An organic nanocomposite hydrogel, its preparation method and application

Abstract

This invention discloses an organic nanocomposite hydrogel, its preparation method, and its applications, belonging to the field of biomedical delivery technology. The organic nanocomposite hydrogel of this invention improves the penetration ability of polysaccharide-based nanoprodrugs into tumor tissues. The polysaccharide-based nanoprodrugs in the composite hydrogel can be protonated on the surface under the stimulation of the slightly acidic extracellular environment of tumor cells, promoting cellular uptake and responding to the low pH / high concentration of glutathione within tumor cells. This achieves intracellular aggregation and controlled release of the nanodrugs, enhancing the drug's retention time within cells and realizing the technical effect of combined chemotherapy in killing tumor cells. The fluorinated orthoester used in this invention accelerates the release of polysaccharide-based nanoprodrugs from the hydrogel and enhances tissue penetration, increasing the penetration of the polysaccharide-based nanoprodrugs into cells. Furthermore, the encapsulation of the drug-loaded complex with gellan gel enhances the sustained-release effect of the polysaccharide-based nanoprodrugs, thereby achieving efficient enrichment of the drug at the tumor site.

Description

Technical Field

[0001] This invention relates to the field of biomedical delivery technology, specifically to an organic nanocomposite hydrogel, its preparation method, and its application. Background Technology

[0002] With the rapid development of nanotechnology, nanomedicines have been widely researched and applied in the biomedical field, bringing new hope to cancer patients. Compared with drug molecules, nanoparticles can increase drug solubility, enhance drug stability, prolong metabolic time, and reduce the toxic side effects of chemotherapy drugs. However, recent studies have shown that the low tumor cell penetration ability and low accumulation at tumor lesion sites of nano-drug delivery systems severely limit their anti-tumor efficacy. Therefore, how to enhance the accumulation and penetration of nanomedicines at tumor sites and the effective release of chemotherapy drugs remains a serious challenge and an urgent problem to be solved.

[0003] Hydrogels are polymer networks formed by the physical or chemical cross-linking of individual polymer chains. The aqueous environment of hydrogels is more conducive to protecting cells and easily inactivated drugs, making them excellent drug carrier materials. The structure of hydrogels combines the properties of both solids and liquids. This structure allows for drug loading within pores and release through diffusion, permeation, and degradation. Simultaneously, the solid properties of hydrogels ensure drug release at local tumor lesions. Furthermore, the aqueous environment of hydrogels is extremely similar to human tissue; their soft, wettable surface and tissue affinity greatly reduce irritation to surrounding tissues, giving hydrogels excellent biocompatibility.

[0004] Incorporating nanomaterials into 3D hydrogel networks through physical or chemical covalent interactions is an effective method to avoid the burst release, rapid elimination, and poor bioadhesion of nanoparticles, as well as the shortcomings of hydrogels in biomedical applications, such as poor antitumor activity, weak bioimaging capabilities, and limited reactivity. In nanocomposite hydrogel systems, nanomaterials typically serve as the functional core, endowing hydrogels with various properties, such as tumor targeting and photothermal conversion. Simultaneously, hydrogels can effectively improve the retention of nanomaterials, giving nanoparticles good plasticity to adapt to various biomedical applications, such as drug delivery. However, the interaction between nanomaterials and hydrogels hinders the effective release of drugs embedded in the hydrogel network. Existing drug-loaded nanogels fail to respond well to the tumor microenvironment, affecting the penetration of nanomedicines into tumor cells and thus weakening the antitumor efficacy of nanomedicines. Summary of the Invention

[0005] This invention provides an organic nanocomposite hydrogel, its preparation method, and its application. It effectively solves the technical problems of existing nano-drug delivery systems, such as low tumor cell penetration and low tumor lesion enrichment capacity. The interaction between nanomaterials and hydrogels hinders the effective release of drugs embedded in the hydrogel network, fails to produce a good response to the tumor microenvironment, affects the penetration of nanomedicines into tumor cells, and thus weakens the anti-tumor efficacy of nanomedicines.

[0006] The first objective of this invention is to provide a method for preparing organic nanocomposite hydrogels, comprising the following steps:

[0007] Divalent platinum is reacted with hydrogen peroxide to obtain a tetravalent platinum compound with dihydroxy coordination. Using the tetravalent platinum compound with dihydroxy coordination and cantharidin compounds as raw materials, a solvent and triethylamine are added, and an esterification reaction is carried out at 50°C~60°C in a light-protected, protective atmosphere to obtain a tetravalent platinum cantharidin complex.

[0008] Using water as a solvent, the carboxyl groups of the tetravalent platinum cantharidin complex were activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain an activated tetravalent platinum cantharidin complex. This activated complex was then coupled with chitosan or its derivatives. The carboxyl groups of the activated tetravalent platinum cantharidin complex underwent an amide reaction with the amino groups of the chitosan compound to obtain a polysaccharide-based nanoprodrug.

[0009] The polysaccharide-based nanoprodrug was dispersed in fluorinated orthoester to improve the release effect of the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex. The drug-loaded complex was then encapsulated with gellan gel to obtain an organic nanocomposite hydrogel.

[0010] In a preferred embodiment, the molar ratio of the dihydroxy-coordinated tetravalent platinum compound to the cantharidin compound is 1:2~4.

[0011] In a preferred embodiment, the molar ratio of the tetravalent platinum cantharidin complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:2~2.5:2~2.5; and the mass ratio of the tetravalent platinum cantharidin complex to the chitosan compound is 1:1.48~2.8.

[0012] In a preferred embodiment, the ratio of the polysaccharide-based nanoprodrug to the fluorinated orthoester is 10 mg to 120 mg: 1 mL.

[0013] As a preferred embodiment, the preparation method of the polysaccharide-based nanoprodrug is as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are dissolved in water to obtain an activation solution; a tetravalent platinum cantharidin complex is dissolved in water, and then the activation solution is added. The mixture is stirred at room temperature in the dark, and then a carboxymethyl chitosan aqueous solution is added. The mixture is stirred at room temperature in the dark, and then dialyzed to obtain the polysaccharide-based nanoprodrug.

[0014] As a preferred embodiment, the encapsulation of the drug-loaded complex using gellan gum specifically involves: preparing a low-acyl gellan gum solution by heating and dissolving 1% to 2% by mass of low-acyl gellan gum, and then dispersing the drug-loaded complex in the low-acyl gellan gum.

[0015] In a preferred embodiment, the concentration of the drug-loaded complex in the organic nanocomposite hydrogel is 10 mg / mL to 70.5 mg / mL.

[0016] In a preferred embodiment, the cantharidin compound is cantharidin or norcantharidin.

[0017] The second objective of this invention is to provide an organic nanocomposite hydrogel prepared by the above-described preparation method.

[0018] A third objective of this invention is to provide an application of the above-mentioned organic nanocomposite hydrogel in the preparation of antitumor drugs.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention provides a method for preparing an organic nanocomposite hydrogel. First, divalent platinum is reacted with hydrogen peroxide to obtain a tetravalent platinum compound with dihydroxy coordination. The tetravalent platinum compound is then reacted with a cantharidin-like compound to obtain a tetravalent platinum cantharidin complex. The tetravalent platinum cantharidin complex is activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups. A chitosan-like compound is then added to initiate a coupling reaction. The activated carboxyl groups react with the amino groups in the chitosan compound to form an amide reaction, yielding a polysaccharide-based nanoprodrug. The formed amide bonds respond to the pH environment of the tumor site and break at the tumor site, thereby improving drug release. The polysaccharide-based nanoprodrug is dispersed in a fluorinated orthoester and freeze-dried to obtain a drug-loaded complex. The drug-loaded complex is then encapsulated using gellan gel to obtain an organic nanocomposite hydrogel that responds to the tumor microenvironment. The organic nanocomposite hydrogel prepared in this invention contains nanomedicines of suitable size, thereby improving the penetration ability of polysaccharide-based nanoprodrugs into tumor tissues. The polysaccharide-based nanoprodrugs in the organic nanocomposite hydrogel can undergo surface protonation under the stimulation of the slightly acidic extracellular environment of tumor cells, promoting cellular uptake and responding to the low pH / high concentration of glutathione within tumor cells. This achieves intracellular aggregation and controlled release of the polysaccharide-based nanoprodrugs, enhancing the drug's retention time within cells and realizing the technical effect of combined chemotherapy in killing tumor cells. The fluorinated orthoester used in this invention accelerates the release of polysaccharide-based nanoprodrugs from the organic nanocomposite hydrogel and enhances tissue penetration, increasing the penetration of polysaccharide-based nanoprodrugs into cells. Furthermore, the encapsulation of the drug-loaded complex with gellan gel enhances the sustained-release effect of the polysaccharide-based nanoprodrugs, thereby achieving efficient drug accumulation at the tumor site. Attached Figure Description

[0021] Figure 1 The tetravalent platinum cantharidin complex prepared in Example 1 of this invention 1 H NMR spectrum.

[0022] Figure 2 The polysaccharide-based nanoprodrug prepared in Example 1 of this invention 1 H NMR spectrum.

[0023] Figure 3 The average particle size distribution diagram is shown for the polysaccharide-based nanoprodrug prepared in Example 1 of this invention.

[0024] Figure 4 The figure shows the isoelectric point titration curve of the polysaccharide-based nanoprodrug prepared in Example 1 of this invention; the inset is a second derivative plot.

[0025] Figure 5 Potentiograms of the polysaccharide-based nanoprodrug prepared in Example 1 of this invention under different environments.

[0026] Figure 6 This is a graph showing the particle size variation of the polysaccharide-based nanoprodrug prepared in Example 1 of the present invention under different environments.

[0027] Figure 7 The images shown are scanning electron microscope (SEM) images of the organic nanocomposite hydrogel of Example 1 and the gellan gel of Comparative Example 2, wherein image A is the gellan gel of Comparative Example 2 and image B is the organic nanocomposite hydrogel of Example 1.

[0028] Figure 8 This is a dynamic frequency scan of the organic nanocomposite hydrogel of Example 1 of the present invention.

[0029] Figure 9 This refers to the shear-thinning property of the organic nanocomposite hydrogel in Example 1 of the present invention.

[0030] Figure 10 This demonstrates the injectability of the organic nanocomposite hydrogel in Example 1 of this invention.

[0031] Figure 11 The storage modulus and loss modulus of the organic nanocomposite hydrogel of Example 1 of the present invention are measured during strain scanning tests at γ = 0.1%~12%, and during cyclic strain scanning tests at γ = 1%~12%, where Figure A represents γ = 0.1%~12% and Figure B represents γ = 1%~12%.

[0032] Figure 12 This describes the macroscopic self-healing property of the organic nanocomposite hydrogel in Example 1 of the present invention.

[0033] Figure 13 This is a test diagram of the physical adhesion of the organic nanocomposite hydrogel of Example 1 of the present invention to different materials and tissues.

[0034] Figure 14 The degradation curves of the organic nanocomposite hydrogel of Example 1 of the present invention under different environments are shown.

[0035] Figure 15 This is an in vitro release curve of the polysaccharide-based nanoprodrug in the organic nanocomposite hydrogel of Example 1 of the present invention.

[0036] Figure 16 This is an in vitro release curve of cisplatin in the organic nanocomposite hydrogel of Example 1 of the present invention.

[0037] Figure 17 The image shows a hemolysis experiment of the organic nanocomposite hydrogel in Example 1 of this invention, where the saline group is the negative control and H2O is the positive control.

[0038] Figure 18This is a hemolysis rate diagram of the organic nanocomposite hydrogel of Example 1 of the present invention.

[0039] Figure 19 The figures show the qualitative uptake results of the organic nanocomposite hydrogel of Example 1 and the nanocomposite hydrogel of Comparative Example 1 by tumor cells HepG2 and H22, respectively.

[0040] Figure 20 The figures show the quantitative uptake results of the organic nanocomposite hydrogel of Example 1 and the nanocomposite hydrogel of Comparative Example 1 by tumor cells HepG2 and H22, respectively. Figure A represents HepG2 and Figure B represents H22.

[0041] Figure 21 The figures show the cytotoxicity of the organic nanocomposite hydrogel of Example 1, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide nanoprodrug of Example 1, and the gel of Comparative Example 2 to different cells. In the figures, A represents CT26, B represents A549, C represents H22, and D represents HepG2.

[0042] Figure 22 The images show the staining of live and dead cells after incubation with HepG2 or H22 tumor cells using the organic nanocomposite hydrogel of Example 1, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide nanoprodrug of Example 1, and the Gel of Comparative Example 2.

[0043] Figure 23 The figures show the apoptosis results of HepG2 or H22 induced by the organic nanocomposite hydrogel of Example 1, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide nanoprodrug of Example 1, and the Gel of Comparative Example 2, respectively.

[0044] Figure 24 This is a live mouse imaging image of the present invention, wherein ICG is a near-infrared dye - indocyanine green, ICG-NPs is an ICG-grafted polysaccharide-based nanoprodrug, ICG-NPs@Gel is a nanocomposite hydrogel of ICG-grafted polysaccharide-based nanoprodrug, and FOE / ICG-NPs@Gel is an organic nanocomposite hydrogel of ICG-grafted polysaccharide-based nanoprodrug.

[0045] Figure 25 This is a semi-quantitative statistical analysis chart of mouse in vivo imaging according to the present invention.

[0046] Figure 26 The images show the antitumor effects of the organic nanocomposite hydrogel of Example 1, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the gel of Comparative Example 2. In the images, Figure A is a line graph of tumor volume, Figure B is a graph of tumor volume change, Figure C is a graph of tumor weight change, and Figure D is a graph of mouse body weight change.

[0047] Figure 27 The images show hematoxylin and eosin staining of different tissues and tumors in mice using the organic nanocomposite hydrogel of Example 1, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the gel of Comparative Example 2.

[0048] In the above figures, Saline is physiological saline, Gel is the hydrogel of Comparative Example 2, NPs is the polysaccharide-based nanoprodrug of Example 1, NPs@Gel is the nanocomposite hydrogel of Comparative Example 1, and FOE / NPs@Gel is the organic nanocomposite hydrogel of Example 1. Detailed Implementation

[0049] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention is further described below with reference to specific embodiments. However, the embodiments are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0050] The background section of this invention mentions two main issues: First, existing nanoparticle-based drug delivery systems exhibit low tumor cell penetration and low accumulation capacity at tumor lesion sites. Second, for existing drug-loaded composite hydrogels, the interaction between the nanomaterial and the hydrogel hinders the effective release of drugs embedded in the hydrogel network, failing to provide a good response to the tumor microenvironment and affecting the penetration of nanomedicines into tumor cells, thereby weakening the anti-tumor efficacy of nanomedicines. To address these technical problems, this invention provides an organic nanocomposite hydrogel, its preparation method, and its applications.

[0051] The technical solution of the present invention will be described in detail below.

[0052] This invention first provides a method for preparing organic nanocomposite hydrogels, comprising the following steps:

[0053] S1. Divalent platinum is reacted with hydrogen peroxide to obtain a tetravalent platinum compound with dihydroxy coordination. Using the tetravalent platinum compound with dihydroxy coordination and cantharidin compounds as raw materials, a solvent and triethylamine are added, and an esterification reaction is carried out at 50°C~60°C in a light-protected, protective atmosphere to obtain a tetravalent platinum cantharidin complex.

[0054] It should be noted that since cantharidin compounds are small molecule anticancer drugs, they form complexes after esterification with tetravalent platinum compounds coordinated with dihydroxyl groups. These complexes can respond to the high concentration of glutathione environment at the tumor site and be reduced to release the drug at the tumor site, thereby playing a synergistic antitumor role.

[0055] S2, using water as a solvent, activates the carboxyl groups of the tetravalent platinum cantharidin complex with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain an activated tetravalent platinum cantharidin complex. This activated complex then undergoes a coupling reaction with a chitosan compound. The carboxyl groups of the activated tetravalent platinum cantharidin complex react with the amino groups of the chitosan compound to form an amide, yielding a polysaccharide-based nanoprodrug, denoted as NPs, with the following structure: ,in, This refers to chitosan-like compounds. This indicates a tetravalent platinum cantharidin complex.

[0056] The chitosan compounds used in this invention are selected from chitosan, carboxymethyl chitosan, hydroxypropyl chitosan, or chitosan sulfate, with carboxymethyl chitosan being preferred. Chitosan is a natural cationic polysaccharide with a large number of amino groups, exhibiting good biocompatibility. The amino groups on chitosan are beneficial for chemical modification or alteration, and can also adhere to the negatively charged cancer cell membranes, promoting cancer cell endocytosis. Therefore, it can be used to formulate nanomedicines, enhancing the delivery effect of antitumor drugs.

[0057] S3, the polysaccharide-based nanoprodrug is dispersed in fluorinated orthoester to improve the release effect of the polysaccharide-based nanoprodrug, and a drug-loaded complex is obtained, denoted as FOE / NPs. The drug-loaded complex is encapsulated with gellan gel to obtain an organic nanocomposite hydrogel, denoted as FOE / NPs@Gel.

[0058] Regarding the fluorinated orthoesters used in the above schemes, the structural formula of the fluorinated orthoesters used in this invention is as follows: .

[0059] Since the polysaccharide-based nanoprodrugs obtained from the reaction are in an aqueous solution state, their storage stability is poor. The polysaccharide-based nanoprodrugs are dispersed in a fluorinated orthoester and freeze-dried. After freeze-drying into a powder, they are difficult to redissolve in water. The polysaccharide-based nanoprodrugs are then emulsified and mixed with the fluorinated orthoesters, and freeze-dried to remove the water carried in the polysaccharide-based nanoprodrugs. This process improves the poor water solubility and difficulty in redissolving the polysaccharide-based nanoprodrugs, resulting in a drug-loaded complex. The dynamic changes of the fluorinated orthoesters in the hydrogel facilitate the release of the polysaccharide-based nanoprodrugs from the hydrogel. The fluorinated orthoesters also have excellent tissue permeability, thereby increasing the penetration of the polysaccharide-based nanoprodrugs into tumor tissue.

[0060] The gellan gum used in this invention has high biocompatibility and is injectable. As a drug carrier, it delivers polysaccharide-based nanoprodrugs to the tumor site via in-situ injection, forming a drug reservoir at the tumor site. This enhances the accumulation and retention capacity of the polysaccharide-based nanoprodrugs at the tumor site, prevents the polysaccharide-based nanoprodrugs from being cleared, thereby improving the utilization rate of the polysaccharide-based nanoprodrugs, enhancing the anti-tumor effect, and effectively avoiding the systemic toxicity caused by polysaccharide-based nanoprodrugs.

[0061] According to the above-mentioned technical solution provided by the present invention, the polysaccharide-based nanoprodrugs in the organic nanocomposite hydrogel can be protonated on the surface under the stimulation of the microacidic environment outside the tumor cells, promoting cell uptake and responding to the low pH / high concentration of glutathione inside the tumor cells, thereby realizing the intracellular aggregation and controlled release of NPs, enhancing the drug's residence time in the cells, and achieving the technical effect of killing tumor cells in combination with chemotherapy. Fluoroformate accelerates the release of polysaccharide-based nanoprodrugs from the organic nanocomposite hydrogel and enhances their tissue penetration ability, increasing the penetration of polysaccharide-based nanoprodrugs into tumor cells. Furthermore, the encapsulation of the drug-loaded complex with gellan gel enhances the sustained-release effect of the polysaccharide-based nanoprodrugs, thereby achieving efficient enrichment of the drug at the tumor site.

[0062] In a preferred embodiment, the molar ratio of the dihydroxy-coordinated tetravalent platinum compound to the cantharidin compound is 1:2 to 4. When the molar amount of the cantharidin compound is less than 2 (as defined herein), the dihydroxy-coordinated tetravalent platinum compound may not fully bind to the cantharidin compound, resulting in some platinum centers remaining unoccupied and reducing product yield. When the molar amount of the cantharidin compound is greater than 4 (as defined herein), excess cantharidin compound is difficult to completely remove through subsequent purification steps, potentially leading to residual free cantharidin impurities in the product. The theoretical reaction molar ratio of the dihydroxy-coordinated tetravalent platinum compound to the cantharidin compound is 1:2, with the platinum center binding to two cantharidin molecules. A molar ratio of 1:3 can shift the reaction equilibrium towards the product, ensuring high conversion rate, while also facilitating the removal of small amounts of unreacted cantharidin through purification, resulting in a final product with high purity and a well-defined structure.

[0063] To achieve effective activation of the carboxyl group in the tetravalent platinum cantharidin complex, the molar ratio of the tetravalent platinum cantharidin complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:2~2.5:2~2.5. Insufficient amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide reduce the carboxyl group activation efficiency. Insufficiently activated carboxyl groups may lead to a decrease in the coupling rate between the drug molecule and the polysaccharide, reducing the drug loading. Inhomogeneous formation of drug-carrier bonds, such as amide bonds, results in unstable release rates. Excessive amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide can trigger side reactions, affecting drug stability and release controllability.

[0064] To further enhance drug release, the mass ratio of the tetravalent platinum cantharidin complex to the chitosan compound is 1:1.48–2.8. The carboxyl group of the tetravalent platinum cantharidin complex reacts with the amino group of the chitosan compound to form an amide bond. This amide bond responds to the pH environment of the tumor site and breaks at the tumor site, thereby improving drug release. If the amount of chitosan compound used is less than the specified 1.48, sufficient amide bonds cannot be generated, reducing the amount of amide bonds formed and decreasing the pH-responsive release efficiency. This affects the drug release effect in the tumor microenvironment and reduces the efficacy of the drug against the tumor. Furthermore, the limited carrier function of chitosan can lead to premature drug leakage or release at non-target sites. Excessive free tetravalent platinum complexes may degrade or be cleared due to ineffective encapsulation, reducing tumor accumulation. Unbound complexes may be directly exposed to normal tissues, increasing systemic toxicity. If the amount of chitosan compounds used exceeds the limit of 2.8, excessive chitosan will lead to insufficient drug content per unit carrier. An overly dense chitosan network will hinder drug release, increase formulation volume or viscosity, affect in vivo delivery, increase injection difficulty, or cause local retention. Excessive use of chitosan compounds coating the drug surface can actually impair drug release, thus significantly affecting the drug's efficacy. In the experiments of this invention, a better drug release effect was achieved when the mass ratio of tetravalent platinum cantharidin complex to chitosan compound was 20.1 mg:53.7 mg, i.e., 1:2.67.

[0065] To improve the permeability of the polysaccharide-based nanoprodrug to tumor tissue, the ratio of the polysaccharide-based nanoprodrug to fluorinated orthoester is 10 mg to 120 mg: 1 mL. If the mass ratio of the polysaccharide-based nanoprodrug is less than the specified 10 mg, i.e., the amount of fluorinated orthoester is too high, the drug loading is low, and excessive fluorinated orthoester added to gellan gum makes it difficult to form a gel, resulting in unstable gel properties. If the mass ratio of the polysaccharide-based nanoprodrug is greater than the specified 120 mg, i.e., the amount of fluorinated orthoester is too low, the drug release from gellan gum will be limited, and a small amount of fluorinated orthoester will not be enough to increase the drug's permeability to tissue. During the experimental investigation, this invention found that dispersing 60 mg of the polysaccharide-based nanoprodrug in 1 mL of fluorinated orthoester can achieve a better permeation effect of the polysaccharide-based nanoprodrug to tumor tissue.

[0066] To further enhance the therapeutic effect of polysaccharide-based nanoprodrugs in organic nanocomposite hydrogels on tumors, the concentration of the drug-loaded complex in the organic nanocomposite hydrogel is between 10 mg / mL and 70.5 mg / mL. When the drug concentration is below 10 mg / mL, the effective dose of the drug is insufficient, which will lead to a significant reduction in anti-tumor efficacy and failure to achieve the expected therapeutic effect. When the drug concentration exceeds 70.5 mg / mL, systemic toxicity may occur due to drug overdose, causing irreversible damage to normal tissues and organs, and may also increase the risk of adverse reactions.

[0067] It should be noted that the cantharidin compounds used in this invention are cantharidin or norcantharidin; the gellan gum is a low-acyl gellan gum with a mass fraction of 1% to 2%.

[0068] The technical effects of the present invention will be described below through specific embodiments and comparative examples.

[0069] Example 1

[0070] A method for preparing an organic nanocomposite hydrogel includes the following steps:

[0071] S1. 200 mg (0.67 mmol) of cisplatin was dispersed in 12 mL of deionized water. 13 mL of 30 wt% hydrogen peroxide was added dropwise through a constant-pressure dropping funnel. The mixture was stirred at 55 °C in the dark for 5 h. After the reaction, the solution was concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow tetravalent cisplatin powder, i.e., DHP. 0.1 g (0.3 mmol) of DHP and 0.15 g (0.89 mmol) of norcantharidin (DMC) were dissolved in 2 mL of anhydrous N,N-dimethylformamide. Then, anhydrous triethylamine was added dropwise. The mixture was stirred at 60 °C in a dark nitrogen atmosphere for 24 h. After the reaction, the mixture was precipitated with ice-cold anhydrous diethyl ether, washed with acetone, filtered, and finally dried in a vacuum drying oven to obtain a pale yellow powder, i.e., a tetravalent platinum cantharidin complex.

[0072] S2, 20.1 mg of tetravalent platinum cantharidin complex was dissolved in 2 mL of deionized water, and then 8.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14.4 mg of N-hydroxysuccinimide dissolved in 1 mL of deionized water were added to activate the tetravalent platinum cantharidin complex. After stirring in the dark at room temperature for 5 h, 5 mL of carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued in the dark at room temperature for 24 h. After dialyzing, polysaccharide-based nanoprodrugs were obtained, denoted as NPs.

[0073] S3, 60 mg of the polysaccharide-based nanoprodrug is dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex, denoted as FOE / NPs. A gellan gel solution is prepared by dissolving 1.5% by mass of low-acyl gellan gel in an oil bath at 80°C. The drug-loaded complex is then dispersed in 1.5% by mass of low-acyl gellan gel and encapsulated with the low-acyl gellan gel to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, denoted as FOE / NPs@Gel.

[0074] Example 2

[0075] A method for preparing an organic nanocomposite hydrogel includes the following steps:

[0076] S1. 200 mg (0.67 mmol) of cisplatin was dispersed in 12 mL of deionized water. 13 mL of 30 wt% hydrogen peroxide was added dropwise through a constant-pressure dropping funnel. The mixture was stirred at 55 °C in the dark for 5 h. After the reaction, the solution was concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow powder, i.e., tetravalent cisplatin. 0.1 g (0.3 mmol) of tetravalent cisplatin and 0.15 g (0.89 mmol) of norcantharidin were dissolved in 2 mL of anhydrous N,N-dimethylformamide. Then, anhydrous triethylamine was added dropwise. The mixture was stirred and reacted at 60 °C in a dark nitrogen atmosphere for 24 h. After the reaction, the mixture was precipitated with ice-cold anhydrous diethyl ether, washed with acetone, filtered, and finally dried in a vacuum drying oven to obtain a pale yellow powder, i.e., a tetravalent platinum cantharidin complex.

[0077] S2, 20.1 mg of tetravalent platinum cantharidin complex was dissolved in 2 mL of deionized water, and then 8.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14.4 mg of N-hydroxysuccinimide dissolved in 1 mL of deionized water were added to activate the tetravalent platinum cantharidin complex. After stirring in the dark at room temperature for 5 h, 5 mL of carboxymethyl chitosan aqueous solution with a concentration of 6 mg / mL was added, and stirring was continued in the dark at room temperature for 24 h. After dialyzing, polysaccharide-based nanoprodrugs were obtained, denoted as NPs.

[0078] S3, 60 mg of the polysaccharide-based nanoprodrug is dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex, denoted as FOE / NPs. A gellan gel solution is prepared by dissolving 1.5% by mass of low-acyl gellan gel in an oil bath at 80°C. The drug-loaded complex is then dispersed in 1.5% by mass of low-acyl gellan gel and encapsulated with the low-acyl gellan gel to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, denoted as FOE / NPs@Gel.

[0079] Example 3

[0080] A method for preparing an organic nanocomposite hydrogel includes the following steps:

[0081] S1. 200 mg (0.67 mmol) of cisplatin was dispersed in 12 mL of deionized water. 13 mL of 30 wt% hydrogen peroxide was added dropwise through a constant-pressure dropping funnel. The mixture was stirred at 55 °C in the dark for 5 h. After the reaction, the solution was concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow powder, i.e., tetravalent cisplatin. 0.1 g (0.3 mmol) of tetravalent cisplatin and 0.15 g (0.89 mmol) of norcantharidin were dissolved in 2 mL of anhydrous N,N-dimethylformamide. Then, anhydrous triethylamine was added dropwise. The mixture was stirred and reacted at 60 °C in a dark nitrogen atmosphere for 24 h. After the reaction, the mixture was precipitated with ice-cold anhydrous diethyl ether, washed with acetone, filtered, and finally dried in a vacuum drying oven to obtain a pale yellow powder, i.e., a tetravalent platinum cantharidin complex.

[0082] S2, 20.1 mg of tetravalent platinum cantharidin complex was dissolved in 2 mL of deionized water, and then 8.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14.4 mg of N-hydroxysuccinimide dissolved in 1 mL of deionized water were added to activate the tetravalent platinum cantharidin complex. After stirring in the dark at room temperature for 5 h, 5 mL of carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued in the dark at room temperature for 24 h. After dialyzing, polysaccharide-based nanoprodrugs were obtained, denoted as NPs.

[0083] S3, 40 mg of the polysaccharide-based nanoprodrug is dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex, denoted as FOE / NPs. A gellan gel solution is prepared by dissolving 1.5% by mass of low-acyl gellan gel in an oil bath at 80°C. The drug-loaded complex is then dispersed in 1.5% by mass of low-acyl gellan gel and encapsulated with the low-acyl gellan gel to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 11.4 mg / mL, denoted as FOE / NPs@Gel.

[0084] Example 4

[0085] A method for preparing an organic nanocomposite hydrogel includes the following steps:

[0086] S1. 200 mg (0.67 mmol) of cisplatin was dispersed in 12 mL of deionized water. 13 mL of 30 wt% hydrogen peroxide was added dropwise through a constant-pressure dropping funnel. The mixture was stirred at 55 °C in the dark for 5 h. After the reaction, the solution was concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow powder, i.e., tetravalent cisplatin. 0.1 g (0.3 mmol) of tetravalent cisplatin and 0.15 g (0.89 mmol) of norcantharidin were dissolved in 2 mL of anhydrous N,N-dimethylformamide. Then, anhydrous triethylamine was added dropwise. The mixture was stirred and reacted at 60 °C in a dark nitrogen atmosphere for 24 h. After the reaction, the mixture was precipitated with ice-cold anhydrous diethyl ether, washed with acetone, filtered, and finally dried in a vacuum drying oven to obtain a pale yellow powder, i.e., a tetravalent platinum cantharidin complex.

[0087] S2, 20.1 mg of tetravalent platinum cantharidin complex was dissolved in 2 mL of deionized water, and then 8.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14.4 mg of N-hydroxysuccinimide dissolved in 1 mL of deionized water were added to activate the tetravalent platinum cantharidin complex. After stirring in the dark at room temperature for 5 h, 5 mL of carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued in the dark at room temperature for 24 h. After dialyzing, polysaccharide-based nanoprodrugs were obtained, denoted as NPs.

[0088] S3, 60 mg of the polysaccharide-based nanoprodrug is dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex, denoted as FOE / NPs. A gellan gel solution is prepared by dissolving 1% by mass of low-acyl gellan gel in an oil bath at 80°C. The drug-loaded complex is then dispersed in 1% by mass of low-acyl gellan gel and encapsulated with the low-acyl gellan gel to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, denoted as FOE / NPs@Gel.

[0089] To further demonstrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0090] Comparative Example 1

[0091] A method for preparing a nanocomposite hydrogel includes the following steps:

[0092] S1. 200 mg (0.67 mmol) of cisplatin was dispersed in 12 mL of deionized water. 13 mL of 30 wt% hydrogen peroxide was added dropwise through a constant-pressure dropping funnel. The mixture was stirred at 55 °C in the dark for 5 h. After the reaction, the solution was concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow tetravalent cisplatin powder, i.e., DHP. 0.1 g (0.3 mmol) of DHP and 0.15 g (0.89 mmol) of norcantharidin (DMC) were dissolved in 2 mL of anhydrous N,N-dimethylformamide. Then, anhydrous triethylamine was added dropwise. The mixture was stirred at 60 °C in a dark nitrogen atmosphere for 24 h. After the reaction, the mixture was precipitated with ice-cold anhydrous diethyl ether, washed with acetone, filtered, and finally dried in a vacuum drying oven to obtain a pale yellow powder, i.e., a tetravalent platinum cantharidin complex.

[0093] S2, 20.1 mg of tetravalent platinum cantharidin complex was dissolved in 2 mL of deionized water, and then 8.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 14.4 mg of N-hydroxysuccinimide dissolved in 1 mL of deionized water were added to activate the tetravalent platinum cantharidin complex. After stirring in the dark at room temperature for 5 h, 5 mL of carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued in the dark at room temperature for 24 h. After dialyzing, polysaccharide-based nanoprodrugs were obtained, denoted as NPs.

[0094] S3, a gellan gum solution was prepared by heating and dissolving 1.5% low-acyl gellan gum in an oil bath at 80°C. Then, 60 mg of the polysaccharide-based nanoprodrug was dispersed in the 1.5% low-acyl gellan gum solution. The polysaccharide-based nanoprodrug was encapsulated in the low-acyl gellan gum to obtain a nanocomposite hydrogel with a polysaccharide-based nanoprodrug concentration of 17 mg / mL, denoted as NPs@Gel.

[0095] Comparative Example 2

[0096] A method for preparing a hydrogel includes the following steps:

[0097] A gellan gum solution was prepared by heating and dissolving a 1.5% (w / w) low-acyl gellan gum in an oil bath at 80°C. After cooling, the solution solidified into a hydrogel, denoted as Gel.

[0098] The structure of the synthesized polysaccharide-based nanoprodrug was verified by proton nuclear magnetic resonance spectroscopy, such as... Figure 1 and Figure 2 As shown, the synthesized tetravalent platinum cantharidin complex and NPs have accurate structures. The particle size of the NPs at 37℃ was determined using a dynamic light scattering particle size analyzer (DLS), and the results are as follows. Figure 3As shown, the particle size of the NPs is approximately 134 nm, and scanning electron microscopy reveals that the NPs are regularly shaped spheres. To determine the isoelectric point of the NPs, potentiometric titration was used, and the results are as follows. Figure 4 As shown, the isoelectric point of the NPs is approximately pH=5. The potential changes of the NPs were also measured at different pH values, namely pH=7.4, pH=6.8, and pH=5.0. Figure 5 As shown, the particle size changes under different environments (pH=7.4, pH=6.8, pH=5.0, and pH=5.0 / GSH) are as follows. Figure 6 As shown, the surface potential of NPs increases as the pH value decreases, which is conducive to the uptake of NPs by tumor cells. The particle size can remain stable at pH=7.4 and pH=6.8, but in the environment of pH=5.0 in tumor cells, due to reaching the isoelectric point, aggregation and precipitation occur, and then it is gradually degraded by glutathione (GSH).

[0099] A 1.5% (w / w) low-acyl gellan gum was dissolved in an 80°C oil bath. After gelation, the gel was frozen and fractured. Simultaneously, FOE / NPs@Gel was prepared and frozen and fractured. The morphology of the two hydrogels was observed using scanning electron microscopy. Figure 7 As shown, by Figure 7 As shown in Figure A, gellan gum possesses abundant pores, enabling it to load and release drugs. Furthermore, after loading FOE / NPs, as... Figure 7 As shown in Figure B, the pores of gellan gum are filled with FOE / NPs, and the porous structure is reduced or even disappears, thus proving that FOE / NPs are successfully loaded into gellan gum.

[0100] Hydrogel rheological property testing: The rheological properties of FOE / NPs@Gel prepared in Example 1 of this invention were analyzed using a Kinexus Lab rotational rheometer. First, FOE / NPs@Gel was subjected to a frequency scan of 0.15Hz to 10Hz at a constant temperature of 37℃ and a constant stress of 1%, to detect its storage modulus and loss modulus, and its stability at different frequencies was also tested. Figure 8 As shown. Then for 0.1s... -1 ~100 s -1 The viscosity of FOE / NPs@Gel was measured at different shear rates, such as... Figure 9 As shown. Finally, maintaining a constant frequency of 1Hz, strain scans of 0.1% to 12% were performed on FOE / NPs@Gel to determine its linear viscoelastic region. Subsequently, strain scans were performed alternately with high strain of 12% and low strain of 1% to detect the trends of storage modulus and loss modulus of FOE / NPs@Gel as a function of strain. Figure 11As shown, its injectability was tested macroscopically, such as Figure 10 As shown, the undried FOE / NPs@Gel solution was placed in a 1mL syringe. After the FOE / NPs@Gel gelled, it was observed that it could be expelled through a 25G needle.

[0101] The organic nanocomposite hydrogel of this invention was stained with ICG and Rhodamine B, respectively, and each was cut in half. Then, two pieces of FOE / NPs@Gel of different colors were attached along the cut surface and placed in a mold. After 6 hours at room temperature, photographs were taken to record whether the FOE / NPs@Gel could self-heal. The results are as follows: Figure 12 As shown.

[0102] from Figure 8 It can be seen that at different frequencies, the storage modulus of FOE / NPs@Gel is always much higher than the loss modulus, indicating that the organic nanocomposite hydrogel is always in a stable state with high elasticity. Figure 9 It is known that the viscosity of organic nanocomposite hydrogels decreases rapidly with increasing shear rate, exhibiting typical shear-thinning behavior. For example... Figure 11 As shown in Figures A and B, amplitude-strain scanning was performed to determine the linear viscoelastic region of the hydrogel. As the strain gradually increased, the storage modulus of the hydrogel gradually decreased, while the loss modulus gradually increased. When the strain was greater than 12%, the storage modulus was less than the loss modulus, indicating that the hydrogel transitioned from a gel state to a sol state. Subsequently, cyclic strain scanning was performed with a low strain of 1% and a high strain of 12%. When the strain was lower, the storage modulus was greater than the loss modulus, indicating a gel state; when the strain was higher, the storage modulus was less than the loss modulus, indicating a sol state. When the strain returned to 1%, the modulus returned to its initial value, indicating a return to a gel state. From a macroscopic perspective, as... Figure 10 and Figure 12 As shown, this further verifies that the hydrogel is injectable through a 25G needle and exhibits self-healing behavior.

[0103] The organic nanocomposite hydrogel was attached to different materials to evaluate its adhesion ability to different materials, such as... Figure 13 As shown, the organic nanocomposite hydrogel exhibits strong adhesion to various materials, such as polypropylene plastics, metals, glass, and nitrile rubber. Furthermore, the tissue adhesion properties of FOE / NPs@Gel were investigated. After folding, stretching, and repeated torsion, FOE / NPs@Gel remained adhered to pig skin, pig muscle, and pig liver tissue, without separating from these tissues, maintaining strong adhesion.

[0104] Phosphate buffer solutions with pH values ​​of 7.4 and 6.8 were prepared to simulate the degradation of FOE / NPs@Gel under physiological conditions and in the tumor microenvironment in vitro. Figure 14 As shown. Under pH=7.4 and pH=6.8 conditions, in the first 3 hours, FOE / NPs@Gel swelled due to water absorption, increasing its mass. Subsequently, FOE / NPs@Gel stopped swelling and began to degrade. The degradation rate of FOE / NPs@Gel was relatively slow, but the addition of fluorinated orthoesters increased its degradation rate, which was more conducive to the release of NPs.

[0105] This invention investigates the release of NPs in the FOE / NPs@Gel of Comparative Example 1 and Example 1, such as... Figure 15 As shown, NPs can be released from the hydrogel, and the release amount of FOE / NPs@Gel is greater than that of NPs@Gel. This invention further tested whether the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1 could effectively release NPs of suitable particle size. The results are as follows... Figure 7 Figure B in the figure demonstrates the presence of NPs with the expected particle size on the surface of the organic nanocomposite hydrogel after 48 h, and NPs of the corresponding size were detected in the degradation solution by DLS, thus proving that NPs can be effectively released from the organic nanocomposite hydrogel. Further investigation was conducted on the cisplatin drug release from the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1, and the results are as follows. Figure 16 At pH 7.4 and pH 6.8, cisplatin release was low, while at pH 5.0 / GSH, 77% of the total drug was released. Upon stimulation at pH 5.0 / GSH, tetravalent cisplatin was reduced to divalent cisplatin, disrupting the NP structure and subsequently releasing cisplatin. These results indicate that the presence of fluorinated orthoesters accelerates the effective escape of NPs from the organic nanocomposite hydrogel in nanoparticle form. The escaped NPs respond to the pH 5.0 / GSH environment, achieving precise intracellular drug release in tumor cells.

[0106] The biocompatibility of the organic nanocomposite hydrogel prepared in this invention was tested using a hemolysis experiment, such as... Figure 17 and Figure 18 It can be seen that there is no significant difference between using different concentrations of FOE / NPs@Gel and the negative control group. The hemolytic activity of all experimental groups is less than 5%, indicating that the organic nanocomposite hydrogel prepared in this invention has no obvious hemolytic activity, high blood compatibility, and excellent biosafety.

[0107] Cell uptake assay: HepG2 and H22 tumor cells were seeded into 6-well plates. NPs were first labeled with fluorescein isothiocyanate (FITC). After the tumor cells adhered, 200 μL of NPs@Gel (Comparative Example 1) and FOE / NPs@Gel (Example 1) were added to each well. The NP concentration was 63.5 μM, and the gel concentration was 72 mg / mL. Co-culturing continued for 4 h and 8 h. A portion of the cells were fixed with paraformaldehyde for 10 min, stained with the fluorescent dye 4',6-diamidinyl-2-phenylindole (DAPI) for 3 min, washed three times with fresh PBS, and observed and photographed using a confocal laser scanning microscope. Figure 19 As shown. Another portion was digested with trypsin, centrifuged at 1000 rpm for 10 min, dispersed in 0.5 mL PBS, and the uptake content of tumor cells after incubation with HepG2 or H22 cells for 4 h using flow cytometry was measured for Comparative Example 1 (NPs@Gel) and Example 1 (FOE / NPs@Gel). The quantitative cell uptake results are shown below. Figure 20 As shown, the uptake of both types of tumor cells over 4 hours was higher in FOE / NPs@Gel than in NPs@Gel. The confocal qualitative uptake results are as follows. Figure 19 As shown, obvious green fluorescence appeared in tumor cells, indicating that NPs were internalized by cells. At the same time, the green fluorescence intensity of the NPs@Gel group was significantly weaker than that of the FOE / NPs@Gel group. Since FOE / NPs@Gel contains fluorinated orthoesters, which encapsulate NPs, the escape of fluorinated orthoesters accelerated the escape of NPs from FOE / NPs@Gel, allowing more NPs to be released and taken up by tumor cells. After 8 hours, the green fluorescence intensity in tumor cells further increased, and clustered fluorescence was produced.

[0108] Cytotoxicity assay: Four different tumor cell lines, HepG2, H22, CT26, and A549, were selected and co-incubated for 48 h with FOE / NPs@Gel from Example 1, NPs from Example 1, NPs@Gel from Comparative Example 1, and blank hydrogel from Comparative Example 2, respectively. Figure 21 As shown, the blank hydrogel in Comparative Example 2 exhibited higher cell survival rates, indicating that the hydrogel without NP loading has good biocompatibility. In contrast to the NPs@Gel in Comparative Example 1, the FOE / NPs@Gel in Example 1 showed lower cell survival rates, and FOE / NPs@Gel also exhibited higher cytotoxicity. This suggests that the escape of fluorinated orthoesters from the hydrogel accelerated the escape of NPs from the composite hydrogel, allowing more NPs to be taken up by tumor cells, thus resulting in higher cytotoxicity.

[0109] Double staining of live and dead cells: HepG2 and H22 tumor cells were seeded into 24-well plates at 40,000 cells / well. Blank hydrogel (Comparative Example 2), NPs (Example 1), NPs@Gel (Comparative Example 1), and FOE / NPs@Gel (Example 1) were added. The NPs concentration was 63.5 μM, and the gel concentration was 72 mg / mL. The cells were co-cultured with HepG2 or H22 cells for 48 h, respectively. Staining was performed according to the Calcein / PI cell viability and cytotoxicity assay kit, followed by observation under an inverted fluorescence microscope. Results are as follows: Figure 22 As shown, FOE / NPs@Gel exhibited higher cytotoxicity than other groups, demonstrating significant cytotoxicity against tumor cells.

[0110] Apoptosis detection: HepG2 and H22 cells were seeded into 6-well plates. When the cell density reached 50,000 cells / well, Gel from Comparative Example 2, NPs from Example 1, NPs@Gel from Comparative Example 1, and FOE / NPs@Gel from Example 1 were added, respectively. The concentration of NPs was 63.5 μM, and the concentration of Gel was 72 mg / mL. Cells were co-cultured with HepG2 or H22 for 48 h, respectively. Afterward, each well was washed with PBS and transferred to centrifuge tubes. All cells were stained with fluorescein isothiocyanate (FITC) and propidium iodide (PI), and the apoptosis rate was detected by flow cytometry. The results are shown below. Figure 23 As shown, the apoptosis rate of FOE / NPs@Gel reached over 90% in both types of tumor cells, which is consistent with the results of the cytotoxicity experiment. Therefore, after NPs in FOE / NPs@Gel are taken up by HepG2 and H22, they disintegrate in the cell to release small molecule drugs, namely tetravalent platinum cantharidin complexes, thereby inducing apoptosis of HepG2 and H22.

[0111] In vivo imaging experiment: ICG aqueous solution, ICG-NPs aqueous solution, ICG-NPs@Gel, and FOE / ICG-NPs@Gel were injected orally into the tumor sites of H22 tumor-bearing mice. The dosage of ICG was 2 mg / kg. Mice were shaved on days 1, 3, 5, 7, 9, and 13 post-administration. After anesthesia, fluorescence imaging and fluorescence intensity were detected at 790 nm using an in vivo imaging system. Figure 24 and Figure 25 As shown.

[0112] from Figure 24 and Figure 25As can be seen, due to the lack of adhesiveness and poor retention capacity of the ICG solution, it enters the systemic bloodstream through the abundant microvessels of the tumor site and is eventually metabolized, with fluorescence disappearing after day 7. Therefore, its retention time at the tumor site is relatively short. Over time, compared to other control groups, the fluorescence signal decay rate of FOE / ICG-NPs@Gel prepared in Example 1 of this invention is slower, mainly producing high-intensity fluorescence signals in tumor tissue, and maintaining this signal for a longer period. This result indicates that FOE / ICG-NPs@Gel can remain in the tumor site for a long time and gradually release NPs for uptake by tumor cells.

[0113] In vivo anti-tumor experiment in mice: When the solid tumor volume of H22 tumor-bearing mice was approximately 200 mm... 3 Mice were randomly divided into 5 groups of 6 mice each: saline (blank control), gel (Comparative Example 2), NPs (Example 1), NPs@Gel (Comparative Example 1), and FOE / NPs@Gel (Example 1). Pt was injected orally into the solid tumor site at a dose of 8.56 mg / kg. For the next 14 days, mouse body weight, tumor volume, and mental status were recorded. After 14 days, the mice were euthanized, and major organs (heart, liver, spleen, lungs, kidneys, and tumors) were dissected. The tissues were then stained with hematoxylin and eosin (H&E).

[0114] The results are as follows Figure 26 As shown, by Figure 26 Figure A shows that on day 14, the FOE / NPs@Gel group exhibited a more significant inhibitory effect, successfully controlling the tumor volume within the expected range. From Figure 26 Figures B and C show that, both in terms of visual images of the tumors and their weight, tumor growth was inhibited in all groups of mice compared to the saline group. Figure 26 As shown in Figure D, the weight gain trend of the mice is relatively slow. H&E staining results are as follows... Figure 27 As shown in the pathological section analysis, in terms of solid tumor tissue, the NPs, NPs@Gel, and FOE / NPs@Gel group samples all showed varying degrees of damage. The FOE / NPs@Gel group exhibited sparser cell density compared to the other two groups, with obvious signs of tissue damage. No obvious damage was found in normal tissues of the heart, liver, spleen, lungs, and kidneys, maintaining structural integrity. This further verifies that the organic nanocomposite hydrogel prepared in this invention specifically damages tumor tissue, exhibits minimal systemic toxicity, and demonstrates high biocompatibility.

[0115] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing an organic nanocomposite hydrogel, characterized in that, Includes the following steps: Divalent platinum is reacted with hydrogen peroxide to obtain a tetravalent platinum compound with dihydroxy coordination. Using the tetravalent platinum compound with dihydroxy coordination and cantharidin compounds as raw materials, a solvent and triethylamine are added, and an esterification reaction is carried out at 50°C~60°C in the dark and under a protective atmosphere to obtain a tetravalent platinum cantharidin complex. Using water as a solvent, the carboxyl groups of the tetravalent platinum cantharidin complex were activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain an activated tetravalent platinum cantharidin complex. This activated complex was then coupled with chitosan, where the carboxyl groups of the activated complex reacted with the amino groups of chitosan via an amide reaction to obtain a polysaccharide-based nanoprodrug. The cantharidin compound was either cantharidin or norcantharidin. The polysaccharide-based nanoprodrug was dispersed in a fluorinated orthoester to obtain a drug-loaded complex. The drug-loaded complex was then encapsulated with gellan gel to obtain an organic nanocomposite hydrogel.

2. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The molar ratio of the dihydroxy-coordinated tetravalent platinum compound to the cantharidin compound is 1:2~4.

3. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The molar ratio of the tetravalent platinum cantharidin complex, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide is 1:2~2.5:2~2.5; the mass ratio of the tetravalent platinum cantharidin complex to chitosan is 1:1.48~2.

8.

4. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The ratio of the polysaccharide-based nanoprodrug to fluorinated orthoester is 10 mg to 120 mg: 1 mL.

5. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The preparation method of the polysaccharide-based nanoprodrug is as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are dissolved in water to obtain an activation solution; a tetravalent platinum cantharidin complex is dissolved in water, and then the activation solution is added. The mixture is stirred at room temperature in the dark, and then a carboxymethyl chitosan aqueous solution is added. The mixture is stirred at room temperature in the dark and then dialyzed to obtain the polysaccharide-based nanoprodrug.

6. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The drug-loaded complex is encapsulated using gellan gum by heating and dissolving 1% to 2% by mass of low-acyl gellan gum to obtain a low-acyl gellan gum solution, and then dispersing the drug-loaded complex in the low-acyl gellan gum solution.

7. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that, The concentration of the drug-loaded complex in the organic nanocomposite hydrogel is 10 mg / mL to 70.5 mg / mL.

8. An organic nanocomposite hydrogel, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the organic nanocomposite hydrogel according to claim 8 in the preparation of antitumor drugs.

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