Organic nano composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses organic nano composite hydrogel as well as a preparation method and application of the organic nano composite hydrogel, and belongs to the technical field of biological medicine delivery. The polysaccharide-based nano prodrug in the composite hydrogel can be subjected to surface protonation under the stimulation of a tumor extracellular slightly acidic environment, so that cellular uptake is promoted, low-pH / high-concentration glutathione in tumor cells is responded, intracellular aggregation and controllable release of the nano drug are realized, the retention time of the drug in the cells is prolonged, and the bioavailability of the drug is improved. The technical effect of killing tumor cells through combined chemotherapy is achieved. According to the preparation method disclosed by the invention, the release of the polysaccharide-based nano prodrug from the hydrogel and the tissue infiltration capacity are accelerated by adopting fluorinated orthoester, the infiltration of the polysaccharide-based nano prodrug to cells is increased, and then the drug-loaded compound is encapsulated by using gellan gum, so that the slow-release effect of the polysaccharide-based nano prodrug is enhanced; therefore, the efficient enrichment of the medicine at the tumor part is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine delivery technology, and in particular to an organic nanocomposite hydrogel and a preparation method and application thereof. Background Art

[0002] With the rapid development of nanotechnology, nanomedicines have been widely researched and applied in the biomedical field, offering new hope for cancer patients. Compared to molecular drugs, nanoparticles can increase drug solubility, enhance drug stability, prolong metabolism, and reduce the toxic side effects of chemotherapy drugs. However, recent studies have shown that the low tumor cell penetration and accumulation of nanoparticle drug delivery systems severely limit their anti-tumor efficacy. Therefore, enhancing 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 physical or chemical crosslinking of single polymer chains. The aqueous solution environment of hydrogels is more conducive to protecting cells and easily inactivated drugs, and it is an excellent drug carrier material. The structure of hydrogels has the properties of both solid and liquid. This structure can realize the loading of drugs in pores and release drugs through diffusion, penetration, degradation, etc. At the same time, the properties of hydrogel solids can ensure the release of drugs in local tumor lesions. In addition, the environment of hydrogel aqueous solution is extremely similar to human tissue. The soft, moist surface and affinity with tissue greatly reduce the irritation of the material to surrounding tissues, making hydrogels have good biocompatibility.

[0004] Incorporating nanomaterials into 3D hydrogel networks through physical or chemical covalent interactions is an effective approach to avoid the explosive release, rapid elimination, and poor bioadhesion of nanoparticles, as well as the shortcomings of hydrogels in biomedical applications, such as poor anti-tumor ability, weak bioimaging capabilities, and limited reactivity. In nanocomposite hydrogel systems, nanomaterials typically serve as the functional core, endowing the hydrogel with various properties, such as tumor targeting and photothermal conversion. At the same time, hydrogels can effectively improve the retention of nanomaterials and impart good plasticity to nanoparticles for 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 nano-drug-loaded hydrogels fail to respond well to the microenvironment of the tumor site, affecting the penetration of nanodrugs into tumor cells and thus weakening the anti-tumor efficacy of nanodrugs. Summary of the Invention

[0005] The present invention provides an organic nanocomposite hydrogel and its preparation method and application, which effectively solves the technical problems of low tumor cell penetration ability and low tumor lesion site enrichment ability of existing nano drug delivery systems, the interaction between nanomaterials and hydrogels hinders the effective release of drugs embedded in the hydrogel network, and fails to respond well to the microenvironment of the tumor site, affecting the penetration of nano drugs into tumor cells, thereby weakening the anti-tumor efficacy of nano drugs.

[0006] The first object of the present invention is to provide a method for preparing an organic nanocomposite hydrogel, comprising the following steps: Divalent platinum is reacted with hydrogen peroxide to obtain a dihydroxy-coordinated tetravalent platinum compound; the dihydroxy-coordinated tetravalent platinum compound and a cantharidin compound are used as raw materials, a solvent and triethylamine are added, and an esterification reaction is carried out at 50° C. to 60° C. in a dark and protective atmosphere to obtain a tetravalent platinum cantharidin complex.

[0007] Water is used as a solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are used to activate the carboxyl group of the tetravalent platinum cantharidin complex to obtain an activated tetravalent platinum cantharidin complex, and then a coupling reaction is carried out with chitosan or a derivative thereof. The carboxyl group of the activated tetravalent platinum cantharidin complex undergoes an amide reaction with the amino group of the chitosan compound to obtain a polysaccharide-based nano-prodrug.

[0008] The polysaccharide-based nano prodrug is dispersed in fluorinated orthoester to improve the release effect of the polysaccharide-based nano prodrug to obtain a drug-loaded complex, and gellan gum is used to encapsulate the drug-loaded complex to obtain an organic nano composite hydrogel.

[0009] As a preferred embodiment, the molar ratio of the dihydroxy-coordinated tetravalent platinum compound to the cantharidin compound is 1:2-4.

[0010] As 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; the mass ratio of the tetravalent platinum cantharidin complex to the chitosan compound is 1:1.48~2.8.

[0011] As a preferred embodiment, the usage ratio of the polysaccharide-based nano-prodrug to the fluorinated orthoester is 10 mg~120 mg:1 mL.

[0012] As a preferred embodiment, the preparation method of the polysaccharide-based nano-prodrug 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, the activation solution is added, and the mixture is stirred in the dark at room temperature; then, a carboxymethyl chitosan aqueous solution is added, and the mixture is stirred in the dark at room temperature, and dialyzed to obtain a polysaccharide-based nano-prodrug.

[0013] As a preferred embodiment, gellan gum is used to encapsulate the drug-loaded complex, specifically: 1% to 2% by mass of low-acyl gellan gum is heated and dissolved to prepare a low-acyl gellan gum solution, and the drug-loaded complex is dispersed in the low-acyl gellan gum.

[0014] As a preferred embodiment, the concentration of the drug-loaded complex in the organic nanocomposite hydrogel is 10 mg / mL to 70.5 mg / mL.

[0015] As a preferred embodiment, the cantharidin compound is cantharidin or norcantharidin.

[0016] The second object of the present invention is to provide an organic nanocomposite hydrogel prepared by the above preparation method.

[0017] The third object of the present invention is to provide a use of the above organic nanocomposite hydrogel in the preparation of anti-tumor drugs.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a preparation method of an organic nanocomposite hydrogel. The method comprises the following steps: firstly reacting divalent platinum with hydrogen peroxide to obtain a dihydroxy-coordinated tetravalent platinum compound; reacting the tetravalent platinum compound with a cantharidin compound to obtain a tetravalent platinum cantharidin complex; activating the tetravalent platinum cantharidin complex with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the carboxyl groups in the tetravalent platinum cantharidin complex; then adding a chitosan compound to carry out a coupling reaction; the activated carboxyl groups react with amino groups in the chitosan compound to produce an amide reaction to obtain a polysaccharide-based nanoprodrug; the formed amide bond can respond to the pH environment of a tumor site and break at the tumor site, thereby improving the drug release effect; dispersing the polysaccharide-based nanoprodrug in a fluorinated orthoester, freeze-drying the mixture to obtain a drug-loaded complex; and encapsulating the drug-loaded complex with gellan gum to obtain an organic nanocomposite hydrogel capable of responding to the tumor microenvironment. The nanodrugs in the organic nanocomposite hydrogel prepared by the present invention have a suitable size, thereby improving the penetration ability of polysaccharide-based nanoprodrugs into tumor tissue. The polysaccharide-based nanoprodrugs in the organic nanocomposite hydrogel can be surface protonated under the stimulation of the slightly acidic environment outside the tumor cell, promoting cellular uptake and responding to the low pH / high concentration of glutathione inside the tumor cell, achieving intracellular aggregation and controlled release of the polysaccharide-based nanoprodrug, enhancing the drug's intracellular retention time, and achieving the technical effect of killing tumor cells in combination with chemotherapy. The fluorinated orthoester used in the present invention accelerates the release of the polysaccharide-based nanoprodrug from the organic nanocomposite hydrogel and its tissue penetration ability, increasing the penetration of the polysaccharide-based nanoprodrug into cells. The encapsulation of the drug-loaded complex with gellan gum enhances the sustained release effect of the polysaccharide-based nanoprodrug, thereby achieving efficient drug enrichment at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The tetravalent platinum cantharidin complex prepared in Example 1 of the present invention 1 H NMR spectrum.

[0020] Figure 2 The polysaccharide-based nano prodrug prepared in Example 1 of the present invention 1 H NMR spectrum.

[0021] Figure 3 This is the average particle size distribution diagram of the polysaccharide-based nanoprodrug prepared in Example 1 of the present invention.

[0022] Figure 4 This is the isoelectric point titration curve of the polysaccharide-based nanoprodrug prepared in Example 1 of the present invention; the inset is the second-order derivative graph.

[0023] Figure 5 This is the potential diagram of the polysaccharide-based nanoprodrug prepared in Example 1 of the present invention under different environments.

[0024] Figure 6 This is a graph showing the particle size changes of the polysaccharide-based nanoprodrug prepared in Example 1 of the present invention under different environments.

[0025] Figure 7 These are scanning electron microscope images of the organic nanocomposite hydrogel of Example 1 of the present invention and the gellan gum of Comparative Example 2, wherein Figure A is the gellan gum of Comparative Example 2, and Figure B is the organic nanocomposite hydrogel of Example 1.

[0026] Figure 8 This is a dynamic frequency scanning diagram of the organic nanocomposite hydrogel according to Example 1 of the present invention.

[0027] Figure 9 This is the shear thinning property of the organic nanocomposite hydrogel of Example 1 of the present invention.

[0028] Figure 10 This is the injectability of the organic nanocomposite hydrogel of Example 1 of the present invention.

[0029] Figure 11 These are the storage modulus and loss modulus of the organic nanocomposite hydrogel of Example 1 of the present invention during the strain sweep test of γ = 0.1% to 12%, as well as the storage modulus and loss modulus under the cyclic strain sweep of γ = 1% to 12%, where Figure A is γ = 0.1% to 12% and Figure B is γ = 1% to 12%.

[0030] Figure 12 This is the macroscopic self-healing property of the organic nanocomposite hydrogel of Example 1 of the present invention.

[0031] 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.

[0032] Figure 14 This is a degradation curve diagram of the organic nanocomposite hydrogel in Example 1 of the present invention under different environments.

[0033] 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.

[0034] Figure 16 This is an in vitro release curve of cisplatin in the organic nanocomposite hydrogel of Example 1 of the present invention.

[0035] Figure 17 This is a diagram of the hemolysis experiment of the organic nanocomposite hydrogel according to Example 1 of the present invention, wherein the physiological saline group is a negative control and H2O is a positive control.

[0036] Figure 18This is a graph showing the hemolysis rate of the organic nanocomposite hydrogel according to Example 1 of the present invention.

[0037] Figure 19 Graphs showing the qualitative uptake results of the organic nanocomposite hydrogel of Example 1 of the present invention and the nanocomposite hydrogel of Comparative Example 1 by tumor cells HepG2 and H22, respectively.

[0038] Figure 20 These are the quantitative uptake results of the organic nanocomposite hydrogel of Example 1 of the present invention and the nanocomposite hydrogel of Comparative Example 1 by tumor cells HepG2 and H22, respectively, wherein Figure A is for HepG2 and Figure B is for H22.

[0039] Figure 21 These are graphs showing the cytotoxicity of the organic nanocomposite hydrogel of Example 1 of the present invention, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the Gel of Comparative Example 2 to different cells, wherein Figure A is for CT26, Figure B is for A549, Figure C is for H22, and Figure D is for HepG2.

[0040] Figure 22 Live and dead cell staining images after incubation of the organic nanocomposite hydrogel of Example 1 of the present invention, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the Gel of Comparative Example 2 with tumor cells HepG2 or H22, respectively.

[0041] Figure 23 The graph shows the results of apoptosis of HepG2 or H22 induced by the organic nanocomposite hydrogel of Example 1 of the present invention, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the Gel of Comparative Example 2, respectively.

[0042] Figure 24 This is the in vivo imaging image of mice of the present invention, wherein ICG is a near-infrared dye - indocyanine green, ICG-NPs is 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.

[0043] Figure 25 This is a semi-quantitative statistical analysis chart of the in vivo imaging of mice of the present invention.

[0044] Figure 26 These are graphs showing the anti-tumor effects of the organic nanocomposite hydrogel of Example 1 of the present invention, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1, and the Gel of Comparative Example 2, wherein Figure A is a line graph of tumor volume, Figure B is a graph showing changes in tumor volume, Figure C is a graph showing changes in tumor weight, and Figure D is a graph showing changes in mouse body weight.

[0045] Figure 27 The figures are hematoxylin & eosin staining of different tissues and tumors of mice using the organic nanocomposite hydrogel of Example 1 of the present invention, the nanocomposite hydrogel of Comparative Example 1, the polysaccharide-based nanoprodrug of Example 1 and the Gel of Comparative Example 2.

[0046] 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 DESCRIPTION

[0047] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention is further described below with reference to specific examples, but the examples are not intended to limit the present invention. The following experimental and detection methods are conventional methods unless otherwise specified; the reagents and raw materials are commercially available unless otherwise specified.

[0048] The background of this invention mentions: First, existing nano-drug delivery systems have low tumor cell penetration and low tumor lesion accumulation capabilities; 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, preventing them from responding well to the microenvironment of the tumor site, affecting the penetration of nano-drugs into tumor cells, and thus weakening the anti-tumor efficacy of nano-drugs. To address the above technical issues, the present invention provides an organic nano-composite hydrogel, a preparation method, and applications thereof.

[0049] The technical solution of the present invention is described in detail below.

[0050] The present invention first provides a method for preparing an organic nanocomposite hydrogel, comprising the following steps: S1, reacting divalent platinum with hydrogen peroxide to obtain a dihydroxy-coordinated tetravalent platinum compound, using the dihydroxy-coordinated tetravalent platinum compound and a cantharidin compound as raw materials, adding a solvent and triethylamine, and performing an esterification reaction at 50° C. to 60° C. in a dark and protective atmosphere to obtain a tetravalent platinum cantharidin complex.

[0051] It should be noted that since cantharidin compounds are small molecule anticancer drugs, they form complexes after esterification with dihydroxy-coordinated tetravalent platinum compounds. The complexes can respond to the high concentration of glutathione in the tumor site and be reduced to release drugs at the tumor site, thereby playing a synergistic anti-tumor role.

[0052] S2, using water as a solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are used to activate the carboxyl group of the tetravalent platinum cantharidin complex to obtain an activated tetravalent platinum cantharidin complex, which is then coupled with a chitosan compound. The carboxyl group of the activated tetravalent platinum cantharidin complex undergoes an amide reaction with the amino group of the chitosan compound to obtain a polysaccharide-based nanoprodrug, denoted as NPs, whose structure is: ,in, Chitosan compounds, Represents a tetravalent platinum cantharidin complex.

[0053] The chitosan compounds used in the present invention are selected from chitosan, carboxymethyl chitosan, hydroxypropyl chitosan, or chitosan sulfate, preferably carboxymethyl chitosan. Chitosan is a natural cationic polysaccharide with a large number of amino groups and exhibits excellent biocompatibility. The amino groups on chitosan facilitate chemical modification or modification, and can also adhere to the negatively charged surface membranes of cancer cells and promote their endocytosis, thereby enabling the formulation of nanomedicines and enhancing the delivery of anti-tumor drugs.

[0054] S3, dispersing the polysaccharide-based nanoprodrug in fluorinated orthoester to improve the release effect of the polysaccharide-based nanoprodrug, obtaining a drug-loaded complex, recorded as FOE / NPs, and encapsulating the drug-loaded complex with gellan gum to obtain an organic nanocomposite hydrogel, recorded as FOE / NPs@Gel.

[0055] For the fluorinated orthoester used in the above scheme, the structural formula of the fluorinated orthoester used in the present invention is .

[0056] Because the resulting polysaccharide-based nanoprodrug is in an aqueous solution with poor storage stability, the polysaccharide-based nanoprodrug is dispersed in a fluorinated orthoester and freeze-dried. Once freeze-dried into a powder, it is difficult to re-dissolve in water. The polysaccharide-based nanoprodrug and the fluorinated orthoester are emulsified and mixed, and freeze-dried to remove the water carried by the polysaccharide-based nanoprodrug, thereby improving the poor water solubility and difficulty in re-dissolving the polysaccharide-based nanoprodrug, resulting in a drug-loaded complex. The dynamic changes of the fluorinated orthoester in the hydrogel facilitate the release of the polysaccharide-based nanoprodrug from the hydrogel. The fluorinated orthoester has excellent tissue permeability, thereby increasing the penetration of the polysaccharide-based nanoprodrug into tumor tissue.

[0057] The gellan gum used in the present invention has high biosafety and is injectable. It can be used as a drug carrier to deliver polysaccharide-based nano-prodrugs to the tumor site through in situ injection, forming a drug storage reservoir at the tumor site, thereby enhancing the enrichment and retention capacity of the polysaccharide-based nano-prodrugs at the tumor site, preventing the polysaccharide-based nano-prodrugs from being cleared, thereby improving the utilization rate of the polysaccharide-based nano-prodrugs, enhancing the anti-tumor effect, and effectively avoiding the systemic toxicity caused by the polysaccharide-based nano-prodrugs.

[0058] According to the technical solution provided by the present invention, the polysaccharide-based nanoprodrug in the organic nanocomposite hydrogel can be surface protonated under the stimulation of the slightly acidic environment outside the tumor cell, promoting cellular uptake and responding to the low pH / high concentration of glutathione inside the tumor cell, achieving intracellular aggregation and controlled release of NPs, enhancing the drug's intracellular retention time, and achieving the technical effect of killing tumor cells in combination with chemotherapy. The fluorinated orthoester accelerates the release of the polysaccharide-based nanoprodrug from the organic nanocomposite hydrogel and its tissue penetration ability, increasing the penetration of the polysaccharide-based nanoprodrug into tumor cells. The encapsulation of the drug-loaded complex with gellan gum enhances the sustained release of the polysaccharide-based nanoprodrug, thereby achieving efficient enrichment of the drug at the tumor site.

[0059] As a preferred embodiment, the molar ratio of the tetravalent platinum compound of the dihydroxy coordination to the cantharidin compound is 1:2~4. When the molar dosage of the above-mentioned cantharidin compound is less than 2 defined herein, the tetravalent platinum compound of the dihydroxy coordination may not be able to fully combine with the cantharidin compound, resulting in some platinum centers being unoccupied, reducing the product yield. When the molar dosage of the cantharidin compound is greater than 4 defined herein, the excess cantharidin compound is difficult to completely remove by subsequent purification steps, which may result in free cantharidin impurities remaining in the product. The theoretical reaction molar ratio of the tetravalent platinum compound of the dihydroxy coordination to the cantharidin compound is 1:2, and the platinum center is combined with two cantharidin molecules. A molar ratio of 1:3 can drive the reaction equilibrium to move toward the product direction, ensuring a high conversion rate, while being easy to remove a small amount of unreacted cantharidin by purification, and the final product has high purity and a clear structure.

[0060] To effectively activate the carboxyl groups 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. Excessive amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide can reduce the efficiency of carboxyl group activation. Insufficiently activated carboxyl groups can lead to a decrease in the coupling rate between the drug molecule and the polysaccharide, reducing drug loading. The formation of drug-carrier bonds, such as amide bonds, can be uneven, leading to unstable release rates. Excessive amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide can trigger side reactions, affecting drug stability and release controllability.

[0061] To further enhance drug release, the mass ratio of the tetravalent platinum cantharidin complex to the chitosan compound is 1:1.48 to 2.8. The carboxyl groups of the tetravalent platinum cantharidin complex react with the amino groups of the chitosan compound to form amide bonds, which can respond to the pH environment of the tumor site and break at the tumor site, thereby enhancing drug release. If the amount of chitosan compound used is less than the 1.48 ratio specified herein, insufficient amide bonds will be generated, resulting in a reduced amount of amide bond formation and a decrease in pH-responsive release efficiency, thereby affecting drug release in the tumor microenvironment and reducing the drug's efficacy against tumors. Furthermore, the limited carrier function of chitosan can lead to premature drug leakage or release in non-target areas. Excess free tetravalent platinum complex, due to ineffective encapsulation, degrades or is cleared, reducing tumor accumulation. Unbound complexes may be directly exposed to normal tissues, increasing systemic toxicity. If the amount of chitosan compound used is greater than 2.8 as defined herein, excessive chitosan can result in too low a drug content per unit carrier, and an overly dense chitosan network can hinder drug release, increase the volume or viscosity of the formulation, affect in vivo delivery, increase injection difficulty, or cause local retention. Excessive chitosan compound coating the drug surface can affect the release effect of the drug, thereby also having a significant impact on the drug's efficacy. During the experimental process of the present invention, when the mass ratio of the tetravalent platinum cantharidin complex to the chitosan compound was 20.1 mg:53.7 mg, i.e., 1:2.67, a good drug release effect was achieved.

[0062] In order to improve the permeability of polysaccharide-based nano prodrugs to tumor tissue, the amount ratio of the polysaccharide-based nano prodrug to the fluorinated orthoester is 10mg~120mg:1mL. If the mass ratio of the polysaccharide-based nano prodrug is less than 10mg defined herein, that is, the amount of the fluorinated orthoester is too much, the drug loading is less, and too much fluorinated orthoester is added to the gellan gum so that it is difficult to form a gel, and the gel property is unstable; If the mass ratio of the polysaccharide-based nano prodrug is greater than 120mg defined herein, that is, the amount of the fluorinated orthoester is too little, it will cause the drug to be released from the gellan gum and be restricted, and a small amount of fluorinated orthoester is difficult to increase the permeability of the drug to the tissue. The present invention learned during the experimental exploration process that dispersing 60mg of polysaccharide-based nano prodrug in 1mL of fluorinated orthoester can achieve a better penetration effect of polysaccharide-based nano prodrug on tumor tissue.

[0063] To further enhance the therapeutic efficacy of polysaccharide-based nanoprodrugs in organic nanocomposite hydrogels against tumors, the concentration of the drug-loaded complex in the organic nanocomposite hydrogel is 10 mg / mL to 70.5 mg / mL. When the drug-loaded concentration is lower than 10 mg / mL, the effective drug dose is insufficient, significantly reducing the anti-tumor efficacy and failing to achieve the desired therapeutic effect. When the drug-loaded concentration exceeds 70.5 mg / mL, systemic toxic reactions can be triggered due to drug overdose, causing irreversible damage to normal tissues and organs and potentially increasing the risk of adverse reactions.

[0064] It should be noted that the cantharidin compound used in the present invention is cantharidin or norcantharidin; and the gellan gum is a low-acyl gellan gum with a mass fraction of 1% to 2%.

[0065] The technical effects of the present invention are described below through specific embodiments and comparative examples.

[0066] Example 1 A method for preparing an organic nanocomposite hydrogel comprises the following steps: S1: Disperse 200 mg (0.67 mmol) of cisplatin in 12 mL of deionized water. Add 13 mL of 30 wt% hydrogen peroxide dropwise via a constant pressure dropping funnel. Stir and react at 55°C in the dark for 5 h. After the reaction, the solution is concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow powder of tetravalent cisplatin (DHP). Dissolve 0.1 g (0.3 mmol) of DHP and 0.15 g (0.89 mmol) of norcantharidin (DMC) in 2 mL of anhydrous N,N-dimethylformamide. Then, add anhydrous triethylamine dropwise. Stir and react at 60°C in the dark under a nitrogen atmosphere for 24 h. After the reaction, precipitate with ice-cold anhydrous ether, wash the powder with acetone, filter, and dry in a vacuum oven to yield a pale yellow powder of the tetravalent platinum-cantharidin complex.

[0067] 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 at room temperature in the dark for 5 h, 5 mL of a carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued at room temperature in the dark for 24 h. The solution was dialyzed to obtain a polysaccharide-based nanoprodrug, which was recorded as NPs.

[0068] S3, 60 mg of the polysaccharide-based nanoprodrug was dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug to obtain a drug-loaded complex, recorded as FOE / NPs, and 1.5% by mass of low-acyl gellan gum was heated and dissolved in an 80°C oil bath to prepare a gellan gum solution, and then the drug-loaded complex was dispersed in 1.5% by mass of low-acyl gellan gum, and the low-acyl gellan gum was used to encapsulate the drug-loaded complex to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, recorded as FOE / NPs@Gel.

[0069] Example 2 A method for preparing an organic nanocomposite hydrogel comprises the following steps: S1: Disperse 200 mg (0.67 mmol) of cisplatin in 12 mL of deionized water. Add 13 mL of 30 wt% hydrogen peroxide dropwise via a constant pressure dropping funnel. Stir and react at 55°C in the dark for 5 h. After the reaction, concentrate the solution by rotary evaporation and lyophilize to remove water, yielding a pale yellow powder (quaternary cisplatin). Dissolve 0.1 g (0.3 mmol) of quaternary cisplatin and 0.15 g (0.89 mmol) of norcantharidin in 2 mL of anhydrous N,N-dimethylformamide. Then, add anhydrous triethylamine dropwise. Stir and react at 60°C in the dark under a nitrogen atmosphere for 24 h. After the reaction, precipitate with ice-cold anhydrous ether, wash the powder with acetone, filter, and dry in a vacuum oven to yield a pale yellow powder (quaternary platinum-cantharidin complex).

[0070] 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 at room temperature in the dark for 5 h, 5 mL of a 6 mg / mL carboxymethyl chitosan aqueous solution was added, and stirring was continued at room temperature in the dark for 24 h. The solution was dialyzed to obtain a polysaccharide-based nanoprodrug, which was recorded as NPs.

[0071] S3, 60 mg of the polysaccharide-based nanoprodrug was dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug to obtain a drug-loaded complex, recorded as FOE / NPs, and 1.5% by mass of low-acyl gellan gum was heated and dissolved in an 80°C oil bath to prepare a gellan gum solution, and then the drug-loaded complex was dispersed in 1.5% by mass of low-acyl gellan gum, and the low-acyl gellan gum was used to encapsulate the drug-loaded complex to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, recorded as FOE / NPs@Gel.

[0072] Example 3 A method for preparing an organic nanocomposite hydrogel comprises the following steps: S1: Disperse 200 mg (0.67 mmol) of cisplatin in 12 mL of deionized water. Add 13 mL of 30 wt% hydrogen peroxide dropwise via a constant pressure dropping funnel. Stir and react at 55°C in the dark for 5 h. After the reaction, concentrate the solution by rotary evaporation and lyophilize to remove water, yielding a pale yellow powder (quaternary cisplatin). Dissolve 0.1 g (0.3 mmol) of quaternary cisplatin and 0.15 g (0.89 mmol) of norcantharidin in 2 mL of anhydrous N,N-dimethylformamide. Then, add anhydrous triethylamine dropwise. Stir and react at 60°C in the dark under a nitrogen atmosphere for 24 h. After the reaction, precipitate with ice-cold anhydrous ether, wash the powder with acetone, filter, and dry in a vacuum oven to yield a pale yellow powder (quaternary platinum-cantharidin complex).

[0073] 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 at room temperature in the dark for 5 h, 5 mL of a carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued at room temperature in the dark for 24 h. The solution was dialyzed to obtain a polysaccharide-based nanoprodrug, which was recorded as NPs.

[0074] S3, 40 mg of the polysaccharide-based nanoprodrug was dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug to obtain a drug-loaded complex, recorded as FOE / NPs, and 1.5% by mass of low-acyl gellan gum was heated and dissolved in an 80°C oil bath to prepare a gellan gum solution, and then the drug-loaded complex was dispersed in 1.5% by mass of low-acyl gellan gum, and the drug-loaded complex was encapsulated with the low-acyl gellan gum to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 11.4 mg / mL, recorded as FOE / NPs@Gel.

[0075] Example 4 A method for preparing an organic nanocomposite hydrogel comprises the following steps: S1: Disperse 200 mg (0.67 mmol) of cisplatin in 12 mL of deionized water. Add 13 mL of 30 wt% hydrogen peroxide dropwise via a constant pressure dropping funnel. Stir and react at 55°C in the dark for 5 h. After the reaction, concentrate the solution by rotary evaporation and lyophilize to remove water, yielding a pale yellow powder (quaternary cisplatin). Dissolve 0.1 g (0.3 mmol) of quaternary cisplatin and 0.15 g (0.89 mmol) of norcantharidin in 2 mL of anhydrous N,N-dimethylformamide. Then, add anhydrous triethylamine dropwise. Stir and react at 60°C in the dark under a nitrogen atmosphere for 24 h. After the reaction, precipitate with ice-cold anhydrous ether, wash the powder with acetone, filter, and dry in a vacuum oven to yield a pale yellow powder (quaternary platinum-cantharidin complex).

[0076] 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 at room temperature in the dark for 5 h, 5 mL of a carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued at room temperature in the dark for 24 h. The solution was dialyzed to obtain a polysaccharide-based nanoprodrug, which was recorded as NPs.

[0077] S3, 60 mg of the polysaccharide-based nanoprodrug was dispersed in 1 mL of fluorinated orthoester and emulsified to improve the release effect of the polysaccharide-based nanoprodrug to obtain a drug-loaded complex, recorded as FOE / NPs, and 1% by mass of low-acyl gellan gum was heated and dissolved in an 80°C oil bath to prepare a gellan gum solution, and then the drug-loaded complex was dispersed in 1% by mass of low-acyl gellan gum, and the drug-loaded complex was encapsulated with the low-acyl gellan gum to obtain an organic nanocomposite hydrogel with a drug-loaded complex concentration of 17 mg / mL, recorded as FOE / NPs@Gel.

[0078] In order to further demonstrate the technical effect of the present invention, the present invention also provides a comparative example, which is as follows: Comparative Example 1 A method for preparing a nanocomposite hydrogel comprises the following steps: S1: Disperse 200 mg (0.67 mmol) of cisplatin in 12 mL of deionized water. Add 13 mL of 30 wt% hydrogen peroxide dropwise via a constant pressure dropping funnel. Stir and react at 55°C in the dark for 5 h. After the reaction, the solution is concentrated by rotary evaporation and lyophilized to remove water, yielding a pale yellow powder of tetravalent cisplatin (DHP). Dissolve 0.1 g (0.3 mmol) of DHP and 0.15 g (0.89 mmol) of norcantharidin (DMC) in 2 mL of anhydrous N,N-dimethylformamide. Then, add anhydrous triethylamine dropwise. Stir and react at 60°C in the dark under a nitrogen atmosphere for 24 h. After the reaction, precipitate with ice-cold anhydrous ether, wash the powder with acetone, filter, and dry in a vacuum oven to yield a pale yellow powder of the tetravalent platinum-cantharidin complex.

[0079] 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 at room temperature in the dark for 5 h, 5 mL of a carboxymethyl chitosan aqueous solution with a concentration of 10.74 mg / mL was added, and stirring was continued at room temperature in the dark for 24 h. The solution was dialyzed to obtain a polysaccharide-based nanoprodrug, which was recorded as NPs.

[0080] S3, heating and dissolving low-acyl gellan gum with a mass fraction of 1.5% in an oil bath at 80°C to prepare a gellan gum solution, then dispersing 60 mg of the polysaccharide-based nanoprodrug in the low-acyl gellan gum with a mass fraction of 1.5%, and using the low-acyl gellan gum to encapsulate the polysaccharide-based nanoprodrug to obtain a nanocomposite hydrogel with a polysaccharide-based nanoprodrug concentration of 17 mg / mL, which is recorded as NPs@Gel.

[0081] Comparative Example 2 A method for preparing a hydrogel comprises the following steps: The gellan gum solution was prepared by heating and dissolving low-acyl gellan gum with a mass fraction of 1.5% in an oil bath at 80°C. The gellan gum solution was then cooled to form a gel to obtain a hydrogel, which was recorded as Gel.

[0082] The structures of the synthesized polysaccharide-based nanoprodrugs were verified by hydrogen nuclear magnetic resonance spectroscopy. Figure 1 and Figure 2 As shown in Figure 2, the structure of the synthesized tetravalent platinum cantharidin complex and NPs is accurate. The particle size of NPs at 37°C was detected by dynamic light scattering particle size analyzer, i.e. DLS. Figure 3 As shown in Figure 2, the particle size of NPs is about 134 nm, and the NPs are spherical with regular morphology under scanning electron microscopy. In order to determine the isoelectric point of NPs, potentiometric titration was used to detect the results. Figure 4 As shown in Figure 2, the isoelectric point of NPs is approximately pH = 5. At the same time, the potential changes of NPs at different pH values, i.e., pH = 7.4, pH = 6.8, and pH = 5.0, were detected. Figure 5 As shown in Figure 2, the particle size changes under different environments of pH = 7.4, pH = 6.8, pH = 5.0 and pH = 5.0 / GSH. Figure 6 As shown in the figure, the surface potential of NPs increases with the decrease of pH value, which is conducive to the uptake of NPs by tumor cells; the particle size can remain stable under the environment of pH=7.4 and pH=6.8, but under the environment of pH=5.0 in tumor cells, aggregation and precipitation occur due to reaching the isoelectric point, and then they are reduced and gradually degraded under the action of glutathione, namely GSH.

[0083] The low-acyl gellan gum with a mass fraction of 1.5% was heated and dissolved in an oil bath at 80℃, and then frozen and fractured after gelation. At the same time, FOE / NPs@Gel was prepared and frozen and fractured. The morphologies of the two hydrogels were observed by scanning electron microscopy. Figure 7 As shown by Figure 7 As can be seen from Figure A, gellan gum has abundant pores, which can load drugs and realize drug release. After loading FOE / NPs, 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, which proves that FOE / NPs are successfully loaded in gellan gum.

[0084] Rheological properties of hydrogels: The rheological properties of FOE / NPs@Gel prepared in Example 1 of the present invention were analyzed using a rotational rheometer from Kinexus Lab, a NETZSCH brand. First, the FOE / NPs@Gel was subjected to a frequency sweep of 0.15 Hz to 10 Hz at a constant temperature of 37°C and a constant stress of 1%, and the storage modulus and loss modulus of the FOE / NPs@Gel were tested to determine its stability at different frequencies. Figure 8 As shown. Then 0.1s -1 ~100 s -1 The viscosity of FOE / NPs@Gel was tested at different shear rates, such as Figure 9 Finally, the strain scan of FOE / NPs@Gel from 0.1% to 12% was performed while maintaining a constant frequency of 1 Hz to determine its linear viscoelastic region. Subsequently, the strain scan was performed alternately with 12% high strain and 1% low strain to detect the trend of the storage modulus and loss modulus of FOE / NPs@Gel with alternating strain, as shown in Figure 2. Figure 11 As shown, and its injectability was tested macroscopically, as Figure 10As shown, the FOE / NPs@Gel solution that has not been cooled and gelled was placed in a 1 mL syringe. After the FOE / NPs@Gel gelled, it was observed that it could be pushed out through a 25G needle.

[0085] The organic nanocomposite hydrogel of the present invention was stained with ICG and rhodamine B respectively, and each was cut into two halves. Then, two pieces of FOE / NPs@Gel with different colors were attached along the cut surface and placed in a mold. After 6 hours at room temperature, photos were taken to record whether the FOE / NPs@Gel could self-heal. The results are shown in FIG. Figure 12 shown.

[0086] 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 can be seen that the viscosity of organic nanocomposite hydrogel decreases rapidly with the increase of shear rate, showing a typical shear thinning behavior. Figure 11 As shown in Figures A and B, in order to determine the linear viscoelastic region of the hydrogel, an amplitude strain sweep was performed. 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 transformed from a gel state to a sol state. Subsequently, the low strain was set to 1% and the high strain was set to 12%, and high and low cyclic strain sweeps were performed. When the strain was low, the storage modulus was greater than the loss modulus, indicating a gel. When the strain was high, the storage modulus was less than the loss modulus, indicating a sol. When the strain was restored to 1%, its modulus returned to its initial value and transformed back into a gel. From a macroscopic perspective, as shown in Figure 1, Figure 10 and Figure 12 As shown, it was once again verified that the hydrogel was injectable through a 25G needle and had self-healing behavior.

[0087] The organic nanocomposite hydrogel was attached to different materials to evaluate the adhesion ability of the organic nanocomposite hydrogel prepared by the present invention to different materials. Figure 13 As shown in Figure 2, the organic nanocomposite hydrogel exhibits strong adhesion to a variety of materials, including polypropylene plastic, metal, glass, and nitrile rubber. Furthermore, the tissue adhesion properties of the FOE / NPs@Gel were investigated. After folding, stretching, and repeated twisting, the FOE / NPs@Gel remained adherent to porcine skin, muscle, and liver tissues, maintaining strong adhesion without detaching from these tissues.

[0088] Phosphate buffer solutions with pH = 7.4 and pH = 6.8 were prepared to simulate the degradation of FOE / NPs@Gel under physiological conditions and tumor microenvironment in vitro. Figure 14Under the conditions of pH = 7.4 and pH = 6.8, in the first 3 h, the FOE / NPs@Gel absorbed water and swelled, causing the mass of the FOE / NPs@Gel to increase. Subsequently, the FOE / NPs@Gel stopped swelling and began to degrade. The degradation rate of the FOE / NPs@Gel was slow, but the addition of fluorinated orthoesters increased its degradation rate, which was more conducive to the release of NPs.

[0089] The present invention investigates the release of NPs in the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1. Figure 15 As shown in Figure 2, NPs can be released from the hydrogel, and the release amount of FOE / NPs@Gel is greater than that of NPs@Gel. The present invention further tested whether the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1 can effectively release NPs with appropriate particle size. The results are shown in Figure 2. Figure 7 Figure B in Figure 1 shows that NPs with the expected particle size are present on the surface of the organic nanocomposite hydrogel after 48 hours, and NPs of the corresponding size are detected in the degradation solution by DLS, thus proving that NPs can be effectively released from the organic nanocomposite hydrogel. The drug release of cisplatin was further investigated for the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1. The results are shown in Figure 1. Figure 16 Under the conditions of pH = 7.4 and pH = 6.8, the drug release of cisplatin was low, while under the condition of pH = 5.0 / GSH, the cisplatin drug release reached 77% of the total drug. Under the stimulation of pH = 5.0 / GSH, tetravalent cisplatin was reduced to divalent cisplatin, the NPs structure was destroyed, and cisplatin was subsequently released. The above results indicate that the presence of fluorinated orthoesters accelerated the effective escape of NPs from the organic nanocomposite hydrogel in the form of nanoparticles. The escaped NPs can respond to the pH = 5.0 / GSH environment, achieving precise drug release inside tumor cells.

[0090] The biosafety of the organic nanocomposite hydrogel prepared by the present invention was tested by hemolysis experiment. Figure 17 and Figure 18 It can be seen that there is no significant difference between the FOE / NPs@Gel at different concentrations and the negative control group, and the hemolytic activity of all experimental groups is less than 5%, indicating that the organic nanocomposite hydrogel prepared by the present invention has no obvious hemolytic activity, high blood compatibility, and excellent biosafety.

[0091] Cell uptake experiment: Tumor cells HepG2 and H22 were inoculated into 6-well plates respectively. First, NPs were labeled with fluorescein isothiocyanate, i.e. FITC. After the tumor cells adhered to the wall, 200 μL of NPs@Gel of Comparative Example 1 and FOE / NPs@Gel of Example 1 were added to each well, with NPs concentration of 63.5 μM and Gel of 72 mg / mL, and co-culture was continued for 4 h and 8 h. Among them, a part was fixed with paraformaldehyde for 10 min, stained with fluorescent dye 4',6-diamidino-2-phenylindole, i.e. DAPI, for 3 min, washed 3 times with fresh PBS, and observed and photographed with a confocal laser scanning microscope, as shown in the figure. Figure 19 The other part was digested with trypsin, centrifuged at 1000 rpm for 10 min, and dispersed in 0.5 mL PBS. The uptake of tumor cells was measured by flow cytometry after the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1 were incubated with tumor cells HepG2 or H22 for 4 h. The results of cell quantitative uptake are shown in Figure 2. Figure 20 As shown in the figure, the uptake of FOE / NPs@Gel by both tumor cells after 4 h was higher than that of NPs@Gel. The qualitative uptake results of confocal microscopy are shown in the figure. Figure 19 As shown in the figure, obvious green fluorescence appeared in the tumor cells, indicating that the NPs were internalized by the 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, the fluorinated orthoesters encapsulate the NPs. As the fluorinated orthoesters escape, the escape of NPs from FOE / NPs@Gel is accelerated, so that more NPs are released and taken up by tumor cells. Moreover, after 8 hours, the green fluorescence intensity in the tumor cells is further enhanced, and there is aggregation and fluorescence generation.

[0092] Cytotoxicity assay: Four different tumor cells, HepG2, H22, CT26 and A549, were selected and incubated with the FOE / NPs@Gel of Example 1, the NPs of Example 1, the NPs@Gel of Comparative Example 1 and the blank hydrogel Gel of Comparative Example 2 for 48 h. Figure 21 As shown, the cell survival rate of the blank hydrogel Gel of Comparative Example 2 is higher, indicating that the hydrogel without NPs loading has good biosafety. The cell survival rates of NPs@Gel of Comparative Example 1 and FOE / NPs@Gel of Example 1 are lower. In comparison, FOE / NPs@Gel has higher cytotoxicity, indicating that the escape of fluorinated orthoesters in the hydrogel accelerates the escape of NPs from the composite hydrogel, allowing more NPs to be taken up by tumor cells, resulting in higher cytotoxicity.

[0093] Live and dead cell double staining: Tumor cells HepG2 and H22 were seeded in 24-well plates, 40,000 cells / well, and the blank hydrogel Gel of Comparative Example 2, NPs of Example 1, NPs@Gel of Comparative Example 1, and FOE / NPs@Gel of Example 1, with NPs concentration of 63.5 μM and Gel of 72 mg / mL, were co-cultured with HepG2 or H22 for 48 hours, and stained according to the procedures of the Calcein / PI cell viability and cytotoxicity detection kit, and then observed under an inverted fluorescence microscope. The results are shown in Figure 2. Figure 22 As shown in the figure, the cytotoxicity of FOE / NPs@Gel was higher than that of other groups, and it had obvious toxicity to tumor cells.

[0094] Apoptosis detection: HepG2 and H22 were seeded in 6-well plates respectively. When the cell density reached 50,000 cells / well, the Gel of Comparative Example 2, the NPs of Example 1, the NPs@Gel of Comparative Example 1 and the FOE / NPs@Gel of Example 1 were added respectively, with the NPs concentration of 63.5 μM and the Gel concentration of 72 mg / mL. They were co-cultured with HepG2 or H22 for 48 hours, and then the cells in each well were washed with PBS and transferred to a centrifuge tube. All cells were stained with fluorescein isothiocyanate, i.e. FITC, and propidium iodide, i.e. PI, and the cell apoptosis rate was detected by flow cytometry. The test results are shown in the figure. Figure 23 As shown in the figure, in two different tumor cells, the apoptosis rate of FOE / NPs@Gel reached more than 90%, and the results were consistent with the results of the cytotoxicity experiment. Therefore, after the NPs in FOE / NPs@Gel were taken up by HepG2 and H22, they disintegrated in the cells to release small molecule drugs, namely tetravalent platinum cantharidin complexes, thereby inducing apoptosis of HepG2 and H22.

[0095] In vivo imaging experiments: ICG aqueous solution, ICG-NPs aqueous solution, ICG-NPs@Gel, and FOE / ICG-NPs@Gel were injected into the tumor site of H22 tumor-bearing mice by in situ injection. The ICG dosage was 2 mg / kg. On the 1st, 3rd, 5th, 7th, 9th, and 13th day after administration, the mice were depilated. After anesthesia, fluorescence imaging and fluorescence intensity detection were performed using an in vivo imager at a wavelength of 790 nm. Figure 24 and Figure 25 shown.

[0096] from Figure 24 and Figure 25It can be seen that since the ICG solution has no adhesion and poor retention ability, it enters the systemic blood circulation through the abundant microvessels in the tumor site and is eventually metabolized. The fluorescence disappears after the 7th day, so the retention time at the tumor site is relatively short. As time goes by, compared with other control groups, the fluorescence signal of FOE / ICG-NPs@Gel prepared in Example 1 of the present invention decays more slowly, mainly producing high-intensity fluorescence signals in tumor tissues, which last longer. This result shows that FOE / ICG-NPs@Gel can be retained in the tumor site for a long time and gradually release NPs to be taken up by tumor cells.

[0097] Anti-tumor experiment in mice: When the volume of solid tumor in H22 tumor-bearing mice was about 200mm 3 The mice were randomly divided into 5 groups, each with 6 mice, namely normal saline, i.e. blank control group, Gel of comparative example 2, NPs of example 1, NPs@Gel of comparative example 1 and FOE / NPs@Gel of example 1, and were injected into the solid tumor site in situ, wherein the Pt dosage was 8.56 mg / kg. The body weight and solid tumor volume of the mice were recorded for the following 14 days, and the mental state of the mice was observed. After 14 days, the mice were sacrificed, and the main organs, heart, liver, spleen, lung, kidney and tumor were dissected out, and the tissues were then stained with hematoxylin & eosin, i.e. H&E staining.

[0098] The results are as follows Figure 26 As shown by Figure 26 As shown in Figure A, on the 14th day, the FOE / NPs@Gel group showed a more significant inhibitory effect and successfully controlled the tumor volume within the expected range. Figure 26 As can be seen from Figures B and C, whether from the visual photos of the tumor or the weight of the tumor, the tumors of the mice in each group were suppressed compared with the normal saline group. Figure 26 As can be seen from Figure D, the weight gain trend of mice is relatively slow. Figure 27 As shown, pathological section analysis revealed that solid tumor tissue samples from the NPs, NPs@Gel, and FOE / NPs@Gel groups all exhibited varying degrees of damage. Cells in the FOE / NPs@Gel group were more sparsely populated than those in the other two groups, showing clear signs of tissue damage. Normal tissues of the heart, liver, spleen, lung, and kidney showed no obvious signs of damage, maintaining their structural integrity. This further demonstrates that the organic nanocomposite hydrogel prepared by this invention specifically damages tumor tissue, exhibits minimal systemic toxicity, and exhibits high biosafety.

[0099] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an organic nanocomposite hydrogel, characterized in that: The following steps are involved: reacting divalent platinum with hydrogen peroxide to obtain a dihydroxy-coordinated tetravalent platinum compound; using the dihydroxy-coordinated tetravalent platinum compound and a cantharidin compound as raw materials, adding a solvent and triethylamine, and performing an esterification reaction at 50° C. to 60° C. in a dark and protective atmosphere to obtain a tetravalent platinum cantharidin complex; Using water as a solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are used to activate the carboxyl group of the tetravalent platinum cantharidin complex to obtain an activated tetravalent platinum cantharidin complex, which is then subjected to a coupling reaction with chitosan or a derivative thereof. The carboxyl group of the activated tetravalent platinum cantharidin complex undergoes an amide reaction with the amino group of the chitosan compound to obtain a polysaccharide-based nano-prodrug; The polysaccharide-based nano prodrug is dispersed in fluorinated orthoester to obtain a drug-loaded complex, and gellan gum is used to encapsulate the drug-loaded complex to obtain an organic nano composite hydrogel.

2. The method for preparing the organic nanocomposite hydrogel according to claim 1, wherein: 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, wherein: 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 the chitosan compound is 1:1.48-2.

8.

4. The method for preparing the organic nanocomposite hydrogel according to claim 1, wherein: The dosage ratio of the polysaccharide-based nano prodrug to the fluorinated orthoester is 10 mg to 120 mg: 1 mL.

5. The method for preparing the organic nanocomposite hydrogel according to claim 1, wherein: The preparation method of the polysaccharide-based nano-prodrug comprises the following steps: dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in water to obtain an activation solution; dissolving a tetravalent platinum cantharidin complex in water, adding the activation solution, stirring at room temperature in the dark, adding a carboxymethyl chitosan aqueous solution, stirring at room temperature in the dark, and dialyzing to obtain the polysaccharide-based nano-prodrug.

6. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that: The method of encapsulating the drug-loaded complex with gellan gum is as follows: 1% to 2% by mass of low-acyl gellan gum is heated and dissolved to prepare a low-acyl gellan gum solution, and the drug-loaded complex is dispersed in the low-acyl gellan gum.

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. The method for preparing the organic nanocomposite hydrogel according to claim 1, characterized in that: The cantharidin compound is cantharidin or norcantharidin.

9. An organic nanocomposite hydrogel, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the organic nanocomposite hydrogel according to claim 9 in the preparation of anti-tumor drugs.

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