Glycerol-encapsulated manganese ion homogenous cross-linked sodium alginate hydrogel loaded with chemotherapeutic drugs and preparation method and application thereof

Abstract

The application discloses a glycerol-wrapped manganese ion homogeneous cross-linking sodium alginate sustained-release hydrogel capable of loading chemotherapeutic drugs and a preparation method and application thereof, and is characterized in that a chemotherapeutic drug is added into an aqueous solution of sodium alginate to uniformly disperse the chemotherapeutic drug, and a glycerol-wrapped manganese ion chelating agent solution is added to cross-link before use, so as to obtain the required hydrogel. The hydrogel has a synergistic treatment effect of chemotherapy and immunotherapy on tumors, has simple synthesis steps, has good clinical application ability, and has a shear thinning ability, so that the hydrogel can not only be implanted in a tumor for treatment, but also be applied to smearing of a lesion site in surgery, thereby greatly increasing the medical clinical application scene of the hydrogel.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and biomedicine, specifically relating to a glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel capable of loading chemotherapy drugs, its preparation method and application. Background Technology

[0002] The incidence and mortality rates of malignant tumors have continued to rise in recent years. Surgical treatment is currently one of the main methods of clinical cancer treatment, primarily aimed at saving patients' lives by removing primary or even metastatic tumors. However, irregular tumor margins, blurred boundaries between normal and tumor tissues, and difficulty in distinguishing important tissue areas where tumors grow and invade can all lead to incomplete tumor removal, further resulting in tumor recurrence.

[0003] The cyclic GMP-AMP synthase (cGAS) and interferon gene stimulating factor (STING) pathways are important components of the cell-induced type I interferon (IFN) innate immune sensor. Studies have confirmed that the cGAS-STING pathway is a component of the human innate immune system, forming the first line of defense against invading pathogens such as bacteria and viruses. Its activation induces the expression of type I IFN, cytokines, and T cell recruitment factors, and activates the adaptive immune system and T cells through a series of cascade reactions. Recent studies have found that the trace element Mn... 2+ It can strongly activate the intracellular cGAS-STING pathway, giving cells a strong antiviral and antitumor capacity. In-depth molecular mechanism studies have confirmed that viral infection can induce the loss of mitochondrial membrane potential and acidification of organelles in host cells, leading to increased Mn in these organelles. 2+ Released into the cytoplasm and extracellular space, including Mn accumulated in the cytoplasm. 2+ Activation of the cGAS-STING pathway leads to the production of large amounts of type I interferon; while extracellular release of Mn 2+ Through the circulatory system, it is taken up by distant natural killer cells, dendritic cells, and lymphocytes, further promoting and activating the innate immune response of these cells. Studies have shown that exogenous addition of Mn... 2+ This can effectively activate the cGAS-STING pathway in human or mouse cells, significantly promoting the expression of host antigens in cells such as dendritic cells (DCs) and... It enhances the ability to present tumor antigens, promotes the infiltration of cytotoxic T cells into tumor tissues, and strengthens the specific killing effect of these cells on tumor cells. 2+ It can promote the survival and proliferation of memory T cells, significantly enhance the tumor-killing ability of NK cells, and promote the host's tumor immune surveillance function.

[0004] Hydrogels with a three-dimensional cross-linked network can encapsulate bioactive substances and retain them within local injection lesions (such as organs and tumors) to achieve sustained drug release. In recent years, hydrogels have been applied in many fields of biomedicine, including cardiology, oncology, immunology, wound healing, and pain management. Similarly, hydrogels have attracted considerable interest in targeted drug delivery due to their excellent feasibility in improving drug bioactivity by providing high drug concentrations with sustained release characteristics locally, minimizing the side effects of systemic exposure. Injectable hydrogels used as photosensitive drug delivery and release platforms have shown significant advantages in phototherapy applications. Therefore, this invention develops a sodium alginate hydrogel with metal ions as a cross-linking agent capable of loading chemotherapeutic drugs and activating systemic immunity, along with its preparation method and applications, which have significant scientific research and clinical application value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a glycerol-encapsulated homogeneous cross-linked sodium alginate sustained-release hydrogel capable of loading chemotherapeutic drugs, and to attempt to utilize Mn 2+ When used in combination with chemotherapy drugs, it can load chemotherapy drugs and activate the whole body's immune system, so as to significantly enhance the anti-tumor treatment effect and inhibit tumor recurrence and metastasis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention first discloses a method for preparing a glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel capable of loading chemotherapy drugs, characterized in that:

[0008] Sodium alginate was added to deionized water and heated and stirred until dissolved to obtain an aqueous solution of sodium alginate; manganese inorganic salt was added to deionized water and sonicated until dissolved, then glycerol was added and mixed evenly to obtain a chelating agent solution of manganese ions encapsulated by glycerol.

[0009] The chelating agent solution was added dropwise to the aqueous solution of sodium alginate, the reaction was stirred, and centrifuged to obtain a homogeneous cross-linked sodium alginate sustained-release hydrogel containing manganese ions encapsulated in glycerol without drug loading.

[0010] Alternatively: Add the antitumor drug to the aqueous solution of sodium alginate and stir until dissolved; then add the chelating agent solution dropwise to the aqueous solution of sodium alginate, stir to react, and centrifuge to obtain the drug-loaded glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel.

[0011] Furthermore, the manganese inorganic salt is MnCl2·4H2O.

[0012] Furthermore, the concentration of sodium alginate in the aqueous solution is 2.0–2.5 wt%. As the concentration of sodium alginate increases, the mechanical properties of the hydrogel improve, while its injectability decreases.

[0013] Further, the volume ratio of glycerol to deionized water in the chelating agent solution is 1:1, and the concentration of manganese inorganic salt is 0.63–2.52 g / 20 mL. The chelating agent contains Mn... 2+ If the molar concentration is too low, a gel cannot be formed; if it is too high, it is difficult to diffuse evenly.

[0014] Furthermore, when the chelating agent solution is added dropwise to the aqueous solution of sodium alginate, the volume ratio of the two is 1:10 to 15.

[0015] Furthermore, the ratio of β-D-mannuronic acid to α-L-guluronic acid in the sodium alginate is 1.0:1.1. The sodium alginate is a commercially available product, and the ratio of β-D-mannuronic acid to α-L-guluronic acid affects the degree of cross-linking, thereby influencing the properties of the hydrogel.

[0016] The glycerol-encapsulated manganese ion homogeneously cross-linked sodium alginate sustained-release hydrogel of this invention can load chemotherapeutic drugs and activate systemic immunity. It exhibits no significant toxicity to mammalian cells and demonstrates good biocompatibility. It can be used to prepare antitumor implants for direct intratumoral injection to inhibit tumor growth, and also for intraoperative application to lesions to prevent tumor recurrence. By combining local injection therapy to the tumor with application to the lesion site after intraoperative tumor resection, many problems such as recurrence after traditional cancer surgery are solved. The chemotherapy treatment of this invention can be used on various diseased tissues and cells, including but not limited to tumors, acne, and various inflammatory conditions. The implant of this invention can encapsulate various types of hydrophilic drugs, which can be modified according to the type of tumor drug resistance, such as carboplatin, doxorubicin hydrochloride, and doxorubicin hydrochloride.

[0017] The hydrogel of this invention has a sustained-release effect on drugs, which can enhance the therapeutic effect of anti-tumor drugs. In addition, the hydrogel of this invention also has a degradation function, allowing the release of manganese ions (the cross-linking agent) to activate the immune system through the cGAS-STING pathway, greatly increasing the anti-tumor therapeutic effect.

[0018] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0019] 1. The hydrogel of the present invention has significantly improved uniformity compared to traditional sodium alginate hydrogels, and its special mechanical properties are more conducive to clinical use.

[0020] 2. The hydrogel of the present invention has excellent biocompatibility with cells and animals.

[0021] 3. The hydrogel of the present invention has good anti-tumor properties. When a mouse cervical cancer model was selected for the experiment, the hydrogel showed significant anti-tumor ability both in vivo and in vitro.

[0022] 4. The hydrogel of the present invention has strong adhesion and can be applied to the lesion site during surgery. It has significant killing properties against residual cells in areas that are difficult to surgically remove.

[0023] 5. The hydrogel of the present invention has good injection performance and self-healing performance, which is very beneficial for clinical application.

[0024] 6. The hydrogel of the present invention has drug sustained-release capability, and the manganese ions used as cross-linking agents also have the function of releasing immune activation.

[0025] 7. The preparation conditions of the hydrogel of the present invention are simple and mild, and it can be prepared and used immediately before treatment. It is also extremely convenient to store and transport. Attached Figure Description

[0026] Figure 1 A schematic diagram of the preparation process for a glycerol-encapsulated manganese ion homogeneously crosslinked sodium alginate sustained-release hydrogel.

[0027] Figure 2 The infrared spectra of the drug-free hydrogel and sodium alginate powder prepared in Example 1 are shown.

[0028] Figure 3 The images show scanning electron microscope (SEM) images (leftmost) and elemental distribution diagrams (middle image shows C elemental distribution, right image shows Mn elemental distribution) of the drug-free hydrogel prepared in Example 1.

[0029] Figure 4 The figure shows a hydrodynamic comparison of the self-healing properties of the drug-free hydrogel prepared in Example 1 with those of traditional sodium alginate calcium ion hydrogel and sodium alginate manganese ion hydrogel.

[0030] Figure 5 This is a diagram showing the in vitro release of manganese ions from the drug-loaded hydrogel in Example 1.

[0031] Figure 6 This is an in vitro release diagram of carboplatin from the drug-loaded hydrogel in Example 1.

[0032] Figure 7 This is a blood solubility test diagram of unloaded hydrogels at different concentrations in Example 1.

[0033] Figure 8 These are skin tissue sections taken at different times after in vivo injection of the drug-free hydrogel in Example 1.

[0034] Figure 9 This is a comparison chart of the killing effects of each experimental group on normal cells and tumor cells in Example 1.

[0035] Figure 10 This is a staining image showing the killing effect of cervical cancer tumor cells on each experimental group in Example 1.

[0036] Figure 11 The tumor volume inhibition curves of each experimental group in Example 1, using the synergistic effect of chemotherapy and immunotherapy in the treatment of mouse cervical cancer models.

[0037] Figure 12 This is a graph showing the levels of type I interferon (corresponding to the left figure) and TNF-α (corresponding to the right figure) in the tumor site after the release of manganese ions enhances immunity through the cGAS-STING pathway in Example 1.

[0038] Figure 13 The figures in Example 1 show the tumor volume inhibition and recurrence curves of each experimental group in the mouse cervical cancer tumor resection model, achieved by the synergistic effect of chemotherapy and immunotherapy. Detailed Implementation

[0039] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0040] The preparation method of sodium alginate manganese ion hydrogel used as a comparison in the following examples is as follows: 2.5g of sodium alginate powder was weighed and added to 100mL of deionized water, heated to 60℃, and stirred until the solution was clear and transparent to obtain an aqueous solution of sodium alginate. 1.257g of MnCl2·4H2O was weighed and added to 10mL of deionized water, and sonicated until completely dissolved to obtain a manganese ion chelating agent solution. 0.3mL of the manganese ion chelating agent was added dropwise to 3.7mL of the sodium alginate aqueous solution at a rate of 3s / drop, and stirred thoroughly at 800rpm / min for 10 minutes. The gel was then placed in a centrifuge tube and centrifuged at 3000rpm for 5 minutes to remove air bubbles, thus obtaining a manganese ion crosslinked sodium alginate sustained-release hydrogel (hereinafter referred to as sodium alginate manganese ion hydrogel).

[0041] The preparation method of sodium alginate calcium ion hydrogel used as a comparison in the following examples is as follows: 2.5g of sodium alginate powder was weighed and added to 100mL of deionized water, heated to 60℃, and stirred until the solution was clear and transparent to obtain an aqueous solution of sodium alginate. 2.2g of CaCl2 was weighed and added to 10mL of deionized water, and sonicated until completely dissolved to obtain a calcium ion chelating agent solution. 0.3mL of the calcium ion chelating agent solution was added dropwise to 3.7mL of the sodium alginate aqueous solution at a rate of 3s / drop, and stirred thoroughly at 800rpm / min for 10 minutes. The gel was then placed in a centrifuge tube and centrifuged at 3000rpm for 5 minutes to remove air bubbles, thus obtaining a calcium ion crosslinked sodium alginate sustained-release hydrogel (hereinafter referred to as sodium alginate calcium ion hydrogel).

[0042] Example 1

[0043] Weigh 2.5g of sodium alginate powder and add it to 100mL of deionized water. Heat to 60℃ and stir until the solution is clear and transparent to obtain an aqueous solution of sodium alginate. Weigh 1.257g of MnCl2·4H2O and add it to 10mL of deionized water. Sonicate until completely dissolved, then add 10mL of glycerol and mix thoroughly to obtain a chelating agent solution of manganese ions encapsulated by glycerol.

[0044] Add 0.6 mL of glycerol-encapsulated manganese ion chelating agent solution to 3.7 mL of sodium alginate aqueous solution dropwise at a rate of 3 s / drop. Stir thoroughly at 800 rpm for 10 minutes. Remove the gel and place it in a centrifuge tube. Centrifuge at 3000 rpm for 5 minutes to remove air bubbles. This yields an injectable, drug-free, glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel (hereinafter referred to as drug-free hydrogel).

[0045] First, dissolve 20 mg of carboplatin in 100 mL of sodium alginate aqueous solution and stir at room temperature in the dark until clear and transparent. Then, add 0.3 mL of the chelating agent solution dropwise to 3.7 mL of the sodium alginate aqueous solution containing the drug at a rate of 3 drops / second. Stir thoroughly at 800 rpm for 10 minutes. Remove the gel and centrifuge at 3000 rpm for 5 minutes to remove air bubbles. This yields a carboplatin-loaded, glycerol-encapsulated, manganese-ion-modified homogeneous cross-linked sodium alginate sustained-release hydrogel (hereinafter referred to as drug-loaded hydrogel).

[0046] Figure 2 The images show the infrared spectra of the drug-free hydrogel and sodium alginate powder obtained in this embodiment. In the figure, 1540-1650 cm⁻¹ -1 and 1488-1350cm -1These are the antisymmetric and symmetric stretching vibration peaks of the carboxyl group on the alginate molecule, respectively. When the difference between the antisymmetric and symmetric stretching vibration peaks is similar, the sodium alginate coordinates with the metal ion in a bridging manner. The results indicate that the structure of the hydroxyl group in alginate directly affects the position and shape of its stretching vibration peak. When the hydroxyl oxygen coordinates with a divalent metal ion, the OH bond energy is weakened due to the formation of the metal-oxygen complex, and the position of the OH stretching vibration peak will redshift.

[0047] Figure 3 The images shown are scanning electron microscope (SEM) images (leftmost) and elemental distribution diagrams (middle image shows C element distribution, right image shows Mn element distribution) of the drug-free hydrogel obtained in this embodiment. The presence of C and Mn elements in the gel can be seen, proving that Mn was successfully crosslinked onto sodium alginate.

[0048] Figure 4 This is a hydrodynamic comparison of the injectability and self-healing properties of the untreated hydrogel obtained in this embodiment compared to traditional sodium alginate manganese ion hydrogels and sodium alginate calcium ion hydrogels. The injectability and self-healing properties of the hydrogel (1 mL) were evaluated using a TADiscoveryDHR-3 rotational rheometer: the test temperature was 25℃, and the test gap was 1000 μm. A 1 mL sample was tested for 120 s at 1% pressure in time-scan mode, then for 60 s at 100% pressure, with the stress magnitude changing four times, thus obtaining the changes in the storage modulus and loss modulus of the implant. Figure 4 It can be seen that the storage modulus and loss modulus of the hydrogel reverse under high pressure, and the hydrogel changes from a colloid to a liquid, and then changes back to a colloid after restoring 1% of the pressure. A comparison of the stress-change rheological properties of the glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel with traditional sodium alginate manganese and calcium ion hydrogels demonstrates that this implant has good injectability and self-healing capabilities.

[0049] Figure 5 The image shows the cumulative manganese ion release curves of the drug-loaded hydrogel obtained in this embodiment at pH values ​​of 6.0, 6.5, and 7.4. The specific characterization method was as follows: 1 mL of the drug-loaded hydrogel was placed in a dialysis bag and immersed in sodium acetate buffer solution at pH values ​​of 6.0, 6.5, and 7.4. 3 mL of solution was taken at regular intervals for testing, and 3 mL of sodium acetate buffer solution at the corresponding pH value was added. The concentration was detected by ICP. Figure 5 As shown, the hydrogel exhibits better manganese ion release in the pH of the tumor microenvironment, demonstrating better pH-responsive drug release performance.

[0050] Figure 6The cumulative release curves of carboplatin from the drug-loaded hydrogel obtained in this embodiment at pH values ​​of 6.0, 6.5, and 7.4 are shown. The specific characterization method is as follows: 1 mL of carboplatin-loaded hydrogel was placed in a dialysis bag and immersed in sodium acetate buffer solution at pH values ​​of 6.0, 6.5, and 7.4. 3 mL of solution was taken at regular intervals for testing, and 3 mL of sodium acetate buffer solution at the corresponding pH value was added. The concentration was determined by high-performance liquid chromatography (HPLC). Figure 6 As shown, the hydrogel exhibits better carboplatin release in the pH of the tumor microenvironment, demonstrating better pH-responsive drug release performance.

[0051] Figure 7 The hemolytic properties of the unloaded hydrogels at different concentrations obtained in this embodiment are shown in the figure. The characterization method is as follows: 200 mg, 400 mg, 600 mg, and 800 mg of hydrogel were placed in 5 mL centrifuge tubes, and respectively mixed with 0.5 mL of treated blood (0.5 L of fresh blood with 4.5 mL of physiological saline, centrifuged 5-8 times at 3000 rpm for 10 minutes until the blood supernatant was clear and transparent, then discarded, and physiological saline was added to 5 mL for later use) and then the mixture was incubated at 37°C for 4 h, centrifuged at 3000 rpm for 10 min, and the absorbance at OD541 nm was measured to calculate the hemolysis rate. As can be seen from the figure, the hemolysis rate of the hydrogels at different concentrations is less than 5%, indicating that the material has good biocompatibility.

[0052] On days 7 and 14 after implantation of the drug-free hydrogel into mice, mouse skin at the injection site was subjected to the following steps: decolorization, rinsing and soaking, antibody application, staining, thorough rinsing, counterstaining, dehydration, clearing, and mounting to prepare H&E and Masson immunohistochemical sections. The results are as follows: Figure 8 As shown, no related inflammatory symptoms were observed in the mouse skin tissue, and the tissue remained intact. This indicates that the material has good biocompatibility and fully meets the injection requirements.

[0053] Figure 9 MTT assays were performed on the cell viability of mammalian normal cells (HUVECs) and mouse cervical cancer cells (U14) using phosphate buffer solution, manganese ion solution, carboplatin solution, manganese ion / carboplatin solution, unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel, and carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel. The experimental groups were as follows: phosphate buffer solution, manganese ion solution (0.045 mmol / mL), carboplatin solution (200 μg / mL), manganese ion / carboplatin solution (Mn...). 2+ 0.045 mmol / mL, carboplatin 200 μg / mL), unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel (Mn 2+0.045 mmol / mL), a sodium alginate hydrogel (Mn) loaded with carboplatin and encapsulated in glycerol and crosslinked with manganese ions. 2+ The above experimental group solutions were prepared to the corresponding concentrations using culture medium solutions containing 0.045 mmol / mL and carboplatin 200 μg / mL. The testing method was as follows: U14 cell suspensions were prepared using culture medium containing 10% fetal calf serum. 10,000 cells were seeded into each well of a 96-well plate (100 μL per well). After the cells had adhered and grown to confluence, 100 μL of the above experimental group solutions were added to each well and incubated for 24 hours and 48 hours, respectively. After incubation, 50 μL of MTT solution (5 mg / mL prepared with PBS, pH 7.4) was added to each well, and incubation continued for 4 hours. The culture was then terminated, and the culture supernatant was carefully aspirated from the wells. For suspended cells, centrifugation was required before aspirating the culture supernatant. 150 μL of LDMSO was added to each well, and the mixture was shaken for 10 min to fully dissolve any crystals. The absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader. The figure shows that the glycerol-encapsulated manganese-crosslinked sodium alginate hydrogel loaded with carboplatin exhibits significantly greater cytotoxicity towards tumor cells than normal cells. This is likely because the carboplatin-loaded drug has a stronger cytotoxic effect on tumor cells during incubation, leading to faster tumor cell division and poorer repair of damage compared to normal cells. The glycerol-encapsulated manganese-crosslinked sodium alginate hydrogel's sustained-release properties result in the continuous release of carboplatin over a period of time, extending its effect on tumor cells. This is consistent with the results showing the effects of unloaded glycerol-encapsulated manganese-crosslinked sodium alginate hydrogel and carboplatin-loaded glycerol-encapsulated manganese-crosslinked sodium alginate hydrogel on cell viability. Furthermore, the incubation time significantly increases the activity of tumor cells.

[0054] Figure 10 The effects of phosphate buffer solution, manganese ions, carboplatin, manganese ion / carboplatin, unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel, and carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel on tumor cell viability were investigated. The experimental groups were as follows: phosphate buffer solution, manganese ion solution (0.045 mmol / mL), carboplatin solution (200 μg / mL), and manganese ion / carboplatin solution (Mn...). 2+ 0.045 mmol / mL, carboplatin 200 μg / mL), unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel (Mn 2+ 0.045 mmol / mL), a sodium alginate hydrogel (Mn) loaded with carboplatin and encapsulated in glycerol and crosslinked with manganese ions. 2+(0.045 mmol / mL carboplatin 200 μg / mL). All solutions in the above experimental groups were prepared to the corresponding concentrations using culture medium. The testing method was as follows: U14 cell suspension was prepared using culture medium containing 10% fetal calf serum. 10,000 cells were seeded per well in a 96-well plate (100 μL per well). After the cells had adhered and grown to confluence, 100 μL of the above experimental group was added to each well and incubated for 24 hours. Then, 50 μL of LAM / PI live / dead dye was added, and after incubation for 30 minutes, images were taken under a fluorescence microscope. Characterization results showed that large green areas in the phosphate buffer solution group indicated live cells, while the area of ​​red areas in other groups gradually increased, consistent with the results of the cell MTT assay.

[0055] Figure 11 To assess the antitumor effect of intratumoral injection, the experimental groups were divided into the following groups: phosphate buffer solution, manganese ion solution (0.045 mmol / mL), carboplatin solution (200 μg / mL), and manganese ion / carboplatin solution (Mn). 2+ 0.045 mmol / mL, carboplatin 200 μg / mL), unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel (Mn 2+ 0.045 mmol / mL), a sodium alginate hydrogel (Mn) loaded with carboplatin and encapsulated in glycerol and crosslinked with manganese ions. 2+ 0.045 mmol / mL carboplatin (200 μg / mL) were used in the above experimental groups, and the solutions were prepared to the corresponding concentrations using culture medium. The anti-tumor effect of intratumoral injection in a mouse cervical cancer U14 model was characterized by measuring the tumor volume of mice in each group at different time points (0, 2, 4, 6, 8, 10, 12, 14, 16) using calipers, with an injection dose of 100 μL. Figure 11 As shown, the tumor volume changes indicate that the tumor volume in mice in the carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel group was significantly reduced and disappeared, demonstrating an excellent synergistic killing effect. This reflects that the carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel has good anti-tumor ability.

[0056] In a mouse model of cervical cancer (U14 tumor), intratumoral injection of manganese ions released manganese into damaged DNA within tumor cells. This manganese, through the cGAS-STING pathway, increased the release of related cytokines, thereby recruiting T cells, dendritic cells, and natural killer cells to activate the autoimmune response against tumor cell invasion. Results were as follows: Figure 12 As shown, the experimental groups consisted of the following: phosphate buffer solution, manganese ion solution (0.045 mmol / mL), carboplatin solution (200 μg / mL), and manganese ion / carboplatin solution (Mn). 2+0.045 mmol / mL carboplatin 200 μg / mL), unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel (Mn 2+ 0.045 mmol / mL), a sodium alginate hydrogel (Mn) loaded with carboplatin and encapsulated in glycerol and crosslinked with manganese ions. 2+ (0.045 mmol / mL carboplatin 200 μg / mL), and all solutions in the above experimental groups were prepared to the corresponding concentrations using culture medium. After intratumoral injection of 100 μL into the mouse cervical cancer U14 model, tumor tissue from the corresponding group was extracted, and the levels of type I interferon and TNF-α were detected using an ELISA kit. Results are as follows... Figure 12 As shown, the group of sodium alginate hydrogels loaded with carboplatin and encapsulated with manganese ions and crosslinked with glycerol showed a significant increase in type I interferon and TNF-α, demonstrating an effective immune activation effect.

[0057] Figure 13 In this embodiment, the hydrogel was used in a mouse model of intraoperative resection of cervical cancer tumor U14 to inhibit tumor volume using chemotherapy-assisted immune killing. The experimental groups included: phosphate buffer solution, manganese ion solution (0.045 mmol / mL), carboplatin solution (200 μg / mL), and manganese ion / carboplatin solution (Mn). 2+ 0.045 mmol / mL, carboplatin 200 μg / mL), unloaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel (Mn 2+ 0.045 mmol / mL), a sodium alginate hydrogel (Mn) loaded with carboplatin and encapsulated in glycerol and crosslinked with manganese ions. 2+ The solutions in the above experimental groups (0.045 mmol / mL carboplatin, 200 μg / mL) were all prepared to the corresponding concentrations using culture medium. The specific characterization method was as follows: mouse tumors of 50 mm... 3 In mice with U14 cervical cancer tumors, tumor resection was performed. The anti-tumor characteristics of each experimental group in the mouse U14 cervical cancer tumor model were assessed by applying a topical treatment to the lesions. The volume of the mouse tumors was measured using calipers at different time points (0, 2, 4, 6, 8, 10, 12, 14, 16), with a topical dose of 100 μL. Figure 13 As shown in the figure, the tumor volume changes indicate that the tumor volume in the group of mice with carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel no longer increased or even disappeared, demonstrating an excellent synergistic killing effect. This reflects that carboplatin-loaded glycerol-encapsulated manganese ion crosslinked sodium alginate hydrogel has a good ability to prevent tumor recurrence.

[0058] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make corresponding adjustments and improvements without departing from the principle of the present invention, and these adjustments and improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel capable of loading chemotherapeutic drugs, characterized in that: Sodium alginate was added to deionized water and heated and stirred until dissolved to obtain an aqueous solution of sodium alginate with a concentration of 2.0-2.5 wt%. Manganese inorganic salt MnCl2·4H2O was added to deionized water and sonicated until dissolved. Glycerol was then added and mixed evenly to obtain a chelating agent solution in which glycerol encapsulates manganese ions. The volume ratio of glycerol to deionized water in the chelating agent solution was 1:1, and the concentration of manganese inorganic salt was 0.63-2.52 g / 20 mL. The chelating agent solution was added dropwise to the sodium alginate aqueous solution at a volume ratio of 1:10-15, the reaction was stirred, and the mixture was centrifuged to obtain a glycerol-encapsulated homogeneous cross-linked sodium alginate sustained-release hydrogel without drug loading. Alternatively: Add the antitumor drug to the aqueous solution of sodium alginate and stir until dissolved; then add the chelating agent solution dropwise to the aqueous solution of sodium alginate, stir to react, and centrifuge to obtain the drug-loaded glycerol-encapsulated manganese ion homogeneous cross-linked sodium alginate sustained-release hydrogel.

2. The preparation method according to claim 1, characterized in that: The ratio of β-D-mannuronic acid to α-L-guluronic acid in the sodium alginate is 1.0:1.

1.

3. A glycerol-encapsulated manganese ion homogeneous crosslinked sodium alginate sustained-release hydrogel prepared by the preparation method according to any one of claims 1 to 2.

4. The application of the glycerol-encapsulated manganese ion homogeneous crosslinked sodium alginate sustained-release hydrogel as described in claim 3, characterized in that: Used for the preparation of antitumor implants for direct intratumoral injection and / or antitumor implants for intraoperative application to the lesion site.

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