Anti-tumor hydrogels based on starvation therapy and preparation and use thereof

Abstract

The application discloses an anti-tumor hydrogel based on a starvation therapy and a preparation and application thereof. After simple mixing of a DHA solution, an AI solution, an Ink solution and an ALG solution, a DHA / AI / Ink@ALG is formed, an in-situ hydrogel is formed by simple injection in a tumor tissue, under 1064nm laser irradiation, alkyl radicals are generated by starting AI decomposition by using a low-temperature photothermal effect, damage of DHA-induced mitochondrial oxidative stress is intensified by using the alkyl radicals, and glucose uptake is reduced by strengthening DHA inhibition on GLUT1, so that DHA-induced cell starvation therapy is enhanced, the effect of anti-tumor treatment is significantly improved, and a new way is provided for high-efficiency and low-toxicity cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to antitumor hydrogels based on starvation therapy, their preparation and application. Background Technology

[0002] Abnormal cellular metabolism and excessive nutrient consumption are key physiological characteristics of tumors. Therefore, starvation therapy, which involves blocking blood supply and depleting key nutrients, has been extensively studied and has become an attractive cancer treatment method. Warburg discovered that tumor cells have a high glucose uptake requirement, and tumor growth and proliferation are promoted through anaerobic glycolysis. Tumor cells strongly rely on glycolysis for energy rather than oxygen. Based on this metabolic characteristic of tumor cells, researchers have developed two starvation therapies: glucose starvation therapy and vascular embolization therapy, for treating tumors. Vascular embolization therapy is a strategy of starvation treatment by blocking blood flow; however, its rapid blood flow may affect plaque formation or lead to ineffective embolization. Glucose starvation therapy uses glucose oxidase (GOx) as a starvation inducing agent, which can effectively deplete glucose in tumor cells; this therapy has received increasing attention.

[0003] GOX has demonstrated remarkable efficacy in cancer treatment, mediating various anticancer therapies. However, it also faces unavoidable limitations in tumor therapy: GOX, being a protein, is prone to degradation, leading to low bioavailability; it inhibits glutathione reductase in vitro in both cancer and non-cancer cells, potentially causing systemic toxicity with non-specific administration; and it lacks targeted delivery capabilities. The significant toxicity and instability of GOX limit its antitumor effects. Furthermore, excessive H2O2 produced by glucose oxidation not only accelerates tumor metastasis and invasion but also causes irreversible oxidative damage to surrounding normal tissues. Therefore, researchers are focusing on designing rationally designed drug delivery systems with targeted capabilities to deliver GOX directly to cancer cell sites, minimizing degradation and loss during transport.

[0004] For example, Chinese invention patent application No. 202111526167.8 discloses a Mn-based biodegradable MOF nanoreactor, its preparation method and application. The nanoreactor includes core Mn-based MOF nanoparticles with a pH-responsive shell copolymer PEG-CDM-PEI grafted onto its surface. The Mn-based MOF nanoparticles are loaded with the bioenzyme GOx and IDO immunosuppressant. Through pH / ROS dual-responsive drug controlled release, the combined therapy of tumor starvation / oxidation / IDO immunotherapy is regulated.

[0005] For example, Chinese invention patent application No. 202110395713.2 discloses a porphyrin-based metal-organic framework nanocarrier, which is obtained by co-loading glucose oxidase and a metal nanozyme with catalase-like activity into porphyrin-based metal-organic framework nanoparticles encapsulated in a cell membrane. Cell membrane modification enhances tumor accumulation at the tumor site; porphyrin molecules with sonodynamic effects form a metal-organic framework structure through coordination to improve the sonodynamic effect; the metal nanozyme decomposes hydrogen peroxide in tumor tissue to produce oxygen; and the glucose oxidase can consume glucose in tumor tissue, cutting off the energy supply to tumor cells, ultimately achieving the combined effect of sonodynamic and starvation therapy to enhance tumor killing.

[0006] For example, Chinese invention patent application number 201911083865.8 discloses a degradable antitumor nanomedicine based on starvation therapy, its preparation method, and its application. This invention dissolves dopamine hydrochloride in Tris-HCl buffer solution, adds glucose oxidase, and stirs rapidly. After secondary filtration, the liquid is centrifuged with an equal volume of a silver cube solution, and the precipitate is mixed. The mixture is then sonicated, allowed to stand, centrifuged again, and the precipitate is collected. The precipitate is resuspended in PBS buffer solution to obtain silver nanocubes loaded with glucose oxidase. The glucose oxidase loading of these particles is 10%–25%, with an average particle size of 100–150 nm, and they can degrade in the acidic and hydrogen peroxide environment of the tumor site. This nanomedicine combines the starvation therapy of glucose oxidase consuming glucose at the tumor site with the photothermal therapy of the silver nanocubes to achieve a tumor treatment effect.

[0007] The aforementioned nanoreactors all directly load glucose oxidase (GOx), making the systems relatively complex, and the limitations of GOX application cannot be completely avoided. Summary of the Invention

[0008] Given the problems existing in current technologies, and unlike the common approach of using glucose oxidase (GOx) to synergistically treat malignant tumors, the inventors have proposed a new solution: injecting DHA / AI / Ink@ALG into tumor tissue, where sodium alginate (ALG) interacts with calcium in the body. 2+ The reaction forms an in-situ hydrogel in tumor tissue. Under 1064nm laser irradiation, the low-temperature photothermal effect activates the decomposition of azodiisopropylimidazoline hydrochloride (AI) to generate alkyl free radicals. These alkyl free radicals exacerbate the damage of dihydroartemisinin (DHA)-induced mitochondrial oxidative stress and enhance DHA's inhibition of glucose transporter-1 (GLUT1) (leading to reduced glucose uptake). This enhances DHA-induced cell starvation therapy, significantly improving the efficacy of anti-tumor treatment and providing a new approach for highly effective and low-toxicity cancer therapy.

[0009] Therefore, a first aspect of the present invention is to provide an injectable hydrogel of the form DHA / AI / Ink@ALG.

[0010] In some specific embodiments of the present invention, the injectable hydrogel is a DHA / AI / Ink@ALG mixed solution obtained by ultrasonic homogenization of ALG solution with AI solution, DHA solution and Ink solution.

[0011] In some specific embodiments of the present invention, in the injectable hydrogel, the ALG solution is prepared by dissolving ALG powder in ultrapure water, the AI ​​solution is prepared by dissolving AI powder in ultrapure water, the DHA solution is prepared by dissolving DHA powder in dimethyl sulfoxide to obtain DHA stock solution and then diluting it with ultrapure water to the target concentration, and the Ink solution is obtained by diluting the Ink stock solution with ultrapure water. The Ink stock solution is prepared by the following steps: Ink powder is added to an aqueous solution of polyvinylpyrrolidone with a concentration of 8-10 mg / mL under ultrasonication, and after complete dispersion, it is transferred to a hydrothermal reactor and heated at 140-170°C for 5-7 hours, and then cooled to room temperature.

[0012] In some specific embodiments of the present invention, the concentration of ALG in the injectable hydrogel is 5-8 mg / mL, the concentration of AI is 1-2 mg / mL, the concentration of DHA is 1-2 mmol / L, and the concentration of Ink is 150-400 μg / mL.

[0013] A second aspect of the present invention is to provide the use of the above-described injectable hydrogel in the preparation of light-controlled tumor therapeutic drugs.

[0014] In some specific embodiments of the present invention, the tumor treatment is a starvation therapy for tumors.

[0015] In some specific examples of the present invention, the tumor is colorectal cancer, breast cancer, melanoma, or liver cancer.

[0016] In some specific embodiments of the present invention, the drug is an injectable reagent used for in situ injection into tumor tissue.

[0017] In some specific embodiments of the present invention, the light control refers to near-infrared light irradiation in the wavelength range of 1000 to 1700 nm.

[0018] In some specific embodiments of the present invention, the light control refers to 1064nm laser irradiation.

[0019] In this invention, the room temperature is 22-27℃.

[0020] The injectable hydrogel of this invention uses ALG as a drug carrier and contains Ink, AI, and DHA. This hydrogel can utilize the multifunctional ALG to interact with Ca in the tumor microenvironment. 2+Rapidly reacts, enabling the formation of an in-situ hydrogel DHA / AI / Ink@ALG-Ca within the tumor tissue. 2+ This invention immobilizes Ink, AI, and DHA within tumor tissue, preventing their diffusion into surrounding normal tissue, thus achieving drug immobilization. The drugs in the injectable hydrogel of this invention do not infiltrate surrounding normal tissue, thereby avoiding damage to adjacent normal tissue.

[0021] Simultaneously, under 1064nm laser irradiation, DHA / AI / Ink@ALG generates mild heat energy due to photothermal effects. This low heat promotes the decomposition of AI into alkyl free radicals, resulting in a significant decrease in mitochondrial membrane potential, a marked reduction in GULT1 expression, and a significant increase in apoptosis within tumor cells. Based on this research, the anti-tumor mechanism of this injectable hydrogel is hypothesized as follows: DHA not only inhibits the expression of glucose transporter-1 (GLUT1) on the cell membrane, thereby reducing cellular glucose uptake, but also generates ROS. Under 1064nm laser irradiation, Ink generates mild heat energy by absorbing light energy. The increased temperature causes AI to decompose into alkyl free radicals, thereby inducing mitochondrial damage within tumor cells. Therefore, when the injectable hydrogel forms an in-situ hydrogel within the tumor, under 1064nm laser irradiation, the alkyl free radicals from AI decomposition and the reactive oxygen species generated by DHA synergistically trigger further damage to mitochondria within tumor cells, leading to impaired ATP synthesis. This reduced ATP synthesis, in turn, enhances the DHA-induced cell starvation therapy. Moreover, the alkyl radicals decomposed by AI can enhance the inhibition of GLUT1 expression by DHA, thereby amplifying the control function of DHA on the glucose uptake "valve" of tumor starvation therapy, resulting in a reduction in glucose uptake, strengthening DHA-induced starvation therapy, and thus enhancing the anti-tumor effect of starvation therapy.

[0022] Compared with the prior art, the injectable hydrogel of the present invention has the following beneficial technical effects:

[0023] 1) The injectable hydrogel of this invention is injected into the tumor via a minimally invasive method, where it interacts with calcium in the body. 2+ The formation of an in-situ hydrogel allows for the immobilization of the drug for therapeutic purposes. Because the drug does not penetrate into surrounding normal tissue, damage to adjacent normal tissues is avoided.

[0024] 2) After the injectable hydrogel of the present invention forms an in-situ hydrogel within the tumor, under irradiation with a 1064nm laser, the alkyl radicals from AI decomposition and the reactive oxygen species generated by DHA synergistically induce further damage to mitochondria within tumor cells, leading to impaired ATP synthesis and reduced ATP production. This reduced ATP synthesis, in turn, enhances the DHA-induced cell starvation therapy. Furthermore, the alkyl radicals from AI decomposition can strengthen the inhibition of GLUT1 expression by DHA, thereby amplifying the control function of DHA on the glucose uptake "valve" of tumor starvation therapy, resulting in reduced glucose uptake and strengthening the DHA-induced starvation therapy, thus enhancing the anti-tumor effect of starvation therapy. In other words, under irradiation with a 1064nm laser, the injectable hydrogel of the present invention exhibits a significant and highly effective anti-tumor effect.

[0025] 3) The injectable hydrogel of the present invention is simple to prepare and has good biocompatibility, low cytotoxicity and stability. Attached Figure Description

[0026] Figure 1 The image shows a SEM image of the hydrogel ① prepared in Example 1.

[0027] Figure 2 The images show the UV absorption spectra of the solutions in Example 2.

[0028] Figure 3 The images show the solution and hydrogel ③ in the test tube of Example 3 after 0 hours and 48 hours.

[0029] Figure 4A This is a graph showing the temperature change of each solution in Example 4 after laser irradiation for different times.

[0030] Figure 4B These are near-infrared thermal images of the solutions in Example 4 after laser irradiation for different times.

[0031] Figure 5 The graph shows the temperature changes at different time points in the Ink solution and DHA / AI / Ink@ALG mixed solution ④ in Example 4 during 5 cycles consisting of laser irradiation and laser off.

[0032] Figure 6 The graph shows the absorbance curves of the ABTS and AI mixed solution at different temperatures and times in Example 5.

[0033] Figure 7 The graph shows the absorbance curves of the ABTS and DHA / AI / Ink@ALG mixed solution in Example 5 after laser irradiation for different times.

[0034] Figure 8ACCK-8 assay results for HepG2 cells treated with Ink solutions of different concentrations.

[0035] Figure 8B The results of CCK-8 assays were obtained by treating HepG2 cells with a mixture containing 25 μg / mL Ink and AI solutions of different concentrations.

[0036] Figure 8C CCK-8 assay results for HepG2 cells treated with DHA solutions of different concentrations.

[0037] Figure 8D The results of CCK-8 assays were obtained by treating HepG2 cells with a mixture containing 25 μg / mL Ink, 100 μg / mL AI solution and DHA solution of different concentrations.

[0038] Figure 8E The results of CCK-8 assays were obtained by treating HepG2 cells with a mixture containing 25 μg / mL Ink, 100 μg / mL AI solution, 500 μg / mL ALG solution, and DHA solution of different concentrations.

[0039] Figure 8F CCK-8 assay results for HepG2 cells treated with different concentrations of ALG solution.

[0040] Figure 8G CCK-8 assay results for HepG2 cells treated with AI solutions of different concentrations.

[0041] Figure 9 These are confocal fluorescence images of HepG2 cells treated in different experimental groups in Example 7, obtained by Calcein-AM / PI double staining to detect live / dead cells.

[0042] Figure 10 These are confocal fluorescence images of ROS generated in HepG2 cells treated with different experimental group solutions in Example 8.

[0043] Figure 11 The fluorescence intensity of DCF in HepG2 cells treated with different experimental group solutions in Example 8 was measured by flow cytometry.

[0044] Figure 12 The changes in mitochondrial membrane potential in HepG2 cells treated with different experimental group solutions in Example 9 were detected by JC-1 flow cytometry.

[0045] Figure 13 The fluorescence intensity of the glucose uptake probe 2-NBDG in HepG2 cells treated with different experimental group solutions in Example 10 was measured by flow cytometry.

[0046] Figure 14 The results show the detection of intracellular ATP levels in HepG2 cells treated with different experimental group solutions in Example 11.

[0047] Figure 15A To detect the expression of GLUT1 in HepG2 cells treated with different experimental group solutions in Example 12 using immunoblotting.

[0048] Figure 15B To detect the relative levels of GLUT1 expression in HepG2 cells treated with different experimental group solutions in Example 12 using Western blotting.

[0049] Figure 16A The curves show the temperature changes during laser irradiation of mice inoculated with HepG2 cells in Example 13, as they were treated in different experimental groups.

[0050] Figure 16B Near-infrared imaging images of mice inoculated with HepG2 cells in Example 13 during laser irradiation treatment in different experimental groups.

[0051] Figure 17A This is a comparison of tumor volume in mice in Example 13 after treatment with different experimental groups following HepG2 cell inoculation.

[0052] Figure 17B This is a comparison of tumor weight in mice in Example 13 after treatment with different experimental groups following HepG2 cell inoculation.

[0053] Figure 17C These are comparison figures showing the size of tumors in mice in Example 13 after being treated with different experimental groups following HepG2 cell inoculation.

[0054] Figure 18 The tumor inhibition rate curves of mice inoculated with HepG2 cells in Example 13 after treatment in different experimental groups.

[0055] Figure 19 The results of H&E and TUNEL staining of tumor tissues from mice inoculated with HepG2 cells in Example 13 after treatment in different experimental groups.

[0056] Figure 20 The graph shows the weight changes of mice in different experimental groups during the treatment of mice inoculated with HepG2 cells in Example 13. Detailed Implementation

[0057] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be understood that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0058] Unless otherwise specified, the techniques or conditions described in the following examples were performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0059] DHA (dihydroartemisinin) was obtained from MedChemExpress. Ink was purchased from HuKai Wen Ink Factory. AI (2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, CAS: 27776-21-2) and ABTS (2,2-azo(3-ethylbenzothiazoline-6-sulfonic acid)) were purchased from Macklin. Sodium alginate (ALG) and calcium chloride were supplied by Aladdin. Cell counting kit-8 (CCK-8) was supplied by Solarbio Technologies. FITC Annexin V Apoptosis assay kit, ATP and BCA protein assay kit were supplied by Beyotime. Calcein-AM / PI live / dead dye kit and Hoechst 33258 were products of Key Gen Biotechnology. 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) was purchased from Amgicam. DMEM cell culture medium and glucose-free DMEM cell culture medium were purchased from Shanghai Yuanpei Biotechnology Co., Ltd. The DMEM cell culture medium contained 10% fetal bovine serum and 1% penicillin-dextrose antibody. HepG2 cells were human liver cancer cells, obtained from the ATCC cell bank.

[0060] In the following examples, 1.8 mmol / L Ca was used. 2+Solutions were used to simulate physiological concentrations in the human body, and various stock solutions were prepared using the following methods: ABST powder was dissolved in water to prepare an ABST stock solution with a concentration of 2 mg / mL; DHA powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a DHA stock solution with a concentration of 5 mmol / L; AI powder was dissolved in ultrapure water to prepare an AI stock solution with a concentration of 5 mg / mL; and ALG powder was dissolved in ultrapure water to prepare an ALG stock solution with a concentration of 10 mg / mL. 100 mg of commercially available Ink powder was added to 10 mL of a 10 mg / mL aqueous solution of polyvinylpyrrolidone (PVP) under ultrasonication. After the Ink was completely dispersed, the resulting solution was transferred to a 30 mL stainless steel autoclave lined with polytetrafluoroethylene and heated at 160 °C for 6 hours. After cooling the autoclave to room temperature, the resulting Ink solution was the Ink stock solution, and its concentration was measured to be 8 mg / mL. Dynamic light scattering (DLS) detectors were used to measure the hydrodynamic diameter and potential of the Ink mother liquor. The results showed that its particle size and potential were approximately 181.78 nm and -19.80 mV, respectively. The ALG, AI, DHA, Ink, and ABTS solutions used in the examples can be obtained directly from the corresponding mother liquors prepared above, or by diluting the mother liquors with ultrapure water to obtain solutions of the target concentration.

[0061] In the following examples, mM is an abbreviation for mmol / L and μM is an abbreviation for μmol / L.

[0062] Example 1

[0063] ALG solution was mixed with AI solution, DHA solution and Ink solution, and homogenized by ultrasonic treatment to obtain DHA / AI / Ink@ALG mixed solution ①. The concentration of ALG in mixed solution ① was 5 mg / mL, the concentration of AI was 200 μg / mL, the concentration of DHA was 200 μM, and the concentration of Ink was 25 μg / mL.

[0064] Take 100 μL of DHA / AI / Ink@ALG mixed solution① and add it to a beaker containing 7 mL of 1.8 mM calcium chloride solution to form a hydrogel①.

[0065] Electron micrographs of the above hydrogel ① were taken using a Zeiss Gemini 300 scanning electron microscope (SEM), as shown below. Figure 1 As shown. By Figure 1 It is evident that a large number of Ink nanoparticles aggregate on the surface of the hydrogel, indicating that ALG and Ca... 2+ Cross-linking forms a hydrogel, which can effectively immobilize Ink.

[0066] Example 2

[0067] The ALG solution was mixed with the AI ​​solution, DHA solution and Ink solution, and homogenized by ultrasonic treatment to obtain a DHA / AI / Ink@ALG mixed solution ②. The concentration of ALG in mixed solution ② was 1 mg / mL, the concentration of AI was 5 mg / mL, the concentration of DHA was 175.8 μM, and the concentration of Ink was 25 μg / mL.

[0068] Ink solution (25 μg / mL), AI solution (5 mg / mL), DHA solution (175.8 μM), ALG solution (1 mg / mL), and a DHA / AI / Ink@ALG mixed solution② were used as different experimental groups. Each group of solutions was scanned using ultraviolet spectrophotometry (UV-Vis) at wavelengths of 300-1000 nm. The results are as follows: Figure 2 As shown.

[0069] from Figure 2 As can be seen, the AI ​​solution exhibits a typical absorption peak at 360 nm. The DHA and ALG solutions do not show obvious absorption peaks. The Ink solution exhibits spectral absorption characteristics in the 300-1000 nm range, suggesting that Ink could act as a photothermal agent in the second near-infrared (NIR-II, 1000-1700 nm) region. The DHA / AI / Ink@ALG mixed solution ② simultaneously displays characteristic absorption peaks of both AI and Ink, indicating that AI and Ink are encapsulated within ALG in mixed solution ②.

[0070] Example 3

[0071] ALG solution was mixed with AI solution, DHA solution and Ink solution, and homogenized by ultrasonic treatment to obtain DHA / AI / Ink@ALG mixed solution ③. The concentration of ALG in mixed solution ③ was 5 mg / mL, the concentration of AI was 200 μg / mL, the concentration of DHA was 200 μM, and the concentration of Ink was 25 μg / mL.

[0072] 100 μL of the above mixed solution ③ was added to a test tube containing 7 mL of a 1.8 mM calcium chloride solution, and a hydrogel ③ was formed in the solution.

[0073] A constant-temperature shaker experiment was used to simulate the drug's pharmacokinetic properties in vivo. Photos of the solution and hydrogel ③ in the test tubes were taken after 0 hours and 48 hours to observe the drug release. (Photos are shown below.) Figure 3 As shown.

[0074] Depend on Figure 3As can be seen, the supernatant of the solution after 0 hours was colorless, while the supernatant of the solution after 48 hours was black. This indicates that the drug was encapsulated within hydrogel ③ and not released at 0 hours. After 48 hours of constant-temperature shaking at 37°C (100 rpm), some drug was released from hydrogel ③. This suggests that ALG and Ca2+ in the hydrogel... 2+ Cross-linking forms a non-compact structure, which can be used as a carrier for drug delivery.

[0075] Example 4

[0076] The ALG solution was mixed with the AI ​​solution, DHA solution and Ink solution, and homogenized by ultrasonic treatment to obtain a DHA / AI / Ink@ALG mixed solution ④. The concentration of ALG in mixed solution ④ was 5 mg / mL, the concentration of AI was 100 μg / mL, the concentration of DHA was 200 μM, and the concentration of Ink was 25 μg / mL.

[0077] H₂O, AI (100 μg / mL) solution, ALG (5 mg / mL) solution, Ink (25 μg / mL) solution, DHA (200 μM) solution, and DHA / AI / Ink@ALG mixed solution ④ were used as different experimental groups, and were tested with a power of 0.5 W / cm². 2 Irradiate with a 1064nm laser for 10 minutes, and record the temperature of different experimental groups at different time points under laser irradiation (e.g., Figure 4A As shown), and images of temperature changes were recorded using a near-infrared thermal imager (e.g. Figure 4B (As shown).

[0078] Depend on Figure 4A and Figure 4B It can be seen that the temperature rise of H2O, AI solution, DHA solution and ALG solution is not significant, while the temperature rise curves of Ink solution and DHA / AI / Ink@ALG mixed solution ④ are obvious and basically consistent: in the first 5 minutes of irradiation, the temperature rises from 28.90℃ to about 44.90℃; in the last 5 minutes of irradiation, the temperature basically stabilizes at about 44℃.

[0079] Meanwhile, the Ink solution and the DHA / AI / Ink@ALG mixed solution ④ were treated as follows: irradiated with a 1064nm laser for 5 minutes, then the laser was turned off and the solution was cooled to room temperature for 5 minutes. This process was repeated 5 times, and the temperature changes at different time points were recorded. The results are as follows: Figure 5 As shown.

[0080] Depend on Figure 5It is evident that the performance of the Ink solution and the DHA / AI / Ink@ALG mixed solution ④ is almost identical: throughout the process, neither the Ink solution nor the DHA / AI / Ink@ALG mixed solution ④ showed significant degradation, which proves that the DHA / AI / Ink@ALG mixed solution ④, like the Ink solution, has good photothermal stability.

[0081] It is evident that the DHA / AI / Ink@ALG mixed solution ④ has the same photothermal properties as the Ink solution, and Ink in the DHA / AI / Ink@ALG mixed solution ④ can still act as a photothermal agent.

[0082] Example 5

[0083] The ALG solution was mixed with the AI ​​solution, DHA solution and Ink solution, and homogenized by ultrasonic treatment to obtain a DHA / AI / Ink@ALG mixed solution ⑤. The concentration of ALG in the mixed solution ⑤ was 500 μg / mL, the concentration of AI was 200 μg / mL, the concentration of DHA was 200 μM, and the concentration of Ink was 25 μg / mL.

[0084] ABTS will be used as a probe to detect alkyl radicals (R·) generated in the AI ​​solution and mixed solution ⑤. When ABTS reacts with alkyl radicals, it will form green ABTS+·, which has a characteristic ultraviolet absorption peak in the range of 400-900 nm.

[0085] 0.5 mL of ABTS solution (concentration 2 mg / mL) and 0.5 mL of AI solution (concentration 200 μg / mL) were mixed thoroughly. The first group was reacted in a water bath at 37 °C, and the second group was reacted in a water bath at 44 °C. The absorbance of each solution in the 400-900 nm range was measured using UV-Vis at 2, 4, and 6 hours of reaction. The results are as follows. Figure 6 As shown. By Figure 6 It is evident that, within the same incubation time, the absorbance of ABTS+· at 44℃ was significantly higher than that at 37℃; and the absorbance of ABTS+· at 6 hours was significantly higher than that at 2 hours and 4 hours. This indicates that AI can decompose more alkyl radicals at higher temperatures or for longer periods, meaning that the decomposition of AI (generation of alkyl radicals) exhibits both temperature- and time-dependent characteristics.

[0086] Mix 0.2 mL of ABTS solution (concentration 2 mg / mL) and 0.2 mL of DHA / AI / Ink@ALG mixed solution⑤, and then use a 1064 nm laser (0.5 W / cm²) to analyze the mixture. 2Irradiation was performed for 0, 2, 4, 6, 8, and 10 minutes, respectively. The absorbance of each solution in the 400-900 nm range was measured using UV-Vis. The results are as follows: Figure 7 As shown. By Figure 7 It can be seen that the absorbance of ABTS+· gradually increases with the extension of 1064nm laser irradiation time, indicating that the generation of alkyl free radicals in the DHA / AI / Ink@ALG mixed solution ⑤ increases with the extension of laser irradiation time.

[0087] Example 6

[0088] HepG2 cells were planted at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured in DMEM medium for 24 hours. Then, different experimental group solutions were added to each well for 12 hours, followed by treatment with or without a 1064nm laser (0.5W / cm²). 2 The cells were cultured under irradiation for 12 hours. Then, the old cell culture medium was discarded, and fresh DMEM cell culture medium and CCK-8 reagent (cell culture medium: CCK-8 = 10:1, volume ratio) were added to each well. The cells were then incubated at 37°C for 1 hour. Afterward, the absorbance at 450 nm was measured using a microplate reader.

[0089] The first group consisted of different concentrations of Ink solution (0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 10 μg / mL, 25 μg / mL). The results are as follows: Figure 8A As shown. By Figure 8A As can be seen, no obvious cell-killing effect was observed after treatment with Ink without laser irradiation; however, cell viability decreased slightly after treatment with Ink under 1064nm laser irradiation.

[0090] The second group consisted of different concentrations of AI solution (0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). The results are as follows: Figure 8G As shown. By Figure 8G It is evident that AI treatment did not exhibit significant cytotoxicity regardless of whether laser irradiation was present. This indicates that AI alone cannot decompose into R· in the absence of a photothermal agent.

[0091] The third group consisted of mixtures containing 25 μg / mL Ink and different concentrations of AI solutions (0 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). The results are as follows: Figure 8B As shown. By Figure 8BIt is evident that no significant cell-killing effect was observed after treatment with AI / Ink without laser irradiation; however, under 1064nm laser irradiation, cell viability gradually decreased with increasing AI concentration, demonstrating that Ink can induce AI decomposition to produce R·.

[0092] The fourth experimental group consisted of DHA solutions of different concentrations (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM). The results are as follows: Figure 8C As shown. By Figure 8C It is evident that DHA can exert anti-tumor effects; when the concentration reaches 80 μM, cell viability decreases to approximately 56.64%. The presence or absence of 1064 nm laser irradiation has little impact on the anti-tumor effect of DHA.

[0093] The fifth group consisted of mixtures containing 25 μg / mL Ink, 100 μg / mL Al solutions, and different concentrations of DHA solutions (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM). The results are as follows: Figure 8D As shown. By Figure 8D It can be seen that, without 1064nm laser irradiation, the half-inhibitory concentration (IC50) of DHA / AI / Ink is... 50 The half-maximum concentration (IC50) of DHA / AI / Ink-L was 111.35 μM; under 1064 nm laser irradiation, the half-maximum inhibitory concentration (IC50) of DHA / AI / Ink-L was... 50 The concentration of DHA was 36.53 μM. When the concentration of DHA reached 80 μM, the cell viability decreased to 14.37%, which was much lower than that of DHA treatment alone. This proves that DHA / AI / Ink have a synergistic effect under laser irradiation. It is speculated that the alkyl free radicals from the decomposition of AI promote the anti-tumor effect of DHA.

[0094] The sixth experimental group consisted of mixtures containing 25 μg / mL Ink, 100 μg / mL Al solution, 500 μg / mL ALG solution, and different concentrations of DHA solution (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM). The results are as follows: Figure 8E As shown. By Figure 8E It can be seen that, without laser irradiation, the half-inhibitory concentration (IC50) of DHA / AI / Ink@ALG is... 50 The half-maximum concentration (IC50) of DHA / AI / Ink@ALG-L was 98.57 μM; under 1064 nm laser irradiation, the half-maximum inhibitory concentration (IC50) of DHA / AI / Ink@ALG-L was 98.57 μM. 50The concentration was 27.48 μM. Compared with the results of the fifth group of experiments, it can be seen that the addition of ALG did not weaken the anti-tumor synergistic effect of DHA / AI / Ink, which indicates that ALG has good biocompatibility. Moreover, compared with the case without laser irradiation, the anti-tumor effect of DHA / AI / Ink@ALG under 1064nm laser irradiation was significantly increased.

[0095] The seventh experimental group consisted of different concentrations of ALG solution (0 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1000 μg / mL). The results are as follows... Figure 8F As shown. By Figure 8F It is evident that even at a concentration of 1000 μg / mL, ALG did not exhibit significant cytotoxicity. This demonstrates that ALG possesses good biocompatibility and can serve as a drug delivery carrier.

[0096] Based on the above results, the IC of each group can also be calculated separately. 50 They are respectively:

[0097] IC50 of DHA solution 50 IC50 of 99.27 μM DHA-L solution 50 The concentration was 91.8 μM; the IC50 of the DHA / AI / Ink solution was... 50 IC50 of 111.35 μM DHA / AI / Ink-L solution 50 The concentration was 36.53 μM; the IC of DHA / AI / Ink@ALG was... 50 The IC of DHA / AI / Ink@ALG-L is 98.57μM. 50 The value was 27.48 μM. This indicates the IC50 values ​​of DHA / AI / Ink solution and DHA / AI / Ink@ALG solution under laser irradiation. 50 The significant decrease suggests a more significant anti-tumor effect. It is speculated that the photothermal agent Ink increases the temperature under laser irradiation, and AI decomposes under low-temperature photothermal conditions to generate alkyl radicals, which promote DHA-induced cell death.

[0098] In Examples 7 through 12 below, HepG2 cells will be treated with different experimental solutions. The solutions used for each experimental group are described below:

[0099] The control group refers to the group that does not contain the experimental solution, meaning the experimental solution is blank; it is denoted as Control.

[0100] PBS solution, denoted as PBS;

[0101] Without laser irradiation: Ink solution, denoted as Ink; AI solution, denoted as AI; DHA solution, denoted as DHA; AI / Ink solution, denoted as AI / Ink; DHA / AI / Ink solution, denoted as DHA / AI / Ink; DHA / AI / Ink@ALG solution, denoted as DHA / AI / Ink@ALG solution.

[0102] Laser irradiation (1064nm laser, 0.5W / cm²) 2 (Irradiated for 10 minutes): PBS solution + laser, denoted as PBS-L; Ink solution + laser, denoted as Ink-L; AI / Ink solution + laser, denoted as AI / Ink-L; DHA / AI / Ink solution + laser, denoted as DHA / AI / Ink-L; DHA / AI / Ink@ALG solution + laser, denoted as DHA / AI / Ink@ALG-L.

[0103] Among them, AI / Ink solution is a mixture of Ink solution and AI solution, DHA / AI / Ink solution is a mixture of AI solution, DHA solution and Ink solution, and DHA / AI / Ink@ALG solution is a mixed solution obtained by mixing ALG solution with AI solution, DHA solution and Ink solution and then homogenizing by ultrasonic treatment.

[0104] In each solution, the concentration of ALG was 500 μg / mL, the concentration of AI was 100 μg / mL, the concentration of DHA was 35 μmol / L, and the concentration of Ink was 25 μg / mL.

[0105] Example 7

[0106] The Calcein-AM / PI assay kit was used to observe the number of live and dead cells in different groups after HepG2 treatment. Specifically, HepG2 cells were cultured at 1.5 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal dish and cultured in DMEM medium for 24 hours. Different experimental group solutions were then added to the confocal dish and co-cultured with HepG2 cells for 6 hours. Then, the cells were cultured with or without a 1064nm laser (0.5W / cm²). 2 Irradiate the cells for 10 minutes, then culture for 6 hours. Finally, wash the cells twice with PBS and incubate them at 37°C in the dark with Calcein-AM / PI for 30 minutes. Afterward, wash away excess dye with PBS and observe the cells using a CLSM scanner.

[0107] Confocal fluorescence images of HepG2 cells from different experimental groups after co-staining with Calcein-AM (green) and PI (red) (scale bar = 50 μm) are shown below. Figure 9As shown, green fluorescence represents live cells, and red fluorescence represents dead cells. Figure 9 Compared to the Control group, no significant red fluorescence was observed in the AI, Ink, and AI / Ink groups. A small number of PI-stained erythrocytes were observed in the Ink-L and AI / Ink-L groups. Significant red fluorescence signals were observed in the DHA and DHA / AI / Ink groups, and large-area red fluorescence signals were observed in the DHA / AI / Ink-L and DHA / AI / Ink@ALG-L groups. This indicates that Ink has good biocompatibility, AI alone cannot decompose to produce alkyl radicals, and both DHA and DHA / AI / Ink have mild antitumor effects. Both Ink and AI / Ink exhibit mild antitumor effects under laser irradiation. Both DHA / AI / Ink and DHA / AI / Ink@ALG show significant antitumor effects under laser irradiation. It is speculated that in DHA / AI / Ink and DHA / AI / Ink@ALG, Ink generates low-temperature photothermal activity under laser irradiation, promoting the decomposition of AI to produce R·, which further promotes DHA starvation therapy, resulting in a large number of dead cells. That is, the synergistic effect of R· and DHA can significantly enhance the killing power against tumor cells; moreover, the addition of ALG does not affect the synergistic effect of DHA and R·.

[0108] Example 8

[0109] DCFH-DA was used as a probe to detect ROS generation in HepG2 cells treated with different experimental solutions (scale bar = 20 μm). Specifically, HepG2 cells were cultured at 1.5 × 10⁶ cells per dish. 5 Cells were seeded at a density of [number] cells per confocal dish and cultured in DMEM medium for 24 hours. Different experimental group solutions were then added to the confocal dish and co-cultured with HepG2 cells for 6 hours. Then, the cells were cultured with or without a 1064nm laser (0.5W / cm²). 2 Cells were irradiated for 10 minutes and then cultured for 2 hours. The supernatant was removed, and the DCFH-DA probe was added. The cells were incubated at 37°C in the dark for 30 minutes. Cells were washed twice with PBS and finally stained with DAPI for 10 minutes. The fluorescence of DCF was observed by CLSM imaging or flow cytometry. The results are shown below. Figure 10 and Figure 11 As shown.

[0110] Figure 10 In this study, the production of ROS is directly proportional to the intensity of green fluorescence. Figure 10As can be seen, no obvious green fluorescence was detected in the AI ​​group, Ink group, AI / Ink group, and Ink-L group. Low green fluorescence signals were observed in the AI / Ink-L group, DHA group, and DHA / AI / Ink group. The DHA / AI / Ink-L group and DHA / AI / Ink@ALG-L group showed similar results, with bright green fluorescence signals observed. This indicates that DHA also produces a small amount of ROS. Under low-temperature photothermal conditions, AI decomposes and produces a small amount of ROS. Without laser irradiation, DHA / AI / Ink does not exhibit a synergistic effect. Only under laser irradiation does Ink in DHA / AI / Ink act as a photothermal agent, causing AI to decompose alkyl radicals under low-temperature photothermal conditions, thus significantly promoting DHA ROS production. This also demonstrates that ALG has good biocompatibility, and the ROS production in DHA / AI / Ink@ALG is not affected by the addition of ALG.

[0111] Figure 11 In the diagram, 1 represents Control, 2 represents Ink-L, 3 represents AI / Ink-L, 4 represents AI, 5 represents Ink, 6 represents DHA / AI / Ink@ALG, 7 represents AI / Ink, 8 represents DHA, 9 represents DHA / AI / Ink-L, and 10 represents DHA / AI / Ink@ALG-L. A rightward shift of the peak indicates increased fluorescence intensity. Figure 11 As can be seen, the fluorescence intensity of the DHA / AI / Ink@ALG-L group was approximately 3.8 times that of the Control group, significantly higher than that of the DHA group or the AI / Ink-L group. This is consistent with... Figure 10 The results from the confocal images are consistent, indicating that this group generated a large amount of ROS.

[0112] Example 9

[0113] JC-1 was used as a probe to detect changes in mitochondrial membrane potential (ΔΨm). Details are as follows:

[0114] HepG2 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a concentration of [number] cells / well in 12-well plates and cultured overnight in DMEM cell culture medium. After co-culturing for 24 hours with different experimental group solutions, the culture medium was removed, and each well was stained with 200 μL of JC-1 staining solution at 37°C for 20 minutes. Cells were then centrifuged and analyzed by flow cytometry. When ΔΨm was high, JC-1 aggregated in the mitochondrial matrix and formed polymers, producing red fluorescence; when ΔΨm was low, JC-1 did not aggregate and produced green fluorescence as monomers.

[0115] Test results as follows Figure 12 As shown. Figure 12In the control group, AI group, Ink group, and AI / Ink group, the red / green fluorescence ratios (Q2 / Q3) after treatment were 5.71, 4.16, 4.42, and 5.58, respectively; after treatment with Ink-L group, DHA group, DHA / AI / Ink group, and AI / Ink-L group, the red / green fluorescence ratios (Q2 / Q3) were 3.37, 2.51, 2.49, and 1.95, respectively; after treatment with DHA / AI / Ink-L group and DHA / AI / Ink@ALG-L group, the red / green fluorescence ratios (Q2 / Q3) were 0.36 and 0.43, respectively. It can be seen that both AI / Ink-L and DHA-only treatments resulted in a partial decrease in ΔΨm, indicating that both AI-derived R· and DHA-induced mitochondrial damage can cause mitochondrial damage. Under no laser irradiation, there was no significant difference between the DHA / AI / Ink and DHA groups, suggesting that AI / Ink did not promote mitochondrial damage. However, treatment with the DHA / AI / Ink-L or DHA / AI / Ink@ALG-L groups significantly reduced mitochondrial membrane potential, with a clear transition from red to green fluorescence, demonstrating a significant synergistic effect between DHA and AI / Ink-L, as well as the good biocompatibility of ALG. Considering the complex mechanisms of ROS and mitochondrial damage, it can be inferred that in the DHA / AI / Ink-L and DHA / AI / Ink@ALG-L groups, AI-derived R· (ROS) and DHA-induced mitochondrial damage mutually promoted each other, exacerbating mitochondrial damage and leading to a more significant decrease in mitochondrial membrane potential.

[0116] Example 10

[0117] 2-NBDG was used as a fluorescent probe to detect intracellular glucose uptake. Specifically, HepG2 cells were inoculated at 1.5 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a concentration of [number] cells / well in 12-well plates and cultured overnight in DMEM cell culture medium. After co-culturing with different experimental group solutions for 24 hours, cells were treated with glucose-free DMEM cell culture medium for 12 hours. The medium was then removed, and 2-NBGD was added. The cells were incubated at 37°C in the dark for 30 minutes. Finally, the cells were washed twice with PBS, and then scraped off with a spatula using 1 mL of PBS. Cells were collected by centrifugation at 3000 rpm for 5 minutes, and flow cytometry was used to detect glucose uptake by the treated cells. The results are shown below. Figure 13 As shown.

[0118] Figure 13In the diagram, 1 represents Control, 2 represents Ink-L, 3 represents AI / Ink-L, 4 represents AI, 5 represents Ink, 6 represents DHA / AI / Ink, 7 represents AI / Ink, 8 represents DHA, 9 represents DHA / AI / Ink-L, and 10 represents DHA / AI / Ink@ALG-L. A leftward shift of the peak indicates a decrease in fluorescence intensity. Compared with the DHA group, the fluorescence intensity of cells treated with the DHA / AI / Ink-L group (or the DHA / AI / Ink@ALG-L group) was significantly reduced, indicating a sharp decrease in glucose uptake by HepG2 cells in the DHA / AI / Ink-L group (or the DHA / AI / Ink@ALG-L group). This suggests that alkyl radicals promoted the inhibition of DHA-induced glucose uptake.

[0119] Example 11

[0120] The ATP levels in HepG2 cells after different treatments were detected using an ATP kit. In short, HepG2 cells were treated at 1.5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a concentration of [number] cells / well in 12-well plates and cultured overnight in DMEM cell culture medium. After treatment with different experimental group solutions for 24 hours, cells were collected and lysed. ATP assay reagent was added according to the ATP kit instructions. Finally, the amount of luminescence was measured using a microplate reader to quantify the ATP content. Results are shown below. Figure 14 As shown.

[0121] Figure 14 In the study, compared with the control group, no significant decrease in ATP levels was observed after treatment with AI, Ink, and AI / Ink. However, when DHA was used to treat cells, intracellular ATP levels decreased slightly, which may be due to ATP synthesis disorders caused by DHA-induced mitochondrial damage. In addition, compared with the DHA group, the ATP levels in the DHA / AI / Ink-L and DHA / AI / Ink@ALG-L groups were significantly reduced. This may be because the R· produced by AI / Ink-L works synergistically with DHA-induced mitochondrial damage, leading to ATP synthesis disorders. At the same time, the ATP synthesis disorders lead to a reduction in ATP production, thereby inducing cellular starvation.

[0122] Example 12

[0123] Immunoblot detection of GLUT1 expression in cells after different treatments: HepG2 cells were treated with 1.5 × 10⁻⁶ cells / cells. 5Cells were seeded at a concentration of [number] cells / well in 6-well plates and cultured in DMEM medium for 24 hours. Then, different experimental groups (Control group, DHA solution, Ink-L solution, AI / Ink-L solution, DHA / AI / Ink-L solution) were added for 12 hours each, followed by treatment with or without a 1064nm laser (0.5W / cm²). 2 Cells were cultured under irradiation for 12 hours. Cells were collected, and 100 μL of tissue cell lysis buffer (Norlen Bio) was added to each cell sample. The mixture was thoroughly lysed using a pipette, and the lysate was transferred to a new centrifuge tube. 10 μL of the sample was directly added to 10 μL of 2×SDS-PAGE loading buffer, mixed, heated at 100°C for 5 minutes, cooled on ice, and centrifuged at 12000g for 5 minutes to remove insoluble precipitates. Samples were separated using 10% SDS-PAGE, with a maximum loading volume of 20 μL per well. After electrophoresis, the PVDF membrane was soaked in methanol for 1 minute, then the gel, filter paper, and methanol-soaked PVDF membrane were soaked in Transfer Buffer and incubated at 4°C for 10 minutes to prepare a transfer sandwich. Note: Semi-dry cell electrophoresis was used for this experiment, with transfer conditions of 30 mA for 60 min. The transfer membrane was blocked with Blocking Buffer and incubated overnight at 4°C. The next day, it was washed three times with 1×TBST for 15 minutes each time. Add diluted primary antibody and incubate at 37°C for 2 hours. Wash four times with 1×TBST, 10 minutes each time. Add diluted secondary antibody and incubate at 37°C for 2 hours. Wash four times with 1×TBST, 10 minutes each time. Perform chemiluminescence detection using Super-GL ECL ultrasensitive luminescent buffer and expose the X-ray film. After development and fixing, the dried film was photographed using a gel imaging analysis system; in this experiment, Gel-ProAnalyzer software was used for analysis. β-actin was used as an internal reference for comparison.

[0124] Immunoblotting results as follows Figure 15A and Figure 15B As shown. By Figure 15A and Figure 15B It is evident that, compared to the Control group, the DHA treatment resulted in a relative reduction in GLUT1 expression, indicating that DHA can inhibit GLUT1 expression. Simultaneously, the DHA / AI / Ink-L treatment significantly reduced GLUT1 expression, suggesting that the R· from AI degradation promoted DHA's inhibition of GLUT1 expression. This implies that the DHA / AI / Ink-L treatment amplified DHA's control over the glucose uptake "valve" in starvation therapy.

[0125] Example 13

[0126] HepG2 cells were cultured and subcutaneously inoculated into mice. When the subcutaneous tumor reached 100 cm, 3 Around [time period missing], mice underwent their first treatment: mice were randomly divided into 5 groups (n=5). On day 0 and day 3, the experimental group solutions were subcutaneously injected and irradiated with a 1064nm laser for 10 minutes. The 5 experimental group solutions were: PBS solution, DHA@ALG solution, Ink@ALG solution, AI / Ink@ALG solution, and DHA / AI / Ink@ALG solution. The concentrations of ALG, AI, DHA, and Ink were 5 mg / mL, 2 mg / mL, 1 mM, and 200 μg / mL, respectively.

[0127] Near-infrared imaging equipment was used to record temperature changes during the first laser irradiation treatment. For example... Figure 16A and Figure 16B As shown. By Figure 16A and Figure 16B As can be seen, the temperature of the DHA@ALG-L group was slightly higher than that of the PBS-L group, which may be due to the thermostatic effect of ALG. Encouragingly, both the AI / Ink@ALG-L and DHA / AI / Ink@ALG-L groups showed a significant temperature increase compared to the Ink@ALG-L group, and remained at approximately 44°C after 10 minutes of laser irradiation, indicating that Ink in the hydrogel formed after injection into the body plays the role of a photothermal agent.

[0128] After the first treatment, tumor size and mouse weight were recorded every other day for 15 consecutive days, and tumor volume (V) and tumor inhibition rate (IR) were calculated using the following formulas:

[0129] V(mm 3 = (length × width) 2 ) / 2

[0130] IR = (W control -W treat ) / W control ×100%

[0131] On day 15, the tumor and major organs were removed. The tumor was extracted, photographed, and weighed. The removed tumor and major organs were stained with H&E and TUNEL to evaluate the antitumor effect and biosafety.

[0132] The volume, weight, and shape of tumors obtained from different experimental groups are as follows: Figure 17A , Figure 17B and Figure 17C As shown in the figure. The tumor inhibition rate was calculated based on the data obtained above, and a curve was plotted as shown in the figure. Figure 18 As shown. Figure 18As can be seen, compared with the PBS-L group, the tumor inhibition rates of the Ink@ALG-L group, AI / Ink@ALG-L group, DHA@ALG-L group, and DHA / AI / Ink@ALG-L group were 19.05%, 44.60%, 59.64%, and 88.50%, respectively. This indicates that: the low-temperature heat generated by Ink under laser irradiation can delay tumor growth, but is insufficient to eliminate tumors; the R· generated by AI under low-temperature heat has a mild anti-tumor effect, and DHA alone has a mild anti-tumor effect. However, in the DHA / AI / Ink@ALG-L group, the R· generated by AI and DHA can synergistically interact. R· promotes the inhibition of GLUT1 expression by DHA, and by amplifying the control function of DHA on the glucose uptake "valve" of starvation therapy, it enhances the anti-tumor effect of starvation therapy, achieving a powerful tumor inhibition effect.

[0133] The results of H&E and TUNEL staining assessment of tumor histopathological damage are as follows: Figure 19 As shown. By Figure 19 As can be seen, compared with the PBS-L group, most cells in Ink@ALG-L, DHA@ALG-L, and AI / Ink@ALG-L maintained normal cell morphology. However, in the DHA / AI / Ink@ALG-L group, a large number of apoptotic cells were observed, exhibiting significant nuclear pyknosis and sparse cell arrangement. Therefore, among all groups, DHA / AI / Ink@ALG showed the strongest killing effect on tumor cells.

[0134] During treatment, the body weight of mice in each group was as follows: Figure 20 As shown. According to Figure 20 During the treatment, the mice did not experience a significant decrease in body weight, indicating that the hydrogels formed in each experimental group did not exhibit significant systemic toxicity.

[0135] Furthermore, pathological morphological analysis of the major organs (heart, liver, spleen, lungs, and kidneys) 15 days after treatment showed that the treated mice did not have significant tissue damage or inflammatory response.

[0136] In the above embodiments, DHA solution, AI solution, Ink solution, and ALG solution are simply mixed to form an injectable DHA / AI / Ink@ALG solution, which is then injected into tumor tissue. The multifunctional ALG reacts with Ca in the tumor microenvironment. 2+It reacts rapidly, forming an in-situ hydrogel within tumor tissue, immobilizing Ink, AI, and DHA within the tumor tissue to prevent degradation, thus achieving drug immobilization. In the injectable solution for use in tumor tissue, the concentrations are 5–8 mg / mL for ALG, 1–2 mg / mL for AI, 1–2 mM for DHA, and 150–400 μg / mL for Ink. Dissection of the skin tissue 48 hours after subcutaneous injection of Ink@ALG revealed excellent drug immobilization properties, while using Ink alone showed significant diffusion into surrounding tissues.

[0137] Meanwhile, based on the above experimental results, under 1064nm laser irradiation, DHA / AI / Ink@ALG generates mild heat due to the photothermal effect and produces alkyl free radicals and ROS. Treatment of tumor cells significantly reduces mitochondrial membrane potential, significantly decreases GULT1 expression, and significantly increases apoptosis, demonstrating a significant anti-tumor effect. Based on the analysis of the above experimental results, the anti-tumor mechanism of DHA / AI / Ink@ALG is hypothesized as follows: DHA not only inhibits GLUT1 expression, thereby reducing cellular glucose uptake, but also generates ROS. Under 1064nm laser irradiation, Ink absorbs light energy to generate mild heat; the increased temperature causes AI to decompose into alkyl free radicals, thereby inducing mitochondrial damage in tumor cells. Therefore, when the injectable hydrogel forms an in-situ hydrogel within the tumor, under 1064nm laser irradiation, the alkyl radicals from AI decomposition and the reactive oxygen species generated by DHA synergistically induce further damage to mitochondria within tumor cells, leading to impaired ATP synthesis. This reduced ATP synthesis, in turn, enhances DHA-induced cell starvation therapy. Furthermore, the alkyl radicals from AI decomposition can strengthen the inhibition of GLUT1 expression by DHA, thereby amplifying DHA's control over glucose uptake in tumor starvation therapy, resulting in reduced glucose uptake and further enhancing DHA-induced starvation therapy, thus strengthening the anti-tumor effect of starvation therapy.

Claims

1. An injectable hydrogel, characterized in that, The injectable hydrogel is dihydroartemisinin DHA / 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride AI / Ink@sodium alginate ALG, which is obtained by ultrasonic homogenization of sodium alginate ALG solution with 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride AI solution, dihydroartemisinin DHA solution and Ink solution. The sodium alginate (ALG) solution is prepared by dissolving sodium alginate (ALG) powder in ultrapure water. The 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AI) solution is prepared by dissolving 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AI) powder in ultrapure water. The dihydroartemisinin DHA solution is prepared by dissolving dihydroartemisinin DHA powder in dimethyl sulfoxide to obtain a dihydroartemisinin DHA stock solution, which is then diluted to the target concentration with ultrapure water. The Ink solution is obtained by diluting the Ink stock solution with ultrapure water. The Ink stock solution is prepared by the following steps: Ink powder is added to an aqueous solution of polyvinylpyrrolidone with a concentration of 8-10 mg / mL under ultrasonication. After complete dispersion, the solution is transferred to a hydrothermal reactor and heated at 140-170°C for 5-7 hours, and then cooled to room temperature.

2. The injectable hydrogel as described in claim 1, characterized in that, In the dihydroartemisinin DHA / 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride AI / Ink@sodium alginate ALG mixed solution, the concentration of sodium alginate ALG is 5~8 mg / mL, the concentration of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride AI is 1~2 mg / mL, the concentration of dihydroartemisinin DHA is 1~2 mmol / L, and the concentration of Ink is 150~400 mg / mL.

3. The application of the injectable hydrogel as described in claim 1 or 2 in the preparation of light-controlled adjuvant tumor therapy drugs, wherein, The tumor treatment is a starvation therapy for the tumor, and the tumor is liver cancer.

4. The application as described in claim 3, characterized in that, The drug is an injectable reagent used for in-situ injection into tumor tissue.

5. The application as described in claim 3, characterized in that, The light control refers to near-infrared light irradiation within the wavelength range of 1000~1700 nm.

6. The application as described in claim 3, characterized in that, The light control refers to 1064 nm laser irradiation.

Images