A biomimetic nanohydrogel delivery system, and a preparation method and application thereof

Abstract

The application provides a kind of biomimetic nanogel delivery system and its preparation method and application.The biomimetic nanogel delivery system of the application includes: (1) temperature-sensitive hydrogel matrix, which includes carbomer 974, poloxamer 407, poloxamer 188 and bletilla striata polysaccharide dispersed in water;(2) biomimetic nanoparticles dispersed in the temperature-sensitive hydrogel matrix, which includes: an inner core composed of zeolite imidazolate framework-8 (ZIF-8) and its loaded active ingredients;Lipid and platelet membrane layer coated outside the inner core;Wherein, the lipid is composed of lecithin and cholesterol.The biomimetic nanogel delivery system of the application can realize targeted therapy for tumor surgical resection site by combining platelet biomimetic nanoparticles with damaged blood vessels and tumor characteristics targeting and hydrogel.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomimetic nanohydrogel delivery system, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide. While radical surgery is ideal for early-stage HCC, residual tumor, cancer stem cells (CSCs), tumor angiogenesis, and the tumor immunosuppressive microenvironment can lead to postoperative recurrence. Therefore, innovative strategies are urgently needed to minimize HCC recurrence. The combined use of bufotoxin (BFL) and sorafenib (SFN) can prevent HCC recurrence by inhibiting tumor cell proliferation, anti-angiogenesis, suppressing hepatocellular carcinoma stem cells (LCSCs), and improving the tumor immunosuppressive microenvironment. However, the combined use of BFL and SFN is limited by several factors, such as poor drug water solubility, certain toxicity, and poor tumor accumulation. Summary of the Invention

[0003] To address the issues of bleeding and postoperative recurrence in hepatectomy for hepatocellular carcinoma (HCC), the inventors have developed a biomimetic nano-hydrogel delivery system using Bletilla striata polysaccharide (Bsp) thermosensitive hydrogel. This system ensures sufficient viscosity while maintaining hemostatic efficacy. The biomimetic nano-hydrogel delivery system of this invention can release drugs locally and continuously in situ while possessing sufficient viscosity and hemostatic function.

[0004] Therefore, in a first aspect, the present invention provides a biomimetic nanohydrogel delivery system, comprising: (1) Thermosensitive hydrogel matrix, wherein the thermosensitive hydrogel matrix comprises Carbomer 974, Poroxam 407, Poroxam 188 and Bletilla striata polysaccharide (Bsp) dispersed in water. (2) Biomimetic nanoparticles dispersed in a thermosensitive hydrogel matrix, wherein the biomimetic nanoparticles comprise: a core composed of zeolite imidazole ester backbone-8 (ZIF-8) and its loaded active ingredients; and lipid and platelet membrane layers covering the core; wherein the lipids are composed of lecithin and cholesterol.

[0005] In the thermosensitive hydrogel matrix of the present invention, Carbomer 974 can be purchased from Shanghai Guoyao Reagent Co., Ltd.

[0006] In this invention, Poroxam 407 can be purchased from Beijing Sorapio Technology Co., Ltd.

[0007] In this invention, Poroxam 188 can be purchased from Beijing Sorapio Technology Co., Ltd.

[0008] In this invention, Bletilla striata polysaccharide (Bsp) can be purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0009] In some embodiments, the temperature-sensitive hydrogel matrix may also contain other common hydrogel excipients, such as fibrinogen.

[0010] In some embodiments, the thermosensitive hydrogel matrix contains, based on the volume of water used, 10% (g / ml) of Carbomer 974, 19% (g / ml) of Poroxam 407, 4% (g / ml) of Poroxam 188, and 10% (g / ml) of a 10% (g / ml) Bsp aqueous solution.

[0011] There are no particular limitations on the method for preparing the thermosensitive hydrogel matrix, as long as the components are evenly dispersed in water. In some embodiments, the thermosensitive hydrogel matrix is ​​prepared by a method including the following steps: Carbomer974 is added at 10% (g / ml) to distilled water and incubated overnight at 4°C to allow it to fully swell; then, Poroxam407 is added at 19% (g / ml), Poroxam188 is added at 4% (g / ml), and the mixture is incubated overnight at 4°C to allow it to fully mix; finally, a 10% (g / ml) aqueous solution of Bletilla striata polysaccharide is added at 20% (ml / ml), and the mixture is incubated overnight to obtain the thermosensitive hydrogel matrix.

[0012] In the biomimetic nanoparticles of this invention, ZIF-8 is used to provide drug loading; the active ingredient is used to inhibit tumor cell proliferation, anti-angiogenesis, and inhibit liver cancer stem cell growth; lipids are used to provide conditions for biomimetic coating; and platelet membranes are used to provide the biomimetic nanoparticles with targeting effects on damaged blood vessels and tumors. Thus, when the biomimetic nanoparticles are released from the gel matrix and come into contact with the target tissue, they can target damaged blood vessels and tumors, release drugs, inhibit tumor cell proliferation, anti-angiogenesis, and inhibit liver cancer stem cell growth.

[0013] ZIF-8 is a product made from zinc ions (Zn). 2+ Metal-organic frameworks (MOFs) composed of α-methylimidazole ligands possess high specific surface area, high thermal stability (>400℃), and excellent chemical stability (stable in water and strong alkalis). They are commonly used in gas adsorption and separation, catalysis, and drug delivery, and are among the most widely studied MOF materials.

[0014] In this invention, there are no particular limitations on the preparation method of ZIF-8. In some embodiments, ZIF-8 can be prepared by reacting Zn(NO3)2 and 2-methylimidazole (2-MIM) in a methanol solvent. In some embodiments, ZIF-8 is prepared as follows: Zn(NO3)2 and 2-MIM are dissolved separately in methanol, then the 2-MIM solution is added dropwise to the Zn(NO3)2 solution to carry out the reaction, and then solid-liquid separation (e.g., centrifugation) is performed to obtain ZIF-8.

[0015] In this invention, the active ingredient refers to a drug that can produce beneficial therapeutic effects in clinical practice. The beneficial therapeutic effects refer to the positive physiological changes produced by the drug in the human body, which can alleviate symptoms, reverse the disease, or promote recovery, with the effects significantly outweighing any potential toxic side effects. There are no particular limitations on the active ingredient, as long as it can be loaded into a hydrogel. The corresponding therapeutic purpose can be achieved according to the function of the active ingredient. For example, the active ingredient can be paclitaxel, thereby killing tumor cells; or the active ingredient can be gambogeylic acid, thereby killing tumor cells, inhibiting angiogenesis, inducing ferroptosis, etc.

[0016] There are no particular limitations on the loading amount of the active ingredient on ZIF-8, which is generally an effective amount for prevention or treatment. In this embodiment, the loading amount of the active ingredient on ZIF-8 can be 5.87%, but is not limited to this.

[0017] There are no particular restrictions on the method for loading active ingredients onto ZIF-8, as long as it can be loaded onto ZIF-8 and does not significantly degrade the beneficial therapeutic effects of the active ingredient. For example, the active ingredient and ZIF-8 can be simultaneously dispersed in an organic solvent, and after loading, solid-liquid separation can be performed to obtain ZIF-8 loaded with the active ingredient (hereinafter sometimes referred to as the ZIF-8 core).

[0018] In some embodiments, the active ingredients are BFL and SFN. In some embodiments, the ratio of BFL to SFN can be 5.3. In some embodiments, the total loading of BFL and SFN based on the mass of ZIF-8 can be 6.89%, but is not limited thereto.

[0019] In some embodiments, the mass ratio of lecithin to cholesterol in the lipids may be 8:1.

[0020] In this invention, lecithin refers to egg yolk lecithin, such as egg yolk lecithin purchased from Shanghai Guoyao Reagent Co., Ltd.

[0021] In this invention, cholesterol is also known as 3β-hydroxy-5-cholestene, 5-cholestene-3β-ol, CAS number 57-88-5, and its molecular formula is C. 27 H46 O, with a molecular weight of 386.65, such as cholesterol that can be purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd.

[0022] In some embodiments, the biomimetic nanoparticles are prepared by a method comprising the following steps: (1) ZIF-8 nanoparticles are mixed with active ingredients (e.g., BFL and SFN) in a solvent (e.g., methanol) and then centrifuged to obtain a core consisting of ZIF-8 and its loaded active ingredients (active ingredients@ZIF-8, sometimes referred to as ZIF-8 core). (2) The core is coated with lipids formed by lecithin and cholesterol to obtain a lipid-coated core; (3) The platelet membrane and lipid-coated nanoparticles are mixed in a solvent to fuse the platelet membrane and lipid to form a lipid and platelet membrane layer that coats the core, thus obtaining biomimetic nanoparticles.

[0023] In some embodiments, the weight ratio of ZIF-8 to the lipid in the biomimetic nanoparticles of the present invention can be 16:9.

[0024] There are no particular limitations on the method for coating the ZIF-8 core with lipids. For example, atmospheric pressure hydration can be used, first forming a lipid membrane, then adding the ZIF-8 core and hydrating it under atmospheric pressure to obtain lipid-coated ZIF-8 cores. To ensure uniform dispersion, sonication can be used. For example, sonication can be performed using a probe (80 W, 3 s sonication, 2 s pause, total processing time 10 min).

[0025] In some embodiments, the method of coating the ZIF-8 core with lipids is as follows: lecithin and cholesterol are weighed in a container at a mass ratio of 8:1, dissolved in chloroform, and then evaporated under reduced pressure in a water bath at 45°C to form a lipid film; the ZIF-8 core is ultrasonically dispersed in a 5% glucose solution, added to a container containing the lipid film for hydration (e.g., hydration in a water bath at 50°C under normal pressure for 10 min), and then ultrasonically treated to obtain the lipid-coated ZIF-8 core.

[0026] In some embodiments, the platelet membrane (PM) can be derived from whole blood or platelets. In some embodiments, whole blood can be used. It is obtained by extraction after repeated freeze-thaw cycles and ultrasonic disruption.

[0027] In some embodiments, the amount of platelet membrane used in the biomimetic nanoparticles of the present invention can be 5%-10% based on the mass of ZIF-8, for example, 5%, 8%, or 10%.

[0028] There are no particular restrictions on the methods for fusing platelet membranes with lipids. For example, platelet membranes can be fused with lipid-coated ZIF-8 cores in solution to obtain biomimetic nanoparticles.

[0029] In some embodiments, the biomimetic nanohydrogel delivery system is prepared by dispersing biomimetic nanoparticles in a thermosensitive hydrogel matrix.

[0030] Secondly, the present invention provides a method for preparing the above-mentioned biomimetic nanohydrogel delivery system, comprising the following steps: S1, preparation of biomimetic nanoparticles; S2, Preparation of temperature-sensitive hydrogel matrix; S3. Add biomimetic nanoparticles to a temperature-sensitive hydrogel matrix and mix to obtain a biomimetic nano-hydrogel delivery system.

[0031] In the method of the present invention, the biomimetic nanoparticles and the thermosensitive hydrogel matrix are as described in the first aspect, and will not be repeated here.

[0032] In some implementations, ultrasonic treatment can be used in S3 to ensure uniform dispersion. For example, ultrasonic treatment can be performed using a probe (80 W, 3 s for 3 seconds, 2 s for 2 seconds, for a total processing time of 10 minutes).

[0033] Thirdly, the present invention provides the use of the above-described biomimetic nanohydrogel delivery system in the preparation of medicaments for targeted therapy of surgical resection sites of tumors (particularly liver cancer (HCC)).

[0034] In some embodiments, the active pharmaceutical ingredients in the above-described biomimetic nanohydrogel delivery system are BFL and SFN.

[0035] In particular, in some embodiments, the treatment can produce an effect selected from one or more of the following: Hemostasis and blood clotting; Inhibit tumor cell stemness; Inhibit tumor cell proliferation; Inhibits the growth of liver cancer stem cells; Inhibit tumor angiogenesis; Improve the tumor immunosuppressive microenvironment.

[0036] Beneficial effects The biomimetic nanohydrogel delivery system of this invention can achieve targeted therapy to surgical resection sites of tumors (e.g., HCC) by combining platelet-inspired biomimetic nanoparticles with targeted properties of damaged blood vessels and tumors with hydrogel. Using a Bsp hydrogel at the surgical incision site to enhance adhesion and hemostasis ensures effective drug delivery and hemostasis. In particular, in this invention, the combined use of BFL and SFN can inhibit tumor cell proliferation, anti-angiogenesis, and inhibit the growth of liver cancer stem cells, thus treating postoperative recurrence of HCC. In this invention, by combining Bletilla striata polysaccharide (Bsp) and a thermosensitive hydrogel matrix, the biomimetic nanohydrogel delivery system of this invention significantly improves efficacy compared to nanogel delivery systems that do not use Bletilla striata polysaccharide (Bsp) and a thermosensitive hydrogel matrix. For example, in the embodiments, the efficacy of a single application of B / S@PM-ZIF-8-Bsp / Gel is superior to the efficacy of multiple injections of B / S@PM-ZIF-8. Attached Figure Description

[0037] Figure 1 The particle size distribution, zeta potential, and transmission electron microscopy (TEM) results of the drug-loaded nanoformulations B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 in Example 1 are shown. (a) Particle size distribution of B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8; (b) Zeta potential of B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8; (c) TEM images of B / S@LP-ZIF-8 and B / S@PM-ZIF-8.

[0038] Figure 2 The images are scanning electron microscope (SEM) images of Bsp / Gel and B / S@PM-ZIF-8-Bsp / Gel in Example 1.

[0039] Figure 3 The adhesion performance results of the hydrogel in Example 1 are shown. Among them: (a) viscosity change of B / S@PM-ZIF-8 gel prepared with and without Bsp at 37°C; (b) temperature viscosity curves of B / S@PM-ZIF-8 gel prepared with and without Bsp from 20°C to 40°C; (c) adhesion ability of B / S@PM-ZIF-8-Bsp / Gel to liver.

[0040] Figure 4 The rheological properties of the hydrogel in Example 1 are shown. Among them: (a) oscillatory strain scan of B / S@PM-ZIF-8-Bsp / Gel; (b) temperature scan test of B / S@PM-ZIF-8-Bsp / Gel; (c) shear recovery test of B / S@PM-ZIF-8-Bsp / Gel.

[0041] Figure 5 The results of the hemostatic effect study of Bletilla striata gel in Example 2 are shown. Among them: (a) photographs of different formulations inhibiting bleeding in hepatectomy wounds; (b) blood loss; and (c) statistical analysis of hemostasis time. (n=3) P <0.05, P <0.01, P <0.001, n = 3).

[0042] Figure 6 The results of the coagulation study using Bletilla striata gel in Example 2 are shown. (a) In vitro dynamic whole blood coagulation assessment of the untreated group (Control), B / S@PM-ZIF-8-Gel group, and B / S@PM-ZIF-8-Bsp / Gel group; (b) BCI values ​​for each group. P <0.001, n = 3).

[0043] Figure 7 The results of flow cytometry analysis of stemness markers in Example 3 are shown. Specifically: (a) flow cytometry analysis of the expression of CD133 and CD44 stemness markers in LCSCs treated with different formulations, and (b) quantitative results. P <0.01, P <0.001, n = 3).

[0044] Figure 8 The results of the microsphere formation rate experiment in Example 3 are shown. (a) Microsphere formation after 7 days of treatment with different formulations and (b) Quantitative results of microsphere quantity. P <0.001, n = 3).

[0045] Figure 9 The study illustrates the effects of drug administration on tubulogenesis and the corresponding semi-quantitative blood vessel numbers under the conditions of inhibiting Huvec angiogenesis receptor activity (a) and inhibiting Huh7 cell angiogenesis secretion (b) in Example 3. P <0.001, P <0.01, P <0.05, n = 3).

[0046] Figure 10 The survival rates of Huh7 cells after treatment with BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8 and B / S@PM-ZIF-8 are shown in Example 3.

[0047] Figure 11 The results of evaluating the efficacy of different formulations in preventing postoperative recurrence of HCC in Example 4 are shown. Among them: (a) in vivo bioluminescence imaging of recurrent tumors after hepatectomy; (b) morphological images of ex vivo tumors in mice of each group; (c) bioluminescence imaging of ex vivo tumors in mice of each group; (d) mean curves of changes in fluorescence intensity of in situ tumors in mice of each group during drug administration; (e) changes in body weight of mice of each group during drug administration. P <0.001, P <0.01, P <0.05, n = 5). Detailed Implementation

[0048] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0050] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0051] instrument

[0052] Material

[0053] cell

[0054] Example Example 1 1. Preparation of biomimetic nanoparticles: Dissolve 150 mg of Zn(NO3)2•6H2O and 330 mg of 2-methylimidazole (2-MIM) in 7.5 mL and 7.15 mL of methanol respectively. Then, add the 2-MIM solution dropwise to the Zn(NO3)2•6H2O solution, stir at 500 r / min for 30 min at room temperature, centrifuge at 15000 r / min for 10 min to obtain ZIF-8, and then wash it three times with methanol.

[0055] Disperse 10 mg of the prepared ZIF-8 in 2 mL of methanol solution containing 20 mg / mL of BFL and 4 mg / mL of SFN, stir at 500 r / min for 24 h at room temperature, and then centrifuge at 15000 r / min for 10 min to obtain ZIF-8 loaded with BFL and SFN (hereinafter abbreviated as B / S@ZIF-8).

[0056] Weigh the lipid materials (i.e., lecithin and cholesterol) in a mass ratio of 8:1 of lecithin to cholesterol into an eggplant-shaped flask, dissolve them with chloroform, and then evaporate under reduced pressure in a 45°C water bath to form a lipid film.

[0057] Disperse 80 mg of B / S@ZIF-8 by ultrasound in 10 mL of 5% (g / ml) glucose solution, add it to the eggplant-shaped flask containing the lipid film, hydrate it at normal pressure in a 50°C water bath for 10 min, and then perform probe sonication (power 80 W, sonication for 3 s with an interval of 2 s) in an ice-water bath until the solution becomes light yellow and translucent to obtain lipid-coated nanoparticles B / S@LP-ZIF-8.

[0058] Collect whole blood from BALB / c mice (Shanghai Slake Experimental Animal Co., Ltd., license number SYXK (Shanghai) 2022-0012, and all animals are raised under standard conditions in the Animal Experiment Center of Shanghai University of Traditional Chinese Medicine), and extract the cell membrane of platelets (abbreviated as platelet membrane) by centrifugation, resuspension, repeated freezing and thawing, ultrasonic fragmentation and ultracentrifugation; Mix a 5% (g / ml) glucose suspension of platelet membrane with a mass ratio of 8% and a 5% (g / ml) glucose suspension of B / S@LP-ZIF-8, and perform ultrasonic treatment to make them fuse to obtain biomimetic nanoparticles B / S@PM-ZIF-8.

[0059] 2. Preparation of thermosensitive gel matrix: Carbomer 974 (10% g / ml) was dissolved in distilled water and incubated overnight at 4°C to allow for full swelling. Then, Poroxam 407 (19% g / ml) and Poroxam 188 (4% g / ml) were added and incubated overnight at 4°C to ensure thorough mixing. A 10% (g / ml) aqueous solution of Bletilla striata polysaccharide (Bsp) was added at a concentration of 20% (ml / ml), and the mixture was incubated overnight to obtain the Bsp / Gel gel matrix. By adding nanoparticles to the Bsp / Gel and stirring until homogeneous, B / S@ZIF-8-Bsp / Gel, B / S@LP-ZIF-8-Bsp / Gel, and B / S@PM-ZIF-8-Bsp / Gel were prepared.

[0060] 3. Fabrication of a biomimetic nanohydrogel delivery system: The B / S@PM-ZIF-8 biomimetic nanoparticles obtained in step 1 were added to the gel matrix Bsp / Gel prepared in step 2 at 4°C and vortexed for 15 min to obtain the biomimetic nanohydrogel delivery system B / S@PM-ZIF-8-Bsp / Gel.

[0061] 4. Characterization of B / S@PM-ZIF-8-Bsp / Gel Particle size, potential, and morphology characterization of the formulation: Under room temperature conditions, an appropriate amount of the formulation was diluted 20 times with distilled water, and its particle size, PDI, and potential were measured. An appropriate amount of the formulation was diluted 2 times with double-distilled water, dropped onto a copper grid, stained with 2% phosphotungstic acid solution (pH 6.8), and the morphology of the formulation was observed and photographed under TEM.

[0062] Characterization of Bletilla striata gel morphology: To avoid structural collapse, the Bsp / Gel and B / S@PM-ZIF-8-Bsp / Gel hydrogels prepared above were stored at -80℃ for 12 h before freeze drying, and then dried in a freeze dryer for 2 days. The pore size and morphology of the hydrogels were determined by SEM.

[0063] Viscosity study of Bletilla striata gel: A rotational viscometer was used to record the viscosity changes of B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel hydrogels within a temperature range of 20-40℃. The viscosity difference between B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel was measured within 10 minutes at 37℃. At 37℃, B / S@PM-ZIF-8-Bsp / Gel was applied between two fresh liver lobes to examine the gel's viscosity on the actual liver lobes.

[0064] Rheological properties of Bletilla striata gel were investigated: The rheological properties of B / S@PM-ZIF-8-Bsp / Gel were measured using a rheometer. The storage modulus G' and loss modulus G" of the gel under different shear rates and temperatures were analyzed. An appropriate amount of B / S@PM-ZIF-8-Bsp / Gel was placed on a plate. Under oscillating scanning mode, the scanning conditions were set as follows: strain range 0.01%–100%, angular frequency fixed at 10 rad / s, and temperature 37℃. The relationship curve between G' and G" of the gel was measured. The phase transition temperature was determined under the conditions of 10 rad / s and 1% strain. Place an appropriate amount of B / S@PM-ZIF-8-Bsp / Gel on a flat plate, set the temperature range to 20-40℃, and test the curves of G' and G” as a function of temperature. Measure the phase transition temperature; the temperature at the intersection of the curves is considered the gelation temperature. Also test the shear recovery performance of B / S@PM-ZIF-8-Bsp / Gel. With a fixed angular frequency of 10 rad / s, each amplitude oscillation shifts the strain from low strain (γ=1%, 100 s) to high strain (γ=1000%, 100 s), with each test lasting 100 s. Record the shear viscosity (η).

[0065] The particle size, PDI, and potential results for the drug-loaded nanoformulations B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 are shown in [reference needed]. Figure 1 The particle size of B / S@ZIF-8 was 133.2±2.9 nm, the PDI was 0.176, and the Zeta potential was 17.2±5.22 mV. The particle sizes of the obtained B / S@LP-ZIF-8 and B / S@PM-ZIF-8 increased to 140.2±5.98 nm and 144.6±7.4 nm, respectively, and the potentials changed from positive to negative to -7.14±3.15 mV and -15.1±6.32 mV, respectively. Electron microscopy revealed a perfectly round phospholipid film coating on B / S@LP-ZIF-8, and the B / S@PM-ZIF-8 film was significantly thicker. Combined with the particle size and potential results, this demonstrates the successful coating of B / S@ZIF-8 by the phospholipid film-PM coating.

[0066] 2 mL of each of the Bsp / Gel and B / S@PM-ZIF-8-Bsp / Gel were observed under SEM. The results are shown in the figure. Figure 2 The hydrogel scaffold possesses a porous network structure with an irregular three-dimensional network structure, providing a structural basis for drug storage and in vivo delivery. Its internal pore size is Bsp / Gel < B / S@PM-ZIF-8-Bsp / Gel, indicating that the higher the cross-linking density, the smaller the internal pore size of the hydrogel. The presence of nanoparticles in B / S@PM-ZIF-8-Bsp / Gel is clearly visible on a 500 nm scale, indicating that Bsp / Gel does not disrupt the nanostructure of B / S@PM-ZIF-8.

[0067] like Figure 3 As shown in Figure a, B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel were equilibrated at 37 ℃ for 600 s. Over time, the viscosity of the hydrogel gradually increased and then tended to stabilize. When gelation was constant, the maximum viscosities of B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel were 4989 mPa•s and 8267 mPa•s, respectively, indicating that adding Bsp can increase the viscosity of the hydrogel. Changes in hydrogel viscosity were observed within the temperature range of 20–40 ℃. Figure 3 In section b), B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel underwent phase transitions at temperatures of 35℃ and 32.5℃, respectively. Simultaneously, the formed B / S@PM-ZIF-8-Bsp / Gel was able to bind two slices of fresh liver together. Figure 3 (c) Ensure that the hydrogel adheres closely to the bleeding surface of the liver surgical incision.

[0068] When the strain is less than 1%, the gel maintains constant G' and G'' values, indicating that its structure has completely gelled; when the strain is between 1% and 10%, the gel structure is gradually destroyed; when the strain exceeds 10%, i.e., G' and G'' no longer remain constant, the gel network collapses. Figure 4 (a). The relationship between gel modulus and temperature provides the temperature at which viscosity jumps ( Figure 4 (b) As the temperature continues to rise, the viscosity of the thermosensitive hydrogel changes significantly within the range of 30℃ to 35℃. The gel undergoes a phase transition, changing from a solution state to a semi-solid state, directly manifested as an increase in viscosity. The temperature at which B / S@PM-ZIF-8-Bsp / Gel exhibits a viscosity jump is 30.5℃. Since frequent movement and compression within the peritoneal cavity may lead to hydrogel collapse and rapid degradation, it is necessary to examine whether the hydrogel possesses sufficient viscoelasticity and self-healing ability to ensure the continuous and stable release of the drug. During the destruction and recovery process, B / S@PM-ZIF-8-Bsp / Gel did not show significant viscoelastic changes, and the η value could recover to its initial value, indicating that the cross-linking network had been restored. Figure 4 (c) indicates that the gel has sufficient viscoelasticity and self-healing ability, which is conducive to the diffusion and release of drugs into the surrounding environment through the hydrophilic network structure of the gel network structure.

[0069] Example 2 1. Investigation on the hemostatic effect of Bletilla striata gel BALB / c nude mice were purchased from Shanghai Slack Experimental Animal Co., Ltd. (use license SYXK (Shanghai) 2022-0012) and were 4-5 weeks old. The animals were all raised under standard conditions in the Animal Experiment Center of Shanghai University of Traditional Chinese Medicine. All experimental operations complied with the regulations of the Animal Experiment Center of Shanghai University of Traditional Chinese Medicine. All animals used in this study were treated according to the procedures approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (ethical number PZSHUTCM2404050003).

[0070] Balb / c mice (male, 20-25 g, n = 3) were used to evaluate the hemostatic effect of B / S@PM-ZIF-8-Bsp / Gel. The mice were anesthetized with isoflurane, and the liver of the mice was exposed through an abdominal incision. Subsequently, a part of the liver of the same size was resected with a scalpel. A filter paper that had been weighed in advance was placed under the free liver, and then the bleeding sites of the liver were hemostatized with B / S@PM-ZIF-8-Gel (1 mL) and B / S@PM-ZIF-8-Bsp / Gel (1 mL) respectively. The filter paper was removed and weighed to estimate the change in blood loss, and the hemostasis time was recorded.

[0071] As Figure 5 shown, after the mice were treated with different groups, the hemostatic effects of each group were judged according to the amount of blood absorbed by the filter paper. The blood loss in the control group was the highest, reaching 58 mg at 8.5 min, while the blood loss in the B / S@PM-ZIF-8-Gel group decreased to 47 mg at 6.5 min. The hemostatic rate of the B / S@PM-ZIF-8-Bsp / Gel group was significantly better than that of the B / S@PM-ZIF-8-Gel group and the control group, and the blood loss at 2.4 min was 21 mg. This indicates that the addition of Bsp can effectively improve the hemostatic and coagulation functions of B / S@PM-ZIF-8-Bsp / Gel.

[0072] 2. Investigation of the coagulation of Bletilla striata gel The coagulation effect of B / S@PM-ZIF-8-Bsp / Gel was evaluated in vitro. B / S@PM-ZIF-8-Gel (1 mL) and B / S@PM-ZIF-8-Bsp / Gel (1 mL) were spread evenly on the bottom of the culture dish. Fifty microliters of fresh blood was dropped onto the blank culture dish and the samples. After 5 min, without stirring the coagulated blood, 10 mL of normal saline was slowly added, and it was gently shaken and observed for 10 min. The absorbance of hemoglobin in the supernatant of each sample was measured at a wavelength of 542 nm with an ultraviolet spectrophotometer, and the blood coagulation index (BCI) was calculated. As Figure 6As shown, both B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel hydrogels exhibited effective in vitro coagulation capabilities, with BCI values ​​of 35.6% and 12.5% ​​for the B / S@PM-ZIF-8-Gel and B / S@PM-ZIF-8-Bsp / Gel groups, respectively. This difference indicates that B / S@PM-ZIF-8-Bsp / Gel has better coagulation performance.

[0073] Example 3 1. Inhibits tumor cell stemness Flow cytometry detection of stemness markers: LCSCs were divided into groups of 2 × 10⁻⁶ cells. 3 Cells were seeded at a density of [number] cells / well in 6-well ultra-low adsorption plates and co-incubated for 7 days with BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 (BFL concentration 0.1 μM, SFN concentration 0.5 μM). After drug treatment, cells were collected by trypsin digestion with EDTA-free enzyme, washed twice with PBS, and stained with anti-CD44 rabbit anti-pharmaceutical (1:500 diluted in PBS) (Abcam) and anti-CD133 mouse anti-pharmaceutical (1:1000 diluted in PBS) (Abcam) and the corresponding fluorescent secondary antibodies. The stem cell ratio of each group was detected by flow cytometry. Experimental results are shown below. Figure 7 .like Figure 7 As shown, the proportions of CD133 and CD44 in stemness-induced LCSCs were significantly higher than those in Huh7 (P<0.05), indicating successful establishment of LCSCs. The B / S@PM-ZIF-8 group showed the best stemness reduction effect, significantly reducing the expression of CD133 and CD44 in LCSCs.

[0074] Microsphere formation rate experiment: LCSCs cells were divided into groups of 2 × 10⁻⁶. 3 Cells were seeded at a density of cells / well in 6-well ultra-low adsorption plates and co-incubated for 7 days with BFL, SFN, BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 (BFL concentration 0.1 μM, SFN concentration 0.5 μM). Random photographs were taken under a 40x optical microscope. The number of spheroids in each group was counted in five randomly selected fields of view. The spheroidization of each group was then photographed and compared under a 200x optical microscope. Experimental results are shown below. Figure 8 .like Figure 8As shown, the number of spheres in the blank control group (Control), BFL group, SFN group, BFL+SFN group, BFL@PM-ZIF-8 group, SFN@PM-ZIF-8 group, B / S@ZIF-8 group, B / S@LP-ZIF-8 group, and B / S@PM-ZIF-8 group were 28.00±1.00, 15.67±0.58, 20.67±2.08, 11.00±1.00, 9.67±0.58, 13.67±1.15, 7.00±1.00, 5.33±0.58, and 1±1, respectively. The difference between B / S@PM-ZIF-8 and B / S@ZIF-8 was statistically significant (P<0.05). The sphere volume clearly shows that BFL can inhibit the size of LCSCs spheres and synergistically enhance the inhibitory effect of SFN on LCSCs.

[0075] 2. Inhibits tumor angiogenesis The study investigated the direct inhibition of vascular endothelial growth factor receptor (VEGF) activity in Huvec cells (Shanghai Saolaxon Biopharmaceutical Co., Ltd.). Huh7 cells were seeded into 6-well plates pre-cultured with collagen and allowed to grow to 80%-90% confluence. The culture medium was changed after 24 h. Huvec cells were then seeded into 6-well plates pre-cultured with collagen and, when the cells reached 80%-90% confluence, co-incubated with drug-containing ECM medium: BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 (where BFL was administered at a concentration of 0.1 μM and SFN at a concentration of 0.5 μM). After 24 h of culture, the drug-treated Huvec cells were digested with EDTA-free trypsin, centrifuged, and resuspended in the collected Huh7 cell DMEM supernatant at a 1:1 ratio with ECM medium. Spread 50 μL of melted Matrigel:ECM medium (1:1) onto 96-well plates on ice and preheat in a 37°C cell culture incubator for approximately 30 min. Seed 100 μL of resuspended Huvec cell mixture onto the Matrigel surface at a seeding density of 2 × 10⁶ cells per well. 4 The cells were cultured in wells for 12 hours, and images were acquired using an inverted microscope and captured using ImageMicroscope. The formed tubular structures were quantified using Image-J.

[0076] The inhibitory effect of the formulation on angiogenesis factor secretion in Huh7 cells was investigated. Huh7 cells were co-incubated with drug-containing DMEM medium in the following formulations: BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8, or B / S@PM-ZIF-8 (where the concentration of BFL was 0.1 μM and the concentration of SFN was 0.5 μM). The culture medium was collected after 24 h. 50 μL of melted Matrigel:ECM medium (1:1) was seeded into 96-well plates and incubated at 37°C for approximately 30 min. Huvec cells that had adhered to the plate to 80%-90% were subjected to routine cell digestion and resuspended in the collected DMEM:ECM medium (1:1) collected after drug administration. The cells were then cultured at 2 × 10⁻⁶ cells / well. 4 At a density of 100 μL per well, Matrigel surface was inoculated and incubated at 37°C for 12 h. Images were acquired using an inverted microscope and captured using ImageMicroscope. The formed tubular structures were quantified using Image-J.

[0077] The experimental results are shown in Figure 9 .like Figure 9 As shown, after the cell tubes are formed, the preparation or Huh7 drug culture medium is added to these cell culture dishes. Twelve hours after drug treatment, the Control group (no preparation, no serum) showed clear tubules with adjacent endothelial cells extending and connecting with each other, and the tubular structure filled the field of view in a relatively loose network. After the drug was applied directly or indirectly to Huvec and Huh7, the number of vascular branches in the BFN@PM-ZIF-8 group (39.3±3.06 and 53.33±1.53), SFN@PM-ZIF-8 group (46.00±3.00 and 72.00±2.00), B / S@ZIF-8 group (23.67±1.53 and 32.67±2.08), and B / S@LP-ZIF-8 group (6.00±1.00 and 22.00±2.00) was higher than that in the B / S@PM-ZIF-8 group (0.00±0.00 and 12.33±2.08).

[0078] 3. Inhibits tumor cell proliferation The inhibitory effects of free drugs and different formulations on the growth of Huh7 cells were detected using a CCK8 assay kit. (8 × 10⁸ cells / cells) 3Huh7 cells were cultured at a density of cells / well. Different concentrations of BFL, SFN, BFL+SFN, BFL@PM-ZIF-8, SFN@PM-ZIF-8, B / S@ZIF-8, B / S@LP-ZIF-8, and B / S@PM-ZIF-8 were added. After discarding the supernatant, 10 μL of CCK-8 solution and 100 μL of fresh culture medium were added, and the cells were incubated at 37°C for 1 h. The absorbance (A) of each well was measured at 450 nm. Six replicates were set up for each group. Cell viability was calculated, and IC50 was calculated using Compusyn software. 50 The survival rate of Huh7 cells after treatment with different agents is as follows: Figure 10 As shown in the figure, compared with BFL and SFN alone, BFL+SFN treatment significantly enhanced the cytotoxicity of Huh7 cells (CI value 0.62), indicating that BFL and SFN have a good synergistic effect. The synergistic effect of the co-loaded drug systems on Huh7 cells from weakest to strongest is as follows: B / S@ZIF-8 (IC50: 0.09±0.01 μM).

[0079] Example 4 A mouse model of orthotopic hepatocellular carcinoma (HCC) resection was established to evaluate the efficacy of different agents in preventing postoperative recurrence of HCC. Healthy Huh7-luc cells (Shanghai Saolaxon Biopharmaceutical Co., Ltd.) were digested, collected, and centrifuged. After removing the supernatant, the cell concentration was adjusted with PBS solution and injected into the vascularized area of ​​the right axilla of mice. The injection volume was 100 µL (containing 1 × 10⁶ cells). 6 (one), when the tumor grows to 500 mm 3 After euthanasia by cervical dislocation, fresh tumor tissue was removed, rinsed twice with physiological saline, and the tumor tissue block (1.5 mm) was collected. 3 ​The incision was sutured to the left lower lobe of the mouse liver. After careful inspection to ensure there was no active bleeding, the abdomen was closed at full thickness, and antibiotics were applied topically to the incision site. An in situ HCC resection mouse model was established. Seven days after inoculation, the tumor was removed (leaving approximately 1% residual tumor postoperatively to simulate residual microscopic tumors) to establish an in situ recurrence mouse model. A total of 40 mice were randomly divided into 8 groups: Control (Bsp / Gel), SFN-Bsp / Gel, BFL+SFN-Bsp / Gel, BFL@PM-ZIF-8-Bsp / Gel, SFN@PM-ZIF-8-Bsp / Gel, B / S@ZIF-8-Bsp / Gel, B / S@PM-ZIF-8-Bsp / Gel, and B / S@PM-ZIF-8 (injection) (n=5, BFL=1 mg / kg, SFN=5 mg / kg). Immediately after tumor resection, hydrogels containing various formulations (100 μL) were injected into the liver resection margin or via tail vein injection. After observing for bleeding, the abdomen was closed, and the wound was treated with local antibiotics. Postoperatively, the animals continued to be fed freely, and in vivo imaging was used regularly to monitor the fluorescence intensity of the tumor in situ and changes in body weight. Tumor recurrence was monitored by intraperitoneal injection of d-fluorescein potassium (150 mg / kg) every 3 days using an in vivo biofluorescence imaging system, and the recurrence was assessed based on the fluorescence intensity. Body weight was monitored every 3 days. Results are as follows: Figure 11 As shown, monitoring tumor recurrence using bioluminescent signals from Huh7-luc cells revealed that B / S@PM-ZIF-8-Bsp / Gel showed the best effect in preventing HCC recurrence, with the weakest tumor fluorescence in all five mice. This may be due to the long-term sustained release of the hydrogel and the fact that B / S@PM-ZIF-8 promoted drug retention and accumulation within the tumor. Notably, compared to B / S@PM-ZIF-8-Bsp / Gel, the average tumor fluorescence intensity of BFL@PM-ZIF-8-Bsp / Gel and SFN@PM-ZIF-8-Bsp / Gel was 23.49 times and 37.50 times higher, respectively, highlighting the potential of the combined use of BFL and SFN to inhibit postoperative HCC recurrence. Compared to the group receiving multiple injections of B / S@PM-ZIF-8, the B / S@PM-ZIF-8-Bsp / Gel group showed a significant difference in inhibiting postoperative recurrence (P<0.05). The mean tumor fluorescence intensity in the 8-injection group was 13.68 times that of the B / S@PM-ZIF-8-Bsp / Gel group, indicating the advantage of hydrogel in postoperative HCC treatment. In addition, the tumor-suppressing effect of B / S@PM-ZIF-8-Bsp / Gel was significantly stronger than that of B / S@ZIF-8-Bsp / Gel, with a mean tumor fluorescence intensity 5.91 times that of B / S@PM-ZIF-8-Bsp / Gel (P<0.05). These results indicate that B / S@PM-ZIF-8-Bsp / Gel treatment can effectively inhibit the recurrence of HCC after resection.

Claims

1. A biomimetic nanohydrogel delivery system, comprising: (1) Thermosensitive hydrogel matrix, wherein the thermosensitive hydrogel matrix comprises Carbomer 974, Poroxam 407, Poroxam 188 and Bletilla striata polysaccharide (Bsp) dispersed in water. (2) Biomimetic nanoparticles dispersed in a thermosensitive hydrogel matrix, wherein the biomimetic nanoparticles comprise: a core composed of zeolite imidazole ester backbone-8 (ZIF-8) and its loaded active ingredients; and lipid and platelet membrane layers covering the core; wherein the lipids are composed of lecithin and cholesterol.

2. The biomimetic nanohydrogel delivery system according to claim 1, wherein, In the thermosensitive hydrogel matrix, based on the volume of water used, the mass-volume percentage concentration of Carbomer 974 is 10%, g / ml, the mass-volume percentage concentration of Poroxam 407 is 19%, g / ml, the mass-volume percentage concentration of Poroxam 188 is 4%, g / ml, and a Bsp aqueous solution with a concentration of 10%, g / ml is added at a concentration of 10%~20%, ml / ml.

3. The biomimetic nanohydrogel delivery system according to claim 1, wherein, The ZIF-8 was prepared by reacting Zn(NO3)2 and 2-methylimidazole (2-MIM) in methanol solvent. The active ingredient is selected from one or more of paclitaxel, gambogeylic acid, bufotalin (BFL), and sorafenib (SFN); The platelet membrane is derived from whole blood or platelets.

4. The biomimetic nanohydrogel delivery system according to claim 1, wherein, The active ingredients are BFL and SFN.

5. The biomimetic nanohydrogel delivery system according to any one of claims 1-4, wherein, The biomimetic nanoparticles are prepared by a method comprising the following steps: (1) ZIF-8 nanoparticles and active ingredients were mixed in a solvent and then centrifuged to obtain a core composed of ZIF-8 and its loaded active ingredients; (2) The core is coated with lipids formed by lecithin and cholesterol to obtain a lipid-coated core; (3) The platelet membrane and the lipid-coated core are mixed in a solvent to fuse the platelet membrane and lipid to form a lipid and platelet membrane layer that coats the core, thus obtaining biomimetic nanoparticles.

6. The biomimetic nanohydrogel delivery system according to claim 5, wherein, The lipid-coated core is prepared by a method including the following steps: first, a lipid membrane is formed using atmospheric pressure hydration, and then a ZIF-8 core is added and hydrated at atmospheric pressure to obtain the lipid-coated core.

7. The biomimetic nanohydrogel delivery system according to claim 5, wherein, The lipid-coated core is prepared by a method including the following steps: lecithin and cholesterol are weighed in a container at a mass ratio of 8:1, dissolved in chloroform, and then evaporated under reduced pressure in a water bath at 45°C to form a lipid film; the ZIF-8 core is ultrasonically dispersed in a 5% glucose solution, added to a container containing the lipid film for hydration, and then ultrasonically treated to obtain the lipid-coated core.

8. A method for preparing a biomimetic nanohydrogel delivery system according to any one of claims 1-7, comprising the following steps: S1, preparation of biomimetic nanoparticles; S2, Preparation of temperature-sensitive hydrogel matrix; S3. Add biomimetic nanoparticles to a temperature-sensitive hydrogel matrix and mix to obtain a biomimetic nano-hydrogel delivery system.

9. Use of the biomimetic nanohydrogel delivery system according to any one of claims 1-7 in the preparation of a medicament for targeted therapy to the surgical resection site of a tumor.

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