Physiological response nano-enzyme particle for catalyzing ozone by doping rare earth elements in metal organic framework and preparation method of physiological response nano-enzyme particle
By doping rare earth elements into metal organic frameworks, a physiologically responsive nanoenzyme particles were designed, which solved the problem of insufficient catalytic efficiency and selectivity in the ozone catalytic reaction of existing catalytic systems, and achieved a sensitive response to physiological mechanical pressure and efficient catalysis ensures safety and therapeutic effects in vivo application.
Patent Information
- Application Number
- CN202411580696.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-02-18
Smart Images
Figure CN119454598A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomaterials, and in particular relates to a metal organic framework doped with rare earth elements for catalyzing ozone physiological response nanozyme particles and a preparation method thereof. Background Art
[0002] Metal-organic framework (MOF) is a porous material self-assembled by metal ions or metal clusters and organic ligands through coordination bonds. Due to its highly ordered structure, large specific surface area and adjustable pore characteristics, it is widely used in piezoelectric catalysis, gas storage, separation and sensing. However, existing catalytic systems often have deficiencies in catalytic efficiency and selectivity, which limits their widespread use in medical applications. Rare earth elements have high catalytic activity and good thermal stability in catalytic reactions. Their doping can effectively regulate the electronic structure of MOF, improve the adsorption capacity and conversion efficiency of reactants, and therefore play an important role in ozone catalytic reactions. At the same time, the rise of physiologically responsive nanozyme systems has provided new ideas for disease monitoring and treatment. These systems achieve real-time monitoring and intervention by designing nanomaterials that catalyze reactions under specific physiological conditions. However, there is still a lack of sensitive piezoelectric catalytic nanozyme systems that respond to physiological weak pressure signals for physiological mechanical pressure stimulation. Although studies have been conducted to dope rare earth elements into MOF to improve its catalytic performance, further exploration is still needed in terms of catalytic efficiency and medical applications to meet the challenges in ozone catalytic reactions.
[0003] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention
[0004] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a metal organic framework doped with rare earth elements for catalyzing ozone physiological response nanozyme particles, the preparation method comprising the following steps:
[0005] (1) Hafnium tetrachloride (HfCl 4 ), 2-amino-1,4-phthalic acid (BDC-NH 2 ) was mixed with N,N-dimethylformamide (DMF) and deionized water, stirred until uniform, and then ytterbium chloride (YbCl 3 ) continue stirring until dissolved to obtain a mixture;
[0006] (2) heating the mixture for reaction and then centrifuging it, and then washing and drying the precipitate in sequence to obtain Yb-UIO-66 (a metal organic framework doped with the rare earth element ytterbium);
[0007] (3) mixing the Yb-UIO-66 with PBS buffer by ultrasonication to obtain a suspension;
[0008] (4) dissolving cholesterol and egg yolk lecithin in chloroform, adding perfluorohexane (PFD), stirring evenly and then rotary evaporating to form a lipid film; then adding the suspension to hydrate at low temperature to obtain PFD@LP-Yb-UIO-66 (liposome-encapsulated Yb-UIO-66);
[0009] (5) Continue to introduce ozone to saturate the ozone, and after completion, obtain O 3 P@LPYU (liposome-encapsulated ozone-loaded Yb-UIO-66) is a physiologically responsive nanozyme particle that catalyzes ozone by doping the metal organic framework with rare earth elements.
[0010] Preferably, in step (2), the heating reaction temperature is 120° C. and the reaction time is 18 h.
[0011] Preferably, in step (2), the centrifugation parameters are: centrifugation at 8000 rpm for 10 min at room temperature.
[0012] Preferably, the washing method is: washing with N,N-dimethylformamide and ethanol for 3 times.
[0013] Preferably, in step (2), the drying method is: vacuum drying at 60°C.
[0014] Preferably, in step (4), the low-temperature hydration temperature is 40° C. and the time is 1 hour.
[0015] Based on the same technical concept, another embodiment of the present invention is to provide a metal organic framework doped with rare earth elements obtained by the above preparation method and capable of catalyzing ozone with physiological response nanozyme particles.
[0016] The beneficial effects of the present invention are:
[0017] 1. Improvement of catalytic efficiency: Traditional piezoelectric catalytic nanozymes usually have low conversion efficiency in the catalytic process of ozone, resulting in insufficient generation of reactive oxygen. By introducing rare earth elements, the present invention aims to significantly improve the piezoelectric catalytic performance of MOF, so that it exhibits higher activity and selectivity in the ozone catalytic reaction, thereby improving the generation efficiency of reactive oxygen.
[0018] 2. Enhanced physiological response capability: Existing piezoelectric catalytic systems often lack the ability to adaptively adjust physiological conditions (such as pleural pressure), which makes them respond slowly or have unsatisfactory effects in practical applications. The present invention designs an integrated nanozyme system to enable it to sensitively adjust catalytic activity in a specific physiological environment, achieve rapid response to physiological changes, and thus improve the accuracy and timeliness of treatment.
[0019] 3. Improved safety and biocompatibility: In medical applications, the safety and biocompatibility of catalysts are crucial. By optimizing the synthesis of MOF and the doping of rare earth elements, the present invention aims to ensure that the nanozyme system does not produce significant toxicity or side effects when used in vivo, thus ensuring the safety of patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0021] Figure 1 Yes 3 Synthesis route of P@LPYU.
[0022] Figure 2 Yes 3 Piezoelectric properties detection diagram of P@LPYU.
[0023] Figure 3 Yes 3 Diagram and cell photos of P@LPYU's cell killing ability with and without mechanical pressure.
[0024] Figure 4 Yes 3 Diagram of P@LPYU piezoelectric catalytic treatment of malignant pleural effusion animal model.
[0025] Figure 5 Yes 3 P@LPYU treats malignant pleural effusion and tumors, and photos of pleural effusion under intrapleural pressure. DETAILED DESCRIPTION
[0026] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.
[0027] Example 1
[0028] This embodiment provides a method for preparing a metal organic framework doped with a rare earth element to catalyze ozone physiological response nanozyme particles, characterized in that the preparation method comprises the following steps:
[0029] (1) Synthesis of Yb-UIO-66 (Hf:Yb=3:1): In a 100 mL pear-shaped bottle, 0.206 g of hafnium tetrachloride (HfCl 4 ) and 0.155 g 2-amino-1,4-phthalic acid (BDC-NH 2 ) was mixed with 50 mL of N,N-dimethylformamide (DMF) and 140 μL of deionized water and stirred thoroughly until dissolved. Then 0.06 g of ytterbium chloride (YbCl 3 ), and continue stirring until completely dissolved. The mixture was transferred to an autoclave lined with polytetrafluoroethylene and reacted in an oil bath at 120°C for 18 hours. The product was collected by centrifugation, washed three times with DMF and ethanol, and finally dried in vacuum at 60°C to obtain Yb-UIO-66 (Hf:Yb=3:1).
[0030] (2) Preparation of PFD@LP-Yb-UIO-66: 10 mg Yb-UIO-66 was dispersed in 10 mL PBS and a stable suspension was formed by ultrasonic treatment. At the same time, 50 mg cholesterol and 150 mg egg yolk lecithin were dissolved in 10 mL chloroform, 400 μL perfluorohexane (PFD) was added, and a lipid film was formed using a vacuum rotary evaporator after stirring. After that, the PBS solution containing Yb-UIO-66 was added to the flask and hydrated at low temperature for 1 hour to form PFD@LP-Yb-UIO-66.
[0031] (3) Preparation of Ozone by Ozone Saturation 3 P@LPYU: Ozone is continuously injected into the solution obtained in step (2) until ozone is saturated, and finally O 3 P@LPYU.
[0032] O 3 The synthesis route of P@LPYU is as follows Figure 1 shown.
[0033] Example 2
[0034] (1) Synthesis of Yb-UIO-66 (Hf:Yb=2:1): In a 100 mL pear-shaped bottle, 0.183 g of hafnium tetrachloride (HfCl 4 ) and 0.155 g 2-amino-1,4-phthalic acid (BDC-NH 2 ) was mixed with 50 mL of N,N-dimethylformamide (DMF) and 140 μL of deionized water and stirred thoroughly until dissolved. Then 0.08 g of ytterbium chloride (YbCl 3), and continue stirring until completely dissolved. The mixture was transferred to an autoclave lined with polytetrafluoroethylene and reacted in an oil bath at 120°C for 18 hours. The product was collected by centrifugation, washed three times with DMF and ethanol, and finally dried in vacuum at 60°C to obtain Yb-UIO-66 (Hf:Yb=2:1).
[0035] (2) Preparation of PFD@LP-Yb-UIO-66: 10 mg Yb-UIO-66 was dispersed in 10 mL PBS and a stable suspension was formed by ultrasonic treatment. At the same time, 50 mg cholesterol and 150 mg egg yolk lecithin were dissolved in 10 mL chloroform, 400 μL perfluorohexane (PFD) was added, and a lipid film was formed using a vacuum rotary evaporator after stirring. After that, the PBS solution containing Yb-UIO-66 was added to the flask and hydrated at low temperature for 1 hour to form PFD@LP-Yb-UIO-66.
[0036] (3) Preparation of Ozone by Ozone Saturation 3 P@LPYU: Ozone is continuously injected into the solution obtained in step (2) until ozone is saturated, and finally O 3 P@LPYU.
[0037] Example 3
[0038] (1) Synthesis of Yb-UIO-66 (Hf:Yb=1:1): In a 100 mL pear-shaped bottle, 0.138 g of hafnium tetrachloride (HfCl 4 ) and 0.155 g 2-amino-1,4-phthalic acid (BDC-NH 2 ) was mixed with 50 mL of N,N-dimethylformamide (DMF) and 140 μL of deionized water and stirred thoroughly until dissolved. Then 0.12 g of ytterbium chloride (YbCl 3 ), and continue stirring until completely dissolved. The mixture was transferred to an autoclave lined with polytetrafluoroethylene and reacted in an oil bath at 120°C for 18 hours. The product was collected by centrifugation, washed three times with DMF and ethanol, and finally dried in vacuum at 60°C to obtain Yb-UIO-66 (Hf:Yb=1:1).
[0039] (2) Preparation of PFD@LP-Yb-UIO-66: 10 mg Yb-UIO-66 was dispersed in 10 mL PBS and a stable suspension was formed by ultrasonic treatment. At the same time, 50 mg cholesterol and 150 mg egg yolk lecithin were dissolved in 10 mL chloroform, 400 μL perfluorohexane (PFD) was added, and a lipid film was formed using a vacuum rotary evaporator after stirring. After that, the PBS solution containing Yb-UIO-66 was added to the flask and hydrated at low temperature for 1 hour to form PFD@LP-Yb-UIO-66.
[0040] (3) Preparation of Ozone by Ozone Saturation 3 P@LPYU: Ozone is continuously injected into the solution obtained in step (2) until ozone is saturated, and finally O 3 P@LPYU.
[0041] Verification Example
[0042] (I) In vitro experiments
[0043] Cell internalization: FITC-labeled O 3 P@LPYU was added to LLC and 4T1 cells, and the cellular internalization levels at different time points were analyzed by flow cytometry.
[0044] Cytotoxicity assay (CCK8 method): LLC and 4T1 cells were seeded in 96-well plates, with 1500 cells per well. After culturing for 24 hours, different concentrations of O 3 P@LPYU. For the mechanical pressure stimulation group, a reciprocating plunger pump was used to pressurize the piezoelectric material to simulate pleural pressure (frequency 120 times / min, pressure 2 kPa). After 24 hours of culture, 10 μL of CCK-8 reagent was added, and the absorbance was measured after 2 hours of incubation.
[0045] Colony formation assay: LLC and 4T1 cells were seeded in 6-well plates with 5000 cells per well and cultured for 14 days after different treatment conditions. They were washed with PBS, fixed with 4% paraformaldehyde and stained with 0.05% crystal violet.
[0046] Detection of reactive oxygen species (ROS) levels: The DCFH-DA probe was used to determine the intracellular ROS level. LLC and 4T1 cells were seeded in 24-well plates and treated according to the treatment steps of the CCK8 experiment. DCFH-DA (5 μM) was then added and observed using a confocal laser scanning microscope after incubation for 30 minutes.
[0047] Apoptosis analysis: LLC and 4T1 cells were seeded in 12-well plates. After different treatments, Annexin V-FITC / PI double staining was used to detect cell apoptosis by flow cytometry.
[0048] Tumor immunogenic death detection: ICD level was evaluated by detecting three indicators: CRT membrane translocation (immunofluorescence method), HMGB1 secretion (ELISA method) and ATP release (luciferase method).
[0049] Depend on Figure 2 and Figure 3 It can be seen that: in the absence of mechanical pressure, O 3P@LPYU showed certain cytotoxicity, which may be due to the effects of ozone and nanomaterials themselves on tumor cell growth, while mechanical pressure stimulation enhanced the O 3 The cell-killing effect of P@LPYU may be due to the piezoelectric catalytic system generating reactive oxygen species under the action of mechanical pressure, which kills tumor cells. 3 The generation of reactive oxygen species in tumor cells in the P@LPYU group increased significantly, and tumor cells died immunogenically. 3 P@LPYU generates reactive oxygen species through piezoelectric catalysis, which significantly enhances its killing effect on tumor cells and leads to immunogenic death of tumor cells.
[0050] (II) In vivo experiments
[0051] Establishment of malignant pleural effusion model: Make a hole at the bottom of a 50 ml centrifuge tube and insert enough clean cotton from the tube mouth to block the hole at the bottom. The tube is used for mouse anesthesia and needs to be sterilized before use. Prepare a clean and simple operating table, such as a foam board sterilized with ultraviolet light. Place the mouse near the tube with its head facing the tube mouth, pull its tail, and use the mouse's nature, it will enter the tube by itself. Fix the mouse in the tube and inject 300 microliters of isoflurane into the cotton through the hole at the bottom of the tube with a syringe (subsequent mice only need to inject 100 microliters to maintain the amount of isoflurane in the cotton). The mouse will be anesthetized quickly and then moved to the operating table. During the operation, the tube should be kept covering the mouse's head to maintain anesthesia. Shave the hair on the anterior chest wall of the mouse to fully expose the skin. Disinfect the puncture site (right chest wall) with an alcohol cotton swab and use a 26-gauge intravenous catheter to puncture the right pleural cavity through the 10th or 11th intercostal space along the mid-axillary line. The resistance will suddenly disappear when entering the pleural cavity. Remove the needle stylet and inject 50 μl LLC-Luc (1.2×10 5 ) or 4T1-Luc (1.2×10 5 ) cell suspension. After injection, the catheter was removed and local pressure was applied to stop bleeding and prevent leakage. Bioluminescence imaging or ultrasound was used to track the progression of malignant pleural effusion (MPE).
[0052] In vivo evaluation of therapeutic effects: The mice randomly divided into groups were injected with 50 μL of nanomaterial suspension (concentration of 10 mg / mL) through the pleural cavity under anesthesia, and the control group was injected with PBS. The changes of tumors and pleural effusion before and after treatment were monitored by IVIS, and the mice were dissected after the treatment to measure the volume of pleural effusion and collect tumor tissue for analysis.
[0053] Depend on Figure 4 and Figure 5It can be seen that the animal model of malignant pleural effusion was successfully established by injecting therapeutic cells into the pleural cavity. Based on the above model, after intrapleural injection of the nanozyme system, obvious biodistribution was observed in the pleural effusion and tumor. Due to the presence of ozone substrate and the rich hydrogen peroxide substrate in the oxygen-rich environment of the pleural cavity, O 3 P@LPYU particles can produce a large amount of reactive oxygen species by piezoelectric catalysis under the action of intrapleural pressure, thereby effectively reducing the volume of malignant pleural effusion, persistently inhibiting the growth of pleural and lung parenchymal tumors, and prolonging the survival time of mice. 3 The P@LPYU nanozyme system, which uses piezoelectric catalysis to generate reactive oxygen species, significantly reduced the volume of pleural effusion, inhibited tumor growth and prolonged the survival time of mice.
[0054] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A method for preparing a physiologically responsive nanozyme particle doped with a metal organic framework and catalyzing ozone with rare earth elements, characterized in that: The preparation method comprises the following steps: (1) mixing hafnium tetrachloride, 2-amino-1,4-phthalic acid, N,N-dimethylformamide and deionized water, stirring until uniform, and then adding ytterbium chloride and continuing to stir until dissolved to obtain a mixture; (2) heating the mixture for reaction and then centrifuging it, and then washing and drying the precipitate in sequence to obtain Yb-UIO-66; (3) mixing the Yb-UIO-66 with PBS buffer by ultrasonication to obtain a suspension; (4) dissolving cholesterol and egg yolk lecithin in chloroform, adding perfluorohexane, stirring evenly and then rotary evaporating to form a lipid film; then adding the suspension to hydrate at low temperature to obtain PFD@LP-Yb-UIO-66; (5) Ozone is continuously introduced to achieve ozone saturation, and upon completion, O3P@LPYU is obtained, which is the physiologically responsive nanozyme particle that catalyzes ozone by doping the metal organic framework with rare earth elements.
2. The method for preparing the physiologically responsive nanozyme particles doped with rare earth elements to catalyze ozone according to claim 1, characterized in that: In step (2), the heating reaction temperature is 120° C. and the reaction time is 18 h.
3. The method for preparing the physiologically responsive nanozyme particles doped with rare earth elements to catalyze ozone according to claim 1, characterized in that: In step (2), the centrifugation parameters are: centrifugation at 8000 rpm for 10 min at room temperature.
4. The method for preparing the physiologically responsive nanozyme particles doped with rare earth elements to catalyze ozone according to claim 1, characterized in that: In step (2), the washing method is: washing with N,N-dimethylformamide and ethanol for 3 times.
5. The method for preparing the physiologically responsive nanozyme particles of metal organic framework doped with rare earth elements for catalyzing ozone according to claim 1, characterized in that: In step (2), the drying method is: vacuum drying at 60°C.
6. The method for preparing the physiologically responsive nanozyme particles doped with rare earth elements to catalyze ozone according to claim 1, characterized in that: In step (4), the low-temperature hydration temperature is 40° C. and the time is 1 hour.
7. Physiologically responsive nanozyme particles that catalyze ozone and are doped with rare earth elements and obtained by the preparation method according to any one of claims 1 to 6.