MOF-based calcium ion nano regulator as well as preparation method and application thereof

Abstract

The invention discloses an MOF-based calcium ion nano regulator as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, preparing PCN-224; 2, preparing PCN-LA, wherein the PCN-LA is obtained by loading L-arginine (LA) on the surface of the nano material obtained in the step 1; 3, preparing PCN-LA-coated CaCO3, wherein the PCN-LA-coated CaCO3 is obtained by wrapping calcium carbonate (CaCO3) on the surface of the nano material obtained in the step 2; and 4, preparing the MOF-based calcium ion nano regulator: continuously modifying folic acid (FA) on the surface of the nano material obtained in the step 3 to obtain the MOF-based calcium ion nano regulator, which is called PLCF for short. According to the MOF-based calcium ion nano regulator, mitochondrial respiration can be inhibited by generating nitric oxide (NO), so that more intracellular oxygen is reserved for photodynamic therapy (PDT), calcium overload is continuously and efficiently induced, and calcium overload-photodynamic synergistic anti-tumor is realized. In a mouse in-vivo test, the nanoreactor can overcome the hypoxia environment of solid tumors and generate an excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and particularly relates to a MOF-based calcium ion nano-regulator, a preparation method thereof, and an application thereof. Background Art

[0002] Ca 2+ is the most abundant metal ion in the human body and has been identified as a key regulator of many cellular processes, including proliferation, metabolism, migration, and cell death. Studies have shown that calcium overload characterized by an increase in intracellular Ca 2+ concentration can cause oxidative stress and mitochondrial dysfunction, leading to the release of apoptotic signals. Calcium overload is a highly promising cancer treatment strategy. At the same time, calcium overload induces immunogenic cell death, stimulates dendritic cell maturation, and activates the immune system. Existing studies have shown that tumor cells can induce calcium overload through various pathways, such as the degradation and release of calcium-based nanomaterials, intracellular calcium flux, and the release of calcium ions from calcium stores such as the endoplasmic reticulum, so as to achieve the purpose of effectively treating tumors.

[0003] Currently, the induction and enhancement of calcium overload by photodynamic therapy (PDT) have been widely used in tumor treatment, which mainly relies on the generation of singlet oxygen to open calcium ion channels on the cell membrane, such as TRPA1, to promote intracellular calcium ion flow. However, the lifetime of singlet oxygen is often very short (3.6 μs), and the diffusion distance is very limited (tens of nanometers), which will not only greatly reduce the treatment effect of PDT, but also, due to its inherent complex calcium ion regulation mechanism, it is often difficult to induce calcium overload solely by this method. In addition, tumor hypoxia is a key factor limiting the efficiency of PDT, which obviously further affects the accumulation of intracellular calcium ions. At the same time, it is very difficult for the generation of singlet oxygen to induce the release of calcium ions in the endoplasmic reticulum, which is the second largest calcium store. The above factors are not conducive to inducing calcium overload and ultimately lead to poor tumor treatment effects.

[0004] Nitric oxide (NO), as a typical reactive oxygen species, has a relatively long half-life (~5 s) and a relatively long diffusion radius (40 - 200 μm). At the same time, NO can release calcium ions by acting on the ryanodine receptor (RyRs) of the endoplasmic reticulum calcium channel, promoting intracellular calcium accumulation. More importantly, NO can relieve hypoxia fundamentally by inhibiting cell respiration and reducing intracellular oxygen consumption, and use the saved oxygen for PDT to achieve sustainable singlet oxygen generation. All of these provide powerful conditions for effectively inducing calcium overload.

[0005] Based on this, we designed and constructed a simple and ingenious porphyrin-MOF-based nano-reaction platform for calcium overload and immune synergistic cancer therapy. Due to the advantages of porphyrin MOF (PCN-224) such as high porosity, large specific surface area, and adjustable structure, it was selected as the photosensitizer. Subsequently, the NO donor L-arginine was encapsulated by porphyrin MOF, and calcium carbonate was encapsulated by MOF. To enhance the targeting of the system to tumors, folic acid was modified on the material surface. Once endocytosed by tumor cells, the acidic environment in lysozyme degrades the calcium carbonate on the surface of the nano-reactor and releases calcium ions. Subsequently, under near-infrared light irradiation, the singlet oxygen generated by PDT can, on the one hand, promote the flow of intracellular calcium ions, and on the other hand, generate NO with a long lifespan and long diffusion distance by oxidizing L-arginine. At the same time, NO can activate the endoplasmic reticulum calcium channel to release calcium ions, thereby promoting intracellular calcium ion accumulation. Most importantly, in the absence of light irradiation, L-arginine reacts with endogenous H2O2 to generate NO in PLCF. NO fundamentally alleviates tumor hypoxia by inhibiting tumor cell respiration and reducing its own oxygen consumption, and the saved oxygen is used for PDT, realizing the cyclic generation of singlet oxygen and downstream NO, and ultimately continuously inducing calcium overload to achieve calcium overload-photodynamic synergistic cancer therapy.

[0006] Glossary: EDC: Carbodiimide.

[0007] NHS: N-Hydroxysuccinimide.

[0008] TCPP: Tetracarboxyporphyrin.

[0009] DMF: N,N-Dimethylformamide.

[0010] FA: Folic acid. Summary of the Invention

[0011] The purpose of the present invention is to provide a MOF-based calcium ion nano-regulator and its preparation method and application.

[0012] To solve the above technical problems, the present invention is realized through the following technical solutions: A preparation method of a MOF-based calcium ion nano-regulator, comprising the following steps: Step 1, prepare porphyrin metal-organic framework PCN-224, and disperse PCN-224 into H2O to obtain a PCN-224 solution; Step 2, mix L-arginine with the PCN-224 solution evenly to prepare PCN-LA; Step 3: Disperse PCN-LA in ethanol, then add CaCl2·2H2O and mix well in the dark; then place it together with NH4HCO3 in another container into a vacuum drying chamber and store it under vacuum at room temperature to obtain PCN-LA coated with CaCO3: PCN-LA@CaCO3; Step 4: React PCN-LA@CaCO3, EDC, and NHS in an aqueous solution, and then add folic acid and stir and mix at room temperature to obtain the MOF-based calcium ion nanoregulator.

[0013] Further improvement, in Step 1, the preparation method of the porphyrin metal-organic framework PCN-224 is as follows: Dissolve TCPP, ZrOCl2·8H2O, and benzoic acid in a DMF solution, ultrasonically disperse it, and react at 90 °C for 5 hours, and centrifuge to collect the precipitate to obtain the porphyrin metal-organic framework PCN-224.

[0014] Further improvement, the mass ratio of TCPP, ZrOCl2·8H2O, and benzoic acid is 25:75:700.

[0015] Further improvement, in Step 2, the mass ratio of L-arginine to PCN-224 is 2.5:5.

[0016] Further improvement, in Step 2, L-arginine and the PCN-224 solution are stirred and mixed evenly in the dark at room temperature for 12 h.

[0017] Further improvement, in Step 3, the mass ratio of PCN-LA to CaCl2·2H2O is 2.5:44; the time for vacuum storage at room temperature is 24 h.

[0018] Further improvement, in Step 4, the mass ratio of PCN-LA@CaCO3, EDC, NHS, and folic acid is 5:10:10:5, and the stirring time at room temperature is 12 h.

[0019] A MOF-based calcium ion nanoregulator, and the preparation method of the MOF-based calcium ion nanoregulator is as described above.

[0020] The use of a MOF-based calcium ion nanoregulator, the preparation method of the MOF-based calcium ion nanoregulator is as described above, and the MOF-based calcium ion nanoregulator is used as a drug for inducing intracellular calcium overload.

[0021] Further improvement, the MOF-based calcium ion nanoregulator is used as a drug for treating tumors or a drug for treating cancer.

[0022] The present invention has the following beneficial effects: (1)The present invention utilizes calcium-based nanomaterials, which can achieve mutual enhancement and synergistic anti-tumor effects with PDT.

[0023] (2)The MOF-based calcium nanoregulator prepared by the present invention has rich raw material sources, simple and convenient operation, and easy preparation conditions.

[0024] (3)The MOF-based nanoregulator prepared by the present invention uses arginine as a nitric oxide donor, which not only realizes the efficient release of nitric oxide, thereby inhibiting cell respiration, relieving hypoxia, and enhancing PDT, but also can efficiently induce intracellular calcium overload, enabling safe and efficient photodynamic therapy, and having broad application prospects in the field of cancer treatment. Description of the Drawings

[0025] Figure 1 It is a transmission electron microscope image of the prepared PCN-224.

[0026] Figure 2 It is a transmission electron microscope image of the prepared PCN-LA.

[0027] Figure 3 It is a transmission electron microscope image of the prepared PCN-LA@CaCO3.

[0028] Figure 4 It is a transmission electron microscope image of the prepared PLCF.

[0029] Figure 5 It is the in vitro NO release of PLCF.

[0030] Figure 6 It is a schematic diagram of the detection of intracellular oxygen content and the detection of oxygen consumption of cells treated with PLCF.

[0031] Figure 7 Intracellular calcium ion content map of cells treated with PLCF.

[0032] Figure 8 It is a solid figure of the tumor treatment effect after 14 days of treating mouse tumors with PLCF.

[0033] Figure 9 It is a physical figure of the tumor treatment effect after 14 days of treating mouse tumors with PLCF. Detailed Embodiments

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Embodiment

[0035] A preparation method of an MOF-based calcium ion nanoregulator includes the following steps: First, porphyrin metal-organic framework (PCN-224) was synthesized as the substrate for subsequent preparation. The general method was to weigh TCPP (25 mg), ZrOCl2·8H2O (75 mg) and benzoic acid (0.7 g), and place them in a round-bottom flask (100 ml). Add DMF (25 ml) and disperse them ultrasonically to make them fully dispersed. Then the mixture was reacted in an oil bath at 90 °C for 5 hours. After the reaction was completed, the mixture was centrifuged (12,000 rpm, 10 min) to separate the PCN-224 precipitate. The precipitate was dispersed in DMF and then centrifuged, repeating three times to remove those molecules that did not participate in the reaction. The PCN precipitate was then dispersed in a DMF solution for subsequent use. As Figure 1 shown.

[0036] L-arginine (2.5 mg) was added to the above-obtained PCN-224 (1 mg / mL, 5 ml) solution. After stirring in the dark at room temperature for 12 h, the PCN@LA nanoparticles were collected by centrifugation and suspended in water for later use. As Figure 2 shown.

[0037] When synthesizing PCN@LA@CaCO3, PCN@LA (5 mg) was suspended in 100 ml of ethanol and placed in a glass bottle. Then, 44 mg of CaCl2·2H2O was added to the mixture. Then, the bottle was placed in a vacuum drying chamber together with other bottles containing NH4HCO3. The whole system was stored in a vacuum environment at room temperature for 24 h. Then, PCN@LA coated with CaCO3 was obtained. As Figure 3 shown.

[0038] PCN@LA@CaCO3 (5 mg), EDC (10 mg) and NHS (10 mg) were dissolved in 5 ml of deionized water. After reacting for 1 h, 5 mL of FA (folic acid) solution (1 mg / mL) was mixed with the above solution and stirred at room temperature for 12 h. The final PCN@LA@CaCO3@FA precipitate was suspended in water for the next use. As Figure 4 shown, the MOF-based calcium nanoregulator (PLCF) prepared by the method of the present invention has good dispersibility and spherical shape with uniform size, and has potential clinical application value.

[0039] NO release of PLCF in vitro: The NO release of PLCF in vitro was detected by a nitric oxide detection kit. PLCF was incubated with 10 mM H2O2 in water. After the reaction was completed, 50 μL of the supernatant was taken for centrifugation, and the relative yield of NO was detected with a NO detection kit.

[0040] As shown in Figure 5, the NO generated by the incubation of PLCF with H2O2 showed time-dependence, and the production amount increased with the increase of the incubation time. PLCF has the ability to produce NO. In the figure, the Control group is the H2O2 solution without adding PLCF.

[0041] Monitor the change of the oxygen content in the PLCF group cells: First, inoculate 4T1 (1×10 5 ) into a 24-well plate, place the plate in a cell incubator with the culture conditions set at 37 °C and 5% CO2 and incubate in an anaerobic chamber for 24 h. Subsequently, dilute different drugs to 16 μg / mL (equivalent to PCN) with cell culture medium containing 10% FBS, discard the original culture medium, add the drug-containing culture medium, and continue to incubate with the cells for 12 h. To facilitate subsequent measurement, cover the culture medium with 2 mL of liquid paraffin, and use a dissolved oxygen meter to detect the dissolved oxygen (DO) in the culture medium every 5 minutes.

[0042] As shown in Figure 6, under anaerobic conditions, compared with Control (no cells, only culture medium), the O2 of the cells treated with Cell Only (cells and culture medium) and PCN@CaCO3@FA (PCF) without modified LA for 30 minutes decreased sharply to 46.9% and 50.7% respectively. In the cells treated with PLCF, the decrease of O2 was slow, and the O2 content was as high as 70.8%. In summary, these results indicate that PLCF can inhibit cell respiration, reduce cell O2 consumption, and relieve hypoxia by producing NO.

[0043] The ability of PLCF to induce intracellular calcium overload: Culture 1.0×10 5 4T1 cells per well in a 24-well plate, incubate overnight under normoxic and hypoxic conditions respectively, then dilute different drugs to 16 μg / mL (equivalent to PCN) with cell culture medium containing 10% FBS, discard the original culture medium, add the drug-containing culture medium, treat the cells for 6 h, irradiate the light group with 660 nm laser for 5 min, and continue to incubate for 2 h. Then aspirate the culture medium, wash the cells three times with PBS. Add Flu-4 AM and incubate for 30 min. Wash the cells three times with PBS. Observe through an inverted fluorescence microscope.

[0044] As Figure 7As shown, visible green fluorescence can be observed in the 4 T1 cells treated with PLCF. Moreover, under hypoxic conditions, the same effect can still be achieved as under normoxic conditions. Therefore, we speculate that under hypoxic conditions, NO can not only inhibit cell respiration, save O2 for PDT to generate ROS to induce Ca 2+ influx, but also induce the endoplasmic reticulum to release Ca 2+ , increasing the intracellular Ca 2+ concentration. Figure 7 In the Control group in Figure 7 , the 4 T1 cells without adding PLCF were used.

[0045] Evaluation of the anti-tumor effect of PLCF Female Balb / c mice (6 weeks old) were used to establish a 4T1 tumor model for in vivo anti-tumor research. When the proximal tumor volume reached about 100 - 150 mm 3 , the mice were randomly divided into 6 groups (n = 3) by simple random sampling method: Saline group and PLCF group. The mice in the experimental groups were injected with drugs via the tail vein on the 1st, 3rd, and 5th days respectively. Laser irradiation was performed 8 h after drug administration. The experiment was terminated on the 14th day, and the tumor size of the mice was observed.

[0046] As Figure 8 shown, on the 14th day, compared with the control group, the tumor size in the PLCF group was significantly smaller, indicating that PLCF can overcome tumor hypoxia and has excellent anti-tumor effects.

[0047] As Figure 9 shown, 14 days after treatment, the tumors were dissected and photographed. Compared with the control group, the tumor size in the PLCF group was significantly smaller, and even the tumors could disappear. This indicates that PLCF can overcome tumor hypoxia and has excellent anti-tumor effects.

[0048] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art in the relevant technical field can understand and utilize the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for preparing a MOF-based calcium ion nanoregulator, characterized in that: The steps include: Step 1: preparing a porphyrin metal organic framework PCN-224, and dispersing PCN-224 in H2O to obtain a PCN-224 solution; Step 2: Evenly mix L-arginine and PCN-224 solution to prepare PCN-LA; Step 3: Disperse PCN-LA in ethanol, then add CaCl2·2H2O and mix in the dark; then put it into a vacuum drying chamber together with NH4HCO3 in other containers and store it in vacuum at room temperature to obtain CaCO3-coated PCN-LA: PCN-LA@CaCO3; Step 4: PCN-LA@CaCO3, EDC and NHS are reacted in an aqueous solution, and then folic acid is added and stirred at room temperature to obtain the MOF-based calcium ion nano-regulator.

2. The method for preparing the MOF-based calcium ion nano-regulator according to claim 1, characterized in that: In the step 1, the preparation method of the porphyrin metal organic framework PCN-224 is as follows: TCPP, ZrOCl2·8H2O and benzoic acid were dissolved in DMF solution, and reacted at 90°C for 5 hours after ultrasonic dispersion. The precipitate was collected by centrifugation to obtain the porphyrin metal organic framework PCN-224.

3. The method for preparing the MOF-based calcium ion nano-regulator according to claim 2, characterized in that: The mass ratio of TCPP, ZrOCl2·8H2O and benzoic acid is 25:75:

700.

4. The method for preparing the MOF-based calcium ion nano-regulator according to claim 1, characterized in that: In the step 2, the mass ratio of L-arginine to PCN-224 is 2.5:

5.

5. The method for preparing the MOF-based calcium ion nano-regulator according to claim 1, characterized in that: In the step 2, L-arginine and PCN-224 solution were stirred in the dark at room temperature for 12 h to mix evenly.

6. The method for preparing the MOF-based calcium ion nano-regulator according to claim 1, characterized in that: In the step 3, the mass ratio of PCN-LA to CaCl2·2H2O is 2.5:44; and the vacuum storage time at room temperature is 24 hours.

7. The method for preparing the MOF-based calcium ion nano-regulator according to claim 1, characterized in that: In the step 4, the mass ratio of PCN-LA@CaCO3, EDC, NHS and folic acid is 5:10:10:5, and the stirring time at room temperature is 12 hours.

8. A MOF-based calcium ion nano-regulator, characterized in that: The preparation method of the MOF-based calcium ion nanoregulator is as described in any one of claims 1-7.

9. A use of a MOF-based calcium ion nano-regulator, characterized in that: The preparation method of the MOF-based calcium ion nano-regulator is as described in any one of claims 1-7, and the MOF-based calcium ion nano-regulator is used as a drug for inducing intracellular calcium overload.

10. The use of the MOF-based calcium ion nano-regulator according to claim 9, characterized in that: The MOF-based calcium ion nano-regulator is used as a drug for treating tumors or a drug for treating cancer.

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