperature; the continued stirring time is 0.5-1.0 hour; the ultrasonic treatment is 150w-300w probe type ultrasound in a 36-38°C water bath, and the ultrasonic treatment time is 5-20min; Step 5: The mixed solution IV is dialyzed continuously in pure water for 8-12 hours, during which the solution is replaced several times; the solution after dialysis is centrifuged at 2500-3500 r / min for 5-10 min to remove the precipitate; then it is passed through a 100kDa liposome propeller, and the continuous dialysis uses a dialysis bag with a molecular cutoff of 10-14kDa. The continuous dialysis time is 8-12 hours, and the obtained supernatant is the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution; Step 6: Heat 10-100 mg of endolysin at 37 degrees and stir for 10-20 minutes, and dissolve it in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution.

[0032] The preparation method of sericin-ethanol solution, hyaluronic acid-ethanol solution and liposome-ethanol solution in step 1 to step 3 is as follows: 1-5 mg of pure sericin, 1-50 mg of HA hyaluronic acid and 10-50 mg of liposome are dissolved in 1-10 ml of anhydrous ethanol respectively, and the pH is adjusted to 6.8-7.2, that is, 1-5 mg / ml sericin solution, 1-50 mg / ml hyaluronic acid solution and 10-50 mg / ml liposome solution are prepared. The pH is adjusted using 1-5% by mass hydrochloric acid.

[0033] Particularly, the step 5 further comprises the steps of adjusting the pH to 6.5-7.5 and adding 1,2-hexanediol. The pH value is adjusted using 3% hydrochloric acid, and the final concentration of 1,2-hexanediol after addition is 0.05-0.10 mg / ml.

[0034] The technical solution of the present invention is further described in detail below in conjunction with embodiments. Example 1

[0035] Step 1: Take 1 mg of sericin, 1 mg of HA hyaluronic acid, and 20 mg of lecithin, dissolve them in 1 ml of ethanol (100%), adjust the pH value to about 7.0 with 3% hydrochloric acid, and prepare 1 mg / ml sericin-ethanol solution, 1 mg / ml HA hyaluronic acid-ethanol solution, and 20 mg / ml lecithin-ethanol solution. Among them: sericin was purchased from Hangzhou Linran Biotechnology Co., Ltd., CAS60650-88-6; HA hyaluronic acid was purchased from MERCK, item number 924474; lecithin was purchased from MERCK, item number 428415. 1 mg / ml HA hyaluronic acid-ethanol solution was first mixed with 20 mg / ml lecithin-ethanol solution to prepare mixed solution I, the mixing volume ratio of the two was 1:1, and the mixing method was magnetic rotation in a water bath for 5 minutes.

[0036] Step 2: Mixture I and 1 mg / ml sericin-ethanol solution were mixed in a volume ratio of 1:1 to prepare mixture II, and the mixture was magnetically rotated in a water bath for 5 min.

[0037] Step 3: Weigh 1 mg of gentiopicroside and add it to 1 ml of anhydrous ethanol. Rotate it magnetically in a warm water bath at 37 degrees for 5-10 minutes to evenly disperse the gentiopicroside in the anhydrous ethanol to obtain a gentiopicroside-ethanol solution with a concentration of 1 mg / ml. Mix the gentiopicroside-ethanol solution with mixed solution II at a volume ratio of 1:5 to prepare mixed solution III. The mixing method is to rotate it magnetically in a water bath for 5 minutes.

[0038] Step 4: During the stirring process, the mixed solution III is added dropwise into the pure water system using a microfluidic chip, and a 300W probe type ultrasound is set for 20 minutes in a 37°C water bath to prepare a mixed solution IV.

[0039] Step 5: Transfer the mixed solution IV to a pre-treated dialysis bag (MWCO: 10-14kDa) and dialyze continuously in pure water for 8 hours, changing the solution several times during the period. After the dialysis is completed, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 3000rpm for 5 minutes to remove the precipitate, and pass through a liposome propeller (100kDa). The resulting supernatant is the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution.

[0040] Step 6: Heat the endolysin to 37°C and stir for 10-20 minutes. Dissolve the endolysin in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 at a mass ratio of 1:1 to gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution, referred to as the nanoparticle complex solution.

[0041] The pH value of the resulting nanoparticle complex solution was adjusted using diluted hydrochloric acid (3%) until the pH value reached about 7, and then 1,2-hexanediol was added at a final concentration of 0.1 mg / ml to prevent the growth of microorganisms. The particle size and potential of the prepared nanoparticle complex were observed using a Brookhaven laser particle size analyzer 90Plus. The results are as follows: Figure 4~Figure 5 The results showed that the average particle size of the nanoparticle complex was 146 nm, the zeta potential was -8.13, the average particle size was small, and the stability was good. Figure 8 The results showed that the particle size of the obtained nanoparticle composites was consistent. Example 2

[0042] Step 1: Take 10 mg of sericin, 10 mg of HA hyaluronic acid, and 50 mg of dipalmitoylphosphatidylcholine, and dissolve them in 1 ml of ethanol (100%) respectively. Use 3% hydrochloric acid to adjust the pH value to about 7.0 to prepare 10 mg / ml sericin-ethanol solution, 10 mg / ml HA hyaluronic acid-ethanol solution, and 50 mg / ml dipalmitoylphosphatidylcholine-ethanol solution. Among them: sericin was purchased from Hangzhou Linran Biotechnology Co., Ltd., CAS60650-88-6; HA hyaluronic acid was purchased from MERCK, item number 924474; dipalmitoylphosphatidylcholine solution was purchased from MERCK, item number P0763. The 10 mg / ml HA hyaluronic acid-ethanol solution was first mixed with the 50 mg / ml dipalmitoylphosphatidylcholine-ethanol solution to prepare mixed solution I, the mixing volume ratio of the two was 1:1, and the mixing method was magnetic rotation in a water bath for 5 minutes.

[0043] Step 2: Mixture I and 10 mg / ml sericin-ethanol solution were mixed in a volume ratio of 1:1 to prepare mixture II, and the mixture was magnetically rotated in a water bath for 5 min.

[0044] Step 3: Weigh 10 mg of gentiopicroside and add it to 1 ml of anhydrous ethanol, and rotate it magnetically in a warm water bath at 37 degrees for 5-10 minutes to evenly disperse the gentiopicroside in the anhydrous ethanol to obtain a gentiopicroside-ethanol solution with a concentration of 10 mg / ml. The above gentiopicroside-ethanol solution is mixed with mixed solution II at a volume ratio of 1:5 to prepare mixed solution III, and the mixing method is to rotate it magnetically in a water bath for 5 minutes.

[0045] Step 4: During the stirring process, the mixed solution III is added dropwise into the pure water system using a microfluidic chip, and a 300W probe type ultrasound is set for 20 minutes in a 37°C water bath to prepare a mixed solution IV.

[0046] Step 5: Transfer the mixed solution IV to a pre-treated dialysis bag (MWCO: 10-14kDa) and dialyze continuously in pure water for 8 hours, changing the solution several times during the period. After the dialysis is completed, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 3000rpm for 5 minutes to remove the precipitate, and pass through a liposome propeller (100kDa). The resulting supernatant is the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution.

[0047] Step 6: Heat the endolysin to 37°C and stir for 10-20 minutes. Dissolve the endolysin in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 at a mass ratio of endolysin to gentiopicroside + sericin + hyaluronic acid liposome nanoparticles of 1:2 to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution.

[0048] Example 3

[0049] Step 1: Take 800 mg of sericin, 200 mg of HA hyaluronic acid, and 1000 mg of dipalmitoyl phosphatidylcholine, respectively, dissolve them in 100 ml of ethanol (100%), adjust the pH value to about 7.0 with 3% hydrochloric acid, and prepare 8 mg / ml sericin-ethanol solution, 2 mg / ml HA hyaluronic acid-ethanol solution, and 10 mg / ml dipalmitoyl phosphatidylcholine-ethanol solution. Among them: sericin was purchased from Hangzhou Linran Biotechnology Co., Ltd., CAS60650-88-6; HA hyaluronic acid was purchased from MERCK, item number 924474; dipalmitoyl phosphatidylcholine solution was purchased from MERCK, item number P0763. The 2 mg / ml HA hyaluronic acid-ethanol solution was first mixed with the 10 mg / ml dipalmitoyl phosphatidylcholine-ethanol solution to prepare mixed solution I, the mixing volume ratio of the two was 1:1, and the mixing method was magnetic rotation in a water bath for 5 minutes.

[0050] Step 2: Mixture I and 8 mg / ml sericin-ethanol solution were mixed in a volume ratio of 1:1 to prepare mixture II, and the mixture was magnetically rotated in a water bath for 5 min.

[0051] Step 3: Weigh 100 mg of gentiopicroside and add it to 10 ml of anhydrous ethanol, and rotate it magnetically in a warm water bath at 37 degrees for 5-10 minutes to evenly disperse the gentiopicroside in the anhydrous ethanol to obtain a gentiopicroside-ethanol solution with a concentration of 10 mg / ml. The above gentiopicroside-ethanol solution is mixed with mixed solution II at a volume ratio of 1:5 to prepare mixed solution III, and the mixing method is to rotate it magnetically in a water bath for 5 minutes.

[0052] Step 4: During the stirring process, the mixed solution III is added dropwise into the pure water system using a microfluidic chip, and a 300W probe type ultrasound is set for 20 minutes in a 37°C water bath to prepare a mixed solution IV.

[0053] Step 5: Transfer the mixed solution IV to a pre-treated dialysis bag (MWCO: 10-14kDa) and dialyze continuously in pure water for 8 hours, changing the solution several times during the period. After the dialysis is completed, transfer the solution in the dialysis bag to a centrifuge tube, centrifuge at 3000rpm for 5 minutes to remove the precipitate, and pass through a liposome propeller (100kDa). The resulting supernatant is the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution.

[0054] Step 6: Heat the endolysin to 37°C and stir for 10-20 minutes. Dissolve the endolysin in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 at a mass ratio of endolysin to gentiopicroside + sericin + hyaluronic acid liposome nanoparticles of 1:2 to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution.

[0055] The antibacterial effect was verified using the nanoparticle complex prepared in Example 1.

[0056] The two strains were inoculated in a clean bench into LB liquid culture medium, placed in a constant temperature biological shaker, and cultured at 37°C for 12 hours.

[0057] The nanoparticle complex solution obtained in Example 1 was preliminarily diluted with PBS buffer to prepare drug solutions with nanoparticle complex concentrations of 20 mg / ml, 15 mg / ml, 10 mg / ml, 5 mg / ml and 2.5 mg / ml, respectively.

[0058] In a UV-irradiated biosafety cabinet, pour the solid culture medium into the plate. After solidification, draw 500ul of bacterial solution into the plate and spread it evenly with a coating stick. After the bacterial solution is absorbed by the culture medium, use tweezers to pick up the sterilized Oxford cup and place it on the plate. Press it gently to make it fit tightly with the culture medium. Place 4 Oxford cups in each plate. Add 200ul of drug to the Oxford cup in a clockwise direction. Use PBS buffer as a negative control. Make three replicates for each concentration.

[0059] The culture medium was placed in a constant temperature incubator under standard conditions (37°C, 5% CO2), and the size of the inhibition zone was observed and measured at 24h and 48h, and photographed and recorded. Figure 6-Figure 7 .

[0060] The inhibition zone was determined by the Oxford cup method, and the antibacterial effect was determined according to its size. Figure 6 , Figure 7 The graph shows the determination of the antibacterial effect of nanoparticle complexes at different concentrations on two strains at 24h and 48h. The results show that the antibacterial effect of the nanoparticle complex on Staphylococcus aureus is more obvious at high concentrations of 20mg / ml and 30mg / ml, while the effect is poor at medium and low concentrations. The antibacterial effect on Pseudomonas aeruginosa gradually increases with the increase of concentration, and the process is linearly correlated. See Table 1 The data show that the antibacterial effect of nanoparticle complexes on Staphylococcus aureus is stronger at high concentrations, and the antibacterial effect of nanoparticle complexes on Pseudomonas aeruginosa is stronger at low concentrations. Fig. 9 Table 1. Antibacterial effects of nanoparticle complexes at different concentrations on different bacterial species (unit: mm)

[0061] An inhibition zone diameter of >10mm indicates an antibacterial effect, an inhibition zone diameter of 10mm<inhibition zone diameter<15mm indicates a moderate antibacterial effect, and an inhibition zone diameter of >15mm indicates a high antibacterial effect.

[0062] The results in Table 1 show that the nanoparticle complexes at different concentrations have an antibacterial effect on Pseudomonas aeruginosa, while the antibacterial effect on Staphylococcus aureus is only manifested at high concentrations of nanoparticle complexes.

[0063] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nanoparticle complex for inhibiting bone and soft tissue infection, characterized in that: The nanoparticle complex is gentiopicroside+sericin+hyaluronic acid+endolysin liposome nanoparticles.

2. A method for preparing a nanoparticle complex for inhibiting bone and soft tissue infection, characterized in that: The following steps are involved: Step 1: prepare a mixed solution I by mixing 1-1000 mg / ml hyaluronic acid-ethanol solution and 1-2000 mg / ml liposome-ethanol solution at a volume ratio of 1:(1-60); Step 2: adding 1-10 mg / ml sericin-ethanol solution to mixed solution I to prepare mixed solution II, wherein the volume ratio of the sericin-ethanol solution to the mixed solution I is 1:(1-25); Step 3: Add 1-5 mg / ml gentiopicroside-ethanol solution to mixed solution II to prepare mixed solution III, wherein the volume ratio of the mixed solution II to the gentiopicroside-ethanol solution is 5:(1-20); Step 4: During the stirring process, the mixed solution III is added dropwise into the pure water system through a microfluidic chip to prepare a mixed solution IV; Step 5: Continuously dialyze the mixed solution IV in pure water; centrifuge the solution after the dialysis to remove the precipitate; then pass it through a liposome propeller, and the resulting supernatant is a solution of gentiopicroside + sericin + hyaluronic acid liposome nanoparticles; Step 6: dissolving endolysin in the gentiopicroside + sericin + hyaluronic acid liposome nanoparticle solution described in step 5 to obtain a gentiopicroside + sericin + hyaluronic acid + endolysin liposome nanoparticle solution.

3. The preparation method according to claim 2, characterized in that: The liposome in step 1 is any one or more of soybean lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylcholine, bletilla striata polysaccharide, glycyrrhizic acid, hyaluronic acid, and sericin.

4. The preparation method according to claim 2, characterized in that: The mixing volume ratio of the hyaluronic acid-ethanol solution to the liposome-ethanol solution is 1:

5.

5. The preparation method according to claim 2, characterized in that: In step 2, the volume ratio of the sericin-ethanol solution to the mixed solution I is 1:

1.

6. The preparation method according to claim 2, characterized in that: In step 3, the volume ratio of the mixed solution II to the gentiopicroside-ethanol solution is 1:

1.

7. The preparation method according to claim 2, characterized in that: In the step 4, the speed of the mixed solution III passing through the microfluidic chip is 10 s / drop; after the mixed solution IV is prepared, it also includes the processes of magnetic stirring dispersion, continued stirring and ultrasonic treatment; the magnetic stirring dispersion is carried out at room temperature; the continued stirring time is 0.5-1.0 hour; the ultrasonic treatment is 150w-300w probe type ultrasound in a 36-38°C water bath, and the ultrasonic treatment time is 5-20min.

8. The preparation method according to claim 2, characterized in that: In step 5, the mixed solution IV is dialyzed continuously in pure water for 8-12 hours, during which the solution is replaced several times; after the dialysis is completed, the solution is centrifuged at 2500-3500 r / min for 5-10 min to remove the precipitate; then it is passed through a 100kDa liposome propeller, and the continuous dialysis uses a dialysis bag with a molecular cutoff of 10-14kDa, and the continuous dialysis time is 8-12 hours.

9. The preparation method according to claim 2, characterized in that: The step 6 further includes the steps of adjusting the pH to 6.5-7.5 and adding 1,2-hexanediol.

10. Use of the nanoparticle complex according to claim 1 in the preparation of drugs or other medical materials for inhibiting bone and soft tissue infection.

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