A plant-derived nanoparticle-modified sodium thiosulfate drug and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a sodium thiosulfate drug modified with plant-derived nanoparticles, and its preparation method and application. The present invention relates to a sodium thiosulfate drug modified with plant-derived nanoparticles, which uses plant-derived nanoparticles as carriers to encapsulate sodium thiosulfate. Through distearoylphosphatidylethanolamine-polyethylene glycol-linked polypeptide SP5-52, it has been shown through experiments that STS can be targeted and delivered to vascular calcification lesions, significantly reducing the drug dosage of STS and reducing toxic side effects; it has shown significant anti-vascular calcification effects on mouse vascular smooth muscle cells induced by high-phosphate calcification agents and mouse vascular calcification models induced by high vitamin D3; and the sodium thiosulfate drug modified with plant-derived nanoparticles has good stability and high safety, and has good application prospects in the treatment of vascular calcification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a sodium thiosulfate drug modified with plant-derived nanoparticles, and its preparation method and application. Background Art

[0002] Vascular calcification is an abnormal mineralization that occurs in the cardiovascular system, which can lead to reduced elasticity of the blood vessel walls and impaired vascular structural integrity. Vascular calcification is a common vascular lesion in diabetes, calcific aortic valve disease, chronic kidney disease, and chronic inflammatory diseases, increasing the incidence and mortality of hypertension, aortic valve stenosis, myocardial hypertrophy, myocardial ischemia, and congestive heart failure. As human life expectancy increases, the incidence of vascular calcification caused by aging increases year by year. Currently, vascular calcification has become one of the common and difficult-to-treat diseases in clinical medicine, and there is still a lack of effective treatments for vascular calcification.

[0003] Studies have found that sodium thiosulfate (STS) can effectively inhibit vascular calcification. When treated by intravenous injection, on the one hand, the aorta is located deep, making local administration difficult to achieve, and the aorta blood flow velocity is fast, making it difficult for the drug to stay in the lesion site to exert a therapeutic effect; on the other hand, if the STS dose is increased in order to improve the efficacy, it will cause more serious toxic side effects such as syncope, gastrointestinal reactions, and liver and kidney damage, which greatly limits the application of STS in the treatment of vascular calcification ([1] Jiang Yubo, Wang Shaoqing, Lai Weijing, Dai Xiaozhen. Progress in the clinical application of sodium thiosulfate in the treatment of vascular calcification in patients with chronic kidney disease [J]. Chinese Journal of Practical Internal Medicine, 2021, 41(01): 72-75. DOI: 10.19538 / j.nk2021010117. [2] Jiao Yongyi, Zhang Xiaoliang. Research progress and controversy on the mechanism of sodium thiosulfate in the treatment of vascular calcification and defense against calcification [J]. Journal of Clinical Nephrology, 2022, 22(01): 63-66.). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing sodium thiosulfate in treating vascular calcification, such as the difficulty of local administration of the drug, the difficulty of the drug staying in the lesion, and the toxic side effects caused by increasing the dose. The present invention provides a sodium thiosulfate drug modified with plant-derived nanoparticles (GNVs) that can better act on vascular calcification lesions, achieve better therapeutic effects using a smaller dose of STS, and has higher safety.

[0005] The purpose of the present invention is to provide a method for preparing the plant-derived nanoparticle-modified sodium thiosulfate medicine.

[0006] Another object of the present invention is to provide the application of the sodium thiosulfate medicine modified with the plant-derived nanoparticles.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A plant-derived nanoparticle-modified sodium thiosulfate drug is provided. The plant-derived nanoparticle-modified sodium thiosulfate drug uses plant-derived nanoparticles as carriers to encapsulate sodium thiosulfate, and is linked to polypeptide SP5-52 via distearoylphosphatidylethanolamine-polyethylene glycol to obtain the plant-derived nanoparticle-modified sodium thiosulfate drug.

[0009] Furthermore, the particle size of the sodium thiosulfate drug modified by the plant-derived nanoparticles is 177.3 to 213.6 nm.

[0010] Furthermore, the method for preparing the plant-derived nanoparticles comprises the following steps:

[0011] Grapefruit juice is squeezed, and the juice is subjected to differential centrifugation. The supernatant obtained by removing the pomace is subjected to ultrahigh-speed centrifugation. The separated precipitate is resuspended in a buffer solution and purified by sucrose density gradient centrifugation to obtain grapefruit-derived nanoparticles.

[0012] Furthermore, the ultrahigh-speed centrifugation is performed at 100,000 to 160,000 g for 1.5 to 2.5 h. Preferably, the ultrahigh-speed centrifugation is performed at 100,000 g for 2 h.

[0013] Furthermore, the centrifugation is performed at 3-5°C.

[0014] Furthermore, the differential centrifugation is divided into five stages, which are: first stage centrifugation at 400-600g for 8-12 min, second stage centrifugation at 1800-2200g for 15-25 min, third stage centrifugation at 4500-5500g for 30 min, fourth stage centrifugation at 8000-12000g for 1-1.5 h, and fifth stage centrifugation at 80000-120000g for 1.5-2.5 h. Preferably, the differential centrifugation is divided into five stages, which are: first stage centrifugation at 500g for 10 min, second stage centrifugation at 2000g for 20 min, third stage centrifugation at 5000g for 30 min, fourth stage centrifugation at 10000g for 1 h, and fifth stage centrifugation at 100000g for 2 h.

[0015] Preferably, the buffer is phosphate buffered saline (PBS).

[0016] Furthermore, the sucrose density gradient centrifugation method is as follows: sucrose with densities of 8%, 30%, 45% and 60% are sequentially added to a centrifuge tube to form a sucrose density gradient, the suspension is transferred to the top layer of the sucrose gradient with densities of 8%, 30%, 45% and 60%, and centrifuged at 140,000-160,000 g for 1.5-2.5 hours. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

[0017] In addition, the present invention also provides a method for preparing the plant-derived nanoparticle-modified sodium thiosulfate drug, which specifically comprises the following steps:

[0018] S1. Thoroughly mixing the plant-derived nanoparticles with sodium thiosulfate to completely encapsulate the nanoparticles, centrifuging the mixture, and collecting the precipitate to obtain the plant-derived nanoparticles encapsulating the sodium thiosulfate;

[0019] S2. Completely react distearoylphosphatidylethanolamine-polyethylene glycol with polypeptide SP5-52, add the plant-derived nanoparticles loaded with sodium thiosulfate obtained in step S1, complete the reaction, centrifuge, and the precipitate is the plant-derived nanoparticle-modified sodium thiosulfate drug.

[0020] Furthermore, the mass ratio of the plant-derived nanoparticles to the sodium thiosulfate loading is 1:(30-35); when the plant-derived nanoparticles are loaded with more sodium thiosulfate, cell survival rate is reduced, affecting drug safety; when the plant-derived nanoparticles are loaded with less sodium thiosulfate, the anti-vascular calcification effect is poor. Preferably, the mass ratio of the plant-derived nanoparticles to the sodium thiosulfate loading is 1:32.

[0021] Furthermore, in step S2, the reaction temperature is 3-5° C. Preferably, the reaction time with the polypeptide SP5-52 is 20-30 hours, and the reaction time with the plant-derived nanoparticles loaded with sodium thiosulfate is 2-4 hours.

[0022] Furthermore, in step S2, the molar ratio of the product obtained by the reaction of the distearoylphosphatidylethanolamine-polyethylene glycol with the polypeptide SP5-52 to the plant-derived nanoparticles loaded with sodium thiosulfate obtained in S1 is 1:1.

[0023] Furthermore, in steps S1 and S2, the centrifugation is performed at 3-5°C and 140,000-160,000 g for 1.5-2.5 hours.

[0024] In addition, the present invention also provides the use of the GNVs-modified sodium thiosulfate drug in the preparation of drugs for preventing and treating vascular calcification.

[0025] The present invention has the following beneficial effects:

[0026] The present invention discloses a sodium thiosulfate drug modified with plant-derived nanoparticles. The plant-derived nanoparticles are used as carriers to encapsulate sodium thiosulfate. The distearoylphosphatidylethanolamine-polyethylene glycol linked polypeptide SP5-52 is used. Experiments have shown that the drug STS can be delivered in a targeted manner to vascular calcification lesions, significantly reducing the drug dosage of STS and reducing toxic side effects. The drug exhibits significant anti-vascular calcification effects in both mouse vascular smooth muscle cell models induced by a high-phosphate calcifier and mouse vascular calcification models induced by high vitamin D3. Furthermore, the sodium thiosulfate drug modified with GNVs has good stability and high safety, and has good application prospects in the treatment of vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a synthetic related picture of GS and GSTP in Example 2 of the present invention, wherein: Figure 1 a is the standard curve of STS absorbance and concentration measured by UV spectrophotometer at 215 nm; Figure 1 b is the statistical diagram of the GS encapsulation efficiency obtained by co-incubating STS with GNVs at different concentrations; Figure 1 c and 1d are statistical graphs of the binding rate and binding amount of SP5-52 and GS in SP5-52-modified GS (GSTP) obtained by co-incubating SP5-52 and GS with different protein ratios of SP5-52:GS.

[0028] Figure 2 This is a picture related to the identification of nanoparticles (GNVs, GS, GSTP) in Example 3 of the present invention, wherein: Figure 2 a is the Western blot identification of GNVs, GS, and GSTP markers. Figure 2 b is the morphological characteristics of GNVs, GS, and GSTP under an electron microscope. Figure 2 c is the particle size distribution diagram of GNVs, GS, and GSTP measured by DLS. Figure 2 d is the membrane surface Zeta potential diagram of GNVs, GS, and GSTP.

[0029] Figure 3 These are pictures related to the stability determination of GNVs, GS, and GSTP in Example 4 of the present invention, wherein: Figure 3 a, 3b are the particle size and Zeta potential diagrams of GNVs, GS, and GSTP at different time points in PBS buffer containing 50% fetal bovine serum (FBS). Figure 3 c, 3d are the particle size and Zeta potential diagrams of GNVs, GS, and GSTP in PBS buffer at different time points at 4°C.

[0030] Figure 4 This is the in vitro drug release curve of free STS and GNVs-encapsulated STS (GS) in Example 5 of the present invention.

[0031] Figure 5 This is a picture related to the CCK8 assay for cell viability in Example 6 of the present invention, wherein: Figure 5 a is a statistical graph showing the effects of different concentrations of STS on the survival rate of vascular smooth muscle cells; Figure 5 b is a statistical graph showing the effects of different concentrations of GNVs, GS, and GSTP on the survival rate of vascular smooth muscle cells.

[0032] Figure 6 This is a statistical graph showing the uptake of GNVs, GS, and GSTP by calcified vascular smooth muscle cells measured by flow cytometry in Example 7 of the present invention.

[0033] Figure 7 This is a diagram showing the anti-calcification effect of STS and nanoparticles (GNVs, GS, GSTP) in Example 8 of the present invention on high phosphorus-induced mouse aortic smooth muscle cells (MVSMCs).

[0034] Figure 8 This is a statistical graph showing the in vivo targeted tracing results of GS and GSTP on the high vitamin D3-induced calcification group of C57BL / 6 mice in Example 9 of the present invention.

[0035] Figure 9 This is a vascular staining image of the study on the anti-vascular calcification effect of STS, 8 times the dose of STS (8STS) and nanoparticles (GNVs, GS, GSTP) in Example 10 of the present invention on the high vitamin D3-induced C57BL / 6 mouse vascular calcification model.

[0036] Figure 10 This is a statistical graph showing the effects of STS and nanoparticles (GNVs, GS, GSTP) on the survival rate of C57BL / 6 mice in the high vitamin D3-induced calcification group in Example 10 of the present invention.

[0037] Figure 11 Figure 11 shows the in vitro hemolysis rates of STS and nanoparticles (GNVs, GS, GSTP) at different concentrations in Example 11 of the present invention. Figure 11 a is the statistical graph of the in vitro hemolysis rate of STS drugs at different concentrations. Figure 11 b is the statistical graph of in vitro drug hemolysis rate of nanoparticles (GNVs, GS, GSTP) with different concentrations.

[0038] Figure 12 HE staining of the main organs (heart, liver, spleen, lung, and kidney) of C57BL / 6 mice induced by high vitamin D3 by STS, 8 times the dose of STS (8STS), and nanoparticles (GNVs, GS, and GSTP) in Example 12 of the present invention.

[0039] Figure 13 CT scans of the effects of STS, 8-fold dose of STS (8STS), and nanoparticles (GNVs, GS, GSTP) on high vitamin D3-induced osteoporosis of the fifth lumbar vertebra in C57BL / 6 mice in Example 12 of the present invention.

[0040] Figure 14 This is a picture of the preliminary experimental results involved in Example 2 of the present invention, where Figure 14 a is the anti-calcification effect of different concentrations of STS on high phosphorus-induced mouse aortic smooth muscle cells (MVSMCs); Figure 14 b is the anti-calcification effect of different concentrations of GNVs on high phosphorus-induced mouse aortic smooth muscle cells (MVSMCs). DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0043] Cell culture: Mouse aortic smooth muscle cells (MVSMCs) were isolated from the tunica media of aortic tissue of male C57BL / 6 mice; MVSMCs were cultured in DMEM (4.5Glu) medium containing 10% FBS (Bi), 100 U / ml penicillin, and 100 μg / ml streptomycin, and cells from passages 3 to 8 were used in the experiments.

[0044] Male C57BL / 6 mice aged 6–8 weeks (18–22 g) were purchased from the Guangdong Medical Laboratory Animal Center, and all animal experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University, Guangzhou, China.

[0045] Alizarin red staining of cells: remove the culture medium from the cells, wash three times with PBS, fix with 4% paraformaldehyde solution at room temperature for 15 min, wash three times with distilled water, stain with 2% Alizarin Red S (PH=4.2) for 5 min, wash away the floating color with distilled water, and photograph using an optical microscope.

[0046] Tissue Alizarin Red Staining: The collected aorta tissue was fixed with 4% paraformaldehyde solution at room temperature for 48 h, stained with 0.004% Alizarin Red S overnight, washed with 2% potassium hydroxide for 5 minutes to remove floating color, and scanned and photographed.

[0047] Statistical analysis: All values ​​are expressed as mean ± SD; statistical differences were determined using the unpaired Student-t test with a two-tailed P value when comparing two independent groups, and one-way analysis of variance was performed using the Student-Newman-Keuls (SNK) or Dunnett post hoc test when comparing three or more independent groups, using IBM SPSS 19.0 software; survival analysis was determined using the Kaplan-Meier estimate (log-rank test); differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001.

[0048] Example 1 Extraction and purification of grapefruit-derived plant nanoparticles (GNVs)

[0049] The grapefruit was washed and peeled, and the juice was squeezed at room temperature. After the juice was collected, it was pre-cooled in a 4°C refrigerator and subjected to differential centrifugation at 4°C (500g for 10 minutes, 2000g for 20 minutes, 5000g for 30 minutes, and 10000g for 1 hour). After removing the pomace, the supernatant was centrifuged at ultra-high speed 100000g for 2 hours. The collected precipitate was resuspended in PBS buffer, and the suspension was separated and purified by sucrose density gradient centrifugation to obtain grapefruit-derived plant nanoparticles (GNVs). The suspension was resuspended in PBS to prepare a grapefruit-derived plant nanoparticle suspension for use. The protein concentration measured by BCA was 1 mg / ml. The protein concentration measured by BCA was used to quantify GNVs in subsequent experiments.

[0050] The sucrose density gradient centrifugation method is as follows: sucrose is dissolved in a 20mM Tris.HCl (pH 7.2) solution to prepare sucrose solutions with densities of 8% (4g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), 30% (8g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), 45% (22.5g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), and 60% (30g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g). The sucrose solutions are added sequentially to centrifuge tubes to form a sucrose density gradient, and the suspension is transferred to the top layer of the sucrose gradients with densities of 8%, 30%, 45% and 60%, and centrifuged at 150,000g for 2h. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

[0051] Example 2 Preparation of GNVs-modified sodium thiosulfate drug and determination of encapsulation efficiency

[0052] The preparation of the GNVs-modified sodium thiosulfate drug specifically comprises the following steps:

[0053] S1. Take 1 mg of GNVs and add different concentrations of STS to mix well. Incubate at room temperature overnight. Centrifuge at 150,000 g for 2 h at 4°C. Resuspend the pellet with PBS to obtain STS-encapsulated GNVs (GS).

[0054] The supernatant was recovered and the absorbance of the supernatant before and after synthesis was measured and compared using an ultraviolet spectrophotometer at 215 nm to calculate the encapsulation efficiency: encapsulation efficiency = (absorbance of the supernatant before synthesis - absorbance of the supernatant after synthesis) / percentage of absorbance of the supernatant before synthesis;

[0055] S2, take the same amount of DSPE-(PEG) 2000 After reacting with SP5-52 at 4°C for 24 h, GS with different protein mass ratios was added and reacted at 4°C for 3 h. The mixture was centrifuged at 150,000 g for 2 h at 4°C and the precipitate was resuspended in PBS to obtain SP5-52-modified GS (GSTP).

[0056] The supernatant was recovered and the concentration of SP5-52 in the supernatant before and after synthesis was detected using a BCA kit, and the binding rate of SP5-52 to GS was calculated.

[0057] Results see Figure 1 ,in, Figure 1 a is the standard curve of STS absorbance and concentration measured by UV spectrophotometer at 215 nm. Figure 1 b is the GS encapsulation efficiency obtained by co-incubating STS with GNVs at different concentrations. Figure 1 c, 1d are the binding rates and amounts of SP5-52 and GS in SP5-52-modified GS (GSTP) obtained by co-incubating SP5-52 and GS at different protein ratios; combined with the cell viability assay of sodium thiosulfate drugs modified with STS and GNVs in Example 6 and the results of the inventor's preliminary experiments (the experimental method refers to Example 8, and the results are shown in FIG. Figure 14 ), using 10 μg / ml GNVs and 2 mmol / L STS has a more effective therapeutic effect on calcification without affecting the activity of mouse vascular smooth muscle cells. Therefore, subsequent experiments used 1 mg / mL GNVs and 1600 mol / L STS in equal volumes to synthesize GS (10 μg / ml GNVs-2 mmol / L STS), and SP5-52 and GS were added at a protein ratio of 4:1 to synthesize GSTP.

[0058] Example 3 Identification of Nanoparticles (GNVs, GS, GSTP)

[0059] The protein content in the suspension of nanoparticles (GNVs, GS, GSTP) obtained in Example 2 was determined using a BCA protein quantitative assay kit, and the nanoparticles were verified by Western blotting. The specific method was as follows:

[0060] Proteins from mouse vascular smooth muscle cells (MVSMCs), nanoparticles (GNVs, GS, GSTP), and grapefruit juice were extracted using RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor (P1006, Beyotime Biotechnology, China). The total protein concentration was quantified using a BCA protein quantification kit, and the loading amount was determined to be 50 mg.

[0061] The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were blocked with 5% skim milk in TBST (TBS buffer containing 0.1% Tween-20) for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C: rabbit anti-Alix antibody (dilution 1:2000), rabbit anti-CD9 (dilution 1:1000), rabbit anti-CD63 (dilution 1:1000), and rabbit anti-calnexin (dilution 1:1000). After washing three times with TBST, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (dilution 1:10000) at room temperature for 2 h. The signals were detected by enhanced chemiluminescence (Millipore, USA) and detected using Bio-Rad ChemiDoc. TM The images were taken using the MP imaging system (Bio-Rad, USA).

[0062] Results see Figure 2 a, As shown in the figure, GNVs, GS, and GSTP can highly express exosome-related proteins Alix, CD9, and CD63 compared with grapefruit juice, but do not express Calnexin compared with cells.

[0063] The morphological characteristics of the nanoparticles (GNVs, GS, GSTP) were evaluated by conventional procedures using electron microscopy, and the results are shown in Figure 2 As can be seen from the figure, the GNVs, GS, and GSTP obtained in the present invention are relatively uniform in size and have a double-layered circular structure.

[0064] The particle size and zeta potential of GNVs, GS, and GSTP were measured using DLS. Figure 2 c. Figure 2 As shown in the figure, the particle size of GNVs is 104.5-122.3 nm, and the Zeta potential is -6.4--4.6 mV; the particle size of GS is 126.3-147.9 nm, and the Zeta potential is -5.1--3.6 mV; and the particle size of GSTP is 177.3-213.6 nm, and the Zeta potential is -4.8--2.8 mV.

[0065] Example 4 Stability Test of GNVs-Modified Sodium Thiosulfate Drug

[0066] The nanoparticles (GNVs, GS, GSTP) were placed in PBS buffer containing 50% fetal bovine serum (FBS) and incubated at 37°C. At selected time points (0-168h, every 24h), the particle size and zeta potential of grapefruit-derived plant nanoparticles were measured using DLS.

[0067] The nanoparticles (GNVs, GS, GSTP) were resuspended in PBS buffer and placed at 4°C. At selected time points (0-30 days, every 5 days), the particle size and zeta potential of the grapefruit-derived plant nanoparticles were measured using DLS.

[0068] Results see Figure 3 As can be seen from the figure, no significant changes in the particle size distribution and membrane potential of GNVs, GS, and GSTP were observed in 50% FBS buffer within 168 hours; similarly, no significant changes in the particle size distribution and membrane potential of GNVs, GS, and GSTP were observed in 4°C PBS buffer within 30 days, indicating that the nanoparticles obtained by the present invention have good stability.

[0069] Example 5 In vitro drug release of GNVs modified sodium thiosulfate drug

[0070] Deionized water was poured into a beaker and heated. After the water boiled, the dialysis bag was placed in the dialysis bag and boiled for 15-20 minutes. The dialysis bag was removed, the lower end was clamped, and 0.5 ml of known concentrations of free STS and GS (10 μg / ml GNVs-2 mmol / L STS) were injected respectively. The upper end of the dialysis bag was clamped, and the outer surface of the dialysis bag was rinsed with deionized water. The bag was placed in a 50 ml centrifuge tube (containing 50 ml PBS, pH = 7.4) and incubated in a 37°C constant temperature shaker at 100 rpm for 12 hours. At different time points (0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h), 0.5 ml of release medium was removed for testing, and an equal volume of release medium was added at the same time. The STS concentration of the removed medium was determined by UV spectrophotometry.

[0071] Results see Figure 4 ,As can be seen from the figure, compared with free STS, the release of STS in GS has a certain ,slow-release effect.

[0072] Example 6 Cell Viability Assay of STS and GNVs Modified Sodium Thiosulfate Drugs

[0073] MVSMCs (4 × 10 cells per well in 100 μL culture medium) 3Cells (n = 5) were seeded in 96-well plates and cultured at 37°C for 24 h. The medium in each well was then replaced with 200 μL of DMEM medium or 200 μL of DMEM medium containing free GNVs, GS, or GSTP at varying GNV concentrations (1, 5, 10, 15, 20, 30, or 40 μg / mL), or DMEM medium containing varying STS concentrations (0.5, 1, 2, or 4 mmol / L). Cells cultured in DMEM medium served as the control group. After 48 h of culture, the medium was replaced with 200 μL of DMEM medium containing 10% CCK8 and cultured for an additional 0.5, 1, or 2 h. Absorbance was measured at 450 nm using a microplate reader (Synergy2, Bio-Tek, USA). Cell viability in each group was expressed as the percentage of (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group). Experiments were performed in triplicate, and results show representatives of three independent experiments.

[0074] Results see Figure 5 ,Depend on Figure 5 a It can be seen that STS concentrations less than or equal to 2mmol / L have no significant effect on the activity of MVSMCs cells, while concentrations higher than 2mmol / L can lead to a decrease in the activity of MVSMCs cells. Figure 5 As shown in Figure 2, GNVs, GS, and GSTP concentrations less than or equal to 10 μg / ml (GNVs) did not significantly affect MVSMC cell viability, whereas concentrations above 10 μg / ml (GNVs) decreased MVSMC cell viability. Subsequent experiments used 10 μg / ml GNVs-2 mmol / L STS as the concentration for cell and animal experiments.

[0075] Example 7 Cellular Uptake of GNVs-Modified Sodium Thiosulfate Drug

[0076] MVSMCs cells were seeded into six-well plates (1×10 6Cells were cultured for 24 h to allow attachment. When the cell density reached approximately 60-70%, fresh DMEM medium was replaced and high-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L was added to establish the model. PKH-26-labeled GNVs, GS, and GSTP were added to the wells at different time points (1 h, 3 h, 6 h, 12 h, and 24 h) from the detection time (to a final concentration of 10 μg / ml for GNVs) and incubated at 37°C in the dark. Untreated cells were used as controls. The cells were trypsinized, washed three times with pre-cooled PBS, and resuspended in 500 μL PBS. PKH-26 signals were detected using a flow cytometer (Becton Dickinson, USA). The results were analyzed using FlowJo version 10 software (FlowJo, USA). All experiments were performed in triplicate, and the results show representatives of three independent experiments.

[0077] Results see Figure 6 As can be seen from the figure, compared with GNVs and GS, the uptake of GSTP by vascular smooth muscle induced by high phosphorus was significantly increased.

[0078] Example 8 Anti-calcification effect of GNVs-modified sodium thiosulfate drug on high phosphorus-induced mouse aortic smooth muscle cells (MVSMCs)

[0079] MVSMCs cells were seeded into six-well plates (1×10 6 cells) and cultured for 24 hours to allow the cells to adhere to the wall. Fresh DMEM medium was replaced, and when the cell density reached approximately 60-70%, high-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L was added to the calcification group and the treatment group for modeling, which was recorded as day 0. The medium was changed every other day, and fresh DMEM medium was replaced on day 6 of modeling. High-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L was continued to be added to the calcification group, while STS (2 mmol / l), GNVs (10 μg / ml), GS (10 μg / ml GNVs), and GSTP (10 μg / ml GNVs) were added to the high-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L in the treatment group. An equal amount of PBS was added to serve as a normal control group. After 48 hours of intervention, the degree of calcification of mouse vascular smooth muscle cells was assessed by Alizarin Red staining and optical microscopy.

[0080] Results see Figure 7As can be seen from the figure, STS (2mmol / l), GNVs (10μg / ml), GS (10μg / mlGNVs), and GSTP (10μg / mlGNVs) have therapeutic effects on the high phosphorus-induced anti-calcification model of mouse aortic smooth muscle cells (MVSMCs), among which GNVs-modified sodium thiosulfate nanoparticles (GS, GSTP) have better therapeutic effects.

[0081] Example 9 Distribution of GNVs-modified sodium thiosulfate in the aorta of C57BL / 6 mice in a high vitamin D3-induced vascular calcification model

[0082] Calcification group, treatment group: male C57BL / 6 mice aged 6 to 8 weeks (18 to 22 g) were subcutaneously injected with vitamin D3 (dose: 0.2 mg / g) to establish the model; normal group: an equal amount of PBS was subcutaneously injected; after the model was established, Cy7-labeled GNVs, GS, and GSTP (with equal Cy7 content) were injected intraperitoneally into the mice. The mice were killed at different time points in the dark, and the organs were rinsed with PBS and placed on ice in the dark for examination. The main organs and aorta of the mice were fluorescently quantitatively traced using an IVIS in vivo imager to observe the distribution and content of nanoparticles in the mouse aorta.

[0083] Results see Figure 8 As can be seen from the figure, the amount of GSTP bound to the aorta of the calcified group was significantly higher than that of the normal group, which proved that the aortic calcification model was successfully established. It also confirmed that GSTP has targeting effect on calcified foci, indicating that the introduction of SP5-52 can increase the content of GSTP in vascular calcified foci.

[0084] Example 10: Anti-vascular calcification effect of GNVs-modified sodium thiosulfate drug in vivo

[0085] Calcification group, treatment group: Male C57BL / 6 mice aged 6-8 weeks (18-22 g) were subcutaneously injected with vitamin D3 (dose: 0.2 mg / g) to establish the model. After sacrifice, the aorta of the mice was obtained and stained with Alizarin red to confirm the successful model establishment. Normal group (blank group): An equal amount of PBS was subcutaneously injected.

[0086] C57BL / 6 mice were randomly divided into three groups (n=6) with no weight deviation, designated as a normal group (blank group), a calcification group, and a treatment group. Models were established as described above, with day 0 being the designated day. The treatment groups were administered STS, an 8-fold dose of STS (8STS), GNVs, GS, and GSTP (GNVs content: 6.5 mg / kg) via intraperitoneal injection every other day. The calcification group was given an equal volume of PBS as a control. On day 7, mice were sacrificed, and aortas were harvested and fixed with 4% paraformaldehyde solution at room temperature for 48 hours. They were then stained with 0.004% Alizarin Red S overnight, washed with 2% potassium hydroxide for 5 minutes to remove excess stain, and scanned and photographed. Each group was examined twice daily, and survival time was recorded for plotting survival curves.

[0087] Results see Figure 9 As can be seen from the figure, compared with the calcification group, all treatment groups can reduce vitamin D3-induced vascular calcification in C57B / L6 mice. In comparison, GNVs-modified sodium thiosulfate nanoparticles (GS, GSTP) have better therapeutic effects and are significantly better than 8STS.

[0088] Results see Figure 10 As can be seen from the figure, compared with the calcification group, the drugs in each treatment group can improve the survival rate of the vitamin D3-induced C57B / L6 mouse vascular calcification model, among which GSTP is the most significant.

[0089] Example 11 In vitro hemolysis experiment of sodium thiosulfate drugs modified with STS and GNVs

[0090] One ml of blood was collected from the eyeball of male 6-8 week old C57BL / 6 mice and placed in an EDTA K3 blood collection tube. After inversion and mixing, 500 μl was collected and placed in a 15 ml centrifuge tube containing 5 ml of PBS. The tube was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, 10 ml of PBS was added and mixed, and the tube was centrifuged at 3000 rpm for 5 min. The supernatant was discarded, and this process was repeated 3-4 times until the supernatant was clear. The red blood cells were resuspended in 10 ml of PBS to prepare a 5% red blood cell suspension. 200 μl of the red blood cell suspension was added to 800 μl of different concentrations (5, 10, 20, 50, 100, 200 mmol / L STS) and different concentrations (1, 2.5, 5, 10, 25, 50 μg / ml GNVs, GS, GSTP). 800 μl of distilled water was added to the positive control group, and 800 μl of distilled water was added to the negative control group. PBS was incubated at 37°C for 2 hours, centrifuged at 3000 rpm for 5 minutes, and 200 μl of the supernatant was transferred to a 96-well plate. The absorbance was measured at 576 nm using a microplate reader to calculate the hemolysis rate. The formula is: Hemolysis rate = (absorbance of the test sample - absorbance of the negative control) / (absorbance of the positive control - absorbance of the negative control) as a percentage. Experiments were performed in quintuplicate, and the results shown are representative of five independent experiments.

[0091] Results see Figure 11 ,Depend on Figure 11 a It can be seen that when the concentration of STS is lower than 20mmol / L, it will not cause hemolysis in the body; Figure 11 b It can be seen that GNVs, GS, and GSTP will not cause hemolysis to the body when the concentration is lower than 50 μg / ml of GNVs. This concentration is higher than the drug concentration used in animals, which proves its safety.

[0092] Example 12 Organ safety test of sodium thiosulfate modified with STS and GNVs

[0093] Male C57BL / 6 mice aged 6 to 8 weeks (18 to 22 g) were induced by subcutaneous injection of vitamin D3 (dose: 0.2 mg / g). After modeling, they were treated with STS, 8STS, GNVs, GS, and GSTP (6.5 mg / kg) respectively, and the drugs were administered once every other day by intraperitoneal injection. The body weight changes of 6 mice in each group were measured every day until the mice were killed. On the 8th day after modeling, the mice were killed, and the aorta and major organs (heart, liver, spleen, lungs and kidneys) were removed. The organs (heart, liver, spleen, lungs and kidneys) collected from different treatment groups were stained with HE, and all tissue sections were observed under an optical microscope (Nikon, Japan).

[0094] At the same time, microCT was used to scan the fifth lumbar vertebra of the mice for bone density analysis.

[0095] The results are as follows Figure 12 As shown, the use of GNVs-modified sodium thiosulfate drugs to treat vascular calcification did not cause damage to the main organs of mice (heart, liver, spleen, lungs, and kidneys), and was highly safe. In contrast, 8STS was found to cause damage to the main organs of mice (heart, liver, spleen, lungs, and kidneys).

[0096] The results are as follows Figure 13 As shown in the data, compared with the calcification group, the use of STS, GNVs, GS, and GSTP did not cause further bone density reduction in the treatment group. In contrast, increasing the STS concentration to 8 times caused a significant decrease in bone density in the treatment group.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A plant-derived nanoparticle-modified sodium thiosulfate drug, characterized in that: The plant-derived nanoparticle-modified sodium thiosulfate drug uses plant-derived nanoparticles as carriers to encapsulate sodium thiosulfate, and is linked to polypeptide SP5-52 via distearoylphosphatidylethanolamine-polyethylene glycol to obtain the plant-derived nanoparticle-modified sodium thiosulfate drug; Wherein, the mass ratio of the plant-derived nanoparticles to the sodium thiosulfate loading is 1:(30-35); The preparation method of the plant-derived nanoparticles comprises the following steps: Grapefruit juice is squeezed, and the juice is subjected to differential centrifugation. The supernatant obtained by removing the pomace is subjected to ultrahigh-speed centrifugation. The separated precipitate is resuspended in a buffer solution and purified by sucrose density gradient centrifugation to obtain grapefruit-derived nanoparticles.

2. The plant-derived nanoparticle-modified sodium thiosulfate drug according to claim 1, characterized in that: The particle size of the sodium thiosulfate drug modified by the plant-derived nanoparticles is 177.3 to 213.6 nm.

3. The plant-derived nanoparticle-modified sodium thiosulfate drug according to claim 1, characterized in that: The ultra-high speed centrifugation is performed at 100,000 to 160,000 g for 1.5 to 2.5 hours.

4. The plant-derived nanoparticle-modified sodium thiosulfate drug according to claim 1, characterized in that: The differential centrifugation is divided into five stages, namely: the first stage is centrifugation at 400-600g for 8-12 minutes, the second stage is centrifugation at 1800-2200g for 15-25 minutes, the third stage is centrifugation at 4500-5500g for 30 minutes, the fourth stage is centrifugation at 8000-12000g for 1-1.5 hours, and the fifth stage is centrifugation at 80000-120000g for 1.5-2.5 hours.

5. The plant-derived nanoparticle-modified sodium thiosulfate drug according to claim 1, characterized in that: The sucrose density gradient centrifugation method comprises the following steps: sucrose with densities of 8%, 30%, 45% and 60% are sequentially added to a centrifuge tube to form a sucrose density gradient; the suspension is transferred to the top layer of the sucrose gradient with densities of 8%, 30%, 45% and 60%; and the suspension is centrifuged at 140,000 to 160,000 g for 1.5 to 2.5 hours. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

6. The method for preparing the plant-derived nanoparticle-modified sodium thiosulfate drug according to any one of claims 1 to 5, characterized in that: The specific steps include: S1. Thoroughly mixing the plant-derived nanoparticles with sodium thiosulfate to completely encapsulate the nanoparticles, centrifuging the mixture, and collecting the precipitate to obtain the plant-derived nanoparticles encapsulating the sodium thiosulfate; S2. Completely react distearoylphosphatidylethanolamine-polyethylene glycol with polypeptide SP5-52, add the plant-derived nanoparticles loaded with sodium thiosulfate obtained in step S1, complete the reaction, centrifuge, and the precipitate is the plant-derived nanoparticle-modified sodium thiosulfate drug.

7. The preparation method according to claim 6, characterized in that: In step S2, the reaction temperature is 2-8°C.

8. Use of the sodium thiosulfate drug modified with plant-derived nanoparticles according to any one of claims 1 to 5 in the preparation of drugs for preventing and treating vascular calcification.

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