Phthalocyanine-MOF hybrid sonodynamic material and application thereof in immunotherapy of liver cancer
By combining phthalocyanine-MOF hybrid sonodynamic materials with ultrasound, the problems of poor water solubility and insufficient immune microenvironment reprogramming in sonodynamic therapy in the treatment of HCC were solved, efficient ROS generation and immune microenvironment reprogramming were achieved, and the treatment effect of liver cancer was improved.
Patent Information
- Application Number
- CN202511183962.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-10-10
AI Technical Summary
Existing sonodynamic therapy for the treatment of hepatocellular carcinoma (HCC) has problems such as poor water solubility of sonosensitizers, low ROS generation efficiency, inability to reverse the hypoxic microenvironment, and lack of immune microenvironment reprogramming ability, resulting in poor treatment effects.
A phthalocyanine-MOF hybrid sonodynamic material was developed by modifying the surface of the Zr-MOF carrier with a PEG layer and loading Zn-Pc to form Pc@Zr-MOF. Ultrasonic activation was combined with ROS to produce the material, which was then used in combination with immune checkpoint inhibitors to reprogram the immune microenvironment.
It achieves efficient ROS generation, directly kills tumor cells while reprogramming the immune microenvironment, increasing CD8+ T cell infiltration and the proportion of M1 macrophages, and establishing a long-lasting anti-tumor immune response. It is suitable for unresectable HCC.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a phthalocyanine-MOF hybrid sonodynamic material and a liver cancer immunotherapy application thereof. BACKGROUND
[0002] Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and high mortality worldwide. Most patients have lost the opportunity for surgery at the time of diagnosis. Existing treatment methods such as radiofrequency ablation and transcatheter arterial chemoembolization (TACE) have problems such as large damage to normal liver tissue and inaccurate therapeutic effect. In recent years, immunotherapy has made progress in the treatment of HCC, but due to tumor heterogeneity and hypoxia-induced immunosuppressive microenvironment, the treatment effect varies significantly. Sonodynamic therapy (SDT) is a non-invasive precise treatment method, which kills tumor cells by generating reactive oxygen species (ROS) under the action of ultrasound through a sonosensitizer, but its clinical application is limited by the poor water solubility of the sonosensitizer, low ROS generation efficiency and inability to effectively reverse the immunosuppressive microenvironment.
[0003] At present, metal organic frameworks (MOF) and phthalocyanine compounds (such as zinc phthalocyanine, Zn-Pc) are respectively studied as a sonosensitizer carrier and a sonosensitizer, but MOF has problems of poor biocompatibility and insufficient water solubility, and phthalocyanine compounds are limited in application due to poor light stability and biological toxicity; in addition, the existing SDT technology cannot overcome the treatment resistance caused by the hypoxic microenvironment of HCC, and lacks the ability to reprogram the immune microenvironment. Therefore, in view of the above status, it is urgent to develop a phthalocyanine-MOF hybrid sonodynamic material and a liver cancer immunotherapy application thereof to overcome the deficiencies in current practical applications. SUMMARY
[0004] The present application aims to provide a phthalocyanine-MOF hybrid sonodynamic material and a liver cancer immunotherapy application thereof to solve the problems raised in the background.
[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:
[0006] A phthalocyanine-MOF hybrid sonodynamic material, comprising a Zr-MOF carrier, a surface modified PEG layer and Zn-Pc loaded in the pore channel of the Zr-MOF, wherein:
[0007] The loading efficiency of Zn-Pc is 34%;
[0008] The particle size of the material is 100 nm, and the PDI is less than 0.2.
[0009] As a further solution of the present application, the PEG modification layer is DSPE-PEG, and the modification amount of the DSPE-PEG is 5wt% of the mass of the Zr-MOF.
[0010] A method for preparing the above-mentioned phthalocyanine-MOF hybrid sonodynamic material comprises the following steps:
[0011] (1) Synthesis of Zr-MOF support: H2TCPP, ZrOCl2·8H2O, and benzoic acid were dissolved in DMF, reacted at 90°C for 5 h, and centrifuged and washed to obtain Zr-MOF nanoparticles;
[0012] (2) PEG modification: The Zr-MOF obtained in step (1) was incubated with DSPE-PEG in PBS for 24 h and purified to obtain PEG-Zr-MOF;
[0013] (3) Loading zinc phthalocyanine: PEG-Zr-MOF and Zn-Pc were stirred in deionized water for 24 hours, and free Zn-Pc was removed by centrifugation to obtain the phthalocyanine-MOF hybrid sonodynamic material.
[0014] As a further solution of the present invention: in step (1), the mass ratio of H2TCPP, ZrOCl2·8H2O and benzoic acid is 1:3:29.
[0015] The phthalocyanine-MOF hybrid sonodynamic material is used to prepare a sonodynamic drug for hepatocellular carcinoma. The material is injected into a hepatocellular carcinoma model via the tail vein at a dose of 5 mg / kg. Twelve hours after the injection, the tumor site is irradiated with ultrasound at the following parameters: frequency 1 MHz, intensity 1.5 W / cm 2 , irradiation time 10 minutes.
[0016] As a further solution of the present invention: the ultrasonic irradiation activates the phthalocyanine-MOF hybrid sonodynamic material to produce ROS, and simultaneously achieves the following immune microenvironment reprogramming:
[0017] (1) The proportion of M1 macrophages in tumor tissue increased to more than 2.5 times that of M2 macrophages;
[0018] (2)CD8 + The T cell infiltration ratio increased to more than 25%.
[0019] As a further embodiment of the present invention: the sonodynamic therapy drug is used in combination with an immune checkpoint inhibitor.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1. Efficient ROS generation: The ROS production rate of Pc@Zr-MOF under ultrasound is significantly higher than that of existing sonosensitizers, effectively reversing the hypoxic microenvironment. Pc@Zr-MOF (experimental group) and commonly used sonosensitizers in clinical practice (such as protoporphyrin IX, ZnPc, and TiO2 nanoparticles) were compared under the same ultrasound parameters (1 MHz, 1.5 W / cm 2 ) were used to test the ROS production. Electron spin resonance (ESR) was used to measure the hydroxyl radical (·OH) signal intensity, and the characteristic peak intensity of Pc@Zr-MOF (au2850±120) was significantly higher than that of the control group (protoporphyrin IX: 850±60; ZnPc: 1050±80).
[0022] 2. Dual action mechanism: Directly kills tumor cells while reprogramming the immune microenvironment, transforming "cold tumors" into "hot tumors";
[0023] 3. High biocompatibility: PEG modification and Zr-MOF carrier reduce the toxicity of phthalocyanine, and the hemolysis rate is <5%;
[0024] 4. Long-lasting immune memory: Establishing a lasting anti-tumor immune response by activating M1 macrophages and CD8^+^T cells;
[0025] 5. Clinical application potential: Suitable for unresectable HCC and can be used in combination with immune checkpoint inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Schematic diagram of the synthesis and anti-liver cancer effect of phthalocyanine-MOF hybrid materials in an embodiment of the present invention;
[0027] Among them, a is a schematic diagram of the synthesis of phthalocyanine-MOF hybrid material Pc@Zr-MOF; b is a schematic diagram of the in vivo action of Pc@Zr-MOF.
[0028] Figure 2 Schematic diagram of the analysis of low-responsiveness subgroups to liver cancer immunotherapy and characterization of phthalocyanine-MOF hybrid materials in an embodiment of the present invention;
[0029] Among them, a is a schematic diagram of the analysis of low-responsiveness subpopulations to liver cancer immunotherapy; b and c are single-cell sequencing analysis and identification of low-responsiveness subpopulations to liver cancer immunotherapy; e and f are transmission electron microscopy and element distribution-related characterizations of Pc@Zr-MOF; g and h are Pc@Zr-MOF particle size analysis and zeta potential analysis.
[0030] Figure 3 Schematic diagram of the spectral characterization and anti-liver cancer cell experimental results based on the phthalocyanine-MOF hybrid material Pc@Zr-MOF in an embodiment of the present invention;
[0031] Wherein, a-c are XRD, Fourier infrared spectrum, magnetic resonance hydrogen spectrum analysis of Pc@Zr-MOF and its derivatives; d is a schematic diagram of intracellular action of Pc@Zr-MOF; e and f are cell phagocytosis experiment of Pc@Zr-MOF labeled with cy5.5 and fluorescence quantification thereof; g is comparison of ROS content generated by ultrasound under different groups; h and i are cell live and dead experiment and quantification thereof; j is concentration-dependent cell activity experiment; k and l are ROS generation experiment and fluorescence quantification thereof of DCF probe labeling, size: 50 μm.
[0032] Figure 4 It is a schematic diagram of the in vivo sonodynamic therapy effect of Pc@Zr-MOF on HCC in the embodiment of the present application.
[0033] Wherein, a is a schematic diagram of the treatment process of C57 mouse liver cancer xenograft tumor model; b-d are main treatment results including tumor volume monitoring, tumor mass measurement and mouse weight measurement; e is tumor tissue immunohistochemical analysis, including Tunel and Ki-67 staining, size: 200 μm; f and g are comparison of immunofluorescence and quantification of tumor tissue after sonodynamic therapy of Pc@Zr-MOF solution with direct ultrasound treatment of PBS, size: 200 μm; h is a schematic diagram of possible cytological mechanism of tumor killing and obtaining persistent immunity.
[0034] Figure 5 It is a single cell RNA sequencing result schematic diagram of the in vivo sonodynamic therapy of Pc@Zr-MOF on HCC in the embodiment of the present application.
[0035] Wherein, a is a schematic diagram of tissue digestion and single cell sequencing after treatment of C57 mouse liver cancer xenograft tumor model; b and c are schematic diagrams of tsne distribution and cell proportion of each cell subpopulation before and after treatment; d is a volcano plot of bulk differential gene analysis; e and f are tsne distribution and marker gene distribution heat map of tumor cell subpopulation. DETAILED DESCRIPTION
[0036] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.
[0037] The specific implementation of the present application will be described in detail in combination with specific embodiments.
[0038] Please refer to Figure 1-Figure 5The synergistic sonodynamic therapy (SDT) based on phthalocyanine-MOF hybrid material (Pc@Zr-MOF) provided in the embodiments of the present invention is used for the treatment of hepatocellular carcinoma (HCC) and reprogramming of the immunosuppressive microenvironment. By optimizing material design, ultrasound activation mechanism and immune regulation, it achieves efficient tumor killing and lasting anti-tumor immunity.
[0039] Example 1: Preparation of Pc@Zr-MOF hybrid materials ( Figure 1 a);
[0040] Step 1: Zr-MOF synthesis;
[0041] Tetracarboxylic acid phenylporphyrin (H2TCPP) (100 mg), ZrOCl2·8H2O (300 mg) and benzoic acid (2.9 g) were dissolved in 100 mL N,N-dimethylformamide (DMF) and stirred at 90 °C for 5 hours. H2TCPP was used as a tetracarboxylic acid organic ligand to react with ZrOCl2·8H2O through the carboxyl group (-COOH). 4+ Form Zr-OC coordination bond and build MOF skeleton. ZrOCl2·8H2O provides Zr 4+ The metal node coordinates with H2TCPP to self-assemble to form Zr-MOF. Benzoic acid as a crystal growth regulator: ① Competitive binding of Zr 4+ , inhibiting excessive MOF growth; ② adjusting the reaction pH to optimize crystallinity. According to this ratio, the resulting particle size is approximately in the range of 100-200 nanometers, obtaining a suitable loading pore size.
[0042]
[0043] The precipitate was collected by centrifugation and washed three times with DMF to obtain Zr-MOF nanoparticles;
[0044] Step 2: PEG modification;
[0045] Zr-MOF and DSPE-PEG (5wt%) were incubated in phosphate buffered saline (PBS) for 24 hours, and PEG-Zr-MOF was obtained after purification; DSPE-PEG was embedded in the MOF surface through the hydrophobic end (DSPE), and the hydrophilic end (PEG) extended outward to form a hydration layer, thereby improving the biocompatibility of aqueous systems.
[0046] Step 3: Zn-Pc loading;
[0047] Single-cell sequencing technology was used to screen the subpopulation of HCC tissue with low response to PD-L1 immunomonoclonal antibody therapy ( Figure 2 ac), based on the influence of ROS dynamics on tumor cell differentiation, the Zn-Pc loading scheme was determined to regulate the HCC tumor microenvironment ( Figure 2d). PEG-Zr-MOF (10 mg) and Zn-Pc (10 mg) were stirred in 5 mL of deionized water for 24 h, and the free Zn-Pc was removed by centrifugation to obtain Pc@Zr-MOF.
[0048] Example 2: Material structure and performance characterization;
[0049] 1. Material composition and structure;
[0050] Pc@Zr-MOF is composed of the following core components ( Figure 2 e):
[0051] Zr-MOF carrier: It is synthesized by reacting ZrOCl2·8H2O with H2TCPP (tetracarboxyphenylporphyrin) in DMF, and has high porosity and loading capacity; Zr-MOF is based on the UiO-66 structure, and its chemical formula is [Zr6O4(OH)4(TCPP)3], with the following characteristics: Topological structure: Zr6O4(OH)4 metal clusters connected with H2TCPP ligands form a three-dimensional porous framework. Coordination mode: Each Zr 4+ Coordinated with 8 oxygen atoms (4 from H2TCPP carboxyl groups and 4 from μ3-O / OH bridges). Structural stability: Due to the high bond energy of Zr-O bonds (~800kJ / mol), UiO-66 is stable in water, PBS, and acidic environments (pH 3-10). Specific surface area (m 2 / g) is about 1180±50, and the pore size (nm) is about 1.2±0.2, which provides enough space for loading Zn-Pc.
[0052] PEG modification layer: DSPE-PEG is used to modify the Zr-MOF surface, significantly improving the water solubility and biocompatibility of the material (hemolysis rate <5%).
[0053] Zinc phthalocyanine (Zn-Pc) loading: Zn-Pc is embedded in the Zr-MOF pores through π-π stacking and coordination, with a loading efficiency of 34% (Zn content determined by ICP-OES, loading efficiency = (Zn-Pc mass in the support / initial Zn-Pc mass) × 100%).
[0054] Structural features (such as Figure 2 fh):
[0055] The particle size is approximately 100 nm (DLS detection) and the distribution is uniform (polydispersity index (PDI) < 0.2);
[0056] Element distribution (TEM-EDS): Zr (MOF skeleton), Zn (Pc center), and O (coordination bond) are uniformly dispersed;
[0057] Crystal structure (XRD): characteristic peaks at 7.5° and 12.3° appeared, which proved the MOF structure integrity Figure 3 a). Meanwhile, new characteristic peaks appeared in both infrared spectrum and magnetic resonance hydrogen spectrum, which proved the loading of Zn-Pc Figure 3 b).
[0058] Example 3: In vitro sonodynamic therapy effect verification
[0059] (1) Cell uptake experiment: Hepa1-6 cells were incubated with Cy5.5-labeled Pc@Zr-MOF for 4 hours, and fluorescence microscopy showed that the uptake efficiency was >80% (vs. 35% for free Zn-Pc) Figure 3 d-f);
[0060] (2) SDT killing experiment: CCK-8 experiment showed that the cell survival rate of the ultrasound group (1.5 W / cm 2 , 5 minutes) was <20% (control group >90%) Figure 3 g-j);
[0061] (3) ROS generation mechanism:
[0062] Under the action of ultrasound (1 MHz, 1.5 W / cm 2 ) for 10 minutes, Pc@Zr-MOF triggered the separation of electron-hole pairs through the piezoelectric effect, catalyzing O2 to generate singlet oxygen 1 (O2) and hydroxyl radicals (·OH) (verified by DCFH-DA fluorescent probe) Figure 3 k, l);
[0063] DFT calculation showed that the Zr site of Zr-MOF cooperated with the conjugated structure of Zn-Pc to reduce the energy barrier, and the ROS yield was increased by 3.2 times (vs. free Zn-Pc).
[0064] Example 4: In vivo anti-tumor and immune reprogramming effects (as shown in Figure 4 ), the overall biological effect schematic diagram is shown in Figure 1 b;
[0065] (1) Animal model construction: C57BL / 6 mice were subcutaneously inoculated with Hepa1-6 cells (tumor volume ~100mm 3 ) Figure 4 a);
[0066] (2) Treatment plan: Pc@Zr-MOF (5 mg / kg) was injected into the tail vein, and the tumor site was irradiated with ultrasound (10 minutes) 12 hours later. 5 mg / kg achieved the best balance between high tumor inhibition and safety (no weight loss, liver toxicity), while 10 mg / kg had similar efficacy but caused an increase in ALT. In vitro ROS kinetics showed that ROS generation reached a peak (260 a.u.) at 10 minutes, and only increased by 3.8% after 15 minutes, but cytotoxicity was significantly increased. In vivo thermal imaging confirmed that 10 minutes could increase the local temperature of the tumor to 41.5°C (enhancing the SDT effect) while avoiding the risk of overheating of normal tissue (<42°C).
[0067] (3) Efficacy evaluation: After 14 days, the tumor volume inhibition rate was 87.9% (vs. 15% of the PBS group), and the tumor weight was <0.1 g (1.2 g of the control group) ( Figure 4 bd).
[0068] (4) Immune microenvironment analysis Figure 5 );
[0069] Flow cytometry: CD8 in tumor tissue + The proportion of T cells increased from 5% to 25%;
[0070] scRNA-seq: The proportion of M1 type (high expression of IL-12 and TNF-α) increased by 2.5 times, and the proportion of M2 type (CD163 + ) reduced by 60% ( Figure 5 ac);
[0071] Discovery of CD8 + The expression ratio of T cell subsets (high expression of GZMB and IFN-γ) increased. Key gene expression analysis showed that the expression of GZMB and IFN-γ in the effector subset increased by 15 times and 12 times respectively (p < 0.0001), and the expression of TCF7 in the stem cell-like subset increased by 8 times (p = 0.00067) ( Figure 5 d) These subsets collectively mediate the therapeutic effect of a 5-fold increase in CD8+ T cell infiltration. Effector cells directly kill tumors, stem-like cells maintain long-term immunity, and depletion of precursor cells provides a new target for combination immunotherapy.
[0072] T cell activation: CD8 + T cell infiltration increased 4.1-fold (immunofluorescence verification), Tregs (FoxP3 + ) by 70%;
[0073] Enhanced antigen presentation: CD80 / CD86 expression on the surface of dendritic cells (DCs) is upregulated, promoting T cell priming.
[0074] Changes in differentiation of tumor cell subpopulations: Tumor cells showed high reactivity, and the low reactivity subpopulation was significantly reduced (reduced by 72%) ( Figure 5 e,f).
[0075] It should be noted that, in the present invention, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.
Claims
1. A phthalocyanine-MOF hybrid sonodynamic material, characterized in that: The invention comprises a Zr-MOF carrier, a surface-modified PEG layer, and Zn-Pc loaded in the Zr-MOF pores, wherein: The Zn-Pc loading efficiency is 34%; The material particle size is 100nm and PDI<0.
2.
2. The phthalocyanine-MOF hybrid sonodynamic material according to claim 1, characterized in that The PEG modified layer is DSPE-PEG, and the modification amount of the DSPE-PEG is 5 wt% of the mass of the Zr-MOF.
3. A method for preparing the phthalocyanine-MOF hybrid sonodynamic material according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of Zr-MOF support: H2TCPP, ZrOCl2·8H2O, and benzoic acid were dissolved in DMF, reacted at 90°C for 5 h, and centrifuged and washed to obtain Zr-MOF nanoparticles; (2) PEG modification: The Zr-MOF obtained in step (1) was incubated with DSPE-PEG in PBS for 24 h and purified to obtain PEG-Zr-MOF; (3) Loading zinc phthalocyanine: PEG-Zr-MOF and Zn-Pc were stirred in deionized water for 24 hours, and free Zn-Pc was removed by centrifugation to obtain the phthalocyanine-MOF hybrid sonodynamic material.
4. The method for preparing the phthalocyanine-MOF hybrid sonodynamic material according to claim 3, characterized in that: In step (1), the mass ratio of H2TCPP, ZrOCl2·8H2O and benzoic acid is 1:3:
29.
5. Use of the phthalocyanine-MOF hybrid sonodynamic material according to claim 1 in the preparation of a sonodynamic therapeutic drug for hepatocellular carcinoma, characterized in that: The material was injected into the hepatocellular carcinoma model via tail vein at a dose of 5 mg / kg. Ultrasound irradiation was applied to the tumor site 12 hours after injection. The ultrasound parameters were: frequency 1 MHz, intensity 1.5 W / cm 2 , irradiation time 10 minutes.
6. Use of the phthalocyanine-MOF hybrid sonodynamic material according to claim 5 in the preparation of sonodynamic therapeutic drugs for hepatocellular carcinoma, characterized in that: The ultrasonic irradiation activates the phthalocyanine-MOF hybrid sonodynamic material to generate ROS, and simultaneously achieves the following immune microenvironment reprogramming: (1) The proportion of M1 macrophages in tumor tissue increased to more than 2.5 times that of M2 macrophages; (2)CD8 + The T cell infiltration ratio increased to more than 25%.
7. Use of the phthalocyanine-MOF hybrid sonodynamic material according to claim 6 in the preparation of sonodynamic therapeutic drugs for hepatocellular carcinoma, characterized in that: The sonodynamic therapy drug is used in combination with an immune checkpoint inhibitor.