An organic composite hydrogel, its preparation method and application

Abstract

This invention relates to the field of biomaterials and pharmaceutical preparation technology, specifically to an organic composite hydrogel, its preparation method, and its applications. The specific preparation method includes the following steps: dissolving a targeted drug in an orthoester to obtain a targeted drug-orthoester solution; mixing an oxidized dextran solution, the targeted drug-orthoester solution, and a carboxymethyl chitosan solution, vortexing, and allowing to stand to prepare the organic composite hydrogel. This invention utilizes orthoesters to enhance the solubility of the targeted drug in the hydrogel, and the orthoesters can efficiently carry the targeted drug out of the hydrogel. During the escape process, as the orthoesters degrade, the targeted drug can self-assemble into nanoparticles, thereby enhancing the retention capacity of the targeted drug at the tumor site. While improving drug accumulation capacity, it reduces systemic toxicity, solving the problems of poor solubility of nanomedicines, easy clearance after intravenous administration, and poor accumulation at the tumor site.

Description

Technical Field

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

[0002] Currently, drug delivery systems in cancer treatment suffer from numerous limitations, such as poor sustained-release and controlled-release capabilities, easy drug diffusion, weak penetration, inability to accumulate at the tumor site for extended periods, and insufficient tumor targeting. These shortcomings not only limit the further development and application of clinical drugs but may also lead to poor therapeutic effects and increased systemic toxicity. Therefore, it is necessary to find new drug carriers and modify the administration method to reduce toxicity, improve drug delivery efficiency and therapeutic efficacy, and address the problem of poor drug accumulation.

[0003] Nanomedicines, as an emerging drug delivery system, have shown significant advantages in improving drug solubility, enhancing targeting, prolonging circulation time, achieving sustained and controlled release, reducing toxicity, and improving therapeutic efficacy. For example, nanoparticles can be formed by the self-assembly of two targeted drugs, dasatinib and sunitinib, to enhance their retention capacity at tumor sites; however, these nanomedicines have poor solubility.

[0004] Orthoesters are sensitive, hydrophobic, and biodegradable materials. Since their synthesis, they have been studied for pharmaceutical applications and have achieved widespread use and good therapeutic effects in drug delivery, protein release, postoperative pain management, and postoperative cancer treatment. Therefore, researchers utilize orthoesters to enhance the solubility of nanomedicines. However, current preparation methods for orthoester-nanomedicines still face challenges such as easy clearance after intravenous administration and poor accumulation at tumor sites, which limits the widespread application of nanomedicines in cancer treatment. Summary of the Invention

[0005] To address the issues of poor solubility of nanomedicines, their easy clearance after intravenous administration, and poor accumulation at tumor sites, the present invention aims to provide an organic composite hydrogel, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] The first aspect of this invention provides a method for preparing an organic composite hydrogel, comprising the following steps:

[0008] The targeted drug was dissolved in orthoester to obtain a targeted drug-orthoester solution; the oxidized dextran solution, the targeted drug-orthoester solution and the carboxymethyl chitosan solution were mixed, vortexed and allowed to stand to obtain an organic composite hydrogel; the concentration of the oxidized dextran solution was 10 wt%; the concentration of the carboxymethyl chitosan solution was 5 wt%; the volume ratio of the oxidized dextran solution and the carboxymethyl chitosan solution was 1:0.5-2.

[0009] Preferably, the targeted drug is dasatinib or sunitinib; the drug loading concentration of the targeted drug in the organic composite hydrogel is 0.01 mg / mL to 20 mg / mL.

[0010] The organic composite hydrogel prepared in this invention possesses certain injectability and adaptability. After hydrogel degradation, the orthoester can efficiently carry two targeted drugs for escape, and the orthoester contains fluorinated groups, enhancing its permeability within tumor tissues. As the orthoester degrades, the two targeted drugs self-assemble into nanoparticles, enhancing their retention capacity at the tumor site. After being taken up by tumor cells, the two targeted drugs can act on different targets, thereby achieving a combined and synergistic therapeutic effect.

[0011] Preferably, the volume ratio of the oxidized dextran solution, the targeted drug-orthoester solution, and the carboxymethyl chitosan solution is 1:1:1; and the dosage ratio of the targeted drug to the orthoester is 8 mg: 1 mL.

[0012] This invention utilizes a hydrogel formed from oxidized dextran and carboxymethyl chitosan, which can create a three-dimensional network structure with large porosity and specific surface area. This allows the hydrogel to effectively carry and release targeted drugs. This high drug loading capacity not only increases the amount of targeted drug but also improves release efficiency. This invention enables precise control of targeted drug release by manipulating the hydrogel structure, thereby better meeting the demand for precise targeted drug delivery in tumor treatment.

[0013] Preferably, the chemical structural formula of the orthoester is as follows: .

[0014] Preferably, the molecular weight of carboxymethyl chitosan is 20,000 to 21,000; the degree of carboxyl substitution of carboxymethyl chitosan is 35% to 90%; and the amino unit content of carboxymethyl chitosan is 70% to 90%.

[0015] Preferably, the orthoester is prepared by the following method: 3-amino-1,2-propanediol and ethyl trifluoroacetate are reacted in a solvent system with stirring, followed by rotary evaporation, extraction, and drying to obtain intermediate product 1; under the action of a catalyst, intermediate product 1 and trimethyl orthoformate are reacted in a solvent system with stirring, followed by rotary evaporation, extraction, and drying to obtain the orthoester. Preferably, the molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1.5-2; the molar ratio of intermediate product 1 to trimethyl orthoformate is 1:1-4; the solvent is any one of acetonitrile, dichloromethane, chloroform, tetrahydrofuran, and dioxane; the catalyst is p-toluenesulfonic acid; and the molar ratio of intermediate product 1 to catalyst is 1:0.01-0.03.

[0016] Preferably, oxidized dextran is prepared by the following method: dextran, sodium periodate, and sodium acetate buffer solution are reacted with stirring in the dark, and after the reaction is completed, the mixture is dialyzed and lyophilized to obtain oxidized dextran; the molar ratio of sodium periodate to glucose units in dextran is 1:1-2; and the molecular weight of dextran is 60,000-70,000. Preferably, the temperature of the stirring reaction in the dark is room temperature.

[0017] A second aspect of this invention provides an organic composite hydrogel, prepared using the method described in the first aspect. The organic composite hydrogel of this invention can enhance the accumulation of targeted drugs at tumor sites while reducing systemic toxicity, achieving a good anti-tumor effect.

[0018] A third aspect of this invention provides the application of the organic composite hydrogel described in the second aspect in the preparation of pharmaceutical formulations for antitumor or cancer cell inhibition. Preferably, the pharmaceutical formulation is an injectable dosage form.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention mainly utilizes hydrogels formed by oxidized dextran and carboxymethyl chitosan, while using orthoesters to enhance the solubility of targeted drugs in the hydrogel. Furthermore, orthoesters can carry targeted drugs to escape efficiently from the hydrogel. During the escape process, as the orthoesters degrade, the targeted drugs can self-assemble into nanoparticles, thereby enhancing the retention capacity of targeted drugs at the tumor site.

[0021] 2. This invention utilizes a hydrogel formed by oxidized dextran and carboxymethyl chitosan, combined with orthoester, to improve the solubility of targeted drugs in the hydrogel, enhance the enrichment capacity of targeted drugs, and reduce systemic toxic side effects. This solves the problems of poor solubility of nanomedicines, which are easily cleared after intravenous administration and have poor enrichment effects at tumor sites.

[0022] 3. The preparation method of the present invention is simple and the materials are widely available, making it suitable for widespread application.

[0023] 4. The organic composite hydrogel prepared by this invention has certain injectability and self-adaptability, and achieves efficient enrichment of targeted drugs at the tumor site, providing a new treatment option for tumor treatment, improving the treatment effect while reducing toxic side effects. Attached Figure Description

[0024] Figure 1 The image shows the 1H NMR spectrum of the orthoester prepared in Example 2.

[0025] Figure 2 The infrared and X-ray diffraction spectra of carboxymethyl chitosan, oxidized dextran, Gel from Example 1, FOODS from Example 2, Gel-FOE from Example 3, and Gel-FOEDS from Example 2 are shown. (a) is the infrared spectrum; (b) is the X-ray diffraction spectrum.

[0026] Figure 3 This is a scanning electron microscope image of the gel-FOEDS from Example 2 after lyophilization.

[0027] Figure 4 The images show the adhesion test results of Gel-FOEDS in Example 2. Specifically, (a1) to (a3) ​​show the adhesion test results of Gel-FOEDS in Example 2 on the surface of pig skin; (b1) to (b3) show the adhesion test results of Gel-FOEDS in Example 2 on the surface of pork; and (c1) to (c3) show the adhesion test results of Gel-FOEDS in Example 2 on the surface of pig liver.

[0028] Figure 5 The diagram shows the rheological properties of Gel-FOEDS in Example 2. A represents the change in elastic modulus and viscous modulus of Gel-FOEDS in Example 2 over time; B represents the shear rate-viscosity diagram of Gel-FOEDS in Example 2; the inset of B is the test diagram of the shear rate-viscosity test; C represents the change in elastic modulus and viscous modulus of Gel-FOEDS in Example 2 over strain; and D represents the scan diagram of elastic modulus and viscous modulus of Gel-FOEDS in Example 2 under alternating high and low strain conditions.

[0029] Figure 6 This is a graph showing the in vitro degradation rate of Gel-FOEDS in Example 2.

[0030] Figure 7The following are in vivo degradation diagrams of Gel-FOEDS in Example 2. Among them, (a) to (f) are in vivo degradation comparison diagrams of Gel-FOEDS in Example 2 on day 0, day 0.5, day 1, day 3, day 5, and day 7.

[0031] Figure 8 This is the 1H NMR spectrum of the orthoester ester escaping from the hydrogel.

[0032] Figure 9 This is a graph showing the cumulative release rate of the targeted drug using Gel-FOEDS in different environments under different conditions, as shown in Example 2. In the graph, A represents the cumulative release rate of DAS; B represents the cumulative release rate of SUN.

[0033] Figure 10 Characterization of targeted drug nanoparticles. A shows the particle size distribution of the targeted drug nanoparticles; B shows the particle size variation of the targeted drug nanoparticles from 0h to 48h; and C shows the surface Zeta potential of the targeted drug nanoparticles as a function of pH.

[0034] Figure 11 The diagram shows the uptake capacity of tumor cells for targeted drugs. Among them, (a1) to (a4) are the uptake capacity diagrams of tumor cells for Gel-NPs; (b1) to (b4) are the uptake capacity diagrams of tumor cells for Gel-FOEDS.

[0035] Figure 12 The figures show the cytotoxicity test results for different experimental groups. Specifically, A represents the H22 cytotoxicity test result for different experimental groups; B represents the HepG2 cytotoxicity test result for different experimental groups; C represents the MCF-7 cytotoxicity test result for different experimental groups; and D represents the CT26 cytotoxicity test result for different experimental groups.

[0036] Figure 13 The distribution maps of targeted drugs in mice for different experimental groups are shown. Among them, (a1) to (a6) are the distribution maps of H2O-ICG in mice from day 1 to day 11; (b1) to (b6) are the distribution maps of FOE-ICG in mice from day 1 to day 11; (c1) to (c6) are the distribution maps of FEEDS-ICG in mice from day 1 to day 11; and (d1) to (d6) are the distribution maps of Gel-FOEDS-ICG in mice from day 1 to day 11.

[0037] Figure 14 This is a graph showing the blood compatibility test results for the hydrogel. Figure 14 The illustration is a photograph of a centrifuge tube.

[0038] Figure 15 Images of tumors in mice from different experimental groups.

[0039] Figure 16 The figures show the curves of tumor volume and body weight of mice in different experimental groups over time. Among them, (a) shows the curve of tumor volume of mice in different experimental groups over time; and (b) shows the curve of body weight of mice in different experimental groups over time.

[0040] Figure 17 The images show pathological sections of mice from different experimental groups. (a1)–(a6) are pathological sections of major organs and tumors from mice in the saline control group; (b1)–(b6) are pathological sections of major organs and tumors from mice in the Gel-NPs group; (c1)–(c6) are pathological sections of major organs and tumors from mice in the FEEDS group; and (d1)–(d6) are pathological sections of major organs and tumors from mice in the Gel-FOEDS group. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] In this invention, the molecular weight of carboxymethyl chitosan is 20,000–21,000; the degree of carboxyl substitution is 35%–90%; and the amino unit content is 70%–90%. Due to batch-to-batch variability in the production of carboxymethyl chitosan, the structure of the carboxymethyl chitosan in the following embodiments of this invention was determined experimentally. Specifically, the carboxymethyl chitosan used in the following embodiments has a molecular weight of 21,000, a degree of carboxyl substitution of 41.8%, and an amino unit content of 74.7%.

[0044] In the following embodiments, unless otherwise specified, the methods described are conventional methods; the reagents and materials described are commercially available unless otherwise specified. The room temperature is 25℃±5℃.

[0045] Example 1

[0046] A method for preparing a hydrogel includes the following steps:

[0047] Step 1: Weigh 0.0308 mol of dextran powder and 0.0308 mol of sodium periodate and dissolve them together in 125 mL of sodium acetate buffer solution to obtain a mixed solution; wherein the molar ratio of sodium periodate to glucose units in dextran is 1:2. The molecular weight of dextran is 70,000. Then, stir the mixed solution at room temperature in the dark for 2 hours. After the reaction is complete, quench any unreacted sodium periodate with ethylene glycol. Dialyze the reaction solution in deionized water using a 3.5 kD dialysis bag for 3 days. Finally, freeze-dry the dialysate to obtain oxidized dextran, denoted as O-Dex.

[0048] Step 2: Dissolve oxidized dextran in water to prepare a 10 wt% oxidized dextran solution, denoted as O-Dex solution. Dissolve carboxymethyl chitosan powder with a molecular weight of 21000 in water to prepare a 5 wt% carboxymethyl chitosan solution, denoted as CMCS solution. Testing showed that the carboxymethyl chitosan contained 74.7% amino units and had a carboxyl substitution degree of 41.8%.

[0049] Step 3: Add 10 wt% O-Dex solution and 5 wt% CMCS solution sequentially to centrifuge tubes at volume ratios of 2:1, 1:1, and 1:2, mix, vortex, and let stand for 30 seconds. After the reaction is complete, a hydrogel is prepared.

[0050] Table 1 Comparison of gelation time of hydrogels prepared with different dosage ratios of O-Dex and CMCS

[0051]

[0052] Note: Volume ratio a The volume ratio is 10 wt% O-Dex solution and 5 wt% CMCS solution.

[0053] Experimental results showed that the gelation time of the hydrogel decreased with increasing addition of carboxymethyl chitosan solution. When the volume ratio of 10 wt% O-Dex solution to 5 wt% CMCS solution was 1:1, the hydrogel exhibited a moderate gelation time and good mechanical strength. Therefore, a volume ratio of 10 wt% O-Dex solution to 5 wt% CMCS solution of 1:1 was determined, resulting in the hydrogel Gel-2 with the best performance. Subsequent tests were conducted based on this ratio, and the hydrogel referred to as Gel in these tests will be used in the overall testing process.

[0054] Example 2

[0055] A method for preparing an organic composite hydrogel includes the following steps:

[0056] Step 1: Under nitrogen atmosphere, 0.2 mol of 3-amino-1,2-propanediol and 0.3 mol of ethyl trifluoroacetate were added to a three-necked reaction flask, along with 150 mL of anhydrous acetonitrile as the reaction solvent. The mixture was stirred vigorously for 12 h. After removing the volatile solvent by rotary evaporation under reduced pressure, the crude product was dissolved in ethyl acetate and washed with 10 wt% sodium carbonate aqueous solution and saturated brine. The organic phase was then dried under vacuum after removing the organic solvent by rotary evaporation to obtain intermediate product 1. 0.1 mol of intermediate product 1 and 0.4 mol of trimethyl orthoformate were added to a dry reaction flask, followed by 0.01 mol of p-toluenesulfonic acid as a catalyst and 150 mL of anhydrous acetonitrile as the reaction solvent. The mixture was stirred vigorously for 12 h. After removing the volatile solvent by rotary evaporation under reduced pressure, the crude product was dissolved in ethyl acetate and washed with 10 wt% sodium carbonate aqueous solution and saturated brine. The organic phase was then dried under vacuum after removing the organic solvent by rotary evaporation to obtain the orthoformate ester, denoted as FOE. The specific reaction equation is as follows:

[0057] .

[0058] Depend on Figure 1 The 1H NMR spectrum confirms that the orthoester was successfully synthesized in Example 2 of this invention.

[0059] Step 2: Dissolve the two targeted drugs, dasatinib and sunitinib, in the orthoester ester prepared in Step 1 at a mass ratio of 1:1. The ratio of targeted drug to orthoester ester is 8 mg: 1 mL. The resulting targeted drug-orthoester ester solution is denoted as FOODS solution.

[0060] Step 3: Weigh 0.0308 mol of dextran powder and 0.0308 mol of sodium periodate and dissolve them together in 125 mL of sodium acetate buffer solution to obtain a mixed solution; wherein the molar ratio of sodium periodate to glucose units in dextran is 1:2. The molecular weight of dextran is 70,000. Then, stir the mixed solution in the dark at room temperature for 2 hours. After the reaction is complete, quench any unreacted sodium periodate with ethylene glycol. Dialyze the reaction solution in deionized water using a 3.5 kD dialysis bag for 3 days. Finally, freeze-dry the dialysate to obtain oxidized dextran, denoted as O-Dex.

[0061] Step 4: Dissolve oxidized dextran in water to prepare a 10 wt% oxidized dextran solution, denoted as O-Dex solution. Dissolve carboxymethyl chitosan powder with a molecular weight of 21000 in water to prepare a 5 wt% carboxymethyl chitosan solution, denoted as CMCS solution. Testing showed that the carboxymethyl chitosan contained 74.7% amino units and had a carboxyl substitution degree of 41.8%.

[0062] Step 5: Add 10 wt% oxidized dextran solution, FEODS solution, and 5 wt% carboxymethyl chitosan solution sequentially to a centrifuge tube at a volume ratio of 1:1:1, mix, vortex, and let stand for 30 seconds. After the reaction is complete, an injectable organic composite hydrogel, denoted as Gel-FOEDS, is prepared. The drug loading concentration of the targeted drug in the organic composite hydrogel is 10 mg / mL.

[0063] Example 3

[0064] A method for preparing an organic composite hydrogel differs from Example 2 in that step 2 is omitted. The specific preparation method is as follows: Orthoester ester (FOE) is prepared according to the method in Example 2. Oxidized dextran is prepared according to the method in Example 2, and a 10 wt% oxidized dextran solution is prepared. A 5 wt% carboxymethyl chitosan solution is prepared. At a volume ratio of 1:1:1, 10 wt% O-Dex solution, orthoester ester, and 5 wt% CMCS solution are sequentially added to a centrifuge tube, mixed, vortexed, and allowed to stand for 30 seconds. After the reaction is complete, an injectable organic composite hydrogel, denoted as Gel-FOE, is obtained. The drug loading concentration of the targeted drug in the organic composite hydrogel is 10 mg / mL.

[0065] Example 4

[0066] An organic composite hydrogel preparation method differs from Example 2 in that the FEODS solution prepared in steps 1 and 2 is replaced with a targeted drug solution. Specifically, the preparation method is as follows: Oxidized dextran is prepared according to the method in Example 2, and a 10 wt% oxidized dextran solution is prepared. A 5 wt% carboxymethyl chitosan solution is prepared. Dasatinib and sunitinib, two targeted drugs, are mixed at a mass ratio of 1:1 and dissolved in dimethyl sulfoxide. The dimethyl sulfoxide is then removed by dialysis. The resulting solution is added to the 10 wt% oxidized dextran solution and the 5 wt% carboxymethyl chitosan solution in an equal volume ratio, vortexed, and allowed to stand for 30 seconds. After the reaction is complete, an organic composite hydrogel, denoted as Gel-NPs, is obtained. The drug loading concentration of the targeted drug in the organic composite hydrogel is 10 mg / mL.

[0067] The performance of the hydrogels and organic composite hydrogels prepared in the above embodiments is then tested.

[0068] Carboxymethyl chitosan (CMCS) is the full name of the chemical compound. Oxidized dextran (O-Dex) is the full name of the chemical compound. Orthoesters are abbreviated as FOE. Dasatinib (DAS) is the full name of the chemical compound. Sunitinib (SUN) is the full name of the chemical compound. Targeted drugs—orthoesters—are abbreviated as FOODS.

[0069] Test 1: Infrared spectroscopy test.

[0070] Carboxymethyl chitosan, oxidized dextran, Gel from Example 1, FOODS from Example 2, Gel-FOE from Example 3, and Gel-FOEDS from Example 2 were used as test samples, and the scanning range was 500 cm⁻¹. -1 ~4000cm -1 Infrared spectroscopy was performed at the wavelength, and the results are as follows: Figure 2 Figure (a)

[0071] Depend on Figure 2 As can be observed in Figure (a), oxidized dextran at 1733 cm⁻¹ -1 The presence of a characteristic peak at 1635 cm⁻¹ indicates the stretching vibration of the -C=O bond on the aldehyde group, suggesting that some of the hydroxyl groups in the dextran structure have been converted into aldehyde groups after oxidation, indicating successful dextran oxidation. -1 A new absorption peak also appeared, attributed to the imine bond -C=N-, indicating that Schiff base crosslinking occurred between carboxymethyl chitosan and oxidized dextran. These results demonstrate that Example 2 of this invention successfully prepared oxidized dextran, and that an organic composite hydrogel was ultimately prepared by utilizing the Schiff base crosslinking reaction between oxidized dextran and carboxymethyl chitosan.

[0072] Test 2: X-ray diffraction test.

[0073] Carboxymethyl chitosan, oxidized dextran, Gel from Example 1, FOODS from Example 2, Gel-FOE from Example 3, and Gel-FOEDS from Example 2 were used as test samples, and X-ray diffraction tests were performed in an angle range of 5° to 80°.

[0074] like Figure 2As shown in Figure (b), carboxymethyl chitosan exhibits a sharp peak at 20.5°, while O-Dex shows a broad peak at 21.7°, and Gel shows a broad peak at 22.8°. This indicates that O-Dex and CMCS undergo cross-linking, and the formation of covalent bonds alters the crystal structure of the substances, proving the successful preparation of the organic composite hydrogel. Furthermore, it can be seen that the broad peak positions of FOODS, Gel-FOE, and Gel-FOEDS are the same, indicating that the organic composite hydrogel structure is correct, and the introduction of orthoesters and targeted drugs did not change the structure of the organic composite hydrogel. The mass difference before and after hydrogel formation was measured using the lyophilization method, and the cross-linking degree of the hydrogel was calculated to be 80.9%.

[0075] Test 3: Scanning electron microscopy observation.

[0076] Gel-FOEDS from Example 2 was gelled using a Schiff base reaction with carboxymethyl chitosan and oxidized dextran. Once the morphology of the gel-FOEDS was stable and unchanged, the macroscopic state of the hydrogel was recorded using a camera. The gel-FOEDS was then rapidly frozen in liquid nitrogen to induce brittle fracture, followed by freeze-drying. A thin slice was cut from the brittle fracture surface of the freeze-dried gel-FOEDS, with the brittle fracture surface facing upwards. The brittle fracture surface was then adhered to the silicon wafer surface using conductive tape. The silicon wafer was then attached to the edge of the sample stage. After gold sputtering, the sample was observed using a scanning electron microscope.

[0077] like Figure 3 As shown, the microstructure of Gel-FOEDS in Example 2 was observed using a scanning electron microscope. It can be seen that the freeze-dried Gel-FOEDS exhibits a regular and dense porous structure.

[0078] Test 4: Adhesion test.

[0079] To verify that the organic composite hydrogel Gel-FOEDS of Example 2 has good adhesion, different animal tissues, such as skin, muscle and liver, were selected for adhesion tests; then different materials, such as metal, plastic, glass and rubber, were selected for testing. Gel-FOEDS was attached to the surface of the above different materials and the adhesion was recorded with a camera.

[0080] like Figure 4 As shown, the Gel-FOEDS of Example 2 can adhere well to the surfaces of different animal tissues, such as pigskin, pork, and pig liver, and remains firmly attached to the tissue surface even after twisting, flipping, and folding. This indicates that the good adhesion of the Gel-FOEDS of Example 2 is due to the easy formation of non-covalent forces such as hydrogen bonds between the Gel-FOEDS and different materials.

[0081] Test 5: Elastic modulus and viscous modulus test, and shear thinning ability test.

[0082] The organic composite hydrogel Gel-FOEDS of Example 2 was tested using a rotational rheometer. A single-frequency scan was performed on the Gel-FOEDS to determine its elastic modulus and viscous modulus under isothermal and isostatic stress conditions. The elastic modulus is denoted as G' and the viscous modulus as G''. Shear rate scans were performed on the Gel-FOEDS within a range of 0.1 s⁻¹. -1 ~100s -1 The shear thinning ability of Gel-FOEDS was determined, which is an important indicator for judging the injectability of Gel-FOEDS. Finally, the critical strain of Gel-FOEDS was found under different strain conditions from 1% to 200%. Then, the changes of G' and G” were detected by alternating high and low strain scans to detect the self-healing performance of Gel-FOEDS.

[0083] like Figure 5 As shown in Figure A, the G' of Gel-FOEDS in Example 2 is always greater than G, indicating that Gel-FOEDS is already in a gel state. Over time, Gel-FOEDS gradually stabilizes. Shear thinning refers to the phenomenon that the apparent viscosity of a non-Newtonian fluid decreases with increasing shear rate. This is because the internal structure of the fluid changes under shear force, leading to a decrease in viscosity. This property is extremely important for applications such as gel injection and drug delivery.

[0084] like Figure 5 As shown in Figure B, the viscosity decreases and stabilizes with increasing shear rate, and Gel-FOEDS can pass smoothly through the syringe needle, indicating good injectability. By increasing the oscillatory strain at a constant frequency and observing the changes in G' and G”, the critical strain value required to disrupt the gel network and transition to a solution state is found to be 101.2%. Figure 5 The C-plot was used as a standard to determine alternating scans of high strain (150%) and low strain (1%). Figure 5 The D-plot shows that when under high strain, G” is greater than G’, indicating that Gel-FOEDS is in a sol state. When under low strain, G” is less than G, indicating that Gel-FOEDS becomes a gel state. This change can be repeated with alternation between high and low strain, indicating that Gel-FOEDS has certain self-healing properties.

[0085] Test 6: In vitro degradation test.

[0086] The organic composite hydrogel Gel-FOEDS prepared in Example 2 was weighed and designated as M1. It was then placed in phosphate buffer solutions with pH values ​​of 7.4, 6.8, and 5.0, respectively, with three parallel controls for each group. The solutions were placed in a 37°C constant-temperature shaker. At preset time points—0h, 1h, 3h, 6h, 12h, 24h, 48h, 72h, 96h, and 120h—the phosphate buffer solution was aspirated with a syringe, and the remaining Gel-FOEDS was weighed and denoted as M. r The residual mass ratio of Gel-FOEDS is calculated using the following formula:

[0087] Residual mass ratio = (M r / M1)×100%; where M r M1 represents the mass of the remaining Gel-FOEDS; M2 represents the mass of the initial Gel-FOEDS.

[0088] Gel-FOEDS was added to buffer solutions at pH 5.0, 6.8, and 7.4, respectively. Figure 6 As shown, under different pH conditions, Gel-FOEDS undergoes water absorption and swelling in the first hour, leading to an increase in its mass. Subsequently, the swelling stops, and Gel-FOEDS begins to degrade gradually. The degradation rate is relatively fast in the first 48 hours, followed by slow degradation. Furthermore, the degradation rate of Gel-FOEDS at pH 5.0 is greater than that at pH 6.8 and pH 7.4, mainly because the hydrolysis rate of imine bonds is affected by the ambient pH; the stronger the acidity, the faster the degradation rate.

[0089] Test 7: In vivo degradation test.

[0090] An equal amount of methylene blue-stained Gel-FOEDS was subcutaneously injected into the chest of mice using a syringe. Mice were dissected at preset time points: day 0, day 0.5, day 1, day 3, day 5, and day 7, and the degradation of the organic composite hydrogel was recorded with a camera.

[0091] Over time, the volume of Gel-FOEDS gradually decreases, indicating that the degradation of Gel-FOEDS is good, which is conducive to the stable release of targeted drugs.

[0092] Figure 7 The in vivo degradation observation results showed that Gel-FOEDS was almost completely degraded in vivo by the third day, and the in vivo degradation rate was higher than that in vitro. This may be because the action of enzymes and pH changes in vivo accelerated the collapse of the gel network, resulting in a higher in vivo degradation rate than in vitro.

[0093] First, phosphate buffer solutions with pH values ​​of 6.8 and 7.4 were prepared. Gel-FOEDS were immersed in the buffer solutions and placed in a 37°C constant-temperature shaker. At preset time points, Gel-FOEDS were removed and sectioned in different regions. The morphological changes of FOEs were observed under a microscope. Samples of the buffer solution were then freeze-dried at 0.5h, 1h, and 1.5h. 1 H NMR was used to detect FOE degradation.

[0094] like Figure 8 As shown, FOE was detected in the buffer solution, and it gradually degraded over time, reaching almost complete degradation at 1.5 h. This result indicates that FOE can escape from the organic composite hydrogel, and that the presence of F-type groups in the FOE structure enhances its ability to encapsulate two targeted drugs and penetrate into tumor tissue.

[0095] Test 8: Drug release rate analysis.

[0096] First, prepare 0.2M phosphate buffer solutions with pH values ​​of 7.4, 6.8, and 5.0. Add 1 mL of Gel-FOEDS to a dialysis bag with a molecular weight cutoff of 3500 Da, and immerse the dialysis bag in 5 mL of 0.2M phosphate buffer solutions with different ratios. Replace the buffer solution at preset time points. Finally, use a multi-mode microplate reader to detect the cumulative release rate of DAS and SUN.

[0097] like Figure 9 As shown in Figure A, on the fifth day, the cumulative release rate of SUN reached 83% at pH 5.0, while the cumulative release rates were 61% and 56% at pH 6.8 and pH 7.4, respectively.

[0098] like Figure 9 As shown in Figure B, on day 5, the cumulative release rate of DAS reached 82% at pH 5.0, while the cumulative release rates were 63% and 60% at pH 6.8 and pH 7.4, respectively. This is because the imine bond network in the organic composite hydrogel collapses at different rates under different pH conditions, allowing DAS and SUN to continuously escape from the organic composite hydrogel, thus achieving sustained release of the targeted drug.

[0099] Test 9: Hydrated particle size, polydispersity index and surface zeta potential test.

[0100] Gel-FOEDS were immersed in 0.2 M phosphate buffer solutions at pH 5.0, 6.8, and 7.4, and placed in a constant-temperature shaker at 100 rpm and 37°C. Extracts were collected at preset time points of 0 h, 3 h, 6 h, 12 h, 24 h, and 48 h, and fresh buffer solution was added. The hydrated particle size, polydispersity index, and surface zeta potential of the extracts were determined using dynamic light scattering (DLS), with three replicate measurements performed for each sample.

[0101] The particle size of nanoparticles was determined continuously for 96 hours at pH 6.8 using dynamic light scattering technology. The experiment was conducted in three groups, and the average value of the three groups was taken. The extract was then lyophilized, and the microstructure and size were observed and measured using transmission electron microscopy.

[0102] like Figure 10 As shown in Figure A, the average particle size was measured to be 163.8 nm. Subsequent transmission electron microscopy (TEM) detected the presence of targeted drug nanoparticles, further validating that the targeted drug can escape from Gel-FOEDS in the form of nanoparticles. Figure 10 As shown in Figure B, the particle size of the targeted drug nanoparticles in the extract was detected at preset time points, and remained almost unchanged, indicating that the targeted drug nanoparticles can remain stable under normal physiological conditions. Figure 10 As shown in Figure C, the surface potential gradually increases as the pH value decreases. This is due to the formation of amino protonation on the surface of the targeted drug nanoparticles, which is beneficial for the targeted drug nanoparticles to be taken up by tumor cells.

[0103] In summary, targeted drug nanoparticles can indeed successfully escape from gel-FOEDS and remain stable under physiological conditions. Furthermore, the protonation effect of targeted drug nanoparticles under acidic conditions can enhance their uptake by tumor cells, thereby exerting an anti-tumor effect.

[0104] Test 10: Qualitative uptake by cells.

[0105] H22 mouse liver cancer cells, abbreviated as H22 cells. 4',6-diamidino-2-phenylindole, abbreviated as DAPI, is used to disperse H22 cells in RPMI-1640 cell culture medium after washing, digestion, and pipetting. H22 cells and HepG2 cells were cultured at a ratio of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 100 μL in six-well plates and cultured. They were then placed in a sterile incubator and cultured for an additional 8 hours. After cell attachment, 200 μL of Gel-NPs and 200 μL of Gel-FOEDS were added to each well. After incubation, cells were digested and collected, then fixed with 4% paraformaldehyde for 10 min. After fixation, the paraformaldehyde was removed, and the cells were washed with PBS. The nuclei were then stained with DAPI for 10 min. Finally, the analysis and imaging were performed using a confocal laser scanning microscope. Successful uptake of targeted drugs by tumor cells is crucial for treatment success. H22 cells were used to assess the uptake capacity of targeted drugs by tumor cells under the acidic extracellular environment of pH 6.8. DAS (drug-assisted chemiluminescence) produces an aggregation luminescence effect, which can be used to track intracellular drugs.

[0106] like Figure 11 As shown, obvious green fluorescence was observed in cells after 4 hours. Furthermore, the Gel-FOEDS group showed significantly stronger targeted drug uptake ability than Gel-NPs due to the presence of FOE. This is because the FOE structure contains fluorinated groups, which enhance its permeability.

[0107] Test 11: The MTT assay was used to evaluate the in vitro activity of the organic composite hydrogel.

[0108] The tumor cells used in the experiment were H22, HepG2, MCF-7, and CT26 cells. Experimental groups included Gel, Gel-NPs, FODDS, and Gel-FOEDS. The concentration gradients of the targeted drug nanoparticles were 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. Gel was a blank hydrogel without nanoparticles, and its components, carboxymethyl chitosan and oxidized dextran, had the same concentrations as Gel-FOEDS. 96-well plates were used, and the experimental procedures were strictly performed in a sterile environment. To further evaluate the in vitro antitumor activity of the organic composite hydrogel, H22, HepG2, MCF-7, and CT26 cells were used to construct tumor cell models, which were then co-incubated with different concentrations of the drug for 48 hours.

[0109] like Figure 12As shown in Figures A through D, the cell survival rate of the blank hydrogel group was above 80%, indicating that the blank hydrogel gel has good biocompatibility. However, with increasing concentration, the cell survival rate of the orthoester group fell below 50%, indicating that the orthoester has certain cytotoxicity. This is because the degradation products of the orthoester are toxic, leading to cell death. The cytotoxicity of the Gel-FOEDS group was stronger than that of the Gel-NPs group. The fluorinated groups in the orthoester of the organic composite hydrogel enhance the penetration of the targeted drug into tumor tissues, allowing for greater uptake of the targeted drug by cells. Therefore, the Gel-FOEDS group exhibited higher cytotoxicity, demonstrating that the Gel-FOEDS of Example 2 of this invention has excellent in vitro antitumor activity.

[0110] Test 12: Observe the distribution of targeted drugs at the tumor sites in mice.

[0111] Select tumors with a volume of 200 mm 3 Mice were subjected to hair removal treatment in the left axilla and divided into four groups. H2O-ICG, FOE-ICG, FOODS-ICG, and Gel-FOEDS-ICG were injected into the tumor site of the mice at a dose of 2.5 mg / kg. The distribution of targeted drugs in the tumor site of the mice was observed and photographed at preset time points of 1d, 3d, 5d, 7d, 9d, and 11d using a multi-mode in vivo imaging system.

[0112] Preparation of H2O-ICG: Indocyanine green is dissolved in water to obtain an aqueous solution of indocyanine green, denoted as H2O-ICG; wherein the concentration of indocyanine green is 1 mg / mL.

[0113] Preparation of FOE-ICG: Indocyanine green and orthoester are mixed evenly to obtain an indocyanine green-orthoester solution, denoted as FOE-ICG; wherein the concentration of indocyanine green is 1 mg / mL.

[0114] Preparation of FOODS-ICG: DAS, SUN and indocyanine green were mixed in a mass ratio of 1:1:1, and then the orthoester was added, vortexed and sonicated to obtain an indocyanine green-targeted drug-orthoester solution, denoted as FOODS-ICG; wherein the concentration of indocyanine green was 1 mg / mL.

[0115] Preparation of Gel-FOEDS-ICG: Following the method in Example 2, DAS, SUN, and indocyanine green were mixed in a mass ratio of 1:1:1, and then an orthoester was added to obtain an indocyanine green-targeted drug-orthoester solution, wherein the concentration of indocyanine green was 1 mg / mL. The indocyanine green-targeted drug-orthoester solution was then added dropwise to a 10 wt% oxidized dextran solution, and finally a 5 wt% carboxymethyl chitosan solution was added to form a gel; the volume ratio of the oxidized dextran solution to the carboxymethyl chitosan solution was 1:1, thus preparing an organic composite hydrogel, denoted as Gel-FOEDS-ICG. The drug loading concentration of the targeted drug in the organic composite hydrogel was 10 mg / mL.

[0116] To investigate the enrichment of targeted drugs in mouse tumor sites, H2O-ICG, FOE-ICG, FOODS-ICG, and Gel-FOEDS-ICG were injected into mouse tumor sites in situ at a dose of 100 μL, and their fluorescence intensity was observed using a multimodal in vivo imaging system.

[0117] like Figure 13 As shown, H2O-ICG diffuses rapidly at the tumor site, its fluorescence disappears by day 5, indicating a short residence time at the tumor site. Because orthoesters can enhance the penetration of targeted drugs at the tumor site, FOE-ICG has a longer residence time than H2O-ICG. Furthermore, from... Figure 13 As can be seen, the fluorescence intensity of both FEODS-ICG and Gel-FOEDS-ICG showed a trend of first increasing and then decreasing, and the fluorescence of the Gel-FOEDS-ICG group persisted longest at the tumor site, exhibiting the best retention effect. This conclusion is also verified by the fluorescence decay trend graph in the figure. In summary, organic composite hydrogels can enhance the accumulation of targeted drugs at the tumor site, thereby enabling the sustained release of targeted drugs to kill tumors.

[0118] Test 13: Hemolytic Activity Assessment. Hemolysis refers to the destruction of red blood cells in vitro or in vivo, leading to the release of intracellular hemoglobin and causing a hemolytic reaction, which can result in death in severe cases. Therefore, before conducting in vivo antitumor experiments, it is necessary to assess the hemolytic activity of the organic composite hydrogel Gel-FOEDS from Example 2 to detect its blood compatibility. The specific steps are as follows: First, take 500 μL of whole mouse blood, add a small amount of heparin sodium to prevent blood clotting, disperse it in 5 mL of PBS buffer solution, centrifuge at 2500 rpm for 5 min, collect the lower layer of red blood cells, repeat the above steps until the supernatant is clear, then collect the red blood cells and evenly disperse them in 10 mL of physiological saline to prepare a red blood cell suspension for later use. Experimental group: Mix 500 μL of red blood cell suspension with different concentrations of Gel-FOEDS samples ranging from 31.25 μg / mL to 1000 μg / mL, and incubate at 37°C for 1 h. Positive control group: 500 μL of cell suspension and 500 μL of deionized water were mixed and incubated at 37°C for 1 h. Negative control group: 500 μL of cell suspension and 500 μL of physiological saline were mixed and incubated at 37°C for 1 h. After incubation, the cells were centrifuged at 10,000 rpm for 5 min. The centrifuge tubes were arranged in a row and photographed. 200 μL of the supernatant was added to a 96-well cell culture plate, and the absorbance of each well was measured and recorded using a multi-mode microplate reader at a UV wavelength of 570 nm. The hemolysis rate was calculated using the following formula:

[0119] Hemolysis rate (%) = (OD) 样品组 -OD 阴性对照组 / OD 阳性对照组 -OD 阴性对照组 ) × 100%; where, OD 样品组 Indicates the absorbance value of the experimental group; OD 阴性对照组 This represents the absorbance value of the negative control group; OD 阳性对照组 This represents the absorbance value of the positive control group.

[0120] like Figure 14 As can be seen, in the positive control group, red blood cell membranes ruptured, hemoglobin was released, and the supernatant appeared distinctly red. In contrast, the experimental groups with different concentrations of Gel-FOEDS showed clear supernatants with no obvious hemolysis compared to the negative control group. The absorbance of each experimental group was measured using a multi-functional microplate reader at 540 nm. The hemolysis rate, calculated using the hemolysis rate formula, was less than 5% in each experimental group, indicating that the organic composite hydrogel of Example 2 has high blood compatibility and good biosafety.

[0121] Test 14: In vivo antitumor experiment. Male ICR mice weighing between 18g and 22g were used in the experiment, and all animals were strictly managed according to laboratory animal regulations. The specific procedures were as follows: H22 cell lines were injected intraperitoneally into mice and cultured for 7-10 days. Afterward, ascites fluid was aspirated and transferred to 10mL sterile centrifuge tubes. The supernatant was removed by centrifugation, and the cells were washed three times with sterile PBS solution until the supernatant was clear. The cells were then redispersed with sterile physiological saline. Trypan blue staining and hemocytocyte count showed a cell viability greater than 95%. The cells were then diluted with physiological saline to approximately 1 million / mL. The diluted cell suspension was injected into the left axilla of mice using a syringe, with an injection volume of approximately 100μL. The tumor volume was monitored until it reached 180mm². 3 -220mm 3 Then, subsequent anti-tumor experiments can be conducted. The tumor volume in mice can be calculated using the following formula: Tumor volume (mm²) 3 ) = A × B 2 / 2; where A is the long diameter of the tumor; B is the short diameter of the tumor. When the tumor volume in mice reaches 200 mm... 3 Mice were randomly divided into four groups, with six mice in each group. The control group consisted of: a saline control group (saline was injected orally into the tumor site), a gel-NPs group (gel-NPs were injected orally into the tumor site), a FODDS group (FOEDS was injected orally into the tumor site), and a gel-FOEDS group (Gel-FOEDS was injected orally into the tumor site).

[0122] The mice were administered the drug once on days 1, 3, and 6, with DAS at a dose of 20 mg / kg. Mice were then observed for 12 days, with daily recording of body weight and tumor volume. After 12 days, the mice were sacrificed, and their major organs (heart, liver, spleen, lungs, kidneys) and tumor tissue were harvested. The extracted tumor tissue was weighed and photographed. The removed organ tissues and tumors were fixed in 4% paraformaldehyde, stained with hematoxylin and eosin, and then examined under an inverted microscope to assess the degree of damage.

[0123] This invention utilizes a mouse liver cancer tumor model to verify the in vivo antitumor effect of organic composite hydrogels. Orthotopic injections of DAS were performed at the mouse tumor site on days 1, 3, and 6, with a dosage of 20 mg / kg.

[0124] like Figure 15 The tumor images of mice show that, compared with the saline control group, the Gel-NPs, FOODS, and Gel-FOEDS groups all exhibited varying degrees of tumor inhibition.

[0125] The curve of tumor volume change over time in mice, as shown in... Figure 16 Figure (a) shows that at 12 days, the tumor volume in the saline control group mice reached 1420 mmHg. 3 Around 290 mmHg, the tumor volume in the Gel-NPs group was approximately 290 mmHg. 3 The tumor volume in the Gel-FOEDS group was approximately 160 mm. 3 This indicates that the Gel-FOEDS group has a more significant anti-tumor effect. This is because the organic composite hydrogel accumulates for a long time at the tumor site, and the orthoester enhances the penetration of targeted drugs at the tumor site, thus resulting in a better anti-tumor effect.

[0126] The curve showing the change in body weight of mice over 12 days, as shown below. Figure 16 Figure (b) shows that the mice's weight did not change significantly and they were in good condition with no obvious toxic side effects, indicating that the organic composite hydrogel of Example 2 promoted the long-term accumulation of targeted drugs at the tumor site, thereby reducing systemic toxicity.

[0127] The results of hematoxylin and eosin staining were analyzed. Pathological sections of major organs and tumors in mice stained with hematoxylin and eosin were shown. Figure 17 .

[0128] Depend on Figure 17 As can be seen, compared with the saline control group, tumor sections from both Gel-NPs and FOODS showed varying degrees of damage, with the Gel-FOEDS group exhibiting greater damage. This is because the organic composite hydrogel Gel-FOEDS can achieve long-term enrichment of targeted drugs at the tumor site, and the permeation-enhancing function of the orthoester enhances its anti-tumor effect, resulting in a more significant tumor-killing effect. In contrast, the major organ tissues of mice—heart, liver, spleen, lungs, and kidneys—did not show significant damage compared to the saline group. This further verifies that the organic composite hydrogel prepared in Example 2 of this invention can remain at the tumor site for a long time, thereby achieving long-term enrichment of targeted drugs at the tumor site and exhibiting good biocompatibility.

[0129] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an organic composite hydrogel, characterized in that, Includes the following steps: The targeted drug is dissolved in orthoester to obtain a targeted drug-orthoester solution; An organic composite hydrogel was obtained by mixing oxidized dextran solution, targeted drug-orthoester solution and carboxymethyl chitosan solution, vortexing and then allowing it to stand. The concentration of the oxidized dextran solution was 10 wt%; the concentration of the carboxymethyl chitosan solution was 5 wt%. The volume ratio of oxidized dextran solution to carboxymethyl chitosan solution is 1:0.5-2; Orthoesters are prepared by the following method: 3-Amino-1,2-propanediol and ethyl trifluoroacetate were reacted in a solvent system under stirring, and then subjected to rotary evaporation, extraction and drying to obtain intermediate product 1. Under the action of a catalyst, intermediate 1 and trimethyl orthoformate were stirred and reacted in a solvent system, and the orthoformate was obtained by rotary evaporation, extraction and drying. The targeted drugs are dasatinib and sunitinib; the drug loading concentration of the targeted drugs in the organic composite hydrogel is 0.01 mg / mL to 20 mg / mL.

2. The method for preparing the organic composite hydrogel according to claim 1, characterized in that, The volume ratio of oxidized dextran solution, targeted drug-orthoester solution and carboxymethyl chitosan solution was 1:1:1; the dosage ratio of targeted drug to orthoester was 8 mg: 1 mL.

3. The method for preparing the organic composite hydrogel according to claim 1, characterized in that, The molecular weight of carboxymethyl chitosan is 20,000 to 21,000; the degree of carboxyl substitution of carboxymethyl chitosan is 35% to 90%; and the amino unit content of carboxymethyl chitosan is 70% to 90%.

4. The method for preparing the organic composite hydrogel according to claim 1, characterized in that, The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1.5 to 2; The molar ratio of intermediate 1 to trimethyl orthoformate is 1:1 to 4; The solvent is any one of acetonitrile, dichloromethane, chloroform, tetrahydrofuran, and dioxane; The catalyst is p-toluenesulfonic acid; The molar ratio of intermediate product 1 to catalyst is 1:0.01 to 0.

03.

5. The method for preparing the organic composite hydrogel according to claim 1, characterized in that, Oxidized dextran is prepared by the following method: Dextran, sodium periodate and sodium acetate buffer solution were reacted in the dark with stirring. After the reaction was completed, the mixture was dialyzed and freeze-dried to prepare oxidized dextran. The molar ratio of sodium periodate to glucose units in dextran is 1:1 to 2; The molecular weight of dextran is 60,000 to 70,000.

6. An organic composite hydrogel, characterized in that, The organic composite hydrogel was prepared using the preparation method described in any one of claims 1 to 5.

7. The application of the organic composite hydrogel according to claim 6 in the preparation of antitumor pharmaceutical formulations, characterized in that, The tumor in question is a liver cancer tumor.

8. The use of the organic composite hydrogel of claim 6 in the preparation of a pharmaceutical formulation for inhibiting cancer cells, characterized in that, The cancer cells are at least one of H22 cells, HepG2 cells, MCF-7 cells, and CT26 cells.

9. The application according to claim 7 or 8, characterized in that, The pharmaceutical preparation is an injectable dosage form.

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