Injectable hydrogel with deep tumor infiltration capacity as well as preparation method and anti-breast cancer application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to injectable hydrogel with deep tumor infiltration capacity as well as a preparation method and anti-breast cancer application of the injectable hydrogel. The preparation method comprises the following steps: reacting pectic acid with chitosan to form pectic acid acylated chitosan, loading emodin with the pectic acid acylated chitosan to form nanoparticles, adding the nanoparticles and hyaluronidase into a sodium alginate solution, and forming the injectable hydrogel in a bivalent cation cross-linking agent environment with physiological concentration. On one hand, the emodin is loaded through an acylated compound of pectic acid and chitosan, the solubility and pH responsiveness of the emodin are remarkably improved, so that the emodin can release drugs more efficiently in an acidic tumor microenvironment, and on the other hand, the permeability of the drugs in tumor tissues is enhanced through the degradation effect of hyaluronidase, so that the bioavailability of the emodin is improved. Finally, the prepared injectable hydrogel has a good treatment effect on the triple-negative breast cancer, and an efficient treatment strategy is provided for the triple-negative breast cancer and other types of tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an injectable hydrogel with deep tumor penetration ability, a preparation method thereof, and an application thereof in preventing breast cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors and seriously threatens women's life and health. Among them, triple-negative breast cancer (TNBC) is one of the most challenging breast cancer subtypes. It has the characteristics of strong heterogeneity, strong invasiveness, easy recurrence and metastasis, and lacks the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) on its surface. Due to the lack of these common therapeutic targets, traditional endocrine therapy and HER2 targeted therapy are less effective in TNBC patients. Therefore, the treatment strategy of TNBC is relatively limited, and the prognosis of patients is poor. At present, the main treatment for TNBC is chemotherapy, but the side effects and drug resistance of chemotherapeutic drugs are still major problems in clinical treatment. Therefore, it is urgent to develop a novel and effective TNBC treatment strategy to improve the treatment effect of patients and reduce the side effects of treatment.

[0003] Emodin is a natural anthraquinone compound widely distributed in plants such as rhubarb and knotweed. It has anti-inflammatory, antibacterial, antioxidant and anti-tumor biological activities. Studies in recent years have shown that emodin has shown good anti-tumor effects in the treatment of various cancers. However, the clinical application of emodin is limited by its poor water solubility, which leads to its low bioavailability and poor effect. In addition, emodin may cause toxicity to the liver, kidneys and reproductive system at high concentrations. Therefore, how to improve the water solubility of emodin, reduce its toxicity, and enhance its anti-tumor activity is a hot topic and challenge in current research. In recent years, researchers have tried to use drug delivery systems such as liposomes and micelles to load emodin to improve its therapeutic effect, but poor drug permeability is still a major bottleneck in tumor drug delivery.

[0004] Pectin acid is a natural polysaccharide, which is mainly found in the cell walls of fruits (such as apples, citrus, etc.) and plants, and plays an important role in the composition of peel and pulp. The main structural unit of pectin acid is galacturonic acid, and its molecular chain has abundant carboxyl and hydroxyl functional groups, and has strong hydration and solubility. Since pectin acid has the characteristics of natural origin, biocompatibility, low toxicity and biodegradability, it has important application value in the preparation of drug delivery systems. Chitosan is also a natural polysaccharide with good biodegradability, biocompatibility and low toxicity, and is widely used in drug delivery systems. Sodium alginate is another natural polysaccharide, which can quickly form a hydrogel with good biocompatibility and degradability under the action of divalent cations (such as calcium ions). Sodium alginate hydrogel can not only slowly release drugs, but also reduce the penetration of drugs into surrounding normal tissues and reduce systemic toxic side effects by controlling the local concentration of drugs, thereby improving the efficacy.

[0005] Hyaluronic acid is an important component of the extracellular matrix and is involved in the growth, adhesion, migration of tumor cells and the regulation of the tumor microenvironment. In tumors, the overexpression and accumulation of hyaluronic acid often form a high-viscosity matrix, which hinders the effective penetration of drugs. Hyaluronidase (HAase), an enzyme that can specifically degrade hyaluronic acid, has been shown to significantly improve the permeability of drugs. As an FDA-approved drug diffuser, HAase can degrade hyaluronic acid, enhance drug permeability, and improve drug targeting in tumor treatment, and has gradually become an emerging strategy for cancer treatment.

[0006] In summary, if a novel and effective TNBC treatment drug can be developed using pectin acid, chitosan, roentgenase and rhamnosine, it is expected to provide a new and efficient solution for the treatment of TNBC and have important application value. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a novel injectable hydrogel for deep tumor penetration, which, on the one hand, utilizes chitosan and pectic acid to form a stable acylated complex through electrostatic effects, thereby effectively loading the emodin drug, and significantly improving its solubility and pH responsiveness, so that it can release the drug more efficiently in the acidic tumor microenvironment. On the other hand, the deep penetration of the drug is promoted in the tumor microenvironment through the degradation of hyaluronidase, thereby enhancing its therapeutic effect. Based on this innovative strategy, the novel injectable hydrogel developed by the present invention can not only provide a new and efficient solution for the treatment of triple-negative breast cancer, but also is expected to be used in the treatment of other types of tumors, with broad application prospects.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for preparing an injectable hydrogel with deep tumor penetration ability, specifically: first, pectic acid and chitosan are reacted to form pectic acid acylated chitosan, then emo is loaded on the pectic acid acylated chitosan to form PE NPs nanoparticles, and then the PE NPs nanoparticles and hyaluronidase (HAase) are added to a sodium alginate hydrogel, and finally a PE NPs / HAase@ALG hydrogel is formed in a divalent cation environment at a physiological concentration.

[0010] Preferably, the method for preparing the injectable hydrogel specifically comprises the following steps:

[0011] S1. Add chitosan into 1% acetic acid aqueous solution, and adjust the pH of the solution after it is completely dissolved at room temperature;

[0012] S2, dissolving pectin acid in water to form a pectin acid solution, and adjusting the pH of the solution;

[0013] S3, adding the pectin acid solution to the chitosan solution, and then adding a cross-linking agent, and preparing a pectin acid acylated chitosan solution after stirring at room temperature;

[0014] S4, dissolving rhein in an organic solvent, and filtering to remove insoluble impurities;

[0015] S5, adding sodium tripolyphosphate into water and completely dissolving it at room temperature to obtain a sodium tripolyphosphate solution;

[0016] S6, adding the emodin solution and the sodium tripolyphosphate solution to the pectin acid-acylated chitosan solution at the same time, stirring at room temperature to ensure that the emodin and the pectin acid-acylated chitosan are completely mixed, and then subjecting the mixed solution to high-speed centrifugation, discarding the upper liquid, collecting the precipitate, and obtaining PENPs nanoparticle precipitation;

[0017] S7, resuspending the obtained PE NPs nanoparticle precipitate in water, and then dialyzing the resuspended solution to remove the organic solvent and unreacted monomers;

[0018] S8, adding a lyophilization protective agent to the dialyzed solution, and then drying to obtain PE NPs nanoparticle powder;

[0019] S9, adding PE NPs nanoparticles and hyaluronidase into the sodium alginate solution, and mixing them thoroughly to obtain a hydrogel precursor solution;

[0020] S10. Add the hydrogel precursor solution into a divalent cationic crosslinker solution of physiological concentration, and after standing, PE NPs / HAase@ALG hydrogel can be obtained.

[0021] On the one hand, the present invention loads emodin through the acylated complex of pectin acid and chitosan, significantly improving its solubility and pH responsiveness, so that it can release drugs more efficiently in the acidic tumor microenvironment, and on the other hand, enhances the permeability of drugs in tumor tissues through the degradation of hyaluronidase, and finally through the synergistic effect of the above two mechanisms, the prepared injectable hydrogel has a good therapeutic effect on triple-negative breast cancer. It can be seen that the new injectable hydrogel developed by the present invention provides an efficient treatment strategy for triple-negative breast cancer and other types of tumors, and has broad application prospects.

[0022] More preferably, in S1, the pH of the chitosan solution is adjusted to 5.0-6.0, and the concentration of chitosan is 1-10 mg / mL; in S2, the pH of the pectin solution is adjusted to 7.0-8.0, and the concentration of pectin acid is 1-10 mg / mL.

[0023] More preferably, in S3, the mass ratio of pectin acid to chitosan is 1:1 to 20:1; the cross-linking agent is one of EDC or NHS, and the concentration is 1 to 2 mg / mL.

[0024] More preferably, in S4, the concentration of the rhein solution is 1-20 mg / mL, and the organic solvent is one of DMSO and ethanol; in S5, the concentration of the sodium tripolyphosphate solution is 1-5 mg / mL.

[0025] More preferably, in S6, the mass ratio of chitosan to rhein is 20:1 to 100:1, and the mass ratio of sodium tripolyphosphate to chitosan is 1:2 to 1:10.

[0026] More preferably, in S8, the content of the added lyoprotectant is 2-5% by volume, and the lyoprotectant is one of mannitol, sucrose or trehalose; in S9, the mass ratio of hyaluronidase to rhein in PE NPs nanoparticles is 1:1-1:10.

[0027] More preferably, in S9, the concentration of the sodium alginate solution is 1 to 20 mg / mL; in S10, the divalent cation is Ca 2+ Mg 2+ One of them, the physiological concentration is 1~2mM.

[0028] More preferably, in S6, the stirring time at room temperature is 0.5 to 6 hours, and the rotation speed is 400 to 800 rpm; the centrifugation time of the high-speed centrifugation is 10 to 30 minutes, and the rotation speed is 10000 to 120000 rpm.

[0029] More preferably, in S7, the dialysis time is 20-30 hours to remove the organic solvent and unreacted monomers.

[0030] More preferably, in S10, after the hydrogel precursor solution is added to the divalent cationic crosslinking agent solution, the standing time is 1 to 6 hours.

[0031] The second aspect of the present invention provides an injectable hydrogel with deep tumor penetration capability prepared by the preparation method described in the first aspect.

[0032] The third aspect of the present invention provides use of the injectable hydrogel described in the second aspect in the preparation of an anti-triple-negative breast cancer drug.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The invention discloses a method for preparing an injectable hydrogel with deep tumor penetration ability, which comprises first reacting pectin acid with chitosan to form pectin acid acylated chitosan, then using pectin acid acylated chitosan to load emodin to form nanoparticles, and then adding the nanoparticles and hyaluronidase to a sodium alginate solution to form an injectable hydrogel under a divalent cation crosslinker environment of physiological concentration. The injectable hydrogel PENPs / HAase@ALG with deep tumor penetration ability obtained by the method of the invention, on the one hand, loads emodin through an acylated complex of pectin acid and chitosan, solves the problems of poor water solubility and low bioavailability of emodin, and significantly improves its pH responsiveness, so that it releases drugs more efficiently in an acidic tumor microenvironment; on the other hand, the degradation of hyaluronidase enhances the penetration of drugs into tumor tissues, and through the synergistic effect of the above two mechanisms, NPs / HAase@ALG has a good therapeutic effect on triple-negative breast cancer, and is expected to become an efficient anti-tumor treatment strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the particle size of PE NPs nanoparticles prepared in Example 3.

[0036] Figure 2 This is the zeta potential of the PE NPs nanoparticles prepared in Example 3.

[0037] Figure 3 This is the scanning electron microscope (SEM) image of the PE NPs / HAase@ALG hydrogel prepared in Example 3.

[0038] Figure 4 The inhibitory effect of PE NPs / HAase@ALG hydrogel on the proliferation of 4T1 breast cancer cells measured by in vitro cytotoxicity experiment.

[0039] Figure 5 The tumor morphology and size of tumor-bearing mice after treatment with PE NPs / HAase@ALG hydrogel in the in vivo pharmacodynamic experiment. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0042] Example 1

[0043] This embodiment provides a method for preparing an injectable hydrogel PENPs / HAase@ALG, comprising the following steps:

[0044] (1) Chitosan (1.0 g) was added to 100 mL of 1% acetic acid aqueous solution and stirred at room temperature using a magnetic stirrer until completely dissolved. Then, NaOH solution was added dropwise to the solution to adjust the pH to 5.5. The stirring process was continued for 30 min to ensure that the chitosan was completely dissolved.

[0045] (2) Pectin acid (5.0 g) was dissolved in 1000 mL of deionized water to form a pectin acid solution, and the pH of the solution was adjusted to 7.5.

[0046] (3) adding the pectin acid solution of step (2) to the chitosan solution of step (1), and then adding 1.5 mg / mL EDC as a cross-linking agent, and magnetically stirring at room temperature (speed 600 rpm, 30 min) to obtain a pectin acid acylated chitosan solution.

[0047] (4) Rhein (10 mg) was dissolved in 10 mL of DMSO solvent, and the solution was filtered using a 0.22 μm syringe filter to remove insoluble impurities.

[0048] (5) Sodium tripolyphosphate (0.5 g) was added to 100 mL of deionized water and stirred at room temperature using a magnetic stirrer until the mixture was completely dissolved to obtain a sodium tripolyphosphate solution.

[0049] (6) The emodin solution of step (4) and the sodium tripolyphosphate solution of step (5) are simultaneously added to the pectin acid-acylated chitosan solution of step (3), and magnetic stirring (400 rpm, 30 min) is performed at room temperature to ensure that the emodin and the pectin acid-acylated chitosan are completely mixed. Then, the mixed solution is subjected to high-speed centrifugation (speed 12000 rpm, centrifugation for 10 min), the upper layer of liquid is discarded, and the precipitate is collected to obtain PENPs nanoparticle precipitation.

[0050] (7) The obtained PE NPs nanoparticle precipitate was resuspended in deionized water, and the resuspension was transferred to a dialysis bag (3500 KD) and dialyzed for 24 h to remove the organic solvent and unreacted monomers.

[0051] (8) Adding lyophilization protective agent mannitol (3% by volume) to the dialyzed solution, followed by vacuum freeze drying to obtain PENPs nanoparticle powder.

[0052] (9) PE NPs (100 mg) and hyaluronidase (10 mg) were added to 2 mL of sodium alginate solution (sodium alginate concentration was 20 mg / mL) and stirred with a magnetic stirrer until completely mixed to obtain a hydrogel precursor solution.

[0053] (10) Add the hydrogel precursor solution to the Ca-containing 2+ ions (CaCl2, concentration 1mM) cross-linker solution, mixed evenly, and allowed to stand for 1h to promote the cross-linking reaction, and finally PE NPs / HAase@ALG injectable hydrogel was obtained.

[0054] Example 2

[0055] This embodiment provides a method for preparing an injectable hydrogel PENPs / HAase@ALG, comprising the following steps:

[0056] (1) Chitosan (0.5 g) was added to 100 mL of 1% acetic acid aqueous solution and stirred at room temperature using a magnetic stirrer until completely dissolved. Then, NaOH solution was added dropwise to the solution to adjust the pH to 6.0. The stirring process was continued for 30 min to ensure that the chitosan was completely dissolved.

[0057] (2) Pectin acid (8.0 g) was dissolved in 1000 mL of deionized water to form a pectin acid solution, and the pH of the solution was adjusted to 8.0.

[0058] (3) The pectin acid solution of step (2) was added to the chitosan solution of step (1), and 2.0 mg / mL of EDC was added as a cross-linking agent, and magnetic stirring was performed at room temperature (speed 600 rpm, 1 h) to obtain a pectin acid acylated chitosan solution.

[0059] (4) Rhein (20 mg) was dissolved in 10 mL of DMSO solvent, and the solution was filtered using a 0.22 μm syringe filter to remove insoluble impurities.

[0060] (5) Sodium tripolyphosphate (0.1 g) was added to 100 mL of deionized water and stirred at room temperature using a magnetic stirrer until the mixture was completely dissolved to obtain a sodium tripolyphosphate solution.

[0061] (6) The emodin solution of step (4) and the sodium tripolyphosphate solution of step (5) are simultaneously added to the pectin acid-acylated chitosan solution of step (3), and magnetic stirring (800 rpm, 20 min) is performed at room temperature to ensure that the emodin and the pectin acid-acylated chitosan are completely mixed. Then, the mixed solution is subjected to high-speed centrifugation (speed 10000 rpm, centrifugation for 20 min), the upper layer of liquid is discarded, and the precipitate is collected to obtain PENPs nanoparticle precipitation.

[0062] (7) The obtained PE NPs nanoparticle precipitate was resuspended in deionized water, and the resuspension was transferred to a dialysis bag (3500 KD) and dialyzed for 24 h to remove the organic solvent and unreacted monomers.

[0063] (8) Adding lyophilization protective agent mannitol (addition amount is 4% by volume) to the dialyzed solution, and then performing vacuum freeze drying to obtain PENPs nanoparticle powder.

[0064] (9) PE NPs (50 mg) and hyaluronidase (10 mg) were added to 2 mL of sodium alginate solution (sodium alginate concentration was 10 mg / mL) and stirred with a magnetic stirrer until completely mixed to obtain a hydrogel precursor solution.

[0065] (10) The hydrogel precursor solution was added to the Mg-containing 2+ After being mixed evenly, the mixture was allowed to stand for 2 h to promote the cross-linking reaction, and finally PE NPs / HAase@ALG injectable hydrogel was obtained.

[0066] As in Example 1, the prepared PE NPs nanoparticles also have a suitable size and good uniformity, and have good stability, and are not easy to aggregate or precipitate in the dispersion. At the same time, the prepared PE NPs / HAase@ALG hydrogel also has an irregular porous structure.

[0067] Example 3

[0068] This embodiment provides a method for preparing an injectable hydrogel PENPs / HAase@ALG, comprising the following steps:

[0069] (1) Chitosan (1.0 g) was added to 100 mL of 1% acetic acid aqueous solution and stirred at room temperature using a magnetic stirrer until completely dissolved. Then, NaOH solution was added dropwise to the solution to adjust the pH to 5.0. The stirring process was continued for 30 min to ensure that the chitosan was completely dissolved.

[0070] (2) Pectin acid (3.0 g) was dissolved in 1000 mL of deionized water to form a pectin acid solution, and the pH of the solution was adjusted to 7.0.

[0071] (3) adding the pectin acid solution of step (2) to the chitosan solution of step (1), and then adding 1.0 mg / mL EDC as a cross-linking agent, and magnetically stirring at room temperature (speed 600 rpm, 2 h) to obtain a pectin acid acylated chitosan solution.

[0072] (4) Rhein (40 mg) was dissolved in 10 mL of DMSO solvent, and the solution was filtered using a 0.22 μm syringe filter to remove insoluble impurities.

[0073] (5) Sodium tripolyphosphate (0.1 g) was added to 100 mL of deionized water and stirred at room temperature using a magnetic stirrer until the mixture was completely dissolved to obtain a sodium tripolyphosphate solution.

[0074] (6) The emodin solution of step (4) and the sodium tripolyphosphate solution of step (5) are simultaneously added to the pectin acid-acylated chitosan solution of step (3), and magnetic stirring (600 rpm, 30 min) is performed at room temperature to ensure that the emodin and the pectin acid-acylated chitosan are completely mixed. Then, the mixed solution is subjected to high-speed centrifugation (speed 10000 rpm, centrifugation for 10 min), the upper layer of liquid is discarded, and the precipitate is collected to obtain PENPs nanoparticle precipitation.

[0075] (7) The obtained PE NPs nanoparticle precipitate was resuspended in deionized water, and the resuspension was transferred to a dialysis bag (3500 KD) and dialyzed for 24 h to remove the organic solvent and unreacted monomers.

[0076] (8) Adding lyophilization protective agent mannitol (addition amount is 5% by volume) to the dialyzed solution, and then performing vacuum freeze drying to obtain PE NPs nanoparticle powder. Among them, the nanoparticles formed by coating emodin with chitosan that is not modified with pectin acid are recorded as CE NPs nanoparticle powder, that is, its preparation method only includes steps (1) and steps (4) to (8).

[0077] (9) PE NPs nanoparticles (100 mg) and hyaluronidase (5 mg) were added to 2 mL of sodium alginate solution (sodium alginate concentration was 5 mg / mL) and stirred with a magnetic stirrer until completely mixed to obtain a hydrogel precursor solution.

[0078] (10) Add the hydrogel precursor solution to the Ca-containing 2+ ions (CaCl2, concentration 1.8 mM) cross-linker solution, mixed evenly, and allowed to stand for 6 h to promote the cross-linking reaction, and finally obtained PE NPs / HAase@ALG injectable hydrogel.

[0079] Figure 1 The results show that the particle size of the prepared PE NPs nanoparticles is 118.91nm and the PDI is 0.217, indicating that PE NPs have a suitable size and good uniformity, and are suitable for biomedical applications such as drug delivery and targeted therapy.

[0080] Figure 2 The results show that the zeta potential of the prepared PENPs nanoparticles is 30.64 mV, indicating that PENPs have good stability and are not easy to aggregate or precipitate in the dispersion.

[0081] Figure 3 This is the scanning electron microscope (SEM) image of the prepared PENPs / HAase@ALG hydrogel. It can be seen from the image that PENPs / HAase@ALG hydrogel has an irregular porous structure.

[0082] Experimental Example 1: In vitro cytotoxicity test to test the inhibitory effect of the prepared PE NPs / HAase@ALG hydrogel on the proliferation of 4T1 breast cancer cells

[0083] The PE NPs / HAase@ALG hydrogel of Example 3 was used as an example for testing: the triple-negative breast cancer cell line 4T1 (1×10 4 Cells / well) were inoculated into 96-well plates, and different concentrations of EMO, HAase, CE NPs, PENPs, PE NPs+HAase (the mass ratio of PE NPs to HAase was 20:1), and PE NPs / HAase@ALG were added after 24 h of incubation. After 24 h of incubation, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for another 1 h. Finally, the absorbance at 450 nm was measured using an ELISA reader. Figure 4The cytotoxic effects of each group of drugs on 4T1 breast cancer cells were demonstrated. It can be seen that as the EMO concentration increased from 10 μM to 200 μM, the cell viability of each group gradually decreased, indicating that the higher the concentration, the stronger the inhibitory effect. In contrast, pectin acid acylation modification enhanced the inhibitory effect of chitosan-coated rhein nanoparticles on 4T1 cell proliferation, and HAase further enhanced this inhibitory effect. Therefore, the decrease in cell viability in the PE NPs / HAase@ALG group was relatively more significant, especially at high concentrations of 80 μM and above, the inhibitory effect was most significant, and the killing effect on 4T1 cells was significantly better than that of other groups. It is proved that the PE NPs / HAase@ALG hydrogel prepared by the present invention can significantly inhibit the proliferation of 4T1 breast cancer cells.

[0084] In addition, the PE NPs / HAase@ALG hydrogel prepared in Examples 1 and 2 also had a good inhibitory effect on the proliferation of 4T1 breast cancer cells.

[0085] Experimental Example 2: The prepared PE NPs / HAase@ALG hydrogel has a good therapeutic effect on triple-negative breast tumors tested by in vivo pharmacodynamics experiments

[0086] Taking the PE NPs / HAase@ALG hydrogel of Example 3 as an example, BALB / c mice were subcutaneously injected with 1×106 4T1 breast cancer cells to establish a breast cancer model. The tumor volume calculation formula is: tumor volume = 1 / 2×length×width 2 When the tumor volume reaches 100mm 3 At about 14 h, the mice were randomly divided into the following 6 groups: Control group (model control), EMO group, CE NPs group, PE NPs group, PE NPs+HAase group (the mass ratio of PE NPs to HAase was 20:1), and PE NPs / HAase@ALG group. Each group was administered once every three days, with 50 μL of drug (drug concentration of EMO 40 mg / kg and / or HAase 25 mg / kg) administered intratumorally each time, and the tumor volume and mouse body weight were measured every two days. The treatment cycle was 21 days. Figure 5 The therapeutic effects of each group of drugs on triple-negative breast tumors were demonstrated. It can be seen that the tumor volume of the PE NPs / HAase@ALG group was the smallest and the therapeutic effect was the most significant, far superior to the therapeutic effects of other single or partial components. It is proved that the PE NPs / HAase@ALG hydrogel prepared by the present invention can significantly inhibit the growth of 4T1 breast cancer tumors and is expected to become an efficient anti-tumor treatment strategy.

[0087] In addition, the PE NPs / HAase@ALG hydrogel prepared in Examples 1 and 2 also has a good therapeutic effect on triple-negative breast tumors.

[0088] In summary, the present invention loads emodin through an acylated complex of pectin acid and chitosan to solve the problems of poor water solubility and low bioavailability, while significantly improving pH responsiveness, more efficiently releasing drugs in the acidic tumor microenvironment, and enhancing drug penetration into tumor tissues through the degradation action of hyaluronidase. Ultimately, the obtained injectable hydrogel has a good therapeutic effect on triple-negative breast cancer, and is expected to become an efficient anti-tumor treatment strategy.

[0089] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for preparing an injectable hydrogel with deep tumor penetration ability, characterized in that: Pectin acid reacts with chitosan to form pectin acylated chitosan, and then pectin acylated chitosan is loaded with rhamnosin to form PE NPs nanoparticles. PE NPs nanoparticles and hyaluronidase are then added to sodium alginate hydrogel to finally form PENPs / HAase@ALG hydrogel under a physiological concentration of divalent cations.

2. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 1, characterized in that: The preparation method of the injectable hydrogel specifically comprises the following steps: S1. Add chitosan into 1% acetic acid aqueous solution, and adjust the pH of the solution after it is completely dissolved at room temperature; S2, dissolving pectin acid in water to form a pectin acid solution, and adjusting the pH of the solution; S3, adding the pectin acid solution to the chitosan solution, and then adding a cross-linking agent, and preparing a pectin acid acylated chitosan solution after stirring at room temperature; S4, dissolving rhein in an organic solvent, and filtering to remove insoluble impurities; S5, adding sodium tripolyphosphate into water and completely dissolving it at room temperature to obtain a sodium tripolyphosphate solution; S6, adding the emodin solution and the sodium tripolyphosphate solution to the pectin acid-acylated chitosan solution at the same time, stirring at room temperature to ensure that the emodin and the pectin acid-acylated chitosan are completely mixed, and then subjecting the mixed solution to high-speed centrifugation, discarding the upper liquid, collecting the precipitate, and obtaining PENPs nanoparticle precipitation; S7, resuspending the obtained PE NPs nanoparticle precipitate in water, and then dialyzing the resuspended solution to remove the organic solvent and unreacted monomers; S8, adding a lyophilization protective agent to the dialyzed solution, and then drying to obtain PE NPs nanoparticle powder; S9, adding PE NPs nanoparticles and hyaluronidase into the sodium alginate solution, and mixing them thoroughly to obtain a hydrogel precursor solution; S10. Add the hydrogel precursor solution into a divalent cationic crosslinker solution of physiological concentration, and after standing, PE NPs / HAase@ALG hydrogel can be obtained.

3. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S1, the pH of the chitosan solution is adjusted to 5.0-6.0, and the concentration of chitosan is 1-10 mg / mL; in S2, the pH of the pectin acid solution is adjusted to 7.0-8.0, and the concentration of pectin acid is 1-10 mg / mL.

4. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S3, the mass ratio of pectin acid to chitosan is 1:1 to 20:1; the cross-linking agent is one of EDC and NHS, and the concentration is 1 to 2 mg / mL.

5. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S4, the concentration of the rhein solution is 1-20 mg / mL, and the organic solvent is one of DMSO and ethanol; in S5, the concentration of the sodium tripolyphosphate solution is 1-5 mg / mL.

6. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S6, the mass ratio of chitosan to rhein is 20:1 to 100:1, and the mass ratio of sodium tripolyphosphate to chitosan is 1:2 to 1:

10.

7. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S8, the content of the added lyoprotectant is 2-5% by volume, and the lyoprotectant is one of mannitol, sucrose or trehalose; in S9, the mass ratio of hyaluronidase to rhein in PE NPs nanoparticles is 1:1-1:

10.

8. The method for preparing an injectable hydrogel with deep tumor penetration ability according to claim 2, characterized in that: In S9, the concentration of the sodium alginate solution is 1-20 mg / mL; in S10, the divalent cation is Ca 2+ Mg 2+ One of them, the physiological concentration is 1~2mM.

9. An injectable hydrogel having deep tumor penetration capability prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable hydrogel according to claim 9 in the preparation of an anti-triple-negative breast cancer drug.

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