ROS-responsive core-shell co-delivery nano-drug carrier and preparation method thereof

Abstract

The invention discloses an ROS (reactive oxygen species)-responsive core-shell co-delivery nano-drug carrier, which comprises 22-32 wt% of mesoporous silica nanoparticles as a core in percentage by weight, 2-10 wt% of 2-([1, 1-biphenyl]-4-ylamino formyl) benzoic acid is loaded in the core, and the mesoporous silica nanoparticles and the 2-([1, 1-biphenyl]-4-ylamino formyl) benzoic acid are uniformly distributed in the core, so that the ROS-responsive core-shell co-delivery nano-drug carrier is prepared. 15-20wt% of ultra-small Prussian blue is embedded into the surface of the core to form an ultra-small Prussian blue layer, the ultra-small Prussian blue layer is coated with a shell formed by 50-60wt% of a selenized hyaluronic acid layer, and the average particle size of the ROS-responsive core-shell co-delivery nano-drug carrier is 80-110nm. The ROS-responsive core-shell co-delivery nano-drug carrier disclosed by the invention has an ordered structure, and the ultra-small Prussian blue not only can properly block pore inlets of mesoporous silica nanoparticles, but also can increase the oxidation resistance of the mesoporous silica nanoparticles. The selenized hyaluronic acid layer further enhances the drug loading efficiency to prevent drug leakage, and endows cartilage targeting and ROS reactivity to control drug release, thereby improving the stability and bioavailability of the drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a ROS-responsive core-shell co-delivery nanodrug carrier and a preparation method thereof. BACKGROUND

[0002] Various pathogenic factors during pregnancy can cause fetal cartilage dysplasia, leading to skeletal deformity and even disability, and there is no effective in-utero treatment method. Cartilage dysplasia or regeneration disorder often causes skeletal deformity and even disability. Cartilage development and regeneration depend on the precise regulation of chondroblast differentiation and the structural support of extracellular matrix (ECM) of chondrocytes. Impaired chondrocyte proliferation, abnormal differentiation, and changes in ECM composition and structure lead to cartilage dysplasia and significant disruption of function.

[0003] Kartogenin (KGN, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid) is a small molecule chondroinductive agent that can promote mesenchymal stem cell (MSC) proliferation and chondrogenic differentiation, but its water solubility is low, affecting its bioavailability and in vivo delivery efficiency, which in turn limits its oral treatment effect. Therefore, there is an urgent need for a carrier that can target and control drug release, is biodegradable, and can reach the fetus in the body.

[0004] Mesoporous silica nanoparticles (MSNs) have high surface area, adjustable pore structure pore size, and high biocompatibility, so that hydrophobic or hydrophilic therapeutic drugs can be efficiently encapsulated to protect the drugs from premature degradation. However, due to the difficulty of penetrating deep cartilage, achieving cartilage-specific targeting remains a key obstacle.

[0005] Hyaluronic acid (HA) has good biocompatibility and can target chondrocytes through cluster determinant CD44, and can be enriched in cartilage lesions. HA can also bind to CD44 receptors on the surface of trophoblast cells, and after optimization of particle size and surface charge, it has the potential to actively target and penetrate the placenta. However, hyaluronic acid lacks antioxidant and anti-inflammatory properties.

[0006] Therefore, it is of great significance to develop a ROS-responsive core-shell co-delivery nanodrug carrier that overcomes the above technical defects. SUMMARY

[0007] The purpose of the present application is to overcome the above technical defects and provide a ROS-responsive core-shell co-delivery nanodrug carrier and a preparation method thereof.

[0008] The present application is achieved by the following technical solutions: A ROS-responsive core-shell co-delivery nanomedicine carrier, comprising, in terms of weight percentage of the ROS-responsive core-shell co-delivery nanomedicine carrier, 22-32 wt% of mesoporous silica nanoparticles as a core, 2-10 wt% of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid loaded in the core, 15-20 wt% of ultrasmall Prussian blue embedded on the surface of the core to form an ultrasmall Prussian blue layer, and 50-60 wt% of a selenium hyaluronic acid layer as an outer shell wrapped outside the ultrasmall Prussian blue layer, wherein the ROS-responsive core-shell co-delivery nanomedicine carrier has an average particle size of 80-110 nm.

[0009] The ultrasmall Prussian blue has an average particle size of 2-10 nm.

[0010] The ROS-responsive core-shell co-delivery nanomedicine carrier is prepared by the following steps: Step A: 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (hereinafter referred to as KGN) is dissolved in an alcohol solvent to prepare a solution A1 with a mass concentration of 0.0015-0.0025 g / mL; mesoporous silica nanoparticles (hereinafter referred to as MSN) are dispersed in an alcohol solvent to prepare a solution A2 with a mass concentration of 0.0015-0.0025 g / mL; then 1 volume of the solution A1 and 4-6 volumes of the solution A2 are stirred at 8-35°C for 8-36 hours, and the product MK is collected by washing and freeze-drying; Step B: 1 part by weight of MK and 0.4-1 part by weight of ultrasmall Prussian blue (hereinafter referred to as USPB) are stirred in PBS (phosphate balanced physiological saline, or PBS buffer) for 0.5-5 hours (physical adsorption), and the product MKP is obtained by freeze-drying; Step C: 1 part by weight of MKP and 0.5-1.5 parts by weight of selenium hyaluronic acid (hereinafter referred to as Se-HA) are mixed and stirred at 8-35°C for 6-24 hours, and the ROS-responsive core-shell co-delivery nanomedicine carrier (hereinafter referred to as MKP@Se-HA) is obtained by washing, centrifugation and drying.

[0011] In step A, the alcohol solvent is selected from any one of methanol, ethanol and isopropanol; and the washing is performed by flushing with PBS buffer with a pH of 7.2-7.6.

[0012] In step B, the PBS is used in an amount of 100-1000 times the total weight of the ultrasmall Prussian blue.

[0013] In step C, the washing is performed by flushing with physiological saline or PBS buffer with a pH of 7.2-7.6.

[0014] The mesoporous silica nanoparticles of the present application can be commercially available products or self-made, and the present application further provides a preparation method of the mesoporous silica nanoparticles, which comprises the following steps: dissolving 1 part by weight of cetyltrimethylammonium bromide in 40-60 times of deionized water, then adding 0.5-0.7 parts by weight of a metal hydroxide salt and uniformly mixing, then sequentially adding 1 / 8-1 / 12 times of deionized water of ethyl silicate and 1 / 8-1 / 12 times of deionized water of ethyl acetate, and stirring and reacting at 70-90 DEG C; washing and centrifuging the product with ethanol and / or deionized water, then dispersing and suspending the centrifugal precipitate with ethanol, then adding 0.8-1.2 parts by weight of ammonium nitrate to the suspension, and ultrasonically reacting for 1-3 hours; and obtaining the mesoporous silica nanoparticles through freeze-drying. The metal hydroxide salt is any one selected from the group consisting of sodium hydroxide and potassium hydroxide.

[0015] The selenized hyaluronic acid of the present application can be commercially available products or self-made, and the present application further provides a preparation method of the selenized hyaluronic acid, which comprises the following steps: adding 1 part by weight of hyaluronic acid, 2-3 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-2 parts by weight of N-hydroxysuccinimide into 400-600 times of a solvent, and activating for 12-36 hours, then adding 0.5-1.5 parts by weight of seleno cysteamine dihydrochloride and 0.2-0.4 parts by weight of 1,4-diaminobutane, and stirring at 0-35 DEG C for 24-72 hours, then dialyzing for 2-4 days, and freeze-drying to obtain the selenized hyaluronic acid. The solvent is any one selected from the group consisting of dimethyl sulfoxide, dimethyl sulfoxide / water solution (the weight percentage of dimethyl sulfoxide is less than 10%), and deionized water containing citric acid.

[0016] The ultra-small Prussian blue of the present application can be commercially available products or self-made, and the present application further provides a preparation method of the ultra-small Prussian blue, which comprises the following steps: dissolving 3 parts by weight of polyvinylpyrrolidone and 0.1-0.2 parts by weight of potassium ferricyanide in a 75% ethanol solution containing 0.01M hydrochloric acid, then stirring at 70-90 DEG C for 2-4 hours, purifying through an ultrafiltration tube, washing with methanol and / or deionized water, and freeze-drying to obtain the ultra-small Prussian blue.

[0017] The ROS-responsive core-shell co-delivery nanodrug carrier of the present application is applied to the preparation of a drug for treating chondrodysplasia or regeneration disorder.

[0018] The present application has the following beneficial effects: The ROS-responsive core-shell co-delivery nanodrug carrier of the application has an ordered structure, KGN is loaded on the MSN to form MK, and ultra-small prussian blue is embedded in the MK as a particle core to form MKP, which not only properly closes the pore entrance of the mesoporous silica nanoparticles, but also increases the oxidation resistance thereof. The selenium hyaluronic acid layer (Se-HA) as the particle shell further enhances the drug loading efficiency and prevents drug leakage, and at the same time endows the cartilage targeting and ROS reactivity to control the drug release. Especially, selenium can protect chondrocytes from oxidative damage by scavenging reactive oxygen species (ROS) during the development of cartilage, thereby maintaining the normal physiological function of cartilage. The selenium-modified hyaluronic acid (selenium-HA) not only retains the targeting of HA, but also endows the material with enhanced antioxidant and anti-inflammatory properties, so that it becomes a biomaterial with ROS-driven release characteristics. In summary, the MKP@Se-HA of the application can reduce the hydrophobicity of KGN, and improve the stability and bioavailability of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Schematic diagram of the structure of MKP@Se-HA.

[0020] Figure 2 : Particle size distribution diagram of MSN, MK, MKP and MKP@Se-HA nanoparticles.

[0021] Figure 3 : Transmission electron microscope image of MSN, MK, MKP and MKP@Se-HA nanoparticles.

[0022] Figure 4 : XRD pattern of MSN, MKP and MKP@Se-HA nanoparticles.

[0023] Figure 5 : Release of KGN from MKP@Se-HA nanoparticles in different environments.

[0024] Figure 6 : ABTS clearance rate of MSN, MK, USPB, MKP and MKP@Se-HA nanoparticles.

[0025] Figure 7 : UV-visible spectrum of various nanoparticles detected by ABTS method.

[0026] Figure 8 : DCF fluorescence image of TDC5 cells after being treated with MK, MKP and MKP@Se-HA nanoparticles, respectively.

[0027] Figure 9 : Hemolysis rate of MK, MKP and MKP@Se-HA nanoparticles.

[0028] Figure 10 : Live / dead cell staining of MK, MKP, and MKP@Se-HA nanoparticles.

[0029] Figure 11 : Cell viability of ATDC5 cells treated with MK, MKP, and MKP@Se-HA nanoparticles.

[0030] Figure 12 : Images of ATDC5 cells uptake of MKP@Se-HA after treatment with MKP@Se-HA nanoparticles.

[0031] Figure 13 : Analysis of chondrogenic differentiation of ATDC5 cells after MKP@Se-HA nanoparticle treatment: Alcian blue staining images.

[0032] Figure 14 : Analysis of chondrogenic differentiation of ATDC5 cells after MKP@Se-HA nanoparticle treatment: Quantification of Alcian blue-stained areas.

[0033] Figure 15 : Immunofluorescence staining of SOX9 and Col Ⅱ in ATDC5 cells after treatment with MKP@Se-HA nanoparticles. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0035] The sources of raw materials used in the present invention are as follows: Polyvinylpyrrolidone (PVP, Aladdin, China); Potassium ferrocyanide (K3FeC6N6, Aladdin, China); cetyltrimethylammonium bromide (CTAB, Aladdin, China); tetraethyl silicate (TEOS, Aladdin, China); KGN (Selleck, USA); hyaluronic acid (HA, Maclean, China); 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Maclean, China); n -hydroxysuccinimide (NHS, Maclean, China); selenocysteine ​​dihydrochloride (Adamas, China); Dulbecco's modified Eagle's medium (DMEM, GBICO, USA); Antibodies (CST, USA); CCK8 kit (GLPBIO, USA); Live-dead cell staining kit (APExBIO, USA); Alexa Fluor 488-conjugated phalloidin (Servicebio, China); Alcian blue staining kit (Servicebio, China).

[0036] The following is the preparation process of the product of Example 1 and Comparative Example 1:

[0037] Example 1: Step 1.1, synthesis of USPB: Dissolve 3.0 g of polyvinylpyrrolidone, 0.118 g of potassium ferricyanide in 75% ethanol containing 0.01 M hydrochloric acid. Place the above mixed solution in a water bath at 80°C. After stirring for 3 h, purify the USPB through 120 ultrafiltration tubes (100 kilodaltons). Then wash with methanol and deionized water, and then collect the USPB by freeze-drying, with an average particle size of 7.7 nm.

[0038] Step 1.2, synthesis of MSN: Dissolve 1.0 g of cetyltrimethylammonium bromide in 50 mL of deionized water at 80°C, then mix with 15.6 mmol of NaOH. Then add 5 mL of TEOS and 5 mL of ethyl acetate to the above mixture in turn, and stir at 80°C. After 2 h of reaction, the product is washed with ethanol and deionized water, and finally centrifuged (10000 rpm, 20 min). Resuspend the precipitate with ethanol, and add 1.0 g of ammonium nitrate to the suspension, and treat the reaction by ultrasonic for 130 min. Finally, collect the MSN by freeze-drying.

[0039] Step 1.3, synthesis of Se-HA: Put 0.2 g of hyaluronic acid, 0.5 g of diethyl dithiocarbamate and 0.3 g of N-hydroxysuccinimide into 100 mL of dimethyl sulfoxide, and then activate within 24 hours. Then, add 0.2 g of seleno-cysteamine dihydrochloride and 0.06 g of 1,4-diaminobutane to the mixture, and stir at room temperature for 48 hours. After 3 days of dialysis (1 kDa cutoff membrane, pure water, temperature 4°C, avoid light), collect the Se-HA by freeze-drying.

[0040] Synthesis of MKP@Se-HA: Step A: 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid was dissolved in an alcoholic solvent to prepare a solution A1 with a mass concentration of 0.002 g / mL; mesoporous silica nanoparticles were dispersed in an alcoholic solvent to prepare a solution A2 with a mass concentration of 0.002 g / mL; then 10 mL of solution A1 and 50 mL of solution A2 were stirred at room temperature for 24 hours, and the product MK was collected by washing and freeze-drying; Step B: 1 mg of MK and 0.6 mg of ultra-small Prussian blue were stirred in PBS for 2 hours, and the product MKP was obtained by freeze-drying; Step C: 1 mg / mL of MKP and 1 mg / mL of selenium hyaluronate were mixed and stirred at room temperature for 12 hours, and the ROS-responsive core-shell co-delivery nanodrug carrier (MKP@Se-HA) was obtained by washing, centrifugation and drying, with an average particle size of 97.80 nm.

[0041] Comparative Example 1: Compared with Example 1, Step C does not add Se-HA, and the rest of the procedures are the same.

[0042] Comparative Example 2: Compared with Example 1, Step B does not add ultra-small Prussian blue, and Step C does not add Se-HA, and the rest of the procedures are the same.

[0043] Test items of the examples and comparative examples: (1) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis.

[0044] (2) X-ray powder diffractometer (XRD) analysis: the sample was dried and powdered. The crystal structure of the nanoparticles was observed by XRD.

[0045] (3) In vitro KGN release experiment: MKP@Se-HA nanoparticles were weighed and placed in different pH PBS solutions (some of which contained H2O2). At the predetermined time point, 1.5 milliliters of supernatant were taken and measured by ultraviolet visible spectrophotometer (Shimadzu UV-3600 plus).

[0046] (4) ABTS detection method: ABTS solution was mixed with 168 microliters of the same K2S2O8 solution, and then incubated in the dark for 16 hours. 200 microliters of the experimental sample were mixed with 800 microliters of ABTS working solution. After 10 minutes of ABTS solution, the absorbance of the supernatant was read at 734 nanometers.

[0047] (5) In vitro experiment, Hemolysis test to evaluate cytotoxicity: fresh blood of mice was mixed with prepared nanoparticles and incubated at 37°C for 60 minutes. After centrifugation, the OD value of the solution was measured using the absorbance at 540 nanometers.

[0048] Cell viability assay by live / dead cell staining: Calcein-AM and propidium iodide (PI) solution was added to chondrocyte cell line (ATDC5) and incubated in the dark. After completing the staining, cell viability was detected using a fluorescence microscope.

[0049] (6) In vitro experiment, ATDC5 cells were incubated with Rhodamine B (RhB) labeled nanoparticles. The cellular uptake of MKP@Se-HA nanoparticles was tracked by fluorescent dye.

[0050] (7) In vitro experiment, chondrogenic differentiation experiment: ATDC5 cells were co-cultured with prepared MKP@Se-HA nanoparticles for 7 days. Alcian blue and Safranin-O staining were used to evaluate chondrogenic differentiation, and immunofluorescence (IF) staining and western blot (WB) were used to detect chondrocyte-related markers.

[0051] Test results of examples and comparative examples:

[0052] Example 1: Preparation and characterization of MKP@Se-HA nanoparticles: By loading KGN on MSN and further filling USPB, and coating Se-HA as a responsive trigger point, core-shell co-delivery nanoparticles were prepared. As shown in Figure 2 , the particle size of MSN was (73.85 ± 11.80) nm, and after loading KGN in the internal space of MSN, the particle size increased to (80.76 ± 10.69) nm. USPB was adsorbed onto the pore channels of MK nanoparticles, and the particle size increased slightly. MKP@Se-HA increased a thin and uniform shell layer, and the particle size increased. These results preliminarily confirmed the successful preparation of MKP@Se-HA nanoparticles.

[0053] Transmission electron microscopy images showed that MSN had parallel arranged pores, and drugs could be adsorbed into the pores and internal space thereof. After loading KGN and USPB, the pore structure became dark, indicating that MKP nanoparticles were successfully prepared. A complete Se-HA coating layer Figure 3 ) was observed on the surface of MKP@Se-HA nanoparticles. As shown in Figure 4 , the XRD pattern showed that MSN had a distinct amorphous silica diffraction peak at 2θ value of 46°. Due to the filling of KGN and USPB in the pores, the diffraction peak intensity of MKP nanoparticles was weakened. In the high angle region of MKP@Se-HA nanoparticles, the corresponding diffraction peak of selenium appeared, indicating the presence of selenium crystals in the coating layer. The above results further confirmed that MKP@Se-HA nanoparticles were successfully prepared.

[0054] In vitro drug release behavior and antioxidant performance: The release profile of KGN under different environmental conditions is shown in Figure 5 The release rate of KGN in the environment of pH 3.0 and H2O2 is significantly faster than that in other environments. Under the conditions of acid and excess ROS, the diselenide bond will be oxidized and broken, thereby releasing KGN. MKP@Se-HA exhibits a ROS-responsive release behavior.

[0055] The ABTS scavenging rate of MKP nanoparticles is higher than that of MK nanoparticles, as shown in Figures 6-7 This is because both USPB and selenium prepared have antioxidant properties, so the antioxidant effect of MKP@Se-HA nanoparticles in vitro is stronger than that of MKP nanoparticles. Bioactive materials with antioxidant properties help to promote cartilage regeneration. The results of DCF fluorescent staining show that the fluorescence of the nanoparticle treatment group is weak, indicating that the nanoparticles can reduce the generation of intracellular reactive oxygen species (Figure 8). This is because USPB can mimic the activity of several antioxidant enzymes, thereby catalyzing the decomposition of reactive oxygen species. Compared with the MKP group, the ROS scavenging effect of the MKP@Se-HA group is more significant, because the diselenide bond has high reducibility and can participate in redox reactions. Therefore, MKP@Se-HA nanoparticles have intracellular antioxidant activity.

[0056] Biocompatibility and cell behavior: Hemolysis test Figure 9 It is shown that the positive control group has strong hemolytic activity, and the hemolysis rate of MKP@Se-HA nanoparticles is less than 5% (international standard threshold), indicating good blood compatibility. Figure 10 / 11 shows that the cell viability of MKP@Se-HA nanoparticle-treated cells is higher than that of the untreated group at 24 hours and 48 hours. Since KGN promotes chondrocyte proliferation, HA provides an ideal microenvironment for cell proliferation. Therefore, MKP@Se-HA nanoparticles have good biocompatibility and cell proliferation ability.

[0057] Cell uptake experiments were performed by double-label immunofluorescence. As shown in Figure 12 The cell uptake experiment treated with MKP@Se-HA shows obvious red fluorescence, which is significantly different from the control group, indicating that nanoscale biomaterials are more likely to enter cells through endocytosis. The Se-HA coating of nanoparticles promotes the adsorption of cell membranes due to the targeting of the CD44 receptor of ATDC5 cells by HA. Therefore, MKP@Se-HA has a more outstanding intracellular uptake effect.

[0058] In vitro cartilage differentiation ability: Alcian blue staining was used to detect the acidic polysaccharides in chondrocytes Figure 13In the PBS group, Alcian blue staining revealed only a small amount of blue staining around chondrocytes, indicating a low content of acidic polysaccharides in the cartilage extracellular matrix. The Alcian blue staining area was significantly more pronounced in the MK group compared to the PBS group, suggesting that KGN promotes the synthesis or secretion of acidic polysaccharides in the cartilage extracellular matrix. Staining was further enhanced in the MKP group, indicating that the presence of USPB indirectly promotes chondrogenic differentiation. The MKP@Se-HA group showed the highest staining intensity, indicating that acidic polysaccharides in the cartilage extracellular matrix were most abundant in this group, further confirming the superiority of MKP@Se-HA nanoparticles in promoting the synthesis or secretion of acidic polysaccharides in the cartilage extracellular matrix.

[0059] Immunofluorescence staining was used to detect the expression of cartilage differentiation-related proteins. SOX9 is a core transcription factor in the cartilage differentiation process, which regulates genes related to matrix synthesis. At the same time, ColⅡ is the main collagen protein in the extracellular matrix of cartilage cells and plays a vital role in the normal development and functional maintenance of cartilage tissue. Figure 15 As shown, staining for SOX9 and ColⅡ proteins in chondrocytes in the PBS group showed only a small amount of fluorescence intensity. The IF staining fluorescence intensity in the MK group increased, indicating that KGN significantly promoted the expression of SOX9 and ColⅡ proteins. The protein expression levels of SOX9 and ColⅡ in the MKP@Se-HA group were significantly higher than those in the MK and MKP groups, suggesting that MKP@Se-HA nanoparticles may promote the expression of the cartilage-related gene ColⅡ by activating SOX9 expression, thereby enhancing the synthesis and secretion capacity of chondrocytes.

[0060] In vivo treatment effects of achondroplasia A zebrafish model of chondrodysplasia was established. Alcian blue staining in the model group at 4 days of age showed a narrower distribution of staining, and the staining intensity further weakened at 7 days of age, indicating that the production of cartilage matrix components such as acidic polysaccharides was inhibited or degradation was increased. Compared with the model group, the MKP@Se-HA nanoparticle-treated group showed a wider and deeper Alcian blue staining at 4 days of age, and the difference was more pronounced at 7 days of age, suggesting that the nanoparticle treatment may have affected the synthesis or deposition of acidic polysaccharides and other components at an early stage. The combined effects of KGN (promoting cell differentiation), USPB's antioxidant effects, and HA's ability to enhance extracellular matrix synthesis promote cartilage differentiation.

[0061] H&E staining showed that the model group showed abnormal chondrocyte density, irregular shape or disordered tissue hierarchy, presenting cartilage development arrest. Compared with the model group, the MKP@Se-HA nanoparticle treatment group had dense and uniform cartilage layers, with clearer and more complete hierarchy, proving that MKP@Se-HA nanoparticles had a positive effect on the morphological changes of cartilage tissue during zebrafish development.

[0062] CONCLUSION: MKP@Se-HA nanoparticles are a unique core-shell drug delivery system, and their innovative preparation aims to address the challenges of oral drug delivery. The stable loading of KGN and USPB in MSN nanoparticles enhances the structural robustness and drug loading efficiency. On the other hand, the presence of selenadiselenide bonds endows these nanoparticles with ROS response characteristics, enabling precise drug release. In terms of cartilage formation, MKP@Se-HA nanoparticles are proven to promote the synthesis of cartilage extracellular matrix through Alcian blue staining. In addition, MKP@Se-HA nanoparticles can promote cartilage formation by upregulating the cartilage-related genes SOX9 and ColII. These research results collectively indicate that this core-shell composite delivery nanoparticle can synergistically promote cartilage growth and repair by combining chondrocyte proliferation and differentiation, providing a highly promising strategy for promoting cartilage differentiation and development and treating cartilage diseases.

[0063] Comparative Example 1: The release rate of Example KGN is significantly faster than that of Comparative Example 1 in the environment of pH 3.0 and H2O2, which is due to the oxidation and breakage of diselenide bonds under acidic and excessive ROS conditions, thereby releasing KGN more quickly. Therefore, Example exhibits ROS-responsive release behavior.

[0064] The ABTS clearance rate of Example is higher than that of Comparative Example 1, indicating that the in vitro antioxidant effect of Example is stronger than that of Comparative Example 1, which is due to the antioxidant properties of selenium on the surface.

[0065] Example has a better intracellular uptake effect than Comparative Example 1. The outer-coated HA of Example can target the CD44 receptor of chondrocyte cells, thereby promoting the adsorption of the cell membrane.

[0066] The results of DCF fluorescence staining show that Example has weaker fluorescence than Comparative Example 1, indicating that the ROS scavenging effect of Example is more significant, which is because the diselenide bond has high reducibility and can participate in redox reactions.

[0067] Comparative Example 2: The ABTS clearance rate of Example is higher than that of Comparative Example 2. This is because the prepared USPB has better antioxidant enzyme-like activity.

[0068] The results of DCF fluorescent staining showed that the fluorescence of Example was weaker than that of Comparative Example 2, indicating that Example could reduce the generation of intracellular reactive oxygen species. This is because USPB can simulate the activity of several antioxidant enzymes, thereby catalyzing the decomposition of reactive oxygen species.

Claims

1. A ROS-responsive core-shell co-delivery nanodrug carrier, characterized in that: The ROS-responsive core-shell co-delivery nanodrug carrier comprises, in weight percentage, 22-32 wt% of mesoporous silica nanoparticles as a core, 2-10 wt% of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid loaded inside the core, 15-20 wt% of ultrasmall Prussian blue embedded on the core surface to form an ultrasmall Prussian blue layer, and an outer shell composed of a 50-60 wt% selenized hyaluronic acid layer coated outside the ultrasmall Prussian blue layer. The average particle size of the ROS-responsive core-shell co-delivery nanodrug carrier is 80-110 nm.

2. The ROS-responsive core-shell co-delivery nanodrug carrier according to claim 1, characterized in that The average particle size of the ultra-small Prussian blue is 2-10 nm.

3. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to any one of claims 1 to 2, characterized in that: The following steps are involved: Step A: 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid is dissolved in an alcohol solvent to prepare a solution A1 with a mass concentration of 0.0015-0.0025 g / mL; mesoporous silica nanoparticles are dispersed in an alcohol solvent to prepare a solution A2 with a mass concentration of 0.0015-0.0025 g / mL; then, one volume of solution A1 and four to six volumes of solution A2 are stirred at 8-35° C. for 8-36 hours, and the product MK is collected by washing and lyophilized; Step B: 1 part by weight of MK and 0.4-1 part by weight of ultra-small Prussian blue were stirred in PBS for 0.5-5 hours, and the product MKP was obtained by freeze-drying; Step C: 1 part by weight of MKP and 0.5-1.5 parts by weight of selenized hyaluronic acid are mixed and stirred at 8-35° C. for 6-24 hours, and a ROS-responsive core-shell co-delivery nanodrug carrier is obtained by washing, centrifugation, and drying.

4. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: In step A, the alcohol solvent is selected from any one of methanol, ethanol, and isopropanol; and the washing is performed using a PBS buffer solution with a pH of 7.2-7.

6.

5. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: In step B, the amount of PBS used is 100-1000 times the total weight of the ultra-small Prussian blue.

6. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: In step C, the washing is performed using physiological saline or PBS buffer with a pH of 7.2-7.

6.

7. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: The preparation method of the mesoporous silica nanoparticles comprises: dissolving 1 part by weight of hexadecyltrimethylammonium bromide in 40-60 times the volume of deionized water, adding 0.5-0.7 parts by weight of a metal hydroxide salt and mixing evenly, then sequentially adding ethyl silicate in an amount of 1 / 8-1 / 12 the volume of deionized water and ethyl acetate in an amount of 1 / 8-1 / 12 the volume of deionized water, and stirring and reacting at 70-90° C.; washing the product with ethanol and / or deionized water, centrifuging, and then dispersing and suspending the centrifugal precipitate with ethanol; then adding 0.8-1.2 parts by weight of ammonium nitrate to the suspension, and subjecting the suspension to ultrasonic reaction for 1-3 hours; and freeze-drying to obtain the mesoporous silica nanoparticles. The metal hydroxide salt is selected from any one of sodium hydroxide and potassium hydroxide.

8. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: The preparation method of the selenized hyaluronic acid is as follows: 1 part by weight of hyaluronic acid, 2-3 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1-2 parts by weight of N-hydroxysuccinimide are added to a 400-600 times solvent and activated for 12-36 hours; 0.5-1.5 parts by weight of selenocysteamine dihydrochloride and 0.2-0.4 parts by weight of 1,4-diaminobutane are then added; the mixture is stirred at 0-35° C. for 24-72 hours, dialyzed for 2-4 days, and freeze-dried to obtain the selenized hyaluronic acid; The solvent is selected from any one of dimethyl sulfoxide, dimethyl sulfoxide / water solution, and deionized water containing citric acid.

9. The method for preparing the ROS-responsive core-shell co-delivery nano drug carrier according to claim 3, characterized in that: The preparation method of the ultrasmall Prussian blue comprises: dissolving 3 parts by weight of polyvinyl pyrrolidone and 0.1-0.2 parts by weight of potassium ferrocyanide in a 75% ethanol solution containing 0.01M hydrochloric acid, stirring at 70-90°C for 2-4 hours, purifying through an ultrafiltration tube, washing with methanol and / or deionized water, and freeze-drying to obtain the ultrasmall Prussian blue.

10. Use of the ROS-responsive core-shell co-delivery nano drug carrier according to any one of claims 1-2, characterized in that: Used for preparing oral medicine for treating chondrodysplasia or aplastic disorders.

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