Sulfated polysaccharide hypoxic sterol derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a sulfated polysaccharide hypoxic sterol derivative as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) synthesizing a sterol derivative modified by a hypoxic response group; and (2) synthesizing the sulfated polysaccharide hypoxic sterol derivative. A thrombolytic drug urokinase is used as a model drug, sulfated polysaccharide is used as a raw material, a block copolymer sulfated polysaccharide hypoxic sterol derivative is synthesized through azobenzene bond (hypoxic response) coupling and other amide reactions, and the sulfated polysaccharide hypoxic sterol derivative (at) UK liposome with hypoxic response can be further obtained through a multiple emulsion method. The liposome can be specifically combined with high-expression P-selectin at the thrombus part, so that the liposome has targeting property, and can realize directional administration with loaded urokinase, so that the medicament can be loaded into the focus part in a targeted manner, the bleeding side effect of the thrombolytic medicament urokinase is reduced, the half-life period of the thrombolytic medicament urokinase is prolonged, and the bioavailability of the urokinase is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a sulfated polysaccharide hypoxic sterol derivative, a preparation method and an application thereof. Background Art

[0002] P-selectin is an inflammatory cell adhesion molecule responsible for leukocyte recruitment and platelet binding. It is expressed in endothelial cells and stored in intracellular granules called Weibel-Palade bodies. Recent studies have shown that endothelial cell damage generates reactive oxygen species, which activate platelets and promote platelet recruitment. P-selectin is expressed in activated platelets and participates in thrombus formation. P-selectin may be a target for antithrombotic drug delivery.

[0003] Urokinase is a commonly used thrombolytic drug in clinical practice with the advantages of good efficacy and low price. However, it does not have target specificity for the treatment of thrombosis and is prone to cause bleeding side effects.

[0004] Thrombosis sites exhibit significantly different physiological characteristics from normal tissues, including low pH, high levels of reactive oxygen species, and, under ischemic and hypoxic conditions, elevated expression of hypoxia-inducible factor (HIF-1α) and increased expression of NADPH reductase. Based on these unique physiological characteristics, we have designed a hypoxia-responsive nanodrug delivery system to target the high concentration of NADPH in the thrombus microenvironment. This system enables targeted delivery and intelligent release of thrombolytic drugs in vivo, promoting their further clinical application. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provides a sulfated polysaccharide hypoxic sterol derivative, its preparation method, and application. A sulfated polysaccharide targeting P-selectin is coupled to a sterol derivative via an azobenzene bond (hypoxia-responsive) to obtain a sulfated polysaccharide hypoxic sterol derivative. This derivative is then loaded with a thrombolytic drug to produce a hypoxic nanoformulation. This nanoformulation exhibits targeting, stability, and controlled drug release. The preparation method features a rationally designed synthetic route and is simple to operate.

[0006] In order to achieve the above object, the technical solution of the present invention is: a method for preparing sulfated polysaccharide hypoxic sterol derivatives, comprising the following steps:

[0007] (1) Synthesis of sterol derivatives modified with hypoxia-responsive groups: Weigh a sterol succinate derivative and dissolve it in a first solvent. Then, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) respectively. Finally, add a compound having a hypoxia-responsive group to react and obtain a sterol derivative modified with a hypoxia-responsive group.

[0008] (2) Synthesis of sulfated polysaccharide hypoxic sterol derivatives: The sulfated polysaccharide was dissolved in water and acetonitrile solution was added, followed by addition of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA, stirred for activation, and finally added with the hypoxic responsive group-modified sterol derivative prepared in step (1), and the stirring reaction was continued to obtain the sulfated polysaccharide hypoxic sterol derivatives.

[0009] Furthermore, the preparation method comprises the following steps:

[0010] (1) Synthesis of sterol derivatives modified with hypoxia-responsive groups: Weigh a sterol succinate derivative and dissolve it in a first solvent. Then, add HATU and DIPEA, respectively. Finally, add a compound having a hypoxia-responsive group. Stir and react. After the reaction is completed, separate and purify by column chromatography. After enrichment, dry the product to obtain the hypoxia-responsive group-modified sterol derivative.

[0011] (2) Synthesis of sulfated polysaccharide hypoxic sterol derivatives: The sulfated polysaccharide was dissolved in water and an acetonitrile solution was added. HBTU and DIPEA were then added and stirred for activation. Finally, the hypoxic responsive group-modified sterol derivative prepared in step (1) was added and the reaction was continued with stirring. After the reaction was completed, the reaction solvent was dried by spin drying, dialyzed, concentrated and freeze-dried to obtain sulfated polysaccharide hypoxic sterol derivatives.

[0012] Furthermore, in step (1), the molar ratio of the sterol succinate derivative, HATU, DIPEA, and the compound having an oxygen-responsive group is 1:(2-4):(2-4):1; the first solvent is selected from one of CH2Cl2, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF); the stirring reaction speed is 200-1000 rpm, and the reaction time is 1-3 h; the compound having an oxygen-responsive group is selected from one of p-diaminoazobenzene and nitroimidazole; the sterol succinate derivative is selected from one of cholesterol, β-sitosterol and its analogs, saponin, and cholesterol.

[0013] Furthermore, in step (2), the molar ratio of sulfated polysaccharide, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), DIPEA, and hypoxia-responsive group-modified sterol derivatives is 1:(1-2):1:1.

[0014] Furthermore, in step (2), the activation temperature is 30-50°C, the activation time is 0.5-2h, the stirring reaction temperature is 30-50°C, the reaction time is 20-30h, and the dialysis method is to use a dialysis bag for dialysis in DMSO and water respectively, and the retention capacity of the dialysis bag is 1000-6000kDa; the sulfated polysaccharide is selected from one of polyguluronic acid sulfate (PGS), polymannuronic acid sulfate (PMGS), polymannuronic acid sulfate (PMS), sodium alginate diester (PSS), fucoidan sulfate, heparin, and heparinoid.

[0015] Another technical solution of the present invention is: the sulfated polysaccharide hypoxic sterol derivatives prepared by the preparation method.

[0016] Another technical solution of the present invention is: an application of the sulfated polysaccharide hypoxic sterol derivative in the preparation of nanoformulations loaded with thrombolytic drugs and the preparation of targeted thrombolytic drugs.

[0017] Furthermore, the nanoformulation is one of liposomes, nanogels, nanomicelles, and nanoparticles.

[0018] Furthermore, the liposome preparation method comprises: dissolving soybean lecithin in a second solvent to obtain an oil phase, dissolving a sulfated polysaccharide hypoxic sterol derivative and a thrombolytic drug in a PBS buffer to obtain an aqueous phase, adding the aqueous phase to the oil phase, shearing at high speed to obtain colostrum, adding the colostrum to a PBS buffer, shearing until liposomes are formed, stirring the liposomes to remove the second solvent, and finally extruding the liposome solution through a filter membrane to obtain uniform sulfated polysaccharide hypoxic sterol derivative @ thrombolytic drug liposomes.

[0019] Furthermore, the second solvent is selected from one of chloroform and ethanol, the mass ratio of the soybean lecithin to the sulfated polysaccharide hypoxic sterol derivative is 9: (10-20), the mass volume ratio of the sulfated polysaccharide hypoxic sterol derivative to the thrombolytic drug is 140 mg: (1-2) mL, the pore size of the filter membrane is 0.2-0.3 μm; the thrombolytic drug is selected from one of urokinase (UK), alteplase, and streptokinase.

[0020] Beneficial effects of the present invention: The present invention uses the thrombolytic drug urokinase as a model drug and sulfated polysaccharides as raw materials, and synthesizes block copolymer sulfated polysaccharide hypoxic sterol derivatives through azobenzene bond (hypoxia response) coupling and other amide reactions. The sulfated polysaccharide hypoxic sterol derivatives @UK liposomes with hypoxia response can be further obtained through the double emulsion method. The sulfated polysaccharide hypoxic sterol derivatives @UK liposomes of the present invention can specifically bind to P-selectin highly expressed in the thrombus site, making the liposome targeted, and can achieve directional drug delivery with the loaded urokinase, so that the drug is loaded into the lesion site in a targeted manner, reducing the bleeding side effects of the thrombolytic drug urokinase, extending its half-life, and improving the bioavailability of urokinase. In addition, the hypoxia-responsive liposomes utilize the hypoxia responsiveness of the azobenzene bond to achieve controlled release of the drug. The preparation method of the hypoxia-responsive liposomes of the present invention is simple, easy to operate, has a low preparation cost, and is easy to industrialize, providing a new strategy for precise targeted drug release at the thrombus site. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the Azo-CHEMS H NMR spectrum;

[0022] Figure 2 This is the H-NMR spectrum of the block copolymer PAC;

[0023] Figure 3 Figure 2 is a test diagram of the block copolymer PAC hypoxia response; (A) UV absorption diagram of the block copolymer PAC under normal and Na2S2O4-simulated hypoxia conditions; (B) Color change of the block copolymer PAC solution under normal and hypoxia conditions;

[0024] Figure 4 is the particle size and distribution coefficient graph of PAC@UK liposomes;

[0025] Figure 5 is a transmission electron microscopy image of PAC@UK liposomes;

[0026] Figure 6 This is the in vitro drug release graph of PAC@UK liposomes;

[0027] Figure 7 is the flow cytometric graph of the interaction between PGS and P-selectin;

[0028] Figure 8 Figure 1 shows the in vitro targeting binding of PAC@IR783 liposomes to thrombus clots and carotid artery thrombi; (A) Targeted binding of isolated rat carotid artery thrombi to PAC@IR783 liposomes; (B) Targeted binding of rat blood clots to PAC@IR783 liposomes and IR783; (C) Fluorescence intensity of targeted binding of rat blood clots to PAC@IR783 liposomes and IR783;

[0029] Figure 9 Evaluation of the thrombolytic activity of PAC@UK liposomes in an in vitro clot retraction experiment; (A) Results of fibrin clot dissolution by PAC@UK liposomes and UK; (B) Statistical graph of the volume ratio of retracted fibrin clots;

[0030] Figure 10 Evaluation of the antithrombotic activity of PAC@UK liposomes in a zebrafish thrombosis model; (A) Recovery of zebrafish cardiac blood flow; (B) Statistical chart of circulating platelets in zebrafish;

[0031] Figure 11 Figure 1 shows the recovery of carotid artery blood flow in mice during the evaluation of the thrombolytic activity of PAC@UK liposomes in a carotid artery thrombosis model; (A) shows the changes in vascular blood flow; (B) shows the actual blood vessels on both sides of the carotid artery;

[0032] Figure 12 This is a statistical graph of carotid artery blood flow reperfusion in mice during the evaluation of the thrombolytic activity of PAC@UK liposomes in the carotid artery thrombosis model. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention are further described in detail with reference to the following specific examples. The following describes preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered within the scope of protection of the present invention. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and all materials and reagents used can be purchased from biological or chemical reagent companies.

[0034] The SD rats in the present invention were 8-week-old males weighing 200 g and were provided by Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0035] Kunming mice were 5-week-old male mice weighing 25 g and were provided by Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0036] Example 1:

[0037] The preparation method of polyguluronic acid sulfate-azobenzene cholesterol comprises the following steps:

[0038] (1) Synthesis of Azo-CHEMS

[0039] Cholesterol monosuccinate (CHEMS) (234 mg, 0.481 mmol) was weighed into a 50 mL round-bottom flask and dissolved in CH2Cl2 (15 mL). HATU (457 mg, 1.203 mmol) and DIPEA (212 μL, 1.203 mmol) were then added, followed by the addition of p-diaminoazobenzene (102 mg, 0.481 mmol). The mixture was stirred at 600 rpm for 2 h at room temperature. The reaction progress was monitored by TLC. After the reaction, the mixture was separated and purified by column chromatography on a silica gel column at a flow rate of 10 mL / min and a ratio of dichloromethane to methanol of 300:1. The product Azo-CHEMS was obtained after enrichment and spin-drying. The structural formula of CHEMS is shown in Formula (I):

[0040]

[0041] (2) Synthesis of polyguluronic acid sulfate-azophenyl cholesterol (PAC)

[0042] PGS (94 mg, 0.0481 mmol) was dissolved in 5 mL of water and placed in a 25 mL round-bottom flask. 5 mL of acetonitrile solution was then added, followed by the addition of HBTU (180 mg, 0.0481 mmol) and DIPEA (110 μL, 0.0625 mmol). The mixture was activated at 40°C with magnetic stirring at 600 rpm for 1 h. Finally, Azo-CHEMS (33 mg, 0.0481 mmol) was added, and magnetic stirring was continued at 40°C with 600 rpm for 24 h. After the reaction was completed, the reaction solvent was dried, and the mixture was dialyzed against DMSO and water, respectively, with a cutoff of 3500 kDa. The mixture was concentrated and lyophilized to obtain a block copolymer PAC. The structural formula of PGS is shown in Formula (II), and the structural formula of PAC is shown in Formula (III):

[0043]

[0044]

[0045] In formula (III), x≤n, n represents an integer between 10 and 100, R=H or at least one of SO3Na, and the structural formula of R2 is shown in formula (IV):

[0046]

[0047] Example 2:

[0048] A method for preparing UK-loaded polyguluronic acid sulfate-azophenyl cholesterol (PAC@UK) liposomes.

[0049] Liposomes were prepared by a double emulsion-extrusion method. 9 mg of soybean lecithin was dissolved in 2 mL of chloroform to obtain an oil phase. 14 mg of the PAC block copolymer prepared in Example 1 and 50,000 U / mL and 0.1 mL of urokinase were dissolved in 2 mL of PBS buffer (pH 7.4) to obtain an aqueous phase. 2 mL of the aqueous phase was added to 2 mL of the oil phase, and the mixture was sheared at 12,000 r / min for 10 min using an FJ-200 high-pressure homogenizer in an ice-water bath to obtain colostrum. 2 mL of the colostrum was added to 5 mL of PBS buffer (pH 7.4) and sheared at 5,000 r / min for 60 s to form liposomes. The prepared liposomes were placed in a magnetic stirrer at 600 rpm to stir and remove chloroform. Finally, the liposome solution was extruded through a 0.22 μm filter membrane to obtain a uniform liposome solution, i.e., PAC@UK liposomes.

[0050] Characterization analysis

[0051] 1. NMR analysis and characterization of PAC and its intermediate Azo-CHEMS.

[0052] NMR analysis method: measured on a JNM-ECP 400M superconducting nuclear magnetic resonance spectrometer.

[0053] 10 mg of PAC was accurately weighed and dissolved in 0.5 mL of D2O (99.96%), and NMR analysis was performed at 25°C.

[0054] Accurately weigh 10 mg of Azo-CHEMS and dissolve it in 0.5 mL of CDCl3. Perform NMR analysis at 25°C.

[0055] NMR analysis results:

[0056] Azo-CHEMS NMR 1 H-NMR Figure 1 As shown: deuterated chloroform (7.26 ppm) as solvent, Azo-CHEMS (261 mg, 79.89%), R f =0.50(CH2Cl2 / CH3OH=20:1,v / v). 1HNMR (400 MHz, CDCl3) δ7.83 (d, J = 8.0 Hz, 2H, Ar-H), 7.78 (d, J = 8.0 Hz, 2H, Ar-H), 7.64 (d, J = 8.5 Hz, 2H, Ar-H), 6.74 (d, J = 8.0 Hz, 2H, Ar-H), 5.37 (t, J = 5.0 Hz, 1H, C = CH), 4.66 (m, 1H, OCH), 2.76 (t, J = 6.4 Hz, 2H, CONHCH2CH2), 2.68 (t, J = 6.4 Hz, 2H, CONHCH2CH2), 2.34-0.63 (m, 43H, CH2, CH3). The above results indicate that Azo-CHEMS was successfully synthesized.

[0057] The block copolymer PAC uses D2O as the NMR solvent, and the NMR spectrum is as follows Figure 2 The solvent peak is 4.79ppm, 1.00-2.10ppm is the CH3 signal peak of the cyclopentahydrophenanthrene structure on cholesterol, 3.50-5.50ppm is the hydrogen signal peak on the PGS sugar ring structure, 5.30ppm is the signal peak of H1 on the sugar ring, 3.5-4.5ppm are the signal peaks of H5, H4, H3, and H2 hydrogen on the sugar ring, 7.69ppm is the hydrogen signal peak of the benzene ring a,a’ The signal peak is 7.45ppm, and the H on the benzene ring b,b’,c,c’ The signal peak at 7.82ppm is the benzene ring H d,d’ Signal peak. Based on the above results, it shows that the block copolymer PAC was successfully prepared.

[0058] Hypoxia responsiveness measurement of PAC block copolymers

[0059] Appropriate amounts of hypoxia-responsive block copolymer PAC and Na2S2O4 powder were prepared using PBS buffer solution (pH 7.4) to prepare a 1 mg / mL PAC solution and a 0.1714 mg / mL Na2S2O4 solution, respectively. 1 mL of each solution was mixed in equal volumes and incubated at room temperature for 2 h. After the reaction, 1 mL of the mixed solution was placed in a quartz dish, and its UV absorption spectrum was measured at a wavelength of 300-600 nm using a UV spectrophotometer. Separately, 1 mL of PAC solution (1 mg / mL) was mixed with 1 mL of PBS buffer solution and incubated at room temperature for 2 h. After the reaction, 1 mL of the mixed solution was placed in a quartz dish, and its UV absorption spectrum was measured at a wavelength of 300-600 nm using a UV spectrophotometer to serve as a normal oxygen control.

[0060] Na2S2O4 was used as a reducing agent to catalyze the azobenzene bond of the PAC block copolymer and reduce the azobenzene bond to aniline derivatives. The cleavage of the azobenzene bond led to the destruction of the structural stability of the block copolymer. Figure 3 As shown in (A), the block copolymer PAC reacts with Na2S2O4, and the absorption of its azobenzene bond at 305nm gradually decreases; secondly, Figure 3 In (B), the reaction phenomenon also shows that the block copolymer is light yellow due to the presence of azobenzene bonds, but after reduction, the azobenzene bonds break, causing the solution to become colorless, further verifying that the azobenzene in the block copolymer undergoes hypoxic responsive breakage under the action of the Na2S2O4 reducing agent.

[0061] 2. Characterization of PAC@UK Liposome Particle Size and Potential

[0062] Dynamic light scattering (DLS) was used to detect the particle size, potential and particle size distribution of the polymer micelles at a detection temperature of 25°C.

[0063] Particle Size Measurement: Preheat the Malvern Nano Particle Sizer for 30 minutes. Add 1 mL of PAC@UK liposomes to the particle size sample cell. Add slowly to avoid air bubbles while tilting the cell to fill the sample to the 10 mm position. Place the cell in the measurement chamber, aligning the polished optical surface with the measurement channel. Close the lid and measure the particle size, distribution coefficient, and potential.

[0064] The particle size and distribution of liposomes were detected by dynamic light scattering. Figure 4 As shown in the figure, the particle size distribution of PAC@UK liposomes is uniform, with an average particle size of 183.17 nm and a narrow distribution (PDI of 0.177) and a Zeta potential of -22.67 mV.

[0065] 3. Determination of morphological characteristics of PAC@UK liposomes

[0066] Observation of liposomes by transmission electron microscopy: Take a clean watch glass, take 10 μL of liposomes and drop them onto the surface of the copper mesh, dry to remove moisture, add 10 μL of counterstaining reagent 2% phosphotungstic acid and stain for 20 minutes, dry, and observe the transmission electron microscopy structure of the liposomes at a voltage of 120 kV.

[0067] Morphological observation results of liposomes by TEM Figure 5 As shown, it was demonstrated that PAC@UK liposomes had a spherical morphology with uniform size distribution.

[0068] 4. In vitro drug release analysis of liposomes

[0069] Cumulative drug release was measured using a Franze diffusion cell method. A Franze diffusion cell was assembled with a 500 kDa molecular weight cutoff polyvinylidene fluoride membrane (PVDF membrane, model UF500) placed between the donor and receiver cells, with the smooth surface facing the donor cell and the rough surface facing the receiver cell. 20 mL of release medium (0.01 M phosphate buffer, pH 7.4, containing 1 mg / mL Na₂S₂O₄ (reducing agent)) was placed in the receiver cell. 2 mL of the PAC@UK liposome solution was also added to the donor cell. The cell was then incubated at 37°C in a shaker with constant shaking. 1 mL of samples were withdrawn from the sampling port at various time points (0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h). Fresh 1 mL of 1 mg / mL Na₂S₂O₄ PBS buffer (pH 7.4) was added to the sampling port promptly. PBS buffer served as a control; all other conditions remained unchanged. Finally, the released urokinase at each time point was measured using Coomassie Brilliant Blue G250 to observe the release of urokinase from the Franze diffusion cell, and the cumulative release amount of urokinase was calculated according to the following formula.

[0070]

[0071] Z UK Urokinase release rate; Vb volume of each buffer replacement (1 mL); C i The urokinase concentration of the i-th sampling solution; C n The concentration of urokinase in the system at the time of sampling for the nth time; V0 the total volume of the release system (20 mL); m a Total urokinase content in liposomes.

[0072] The experimental results are as follows Figure 6 As shown, after 24 hours of incubation in a simulated human physiological state (pH 7.4 PBS buffer), PAC@UK liposomes released 49.18% of urokinase. This result indicates that drug leakage is minimized under normal physiological conditions, reducing potential bleeding side effects of thrombolytic drugs. Na₂S₂O₄ simulates the hypoxic microenvironment at the thrombus site. In the presence of 1 mg / mL Na₂S₂O₄, urokinase release from PAC@UK liposomes was significantly increased, with a cumulative release of 96.41%. These results demonstrate that the liposomes are hypoxic-responsive, significantly reducing urokinase leakage from the systemic circulation. Upon reaching the thrombus site, overexpressed NADPH reductase in the hypoxic environment rapidly triggers urokinase release, achieving enhanced thrombolytic efficacy.

[0073] 5. Interaction between polyguluronate sulfate and P-selectin

[0074] Flow cytometry was used to determine the affinity between polyguluronate sulfate and the target protein P-selectin in platelets, and the interaction between the flow cytometric antibody PE-Anti-P-selectin and PGS was used to verify their binding.

[0075] The specific steps are as follows:

[0076] Platelet extraction and washing: 1.2 mL of Kunming mouse blood was collected by orbital blood sampling and centrifuged at 200 g for 5 min to obtain platelet-rich plasma (PRP) solution (0.4 mL). Then, 0.4 mL of platelets (PRP) was placed in an EP tube and centrifuged at 400 g for 5 min at room temperature, and the supernatant was discarded. The platelets were resuspended in 0.4 mL of benchtop solution A, mixed, and centrifuged at 400 g for 5 min at room temperature, and the supernatant was discarded. The platelets were resuspended in 1 mL of benchtop solution B and adjusted to an appropriate concentration with benchtop solution B for later use.

[0077] Preparation of benchtop stock solution: Weigh NaHCO3 (1.0 g), NaCl (8.0 g), NaH2PO4 (0.05 g), KCl (0.2 g) and MgCl2 (0.2 g) in sequence, dissolve in deionized water (1 L), and prepare 1 L benchtop stock solution.

[0078] Preparation of benchtop solution A (pH 6.5): Take 50 mL of the mother solution, add BSA (0.175 g), EDTA (0.5 mol / L, 250 μL) and glucose (0.05 g) and dissolve them in it to obtain 50 mL of benchtop solution A.

[0079] Preparation of benchtop solution B (pH 7.35-7.45): Take 50 mL of the mother solution and add glucose (0.05 g) to dissolve to obtain 50 mL of benchtop solution B.

[0080] After dilution with benchtop solution B, the concentration is 1×10 6To platelets of 50 cells / mL, 0.2 mg / mL 1.25 μL PE-labeled anti-P-selectin (eBioscience 12-0626-80) was added, followed by 0.5 mL 2 mg / mL FITC-PGS and FITC-PG (polyguluronic acid, not targeting P-selectin) (the labeling rates of FITC in PGS and PG were 5.03‰ and 5.16‰, respectively. For specific steps, refer to the literature PL Li, CX Li, Y T Sue, Y. Zhang, HB Liu, X. Zhao, GL Yu, HS Guan, Preparation, characterization and pharmacokinetics of fluorescence labeled propylene glycol alginate sodium sulfate. J Ocean U China, 13 (2014), 683-690.) of the sample to be tested, and then 4 μL Platelets were activated with 30 U / mL thrombin (final concentration 0.12 U / mL) and incubated at 37°C in the dark for 40 min. The binding of FITC-PGS, FITC-PG, and PE-anti-P-selectin to P-selectin on platelets was detected by flow cytometry.

[0081] The results of flow cytometry showed that Figure 7 The binding of platelets activated by thrombin to PE-anti-CD62P (mouse P-selectin) antibodies increased significantly. Since platelets overexpress P-selectin, the proportion of FITC-PGS binding to platelets increased significantly. PGS has the property of targeting P-selectin. The experimental results show that under the stimulation of thrombin, platelets promote the expression of P-selectin on the platelet membrane, and PGS can target and bind to P-selectin, which has reference value for the subsequent construction of PGS nanocarriers for targeted thrombosis treatment.

[0082] 6. PAC@UK liposomes targeting thrombus formation and carotid artery thrombus in vitro

[0083] After SD rats were anesthetized with 2% (w / v) tribromoethanol (15 μL / g), the skin above the rat carotid artery was cut open, and the left carotid artery of the rat was isolated. Filter paper containing ferric chloride (10%) was applied to the carotid artery for 10 minutes to induce thrombosis and isolate the carotid artery containing thrombus. The carotid artery was fixed with 4% paraformaldehyde for 15 minutes and then washed three times with PBS. The blood vessels were divided into two groups, each with three blood vessels: the PAC@IR783 group and the free IR783 group. The blood vessels containing thrombus were placed in 1 mL PAC@IR783 liposome solution (referring to the preparation method of PAC@UK liposomes, IR783 was replaced by UK to prepare PAC@IR783 liposomes, and the IR783 concentration was 0.5 mg / mL) and 1 mL of free 0.5 mg / mL IR783 solution for incubation for 30 minutes. The enrichment of IR783 in the blood vessels in each group was observed. Figure 8 (A) The experimental results show that compared with the free IR783 fluorescent reagent, the PAC@IR783 liposomes are significantly enriched in the vascular thrombus site, further proving that the PGS-modified liposomes have the function of targeted enrichment of thrombus.

[0084] SD rats were anesthetized with 2% (w / v) tribromoethanol (15 μL / g), the abdominal skin was cut open, and blood was collected through the abdominal aorta. 1 mL of SD rat plasma was taken, and 200 μL of platelet-rich plasma (PRP) was separated by centrifugation at 200 g. 50 μL of PRP was incubated with 50 μL of thrombin (0.05 U / mL) and 20 μL of fibrinogen (1 mg / mL) for 90 minutes, and 100 μL of calcium chloride (50 mM) was added and mixed. The mixture was incubated at 37 ° C for 30 minutes to induce clot formation. 100 μL of PAC@IR783 liposome solution and 100 μL of free 0.5 mg / mL IR783 solution were added and incubated for 1 hour. The mixture was washed with PBS 3 times, and the fluorescence enrichment was observed by in vivo animal imaging. Figure 8 (B) Figure 8 As shown in (C), the PAC@IR783 liposomes' targeting of thrombi and the statistical results of their fluorescence intensity demonstrate that the liposomes possess thrombus-targeting properties, consistent with the results of liposome targeting of carotid artery thrombi. Analysis indicates that PGS has a high affinity for P-selectin, which is highly expressed on activated platelets at the thrombus site, confirming the previous flow cytometry experiments demonstrating PGS binding to P-selectin.

[0085] 7. Evaluation of thrombolytic activity of PAC@UK liposomes in vitro blood clot retraction experiment

[0086] SD rats were anesthetized with 2% (w / v) tribromoethanol (15 μL / g). The abdominal skin was cut open and blood was collected from the abdominal aorta. 5 mL of fresh blood was centrifuged at 200 g to separate 1 mL of platelet-rich plasma (PRP). 50 μL of PRP (platelet-rich plasma) was incubated with 40 μL of PAC@UK liposome solution (containing 12.5 U / μL UK) at 37°C for 5 minutes. 1 mL of PBS was added and 200 μL of calcium chloride (50 mM) was added and mixed. Then, 50 μL of thrombin (0.3 U / mL) was added and incubated at 37°C for 2 hours. The clot size was observed and calculated using Image J. In addition, 40 μL of free UK (12.5 U / μL) was used as a control group.

[0087] The in vitro thrombolytic activity of PAC@UK liposomes was evaluated by plasma clot retraction test. Platelet-rich plasma (PRP) of isolated mice was used to induce thrombus formation under the action of thrombin and calcium chloride. The effect of PAC@UK liposomes on clot retraction was observed. Figure 9 (A) and Figure 9 As shown in (B), compared to the untreated group (Model), PAC@UK liposomes exhibited significant clot inhibition and disintegration capabilities. This is due to the thrombolytic activity of UK within the PAC@UK liposomes. PGS, in particular, has antiplatelet aggregation properties, potentially contributing to a therapeutic effect in inhibiting thrombus formation.

[0088] 8. Evaluation of the antithrombotic activity of PAC@UK liposomes in zebrafish thrombosis model

[0089] Transgenic zebrafish (Tg(gata1:dSRed) (marking erythrocytes) and Tg(CD41:EGFP) (marking platelets) (National Zebrafish Resource Center) were cultured at 3 days post-fertilization (dpf) in 12-well plates, with 15 embryos per well. The 3-dpf zebrafish embryos were cultured in embryo culture medium supplemented with 1 μM phenylhydrazine for 12 hours to successfully establish thrombi. After successful modeling, the zebrafish were washed 2-3 times with embryo culture medium. The successfully modeled zebrafish were divided into two groups: the model group (Model) and the PAC@UK group (15 per group). The successfully modeled zebrafish in each group were added to 2 mL of embryo culture medium (Model group) or 2 mL of embryo culture medium containing PAC@UK liposomes (final UK concentration of 100 U / mL) (PAC@UK group). Unmodeled zebrafish were cultured in embryo culture medium as a control group (control group). The three groups of zebrafish were then incubated in a 28°C incubator for 24 hours. The zebrafish were washed 2-3 times with embryo culture medium and finally maintained in 1 mL of embryo culture medium. After treatment, the zebrafish were anesthetized with tricaine at a mass concentration of 0.4‰ for 30 seconds, placed on a slide coated with 4% carboxymethyl cellulose, and fixed in a prone position. Red blood cell aggregation was observed under a Leica M205A stereo microscope (red blood cells appear red). The integrated optical density of the tail vein was detected using Image-proplus. In addition, the circulation of free platelets in the body was observed under a fluorescence microscope, and the number of free platelets in the body was recorded (platelets appear green).

[0090] Preparation of zebrafish embryo culture medium solution: Weigh NaCl (292 mg, 5 mM), KCl (13 mg, 0.17 mM), CaCl2 (37 mg, 0.33 mM), and MgSO4·7H2O (81 mg, 0.33 mM) in sequence, dissolve in ultrapure water, and prepare 1 L of zebrafish embryo culture medium solution.

[0091] The antithrombotic activity of PAC@UK liposomes was evaluated in a zebrafish thrombosis model. Figure 10 As shown in (A), the zebrafish cardiac ischemia in each group was observed. Compared with the control group, the zebrafish cardiac ischemia in the Model group was significantly more pronounced, indicating that the thrombosis model was successfully established under phenylhydrazine induction. The PAC@UK group significantly improved the relief of cardiac ischemia. This is due to the sulfated polysaccharide's inherent antiplatelet aggregation ability, which has a certain therapeutic effect in combating thrombosis and alleviating cardiac ischemia. These results indicate that the liposomes exert their thrombolytic function by releasing urokinase (UK) and PGS, thereby reducing red blood cell aggregation and alleviating the pathological characteristics of zebrafish cardiac ischemia.

[0092] The number of circulating platelets in zebrafish was used to investigate the effects of different administration components on the alleviation of phenylhydrazine-induced thrombosis and the symptoms of decreased circulating platelets. Figure 10 (B) The statistical chart of zebrafish circulating platelet counts shows that the Model group has a significantly suppressed circulating platelet count compared to the Control group. However, after treatment with polyguluronate sulfate-azobenzene cholesterol liposomes, the platelet count in zebrafish is significantly restored. This is consistent with the aforementioned antithrombotic activity in zebrafish cardiac ischemia.

[0093] 9. Evaluation of thrombolytic activity of PAC@UK liposomes in carotid artery thrombosis model

[0094] Kunming mice were anesthetized with 2% (w / v) tribromoethanol (15 μL / g). The neck skin was cut open to expose the left cervical vessels. The vessels were padded with a plastic spacer. The left carotid artery was then injured with filter paper soaked in 10% (w / v) ferric chloride for 5 minutes. The carotid artery turned black, indicating successful thrombosis modeling. The filter paper was then removed, and the vessel surface was washed with PBS to prevent residual residue from affecting local tissue. After successful modeling, the Kunming mice were divided into three groups, namely the model group, the UK group and the PAC@UK group, with 6 mice in each group. 15 minutes later, 200 μL PBS solution (Model group), 200 μL UK solution (100 U / g) (UK group) and 200 μL PAC@UK liposome solution (100 U / g) (PAC@UK group) were injected into the tail vein of the modeled Kunming mice in each group. After 24 hours, the blood flow changes at the thrombus site were observed. The right blood vessels of the mice were used as blank controls, and the blood flow recovery was observed using a laser Doppler blood flow imager (RFLSIZW). The blood flow reperfusion recovery of the vessels was statistically analyzed.

[0095] The in vivo thrombolytic activity of PAC@UK liposomes was evaluated using a carotid artery thrombosis model. Ferric chloride was used to induce thrombosis in the mouse carotid artery. The drug was injected into the tail vein and blood flow recovery was observed after 24 hours. Figure 11 and 12 As shown in the figure, observations of blood flow changes at the thrombus site indicate that PAC@UK liposomes have a significant effect on restoring blood flow compared to the Model group. Compared to the normal right vessel, PAC@UK liposomes have similar effects on restoring blood flow. Within the liposomes, UK exerts thrombolytic activity and PGS exerts antiplatelet aggregation function, exerting a certain therapeutic effect on restoring blood flow at the thrombus site.

[0096] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing sulfated polysaccharide hypoxic sterol derivatives, characterized in that: The following steps are involved: (1) Synthesis of sterol derivatives modified with hypoxia-responsive groups: Weigh a sterol succinate derivative and dissolve it in a first solvent. Then, HATU and DIPEA are added, respectively. Finally, a compound having a hypoxia-responsive group is added, and the reaction results in a sterol derivative modified with a hypoxia-responsive group. (2) Synthesis of sulfated polysaccharide hypoxic sterol derivatives: The sulfated polysaccharide was dissolved in water and acetonitrile solution was added, followed by HBTU and DIPEA, and the mixture was stirred for activation. Finally, the hypoxic responsive group-modified sterol derivative prepared in step (1) was added, and the reaction was continued with stirring to obtain sulfated polysaccharide hypoxic sterol derivatives.

2. The preparation method according to claim 1, characterized in that The following steps are involved: (1) Synthesis of sterol derivatives modified with hypoxia-responsive groups: Weigh a sterol succinate derivative and dissolve it in a first solvent. Then, add HATU and DIPEA, respectively. Finally, add a compound having a hypoxia-responsive group. Stir and react. After the reaction is completed, separate and purify by column chromatography. After enrichment, dry the product to obtain the hypoxia-responsive group-modified sterol derivative. (2) Synthesis of sulfated polysaccharide hypoxic sterol derivatives: The sulfated polysaccharide was dissolved in water and an acetonitrile solution was added. HBTU and DIPEA were then added and stirred for activation. Finally, the hypoxic responsive group-modified sterol derivative prepared in step (1) was added and the reaction was continued with stirring. After the reaction was completed, the reaction solvent was dried by spin drying, dialyzed, concentrated and freeze-dried to obtain sulfated polysaccharide hypoxic sterol derivatives.

3. The preparation method according to claim 2, wherein: The molar ratio of the sterol succinate derivative, HATU, DIPEA, and the compound having an oxygen-responsive group in step (1) is 1:(2-4):(2-4):1; the first solvent is selected from one of CH2Cl2, DMSO, and DMF, the stirring reaction speed is 200-1000 rpm, and the reaction time is 1-3 hours; the compound having an oxygen-responsive group is selected from one of p-diaminoazobenzene and nitroimidazole; and the sterol succinate derivative is selected from one of cholesterol, β-sitosterol and its analogs, saponin, and cholesterol.

4. The preparation method according to claim 2, wherein: In step (2), the molar ratio of the sulfated polysaccharide, HBTU, DIPEA, and the sterol derivative modified with the hypoxia responsive group is 1:(1-2):1:

1.

5. The preparation method according to claim 2, wherein: The activation temperature in step (2) is 30-50°C, the activation time is 0.5-2h, the stirring reaction temperature is 30-50°C, the reaction time is 20-30h, the dialysis method is to use a dialysis bag for dialysis in DMSO and water respectively, and the retention capacity of the dialysis bag is 1000-6000kDa; the sulfated polysaccharide is selected from one of PGS, PMGS, PMS, PSS, fucoidan sulfate, heparin, and heparinoid.

6. A sulfated polysaccharide hypoxic sterol derivative prepared by the preparation method according to any one of claims 1 to 8.

7. Use of the sulfated polysaccharide hypoxic sterol derivative according to claim 6 in the preparation of nanoformulations loaded with thrombolytic drugs and in the preparation of targeted thrombolytic drugs.

8. The use according to claim 7, characterized in that: The nano preparation is one of liposomes, nano gels, nano micelles and nano particles.

9. The use according to claim 8, characterized in that The liposome preparation method comprises the following steps: dissolving soybean lecithin in a second solvent to obtain an oil phase; dissolving a sulfated polysaccharide hypoxic sterol derivative and a thrombolytic drug in a PBS buffer to obtain an aqueous phase; adding the aqueous phase to the oil phase, shearing at high speed to obtain colostrum; adding the colostrum to a PBS buffer, shearing until liposomes are formed; stirring the liposomes to remove the second solvent; and finally extruding the liposome solution through a filter membrane to obtain uniform sulfated polysaccharide hypoxic sterol derivative@thrombolytic drug liposomes.

10. The use according to claim 9, characterized in that: The second solvent is selected from one of chloroform and ethanol, the mass ratio of the soybean lecithin to the sulfated polysaccharide hypoxic sterol derivative is 9:(10-20), the mass volume ratio of the sulfated polysaccharide hypoxic sterol derivative to the thrombolytic drug is 140 mg:(1-2) mL, the pore size of the filter membrane is 0.2-0.3 μm; and the thrombolytic drug is selected from one of urokinase, alteplase, and streptokinase.

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