Responsive composite nanomaterial, and preparation method and use thereof
By designing tumor microenvironment-responsive nanomedicines, utilizing iron-based metal-organic frameworks and mitochondrial transporter inhibitors to generate ROS under photodynamic therapy, and combining folic acid receptor targeting, the immunosuppression problem of hepatocellular carcinoma was solved, achieving tumor microenvironment remodeling and improved immunotherapy efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
- Filing Date
- 2026-04-28
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, drug therapy in the tumor microenvironment is not very effective for hepatocellular carcinoma, especially due to the inefficient response caused by the immunosuppression and antioxidant defense pathways of tumor cells.
Develop a tumor microenvironment-responsive nanomedicine comprising an iron-based metal-organic framework, modified polyethylene glycol/folic acid, and encapsulated mitochondrial transporter inhibitor PK11195. This nanomedicine generates ROS under near-infrared light via photodynamic therapy, binds to folic acid receptors to actively target tumor cells, inhibits Nrf2 and PD-L1 expression, promotes immune cell death, and facilitates immunotherapy.
By enhancing the combination of photodynamic therapy and immunotherapy, the immunosuppressive tumor microenvironment can be reshaped, significantly improving the treatment effect of hepatocellular carcinoma and promoting systemic anti-tumor therapy.
Smart Images
Figure CN122097633A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine and relates to a nanomaterial, specifically a tumor microenvironment-responsive nanomedicine, its preparation method, and its uses. Background Technology
[0002] In recent years, immune checkpoint inhibitor (ICI) therapy has achieved groundbreaking success in the treatment of multiple malignant tumors, and is expected to become a possible approach to radical cancer treatment. The low response to aPD-1 / aPD-L1 therapy results in "cold tumors" in most HCC patients, characterized by an immunosuppressive tumor microenvironment and a lack of T lymphocyte infiltration. Inducing immunogenic cell death (ICD) to transfer "cold" tumors to "hot" tumors is a feasible strategy. Studies have shown that ROS-mediated lipid peroxidation can induce ICD. However, to resist ROS-mediated lipid peroxidation, tumor cells have evolved robust antioxidant defense pathways, among which Nrf2 is an indispensable transcriptional regulator that inhibits tumor cell ferroptosis and the occurrence of ICD.
[0003] Mitochondrial translocator protein (TSPO) is an 18 kDa transmembrane protein primarily located in the outer mitochondrial membrane. It is highly expressed in various tumors, including liver cancer, breast cancer, brain cancer, colorectal cancer, and ovarian cancer. Studies have shown that high TSPO expression in hepatocellular carcinoma (HCC) is associated with poor prognosis. Emerging research indicates that TSPO can inhibit ROS-mediated lipid peroxidation in HCC cells by promoting Nrf2-mediated antioxidant pathways and promote HCC immune escape through Nrf2-dependent transcriptional upregulation of PD-L1 expression. Therefore, inhibiting TSPO to improve intracellular drug delivery (ICD) and reduce immune escape could be beneficial for developing new strategies.
[0004] Photodynamic therapy (PDT), as a clinically recognized cancer treatment method, has seen extensive research into methods for generating reactive oxygen species (ROS), and its development has been rapid due to its selectivity and non-invasiveness. During PDT, photosensitizers (Ps) injected into the tumor are activated by light, generating ROS. Developing Ps with highly efficient ROS-generating capabilities is essential for the success of PDT. Among these, metal-organic frameworks (MOFs) constructed from metal clusters and organic Pss have attracted widespread attention due to their high ROS-generating capacity, biocompatibility, multimodal imaging performance, and drug loading capacity. Notably, the porous structure of MOFs facilitates O2 dispersion, leading to the generation of ROS. 1 O2. Summary of the Invention
[0005] To address the aforementioned technical problems in the prior art, this invention provides a tumor microenvironment-responsive nanomedicine, its preparation method, and its uses. This tumor microenvironment-responsive nanomedicine, its preparation method, and its uses aim to solve the technical problem of poor efficacy of drug treatment for hepatocellular carcinoma in the prior art.
[0006] This invention provides a tumor microenvironment-responsive nanomedicine, comprising an iron-based metal-organic framework, polyethylene glycol / folic acid modified on the iron-based metal-organic framework, and PK11195 encapsulated within the metal framework. The structural formula of PK11195 is shown below.
[0007] .
[0008] Furthermore, the tumor microenvironment-responsive drug is MRI-guided.
[0009] Furthermore, the iron-based metal-organic framework used in the tumor microenvironment responsive nanomedicine is PCN(Fe). PCN(Fe) is a metal-organic framework material that is formed by the self-assembly of iron metal ions and organic ligands through coordination bonds to form a three-dimensional porous structure.
[0010] This invention also provides a method for preparing a tumor microenvironment-responsive nanomedicine, comprising the following steps:
[0011] Step 1: Benzoic acid, methyl-4-tetra(4-carboxyphenyl)porphyrin and ferric chloride hexahydrate are dissolved in N,N-dimethylformamide (DMF). The mixture is stirred and reacted at 80-90°C. The mixture is then centrifuged and washed with N,N-dimethylformamide to remove unreacted raw materials, yielding a metal-organic framework PCN(Fe).
[0012] Step 2: Dissolve PCN(Fe) with PK11195 and PEG / FA in N,N-dimethylformamide and stir at room temperature. After the reaction is complete, centrifuge the mixed solution and wash with N,N-dimethylformamide to remove unreacted raw materials to obtain tumor microenvironment responsive nanomedicine: PCN-PK / FA.
[0013] Furthermore, in step 1, the mass ratio of benzoic acid, methyl-tetra(4-carboxyphenyl)porphyrin, and ferric chloride hexahydrate is 101:41:1124.
[0014] Furthermore, in step 1, the centrifugation speed is 12000rpm~15000rpm, and the centrifugation time is 15min.
[0015] Furthermore, in step 2, the mass ratio of PCN(Fe), PK11195, and polyethylene glycol / folic acid is 1:1:1.
[0016] Furthermore, in step 3, the centrifugation speed is 10000~12000 rpm, and the centrifugation time is 12 min.
[0017] This invention also provides the use of the aforementioned tumor microenvironment-responsive nanomedicines in the preparation of drugs for treating hepatocellular carcinoma. This invention constructs tumor microenvironment-responsive nanomedicines based on metal-organic frameworks using hydrothermal and one-pot methods. These nanomedicines comprise an iron-based metal-organic framework, polyethylene glycol / folate (PEG / FA) modified onto the framework, and a mitochondrial transporter protein (TSPO) inhibitor (PK11195) encapsulated within the metal framework. The nanomedicines of this invention can actively target tumor cells by binding to folate receptors. Under MRI guidance, by actively targeting tumor cells through binding to folate receptors, they generate abundant ROS under near-infrared light irradiation, effectively inducing cellular ICD. Simultaneously, the released PK11195 inhibits Nrf2 and PD-L1 expression. The responsive release of PK11195 can simultaneously inhibit the expression of Nrf2 and PD-L1, thereby inhibiting TSPO and improving therapeutic efficacy, thus enhancing the efficiency of ICD and anti-PD-L1 mediated immunotherapy.
[0018] Compared with existing technologies, the technical effects of this invention are positive and significant. In the treatment of hepatocellular carcinoma, the combination of tumor microenvironment-responsive nanomedicine-mediated enhanced photodynamic therapy (PDT) and aPD-L1-based immunotherapy has the potential to reshape the immunosuppressive tumor microenvironment (TME), thereby doubly improving the efficiency of immunotherapy and eliminating HCC cells, providing a promising prospect for promoting systemic antitumor photodynamic therapy and immunotherapy. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of the design route of the present invention. In the diagram, Near Infrared irradiation refers to near-infrared laser irradiation, Ferrotosis means ferroptosis, aPD-L1 is PD-L1 antibody, ICD is immunogenic cell death, and DAMP is damage-related molecular model.
[0020] Figure 2 This is a transmission electron microscope (TEM) image of the metal-organic framework (PCN(Fe)) in Example 1.
[0021] Figure 3 This is a transmission electron microscope image of the tumor microenvironment-responsive nanomedicine in Example 1.
[0022] Figure 4 This is the UV-Vis absorption spectrum of the tumor microenvironment responsive nanomedicine in Example 1.
[0023] Figure 5 This is an electron microscopy distribution image of the tumor microenvironment-responsive nanomedicine in Example 1.
[0024] Figure 6 This is the XPS measurement spectrum of the tumor microenvironment-responsive nanomedicine in Example 1.
[0025] Figure 7 This is a graph showing the reactive oxygen species (ROS) generation content of the tumor microenvironment-responsive nanomedicine in Example 1; where, Figure 7 A in the figure represents the change in free radical generation over time under 650 nm laser irradiation alone. Figure 7 B in the figure represents the change in free radical generation of PCN-PK / FA dispersion over time in the dark. Figure 7 C in the figure represents the change in free radical generation in PCN-PK / FA dispersion over time under 650 nm laser irradiation. Figure 7 D in the figure represents the curve of free radical generation changing over time under different conditions.
[0026] Figure 8 This is the ESR spectrum of the reactive oxygen species detection for the tumor microenvironment responsive nanomedicine in Example 1.
[0027] Figure 9 This is a magnetic resonance imaging (MRI) image of the tumor microenvironment-responsive nanomedicine in Example 1.
[0028] Figure 10 The extent to which HCCLM3 hepatocellular carcinoma cells produce reactive oxygen species after different treatments with nanomedicines or different components that respond to the tumor microenvironment.
[0029] Figure 11 Cellular uptake rate of HCCLM3 hepatocellular carcinoma cells after different treatments with nanomedicines or different components at different concentrations in response to the tumor microenvironment.
[0030] Figure 12 Survival rate of hepatocellular carcinoma cells HCCLM3 after different treatments with nanomedicines or different components that respond to tumor microenvironment at different concentrations.
[0031] Figure 13 The degree of apoptosis in hepatocellular carcinoma cells HCCLM3 after different treatments with nanomedicines or different components at different concentrations in response to the tumor microenvironment.
[0032] Figure 14 Magnetic resonance imaging (MRI) of mice after treatment with nano-responsive drugs or ferric chloride for different time periods in the tumor microenvironment.
[0033] Figure 15 Tumor images 15 days after treatment with nanomedicines or different components that respond to the tumor microenvironment.
[0034] Figure 16 Changes in tumor volume after treatment with nanomedicines or different components in response to the tumor microenvironment.
[0035] Figure 17 Tumor weight after 15 days of treatment with nanomedicines or different components that respond to the tumor microenvironment.
[0036] Figure 18 Changes in mouse body weight over 15 days of treatment with nanomedicines or different components that respond to the tumor microenvironment.
[0037] Figure 19 Immunohistochemical staining results of HE, ROS, and CD8 in tumor tissues after treatment with nanomedicines or different components to respond to the tumor microenvironment. Detailed Implementation
[0038] 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.
[0039] Example 1: Preparation of tumor microenvironment-responsive nanomedicines
[0040] Step 1:
[0041] 404 mg benzoic acid, 164 mg TCPP (m-tetra(4-carboxyphenyl)porphyrin) and 4496 mg ferric chloride hexahydrate were dissolved in DMF (N,N-dimethylformamide). The mixture was stirred at 90 °C for 5 h. The mixture was then centrifuged at 12000 rpm for 15 min. Unreacted raw materials were removed by washing with N,N-dimethylformamide (DMF) to obtain the metal-organic framework PCN(Fe). PCN(Fe) is a metal-organic framework (MOF) material that is formed by the self-assembly of iron metal ions and organic ligands through coordination bonds to form a three-dimensional porous structure.
[0042] Step 2:
[0043] 5 mg of PCN(Fe) was mixed with 5 mg of PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxylic acid; purchased from MCE) and 5 mg of PEG / FA (carboxylated polyethylene glycol folic acid; purchased from Shanghai Tuoyang Biotechnology Co., Ltd.) and dissolved in DMF. The mixture was stirred at room temperature for 24 h. The solution was centrifuged at 1000 rpm for 12 min, and unreacted raw materials were removed by washing with DMF to obtain the tumor microenvironment responsive nanomedicine: PCN-PK / FA.
[0044] like Figures 1-9 As shown, PCN(Fe) exhibits a classic spindle structure, and the final product framework did not collapse after modification. Ultraviolet light analysis reveals that the structure remained unchanged during modification, and characteristic signals were preserved. XPS and mapping both detected C, N, O, Fe, and Cl elemental signals, with the elemental distribution matching expectations. Furthermore, PCN-PK / FA can effectively generate reactive oxygen species and possesses MRI imaging potential.
[0045] Example 2: Production of reactive oxygen species in the body
[0046] HCCLM3 tumor cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in 6-well plates and randomly divided into four groups (PBS group, PCN(Fe)+Light (L) group, PCN-PK / FA group, and PCN-PK / FA+Light (L) group). Cells in each group were incubated with the corresponding materials and then subjected to laser treatment. Twenty-four hours after laser treatment, cells were stained with DCFH-DA in the dark for 30 minutes. Subsequently, laser confocal microscopy was used to analyze reactive oxygen species (ROS) production.
[0047] like Figure 10 The experimental results show that PCN-PK / FA can effectively generate ROS under laser excitation and significantly increase the level of intracellular oxidative stress.
[0048] Example 3: Tumor cell uptake capacity
[0049] HCCLM3 tumor cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded in confocal dishes, with each dish containing 2 × 10⁶ cells. 4 Cells were cultured in 1 mL of DMEM high-glucose medium. After 24 hours of culture, hepatocellular carcinoma cells were treated with different concentrations of PCN-PK / FA (loaded with Rhodamine B) for another 4 hours, followed by washing with PBS. Cells were then fixed with 4% paraformaldehyde (PFA) for 15 minutes and washed again with PBS. Subsequently, the nuclei were stained with DAPI at 37°C for 30 minutes. Finally, the cells were imaged in PBS.
[0050] like Figure 11 As shown, PCN-PK / Fe can be effectively taken up by HCCLM3.
[0051] Example 4: In vitro toxicity to tumor cells
[0052] HCCLM3 tumor cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded in 96-well plates, with 5 × 10⁶ cells per well. 3Cells were cultured in 100 μL of DMEM high-glucose medium. After 24 hours of culture, hepatocellular carcinoma cells were treated with different concentrations of PCN-PK / FA for another 24 hours, followed by light / no light treatment. Subsequently, 100 μL of DMEM high-glucose medium (without FBS) containing 10% CCK8 (v / v) was added to each well. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a spectrophotometer in the form of a standard enzyme-linked immunosorbent assay (ELISA). The experiment was repeated three times, and the cell viability percentage was calculated based on the average of each experiment.
[0053] HCCLM3 tumor cells (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in 6-well plates and randomly divided into four groups (PBS group, PCN(Fe)+Light (L) group, PCN-PK / FA group, and PCN-PK / FA+Light (L) group). Cells in each group were incubated with the corresponding materials and then subjected to laser treatment. Twenty-four hours after laser treatment, the cell culture supernatant and adherent cells were collected from each well. Cells were stained with FITC-Annexin V and propidium iodide (PI) in the dark for 20 minutes. Apoptosis was then analyzed using flow cytometry.
[0054] like Figure 12 , 13 Experimental results showed that PCN-PK / FA had a significant killing effect on HCCLM3 cells, and this effect was further enhanced under light irradiation. In vitro cell experiments demonstrated the effectiveness of the tumor microenvironment-responsive nanomedicine used in this study.
[0055] Example 5: MRI Imaging
[0056] Seven-week-old mice (Balb / c mice) were selected and cultured for one week to acclimatize. One week later, HCCLM3 tumor cells were injected into the right hind limb, with a cell concentration of approximately 10⁻⁶. 6 Individuals / animals, cultured until tumors reach approximately 100 mm. 3 Subsequently, HCCLM3 tumor-bearing Balb / c mice were injected via the tail vein with PBS, ferric chloride solution, or PCN-PK / FA solution from Example 1. T1-weighted magnetic resonance imaging (MRI) was performed at different time points after injection (0, 2, and 4 hours).
[0057] like Figure 14 As shown, PCN-PK / FA can serve as an effective T1-weighted MRI contrast agent, enabling visualization of tumor accumulation in vivo.
[0058] Example 6: In vivo therapeutic effect
[0059] Seven-week-old mice (Balb / c mice) were selected and cultured for one week to acclimatize. One week later, HCCLM3 tumor cells were injected into the right hind limb, with a cell concentration of approximately 10⁻⁶. 6 Each mouse was cultured until the tumor was about the size of a mung bean, and then treated. The mice were divided into six groups: (1) PBS, (2) PCN(Fe) + Light (abbreviated L), (3) PCN-PK / FA (Example 1), (4) aPD-L1, (5) PCN-PK / FA (Example 1) + Light (abbreviated L), and (6) aPD-L1 + PCN-PK / FA (Example 1) + Light (abbreviated L). The mice were given five doses during the treatment. Each time, the mice were given the first dose and then exposed to light on the second day. The tumor volume was measured during the treatment. After 15 days, the mice were sacrificed, and the tumor tissue was removed for observation and the tumor weight was measured.
[0060] like Figure 15-18 The experimental results showed that the HCCLM3 model group mice exhibited significantly higher anti-tumor activity than other groups after treatment, demonstrating that it can effectively induce cell death and activate the immune system to a certain extent, thereby treating hepatocellular carcinoma in mice. The mice did not experience significant weight loss during treatment, indicating biocompatibility.
[0061] Example 7: Immunohistochemical staining of HE, ROS and CD8 in tumor tissue
[0062] Tumor tissue sections from mice in each treatment group collected in Example 6 were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and adhered to anti-detachment glass slides. Subsequently, HE, ROS, and CD8 staining were performed: sections were baked, dewaxed, hydrated, stained with antibodies, dehydrated, and cleared and mounted. Figure 19 As shown, the combined treatment group was able to induce tumor cell apoptosis, resulting in severe pathological damage to tumor tissue. PCN-PK / FA combined with laser can effectively generate ROS, and can also effectively synergize PDT and immunotherapy.
[0063] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A tumor microenvironment-responsive nanomedicine, characterized in that: It includes an iron-based metal-organic framework, polyethylene glycol / folic acid modified on the iron-based metal-organic framework, and PK11195 encapsulated within the metal framework. The structural formula of PK11195 is shown below. 。 2. The tumor microenvironment-responsive nanomedicine according to claim 1, characterized in that: The tumor microenvironment-responsive drug is MRI-guided.
3. The tumor microenvironment-responsive nanomedicine according to claim 1, characterized in that: The iron-based metal-organic framework used in the tumor microenvironment responsive nanomedicine is PCN(Fe). PCN(Fe) is a metal-organic framework material that is formed by the self-assembly of iron metal ions and organic ligands through coordination bonds to form a three-dimensional porous structure.
4. The method for preparing a tumor microenvironment-responsive nanomedicine according to claim 1, characterized in that: Includes the following steps: Step 1: Benzoic acid, methyl-4-carboxyphenyl)porphyrin and ferric chloride hexahydrate are dissolved in N,N-dimethylformamide. The mixture is stirred at 80-90°C and then centrifuged. Unreacted raw materials are removed by washing with N,N-dimethylformamide to obtain metal-organic framework PCN(Fe). Step 2: Dissolve PCN(Fe) with PK11195 and polyethylene glycol / folic acid in N,N-dimethylformamide and stir at room temperature. After the reaction is complete, centrifuge the mixed solution and wash with N,N-dimethylformamide to remove unreacted raw materials to obtain tumor microenvironment responsive nanomedicine: PCN-PK / FA.
5. The method for preparing a tumor microenvironment-responsive nanomedicine according to claim 4, characterized in that: In step 1, the mass ratio of benzoic acid, methyl-tetra(4-carboxyphenyl)porphyrin, and ferric chloride hexahydrate is 101:41:1124.
6. The method for preparing a tumor microenvironment-responsive nanomedicine according to claim 4, characterized in that: In step 1, the centrifugation speed is 12000rpm~15000rpm and the centrifugation time is 15min.
7. The method for preparing a tumor microenvironment-responsive nanomedicine according to claim 4, characterized in that: In step 2, the mass ratio of PCN(Fe):PK11195:polyethylene glycol / folic acid is 1:1:
1.
8. The method for preparing a tumor microenvironment-responsive nanomedicine according to claim 4, characterized in that: In step 3, the centrifugation speed is 10000~12000 rpm and the centrifugation time is 12 min.
9. Use of the tumor microenvironment responsive nanomedicine of claim 1 in the preparation of a drug for treating hepatocellular carcinoma.