In-situ drug-loaded synergistic treatment gel P-GFe@Gel and preparation method and application thereof

Abstract

The application discloses in-situ drug-loaded synergistic treatment gel P-GFe@Gel and a preparation method and application thereof, and belongs to the field of biological medicines. The application integrates the photothermal properties of mesoporous polydopamine (MPDA), the CDT efficiency of Fe2+, the TME regulation effect of glucose oxidase (GOx) and the in-situ delivery capacity of protein hydrogel, realizes PTT / CDT synergistic treatment, and solves the problems of insufficient targeting, TME limited curative effect and high systemic toxicity in existing tumor treatment. The preparation process is simple and mild, raw materials are easy to obtain, and the in-situ drug-loaded synergistic treatment gel can be produced on a large scale, and has a wide application prospect in the clinical treatment of breast cancer and other malignant tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an in-situ drug-loaded gel P-GFe@Gel that integrates photothermal therapy (PTT) and chemokinetic therapy (CDT) and its preparation method, which is suitable for multimodal precision treatment of malignant tumors such as breast cancer. Background Technology

[0002] Breast cancer, a highly prevalent malignant tumor in women, faces key challenges with conventional treatments, including insufficient targeting, easy development of drug resistance, and limitations in treatment efficacy due to the tumor microenvironment (TME). Chemokinetic therapy (CDT) has become a research hotspot due to its non-invasive nature and low toxicity to normal tissues; however, the high concentration of glutathione (GSH) in the TME quenches reactive oxygen species (ROS), and the insufficient concentration of endogenous hydrogen peroxide (H2O2) severely restricts its efficacy. While photothermal therapy (PTT) offers precise targeting, it is limited by the penetration depth of near-infrared light, making it difficult to completely remove deep tumors.

[0003] Currently, research on synergistic tumor therapy strategies mainly focuses on simple composites of single-functional materials, which suffers from drawbacks such as poor carrier biocompatibility, uncontrolled drug release, and low synergistic efficiency of therapeutic modalities. Existing drug delivery systems cannot effectively regulate the tumor microenvironment (TME), making it difficult to achieve in-situ supply of H2O2 and the construction of an acidic microenvironment. Furthermore, they lack in-situ delivery and sustained-release capabilities, resulting in high systemic toxicity and poor therapeutic effects.

[0004] Therefore, developing a tumor treatment system that combines TME regulation, multimodal synergy, in-situ sustained release, and biocompatibility has become the key to overcoming the current technological bottlenecks. Summary of the Invention

[0005] The purpose of this invention is to provide an in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel and its preparation method. By integrating the photothermal properties of mesoporous polydopamine (MPDA), the CDT efficacy of Fe²⁺, the TME regulatory role of glucose oxidase (GOx), and the in-situ delivery capability of protein hydrogel, PTT / CDT synergistic therapy is achieved, solving the problems of insufficient targeting, TME-limited efficacy, and high systemic toxicity in existing tumor treatments.

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

[0007] In a first aspect, the present invention provides a method for preparing an in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel, wherein the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel is composed of an MPDA-GOx-Fe nanocomposite and a protein hydrogel matrix, wherein the protein hydrogel matrix is ​​a thrombin-fibrinogen system.

[0008] Furthermore, the preparation method of the MPDA-GOx-Fe nanocomposite is as follows:

[0009] (1) Add 0.5 mg / mL glucose oxidase (GOx) solution to mesoporous polydopamine (MPDA) solution, stir for 6 h and remove free enzyme to obtain MPDA-GOx; based on MPDA 1.0 equivalent, the amount of GOx used is 0.05-0.1 equivalent;

[0010] (2) Add 2 mg / mL polyethyleneimine (PEI) and 10 mg / mL Fe²⁺ solution dropwise and stir continuously for 4 h; based on MPDA 1.0 equivalent, the amount of PEI is 0.1-0.2 equivalents and the amount of Fe²⁺ is 0.01-0.03 equivalents;

[0011] (3) MPDA-GOx-Fe nanocomposite was obtained by dialysis purification.

[0012] Furthermore, in step (1), the method for synthesizing MPDA is as follows:

[0013] 0.1 g of copolymer F127, 0.15 g of dopamine hydrochloride, and 0.2 mL of 3,3',5,5'-tetramethylbenzidine (TMB) were ultrasonically dispersed in a mixture of 5 mL of water and 5 mL of ethanol for 30 min to form an emulsion. 0.375 mL of ammonia was added dropwise while stirring, and the reaction was carried out at room temperature for 12 h. The reaction solution was dialyzed against 50 g / L ethanol solution for 4 h, with a molecular weight cutoff of 3500. The solution was then dispensed and heated at 100 °C for 24 h. The solution was dialyzed against ultrapure water again for 2 h, with a molecular weight cutoff of 3500. The solution was centrifuged at 11000 rpm for 20 min, and the supernatant was stored at 4 °C to obtain MPDA.

[0014] Furthermore, step (3) involves dialysis three times, each time for 2 hours, with a molecular weight cutoff of 3500.

[0015] Furthermore, the preparation method of the above-mentioned in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel includes the following steps: dispersing the MPDA-GOx-Fe nanocomposite at a final concentration of 50 μg / mL in a 20 mg / mL fibrinogen solution to complete drug loading, thereby obtaining a drug-loaded fibrinogen solution; mixing 50 U / mL thrombin and the drug-loaded fibrinogen solution at a 1:1 volume ratio using a dual syringe, allowing it to stand at room temperature for 5 min to naturally solidify, thus obtaining the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel.

[0016] Secondly, the present invention provides an in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel, which is obtained by any of the preparation methods described above.

[0017] Thirdly, the present invention provides the application of the above-mentioned in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel in the preparation of multimodal precision therapeutic drugs for malignant tumors.

[0018] Furthermore, the malignant tumor is breast cancer.

[0019] The in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel provided by this invention has the following reaction mechanism:

[0020] After P-GFe@Gel enters the tumor site, GOx catalyzes the conversion of glucose in the tumor into gluconic acid and H2O2 (Glu + O2 + H2O → GlcA + H2O2), reducing the pH and glucose concentration of the tumor microenvironment, endogenously replenishing H2O2, providing sufficient substrate for CDT and optimizing the reaction microenvironment; Fe²⁺ reacts with H2O2 to generate highly toxic •OH, killing tumor cells; under irradiation with 808 nm near-infrared light (1.1 W / cm²), MPDA generates local high temperature (reaching 67.9℃ at an in vitro concentration of 100.0 μg / mL, and above 66.3℃ measured at the tumor site in vivo), which can directly kill tumor cells and accelerate the efficiency of the Fenton reaction between Fe²⁺ and H2O2, enhancing the efficacy of CDT; the hydrogel matrix enables in-situ sustained release of the drug, reducing systemic toxicity, forming an integrated treatment system of "TME regulation - PTT / CDT synergy - in-situ sustained release".

[0021] Compared with the prior art, the present invention has the following significant advantages:

[0022] 1. For the first time, a GOx-Fe²⁺-MPDA cascade catalytic system was constructed to achieve precise TME control, autonomously supply H2O2 and construct an acidic environment, thus solving the problem of limited efficacy of CDT.

[0023] 2. The synergistic treatment of PTT and CDT enhances the near-infrared photothermal effect and chemokinetic effect, significantly improving the tumor killing rate. The survival rate of 4T1 cells at a concentration of 100.0 μg / mL within 24 hours is less than 30%, and the effect is significantly better than single PTT or CDT treatment.

[0024] 3. The hydrogel carrier has good in-situ molding, self-healing and adhesion properties, enabling sustained drug release and targeted delivery. It has a survival rate of over 85% for normal cells (3T3, L929) and excellent biocompatibility.

[0025] 4. The preparation process is simple and mild, the raw materials are readily available, and it can be mass-produced, showing broad application prospects in the clinical treatment of malignant tumors such as breast cancer. Attached Figure Description

[0026] Figure 1 The structure and characterization diagrams of P-GFe@Gel are as follows: A. Material synthesis flowchart; B. Catalytic mechanism diagram; C. Particle size distribution curve; D. Comparison of hydrated particle size of different intermediates; E. Comparison of Zeta potential of different intermediates; F. Long-term stability of the material.

[0027] Figure 2 Catalytic and photothermal properties of P-GFe@Gel: A. pH regulation curve; BC. Peroxidase-like activity detection; D. MB degradation rate (OH generation); E. H2O2 accumulation curve; F. Photothermal heating and cycling stability; G. Thermal imaging.

[0028] Figure 3 Results of in vitro cell experiments: A. DCFH-DA ROS fluorescence staining; BC. MTT cell viability detection.

[0029] Figure 4 The results of in vivo antitumor experiments include: A. Animal experimental procedures; B. Tumor volume changes; C. Mouse body weight changes; D. Thermographic imaging of the tumor site; E. Histopathological examination results. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Synthesis of mesoporous polydopamine (MPDA): Accurately weigh 0.1 g F127, 0.15 g dopamine hydrochloride, and 0.2 mL TMB, add a mixture of 5 mL water and 5 mL ethanol, and sonicate for 30 min to form a homogeneous emulsion; slowly add 0.375 mL ammonia water dropwise under magnetic stirring, and react at room temperature in the dark for 12 h; transfer the reaction solution to a dialysis bag (MWCO=3500), dialyze with 50 g / L ethanol solution for 4 h, aliquot and heat in a 100℃ oven for 24 h; remove and dialyze with ultrapure water for 2 h, centrifuge at 11000 rpm for 20 min, collect the supernatant and store at 4℃ for later use.

[0033] 2. Preparation of MPDA-GOx-Fe complex: Take 10 mL of MPDA supernatant, add 1 mL of 0.5 mg / mL GOx solution, and stir at room temperature for 6 h; remove free enzyme by centrifugation and ultrafiltration, add 1 mL of 2 mg / mL PEI solution and 100 μL of 10 mg / mL Fe²⁺ solution to the precipitate, and stir continuously for 4 h; dialyze the mixture three times (2 h each time) through a dialysis bag (MWCO=3500) to obtain the purified MPDA-GOx-Fe complex.

[0034] 3. Hydrogel formation: The MPDA-GOx-Fe complex was dispersed in a 20 mg / mL fibrinogen solution at a final concentration of 50 μg / mL and mixed thoroughly. 50 U / mL thrombin and the drug-loaded fibrinogen solution were mixed at a 1:1 volume ratio using a dual syringe. The mixture was allowed to stand at room temperature for 5 min to form naturally, thus obtaining P-GFe@Gel.

[0035] Example 2

[0036] 1. Structural Characterization: DLS analysis showed that the hydrodynamic particle sizes of MPDA, MPDA-GOx, and MPDA-GOx-Fe were 120 nm, 150 nm, and 180 nm, respectively, and the Zeta potential gradually increased from -35 mV to +15 mV. Figure 1 As shown.

[0037] 2. Gel properties: The mixture completely solidified after 5 minutes of laser irradiation, showed no collapse after 60 minutes of inversion, and could self-heal after cutting; rheological testing showed that the storage modulus remained stable at 10. 4 Pa exhibits good mechanical stability; adhesion experiments confirm that it can firmly adhere to the skin and tissue surfaces.

[0038] 3. Catalytic and Photothermal Performance: pH testing showed that the system pH decreased to approximately 5.0 over time; TMB oxidation experiments indicated that the absorbance at 652 nm increased significantly with increasing glucose concentration; after 6 min of 808 nm laser irradiation, the temperature of the 100 μg / mL P-GFe@Gel solution rose to 67.9℃, and its performance showed no degradation after 4 cycles. Figure 2 As shown.

[0039] 4. Performance results of the examples and comparative examples are shown in Table 1.

[0040] Table 1

[0041]

[0042] Example 3

[0043] 1. Cell culture: 4T1 breast cancer cells, 3T3 fibroblasts and L929 fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and passaged in a 37°C, 5% CO2 incubator.

[0044] 2. ROS generation detection: After 4T1 cells were treated with different concentrations (0, 25, 50, 75, 100, 250 μg / mL) of P-GFe@Gel, they were incubated with DCFH-DA fluorescent probe for 30 min. Flow cytometry showed that the intracellular ROS level in the 100 μg / mL concentration group was more than 5 times that of the control group.

[0045] 3. Cell killing assay: MTT assay showed that 100 μg / mL P-GFe@Gel killed more than 70% of 4T1 cells within 24 h, which was significantly higher than that of the single PTT or CDT group; PI staining and apoptosis detection confirmed that the sum of the proportion of early apoptotic and late apoptotic cells reached 75%.

[0046] 4. Biocompatibility: MTT assay showed that 100 μg / mL P-GFe@Gel resulted in a survival rate of over 85% for both 3T3 and L929 cells, with no significant cytotoxicity. Figure 3 As shown.

[0047] Example 4

[0048] 1. Animal model establishment: 5 × 10⁵ cells were subcutaneously injected into the right side of female BALB / c mice. 6 Four T1 cells were randomly assigned to groups when the tumor volume reached 150 mm³.

[0049] 2. Treatment regimen: After injecting P-GFe@Gel into the tumor site, mice in the experimental group were irradiated with 808 nm near-infrared laser (1.1W / cm²) for 10 min, once every 3 days, for a total of 3 treatments; the control group was treated with PBS, MPDA, P@Fe, etc.

[0050] 3. Treatment Efficacy: After 14 days, the tumor volume in the experimental group was significantly smaller than that in the control group, and the mice did not experience significant weight loss, confirming its highly effective anti-tumor activity; thermal imaging showed that the temperature at the tumor site could reach above 52.0℃; histopathological examination showed extensive necrosis of tumor tissue, with no significant pathological damage to the heart, liver, spleen, lungs, and kidneys. Figure 4 As shown.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A method for preparing an in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel, characterized in that, The in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel is composed of an MPDA-GOx-Fe nanocomposite and a protein hydrogel matrix, wherein the protein hydrogel matrix is ​​a thrombin-fibrinogen system; the preparation method of the MPDA-GOx-Fe nanocomposite is as follows: (1) Add 0.5 mg / mL GOx solution to MPDA solution, stir for 6 h and remove free enzyme to obtain MPDA-GOx; Based on MPDA 1.0 equivalent, the GOx dosage is 0.05-0.1 equivalent; (2) Add 2 mg / mL PEI and 10 mg / mL Fe²⁺ solution dropwise and stir continuously for 4 h; based on MPDA 1.0 equivalent, the amount of PEI used is 0.1-0.2 equivalents and the amount of Fe²⁺ used is 0.01-0.03 equivalents; (3) MPDA-GOx-Fe nanocomposite was obtained by dialysis purification; The MPDA-GOx-Fe nanocomposite was dispersed in a 20 mg / mL fibrinogen solution to complete drug loading, resulting in a drug-loaded fibrinogen solution. 50 U / mL thrombin and the drug-loaded fibrinogen solution were mixed at a 1:1 volume ratio using a dual syringe. After standing at room temperature for 5 min, the mixture naturally solidified, yielding the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel.

2. The method for preparing the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel according to claim 1, characterized in that, In step (1), the method for synthesizing MPDA is as follows: 0.1 g of copolymer F127, 0.15 g of dopamine hydrochloride, and 0.2 mL of 3,3',5,5'-tetramethylbenzidine were ultrasonically dispersed in a mixture of 5 mL of water and 5 mL of ethanol for 30 min to form an emulsion. 0.375 mL of ammonia was added dropwise while stirring, and the reaction was carried out at room temperature for 12 h. The reaction solution was dialyzed against 50 g / L ethanol solution for 4 h, with a molecular weight cutoff of 3500. The solution was then dispensed and heated at 100 °C for 24 h. The solution was dialyzed against ultrapure water again for 2 h, with a molecular weight cutoff of 3500. The solution was centrifuged at 11000 rpm for 20 min, and the supernatant was stored at 4 °C to obtain MPDA.

3. The method for preparing the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel according to claim 1, characterized in that, Step (3) involves dialysis three times, each time for 2 hours, with a molecular weight cutoff of 3500.

4. A p-GFe@Gel, an in-situ drug-loaded synergistic therapeutic gel, characterized in that... It is obtained by the preparation method described in any one of claims 1-3.

5. The application of the in-situ drug-loaded synergistic therapeutic gel P-GFe@Gel according to claim 4 in the preparation of multimodal precision therapeutic drugs for malignant tumors.

6. The application according to claim 5, characterized in that, The malignant tumor in question is breast cancer.

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