A dextran-based and zwitterionic-based hydrogel delivery system and antitumor preparation

Abstract

The present application relates to a kind of hydrogel delivery system based on dextran base and amphiphilic ion base and antitumor preparation.The present application first prepares two precursors containing double bond and mercapto, which can form an injectable hydrogel with good biocompatibility by "click chemistry" reaction, and the precursor solution can be simply and uniformly mixed to form in-situ injectable hydrogel under physiological conditions.The present application also provides a P-DOX / 1MT@MM, which combines the chemotherapeutic drug DOX with the IDO inhibitor 1MT to achieve multiple mechanisms of antitumor effect.The present application uses hydrogel to encapsulate the above-mentioned P-DOX / 1MT@MM, which greatly inhibits the recurrence and metastasis of tumor and activates the systemic antitumor immune response.

Description

Technical Field

[0001] This invention belongs to the technical field of hydrogel drug delivery systems, specifically relating to a hydrogel delivery system based on dextran and zwitterionic groups, a method for preparing the hydrogel delivery system, and a hydrogel formulation encapsulating antitumor drugs. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer is one of the leading causes of death threatening human life. Currently, the conventional treatments for cancer mainly include surgical resection, radiotherapy, and chemotherapy. However, malignant tumors are prone to metastasis, are insensitive to radiotherapy and chemotherapy, have poor prognoses, and pose a significant threat to human health. Surgical resection is currently one of the main methods of cancer treatment. However, the high rate of local tumor recurrence after surgery severely affects patient prognosis and survival. In most cases, postoperative chemotherapy is necessary. However, conventional systemic chemotherapy often leads to widespread drug distribution in the body, severe adverse reactions, and, more importantly, low treatment efficiency. Radiotherapy can also be accompanied by serious consequences such as gene mutations, even leading to death. Therefore, adopting drug delivery methods that improve efficacy and reduce toxic side effects is of paramount importance in cancer treatment, and the development of novel cancer treatment strategies is imperative. With the development of biotechnology, many new treatment methods have been used to treat cancer, improving the shortcomings of traditional treatments, especially drug combination therapy.

[0004] Hydrogels are soft materials with a three-dimensional network structure. They are three-dimensional polymer networks with high water content and high flexibility, made from natural or synthetic materials, and are considered one of the most attractive in-situ drug delivery systems. However, designing an effective drug combination system is not as simple as encapsulating drugs in a hydrogel. It is necessary to consider whether the hydrogel can simultaneously accommodate hydrophobic and hydrophilic drugs, and whether the drug, after being released from the hydrogel, can remain at the tumor site for a long time rather than being quickly absorbed and cleared by the body.

[0005] Recent studies have revealed that some anthracycline drugs, such as doxorubicin (DOX), can induce tumor cell death not only through direct insertion into cellular DNA strands but also by causing immunogenic cell death (ICD). Tumor cells undergoing ICD upregulate the expression of damage-associated molecular patterns (DAMPs), such as increased exposure of the calreticulin (CRT) surface, extracellular secretion of adenosine triphosphate (ATP), and passive release of high-mobility group box 1 (HMGB1). During ICD, tumor cells undergo autophagy, and CRT, an endoplasmic reticulum calcium-binding protein, can stimulate dendritic cells (DCs) to phagocytose. Simultaneously, HMGB1 is released, promoting stable connections between DCs and dying tumor cells. High concentrations of ATP produced by the vigorous metabolism within tumor cells are also released in large quantities, recruiting DCs into the tumor lesion. The release of these signals alerts the immune system and triggers an anti-tumor immune response, sometimes even exhibiting a distant effect—the spontaneous regression of distant metastases after local tumor treatment. ICDs can also induce a strong inflammatory response by releasing pro-inflammatory cytokines such as TNF-α, IL-6, and IL-10. Although DOX can induce ICDs, its application faces challenges such as low drug delivery efficiency and unavoidable toxicity. In most cases, the anti-tumor immune response it produces is not strong enough, and it is generally used in combination with other therapies.

[0006] Indoleamine 2,3-dioxygenase (IDO), highly expressed in tumor cells, is a key factor in the suppression of the tumor immune microenvironment. IDO is the first and rate-limiting step in the degradation of tryptophan (Trp) into its downstream metabolite kynurenine (Kyn) and subsequent metabolites along the Kynuren pathway. This action can be blocked by adding its inhibitor, 1-methyl-d-tryptophan (1MT). 1MT is a tryptophan derivative and thus inhibits IDO through competitive binding, thereby modulating the immunosuppressive tumor microenvironment. Furthermore, improving the acidity and hypoxia of the tumor microenvironment also enhances the efficacy of tumor immunotherapy. However, 1MT alone has limitations in efficacy and is generally used in combination with other drugs.

[0007] Dendritic macromolecules are a unique class of synthetic macromolecules with highly branched three-dimensional nanoscale structures. Their structural advantages enable them to play a crucial role in nanotechnology, pharmaceuticals, and medicinal chemistry. Polyamide-amine dendritic macromolecules (PAMAMs) are a commercially available type of dendritic macromolecule that has been extensively studied for applications in the biological field. The particle size of PAMAMs tends to increase with each generation because the terminals of PAMAMs are generally amine groups, thus increasing the number of cations and consequently increasing their toxicity. This property can also be utilized through chemical modification to achieve different applications.

[0008] To address the issue of drug clearance, methods mimicking natural components such as cells, proteins, and other biomolecules have become a hot topic in drug delivery. These methods offer advantages such as good biocompatibility, strong escape capabilities, low toxicity, and long in vivo circulation time. Macrophages, being among the most abundant cells in the tumor microenvironment, are directly related to tumor progression and metastasis. To delay drug carrier clearance by the mononuclear phagocytic system (MPS), a macrophage membrane is often coated onto the carrier. Various macrophage-coated nanocarriers have demonstrated superior stability and tumor-targeting capabilities. Summary of the Invention

[0009] The purpose of this invention is to construct a hydrogel-based local drug delivery system for targeted drug distribution at lesion sites and the formation of a drug reservoir. This not only reduces drug loss but also improves drug bioavailability. Injectable hydrogels, due to their in-situ formation and injectability, are a suitable formulation for local drug delivery. By directly delivering drugs to specific sites, they minimize drug accumulation in normal tissues and organs, thereby reducing systemic toxicity.

[0010] Dextran, a natural polysaccharide, is biocompatible and readily available. Zwitterionic materials are materials that contain both anionic and cationic groups in each repeating structural unit, exhibiting electrical neutrality due to equal amounts of positive and negative charges. In recent decades, zwitterionic betaine polymer hydrogels composed of phosphate betaine, sulfobetaine, and carboxybetaine have attracted increasing attention. These zwitterionic hydrogels possess unique biological properties, such as low cytotoxicity and low endotoxin levels. The superhydrophilicity of zwitterionic materials can establish a high-energy hydration barrier with strong electrical neutrality, preventing non-specific protein adsorption. Therefore, zwitterionic hydrogels have been extensively studied in the biomedical field due to their excellent biocompatibility.

[0011] In a first aspect, the present invention provides a hydrogel delivery system based on dextran and zwitterionic groups, wherein the smallest repeating unit of the hydrogel has the structure shown in Formula I:

[0012]

[0013] The hydrogel delivery system described above has been verified to have good biocompatibility and excellent anti-protein adsorption capacity, which can avoid damage to protein structure. In addition, the hydrogel degrades relatively slowly in vivo, which can achieve long-term circulation effect in vivo as a drug carrier.

[0014] In a second aspect, the present invention also provides a method for preparing the hydrogel delivery system, which is formed by crosslinking a precursor 1 based on dextran groups and a precursor 2 based on zwitterionic groups through a "click chemistry" reaction of thiol groups and double bonds; the structure of the precursor 1 is shown in Formula II below, and the structure of the precursor 2 is shown in Formula III below:

[0015]

[0016] The reaction for preparing the hydrogel is shown in the following formula:

[0017]

[0018] Precursor 1 and precursor 2 were dissolved separately in physiological saline, and then mixed and rapidly stirred until no liquid flowed to obtain the hydrogel.

[0019] Preferably, the preparation reaction of the precursor 1 is shown in the following formula:

[0020]

[0021] The preparation method is as follows: Dextran and lithium chloride are dispersed and dissolved in N,N-dimethylformamide (DMF), and methacryloyl chloride and triethylamine (TEA) are added dropwise; after the reaction is completed, the product is added dropwise to ice-cold methanol and filtered, and the resulting precipitate is precursor 1 (Q1).

[0022] Furthermore, the dextran and lithium chloride are dissolved in DMF by heating at a temperature of 105–115°C; after cooling, methacryloyl chloride and triethylamine are added dropwise in an ice bath and reacted for 20–28 hours.

[0023] Furthermore, the mass ratio of dextran to lithium chloride is 1.3–1.7:0.1–0.3.

[0024] Preferably, the preparation reaction of the precursor 2 is shown in the following formula:

[0025]

[0026] The preparation method is as follows: sulfobetaine methacrylate (SBMA) monomer, N,N'-bisacrylcysteine ​​(BAC) and ammonium persulfate (APS) are dispersed in an ethanol solution, and then tetramethylethylenediamine (TMEDA) is added to the solution to obtain a gel; dithiothreitol is added and stirred until it becomes a liquid solution, then placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 70-80 hours, and then dried to obtain precursor 2 (Q2).

[0027] Furthermore, the concentration of the ethanol solution is 20-30%.

[0028] Furthermore, the dialysis medium is changed every 4-8 hours during the dialysis process, and the medium is a deionized aqueous solution.

[0029] Furthermore, the synthesis of the sulfonate betaine methacrylate (SBMA) involves dissolving methacryloyloxyethyl dimethylamine (DMAEMA) in a solvent, adding 1,3-propanesulfonyl lactone, heating under reflux for a period of time to carry out the reaction, and after the reaction is completed, filtering and washing the product to obtain SBMA.

[0030] Furthermore, the mass ratio of the methacryloyloxyethyl dimethylamine to 1,3-propanesulfonyl lactone is 15-18:11-14.

[0031] Furthermore, the temperature of the heating reflux is 50-60°C, and the heating reflux time is 3-5 hours.

[0032] Furthermore, the preparation of N,N'-bisacryloylcysteamine (BAC) includes the following steps: adding a dichloromethane solution of methacryloyl chloride and a sodium hydroxide solution to a cysteamine hydrochloride solution and reacting for 2-5 hours; after the reaction is complete, allowing it to stand for a period of time, collecting the lower layer and drying it to obtain the product BAC.

[0033] Furthermore, the concentration of the cystamine hydrochloride solution is 18-25%, and it is prepared by dissolving cystamine hydrochloride in deionized water in an ice bath.

[0034] In a third aspect, the present invention provides a hydrogel formulation for encapsulating an antitumor drug, wherein the hydrogel formulation uses the hydrogel delivery system described in the first aspect as a drug carrier.

[0035] Preferably, the antitumor drug is a membrane-bound monomolecular micelle in which doxorubicin hydrochloride (DOX) and 1-methyl-tryptophan (1MT) are adsorbed onto dendritic macromolecular polyamide-amine (PAMAM) through a physical process, and the outer layer is coated with macrophage membrane.

[0036] While DOX kills cancer cells, 1MT regulates the tumor suppressor microenvironment. The hydrogel itself, as a drug carrier, does not harm the body, but the loaded drug has a dual therapeutic effect of chemotherapy and immunotherapy. The macrophage membrane coating prevents the drug from being easily cleared, making it easier to accumulate at the tumor site. Furthermore, as the hydrogel degrades, the internal drug is released, thereby improving drug utilization.

[0037] Furthermore, the preparation method of the encapsulated monomolecular micelles is as follows: 1-methyl-tryptophan solution, polyamide-amine solution and doxorubicin are mixed and stirred in the dark for 40-50 h. After stirring and reacting, the clear solution is retained and dialyzed with water. The dialyzed solution is dried to obtain P-DOX / 1MT powder. The P-DOX / 1MT is added to macrophage solution, sonicated and squeezed to obtain the encapsulated monomolecular micelles.

[0038] Furthermore, the preparation method of the hydrogel formulation is as follows: the above-mentioned precursor 1 and precursor 2 are added to the solution of coated monomolecular micelles and ultrasonically mixed to obtain the hydrogel formulation loaded with antitumor drugs.

[0039] Preferably, one way to use the hydrogel formulation containing antitumor drugs is as follows: after physically mixing the encapsulated monomolecular micelles and the precursor in physiological saline, the mixture is injected into the surgical resection site of the tumor using a syringe, thereby forming a hydrogel in situ at the injection site as a drug reservoir. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0041] Figure 1 The 1H NMR spectrum and infrared spectrum of the precursor described in Example 1;

[0042] in, Figure 1 A is dextran and precursor 1 1 H NMR spectrum;

[0043] Figure 1 B is the infrared spectrum of precursor 1;

[0044] Figure 1 C represents SBMA and precursor 2. 1 H NMR spectrum;

[0045] Figure 1 D is the infrared spectrum of precursor 2;

[0046] Figure 2 Infrared spectrum, scanning electron microscope image and rheological test of the hydrogel described in Example 1;

[0047] in, Figure 2 A is the infrared spectrum of the hydrogel;

[0048] Figure 2 B is a scanning electron microscope image of the hydrogel;

[0049] Figure 2 C represents the rheological test structure of the hydrogel;

[0050] Figure 3 The hemolysis rate of hydrogels formed by different concentrations of precursors in Example 1, the CCK-8 assay of hydrogel extract, the staining of live and dead cells (scale bar 20 μm), and the cell embedding assay (scale bar 20 μm) were performed.

[0051] in, Figure 3 A shows a photograph of the hydrogel hemolysis experiment and the hemolysis rate;

[0052] Figure 3 B represents the results of hydrogel biocompatibility testing using the CCK-8 assay;

[0053] Figure 3 C represents the results of fluorescence observation of staining between live and dead cells;

[0054] Figure 3 D represents the results of the cell embedding experiment;

[0055] Figure 4 The proteins adsorbed on the surface of the hydrogel tested in Example 1;

[0056] Figure 5 The images show the H&E images (scale bar 50 μm) of the hydrogel in vivo at different time points tested in Example 1, and the H&E images (scale bar 100 μm) of various major tissues in normal mice and mice four weeks after hydrogel implantation.

[0057] in, Figure 5 Image A shows the H&E staining of the hydrogel;

[0058] Figure 5 B shows H&E staining images of various tissue sections from mice;

[0059] Figure 6 The results of particle size detection, potential detection, and scanning electron microscope images of different monomolecular micelles in Example 2 are shown.

[0060] in, Figure 6 A represents the particle size detection result;

[0061] Figure 6 B represents the potential detection result;

[0062] Figure 6 C is a scanning electron microscope image;

[0063] Figure 7 These are SDS-PAGE images of different monomolecular micelles in Example 2;

[0064] Figure 8 This refers to the cytotoxicity of each formulation at different drug concentrations tested in Example 2;

[0065] in, Figure 8 The left side shows the cell survival rate of B16F10 under high dose;

[0066] Figure 8 The right side shows the cytotoxicity of different concentrations of Free DOX / 1MT, P-DOX / 1MT, and P-DOX / 1MT@MM;

[0067] Figure 9 The in vitro release curve of the drug tested in Example 2;

[0068] Figure 10 The results of the cytotoxicity test of the drug-loaded hydrogel tested in Example 2;

[0069] Figure 11 This is a confocal image (scale bar 50 μm) of the cells taking up DOX as tested in Example 2.

[0070] Figure 12 The average fluorescence intensity of DOX uptake by cells tested in Example 2 (n=3);

[0071] Figure 13 The results of flow cytometry analysis of B16F10 cells tested in Example 2 after treatment with different agents for 12 hours;

[0072] in, Figure 13 A represents the stream cytometry result;

[0073] Figure 13 B represents the statistical results of cell apoptosis;

[0074] Figure 14 The data in Example 2 show the exposure of CRT on the cell membrane surface after treatment with different formulations (scale bar 30 μm) and the release of HMGB1 (scale bar 100 μm).

[0075] in, Figure 14 A represents the exposure status of CRT on the cell membrane surface;

[0076] Figure 14 B represents the release status of HMGB1;

[0077] Figure 15 The level of ATP remaining in cells after treatment with different formulations, as tested in Example 2;

[0078] Figure 16 The distribution of DiR fluorescence in mice as tested in Example 2;

[0079] Figure 17 The distribution of DiR fluorescence tested in Example 2 in isolated mouse organs: heart, liver, spleen, lung, kidney, and tumor;

[0080] in, Figure 17 A represents the distribution of Free DiR in mouse organs;

[0081] Figure 17 B represents the distribution of DiR-Gel in mouse organs;

[0082] Figure 18 The average tumor volume and tumor photographs of mice after different treatments tested in Example 2 (n=3);

[0083] in, Figure 18 A represents the change in tumor volume in mice;

[0084] Figure 18 B represents the change in mouse body weight;

[0085] Figure 19 Images of the major organs of mice after different treatments tested in Example 2 (scale bar 100 μm).

[0086] Figure 20 Images of H&E staining of tumors in mice after different treatments, as tested in Example 2 (scale bar 100 μm).

[0087] Figure 21 Images of tumors stained with TUNEL in mice after different treatments, as tested in Example 2 (scale bar 50 μm). Detailed Implementation

[0088] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0089] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0090] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0091] Example 1: Preparation and performance characterization of hydrogels

[0092] I. Preparation of Hydrogels

[0093] 1. Preparation of precursor 1:

[0094] 1.5 g of dextran and 0.2 g of lithium chloride were dispersed in 20 mL of N,N-dimethylformamide (DMF) and heated at 110 °C until dissolved. After cooling to room temperature, the mixture was placed in an ice bath, and 1.61 mL of methacryloyl chloride and 2.3 mL of triethylamine (TEA) previously dissolved in 5 mL of DMF were added dropwise. After reacting for 24 h, the product was added dropwise to ice-cold methanol and filtered. The resulting precipitate was precursor 1 (Q1).

[0095] 2. Preparation of precursor 2:

[0096] (1) SBMA: Accurately weigh 17.2 g of methacryloyloxyethyl dimethylamine (DMAEMA) and place it in a 250 mL three-necked flask. Then add 40 mL of acetone and stir mechanically to mix. Accurately weigh 12.2 g of 1,3-propanesulfonyl lactone and dissolve it in 10 mL of acetone. Then add it dropwise to the three-necked flask and react at 55 °C for 4 h. Pay attention to reflux to prevent excessive volatilization of acetone. After the reaction is completed, a large amount of white crystals can be observed to precipitate in the three-necked flask. Filter the solution. Wash the product with acetone. Collect the monomer and place it in a glass dish. Dry it under vacuum for 24 h.

[0097] (2) BAC: Weigh 5.3 g of cystamine hydrochloride and add it to 25 mL of deionized water while stirring in an ice bath. Dissolve 4.86 mL of methacryloyl chloride in 10 mL of dichloromethane (DCM), and weigh 4 g of sodium hydroxide in 10 mL of deionized water. Then add all of these solutions dropwise to the cystamine hydrochloride solution and react for 3 h. After the reaction is complete, let it stand for 12 h, collect the lower layer, and evaporate it to dryness. After drying, dissolve the precipitate in a small amount of ethanol and add it dropwise to deionized water. After filtration, dry the resulting white precipitate under vacuum to obtain the product N,N'-bisacryloylcystamine (BAC).

[0098] (3) Precursor 2: Weigh 0.3 g of SBMA monomer, 100 mg of BAC, and 16 mg of ammonium persulfate (APS) and disperse them in 5 mL of 25% ethanol (EtOH) solution. Then add 11 μL of tetramethylethylenediamine (TMEDA) to the solution and stir. After the solution becomes clear and transparent, let it stand at room temperature for 24 h. The solution will turn into a white gel. Break it up and remove the excess ethanol solution with a pipette. Dissolve 0.43 g of dithiothreitol (DTT) in 5 mL of pH 8 PBS buffer solution and add it to the white gel block. Stir until it becomes a liquid solution. Adjust the solution to pH 3 with hydrochloric acid, dialyze, and freeze-dry to obtain precursor 2 (Q2).

[0099] The precursor structure was analyzed using proton nuclear magnetic resonance spectroscopy and infrared spectroscopy, and the results are as follows: Figure 1In the Q1 spectrum, in addition to the characteristic peaks of dextran, three new sets of peaks were observed: the peak with a chemical shift of 1.84 ppm was attributed to the hydrogen on the methyl group adjacent to the double bond, and the peaks at 6.16 ppm and 5.77 ppm were attributed to the hydrogen on the vinyl group, indicating that the double bond was successfully attached to the dextran backbone. As shown in the figure, in the Q2 spectrum, in addition to the characteristic peaks of SBMA, two new sets of peaks were observed: the single peak with a chemical shift of 2.61 ppm was attributed to the hydrogen on the methylene group in the (-CH2-SH) structure; the disappearance of the two single peaks with chemical shifts of 5.67 ppm and 6.06 ppm in SBMA, and the new chemical shifts at 1.79-3.12 ppm, are related to the polymerization of the double bond and BAC in the structure. These results indicate that the thiol group was successfully attached to the monomer SBMA.

[0100] like Figure 1 As shown in B, compared with the raw material dextran, precursor 1 has a growth rate of 1719 cm⁻¹. -1 v appeared C＝O The stretching vibration absorption peak is at 1619 cm⁻¹. -1 v appears at the location C＝C The stretching vibration absorption peak is at 950 cm⁻¹. -1 v appeared at the location C-H The stretching vibration absorption peak indicates the formation of a new ester bond, meaning precursor 1 was successfully synthesized. For example... Figure 1 As shown in C, in the Q2 spectrum, in addition to the characteristic peaks of SBMA, two new sets of peaks were added: a singlet with a chemical shift of 2.62, attributed to the hydrogen on the carbonyl carbon, and two singlets with chemical shifts of 0.98-1.09, attributed to the hydrogens of the two ethyl groups on the chain containing the thiol group. These results indicate that the thiol group was successfully attached to the monomer SBMA. Figure 1 As shown in D, precursor 2 is at 2550cm. -1 v appeared at the location S-H The absorption peak of stretching vibration indicates the successful synthesis of precursor 2.

[0101] 3. Preparation of hydrogels:

[0102] Different masses of precursor 1 and precursor 2 were weighed and dissolved thoroughly by sonication in different volumes of physiological saline, with a total volume of 1 mL. The precursor solutions were mixed in a small vial and rapidly pipetted to ensure homogeneity. The vial was repeatedly inverted until no liquid flow was observed on the vial wall, indicating that hydrogel formation had occurred. The time taken from the complete mixing of the precursor solutions to this point was recorded as the gelation time, and the results are shown in Table 1 below.

[0103] Table 1. Gelation time of hydrogels

[0104]

[0105] As shown in Table 1, the gelation time varied depending on the concentration of the gel precursor. When the precursor concentration was below a certain value, the mixed system could not form a hydrogel or could only form a partial hydrogel, indicating that a certain concentration of precursor was required for gelation. With increasing precursor concentration and changing the ratio of the two precursors, the viscosity of the solution increased continuously, and the gelation time shortened from several hours or even overnight to tens of minutes. This is because as the precursor concentration increases, the probability of collision between the two groups gradually increases, and the cross-linking reaction occurs faster. However, with a fixed solvent volume, if the precursor mass exceeds a certain level, it cannot be completely dissolved, and a stable and homogeneous system cannot be formed. Due to the different solubilities of precursor 1 and precursor 2, experiments determined that the solvent volume for dissolving precursor 1 was 0.3 mL, and the solvent volume for dissolving precursor 2 was 0.7 mL. Therefore, it is necessary to select appropriate precursor concentrations and ratios. The subsequent characterization of the hydrogel properties was performed using hydrogels prepared with the same precursor concentrations of 60, 90, and 120 mg (G60, G90, and G120).

[0106] II. Performance Characterization

[0107] 1. Infrared Spectroscopy

[0108] The hydrogel prepared in Example 1 was characterized by infrared spectroscopy. The specific steps were as follows: The hydrogel was first soaked in deionized water for 48 hours until it was fully swollen, then freeze-dried and ground into powder. An appropriate amount of sample powder was taken and characterized by Fourier transform infrared spectroscopy (FT-IR) in the range of 650-4000 cm⁻¹. -1 Scanning is performed between intervals.

[0109] The results are as follows Figure 2 As shown in Figure A, the characteristic absorption bands of the hydrogel are entirely composed of the absorption bands of Q1 and Q2, indicating that the two components of the precursor coexist in the gel.

[0110] 2. Observation using a scanning electron microscope

[0111] The microstructure of the hydrogel prepared in Example 1 was observed using a scanning electron microscope. The specific steps were as follows: The hydrogel, after being fully swollen in deionized water, was freeze-dried. The freeze-dried gel was completely immersed in liquid nitrogen for 3 minutes, then removed and broken into pieces. The cut pieces of gel were fixed onto a vertical cross-section sample stage using conductive adhesive and sputtered with gold. The pore structure and morphology of the gel cross-section were then observed under a microscope.

[0112] The results are as follows Figure 2As shown in Figure B, after freeze-drying, the water in the hydrogel sublimates, leaving behind a porous structure. These pores indicate the water content inside the hydrogel. As shown in the figure, the freeze-dried hydrogel has a relatively uniformly distributed pore structure, with pore diameters between 20-50 μm and very thin pore walls. The lower right corner shows a magnified view of this area. The pore structure of the freeze-dried hydrogel also indicates that the hydrogel molecular network contains a large amount of water and allows for its free diffusion.

[0113] 3. Rheometer analysis

[0114] The dynamic rheology of the hydrogel was characterized using a rheometer. One mL of the prepared hydrogel precursor mixture was rapidly injected into the center of the sample stage using a pipette. After wiping away any excess mixture with a paper towel, the test was conducted in frequency scanning mode under conditions of 0.1–100 Hz oscillation frequency, 0.1% constant shear strain, and a constant temperature of 37°C. The dynamic changes in storage modulus (G') and loss modulus (G”) were recorded.

[0115] The results are as follows Figure 2 As shown in Figure C, the storage modulus (G') of the hydrogels formed by the three different concentrations of precursors is greater than the loss modulus (G”) of viscous deformation and is in a relatively balanced state, indicating that the system successfully formed a hydrogel, exhibiting the behavior of an elastic solid. Among them, G' G120 At around 15000 Pa, it is much greater than G' at around 7500 Pa. G90 and G' at around 2500Pa G60 This indicates that the mechanical strength of the gel is positively correlated with the precursor concentration, and G120 has the best mechanical strength.

[0116] 4. Hemolysis test analysis

[0117] The biocompatibility of the hydrogel prepared in Example 1 was analyzed by a hemolysis experiment. The specific steps were as follows: First, fresh rabbit blood was centrifuged at 3000 rpm for 10 min to remove plasma and the white cells on the upper layer. The precipitated cells were then mixed with physiological saline, centrifuged again to remove the supernatant, and the process was repeated until the supernatant was colorless, thus obtaining red blood cells (RBCs). 1 mL of red blood cells was diluted 50 times with physiological saline to obtain a red blood cell suspension (2%, v / v). 0.5 mL of hydrogel prepared from different masses of precursors was immersed in 5 mL of physiological saline for 48 h to obtain a hydrogel extract. 0.6 mL of rabbit blood was diluted with 10 mL of physiological saline. 0.1 mL of diluted blood was added to 0.9 mL of the hydrogel extract. After incubation at 37°C for 4 h, the mixture was centrifuged at 2000 rpm for 5 min. The absorbance at 542 nm was measured using a UV spectrophotometer. The hemolysis rate was calculated using the following formula, with each sample group tested in triplicate.

[0118]

[0119] Among them, A sample A represents the absorbance of the experimental group. negative A represents the absorbance of the saline group. positive The absorbance of the pure water treatment group.

[0120] The results are as follows Figure 3 As shown in Figure A, the hemolysis rate of the hydrogel extracts formed from different concentrations of precursors was less than 5%, which meets the requirements for in vivo application. This indicates that the hydrogels also do not significantly interfere with erythrocytes and have good in vivo blood compatibility.

[0121] 5. CCK-8 Detection

[0122] The effect of the hydrogel extract prepared in Example 1 on the viability of B10F16 cells was assessed using the CCK-8 assay to evaluate its biocompatibility. The specific steps were as follows: First, 1 g of hydrogel was soaked in 10 mL of physiological saline for 48 hours to allow it to fully swell. After removing the hydrogel, the surface moisture was gently wiped off with filter paper, and then it was sterilized with ultraviolet light. The sterilized hydrogel was then immersed in 1640 medium and kept at 37°C for 48 hours. The supernatant was collected by centrifugation to obtain the hydrogel extract for cell culture. Pre-incubated and cultured B16F10 cells were seeded at a density of 8000 cells / well in 96-well plates and incubated overnight with normal medium to ensure full adhesion. Then, the normal medium was aspirated, and 100 μL of the hydrogel extract was added to each well for culture. After culturing for 24 h and 48 h respectively, 100 μL of CCK-8 cell staining working solution was added and the cells were incubated at 37 °C. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula.

[0123]

[0124] Where As is the absorbance of the experimental well, Ab is the absorbance of the blank well, and Ac is the absorbance of the control well.

[0125] The results are as follows Figure 3 As shown in Figure B, after 48 hours of culture, the cell survival rate was still above 90%, which proves that the hydrogel has very good cell compatibility.

[0126] 6. Fluorescence observation

[0127] The effect of the hydrogel extract prepared in Example 1 on the viability of B10F16 cells was observed using fluorescence to assess its biocompatibility. The specific steps were as follows: Pre-incubated and cultured B16F10 cells were seeded at a density of 50,000 cells / well in 24-well cell culture plates and incubated overnight in normal culture medium to ensure full adhesion. Then, the normal culture medium was aspirated, and the cells were cultured with the hydrogel extract, 1 mL per well. After 24 hours of culture, the supernatant was aspirated and washed three times with PBS. Following the kit instructions, Calcein-AM (a green fluorescence agent from the Calcein-AM / PI double staining kit) was added for staining. The 24-well plates were incubated at 37°C for 40 minutes, and then observed using an inverted fluorescence microscope.

[0128] The results are as follows Figure 3 As shown in Figure C, the number of cells increased after 48 hours of culture compared to 24 hours, demonstrating that the hydrogel has good biocompatibility.

[0129] 7. Cell embedding experiment

[0130] The effect of the hydrogel prepared in Example 1 on the viability of B10F16 cells was observed through a hydrogel cell embedding experiment to assess its biocompatibility. The specific steps were as follows: Pre-incubated and cultured B16F10 cells were resuspended in a well-mixed precursor solution at a cell concentration of 20,000 cells / mL. Then, 200 μL of the mixed precursor solution was added to each well of a 24-well cell culture plate and incubated at 37°C for 30 min to allow gelation. After gelation, 1 mL of 1640 medium was added to the top layer, and the plate was incubated at 37°C for further incubation. After 24 h and 48 h of incubation, the upper culture medium was aspirated and washed three times with PBS. Following the kit instructions, Calcein-AM / PI double staining kits were used to stain live cells with Calcein-AM, which provides green fluorescence. The 24-well plates were incubated at 37°C for 40 min and then observed using an inverted fluorescence microscope.

[0131] The results are as follows Figure 3 As shown in Figure D, the number of cells after 48 hours of culture was significantly higher than that after 24 hours of culture, indicating that the porous structure inside the hydrogel can maintain good cell growth and proliferation, and also proving the safety of the hydrogel.

[0132] 8. BCA method for determining protein adsorption

[0133] Quantitative detection of protein adsorption by the hydrogel prepared in Example 1 using the BCA method was performed as follows: A hydrogel was prepared in a 48-well cell culture plate, 100 μL per well. After gelation, 400 μL of deionized water was added to the upper layer to swell the hydrogel. After 48 h, the supernatant was discarded, and 400 μL of fresh fetal bovine serum (BSA) solution (1 mg / mL) prepared with PBS was added to each well. The plate was incubated at 37°C for 2 h. A blank polystyrene (TCPS) plate was used as a positive control. After 2 h, the upper layer solution was aspirated, and the plate was washed three times with PBS. Then, 400 μL of sodium dodecyl sulfate solution (SDS, 1 wt%) was added to each well, and the plate was soaked at room temperature for 1 h to elute the BSA adhering to the surface. Following the steps on the BCA assay kit, 20 μL of SDS solution was added to a 96-well cell culture plate, followed by 200 μL of BCA working solution. After incubation at 37°C for half an hour, the absorbance at 562 nm was measured using a microplate reader. The protein content in the corresponding eluent was then calculated by substituting the measured standard curve.

[0134] The results are as follows Figure 4 As shown, the number of proteins adsorbed on G60, G90, and G120 hydrogels is much smaller than that adsorbed on TCPS, indicating that the hydrogels have excellent anti-protein adsorption ability and avoid damage to proteins.

[0135] 9. In vivo degradation and biocompatibility experiments

[0136] The G120 hydrogel prepared in Example 1 was subjected to in vivo degradation and biocompatibility experiments. The specific steps are as follows: The precursors were dissolved in physiological saline and thoroughly mixed. After the precursor solutions were thoroughly mixed, 100 μL was injected subcutaneously into the back of C57BL / 6 mice using a syringe. Mice were sacrificed on days 0, 7, 14, 21, and 28. The skin on the back of the mice was cut open to observe the in vivo degradation of the gel. The skin at the site of hydrogel attachment, along with the heart, liver, spleen, lung, and kidney, was preserved in 4.0% paraformaldehyde solution, paraffin-embedded, sectioned, and stained with H&E to study the in vivo degradation and biocompatibility of the hydrogel.

[0137] The results are as follows Figure 5 As shown, where Figure 5Image A shows the H&E staining of the hydrogel. As can be seen, the hydrogel has a network structure. Cracks gradually appear inside starting on day 7, and large-scale fractures occur within the internal structure on day 21. By day 28, numerous fragments have appeared inside. These fragments from hydrogel degradation are phagocytosed by phagocytes, resulting in a macroscopic reduction in its volume. If a drug is loaded into the hydrogel, its degradation in vivo allows for its full release. The hydrogel injected in this example was G120, which may have a slower degradation rate due to a higher precursor concentration and denser cross-linking sites. Mice were sacrificed after day 28, and H&E staining of the major organs was performed for observation. Figure 5 B shows H&E staining images of various mouse tissue sections, all of which show no abnormalities. These results indicate that the G120 hydrogel prepared in Example 1 has good biocompatibility in vivo and can degrade slowly.

[0138] Example 2: Preparation and performance study of drug-loaded hydrogels

[0139] I. Preparation of drug-loaded hydrogels

[0140] 1. Preparation of PAMAM-based monomolecular micelles

[0141] The specific steps are as follows: Add 4 mg of doxorubicin hydrochloride (DOX·HCl) to 0.5 mL of pH 9 PBS buffer solution and stir overnight at room temperature in the dark. After stirring, centrifuge at 10,000 rpm for 30 minutes to obtain a precipitate. Wash the precipitate with a small amount of deionized water, and centrifuge again at 10,000 rpm for 10 minutes. Repeat this process three times to obtain water-insoluble doxorubicin (DOX). Dissolve 4 mg of 1MT in 8 mL of pH 7.4 PBS buffer solution by sonication, and then add different masses of 20% PAMAM solution. Add the previously obtained DOX precipitate to the solution and stir in the dark for 48 hours. After stirring, centrifuge at 4,000 rpm for 20 minutes, repeating four times to remove unreacted DOX. Transfer the supernatant from centrifugation to a dialysis bag with an MWCO of 3500, and dialyze in deionized water for 24 hours with stirring to remove free 1MT. Freeze-dry the solution to obtain P-DOX / 1MT powder for later use.

[0142] The drug loading and encapsulation efficiency of DOX were calculated using a UV spectrophotometer. The specific calculation formula is as follows:

[0143]

[0144]

[0145] Among them, W loaded For the quality of the encapsulated drug, W total W represents the total mass of the drug delivery system.added This indicates the total amount of drug added.

[0146] The results are shown in Table 2.

[0147] Table 2. Drug loading and encapsulation efficiency of DOX

[0148]

[0149] Therefore, in the subsequent examples, the preparation of P-DOX / 1MT involved adding 4 mg of a 20% PAMAM solution, at which point the concentration of DOX was 134.98 ± 2.76 μg / mL and the concentration of 1MT was 344.20 ± 11.58 μg / mL.

[0150] 2. Extraction of macrophage cell membranes

[0151] The specific steps are as follows: RAW264.7 cells were collected from the cell culture flask and resuspended in ice-cold Tris-magnesium buffer (TM buffer, pH 7.4, 0.01M Tris and 0.001M MgCl2), and 10% protease inhibitor was added. The cells were homogenized at 2000g and centrifuged at 4℃ for 10 min. The supernatant was collected and diluted 1×10⁻⁶ times. 5 The cell membrane was collected by centrifugation at 4°C for 1.5 h. The cell membrane was resuspended in cold physiological saline, and the protein content in the macrophage membrane was determined using a BCA protein assay kit. 1×10⁻⁶ g of purified protein was collected. 8 The content of macrophage cell membrane protein was 0.34 ± 0.0078 mg.

[0152] 3. Preparation of coated monomolecular micelles

[0153] The specific steps are as follows: Extract the 1×10 8 Macrophage membranes were resuspended in 1 mL of cold physiological saline, and 0.1 mg of P-DOX / 1MT was added. After being sonicated and mixed evenly, polycarbonate membranes with a pore size of 400 nm were extruded. The membranes were extruded back and forth seven times to obtain P-DOX / 1MT@MM.

[0154] 4. Preparation of drug-loaded hydrogels

[0155] The precursor 1 and precursor 2 powders prepared by the method in Example 1 were added to the P-DOX / 1MT@MM drug solution and stirred or sonicated to completely dissolve them.

[0156] II. Performance Characterization

[0157] 1. Detection of single-molecule micelle size and potential using a Malvern particle size analyzer

[0158] like Figure 6 As shown, Figure 6A represents the particle size distribution results. The drug-loaded P-DOX / 1MT particle size is larger than that of PAMAM, possibly due to the drug loading causing a more extended conformation of P-DOX / 1MT. The encapsulated P-DOX / 1MT@MM particle size is larger than that of unencapsulated P-DOX / 1MT, indicating successful macrophage membrane coating on its surface. The small particle size error range of P-DOX / 1MT@MM indicates higher stability, possibly because it was first subjected to membrane compression during preparation, and the negatively charged cell membrane after coating increases stability due to the mutual repulsion between charges. Figure 6 B represents the potential detection results. The potential of P-DOX / 1MT is reduced compared to PAMAM, possibly due to the adsorption of the drug causing some charge to be shielded. After the macrophage membrane is coated, the potential of P-DOX / 1MT@MM further decreases to -2.96±0.347mV, which also indicates successful cell membrane coating.

[0159] 2. Observe the morphology of single-molecule micelles using transmission electron microscopy.

[0160] The specific steps are as follows: Immerse the copper mesh into each drug-loaded solution with tweezers, and after the copper mesh is air-dried, observe the morphology of each drug-loaded preparation under accelerating voltage using a transmission electron microscope.

[0161] The results are as follows Figure 6 As shown, transmission electron microscopy images of PAMAM, P-DOX / 1MT, and P-DOX / 1MT@MM reveal that all three exhibit uniform spherical shapes and good monodispersity.

[0162] 3. Verify whether membrane proteins on the macrophage membrane are retained using SDS-PAGE gel electrophoresis.

[0163] Wash the collected cell membranes three times with pre-chilled PBS, place them in a homogenization tube, add two 3mm homogenization beads, and add 10 times the volume of lysis buffer (add protease inhibitor a few minutes before use). Set the homogenization program to homogenize; if a higher protein concentration is needed, the volume of lysis buffer can be reduced appropriately. Remove the homogenized tube and place it on ice for lysis for 30 minutes, shaking every 5 minutes to ensure complete tissue lysis. Centrifuge at 12000 rpm, 4°C for 10 minutes, and collect the supernatant, which is the total protein solution. Clean the glass plates, prepare the gel, and load the samples. Align the glass plates and place them in the gel casting machine, ensuring they are securely in place to prevent leakage. Prepare the separating gel according to experimental needs, add TEMED, and immediately mix before pouring. After 45 minutes, pour off the water on top of the gel and blot away any remaining water with absorbent paper. Prepare a 5% stacking gel, add TEMED, and immediately mix before pouring. Fill the remaining space with the stacking gel, insert the comb, add sufficient electrophoresis buffer, and then load the samples for electrophoresis. Add the sample to the electrophoresis wells and perform electrophoresis. Use a stacking gel of 75V and a separating gel of 120V. Stop electrophoresis when the bromophenol blue is 1cm from the bottom of the glass plate. Place the gel in a glass petri dish, add Coomassie Brilliant staining solution, and incubate at 70°C for 5 minutes. Then, stain on a shaker for 90 minutes. After recovering the Coomassie Brilliant staining solution, wash the gel with water, add Coomassie Brilliant destaining solution, and destain on a shaker overnight. Remove the destaining solution, wash the gel with water, and photograph the target band to obtain SDS-PAGE gel electrophoresis images.

[0164] The results are as follows Figure 7 As shown, P-DOX / 1MT does not contain any protein components. P-DOX / 1MT@MM has the same protein components as macrophages and macrophage membranes, but the protein bands are relatively weak, which proves the retention of proteins on the cell membrane and the successful encapsulation of the cell membrane.

[0165] 4. Anticancer activity of the formulation

[0166] The anticancer activity of different formulations was detected using a CCK-8 assay kit. The specific steps were as follows: B16F10 cells were seeded at a density of 8000 cells / well (containing 100 μL of cell culture medium) into 96-well cell culture plates and incubated overnight. After cell attachment, the supernatant was discarded, and the cells were washed once with PBS. Then, 100 μL of blank medium and different concentrations of Free DOX / 1MT, P-DOX / 1MT, and P-DOX / 1MT@MM medium were added for further incubation. After 24 hours, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of 1640 medium was added. Following the manufacturer's instructions, 10 μL of CCK-8 was added to each well. The cell culture plates were then sterilized with 75% alcohol and returned to the cell culture incubator for 1-2 hours of incubation. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated using the cell viability formula.

[0167] The results are as follows Figure 8 As shown, even at higher concentrations, 1MT had almost no effect on the survival rate of B16F10 cells. Free DOX / 1MT, P-DOX / 1MT, and P-DOX / 1MT@MM all exhibited concentration-dependent cytotoxicity. At the same concentration, Free DOX / 1MT was more toxic than the other two. This is because free DOX is a small molecule that diffuses across the cell membrane into the cell, rapidly entering the nucleus and quickly killing the cell. However, P-DOX / 1MT and P-DOX / 1MT@MM have large particle sizes, requiring endocytosis to enter the cell, followed by slow release of the loaded DOX into the cytoplasm. The DOX then diffuses freely into the nucleus, a process that takes time. Therefore, within 24 hours, the cytotoxicity of the drug-loaded formulations was lower than that of free DOX. At 15 μg / mL, P-DOX / 1MT killed approximately 50% of B16F10 cells, and P-DOX / 1MT@MM killed approximately 60% of B16F10 cells. This shows that P-DOX / 1MT@MM also has strong cell-killing ability at high DOX concentrations and has good application prospects.

[0168] 5. In vitro drug release experiment

[0169] For ease of storage, 5 mL of P-DOX / 1MT was lyophilized into powder. For this experiment, the lyophilized P-DOX / 1MT was dissolved in 6 mL of physiological saline, with 3 mL prepared as P-DOX / 1MT@MM according to the above method. The DOX content was determined to be 112.5 μg / mL based on the drug concentration. Drug-containing precursor 1 and drug-containing precursor 2 solutions were mixed to obtain the drug-loaded hydrogel. A 1 mL syringe was cut off to create a cylindrical container, and 0.5 mL of the mixed precursor solution was added. After gelation, the container was gently pushed out and placed directly into 6 mL of PBS buffer (pH 7.4) containing 0.2% Tween 80. The system was placed in a constant temperature water bath shaker (temperature maintained at 37°C) and incubated at 100 rpm, continuously protected from light. Each group was divided into triplicate. At the set time points, 1 mL of release medium was taken out and 1 mL of fresh release medium was added, and measurements were performed continuously for three days. The samples at each point were measured using a UV spectrophotometer, and the cumulative in vitro release curve of DOX was plotted.

[0170] The results are as follows Figure 9 As shown, the DOX released by Free DOX / 1MT-Gel, P-DOX / 1MT-Gel, and P-DOX / 1MT@MM-Gel were 93%, 79%, and 75%, respectively, verifying the feasibility of hydrogels as drug carriers. Free DOX / 1MT-Gel achieved near-complete release within 12 hours, demonstrating that hydrogel-loaded drugs have a certain sustained-release effect. Compared to Free DOX / 1MT-Gel, the release from P-DOX / 1MT-Gel and P-DOX / 1MT@MM-Gel groups was more gradual and stable, and showed an increasing trend over three days, proving that PAMAM-loaded DOX also has a sustained-release effect.

[0171] 6. Cytotoxicity test of drug-loaded hydrogels

[0172] Following the concentration gradient of the experimental protocol, 100 μL of drug-loaded hydrogels (0.1, 0.5, 1, 5, 10, and 15 μg / mL) of Free DOX / 1MT, P-DOX / 1MT, and P-DOX / 1MT@MM were prepared in advance. The prepared hydrogels were sterilized under UV light. B16F10 cells were seeded into 24-well plates at a density of 50,000 cells / well (containing 1 mL of 1640 cell culture medium) and incubated for 24 h. After cell attachment, the culture medium was discarded, and the cells were washed once with PBS. The sterilized drug-loaded hydrogel was added, followed by 1 mL of 1640 cell culture medium. After 24 h, the culture medium and drug-loaded hydrogel were discarded, and the cells were washed three times with PBS. The cells were then digested with 200 μL of trypsin, and after 40 s, twice the volume of culture medium was added to stop the digestion. The cells were collected by centrifugation at 1000 rpm for 5 min. Discard the supernatant, resuspend the cells in 100 μL of culture medium, and finally transfer the cells to a 96-well cell culture plate. Add 10 μL of CCK-8 to each well, then sterilize the 96-well cell culture plate with 75% alcohol and incubate it in the cell culture incubator for another 2 hours. Measure the absorbance of each well at 450 nm using a microplate reader, and calculate the cell viability using the formula.

[0173] The results are as follows Figure 10 As shown, the killing effect of all three on tumor cells is concentration-dependent. At lower drug concentrations, their killing ability on tumor cells is weak, with cell survival rates exceeding 80%. With increasing DOX content, FreeDOX / 1MT-Gel exhibits more significant toxicity to B16F10 cells. This is because free DOX is a small molecule that diffuses freely across the cell membrane into the cell, rapidly entering the nucleus and killing the cell. However, the released P-DOX / 1MT and P-DOX / 1MT@MM require endocytosis to enter the cell, followed by slow release of the loaded DOX within the cell. The DOX then diffuses freely into the nucleus, a process that takes time. The effect of P-DOX / 1MT@MM-Gel is not significantly different from that of P-DOX / 1MT-Gel because the hydrogel provides sustained drug release, releasing a small amount of drug in a short time, resulting in weaker cell-killing ability.

[0174] 6. Cell uptake experiment

[0175] (1) Fluorescence observation was performed using the following steps: After placing cell slides in 12-well plates, B16F10 cells were seeded at a density of 100,000 per well. After incubation in normal culture medium for 24 hours to allow adherence, the supernatant was discarded, and 1 mL of culture medium containing 5 μg / mL DOX was added, or 100 μL of drug-loaded hydrogel was added to 1 mL of fresh culture medium. Incubation continued in an incubator. After co-incubation with the formulation for a certain period, the supernatant was discarded, the cells were washed three times with PBS, mounted with an anti-fluorescence quencher, and observed and photographed using a confocal laser scanning microscope. DOX appeared green, and DAPI-stained cell nuclei appeared blue.

[0176] The results are as follows Figure 11 As shown, after 1h, 2h, and 4h of co-culture, the fluorescence intensity of free DOX entering the cell nucleus was similar, while the number of cells decreased, indicating that free DOX easily enters the cells and kills tumor cells. The green fluorescence intensity of the P-DOX / 1MT@MM group was higher than that of the P-DOX / 1MT group, indicating that cell membrane encapsulation enhances the micelles' ability to recognize tumor cells, making it easier for DOX to enter the cells. In the drug-loaded gel group, the intensity of DOX entering the cell nucleus was significantly weaker than that of the corresponding drug group during the same co-culture time, demonstrating that the hydrogel has a good sustained-release effect on the drug. Over time, the amount of green fluorescence entering the cell nucleus in the drug-loaded gel group increased, further demonstrating that the hydrogel has a good sustained-release effect on the drug.

[0177] (2) Quantitative analysis of cell uptake was performed using flow cytometry. The specific steps were as follows: B16F10 cells were seeded at a density of 300,000 per well in 6-well plates. 2 mL of 1640 medium was added and incubated for 24 h to allow adherence. The supernatant was discarded, and medium containing 2 mL of 5 μg / mL DOX was added, or 100 μL of drug-loaded hydrogel was added to 2 mL of fresh medium. Incubation continued in an incubator. Cells were digested with trypsin without EDTA at different time points and collected. The collected cells were transferred to EP tubes and centrifuged at 300g for 5 min. The supernatant was removed, and the cells were washed 2-3 times with cold PBS. A cell suspension was prepared with 300 μL of PBS solution, sieved, and placed in flow cytometry tubes for quantitative analysis, detecting the fluorescence intensity of the cells.

[0178] Figure 12 For quantitative analysis of flow cytometry results, at 4 h, the fluorescence intensity of P-DOX / 1MT@MM taken up by cells was approximately 1.28 times that of P-DOX / 1MT, indicating that the macrophage membrane can enhance the ability of drugs to enter tumor cells. Compared with the solution group, the fluorescence intensity of the drug-loaded gel group was weaker, further clarifying the sustained-release effect of the hydrogel on the drug.

[0179] 7. Apoptosis experiment

[0180] B16F10 cells were seeded at a density of 300,000 per well in 6-well plates and incubated with 2 mL of culture medium for 24 h to allow adherence. The supernatant was then discarded. 2 mL of culture medium containing 3 μg / mL DOX was added, or 100 μL of drug-loaded hydrogel was added to 2 mL of fresh culture medium, and the cells were incubated for another 12 h. After 12 h, the supernatant was collected. Adherent cells were digested with trypsin without EDTA and collected. The cells were then centrifuged at 300g for 5 min along with the supernatant to collect the cells. After collection, the cells were resuspended in cold PBS and washed, followed by centrifugation to collect the cells. This process was repeated 2-3 times. The cells were stained according to the instructions of the FITC-7AAD apoptosis detection kit. The stained cells were then transferred to flow cytometry tubes for detection and analysis using a flow cytometer.

[0181] Streaming results as follows Figure 13 As shown in Figure A. Figure 13 B represents the statistical results of apoptosis. The free DOX group had the highest apoptosis rate of 16.03%, significantly higher than other groups. The corresponding drug-loaded gel group also showed the best apoptosis rate of 8.88% among all gel groups. This is because free DOX can rapidly enter cells through free diffusion to exert its effect, while the formulation group... The P-DOX / 1MT@MM group was superior to the P-DOX / 1MT group, demonstrating that the macrophage membrane can enhance the recognition of the formulation by tumor cells, facilitating DOX entry into cells. However, due to the short co-incubation time between the formulation and cells, there was no significant difference between the groups. The apoptosis rate of the gel groups was lower than that of the corresponding solution groups because free drugs can more easily enter cells and exert their effects in a short time, further illustrating the sustained-release properties of hydrogels for drugs.

[0182] 8. Cell immunogenicity death experiment

[0183] (1) Detection of calreticulin: The specific steps are as follows: B16F10 cells were seeded in confocal dishes at a density of 100,000 per well. After incubation with 1 mL of normal culture medium for 24 h to allow them to adhere, the upper culture medium was discarded. Culture medium containing 1 μg / mL DOX was added, or drug-loaded hydrogel was added to fresh culture medium and incubated for another 24 h. After 24 h, the culture medium was discarded, and the cells were washed three times with PBS. Immunostaining fixative was applied at room temperature for 10 minutes, followed by three washes with PBS. Blocking solution of 5% BSA was applied at 37°C for 30 minutes, followed by three washes with PBS. Rabbit anti-mouse CRT primary antibody diluted 400 times was added and incubated overnight at 4°C. The next day, the cells were removed, washed three times with immunostaining detergent, and AF488-labeled goat anti-rabbit secondary antibody diluted 500 times was added and incubated at 37°C for 1 h. The cells were then washed three times with immunostaining detergent. Add DAPI to stain cell nuclei for 3 minutes, wash three times with PBS, add anti-fluorescence quencher, and observe and photograph using a confocal laser scanning microscope.

[0184] The results are as follows Figure 14 As shown in Figure A, the Free DOX / 1MT group exhibited the highest CRT level, and similarly, the Free DOX / 1MT-Gel group also showed the best CRT level. Notably, in both the solution and drug-loaded gel groups, cells treated with P-DOX / 1MT@MM showed higher CRT levels than those treated with P-DOX / 1MT, possibly because the macrophage membrane coating enhanced tumor cell recognition and uptake of the agent. The fluorescence intensity of the drug-loaded gel group was weaker than that of the corresponding solution group, indicating that the sustained-release effect of the hydrogel resulted in a smaller amount of drug released within the same time frame.

[0185] (2) HMGB1 protein detection: The specific steps are as follows: After placing cell smears in 12-well plates, B16F10 cells were seeded at a density of 100,000 per well. After incubation in normal culture medium for 24 hours to allow adhesion, the upper culture medium was discarded. Culture medium containing 1 μg / mL DOX was added, or drug-loaded hydrogel was added to fresh culture medium, and incubation continued for 24 hours. After 24 hours, the culture medium was discarded, and the cells were washed three times with PBS. Immunostaining fixative was applied at room temperature for 10 minutes, followed by three washes with PBS. 0.1% Triton 100 was added to perforate the cells for 10 minutes, followed by three washes with PBS. Blocking buffer (5% BSA) was applied at 37°C for 30 minutes, followed by three washes with PBS. Add 200-fold diluted rabbit anti-mouse HMGB1 primary antibody and incubate overnight at 4°C. The next day, remove the cells and wash three times with immunostaining detergent. Add 500-fold diluted AF488-labeled goat anti-rabbit secondary antibody and incubate at 37°C for 1 hour, then wash three times with immunostaining detergent. Add DAPI to stain cell nuclei for 3 minutes, wash three times with PBS, place a drop of anti-fluorescence quencher on a glass slide, and carefully use tweezers to remove the slide so that the side with cells comes into contact with the anti-fluorescence quencher. Observe and photograph using a confocal laser scanning microscope.

[0186] The results are as follows Figure 14 As shown in Figure B, almost no green fluorescence was observed in the Free DOX / 1MT group, indicating that HMGB1 had been released from the cells in large quantities. The green fluorescence present in the cell nucleus was less in the P-DOX / 1MT@MM group than in the P-DOX / 1MT group, meaning that the HMGB1 protein treated with P-DOX / 1MT@MM migrated more easily, indicating that P-DOX / 1MT@MM was more likely to induce ICD in tumor cells. The drug-loaded gel group also showed the same trend as the solution group, indicating that P-DOX / 1MT@MM retained its superior performance after being released from the hydrogel.

[0187] (3) ATP detection: The specific steps are as follows: B16F10 cells were seeded at a density of 300,000 per well in 6-well plates and incubated with 2 mL of normal culture medium for 24 h to allow them to adhere. The supernatant was then discarded. Culture medium containing 1 μg / mL DOX was added, or drug-loaded hydrogel was added to fresh culture medium, and the cells were incubated for another 24 h. After 24 h, the supernatant was discarded. The cells were lysed using the lysis buffer provided in the ATP detection kit according to the kit instructions to release the remaining ATP in the cells. The absorbance was measured using a microplate reader capable of detecting chemiluminescence. At the same time, a series of ATP solutions of different concentrations were prepared using the standards provided in the kit to establish a standard curve. The measured absorbance was then used to calculate the ATP content.

[0188] The results are as follows Figure 15As shown, after treatment with the Free DOX / 1MT group, the residual ATP in tumor cells was the lowest, 4.93 times that of the control group. This is because DOX can easily diffuse freely into cells, leading to ICD. The P-DOX / 1MT@MM group was the second highest, 2.41 times that of the control group, also significantly reducing ATP levels. After treatment with the formulation, the P-DOX / 1MT group had the highest ATP content. This is because P-DOX / 1MT@MM, due to its macrophage membrane coating, more easily induces ICD in tumor cells, allowing DOX to enter the cells more readily and exert its effects. The ATP content in the drug-loaded gel groups was higher than that in the corresponding formulation groups, which is related to the sustained-release effect of the hydrogel.

[0189] 9. Small animal in vivo imaging experiment

[0190] The specific steps are as follows: After digesting and washing healthy B16F10 cells, add an appropriate amount of sterile physiological saline to prepare a density of 1×10⁻⁶ cells. 7 A cell suspension of 10 cells / mL was stored on ice and inoculated into 6-8 week old female C57BL / 6 mice within 2 hours. Before inoculation, the cell suspension was thoroughly mixed by pipetting with a sterile pipette, and then 0.1 mL of the cell suspension was injected into the right axilla of the mouse using a 1 mL syringe. The successful establishment of the B16F10 tumor-bearing mouse model was indicated by the appearance of a visible tumor mass in the axilla. The transverse and longitudinal diameters of the tumor were measured using calipers, and then calculated using the formula V = (L × W) / (L × W). 2 The tumor volume is calculated using the formula (L / 2) (where L represents the longest tumor diameter and W represents the shortest tumor diameter). The tumor volume is considered to be approximately 60-80 mm. 3 The mice were randomly divided into two groups (n=3). Tumors were surgically removed, but because residual tumor tissue often remains after clinical surgery, 10% of the tumor was left in situ before the wound was sutured. Due to the weak fluorescence of DOX, to obtain stronger fluorescence, 0.1 mL solutions and drug-loaded gels of DiR were prepared at a mouse dosage of 200 μg / kg and injected into the tumor resection site using a syringe. The distribution of the drug in the mice was observed and photographed using a small animal in vivo imaging system at 24, 48, 72, and 120 hours. After 120 hours, the mice were sacrificed, and the tumors and major organs were dissected for fluorescence analysis.

[0191] The results are as follows Figure 16As shown, 24 hours after injection, the fluorescence intensity of DiR was mainly observed at the tumor site. However, the fluorescence intensity at the tumor site gradually weakened over time. After 48 hours, the DiR-Gel group still showed strong fluorescence at the tumor site, while the fluorescence intensity of the DiR group significantly decreased. By 120 hours, the fluorescence of the DiR group at the tumor site had almost disappeared, while the DiR-Gel group still showed strong fluorescence at the tumor site. This indicates that the injectable hydrogel designed in this embodiment, as a drug carrier, can form a drug reservoir at the injection site, thereby achieving long-term drug delivery. Figure 17 A shows the fluorescence distribution of the two DiR formulations in various ex vivo tissues after 120 hours. At this time point, the fluorescence intensity of the free DiR group at the tumor site was weaker than that of the DiR-Gel group, indicating that hydrogels can effectively prolong the drug's residence time at the tumor site, making the drug use long-lasting and targeted. Figure 17 B represents the quantitative analysis of fluorescence intensity in isolated organs. As shown in the figure, DiR exhibits the strongest fluorescence intensity in the liver, indicating that the drug is absorbed into the body and then metabolized and excreted by the liver. The fluorescence intensity of the DiR-Gel group in the tumor is higher than that of the DiR group, indicating that free DiR has been rapidly metabolized within 120 hours. However, due to the slow drug release in DiR-Gel, the metabolism takes longer, suggesting that the sustained-release effect of the hydrogel allows the drug to accumulate at the tumor site for a longer period, thereby enhancing the anti-tumor effect.

[0192] 10. Anti-recurrence experiment of hydrogel tumor recurrence after surgery

[0193] (1) Evaluation of postoperative anti-recurrence effect and safety, the specific steps are as follows: 6-8 week old female C57BL / 6 mice were taken, and 0.1 mL of B16F10 cells (1×10⁻⁶) were subcutaneously injected into the left axilla. 7 (number / mL), until the tumor volume reaches approximately 60-80 mm. 3 Mice were randomly divided into 8 groups (n=3). After establishing a tumor post-operative model, 0.1 mL of the preparation was injected into the surgical site, i.e., the cavity formed after tumor resection. Control group: saline; experimental group: Free DOX / 1MT, P-DOX / 1MT, P-DOX / 1MT@MM, Free DOX / 1MT-Gel, P-DOX / 1MT-Gel, and P-DOX / 1MT@MM-Gel. The dosage of DOX was 4 mg / kg. The body weight and tumor volume of mice were measured every two days. On day 13, mice were sacrificed, and the tumors were dissected and weighed. The dissected tumors were photographed and subjected to immunofluorescence staining and TUNEL staining. They were also stained with hematoxylin and eosin (H&E) for histological analysis, along with other mouse organs. Inguinal lymph nodes, tumors, and spleens were collected for further analysis.

[0194] The results are as follows Figure 18 As shown in the changes in tumor volume in mice, the antitumor effect of the hydrogel administration group was superior to that of the solution group. This is because free drugs are easily cleared, while hydrogels can provide a long-lasting, sustained release of the drug, prolonging the duration of action on the lesion and thus inhibiting tumor growth. The gel group showed the worst effect was the Free DOX / 1MT-Gel group, because free drugs released from the hydrogel are easily cleared by the body. The tumor volume in the P-DOX / 1MT-Gel group was 1.78 times that in the P-DOX / 1MT@MM-Gel group, indicating that macrophage membrane coating increased the drug's antitumor ability. Mouse body weight is an important indicator reflecting the systemic toxicity of the test drug. Figure 18 As shown in Figure B, all mice in the drug solution group showed a trend of weight gain, which may be due to the weight gain caused by the subsequent growth of the tumor. The weight gain of mice in the drug-loaded gel group was relatively slower than that in the solution group. This is because the tumor volume of these mice was relatively small, resulting in a smaller change in weight, which also demonstrates the safety of hydrogel administration and that it did not cause abnormal changes in mouse weight. H&E staining sections of the major organs of the mice after treatment are shown below. Figure 19 As shown, in the Free DOX / 1MT group and the Free DOX / 1MT-Gel group, the morphology of cell nuclei in the H&E-stained sections of the heart differed from that in the saline group. Cell nuclei showed shrinkage and reduced volume, and myocardial fiber disarray was observed. However, the morphology of other groups showed no significant changes compared to the saline group, indicating that free DOX caused some damage to the heart. No significant changes in cell membranes or nuclei were observed in the H&E-stained sections of the liver, spleen, lungs, and kidneys, indicating the absence of significant apoptosis or necrosis. This suggests that hydrogel administration has low in vivo toxicity and excellent biocompatibility and safety. Figure 20 These are the H&E staining results of mouse tumor tissues after different treatments. As shown in the figure, the tissue sections of the control group show large nuclei and tightly packed cells, indicating that the cells are in a rapid proliferation phase. After treatment with the drug solution and drug-loaded gel, the H&E tissue sections show nuclear condensation and karyolysis, suggesting extensive tumor cell necrosis. In the P-DOX / 1MT@MM-Gel group, large areas of nuclear condensation and karyolysis were observed, indicating that the macrophage membrane coating facilitates the entry of the drug into tumor cells and allows it to exert its effect. This also confirms that the hydrogel has an effect on improving the efficacy of drug sustained release. Figure 21As shown, the fluorescence of the drug solution group was weaker than that of the drug-loaded gel group, indicating no significant cell apoptosis. In the solution group, the fluorescence intensity of the P-DOX / 1MT@MM group was higher than that of P-DOX / 1MT, and in the gel group, the fluorescence intensity of P-DOX / 1MT@MM-Gel was significantly higher than that of the P-DOX / 1MT-Gel group, signifying substantial cell apoptosis in the tumor tissue. This is because hydrogels can prolong the drug's action time, and the encapsulated formulation is more likely to target tumor cells, exhibiting greater specificity and better anti-tumor therapeutic effects.

[0195] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hydrogel based on dextran and zwitterionic groups, characterized in that, The smallest repeating unit of the hydrogel has the structure shown in Equation I: Formula I.

2. The method for preparing the hydrogel based on dextran and zwitterionic groups as described in claim 1, characterized in that, It is formed by cross-linking of thiol groups and double bonds between precursor 1 and precursor 2; the structure of precursor 1 is shown in formula II below, and the structure of precursor 2 is shown in formula III below: Formula II Formula III; Precursor 1 and precursor 2 were dissolved separately in physiological saline, and then mixed and rapidly stirred until no liquid flowed to obtain the hydrogel.

3. The method for preparing hydrogels based on dextran and zwitterionic groups as described in claim 2, characterized in that, The preparation reaction of precursor 1 is shown in the following formula: The preparation method is as follows: Dextran and lithium chloride are dispersed and dissolved in N,N-dimethylformamide, and methacryloyl chloride and triethylamine are added dropwise; after the reaction is completed, the product is added dropwise to ice-cold methanol and filtered, and the resulting precipitate is precursor 1.

4. The method for preparing hydrogels based on dextran and zwitterionic groups as described in claim 3, characterized in that, The dextran and lithium chloride were added to DMF and dissolved by heating at a temperature of 105-115°C. After cooling, the mixture was placed in an ice bath and methacryloyl chloride and triethylamine were added dropwise, and the reaction was carried out for 20-28 hours.

5. The method for preparing hydrogels based on dextran and zwitterionic groups as described in claim 3, characterized in that, The mass ratio of dextran to lithium chloride is 1.3~1.7:0.1~0.

3.

6. The method for preparing hydrogels based on dextran and zwitterionic groups as described in claim 2, characterized in that, The preparation reaction of the precursor 2 is shown in the following formula: ； The preparation method is as follows: sulfonate betaine methacrylate monomer, N,N'-bisacrylcysteine ​​and ammonium persulfate are dispersed in an ethanol solution, and then tetramethylethylenediamine is added to the solution to obtain a gel; dithiothreitol is added and stirred until it becomes a liquid solution, then placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 70-80 hours, and then dried to obtain precursor 2.

7. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 6, characterized in that, The concentration of the ethanol solution is 20~30 v / v.

8. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 6, characterized in that, The dialysis medium is changed every 4-8 hours during the dialysis process. The medium is a deionized aqueous solution.

9. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 6, characterized in that, The synthesis method of the sulfonate betaine methacrylate is as follows: dissolve methacryloyloxyethyl dimethylamine in a solvent, add 1,3-propanesulfonyl lactone, heat under reflux for a period of time to carry out the reaction, and after the reaction is completed, filter and wash the product to obtain the product.

10. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 9, characterized in that, The mass ratio of the methacryloyloxyethyl dimethylamine to 1,3-propanesulfonyl lactone is 15~18:11~14.

11. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 9, characterized in that, The heating and reflux temperature is 50~60℃, and the heating and reflux time is 3~5h.

12. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 6, characterized in that, The preparation of N,N'-bisacryloylcysteamine includes the following steps: adding a dichloromethane solution of methacryloyl chloride and a sodium hydroxide solution to a cysteamine hydrochloride solution and reacting for 2-5 h; after the reaction is complete, allowing it to stand for a period of time, collecting the lower layer and drying it to obtain the final product.

13. The method for preparing a hydrogel based on dextran and zwitterionic groups as described in claim 12, characterized in that, The concentration of the cystamine hydrochloride solution is 18~25 w / v, and it is prepared by dissolving cystamine hydrochloride in deionized water in an ice bath.

14. A hydrogel formulation encapsulating an antitumor drug, characterized in that, In the hydrogel formulation, the hydrogel prepared by the method of claim 2 is used as a drug carrier.

15. The hydrogel formulation encapsulating an antitumor drug as described in claim 14, characterized in that, The antitumor drug is a membrane-bound monomolecular micelle in which doxorubicin hydrochloride and 1-methyl-tryptophan are adsorbed onto a dendritic macromolecular polyamide-amine through a physical process, and the outer layer is coated with a macrophage membrane.

16. The hydrogel formulation encapsulating an antitumor drug as described in claim 15, characterized in that, The preparation method of the membrane monomolecular micelles is as follows: 1-methyl-tryptophan solution, polyamide-amine solution and doxorubicin hydrochloride are mixed and stirred in the dark for 40-50 h. After stirring and reaction, the clear solution is retained and dialyzed with water. The dialyzed solution is dried to obtain P-DOX / 1MT powder. The P-DOX / 1MT is added to macrophage membrane solution, sonicated and squeezed to obtain the membrane monomolecular micelles.

17. The hydrogel formulation encapsulating an antitumor drug as described in claim 14, characterized in that, The preparation method of the hydrogel formulation is as follows: Precursor 1 and precursor 2 are added to a solution of encapsulated monomolecular micelles and ultrasonically mixed to obtain the hydrogel formulation encapsulating antitumor drugs.

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