A chemotherapy drug-hydrogel composite treatment system and a preparation method and application thereof

Abstract

The application discloses a chemotherapy drug-hydrogel compound treatment system and a preparation method and application thereof, and the chemotherapy drug-hydrogel compound treatment system comprises an EDTA-LDH / PLEL hydrogel system or an EGTA / PLEL hydrogel system formed by EDTA / LDH or EGTA and a PLEL hydrogel. 2+ The EDTA / LDH and EGTA based on the chelation principle for treating oral cancer are loaded in the PLEL hydrogel respectively for the first time, two drug-hydrogel treatment systems (EDTA-LDH / PLEL and EGTA / PLEL) are constructed, the loading mode not only realizes efficient drug loading, but also realizes tumor dissociation by destroying the intercellular connection through the Ca 2+ The chelation mechanism destroys the intercellular connection to realize tumor dissociation instead of cell apoptosis. The mechanism is different from the traditional chemotherapy drug action mechanism, and is expected to reduce the side effects caused by cell apoptosis and improve the treatment accuracy.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel materials, specifically relating to a chemotherapy drug-hydrogel complex treatment system, its preparation method, and its application. Background Technology

[0002] Injectable temperature-sensitive hydrogels achieve phase transitions by utilizing the temperature difference between the external environment and the application site within the body. The in vivo application site itself can directly trigger the phase transition, thus avoiding interference from external factors and greatly simplifying the injection procedure. Furthermore, the temperature of the external environment is easily controlled, and human body temperature is relatively constant, resulting in more stable gelation time and other properties of injectable temperature-sensitive hydrogels.

[0003] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, ranking second in incidence among head and neck cancers. Currently, OSCC treatment primarily relies on surgical resection, often combined with radiotherapy and chemotherapy to improve efficacy. However, despite diverse treatment modalities, overall survival remains low and fails to meet clinical needs. Therefore, developing novel oral cancer treatment strategies is crucial for improving patient prognosis.

[0004] Most chemotherapy drugs have difficulty adhering to, targeting, and penetrating oral cancer tumor tissue, resulting in a short residence time in the oral cavity. Simultaneously, saliva secretion and swallowing dilute these drugs and accelerate their excretion. To achieve ideal therapeutic effects, frequent and large-volume injections are often required, but this increases the risk of drug resistance and leads to higher toxic side effects. Similarly, free EDTA faces similar limitations in the local treatment of oral cancer, making effective treatment difficult. Especially when free EDTA molecules are not excreted with saliva, they are more likely to enter the cell interior rather than function on the cell membrane surface to chelate calcium. 2+ This further reduces its effectiveness in the local treatment of oral cancer. Summary of the Invention

[0005] Purpose of the Invention: Addressing the shortcomings of existing methods, the purpose of this invention is to provide a chemotherapy drug-hydrogel complex therapeutic system. This invention, for the first time, loads EDTA / LDH and EGTA, which are based on the chelation principle for treating oral cancer, into PLEL hydrogels, constructing two drug-hydrogel therapeutic systems (EDTA-LDH / PLEL and EGTA / PLEL). This loading method not only achieves highly efficient drug encapsulation but also utilizes Ca... 2+ The chelation mechanism disrupts intercellular connections, leading to tumor dissociation, rather than through apoptosis. This mechanism differs from that of traditional chemotherapy drugs and holds promise for reducing the side effects caused by apoptosis while improving the precision of treatment.

[0006] The present invention also provides a method for preparing the chemotherapy drug-hydrogel complex treatment system and its application.

[0007] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a chemotherapy drug-hydrogel complex treatment system, wherein the chemotherapy drug-hydrogel complex treatment system comprises an EDTA-LDH / PLEL hydrogel system or an EGTA / PLEL hydrogel system formed by EDTA / LDH or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid) and PLEL hydrogel, wherein the PLEL hydrogel is formed by polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer, and the EDTA / LDH uses layered double hydroxide LDH as the carrier of EDTA (ethylenediaminetetraacetic acid).

[0008] In the chemotherapy drug-hydrogel complex treatment system, the block ratio of polylactide, polyethylene glycol, and polylactide is 1000-2000:(1000-2000):1000-2000.

[0009] Preferably, the block ratio of polylactide, polyethylene glycol, and polylactide in the chemotherapy drug-hydrogel complex treatment system is 1500:(1000-2000):1500.

[0010] The gelation time of the chemotherapy drug-hydrogel complex treatment system at 37°C is 20-100 seconds.

[0011] Preferably, the gelation time of the chemotherapy drug-hydrogel complex treatment system at 37°C is 30-40 seconds.

[0012] In the EDTA / LDH, LDH is the carrier with a particle size of 100-1000 nm.

[0013] Preferably, in the EDTA / LDH, LDH is the carrier, and its particle size is 400-700 nm.

[0014] The preparation method of the chemotherapy drug-hydrogel complex therapeutic system of the present invention includes the following steps:

[0015] (1) After heating polyethylene glycol, vacuum was applied while stirring. After cooling to room temperature, D,L-lactide (D,L-LA) and catalyst were added. Then, the reaction system was heated and stirred under an inert atmosphere. After the reaction was completed, it was cooled to room temperature. Ultrapure water was added to dissolve the crude product. The solution was stirred at room temperature to obtain a transparent and homogeneous aqueous solution. The solution was then heated to precipitate the copolymer. The upper layer of solution was removed. The above purification steps were repeated. The separated product, namely the copolymer, was freeze-dried to obtain polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer.

[0016] (2) Add polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer to buffer solution and stir at room temperature until the mixture is a uniform and transparent liquid. Then, place the mixture in a low temperature environment to stand, filter and sterilize to obtain PLEL blank hydrogel.

[0017] (3) Dissolve Zn(NO3)2·6H2O, Al(NO3)3·9H2O, NaNO3 and HMT (hexamethylenetetramine) together in deionized water. Heat and stir the reaction under an inert atmosphere. Dissolve EDTA in deionized water and slowly add it to the above reaction system to carry out the reaction. Lower the reaction temperature and continue the reaction. After the reaction is completed, centrifuge to remove the supernatant, collect the precipitate and wash it. Finally, freeze-dry the product to obtain a white solid powder, namely EDTA / LDH.

[0018] (4) Take a blank PLEL hydrogel and add EDTA / LDH or EGTA solution respectively, and mix well.

[0019] In step (1), after heating polyethylene glycol to above 100°C, vacuum is applied while stirring. After cooling to room temperature, D,L-lactide (D,L-LA) and stannous octoate catalyst (Sn(Oct)2) are added. Then, under an inert atmosphere, the reaction system is heated and stirred at 120-200°C for 8-15 hours. After the reaction is completed, it is cooled to room temperature, and ultrapure water is added to dissolve the crude product. The solution is stirred at room temperature to obtain a transparent and homogeneous aqueous solution. The solution is then heated to 70-120°C to precipitate the copolymer. The upper layer of solution is removed, and the above purification steps are repeated. The separated product, i.e., the copolymer, is freeze-dried to obtain polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer.

[0020] Preferably, a poly(D,L-lactide)-polyethylene glycol-poly(D,L-lactide) (PDLLA-PEG-PDLLA, PLEL) triblock copolymer with a block ratio of 1500:1500:1500 was prepared. First, polyethylene glycol 1500 (PEG1500) and a magnetic stir bar were placed in a clean, dry round-bottom flask. After heating to 100°C in an oil bath, a vacuum was applied for 1 hour under magnetic stirring to remove residual moisture from the raw materials. After the round-bottom flask cooled to room temperature, 2 g of D,L-lactide (D,L-LA) and 18 μL of stannous octoate catalyst (Sn(Oct)2, 0.3% w / w) were added. The ratio of PEG to D,L-LA is the block ratio (w / w). Subsequently, under continuous purging of dry nitrogen, the reaction system was heated to 150°C in an oil bath and reacted for 10 hours under magnetic stirring. After the reaction was complete, the round-bottom flask was cooled to room temperature, and 100 mL of ultrapure water was added to dissolve the crude product. The mixture was stirred at room temperature for 12 h to obtain a clear and homogeneous aqueous solution. The solution was then heated to 80 °C to precipitate the copolymer, and the supernatant was removed to remove the low molecular weight water-soluble copolymer and unreacted monomers. The above purification steps were repeated three times. The separated product, i.e., the copolymer, was freeze-dried and then stored at -20 °C for later use.

[0021] In step (2), the polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer is added to the PBS buffer to make the mass fraction of PLEL reach 20-30%.

[0022] Preferably, in step (2), the mass fraction of PLEL is made to reach 25%.

[0023] Preferably, 0.5942 g of Zn(NO3)2·6H2O, 0.3751 g of Al(NO3)3·9H2O, 0.0850 g of NaNO3, and 0.7025 g of HMT were accurately weighed into a clean, dry round-bottom flask, and 200 mL of deionized water was added to dissolve them together. Nitrogen gas was continuously introduced into the flask, and the mixture was magnetically stirred for 20 min to remove air from the flask. Subsequently, the flask was heated to 120°C in an oil bath and stirred continuously under nitrogen for 2 h. Next, 0.3080 g of EDTA was dissolved in 10 mL of deionized water and slowly added to the above reaction vessel. This operation was repeated once after reacting for 1 h. Then, the reaction temperature was lowered to 80°C, and the reaction was continued for 20 h. After the reaction was completed, the supernatant was removed by centrifugation, the precipitate was collected, and washed thoroughly with deionized water, repeated 3 times. Finally, the product was freeze-dried to obtain a white solid powder, namely EDTA / LDH.

[0024] In step (4), EDTA / LDH or EGTA is added to the buffer solution and dispersed evenly to obtain an EDTA / LDH solution or EGTA solution. The EDTA / LDH solution or EGTA solution is then added to a PLEL blank hydrogel and mixed evenly to form a composite hydrogel. The composite hydrogel contains 1-10 mg / mL of the solution. -1 EDTA-LDH or 1-10 mg / mL -1 EGTA.

[0025] Preferably, in step (4), EDTA / LDH or EGTA is added to the buffer solution and dispersed evenly to obtain an EDTA / LDH solution or EGTA solution. The EDTA / LDH solution or EGTA solution is then added to a PLEL blank hydrogel and mixed evenly to form a composite hydrogel. The composite hydrogel contains 1-5 mg / mL of the solution. -1 EDTA-LDH or 6-10 mg / mL -1 EGTA.

[0026] Preferably, in step (4), EDTA / LDH or EGTA is added to the buffer solution and dispersed evenly to obtain an EDTA / LDH solution or EGTA solution. The EDTA / LDH solution or EGTA solution is then added to a PLEL blank hydrogel and mixed evenly to form a composite hydrogel. The composite hydrogel contains 3 mg / mL of the solution. -1 EDTA-LDH or 8 mg / mL -1 EGTA.

[0027] The application of the chemotherapy drug-hydrogel complex treatment system described in this invention in the preparation of drugs for the treatment of oral cancer.

[0028] This invention proposes a local treatment scheme for oral cancer based on the chelation principle of EDTA and Ca²⁺. To this end, this invention designs an injectable thermosensitive hydrogel of polylactide-polyethylene glycol-polylactide (PLEL). Figure 1 This hydrogel possesses the characteristics of being "liquid at room temperature and gel at body temperature," enabling it to form a drug reservoir at the tumor site and achieve slow drug release. Based on this platform, this invention constructs two hydrogel complex therapeutic systems: EDTA-LDH / PLEL and EGTA / PLEL hydrogels. In the EDTA-LDH / PLEL system, LDH nanosheets are used as a carrier for EDTA. LDH carries a positive surface charge and can electrostatically adsorb onto the negatively charged cell membrane without entering the cell interior. For the EGTA / PLEL system, given that EGTA has a larger molecular weight and size than EDTA, its ability to enter cells is relatively weaker, and it tends to remain on the cell membrane surface and in the extracellular environment, exerting its effect on calcium absorption.2+ The chelating effect of EGTA. Based on this property, EGTA was directly physically mixed with PLEL hydrogel to construct a highly efficient drug delivery system. This drug delivery system is not only simple to prepare and easy to scale up for production, but also can fully utilize the properties of EGTA to achieve the chelation of Ca. 2+ Precise regulation provides a new approach and method for related biological research and potential clinical applications.

[0029] This invention designs and successfully constructs an innovative injectable thermosensitive hydrogel system of poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PDLLA-PEG-PDLLA, PLEL). This system cleverly incorporates EDTA-Ca... 2+ EDTA / LDH and EGTA, based on the chelation principle, are loaded into PLEL hydrogels to form a chemotherapy drug-hydrogel complex therapeutic system (EDTA-LDH / PLEL and EGTA / PLEL hydrogels) capable of precisely forming drug reservoirs at tumor sites and achieving slow release. Studies have shown that both systems respond to the microacidic environment of tumors and achieve precise drug release, providing strong support for targeted tumor therapy. In terms of physicochemical properties, the sol-gel phase transition temperature of the drug-loaded gel with a PLEL content of 25 wt% is approximately 34°C, a characteristic that meets the requirements of an injectable thermosensitive hydrogel with a "room temperature sol state and body temperature gel state." Therefore, selecting this PLEL content for subsequent research is not only reasonable but also highly feasible and has significant application potential. Furthermore, this invention establishes an in vitro high-performance liquid chromatography (HPLC) method for content determination, measuring the EDTA and EGTA content in in vitro released samples of the drug-loaded gel. In vitro drug release behavior studies show that both gels exhibit good drug release behavior and can maintain therapeutic efficacy for a long time in tumor treatment. In summary, this invention not only provides a solid theoretical and experimental foundation for subsequent cell and animal anti-tumor experiments, but also paves a new path for the development and optimization of precision treatment strategies for oral cancer. By constructing and validating a drug delivery system based on PLEL hydrogel, the targeted release of EDTA and EGTA at the tumor site was successfully achieved. This innovative treatment system is expected to significantly improve therapeutic efficacy while reducing toxicity to normal tissues in future clinical applications.

[0030] This invention successfully synthesized EDTA-LDH / PLEL and EGTA / PLEL hydrogel composite systems and characterized their physicochemical properties. The results showed that the PLEL hydrogel exhibits excellent temperature responsiveness, remaining liquid at room temperature while rapidly transforming into a gel state at body temperature to form a stable drug reservoir, achieving localized sustained drug release. Both composite systems demonstrated good stability, drug release performance, and biocompatibility, laying a solid foundation for subsequent biological research and potential clinical applications.

[0031] This invention investigates the cellular mechanisms of action of EDTA-LDH / PLEL and EGTA / PLEL hydrogel composite systems, covering multiple aspects such as cytotoxicity and disruption of intercellular connections. Figure 2 The study aims to evaluate the antitumor activity of these two composite materials. Simultaneously, a systematic assessment of the in vivo biosafety of these composite systems will be conducted, including potential toxicity to normal tissues, to ensure their safety and efficacy in clinical applications. These studies aim to provide a novel, highly effective, low-toxicity strategy with clinical translational potential for the precision treatment of oral cancer.

[0032] This invention addresses the current limitations of oral cancer treatment methods in meeting the practical needs of oral cancer patients for efficient, low-toxicity, precise, and minimally invasive treatment. It constructs a hydrogel composite material that combines PLEL triblock copolymer and EDTA-Ca2+ chelation mechanism, and applies it to in vitro and in vivo anti-tumor studies of SCC-7 oral cancer cells.

[0033] The following solutions will be implemented:

[0034] (1) Preparation of injectable thermosensitive hydrogel composite system

[0035] This invention successfully developed an injectable thermosensitive hydrogel (PLEL) suitable for local injection. By physically mixing EDTA / LDH and EGTA, which are used for oral cancer treatment based on chelation principles, with PLEL hydrogels, two chemotherapy drug-hydrogel complex therapeutic systems were constructed: EDTA-LDH / PLEL and EGTA / PLEL. Both drug-loaded gels exhibited good temperature responsiveness, meeting the characteristics of injectable thermosensitive hydrogels that are "liquid at room temperature and transform into a gel at body temperature." Furthermore, they possess excellent physicochemical properties, providing a solid foundation for subsequent validation of their antitumor effects in cell and animal models.

[0036] (2) Injectable thermosensitive hydrogel materials based on the EDTA-Ca2+ chelation mechanism are applied to the treatment of oral cancer.

[0037] This invention delves into the Ca²⁺ chelation mechanism and its antitumor effects at both in vitro cellular and in vivo animal levels. At the cellular level, various experimental methods confirmed that the Ca²⁺ chelation mechanism disrupts intercellular connections, leading to tumor cell dissociation, rather than inducing apoptosis. Further in vivo studies, using C3H tumor-bearing mice as a model, verified the intratumoral injectability of the composite hydrogel material, establishing its feasibility for in situ injection. Simultaneously, in vivo antitumor studies showed that both hydrogel composites exhibited good antitumor properties and excellent biocompatibility. This study proposes a method based on EDTA-Ca²⁺... 2+ A novel chelation-based treatment strategy for oral cancer utilizes PLEL hydrogel as a carrier to disrupt intercellular connections, promoting cancer cell detachment. PLEL hydrogel exhibits room-temperature liquid properties and gelation at body temperature, enabling sustained drug release and long-lasting effects. It also possesses good biocompatibility and non-toxicity, simplifying the treatment process and reducing patient suffering and medical resource consumption. Compared to current clinical applications of oral cancer surgery and radiotherapy / chemotherapy, this treatment strategy based on the EDTA-Ca²⁺ chelation mechanism offers significant advantages, promising a gentle, non-invasive, and promising new approach to oral cancer treatment, providing patients with safer and more effective treatment options.

[0038] The therapeutic system prepared in this invention utilizes the combination of EDTA and LDH: a synergistic enhancement of the carrier and the drug. EDTA (ethylenediaminetetraacetic acid) is a Ca2+... 2+ Chelating agents, when applied directly, are easily carried away by saliva or enter cells, reducing their efficacy. LDH (layered double hydroxide), acting as a nanocarrier, loads EDTA into its interlayer structure through electrostatic adsorption (confirmed by XRD and FT-IR). Figure 7 As shown in the figure, an EDTA / LDH complex is formed. The layered structure of LDH protects EDTA from degradation and responsively releases the drug in the tumor microacidic environment (pH≈6.5) (in vitro release assay results).

[0039] The therapeutic system prepared in this invention combines EDTA-LDH and EGTA with PLEL hydrogel, achieving a synergistic effect of sustained release and local retention. PLEL hydrogel possesses thermosensitive properties (room temperature liquid, body temperature gel), and when combined with EDTA-LDH and EGTA respectively, forms a hydrogel composite system. At body temperature, the PLEL hydrogel forms a gel reservoir, delaying drug release (in vivo distribution experiments). Figure 22This study confirmed that drug-loaded hydrogels can significantly prolong the retention time of drugs at tumor sites. Meanwhile, EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid) has a larger molecular weight than EDTA, making it more easily retained on the cell surface. It can be directly mixed with PLEL hydrogels to form an EGTA / PLEL system. EGTA can be physically mixed with PLEL without a carrier, simplifying preparation while still achieving sustained release (in vitro release experiments).

[0040] Furthermore, this invention demonstrates the synergistic necessity of the carrier and hydrogel through free drug and composite systems. In in vivo antitumor experiments, all groups used intratumoral injection, with consistent tumor models and administration time points, differing only in drug form (free or composite). Compared to the free EDTA and EGTA groups, the composite hydrogel group showed better tumor inhibition. This proves that removing the hydrogel limits the drug's effectiveness due to rapid loss; the composite system, however, utilizes the thermosensitive gel properties of PLEL to form a drug reservoir, prolonging the local action time. Simultaneously, the free EDTA / LDH group showed better efficacy than the free EDTA group but less than the composite system, indicating that the LDH carrier can enhance targeting, but requires combination with a hydrogel to maximize synergy.

[0041] Meanwhile, in the preparation of EDTA / LDH, this invention, by controlling the reaction temperature, time, and EDTA addition method (e.g., adding in two stages), compared with the conventional single-addition method, evaluated the size and dispersibility of the nanosheets (characterized by SEM and DLS). The optimized steps (reacting at 120℃ for 2 hours followed by adding EDTA in two stages, then continuing the reaction at 80℃ for 20 hours) yielded EDTA / LDH nanosheets with a size of approximately 600 nm. Figure 5 However, simplifying the process may lead to aggregation or uneven size. In this invention, the size (600 nm) avoids endocytosis and enhances cell membrane adsorption. If smaller nanosheets (e.g., <200 nm) are used instead, they are more easily endocytosed, reducing the surface interaction effect. This also demonstrates the special nature of the preparation steps in this invention: by precisely controlling variables, the carrier size is ensured to be within the optimized range, thereby maximizing the biological effect.

[0042] This invention is the first to prepare a specific chemotherapy drug-hydrogel complex treatment system for the treatment of oral cancer, and uses injectable thermosensitive hydrogel PLEL as a drug delivery carrier. While achieving cell dissociation, the sustained release and local positioning properties of the hydrogel further enhance the therapeutic effect.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0044] (1) Construction of the composite system: This invention creatively utilizes a physical mixing method to load EDTA / LDH and EGTA, which are based on the chelation principle for treating oral cancer, into PLEL hydrogels for the first time, constructing two drug-hydrogel therapeutic systems (EDTA-LDH / PLEL and EGTA / PLEL). This loading method not only achieves efficient drug loading but also utilizes Ca... 2+ The chelation mechanism disrupts intercellular connections, leading to tumor dissociation, rather than through apoptosis. This mechanism differs from that of traditional chemotherapy drugs and holds promise for reducing the side effects caused by apoptosis while improving the precision of treatment.

[0045] (2) EDTA-Ca 2+ Chelation mechanism: This invention is the first to utilize EDTA-Ca... 2+ The chelation mechanism-based cancer cell removal strategy, applied to the treatment of oral cancer, is fundamentally different from the currently commonly used surgical resection and radiotherapy / chemotherapy methods. This strategy disrupts intercellular connections by chelating calcium ions, prompting cancer cells to detach from the tissue, rather than directly inducing apoptosis. This avoids the trauma of surgery and the severe side effects of radiotherapy and chemotherapy. This gentle, non-invasive treatment method not only promises to significantly alleviate patient suffering and improve their quality of life but also effectively reduces the risk of side effects and complications during treatment. As a novel approach in the field of oral cancer treatment, this strategy offers a highly promising new direction for future clinical treatment and has significant application prospects.

[0046] (3) Application of LDH in the treatment of oral cancer: The positive charge on the surface of LDH enables it to electrostatically adsorb onto the negatively charged cell membrane, thereby forming a stable adsorption layer on the cell surface. This adsorption not only enhances the retention time of drugs on the surface of tumor cells but also prevents drugs from entering the cell interior through endocytosis, thus reducing the toxicity of drugs to normal cells. In addition, LDH is pH sensitive and can responsively release drugs in the slightly acidic environment of the tumor (pH about 6.5). This characteristic enables LDH to achieve precise drug release at the tumor site, further improving drug targeting and therapeutic efficacy. Through ingenious design and experimental verification, this invention has successfully achieved innovation in oral cancer treatment materials and technologies. Attached Figure Description

[0047] Figure 1 A schematic diagram showing the overall preparation and experimentation of EDTA-LDH / PLEL hydrogels and EGTA / PLEL hydrogels.

[0048] Figure 2 A schematic diagram illustrating the antitumor mechanism of EDTA-LDH / PLEL and EGTA / PLEL on SCC-7 cells.

[0049] Figure 3This is a roadmap for the PDLLA-PEG-PDLLA triblock copolymer.

[0050] Figure 4 PDLLA-PEG-PDLLA polymer 1 H NMR spectrum.

[0051] Figure 5 SEM images of EDTA / LDH: EDTA / LDH monodisperse in water (scale bar 100 nm) (A); EDTA / LDH dispersed in water (scale bar 500 nm) (B); Side view of EDTA / LDH dispersion (scale bar 200 nm) (C).

[0052] Figure 6 EDS scan of EDTA / LDH (A); corresponding elemental distribution (B).

[0053] Figure 7 Infrared spectra of LDH and EDTA / LDH (A); XRD patterns of LDH and EDTA / LDH (B).

[0054] Figure 8 Zeta plots for LDH and EDTA / LDH (A); Particle size distribution of EDTA / LDH (B).

[0055] Figure 9 Infrared spectra of EDTA, EDTA / LDH, PLEL, and EDTA-LDH / PLEL (A); Infrared spectra of EGTA, PLEL, and EGTA / PLEL (B).

[0056] Figure 10 Cryo-SEM images of EDTA-LDH / PLEL (scale bars at 10 μm and 3 μm) (A) and (B); Distribution of EDTA / LDH in the PLEL network structure (scale bars at 1 μm and 500 nm) (C) and (D).

[0057] Figure 11 Phase transition diagrams of PLEL blank hydrogel, EDTA-LDH / PLEL and EGTA / PLEL hydrogel (A); reversible sol-gel phase transition photographs of PLEL blank hydrogel, EDTA-LDH / PLEL and EGTA / PLEL hydrogel (25 wt%) (B).

[0058] Figure 12 The gel time and gel effect of EDTA / LDH (A) and EGTA (B) at different concentrations are shown.

[0059] Figure 13Rheological spectra of EDTA-LDH / PLEL and EGTA / PLEL hydrogels (25 wt%): Storage modulus (G′) and loss modulus (G″) of EDTA-LDH / PLEL hydrogel as a function of temperature (A); G′ and G″ of EGTA / PLEL hydrogel as a function of temperature (B); G′ of EDTA-LDH / PLEL and EGTA / PLEL hydrogel as a function of temperature (C).

[0060] Figure 14 The pH changes during the degradation of PLEL blank hydrogel, EDTA-LDH / PLEL, and EGTA / PLEL hydrogel.

[0061] Figure 15 The following are the standard curves for EGTA (A); EDTA (B); EGTA / PLEL release curves at pH 7.4 and 6.5 (C); and EDTA-LDH / PLEL release curves at pH 7.4 and 6.5 (D).

[0062] Figure 16 Simulated docking diagrams for EDTA and LDH, EDTA / LDH and PLEL, and EGTA and PLEL: Active docking site of LDH (A); Active docking site of PLEL (B); Docking conformations of EDTA and LDH, EDTA / LDH and PLEL, and EGTA and PLEL (CE).

[0063] Figure 17 Cell viability of the three hydrogels in L929 cells (A); Cell viability of the three hydrogels in SCC-7 cells (B) and (C) ).

[0064] Figure 18 Micrographs of SCC-7 cells after co-incubation of EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel for 2 h and 4 h.

[0065] Figure 19 Flow cytometry was used to detect apoptosis in SCC-7 cells after co-incubation with EDTA-LDH / PLEL and EGTA / PLEL for 2 h and 4 h.

[0066] Figure 20 Effects of EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel on the expression of calcium-dependent proteins in cells: Schematic diagram of E-cadherin structure (A); Immunoblot detection of E-cadherin and S100A4 protein expression (B), (C), and (D) ).

[0067] Figure 21 Effects of EDTA-LDH / PLEL and EGTA / PLEL on the migration ability of SCC-7 cells: Cell scratch results (scale bar: 100 µm) (A); Cell migration was quantified by migration distance. (B).

[0068] Figure 22 In vivo fluorescence imaging results at different time points after intratumoral injection of Cy5.5 PLEL hydrogel solution (25 wt%) and free Cy5.5 solution into SCC-7 tumor-bearing C3H mice: mouse fluorescence image (A); statistical graph of relative fluorescence intensity at the tumor site in mice (B) ).

[0069] Figure 23 The antitumor effects of EDTA-LDH / PLEL and EGTA / PLEL hydrogels on the subcutaneous heterotopic transplantation model of SCC-7 in C3H mice: Schematic diagram of treatment cycle (A); Tumor volume change curve over time after the first administration of different groups of drugs (NS; PLEL; EDTA; EDTA / LDH; EGTA; EDTA-LDH / PLEL; EGTA / PLEL) into C3H xenograft mice (n=5); Curve of tumor volume change over time after the first administration (B); Curve of mouse body weight change over time after the first administration (C); Photograph of ex vivo tumor tissue after treatment (D); Statistical graph of ex vivo tumor tissue weight after treatment (E). Labels: (I) NS; (II) PLEL; (III) EDTA; (IV) EDTA / LDH; (V) EGTA; (VI) EDTA-LDH / PLEL; (VII) EGTA / PLEL ).

[0070] Figure 24 The endpoint of tumor growth inhibition rate in EDTA-LDH / PLEL and EGTA / PLEL hydrogel treatment ( ).

[0071] Figure 25 H&E staining images of four groups of tumor tissues (normal saline (NS), PLEL hydrogel, EDTA-LDH / PLEL and EGTA / PLEL hydrogel) (scale bar: 50 µm).

[0072] Figure 26 Figure 1 shows the hemolysis safety test results for EDTA-LDH / PLEL hydrogel (A) and EGTA / PLEL hydrogel (B) (25 wt%).

[0073] Figure 27H&E staining images of major organs (heart, liver, spleen, lung and kidney): NS group, EDTA-LDH / PLEL and EGTA / PLEL (scale bar: 50 µm). Detailed Implementation

[0074] The present invention will be specifically illustrated below through examples. These examples are provided to better illustrate the invention and are not intended to limit its scope. Various changes and modifications can be made to the invention without departing from its spirit and scope.

[0075] Unless otherwise specified, the starting materials used in the embodiments of the present invention are all known products that can be obtained by purchasing commercially available products. For details of specific reagents and instruments, please refer to Tables 1-4.

[0076] Table 1 Experimental Materials and Reagents

[0077]

[0078] Table 2 Experimental Instruments

[0079]

[0080]

[0081] Table 3 Experimental Materials and Reagents

[0082]

[0083] Table 4 Experimental Instruments

[0084]

[0085] Example 1

[0086] Preparation of polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer

[0087] A triblock copolymer of poly(D,L-lactide)-polyethylene glycol-poly(D,L-lactide) (PDLLA-PEG-PDLLA, PLEL) with a block ratio of 1500:1500:1500 was prepared by ring-opening polymerization. Specific steps are detailed below. Figure 3 .

[0088] First, 1 g of polyethylene glycol 1500 (PEG1500) and a magnetic stir bar were placed in a clean, dry round-bottom flask. The mixture was heated to 100°C in an oil bath and then evacuated for 1 h under magnetic stirring to remove residual moisture from the raw materials. After the round-bottom flask cooled to room temperature, 2 g of D,L-lactide (D,L-LA) and 18 μL of stannous octoate catalyst (Sn(Oct)2, 0.3% w / w) were added. Subsequently, the reaction system was heated to 150°C in an oil bath under continuous purging of dry nitrogen and reacted for 10 h under magnetic stirring. After the reaction was complete, the round-bottom flask was cooled to room temperature, and 100 mL of ultrapure water was added to dissolve the crude product. The mixture was stirred at room temperature for 12 h to obtain a clear, homogeneous aqueous solution. The solution was then heated to 80°C to precipitate the copolymer, and the supernatant was removed to remove low-molecular-weight water-soluble copolymers and unreacted monomers. After repeating the purification steps three times, the separated product, the copolymer, was freeze-dried and then stored at -20°C for later use. Finally, the freeze-dried product was dissolved in deuterated chloroform and processed... 1 The structure of the product was identified by H NMR spectroscopy.

[0089] like Figure 4 As shown, PLEL's 1 H NMR (CDCl3) spectral analysis: δa 4.27 ppm is (-CH2CH2O-), δb 3.64 ppm is (-CH2CH2O-), δc 5.16 ppm is (-OCH(CH3)CO-), and δd 1.57 ppm is (-OCH(CH3)CO-). (-CH2CH2O-) is a characteristic structure of the polyethylene glycol (PEG) moiety in the PLEL molecule, with a chemical shift typically occurring in the range of 4.0–3.5 ppm. The peaks δa and δb in the figure correspond to methylene hydrogen in different chemical environments within this unit, indicating that the PEG moiety has been successfully incorporated into the PLEL molecule. The (-OCH(CH3)CO-) unit is a characteristic structure of the lactic acid moiety in the PLEL molecule, with a chemical shift typically occurring in the range of 5.0–1.5 ppm. The peaks δc and δd in the figure correspond to methylene hydrogen and methyl hydrogen in this unit, respectively, indicating that the lactic acid moiety has also been successfully attached to the PLEL molecule. By detecting these characteristic peaks, it can be inferred that PLEL has been successfully synthesized.

[0090] Example 2

[0091] Preparation of EDTA-LDH / PLEL and EGTA / PLEL hydrogel composite systems

[0092] 1. Preparation of EDTA / LDH

[0093] Accurately weigh 0.5942 g of Zn(NO3)2·6H2O, 0.3751 g of Al(NO3)3·9H2O, 0.0850 g of NaNO3, and 0.7025 g of HMT into a clean, dry round-bottom flask, and add 200 mL of deionized water to dissolve them together. Nitrogen gas is continuously bubbled into the flask, and the mixture is magnetically stirred for 20 min to remove air from the flask. Then, the flask is heated to 120°C in an oil bath and stirred continuously under nitrogen for 2 h. Next, 0.3080 g of EDTA is dissolved in 10 mL of deionized water and slowly added to the reaction vessel. This process is repeated once after 1 h of reaction (i.e., after 1 hour of reaction, another 0.3080 g of EDTA dissolved in 10 mL of deionized water is slowly added to the reaction vessel). Finally, the reaction temperature is lowered to 80°C, and the reaction continues for 20 h. After the reaction was completed, the supernatant was removed by centrifugation, the precipitate was collected and washed thoroughly with deionized water, and the process was repeated 3 times. Finally, the product was freeze-dried to obtain a white solid powder, namely EDTA / LDH.

[0094] 2. Preparation of PLEL blank hydrogel

[0095] An appropriate amount of PLEL prepared in Example 1 was placed in a weighing bottle, and PBS with a pH of 7.4 was added to bring the PLEL mass fraction to 25%. The mixture was stirred at room temperature until it became a homogeneous and transparent liquid. The mixture was then allowed to stand at 4°C for one day to reduce its viscosity. Afterward, it was sterilized by filtration under ice bath conditions and stored at 4°C for later use.

[0096] 3. Concentration optimization of EDTA / LDH and EGTA

[0097] The effects of EDTA / LDH and EGTA concentrations on the gelation of PLEL at 37℃ were investigated to determine the optimal concentrations. 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, and 5.0 mg of EDTA / LDH, and 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg of EGTA were weighed and added to 1 mL of a 25 wt% PLEL blank hydrogel. The samples were first placed in a 4℃ refrigerator for 30 min to equilibrate, and then transferred to a 37℃ water bath for 10 min. Afterward, the Eppendorf tube containing the samples was inverted 180°, and the flow of the samples was observed and the gelation time was recorded. If the samples did not flow in the tube, they were considered to be in a gel state; otherwise, they were considered to be in a sol state. The gelation behavior of EDTA-LDH / PLEL and EGTA / PLEL hydrogels at 37℃ was studied using the "inversion method".

[0098] 4. Preparation of EDTA / LDH and EGTA solutions

[0099] Weigh a certain amount of EDTA / LDH, add 2 mL of pH 7.4 PBS, and sonicate for 20 min to disperse evenly to obtain the EDTA / LDH solution. Store at 4℃.

[0100] Weigh a certain amount of EGTA, add 2 mL of pH 7.4 PBS, and sonicate for 5 min to completely dissolve it to obtain the EGTA solution. Store at 4℃.

[0101] 5. Preparation of EDTA-LDH / PLEL and EGTA / PLEL hydrogels

[0102] A certain volume of PLEL blank hydrogel was taken, and certain volumes of EDTA / LDH and EGTA solutions were added respectively, to a final hydrogel concentration of 25 wt%. The mixture was sonicated for 10 min, and homogenized by several aspiration and displacement cycles to obtain a composite hydrogel. This composite hydrogel was stored at 4℃ for later use. The composite hydrogel contained 3 mg / mL of PLEL solution. -1 EDTA-LDH or 8 mg / mL -1 EGTA.

[0103] Example 3

[0104] To demonstrate the successful preparation of EDTA / LDH in Example 2, its morphology and composition were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), elemental analysis, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), zeta potential, and particle size analysis to prove its successful synthesis.

[0105] Cryo-scanning electron microscopy (Cryo-SEM) was performed using a FEI Quanta 450. Scanning electron microscopy (SEM) and elemental energy dispersive spectroscopy (EDS) mapping were performed using a Magellan 400 field emission spectrometer. Fourier transform infrared spectroscopy (FT-IR) was performed using an FT-IR spectrophotometer (Shimadzu, Japan). X-ray diffraction (XRD) was performed using a Rigaku Smartlab SE X-ray photoelectron spectrometer. Zeta potential and particle size were measured using a Zetasizer Nano-ZS90 particle size potential analyzer (Malvern, England).

[0106] (1) Scanning electron microscopy results of EDTA / LDH nanosheets

[0107] like Figure 5As shown in (A) and (B), scanning electron microscope images illustrate the morphology of the two-dimensional EDTA / LDH nanosheets prepared in step 1 of Example 2. The images show that the EDTA / LDH nanosheets have a size of approximately 600-700 nm and exhibit a distinct layered structure (e.g., ...). Figure 5 (As shown in (C)). The adsorption and action mechanisms of nanosheets of this size on cell membranes are significantly different from those of smaller nanosheets. Larger EDTA / LDH nanosheets (600-700 nm) can effectively avoid endocytosis. Clathin-mediated endocytosis is generally more effective for smaller nanoparticles (<200 nm), while larger nanosheets tend to adsorb onto the cell membrane surface, thus exerting their effects on the cell membrane for a longer period.

[0108] (2) EDS elemental analysis

[0109] To gain a deeper understanding of the elemental composition of the material, elemental content determination and energy dispersive spectroscopy (EDS) elemental analysis were performed. Figure 6 The scanning electron microscope (SEM) elemental mapping and corresponding EDS energy dispersive spectroscopy (EDS) spectrum of the EDTA / LDH material system are presented. The figures show that zinc (Zn), aluminum (Al), and oxygen (O) are uniformly distributed on the EDTA / LDH surface, indicating that these are the main components of zinc-aluminum layered double hydroxide (LDH). Furthermore, the elemental content data in Table 5 further confirm the successful synthesis of the material.

[0110] Table 5 Elemental content of EDTA / LDH

[0111]

[0112] (3) FT-IR

[0113] The functional groups of LDH and EDTA / LDH were analyzed using FT-IR spectroscopy. Figure 7 (A) shows the infrared spectra of LDH and EDTA / LDH. In the LDH spectrum, 3520 cm⁻¹ -1 and 1620 cm -1 The broad absorption band is due to the hydrogen bond stretching vibrations in LDH and the bending vibrations of water. At 1384 cm⁻¹ -1 The sharp absorption band at 1604 cm⁻¹ is caused by the stretching vibrational mode of nitrate ions in the LDH interlayer. In the infrared spectrum of EDTA / LDH, this band is observed at 1604 cm⁻¹. -1 and 1392 cm -1 The strong absorption peak at this point is caused by the symmetric and antisymmetric vibrations of the COO- group in EDTA, indicating that EDTA has been successfully inserted into LDH.

[0114] 0.5942 g of Zn(NO3)2·6H2O, 0.3751 g of Al(NO3)3·9H2O, 0.0850 g of NaNO3, and 0.7025 g of HMT were accurately weighed into a clean, dry round-bottom flask, and 200 mL of deionized water was added to dissolve them together. Nitrogen gas was continuously introduced into the flask, and the mixture was magnetically stirred for 20 min to remove air from the flask. Subsequently, the flask was heated to 120°C in an oil bath and stirred continuously under nitrogen for 2 h. Afterward, the reaction temperature was lowered to 80°C, and the reaction was continued for 20 h. After the reaction was completed, the supernatant was removed by centrifugation, the precipitate was collected, and washed thoroughly with deionized water, repeated three times. Finally, the product was freeze-dried to obtain a white solid powder, namely LDH.

[0115] (4) XRD

[0116] To investigate the structural characteristics of LDH and EDTA / LDH materials, the positions and intensities of diffraction peaks in pure LDH and EDTA / LDH materials were analyzed using XRD technology. Figure 7 (B) The EDTA / LDH composite system exhibits a layered structure: the (003), (006), and (009) diffraction peaks of the LDH nanosheets are clearly visible, indicating the layered, ordered arrangement of LDH. A significant shift in the diffraction peak positions is observed between the LDH samples before and after EDTA intercalation. Detailed data show that the characteristic diffraction peaks of the EDTA-modified LDH migrate to lower angles compared to the original LDH. This shift is due to the insertion of EDTA molecules into the LDH interlayer. EDTA molecules have a relatively long van der Waals end length, and when inserted into the LDH interlayer via anion exchange, they significantly increase the interlayer spacing of LDH. XRD analysis confirmed that EDTA successfully inserted into the LDH interlayer via anion exchange. The shift of characteristic peaks to smaller angles indicates that the insertion of EDTA led to a significant increase in the LDH interlayer spacing, thus confirming the successful preparation of the EDTA / LDH composite material.

[0117] (5) Zeta potential and particle size

[0118] To comprehensively characterize the surface charge and particle size of LDH and EDTA / LDH materials, dynamic light scattering (DLS) technology was used for Zeta potential analysis and particle size measurement. Figure 8The zeta potential analysis and particle size distribution results of LDH and EDTA / LDH materials are presented. The figures show that the LDH material surface exhibits a significant positive charge, consistent with the positive charge of the metal hydroxide layer in its layered structure. The zeta potential of the EDTA / LDH material is lower than that of LDH. Since EDTA itself carries a negative charge, its insertion into the LDH interlayer via anion exchange partially neutralizes the positive charge of LDH, leading to a decrease in the overall zeta potential. This result is consistent with our expectations, indicating successful insertion of EDTA into the LDH interlayer. The particle size distribution of the EDTA / LDH material is approximately 600 nm. This result is consistent with the findings of scanning electron microscopy (SEM) images, further confirming the size and dispersion of the EDTA / LDH nanosheets.

[0119] (6) Infrared characterization of EDTA-LDH / PLEL and EGTA / PLEL

[0120] The functional groups of EDTA-LDH / PLEL and EGTA / PLEL were analyzed using FT-IR spectroscopy. Figure 9 (A) shows the infrared spectra of EDTA, EDTA / LDH, PLEL, and EDTA-LDH / PLEL in Example 2. In the EDTA spectrum, 1595 cm⁻¹ -1 This is usually related to the antisymmetric stretching vibration of -COO-. The EDTA molecule contains multiple carboxyl groups -COOH, which form a carboxylate ion after deprotonation. 1417 cm⁻¹ -1 This is likely due to the bending vibration of the amino group (-NH2). In the EDTA / LDH spectrum, at 1604 cm⁻¹... -1 and 1398 cm -1 The strong absorption band at 3500 cm⁻¹ is due to the symmetric and antisymmetric vibrations of the -COO- group in EDTA, proving that EDTA has successfully inserted into LDH. In the PLEL infrared spectrum, 3500 cm⁻¹... -1 This peak is typically associated with the stretching vibration of -OH. 1641 cm⁻¹ -1 The position may be related to the stretching vibration of the ester group (-COO-). In the spectrum of the EDTA-LDH / PLEL hydrogel, 3641 cm⁻¹ -1 and 1637 cm -1 These are the stretching vibrations of -OH and -COO-, respectively. 1460 cm⁻¹ -1 and 1375 cm -1 It may be caused by the symmetric and antisymmetric stretching vibrations of -COO-. Figure 9 (B) shows the infrared spectra of EGTA, PLEL, and EGTA / PLEL. In the EGTA spectrum, 1749 cm⁻¹ -1 and 1396cm -1It is usually associated with antisymmetric and symmetric stretching vibrations of (-COO-). In the infrared spectrum of PLEL, 3466 cm⁻¹ -1 This peak is typically associated with the stretching vibration of the hydroxyl group (-OH). 1645 cm⁻¹ -1 The position may be related to the stretching vibration of the ester group -COO-. In the spectrum of the EGTA / PLEL hydrogel, 3487 cm⁻¹ -1 and 1643 cm -1 These are the stretching vibrations of the -OH and -COO- groups, respectively. 1371 cm⁻¹ -1 It may be caused by the symmetrical stretching vibration of -COO-.

[0121] (7) Cryo-scanning electron microscopy of EDTA-LDH / PLEL

[0122] The internal structure of the EDTA-LDH / PLEL hydrogel was characterized using cryo-scanning electron microscopy. Specifically, the overall 3D network structure of the hydrogel was observed, and the distribution of its internal nanosheets was further analyzed. Figure 10 Figures (A) and (B) illustrate the three-dimensional network structure of the EDTA-LDH / PLEL hydrogel. The scale bars in the figures are 10 μm and 3 μm, respectively, visually reflecting the network structure characteristics of the PLEL hydrogel. Further high-resolution images are shown below. Figure 10 As shown in (C) and (D), numerous EDTA / LDH nanosheets (circled in red) exist within the PLEL hydrogel network structure. These nanosheets are uniformly distributed throughout the hydrogel network, forming a composite structure. The scale bars in the figures are 1 μm and 500 nm, respectively. These results demonstrate that the EDTA-LDH / PLEL hydrogel possesses a typical three-dimensional network structure, which provides the hydrogel with excellent mechanical properties.

[0123] Example 3

[0124] The following methods were used to investigate the phase transition behavior of PLEL blank hydrogel, EDTA-LDH / PLEL, and EGTA / PLEL hydrogel.

[0125] 1. Phase diagram drawing using the inverted test tube method

[0126] Blank hydrogels containing 30.0, 25.0, and 20.0 wt% PLEL and hydrogels containing 1-3 mg / mL PLEL were prepared according to the method in Example 2. -1 EDTA-LDH composite hydrogels and those containing 6-10 mg / mL -1EGTA composite hydrogels. All samples were stored at 4℃ for 30 min and then subjected to programmed temperature increases in increments of 1℃ within the 15-65℃ range. After being kept at a constant temperature for 10 min at each temperature point, the rheological behavior was observed over 60 s by inverting the Ep tube: the criteria were (1) phase separation and overall flow as precipitation, (2) stable adhesion as gel, and (3) flow along the tube wall as sol. A phase transition diagram was constructed with PLEL content as the independent variable and phase transition temperature as the dependent variable by recording the critical phase transition temperature. The temperature response characteristics of hydrogels with different components were systematically evaluated using the inversion method during the experiment.

[0127] The effects of different concentrations of EDTA / LDH and EGTA on the gelation properties of PLEL hydrogel (25 wt%) were tested. By precisely controlling the material concentrations, the gelation time of the hydrogel at 37℃ was observed, and the stability of the gel was evaluated using the inverted test tube method. Figure 11 As shown in (A), when the concentration of EDTA / LDH is 3 mg / mL -1 At 37°C, the gelation time of the PLEL hydrogel remained relatively stable at approximately 35 seconds. Observation using the inverted test tube method showed that the gel at this concentration was relatively stable, exhibiting no significant flow or deformation. This indicates that EDTA / LDH at this concentration effectively promotes the rapid formation of the PLEL hydrogel, and the resulting gel possesses good physical stability. Figure 11 As shown in (B), when the concentration of EGTA is 8 mg / mL -1 At 37°C, the gelation time of the PLEL hydrogel stabilized at approximately 32 s. Evaluation using the inverted test tube method showed good gelation performance at this concentration, with the gel exhibiting excellent stability and no obvious liquefaction or stratification. This indicates that EGTA at this concentration can effectively promote the formation of PLEL hydrogels, and the resulting gel possesses excellent physical properties.

[0128] Further experimental data showed that when the PLEL concentration was in the range of 20-30 wt%, the blank and the drug-loaded hydrogel (containing 3 mg / mL) were significantly different. -1 EDTA-LDH composite hydrogel or containing 8 mg / mL -1 The EGTA composite hydrogel exhibits a continuous state transition from sol to gel to precipitation when heated to 30-60℃. Figure 12A). As the PLEL concentration increases, the critical temperature for the sol-gel transition gradually decreases, while the critical temperature for the gel-precipitation transition increases simultaneously, leading to a widening of the gel stability range. After introducing EDTA / LDH and EGTA, the sol-gel transition temperature did not shift significantly, but the gel-precipitation transition temperature decreased by approximately 2.5℃, resulting in a slight contraction of the gel stability range. Notably, when the PLEL concentration is 25%, the sol-gel transition temperature stabilizes around 35℃, a characteristic that meets the application standard of "flowing at room temperature and curing at body temperature" for temperature-sensitive injection hydrogels. Figure 12 (B)).

[0129] 2. Rheological property study

[0130] EDTA-LDH / PLEL (containing 25 wt% PLEL and 3 mg / mL) was prepared according to the method in Example 2. -1 EDTA-LDH) and EGTA / PLEL (containing 25 wt% PLEL with 8 mg / mL) -1 Two types of hydrogels (EGTA) were tested. Samples were pre-equilibrated at 4°C for 30 min before testing. A rotational rheometer equipped with a P20 Ti L-shaped rotor was used for testing: the sample was loaded onto a 20 mm diameter stage, the clamp spacing was adjusted to 1 mm, and the temperature was cycled at 1°C / min within the 20-60°C range. During the test, a constant frequency of 1.0 Hz and a constant stress of 4.0 dyn / cm² were maintained, and the dynamic response of the storage modulus G' and loss modulus G'' as a function of temperature was continuously monitored.

[0131] Rheological properties are important indicators for evaluating the performance of hydrogels in biomedical applications, including their behavior during injection, molding, and in vivo application. This invention investigated the rheological properties of EDTA-LDH / PLEL and EGTA / PLEL hydrogels with a PLEL content of 25 wt% using a rotational rheometer. The results are as follows: Figure 13 As shown. Storage modulus (G′), also known as elastic modulus, represents the energy stored in a material during elastic (reversible) deformation, reflecting the material's elasticity. Loss modulus (G″), also known as viscous modulus, represents the energy lost in a material during viscous (irreversible) deformation, reflecting the material's viscosity. When G′ is much larger than G″, the material mainly exhibits elastic deformation and is solid; when G″ is much larger than G′, the material mainly exhibits viscous deformation and is liquid. When G′ and G″ are similar, the material is semi-solid, and gels are a typical example of semi-solid materials. Therefore, the temperature corresponding to the intersection of the curves of G′ and G″ with temperature is usually defined as the phase transition temperature of temperature-sensitive hydrogels.

[0132] rheological data ( Figure 13The (A)-(C) reveals the modulus law of EDTA-LDH / PLEL (25 wt%) and EGTA / PLEL (25 wt%) hydrogels: below 30°C, the storage modulus (G') and loss modulus (G'') are at a low level and remain stable (G'<G''), indicating that the system has sol properties; after breaking through 30°C, both moduli increase sharply and the growth rate of G' is dominant, indicating that it starts to undergo sol-gel transition; when the temperature rises to 35°C, G'=G'' reaches the gelation threshold, and the system completes the gel state switching; in the subsequent temperature rise, G' continues to grow ahead of G''. Meanwhile, the gradual expansion of the difference between the two moduli confirms the strengthening of the three-dimensional network and the increase in the degree of solidification. Then, when the temperature reaches 39°C, both moduli suddenly decrease and G' decreases more sharply than G''. When the temperature rises to 40°C, the intersection point of G'=G'' reappears, which marks the beginning of precipitation; thereafter, as the temperature rises, G' and G'' continue to decrease and the decrease of G' is more significant, and the difference between the two moduli narrows, which reflects that the precipitation amount is increasing gradually. This dynamic evolution process of modulus is basically consistent with the phase transition observed by the inversion method.

[0133] 3. In vitro degradation performance of hydrogels

[0134] Under simulated physiological conditions, the blank PLEL hydrogel with a content of 25 wt% and EDTA-LDH / PLEL (EDTA-LDH concentration is 3 mg mL -1 ) and EGTA / PLEL hydrogel (EGTA concentration is 8 mg mL -1 ) were prepared according to the method of Example 2. Take 1 mL of each and add to the bottom of a test tube, after equilibrating at 37°C for 30 min, add 10 mL of PBS solution (pH 7.4) to the formed gel, and incubate on a shaker at 37°C at a speed of 100 r / min. Take out 2 mL of the upper buffer on the 1st, 3rd, 7th, and 10th days, measure its pH, and then replace with fresh buffer to maintain the environmental pH.

[0135] Under simulated physiological conditions (37°C, pH 7.4), the in vitro degradation behaviors of blank PLEL hydrogel, EDTA-LDH / PLEL and EGTA / PLEL hydrogels were evaluated ( Figure 14 ). During the degradation process, the pH change of the buffer medium was regularly detected to evaluate the release of acidic products during the degradation of the hydrogels.

[0136] Even with the culture medium changed every 2-3 days, the pH of the buffer solution still showed a slow decreasing trend due to the release of D,L-LA and their low molecular weight oligomers. These acidic products are typical products of ester bond hydrolysis during hydrogel degradation. Nevertheless, the pH of the buffer solutions containing the three hydrogels remained above 4.5, indicating that the hydrogels exhibit a mild acidic effect during degradation, which may be attributed to the high water content within the hydrogels. This high water content allows acidic degradation products to rapidly diffuse from the gel matrix into the external buffer solution, thus avoiding excessive accumulation of localized acidic environments. Furthermore, the degradation process of the hydrogels is mainly achieved through the hydrolysis of stable ester bonds within them. This hydrolysis mechanism not only ensures the controllability and mildness of the degradation process but also guarantees the biocompatibility of the hydrogel under physiological conditions and the safety of the degradation products.

[0137] 4. In vitro release study

[0138] Under simulated physiological conditions, a blank PLEL hydrogel with a content of 25 wt% and an EDTA-LDH / PLEL (EDTA-LDH concentration of 3 mg / mL) were prepared. -1 ) and EGTA / PLEL hydrogel (EGTA concentration of 8 mg / mL) -1 2 mL of each solution was injected into the bottom of a test tube and equilibrated at 37°C for 30 min to form a stable hydrogel. 12 mL of PBS solution (pH 7.4 and 6.5) was added to the formed gel, and the solution was placed in a shaker at 37°C and 100 rpm. 1 mL of the supernatant was collected at specified time points (15 min, 0.5, 2, 4, 8, 24, 48, 72, 96, 120, and 144 h), and fresh PBS was added simultaneously. For the EGTA / PLEL release sample, 2 mL of CuSO4 and 1 mL of NaH2PO4 were added; for the EDTA-LDH / PLEL release sample, 2 mL of FeCl3·6H2O was added, and both were incubated for 30 min. HPLC analysis was used for detection.

[0139] (1) Chromatographic conditions

[0140] The high-performance liquid chromatograph (Shimadzu) is equipped with an LC-20AT pump and an SPD-20A UV detector; the column is a Thermo ODS C18 column (250 mm × 4.6 mm, 6 μm); the mobile phase is methanol-tetrabutylammonium bromide aqueous solution (5 mmol / L). -1 Tetrabutylammonium bromide aqueous solution (pH adjusted to 6.5 with acetic acid) - sodium acetate solution (35 mmol / L) -1Sodium acetate, acetic acid adjusted to pH 6.50 (20:20:60, v / v / v); Detection wavelength: 245 nm; Flow rate: 1 mL / min -1 Column temperature: 40℃; injection volume: 10 μL. All samples were filtered through a 0.22 μm filter membrane.

[0141] (2) Examination of linear relationships

[0142] Accurately measure the EDTA standard solution and add 2 mL of FeCl3·6H2O to obtain EDTA-2Na mass concentrations of 500, 100, 50, 20, and 5 μg / mL. -1 The series of standard curve solutions were injected and determined according to the chromatographic conditions in section (1), with the mass concentration of EDTA-2Na (X, μg / mL) as the standard curve. -1 (x) represents the x-axis, EDTA-Fe 3+ Using the peak area (Y) as the ordinate, a linear regression is performed to obtain the regression equation.

[0143] Accurately measure the EGTA reference solution, add 2 mL of CuSO4 and 1 mL of NaH2PO4 to obtain EGTA mass concentrations of 500, 100, 50, 20, and 5 μg / mL, respectively. -1 The series of standard curve solutions were injected and determined according to the chromatographic conditions under section (1), with EGTA mass concentration (X, μg / mL) as the standard curve. -1 ) is the x-axis, EGTA-Cu 2+ Using the peak area (Y) as the ordinate, a linear regression is performed to obtain the regression equation.

[0144] Under the chromatographic conditions described above, five concentration gradients of EDTA and EGTA solutions were injected sequentially from low to high concentration. The peak area was then compared with the concentration (μg / mL). -1 Linear regression was performed, and the resulting EGTA standard curve equation and correlation coefficient were: y = 3180.70972x - 11616.94209, R² = 0.9982; the EDTA standard curve equation and correlation coefficient were: y = 321.71068x + 2897.48911, R² = 0.9918. The standard curves show good linearity of this content determination method within the concentration range of 5–500 μg mL⁻¹. The standard curves were plotted with concentration on the x-axis and peak area on the y-axis, and the results are as follows: Figure 15 As shown in (A) and 15 (B).

[0145] Release profiles of EDTA-LDH / PLEL and EGTA / PLEL in release media (pH 7.4 and 6.5 PBS) are as follows: Figure 15As shown in (C) and (D), we placed EGTA / PLEL and EDTA-LDH / PLEL in PBS solutions at pH 6.5 and 7.4, respectively, to simulate the release behavior of EGTA and EDTA in the microacidic environment of a tumor. Figure 15 (C) EGTA has a rapid release rate within 10 hours, and the release amount can reach 25% within 144 hours. For example... Figure 15 As shown in (D), the structural stability of LDH nanosheets decreases in the slightly acidic environment of tumors, leading to weakened interlayer electrostatic interactions and thus promoting the rapid release kinetics of EDTA. Experimental data show that EDTA exhibits rapid release within 20 hours, which helps to quickly exert its chelating effect in the early stages of treatment; subsequent release enters a sustained-release phase, with a cumulative release rate of 60% after 144 hours, demonstrating sustained drug delivery capability. This two-stage release characteristic indicates that the EDTA-LDH / PLEL system combines the advantages of rapid response and long-term sustained release, making it more suitable for the time-sensitive needs of tumor treatment compared to the EGTA system.

[0146] 5. Molecular simulation

[0147] To investigate the interaction mechanisms between EDTA and LDH, EDTA / LDH and PLEL, and EGTA and PLEL, this study used Discovery Studio software for molecular docking. First, based on docking site analysis ( Figure 16 (A and B), respectively using three Zn in LDH 2+ / Al 3+ A 6 Å spherical cavity was set as the active docking site, centered on octahedral structural units and PLEL long-chain units. Molecular docking and structural optimization were performed under the CHARMM force field. The acceptor-ligand binding energy was obtained by running the docking energy calculation module, and the conformation corresponding to the maximum value of -CDOCKER-INTERACTION-ENERGY was selected as the optimal docking model. Figure 16 The results of geometric optimization and pharmacophore analysis were combined to evaluate the efficacy of the drug, and the root mean square difference, nonbonded interactions, and affinity were evaluated.

[0148] According to the docking principle, -CDOCKER-INTERACTION-ENERGY represents the combined interaction force between the receptor and the ligand. Therefore, the conformation with the maximum -CDOCKER-INTERACTION-ENERGY value is taken as the simulated docking conformation. Figure 16The results showed that the interaction between EDTA and LDH was mainly electrostatic, while the interactions between EDTA / LDH-PLEL and EGTA-PLEL were mainly dependent on hydrogen bonding (as shown in Table 6). These interaction characteristics reveal the feasibility of the three combinations and suggest their potential application value in the treatment of oral cancer. The study elucidated the specific binding modes between different ligands and receptors through molecular docking, laying a theoretical foundation for subsequent functional verification.

[0149] Table 6. Types and magnitudes of interactions between EDTA and LDH, EDTA / LDH and PLEL, and EGTA and PLEL.

[0150]

[0151] In summary, this invention designed and successfully constructed an innovative injectable thermosensitive hydrogel system of polylactide-polyethylene glycol-polylactide (PDLLA-PEG-PDLLA, PLEL). This system cleverly loads EDTA / LDH and EGTA, based on the EDTA-Ca²⁺ chelation principle, into PLEL hydrogels through physical mixing, forming a chemotherapy drug-hydrogel complex therapeutic system (EDTA-LDH / PLEL and EGTA / PLEL hydrogels) capable of precisely forming drug reservoirs at tumor sites and achieving slow release. Studies have shown that both systems respond to the microacidic environment of tumors and achieve precise drug release, providing strong support for targeted tumor therapy. In terms of physicochemical properties, the sol-gel phase transition temperature of the drug-loaded gel with a PLEL content of 25 wt% is found to be approximately 34°C, a characteristic that meets the requirements of an injectable thermosensitive hydrogel with a "room temperature sol state and body temperature gel state." Therefore, selecting a drug-loaded gel with this PLEL content for subsequent research is not only reasonable but also highly feasible and has significant application potential. Furthermore, an in vitro high-performance liquid chromatography (HPLC) method was established to determine the contents of EDTA and EGTA in the in vitro released samples from the drug-loaded gel. In vitro drug release behavior studies showed that both gels exhibited good drug release behavior and were able to maintain therapeutic efficacy for a prolonged period in tumor treatment. This invention not only provides a solid theoretical and experimental foundation for subsequent cell and animal anti-tumor experiments but also paves a new path for the development and optimization of precision treatment strategies for oral cancer in the future. By constructing and validating a drug-loaded system based on PLEL hydrogels, the targeted release of EDTA and EGTA at the tumor site was successfully achieved. This innovative therapeutic system is expected to significantly improve therapeutic efficacy while reducing toxicity to normal tissues in future clinical applications.

[0152] Example 4

[0153] Experimental use of composite hydrogels for oral cancer treatment

[0154] 1. Cell Culture

[0155] Mouse oral squamous cell carcinoma cells (SCC-7) were purchased from Wuhan Shangen Biotechnology and cultured in DMEM medium containing 10% fetal bovine serum. Mouse fibroblast cells (L929) were cultured in DMEM medium containing 10% fetal bovine serum. All cells were cultured in a constant temperature incubator at 37°C and 5% CO2.

[0156] 2. Cytotoxicity test

[0157] The MTT assay was used to investigate the cytotoxicity of blank PLEL hydrogel, EDTA-LDH / PLEL, and EGTA / PLEL hydrogel on mouse fibroblast L929 and mouse oral squamous cell carcinoma SCC-7 cells.

[0158] (1) Preparation of cell suspension

[0159] Cells were cultured and passaged at 37°C and 5% CO2. Once the cells were stable, the original culture medium was aspirated, and the cells were washed twice with pre-warmed PBS. They were then digested twice with 1 mL of 0.25% trypsin, 30 s each time. Under a microscope, when the intercellular spaces increased and the cells shrank and rounded, 3 mL of complete culture medium was added to stop the digestion, and the cells were thoroughly mixed. The cells were then transferred to centrifuge tubes and centrifuged (1500 rpm, 3 min). After centrifugation, the supernatant was aspirated, and 1 mL of culture medium was added, followed by resuspending the cells. 10 μL of the cell suspension was counted under a microscope using a hemocytometer. The cell suspension was then diluted to a concentration of 5 × 10⁻⁶. 4 Add 100 μL of cell suspension per well to a 96-well plate, and seed the plate to adjust the cell density to 5 × 10⁶ cells / mL. 3 Each well contains one plate, and the edges are filled with PBS. The 96-well plate is incubated in a constant temperature incubator (37℃, 5% CO2) for 24 h.

[0160] (2) Preparation of hydrogel extract

[0161] A blank hydrogel containing 25 wt% PLEL was prepared according to the method in Example 2, as well as a drug-loaded PLEL hydrogel system containing gradient concentrations of EDTA / LDH (0.3, 1, 3 mg mL⁻¹) and EGTA (0.2, 4, 8 mg mL⁻¹). Both extracts were prepared using a pre-equilibration method: 5 mL of gel and an equal volume of preheated serum-free culture medium were placed together in a sterile 50 mL centrifuge tube and extracted at 37°C for 24 h. Prior to the experiment, the gel and culture medium were preheated simultaneously for 30 min to ensure the gel underwent a sol-gel phase transition and the medium temperature reached physiological conditions. After extraction, the collected samples were temporarily stored at 4°C for subsequent analysis.

[0162] (3) Addition of drugs and color development

[0163] Drug treatment of L929 cells: After 24 h, the original culture medium was removed, and 100 μL of blank PLEL hydrogel (25 wt%) and EDTA-LDH / PLEL hydrogel (EDTA / LDH concentration of 3 mg / mL) were added. -1 ) and EGTA / PLEL hydrogel (EGTA concentration of 8 mg / mL) -1 ), 4-6 duplicate holes.

[0164] Drug treatment of SCC-7 cells: After 24 h, remove the original culture medium and add 100 μL of different concentrations of extracted EDTA-LDH / PLEL hydrogel. Add 100 μL of different concentrations of extracted EGTA / PLEL hydrogel to the remaining wells. 4-6 replicates.

[0165] Incubate the 96-well plate in a constant temperature incubator (37℃, 5% CO2) for 12 h. Add 50 μL of MTT reagent to each well and continue incubation for 4 h. Then aspirate the supernatant, but do not remove the formazan. Add 150 μL of DMSO to each well. Shake the plate at 1020 Hz for 30 s to fully dissolve the formazan. Measure the absorbance of each well at 490 nm.

[0166]

[0167] Wherein, OD(sample) is the absorbance of the sample group, OD(blank) is the absorbance of the blank control group, and OD(control) is the absorbance of the control group.

[0168] 3. Detection of disrupted cell junctions

[0169] (1) Apoptosis

[0170] SCC-7 cells were used at a rate of 1×10 6Cells were seeded at a density of 1 / 2 well in 6-well plates and cultured for 24 h at 37°C and 5% CO2. Then, DMEM medium containing 300 ppm EDTA-LDH / PLEL or EGTA / PLEL was added, and the cells were co-cultured at 37°C and 50 rpm for 1 h and 3 h, respectively. Untreated cells were used as a blank control. To differentiate cell detachment mechanisms (cell junction disruption and apoptosis induction), an apoptosis detection experiment was conducted: cell suspensions treated with 300 ppm EDTA-LDH / PLEL or EGTA / PLEL at 80 rpm and 37°C for 2 h and 4 h were collected by centrifugation, washed twice with PBS, resuspended in 400 μL binding buffer, aliquoted, and stained with 5 μL Annexin V-FITC / PI double staining reagent. The cells were stained on ice in the dark for 20 min, and the proportion of apoptotic cells was finally quantitatively analyzed by flow cytometry.

[0171] (2) Western Blot

[0172] SCC-7 cells at 10 6 Cells were seeded at a density of 1 / 2 well in 6-well plates and cultured at 37°C and 5% CO2 for 24 h. The original culture medium was replaced with DMEM containing 300 ppm EDTA-LDH / PLEL or EGTA / PLEL, and co-cultured at 37°C with a shaker (50 rpm) for 4 h. Control group cells maintained their original culture conditions. The experimental and control groups were placed in the same temperature-controlled environment to ensure consistency in all variables except for treatment factors. After culture, cells were washed with PBS, and after removing the PBS, 1 mL of trypsin digest was added to each dish. After digestion for one minute, the trypsin digest was removed, and 1 mL of blank culture medium was added to each dish. Cells were pipetted from the bottom of the dish and transferred to centrifuge tubes. Cells were collected by centrifugation at 1000 rpm for 5 minutes and the supernatant was discarded. Add 1 mL of PBS and mix well. Centrifuge repeatedly, discard the supernatant PBS solution, add 100 μL of cell lysis buffer (protease phosphatase inhibitor: RIPA lysis buffer = 1:50), and lyse on ice for 30 minutes. Then centrifuge at 4°C and 12,000 rpm for 15 minutes and collect the supernatant. Quantify the protein using a BCA assay kit, and then add 1 / 4 volume of loading buffer to the protein extract.

[0173] Prepare the gel according to the instructions of the 12% SDS-PAGE gel rapid preparation kit. After the gel solidifies, place it in an electrophoresis tank and add an appropriate amount of electrophoresis buffer. After removing the comb, clean the fragments of gel from the wells, and then add an appropriate amount of pre-stained marker and sample into the wells. Start electrophoresis at 80 V until the bromophenol blue dye enters the separating gel. Adjust the voltage to 120 V and continue electrophoresis until the dye is close to the bottom of the separating gel, then stop electrophoresis. After removing the glass plate, cut the desired target band according to the marker instructions. Transfer the desired protein onto a methanol-activated polyvinylidene fluoride (PVDF) membrane. Transfer will release a large amount of heat, so it should be performed under ice bath conditions. Adjust the transfer time according to the molecular weight of the protein. After transfer, place the PVDF membrane in a solution containing 5% skim milk powder and shake on a shaker for 2 hours to block. After blocking, wash the PVDF membrane three times with TBST for ten minutes each time. After blotting the membrane dry with filter paper, add it to the corresponding primary antibody and incubate overnight at 4°C. After incubation, wash three times with TBST for 10 min each time. Incubate the membrane with secondary antibody for 1 h, then repeat the washing process with TBST. Remove the PVDF membrane, visualize the bands using ultrasensitive luminescence solution, and perform imaging analysis using a ChemiDOX XRS+ gel imaging system.

[0174] 4. Cell migration resistance test

[0175] SCC-7 cells were loaded at 5 × 10 5 Cells were seeded at a density of [number] cells / well in 6-well plates. After incubation and adherence to the wells, forming a distinct dense monolayer, the culture medium was aspirated, and a straight line was drawn across the monolayer using the tip of a 200 µL pipette. The disrupted cells were then rinsed with PBS, and DMEM containing EDTA-LDH / PLEL and EGTA / PLEL (100 ppm) was added to replace the medium, along with the control medium. At fixed time points (0, 1, and 3 h), the drawn area was photographed under white light using an inverted microscope.

[0176] 5. Construction of tumor-bearing mouse animal model

[0177] SCC-7 cells were cultured in 10 cm diameter dishes. When cells were in good growth condition, they were digested twice with 1 mL of trypsin, followed by the addition of DMEM medium to terminate the digestion process. The cells were gently pipetted and the cell suspension was collected. The cells were centrifuged at 1200 rpm for 3 min, the supernatant was removed, and the cells were resuspended in PBS buffer. The cells were then centrifuged again to wash the cells, and this process was repeated twice. Cell counts were performed during the final operation, and the appropriate amount of PBS was added based on the count results to prepare a cell suspension of suitable concentration. After cell collection, the cell suspension was placed on ice in preparation for the subsequent seeding steps.

[0178] Healthy male C3H mice aged 4-6 weeks were selected, and 100 μL of SCC-7 cell suspension was subcutaneously injected into the right hind limb, so that it contained 2×10⁻⁶ cells. 6 One SCC-7 cell per cell. Tumor volume was monitored daily starting one week after cell seeding. The major and minor axes of the tumor were measured using electronic calipers, and the tumor volume was calculated using the following formula:

[0179]

[0180] When the tumor volume reaches 80-100 mm 3 C3H mice were randomly assigned to groups for subsequent experiments. All animal-related experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of China Pharmaceutical University (ethics approval number: YSL-202503110).

[0181] 6. Changes in tumor distribution over time

[0182] The preparation contains 5 μg mL -1 A 25 wt% PLEL hydrogel precursor solution of Cy5.5 fluorescent dye and a free Cy5.5 control solution were prepared. A C3H mouse SCC-7 oral cancer subcutaneous xenograft model was constructed using the above method, and tumors were allowed to grow until the tumor volume reached 600 mm². 3 Two tumor-bearing mice were randomly assigned to each tumor: the experimental group received an intratumoral injection of 100 μL of PLEL hydrogel precursor solution loaded with Cy5.5, while the control group received an equal volume of free Cy5.5 solution. At time points of 0, 2, 6, 24, 48, and 120 h post-injection, mice were anesthetized by intraperitoneal injection of tribromoethanol. The fluorescence signal intensity and spatial distribution were tracked using a small animal in vivo imaging system to systematically evaluate the retention characteristics of the drug-loaded hydrogel in the tumor microenvironment and the drug release kinetics.

[0183] 7. In vivo antitumor activity experiment

[0184] C3H tumor-bearing mice were randomly divided into 7 groups (n=5): (i) normal saline (NS); (ii) blank PLEL; (iii) EDTA (free solution); (iv) EDTA / LDH (free solution); (v) EGTA (free solution); (vi) EDTA-LDH / PLEL; (vii) EGTA / PLEL. Each group received a dose of 20 mg / kg. -1 The drug was administered via intratumoral injection, with a volume of 100 μL per administration (two-point dosing method), at days 0, 3, 6, and 9. Mouse body weight and tumor volume were recorded every three days after administration, calculated using the formula 3-2. Mice were sacrificed on day 15 after the first administration, and tumor tissue, heart, liver, spleen, lung, and kidney were harvested. Subsequently, the organs and tumors were fixed, embedded, sectioned, and stained with hematoxylin-eosin (H&E staining), and the tissue sections were photographed under a microscope.

[0185] 8. Biosafety Research

[0186] Preparation of 2% erythrocyte suspension: Collect 2 mL of fresh anticoagulated whole blood (rabbit) and add it to a blood collection tube pretreated with heparin. Add a certain amount of physiological saline and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add physiological saline, mix well, and centrifuge at 1000 rpm for 5 min. Repeat the washing step 2-3 times until the supernatant is no longer red. Finally, use 0.9% sodium chloride injection to prepare a 2% erythrocyte suspension.

[0187] Take 1 mL of EDTA-LDH / PLEL and EGTA / PLEL solutions of different concentrations and add them to 500 μL of 2% erythrocyte suspension, then mix well. Incubate in a 37℃ water bath for 1 h, gently shaking to mix every 15 min. Simultaneously, 1 mL of distilled water and 1 mL of 0.9% sodium chloride injection are used as positive and negative controls, respectively. After incubation, centrifuge each group of samples at 1000 rpm for 5 min at room temperature to separate the supernatant. Transfer 100 μL of the supernatant from each group to a 96-well plate. Measure the absorbance (OD value) of the supernatant at 545 nm using a microplate reader, and calculate the hemolysis rate using the following formula.

[0188]

[0189] Wherein, OD (sample) represents the absorbance value of the test sample, and OD (positive) and OD (negative) represent the absorbance values ​​of the positive and negative controls, respectively.

[0190] 9. Statistical Analysis

[0191] GraphPad Prism 8.0 software was used for statistical analysis of the data in this experiment. All values ​​are expressed as mean ± standard deviation. One-way ANOVA or Students's t-test was used to analyze the significance of differences between groups, and P < 0.05 was considered statistically significant.

[0192] result:

[0193] 1. Cytotoxicity test

[0194] Cytotoxicity testing is a crucial step in assessing the safety of biomaterials, especially when developing materials for medical devices or drug delivery systems. The MTT assay was used to systematically evaluate the cytotoxicity of two hydrogel materials (EDTA-LDH / PLEL and EGTA / PLEL). L929 mouse fibroblasts and SCC-7 mouse oral squamous cell carcinoma cells were selected as test cell lines to comprehensively assess the effects of the materials on normal and cancer cells. Hydrogel extracts were prepared according to the International Organization for Standardization (ISO) standard, "Biological evaluation of medical devices – Part 12: Sample preparation and reference samples," for toxicity testing. Figure 17 As shown in (A), blank PLEL hydrogel (25 wt%) and EDTA-LDH / PLEL hydrogel (EDTA / LDH concentration of 3 mg / mL) were compared. -1 ) and EGTA / PLEL hydrogel (EGTA concentration of 8 mg / mL) -1 The cell viability of all samples was above 85%. This result indicates that the material used still exhibits good cell compatibility at high concentrations, meeting the basic requirements of ISO standards for biomaterials.

[0195] Furthermore, the cell viability of different concentrations of EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel in SCC-7 cells was evaluated. The cytotoxicity of EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel showed a clear concentration-dependent effect; cell viability decreased significantly with increasing concentration. Figure 17 As shown in (B) and (C), when the EDTA-LDH concentration is 0.3 mg / mL -1 At that time, the cell survival rate of EDTA-LDH / PLEL hydrogel was 80%, and it decreased with increasing concentration to 1 mg / mL. -1 and 3mg mL -1 Cell viability decreased to below 60% and 40%, respectively. When the EGTA concentration was 0.2 mg / mL... -1 At that time, the cell survival rate was 80%; as the concentration increased to 4 mg / mL-1 and 8 mg mL -1 Cell viability dropped to below 70% and 50%, respectively.

[0196] 2. Disruption of intercellular connections

[0197] In the field of cell biology, the connection system between cells and between cells and the extracellular matrix is ​​a key architecture for maintaining tissue structure and function. Based on its functional characteristics, it can be precisely divided into three core structures: tight junctions, gap junctions, and adhesive junctions. (1) Tight junctions are a highly precise form of intercellular connection, mainly formed by the interaction of a series of specific transmembrane proteins to form a continuous and dense sealing band structure. This structure builds a stable biological barrier between cells, which can effectively block the free diffusion of substances inside and outside the cell. (2) Gap junctions focus on intercellular communication and metabolic coordination. They are composed of channel proteins, which form specific channel structures on the cell membrane, allowing small molecular signaling substances (such as ions, glucose, etc.) to pass through quickly and efficiently, thereby realizing intercellular communication and enabling cell groups to work together in physiological processes such as metabolism to better adapt to changes in the internal and external environment of the body. (3) Adhesive junctions are mainly responsible for the mechanical integration between cells or between cells and the extracellular matrix. They function by means of mechanical anchoring proteins, among which the desmosome complex is a typical mechanical anchoring protein structure. These proteins can bind cells together tightly or anchor them firmly to the extracellular matrix, thus giving the tissue sufficient mechanical strength to maintain structural stability when subjected to external forces.

[0198] (1) Apoptosis

[0199] Changes in cell morphology are an important indicator for assessing the impact of biomaterials on cell behavior. Cell adhesion, morphology, and connectivity can reflect the biocompatibility of materials and their influence on cellular physiological functions. Cell morphology was observed in SCC-7 mouse oral squamous cell carcinoma cells incubated with EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel to evaluate the effects of these two materials on cell connectivity and morphology.

[0200] Microscopic observation results (such as) Figure 18As shown, the study clearly reveals that untreated SCC-7 cells exhibit typical epithelial cell morphology. These cells expand in a spindle-shaped pattern on the culture substrate and adhere tightly to each other, forming a dense and orderly cell network structure. However, after 2 hours of treatment with EDTA-LDH / PLEL and EGTA / PLEL composite hydrogels, the morphological characteristics of SCC-7 cells underwent significant and obvious changes. Specifically, the originally tight intercellular connection network began to disintegrate, leading to a gradual expansion of the intercellular spaces. At the same time, the cell bodies themselves gradually shrank, eventually exhibiting a round shape, and the distribution of cells became discrete, no longer as tightly connected as before. Furthermore, when the treatment time was extended to 4 hours, most cells completely detached from the culture substrate and entered a suspension state. This series of cell morphological changes strongly confirms that EDTA-LDH / PLEL and EGTA / PLEL composite hydrogels can precisely disrupt the cell adhesion system through targeted action, thereby effectively inducing the discrete process of originally adherent cells.

[0201] In the aforementioned experiments, it was confirmed that EDTA-LDH / PLEL hydrogels and EGTA / PLEL hydrogels can induce cell dissociation and detachment from the cell wall. However, apoptosis can also lead to cell detachment. To eliminate the influence of apoptosis on detachment, this study analyzed the viability of suspended cells using an Annexin V-FITC / PI dual-fluorescence labeling strategy. This detection is based on the extracellular exposure of phosphatidylserine, a core biomarker of apoptosis. Flow cytometry was used to quantitatively assess cell membrane integrity and apoptosis-specific signals, thereby accurately distinguishing between mechanical detachment and cell detachment events caused by programmed cell death. Annexin V has a high affinity for PS. When early apoptosis occurs, PS everts to the outer side of the cell membrane, where Annexin V-FITC can bind and emit green fluorescence; while PI can only enter the nucleus of late apoptotic or necrotic cells, causing the nucleus to emit red fluorescence. Based on this, the fluorescence signal of the Annexin V-FITC / PI dual staining solution can be used to indicate the early and late apoptosis status of cells.

[0202] like Figure 19As shown, neither material induced significant early apoptosis within 2 h of co-culturing with cells, and the proportions of Annexin V-FITC single-positive cells were low, at 16.7% and 18.6%, respectively. However, with prolonged incubation, released EDTA and EGTA gradually accumulated and entered the cells, causing damage to the cell membrane and organelles. After 4 h of incubation, the percentage of early apoptotic cells increased, manifested by an increase in the proportion of Annexin V-FITC single-positive cells. Specifically, the proportion of Annexin V-FITC single-positive cells in the EGTA / PLEL treatment group reached 37.3%, significantly higher than that in the EDTA / PLEL treatment group. This result indicates that, compared to EDTA / LDH, EGTA treatment alone causes more cytotoxic effects on the cell membrane and organelles. Furthermore, late apoptotic signals were detected using Annexin V-FITC / PI double staining. The results showed that after 4 h of treatment, the proportion of late apoptotic cells in both materials was low, approximately 10%. However, despite the low proportion of late apoptotic cells, most cells still underwent separation. This phenomenon indicates that the main cause of cell shedding is the disruption of cell junctions, rather than shedding caused by apoptosis.

[0203] (2) Western Blot

[0204] The effects of EDTA-LDH / PLEL and EGTA / PLEL composite hydrogels on the expression of calcium-sensitive adhesion molecules were systematically analyzed using Western blotting (WB). Figure 20 (A) reveals that E-cadherin, a core member of the calcium-dependent adhesion membrane protein family, is significantly controlled by material treatment conditions. Due to its unique calcium ion-responsive properties, this protein plays a crucial role in maintaining epithelial tissue integrity. Through its extracellular repeating domains of five or six calcium ions, E-cadherin mediates tight junctions between cells, maintaining the morphology of cell aggregation and tissue integrity. However, calcium chelators such as EDTA and EGTA can bind to calcium ions, thereby disrupting the protein structure of E-cadherin and affecting its function. Figure 20(B) shows that we treated SCC-7 cells with EDTA-LDH / PLEL hydrogels and EGTA / PLEL hydrogels, respectively, and detected the expression level of E-cadherin by Western blotting. The results showed that the expression of E-cadherin decreased significantly in cells treated with both hydrogels. This result indicates that EDTA-LDH / PLEL and EGTA / PLEL hydrogels disrupt the protein structure of E-cadherin by chelating calcium ions, thereby inhibiting its expression. This inhibition leads to the disruption of intercellular connections, the inability to maintain cell aggregation, and ultimately cell dissociation. This finding reveals the mechanism by which these two hydrogel materials disrupt intercellular connections by affecting the expression of calcium-dependent proteins.

[0205] In addition to E-cadherin, the S100A4 protein, a member of the S100 family of calcium-binding proteins, was also examined. S100A4 is a commonly overexpressed calcium-binding protein in tumors; its double-helix amino acid motif exerts biological effects by binding to calcium ions. Studies have shown that S100A4 can be secreted into the extracellular matrix and is closely related to angiogenesis, cell differentiation, and migration. More importantly, S100A4 plays a crucial role in promoting tumor cell invasion and metastasis, and is therefore considered one of the key factors in tumor metastasis. After treatment, SCC-7 cells, such as... Figure 20 As shown in (C), similar to the expression results of E-cadherin, the expression of S100A4 protein was also significantly reduced in cells treated with EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel. In summary, both materials inhibited the expression of related connective proteins by chelating calcium ions, disrupting intercellular connections and leading to cell dissociation. Furthermore, they also inhibited calcium-dependent S100A4 protein, suggesting that this material system has the potential to prevent tumor metastasis.

[0206] 3. Cell migration resistance test

[0207] Subsequently, a cell scratch assay was used to further investigate the anti-migration ability of EDTA-LDH / PLEL and EGTA / PLEL against SCC-7 tumor cells. Figure 21 As shown, after 3 h of incubation, the scratch distance of cells in the blank culture medium treatment group was shortened by 29%, while the distances in the EDTA-LDH / PLEL and EGTA / PLEL groups increased by 37% and 49%, respectively. This is because both experimental groups had a disruptive effect on intercellular connections with SCC-7 cells. These results indicate that EDTA-LDH / PLEL and EGTA / PLEL hydrogels have significant anti-migration capabilities against SCC-7 tumor cells (P<0.001).

[0208] 4. Changes in intratumoral injection distribution over time

[0209] Intratumoral administration is a common drug delivery method in cancer treatment, aiming to deliver drugs directly to the tumor site to increase drug concentration and reduce systemic side effects. However, the residence time of the drug in the tumor is one of the key factors affecting the tumor-suppressing effect. An ideal drug delivery system should be able to maintain a high concentration at the tumor site for a long time, thereby exerting a sustained therapeutic effect. Typically, while drugs using water as a dispersion medium can be rapidly and uniformly distributed in tumor tissue, their high concentration within the tumor can only be maintained for a short time. This is because the drug is quickly diluted by blood flow and diffuses into the interstitial spaces. This rapid drug loss limits the effective duration of action of the drug at the tumor site, thus affecting the therapeutic effect. The PLEL hydrogel precursor (25 wt%), after intratumoral injection, rapidly transforms into a semi-solid gel state under the influence of body temperature. The drug is slowly released through self-diffusion and the dissolution of the gel skeleton, thereby prolonging the drug residence time at the tumor site. Theoretically, this hydrogel system can effectively prolong the drug's residence time in the tumor, but this hypothesis still needs to be verified experimentally.

[0210] To verify the effectiveness of the PLEL hydrogel precursor in prolonging drug retention time, a water-soluble small-molecule fluorescent probe, Cy5.5, was used as a model drug. Free Cy5.5 solution and Cy5.5-loaded PLEL hydrogel (25 wt%) were prepared. The changes in fluorescence intensity within the tumors of SCC-7 tumor-bearing C3H mice were observed using an in vivo imaging system under two different administration routes (intratumoral injection of free Cy5.5 solution and intratumoral injection of Cy5.5-loaded PLEL hydrogel). The results are as follows: Figure 22 As shown. From Figure 22 As shown in (A), both administration methods resulted in drug accumulation at the tumor site, with fluorescence intensity gradually decreasing over time. However, there was a significant difference in the rate of change of fluorescence intensity between the two groups. Specifically, the fluorescence intensity of the free Cy5.5 solution group decreased rapidly after injection, and no high-intensity fluorescence area was observed after 24 hours, indicating a short retention time of the drug at the tumor site, with the drug quickly being flushed and diluted by the bloodstream and diffused into the interstitial space. In contrast, the fluorescence intensity of the Cy5.5-loaded gel group changed less within 10 hours and showed no decreasing trend within 48 hours, with high-intensity fluorescence areas still observable. This indicates that the drug-loaded gel has a significant drug retention capacity at the tumor site, with the drug being slowly released through gel skeleton erosion and self-diffusion, thereby prolonging the effective duration of drug action at the tumor site. To further verify this conclusion, the fluorescence intensity change curves of the two groups were also compared ( Figure 22(B) Statistical analysis showed that the fluorescence intensity change rate of the Cy5.5-loaded gel group within 10 h was significantly lower than that of the free Cy5.5 solution group, and the fluorescence intensity retention rate within 48 hours was significantly higher than that of the free drug solution group (P<0.001).

[0211] 5. In vivo antitumor activity study

[0212] Previous studies have demonstrated that EDTA-LDH / PLEL and EGTA / PLEL hydrogels significantly disrupt intercellular connections in SCC-7 tumor cells and exhibit good tumor accumulation capacity in in vivo experiments. Based on these findings, this study further investigated the in vivo antitumor activity of these two hydrogel materials using xenograft tumor model mice. When the tumor volume reached 90–120 mm³, all groups were administered the drug via intratumoral injection on days 0, 3, 6, and 9 (e.g., ...). Figure 23 (As shown in (A)). The experiment included multiple control and experimental groups, including saline intratumoral injection group, blank gel intratumoral injection group, free EDTA intratumoral injection group, EGTA intratumoral injection group, and two groups of drug-loaded gel (EDTA-LDH / PLEL and EGTA / PLEL hydrogel) intratumoral injection groups.

[0213] Experimental results showed that the tumor growth curve and the weight of the ex vivo tumor after treatment were as follows: Figure 23 As shown in (B) and (E), the tumor volume in the saline intratumoral injection group and the blank gel intratumoral injection group showed rapid growth within 15 days, increasing to 7.2 times and 6.7 times the initial volume, respectively, after 15 days of administration, with no significant difference between the two groups. In contrast, the tumor volume growth rate in the free EDTA intratumoral injection group and the EGTA group was slightly slower than that in the saline group, but still higher than that in the drug-loaded gel group, increasing to 4.7 times and 3.8 times the initial volume, respectively. This may be because free EDTA lacks adhesiveness and cannot effectively accumulate in the tumor area, thus limiting its antitumor effect. Compared with the control group and the free drug intratumoral injection group, the tumor volume growth rate in both drug-loaded gel intratumoral injection groups was significantly reduced, increasing to approximately 2.3 times and 2 times the initial volume, respectively, and the difference between the two groups was statistically significant (P<0.0001). Although the EDTA / LDH concentration was lower than that of EGTA at the same dosage, the tumor inhibition rates of the two groups were similar, indicating that the EDTA-LDH / PLEL hydrogel composite system has a greater advantage in anti-tumor effect.

[0214] During the treatment, we also monitored the changes in the average body weight of mice in each group, and the results were as follows: Figure 23As shown in (C), during the treatment period, the body weight of mice in the saline group and the blank PLEL hydrogel group remained at 17-20 g, with a slight decrease. This may be because the rapid growth of the tumor had a negative impact on the physiological functions of the mice. The body weight of mice in the other treatment groups remained at 20-25 g, indicating that the EDTA-LDH / PLEL and EGTA / PLEL hydrogel materials have good biocompatibility and no significant negative impact on the normal physiological functions of mice.

[0215] At the treatment endpoint, the morphology of tumors and the tumor growth inhibition rate in each group were as follows: Figure 23 (D) and Figure 24 As shown, the results are consistent with the tumor volume change curve. These results further confirm the significant antitumor effects of EDTA-LDH / PLEL hydrogel and EGTA / PLEL hydrogel in vivo. In particular, the treatment effect of the drug-loaded gel group was significantly better than that of other treatment groups. This advantage may be attributed to the longer retention time of the injectable hydrogel in vivo, allowing it to exert its effect more persistently and thus achieve better therapeutic results.

[0216] To further evaluate the therapeutic effects of EDTA-LDH / PLEL and EGTA / PLEL hydrogels in the SCC-7 tumor-bearing C3H mouse model, tumor tissue from one mouse in each group was randomly selected after treatment and subjected to H&E staining for histopathological study. The H&E staining results of the tumor tissue are shown below. Figure 25 As shown, the cell density of tumor tissue in the EDTA-LDH / PLEL and EGTA / PLEL hydrogel groups was much lower than that in the other control groups, indicating that EDTA-LDH / PLEL and EGTA / PLEL hydrogels have the best in vivo anticancer efficacy compared to other control groups.

[0217] 6. Biosafety Research

[0218] (1) Hemolysis test

[0219] Biocompatibility is a key factor in evaluating the clinical applicability of novel biomaterials, and blood compatibility is an important component of biocompatibility. Blood compatibility primarily examines whether the material causes abnormal reactions in blood components upon contact with blood, such as hemolysis, platelet activation, and coagulation. According to the national standard "Biological Evaluation of Medical Devices Part 4: Selection of Blood Interaction Tests," a hemolysis rate of less than 5% is considered to indicate good blood compatibility. Figure 26 As shown, the hemolysis rate of all concentrations of the materials in EDTA-LDH / PLEL and EGTA / PLEL hydrogels (25 wt%) was less than 5%. This indicates that the hydrogel materials have good blood compatibility and meet the basic requirements for the clinical application of biomaterials.

[0220] (2) In vivo safety studies

[0221] To further evaluate the in vivo safety of EDTA-LDH / PLEL and EGTA / PLEL hydrogels, after completion of in vivo antitumor therapy, one mouse from each group was randomly selected, and its major organs were stained with H&E. The results are as follows: Figure 27 As shown, the cardiomyocytes in all groups exhibited normal morphology, appearing fibrous with clear nuclei and normal density and arrangement, indicating no significant damage or toxicity to the myocardial tissue. The white pulp, marginal zone, red pulp, trabeculae, and central artery of the spleen in all groups were clearly defined, with no pathological changes observed. Compared to the saline (NS) group, H&E staining results of the major organs (heart, liver, spleen, lung, and kidney) in all groups showed no significant acute or chronic toxicity or adverse reactions. These results indicate that both materials have good biocompatibility in vivo.

[0222] In summary, this invention investigated the cellular and in vivo behavioral characteristics of EDTA-LDH / PLEL and EGTA / PLEL hydrogel composite systems, focusing on their cytotoxicity, intercellular junction disruption ability, in vivo distribution, tumor accumulation, antitumor activity, and biosafety. At the cellular level, the good biocompatibility and low cytotoxicity of the two hydrogel composites were verified. It was also revealed that these two materials can chelate calcium ions between cells, disrupting intercellular junctions and thus achieving the dissociation of tumor cells. This mechanism not only facilitates tumor cell separation but may also inhibit tumor invasion and metastasis. In in vivo studies, the distribution and tumor accumulation of the two hydrogel composites were evaluated using a mouse tumor model. Experimental results showed that these materials have a long retention time in tumor tissue and can achieve a sustained drug release effect. This characteristic allows the drug to exert its effects continuously at the tumor site, thereby improving therapeutic efficacy and reducing systemic toxicity. Furthermore, this chapter also evaluated the biosafety of the two hydrogel composites through H&E staining and hemolysis experiments, demonstrating their good blood compatibility.

[0223] Although the antitumor effects of EDTA-LDH / PLEL and EGTA / PLEL hydrogels were similar in cell and animal experiments, the amount of EDTA / LDH added to PLEL was only 3 mg / mL. -1 The amount of EGTA added to PLEL was 8 mg / mL. -1 EDTA-LDH / PLEL exhibits considerable antitumor effects even at relatively low concentrations, indicating higher antitumor efficiency. Based on these advantages, EDTA-LDH / PLEL hydrogel shows promising prospects for clinical applications.

[0224] EDTA-LDH / PLEL and EGTA / PLEL hydrogel composites exhibited excellent performance both in cells and in vivo. They not only effectively disrupted intercellular junctions but also achieved sustained drug release and long-term retention at the tumor site while maintaining good biocompatibility. These properties make this material system of significant potential application in oral cancer treatment, laying a solid foundation for subsequent clinical translational research.

Claims

1. A chemotherapy drug-hydrogel complex treatment system, characterized in that, The chemotherapy drug-hydrogel complex treatment system includes an EDTA-LDH / PLEL hydrogel system or an EGTA / PLEL hydrogel system formed by EDTA / LDH or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid) and PLEL hydrogel. The PLEL hydrogel is formed by polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer. The EDTA / LDH uses layered dihydrogen hydride LDH as the carrier of EDTA (ethylenediaminetetraacetic acid).

2. The chemotherapy drug-hydrogel complex treatment system according to claim 1, characterized in that, In the chemotherapy drug-hydrogel complex treatment system, the block ratio of polylactide, polyethylene glycol, and polylactide is 1000-2000:(1000-2000):1000-2000.

3. The chemotherapy drug-hydrogel complex treatment system according to claim 1, characterized in that, The gelation time of the chemotherapy drug-hydrogel complex treatment system at 37°C is 20-100 seconds.

4. The chemotherapy drug-hydrogel complex treatment system according to claim 1, characterized in that, In the EDTA / LDH, LDH is the carrier, and its particle size is 100-1000 nm.

5. A method for preparing the chemotherapy drug-hydrogel complex therapeutic system according to claim 1, characterized in that, Includes the following steps: (1) After heating polyethylene glycol, vacuum was applied while stirring. After cooling to room temperature, D,L-lactide (D,L-LA) and catalyst were added. Then, the reaction system was heated and stirred under an inert atmosphere. After the reaction was completed, it was cooled to room temperature. Ultrapure water was added to dissolve the crude product. The solution was stirred at room temperature to obtain a transparent and homogeneous aqueous solution. The solution was then heated to precipitate the copolymer. The upper layer of solution was removed. The above purification steps were repeated. The separated product, namely the copolymer, was freeze-dried to obtain polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer. (2) Add polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer to buffer solution and stir at room temperature until the mixture is a uniform and transparent liquid. Then, place the mixture in a low temperature environment to stand, filter and sterilize to obtain PLEL blank hydrogel. (3) Dissolve Zn(NO3)2·6H2O, Al(NO3)3·9H2O, NaNO3 and HMT (hexamethylenetetramine) together in deionized water. Heat and stir the reaction under an inert atmosphere. Dissolve EDTA in deionized water and slowly add it to the above reaction system to carry out the reaction. Lower the reaction temperature and continue the reaction. After the reaction is completed, centrifuge to remove the supernatant, collect the precipitate and wash it. Finally, freeze-dry the product to obtain a white solid powder, namely EDTA / LDH. (4) Take a blank PLEL hydrogel and add EDTA / LDH or EGTA solution respectively, and mix well.

6. The method for preparing the chemotherapy drug-hydrogel complex treatment system according to claim 5, characterized in that, In step (1), after heating polyethylene glycol to above 100°C, vacuum is applied while stirring. After cooling to room temperature, D,L-lactide (D,L-LA) and stannous octoate catalyst (Sn(Oct)2) are added. Then, under an inert atmosphere, the reaction system is heated and stirred at 120-200°C for 8-15 hours. After the reaction is completed, it is cooled to room temperature, and ultrapure water is added to dissolve the crude product. The mixture is stirred at room temperature to obtain a transparent and homogeneous aqueous solution. The solution is then heated to 70-120°C to precipitate the copolymer. The upper layer of solution is removed, and the above purification steps are repeated. The separated product, i.e., the copolymer, is freeze-dried to obtain polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer.

7. The method for preparing the chemotherapy drug-hydrogel complex treatment system according to claim 5, characterized in that, In step (2), polylactide-polyethylene glycol-polylactide (PLEL) triblock copolymer is added to PBS buffer to make the mass fraction of PLEL reach 20-30%.

8. The method for preparing the chemotherapy drug-hydrogel complex treatment system according to claim 5, characterized in that, In step (4), EDTA / LDH or EGTA is added to the buffer solution and dispersed evenly to obtain an EDTA / LDH solution or EGTA solution. The EDTA / LDH solution or EGTA solution is then added to a PLEL blank hydrogel and mixed evenly to form a composite hydrogel. The composite hydrogel contains 1-10 mg / mL of the solution. -1 EDTA-LDH or 1-10 mg / mL -1 EGTA.

9. The method for preparing the chemotherapy drug-hydrogel complex treatment system according to claim 8, characterized in that, In step (4), EDTA / LDH or EGTA is added to the buffer solution and dispersed evenly to obtain an EDTA / LDH solution or EGTA solution. The EDTA / LDH solution or EGTA solution is then added to a PLEL blank hydrogel and mixed evenly to form a composite hydrogel. The composite hydrogel contains 1-5 mg / mL of the solution. -1 EDTA-LDH or 6-10 mg / mL -1 EGTA.

10. The use of the chemotherapy drug-hydrogel complex therapeutic system of claim 1 in the preparation of a drug for the treatment of oral cancer.

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