Ferriporphyrin-based metal organic framework-berberine nano-drug with multienzyme activity

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a ferriporphyrin-based metal organic framework (berberine nano-drug with multienzyme activity. According to the invention, berberine is encapsulated in the ferriporphyrin-based metal organic framework, so that a scheme that the ferriporphyrin-based nano metal organic framework and berberine are synergistically used for treating tumors is realized. According to the invention, the berberine is used for modification, and chemotherapy is added on the basis of the photodynamic curative effect of the ferriporphyrin-based MOFs, so that the dosage of the ferriporphyrin-based MOFs is effectively reduced, the problem of too fast oxygen consumption of the photodynamic therapy is relieved, and finally, the purposes of combining the photodynamic therapy with the chemotherapy and synergistically improving the tumor treatment effect are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicine, and in particular relates to an iron porphyrin-based metal organic framework@berberine nanomedicine with multi-enzyme activity. Background Art

[0002] Iron porphyrin-based nanometal organic frameworks (Fe-TCPP-MOFs, iron porphyrin MOFs or FTMs) have circular porous structures, prominent surface areas, biodegradability, biocompatibility, and excellent photophysical and photochemical properties. However, the potential cytotoxicity, long-term biodistribution in vivo, and excretion of iron porphyrin MOFs limit their development and application as tumor therapeutics. Therefore, it is necessary to find safer, simpler, and more efficient means to treat tumor sites. Photodynamic therapy of iron porphyrin MOFs in combination with other therapies (such as chemotherapy, photothermal therapy, ferroptosis, immunotherapy, etc.) has become a research hotspot for tumor treatment.

[0003] Photodynamic therapy (PDT) is a new method of treating diseases with photosensitive drugs and lasers. By irradiating the lesion with a specific wavelength, the photosensitive drugs that selectively accumulate in the lesion tissue can be activated, triggering a photochemical reaction to destroy the lesion. Iron porphyrin-based MOFs can cause irreversible chemical damage to the tumor site during light-mediated photodynamic therapy, but they can also damage normal cells around the tumor tissue. More importantly, the efficacy of photodynamic therapy is hindered by hypoxia, which makes the bioavailability of the photosensitizer low and limits the scope of photodynamic therapy to the superficial surface of the tumor.

[0004] Berberine (BBR) is an alkaloid isolated from the traditional Chinese medicine Coptis chinensis and is the main active ingredient in its antibacterial properties. The prior art discloses that some berberines have anti-tumor effects. For example, Sun Qiang et al. listed the effects of berberine in inhibiting the proliferation and metastasis of colorectal cancer cells, inducing apoptosis, blocking the cell cycle, regulating inflammatory responses, reversing chemotherapy drug resistance, and regulating intestinal flora in "Research Progress on the Mechanism of Action of Berberine against Colorectal Cancer." Acta Pharmaceutica Sinica 002 (2022): 057. Berberine has a wide range of functions and a complex mechanism of treatment. Currently, no research has clearly demonstrated its specific functions.

[0005] In summary, there is no solution in the prior art for the synergistic use of iron porphyrin-based nanometal organic frameworks and berberine for treating tumors. Summary of the invention

[0006] The purpose of the present invention is to provide a nano drug combining ferroporphyrin-based metal organic framework with berberine. The nano drug provided by the present invention can add chemotherapy on the basis of the photodynamic efficacy of ferroporphyrin-based MOFs and effectively kill tumor cells at a lower dose.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a ferroporphyrin-based metal organic framework@berberine (FTM@BBR) nanomedicine with multi-enzyme activity. The nanomedicine comprises a ferroporphyrin-based metal organic framework encapsulating berberine.

[0009] In light-mediated photodynamic therapy (PDT), laser irradiation of the nanodrug can stimulate the photodynamic efficacy of iron porphyrin-based MOFs and promote the generation of ROS. PDT consumes a lot of oxygen, and a low oxygen environment will affect the efficacy of PDT on tumors.

[0010] Iron porphyrin-based MOFs have multiple enzymatic activities and can catalyze a variety of reactions, including peroxidase-like, PDT / CDT (chemodynamic therapy)-like, and catalase-like reactions to induce free radical production. Endogenous hydrogen peroxide (H2O2) overexpressed in tumors as a reaction substrate can be catalytically decomposed by the catalase properties of iron porphyrin-based MOFs to produce oxygen (O2) in situ, thereby enhancing the generation of reactive oxygen species (ROS) in TCPP (tetrahydroxyporphyrin) and light-mediated photodynamic therapy.

[0011] Berberine has a wide range of pharmacological activities and can reduce tumor oxygen consumption to reverse the hypoxic tumor microenvironment. Berberine can inhibit mitochondrial respiratory complex I, reduce tumor oxygen consumption, and enhance ROS generation during TCPP and light-mediated PDT.

[0012] The present invention also provides a method for preparing the above-mentioned iron porphyrin-based metal organic framework@berberine nanomedicine, comprising the following steps: encapsulating berberine in the iron porphyrin-based metal organic framework.

[0013] Preferably, the preparation method comprises the following steps: adding the iron porphyrin-based metal organic framework (Fe-TCPP-MOFs) to the berberine (BBR) solution, mixing, washing, and obtaining the iron porphyrin-based metal organic framework @ berberine nanomedicine.

[0014] Preferably, the mixing time is more than 12 hours.

[0015] Preferably, the preparation method of Fe-TCPP-MOFs comprises the following steps:

[0016] 5,10,15,20-tetra(4-carboxyl)-21H,23H-porphyrin (TCPP), FeCl3 and 0.1M HCl ethanol solution are dissolved in a solvent to obtain a mixture; the mixture is sealed and mixed evenly, and then heated for more than 48 hours. After naturally cooling to room temperature, the crystals are filtered, collected and washed to obtain Fe-TCPP-MOFs; the solvent is a mixture of N,N-dimethylformamide (DMF) and ethanol.

[0017] Preferably, the molar ratio of TCPP to FeCl3 is 1:3; the molar ratio of TCPP to HCl is 5:12.

[0018] Preferably, the volume ratio of DMF to ethanol in the solvent is 1:2.

[0019] Preferably, the crystal washing agent is DMF and ethanol.

[0020] The present invention also provides the use of the above-mentioned iron porphyrin-based metal organic framework@berberine nanomedicine in the preparation of anticancer drugs.

[0021] Beneficial Effects

[0022] (1) The present invention provides a ferroporphyrin-based metal organic framework @ berberine (FTM@BBR) nanodrug with multi-enzyme activity, which encapsulates berberine in ferroporphyrin MOFs to reduce tumor oxygen consumption and effectively alleviate the problem of excessive oxygen consumption in photodynamic therapy.

[0023] (2) The present invention utilizes the modification of berberine to add chemotherapy treatment on the basis of the photodynamic therapy effect of iron porphyrin-based MOFs, thereby effectively reducing the dosage of iron porphyrin-based MOFs.

[0024] (3) The present invention combines photodynamic therapy with chemotherapy to improve the therapeutic efficacy of tumors.

[0025] (4) The present invention makes full use of the enzyme-like activity of iron porphyrin-based MOFs to spontaneously produce reactants (O2) at the tumor site, effectively reducing the use of other materials, and ultimately achieving the synthesis of nanomedicines with excellent therapeutic effects using less materials and achieving good therapeutic effects.

[0026] (5) The preparation method of the nanomedicine described in the present invention is fast and simple, and is easy to operate and promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 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 labor.

[0028] Figure 1 This is an electron microscope image of the iron porphyrin-based metal-organic framework@berberine nanodrug.

[0029] Figure 2 This is a diagram showing the particle size of FTM and FTM@BBR detected by a particle size analyzer.

[0030] Figure 3 This is a diagram of the ultraviolet absorption of BBR, FTM and FTM@BBR detected by UV-NIR-Vis spectrophotometer.

[0031] Figure 4 This is a diagram showing the verification of the peroxidase-like activity and catalase-like activity of FTM@BBR and the detection of its ROS generation using a UV-NIR-Vis spectrophotometer.

[0032] Figure 5 This is a graph showing the activity of 4T1 tumor cells after different treatments. DETAILED DESCRIPTION

[0033] The invention provides a ferroporphyrin-based metal organic framework@berberine (FTM@BBR) nanomedicine with multi-enzyme activity. The nanomedicine comprises a ferroporphyrin-based metal organic framework encapsulating berberine.

[0034] The present invention also provides a method for preparing the above-mentioned iron porphyrin-based metal organic framework@berberine nanomedicine, comprising the following steps: encapsulating berberine in the iron porphyrin-based metal organic framework.

[0035] The present invention has no particular limitation on the encapsulation method, and the encapsulation step is preferably: adding the iron porphyrin-based metal organic framework (Fe-TCPP-MOFs) to the berberine (BBR) solution, mixing, washing, and obtaining the iron porphyrin-based metal organic framework @ berberine nanomedicine.

[0036] The concentration of the berberine (BBR) solution is preferably 10-100 ug / mL of the berberine solution, and the final concentration of Fe-TCPP-MOFs is preferably 10-100 ug / mL.

[0037] The mixing method in the present invention is not particularly limited, and is preferably mixed on a rotary mixer at room temperature, and the mixing time is preferably more than 12 hours.

[0038] The preparation method of Fe-TCPP-MOFs in the present invention comprises the following steps:

[0039] 5,10,15,20-tetrakis(4-carboxy)-21H,23H-porphyrin (TCPP), FeCl3 and 0.1M HCl ethanol solution are dissolved in a solvent to obtain a mixture, wherein the solvent is a mixture of N,N-dimethylformamide (DMF) and ethanol; the mixture is sealed and mixed evenly, then heated for more than 48 hours, cooled naturally to room temperature, filtered, collected crystals and washed to obtain Fe-TCPP-MOFs.

[0040] In the present invention, the molar ratio of TCPP to FeCl3 is preferably 1:3; the molar ratio of TCPP to HCl is preferably 5:12.

[0041] The volume ratio of DMF to ethanol in the solvent of the present invention is preferably 1:2.

[0042] In the present invention, the method for uniformly mixing the mixture is preferably ultrasonic treatment.

[0043] The crystal detergents in the present invention are preferably DMF and ethanol; the ethanol is preferably anhydrous ethanol with a purity of ≥99.8%; and the DMF is preferably anhydrous DMF with a purity of ≥99.8%.

[0044] The present invention also provides the use of the above-mentioned iron porphyrin-based metal organic framework@berberine nanomedicine in the preparation of anticancer drugs.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0048] Example 1

[0049] Preparation method of iron porphyrin-based metal-organic framework@berberine (FTM@BBR).

[0050] 5,10,15,20-Tetra(4-carboxyl)-21H,23H-porphyrin (TCPP) (0.05mmol) and FeCl3 (0.15mmol) were dissolved in a mixture of 4mL anhydrous DMF (purity ≥99.8%) and 8mL anhydrous ethanol (purity ≥99.8%). Subsequently, the pH of the mixed solution was adjusted to about 3 with 0.1M HCL. The final mixture was sealed in a small cap vial and ultrasonically treated to ensure that the mixture was mixed evenly. Subsequently, the obtained mixture was heated in an oven at 150°C for 48 hours and then slowly cooled to room temperature. Finally, the precipitate was collected by filtration and washed with DMF and ethanol, and then resuspended in PBS to obtain a Fe-TCPP-MOFs solution.

[0051] The obtained Fe-TCPP-MOFs (FTM) was added to a 100ug / mL berberine (BBR) solution, and the final concentration of FTM was 100ug / mL. The mixture was mixed on a rotary mixer at room temperature for 12 hours, and washed to obtain FTM@BBR. The detergent was a PBS buffer solution with a pH of 7.2-7.4. Washing step: After mixing for 12 hours, centrifuge at 8000rpm for 10 minutes, and then resuspend with PBS.

[0052] The obtained FTM@BBR was examined by electron microscopy. The electron microscopy photos are shown in Figure 1 shown.

[0053] The particle size of FTM and FTM@BBR was detected by a particle size analyzer to characterize the change in particle size before and after loading BBR. Figure 2 shown.

[0054] Example 2

[0055] The difference from Example 1 is that the concentration of the BBR solution is 10 ug / mL, and the final concentration of FTM is 10 ug / mL.

[0056] Example 3

[0057] The UV absorption of BBR, FTM and FTM@BBR was detected by UV-NIR-Vis spectrophotometer to characterize the successful preparation of FTM@BBR. 100ug / mL of FTM was added to 10ug / mL of BBR and mixed on a rotary mixer at room temperature for 12 hours, washed, and FTM@BBR was obtained. Figure 3 As shown, the characteristic peak curve of FTM@BBR is similar to that of FTM, and shows similar characteristic peaks to BBR, proving the successful preparation of FTM@BBR.

[0058] Example 4

[0059] The peroxidase activity and catalase activity of FTM@BBR and the generation of ROS were verified by using a UV-NIR-Vis spectrophotometer, with 1 mM TMB (3,3',5,5'-tetramethylbenzidine) as the colorimetric reagent. Figure 4 As shown in the figure, when 100ug / mL of FTM@BBR produces ROS under the condition of 10mM H2O2, a characteristic absorption peak appears at 652nm. This is because the peroxidase activity of FTM@BBR catalyzes H2O2 to produce ROS; in addition, after the introduction of light, the FTM@BBR-H2O2 group shows stronger absorption, proving that FTM@BBR also has catalase activity, which can catalyze H2O2 to produce O2, thereby enhancing the generation of ROS through PDT.

[0060] Example 5

[0061] 4T1 tumor cells were seeded in 96-well plates at 1×10 5 The cells were cultured at 5% CO2 and 37°C for 12 h, and then treated with 1640 medium containing FTM (100 ug / mL), BBR (50 ug / mL) or FTM@BBR (100 ug / mL). After incubation for 4 h, the cells were irradiated with laser and incubated for 24 h. The effects of different treatments on the viability of 4T1 cells were determined using the standard CCK-8 method. The results are shown in Figure 2. Figure 5 shown.

[0062] Depend on Figure 5 It can be seen that FTM has almost no effect on the cell viability of 4T1; BBR has a certain killing ability on 4T1, which is mainly due to the chemotherapeutic effect of BBR; 4T1 cells treated with FTM@BBR showed relatively low cell viability compared with the BBR group, which is due to the synergistic effect of FTM@BBR's catalase-like and peroxidase-like activities on chemotherapy; and FTM@BBR significantly reduced the cell viability of 4T1 after adding light treatment, which further proves that FTM@BBR with catalase-like and peroxidase-like activities can cooperate with PDT and chemotherapy to achieve effective killing of 4T1 tumor cells.

[0063] The above examples illustrate that the present scheme can successfully prepare FTM@BBR, and the prepared FTM@BBR has catalase-like and peroxidase-like activities, and can be enhanced by PDT, that is, can cooperate with PDT.

[0064] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A ferroporphyrin-based metal organic framework@berberine (FTM@BBR) nanodrug with multi-enzyme activity, wherein the nanodrug comprises a ferroporphyrin-based metal organic framework encapsulating berberine.

2. The method for preparing the iron porphyrin-based metal organic framework @ berberine nanomedicine according to claim 1, characterized in that: The method comprises the following steps: encapsulating berberine in an iron porphyrin-based metal organic framework.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: adding the iron porphyrin-based metal organic framework into the berberine solution, mixing, washing, and obtaining the iron porphyrin-based metal organic framework@berberine nanomedicine.

4. The preparation method according to claim 3, characterized in that: The mixing time is more than 12 hours.

5. The preparation method according to claim 3, characterized in that: The preparation method of the iron porphyrin-based metal organic framework comprises the following steps: TCPP, FeCl3 and 0.1M HCl ethanol solution are dissolved in a solvent to obtain a mixture; the mixture is sealed and mixed evenly, then heated for more than 48 hours, naturally cooled to room temperature, filtered to collect crystals, and washed to obtain an iron porphyrin-based metal organic framework; the solvent is a mixture of N,N-dimethylformamide and ethanol.

6. The preparation method according to claim 5, characterized in that: The molar ratio of TCPP to FeCl3 is 1:3; the molar ratio of TCPP to HCl is 5:

12.

7. The preparation method according to claim 5, characterized in that: The volume ratio of N,N-dimethylformamide to ethanol in the solvent is 1:

2.

8. The preparation method according to claim 5, characterized in that: The washing agent of the crystal is N,N-dimethylformamide and ethanol.

9. Use of the iron-porphyrin-based metal-organic framework@berberine nanodrug according to claim 1 or the iron-porphyrin-based metal-organic framework@berberine nanodrug prepared by the method according to any one of claims 2 to 8 in the preparation of anticancer drugs.

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