CN112791185B - Nano medicine for treating tumor by combining photo-thermal treatment with iron agent and preparation method thereof - Google Patents
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Abstract
The invention discloses a nano-drug for treating tumor by combining photo-thermal and iron-based treatment and a preparation method thereof, wherein Fe is compounded on carrier protein of metal-protein nano-particles3+Ions while passing Fe3+Ion-chelating pharmaceutically acceptable polyphenolic compounds. The inventor finds that, through experiments, different from the traditional fact that the Fenton reagent generated in the tumor photothermal treatment process can cause the oxidative degradation failure of drug molecules or the uncontrollable drug effect, after the specific iron death drug is compounded with the metal-protein nanoparticle containing the polyphenol compound and used for tumor photothermal treatment, the two drugs can unexpectedly act in a synergistic manner, and have a better killing effect on tumor cells.
Description
Technical Field
The invention relates to a metal-protein nano-drug carrier, in particular to a metal-protein nano-drug for combined tumor photothermal therapy and iron death, and a preparation method and application thereof.
Background
Cancer is a serious disease which threatens human health worldwide nowadays, and the main means for treating cancer at present are chemotherapy, radiotherapy, surgical resection and the like, however, the methods are often accompanied by higher toxicity and recurrence rate and low targeting. In recent years, various novel biomedical nanomaterials have been applied to cancer therapy, including photothermal and photodynamic therapy materials, nano-drug carrier materials, and nanomaterials that induce iron death of tumor cells by using Fenton reaction (Fenton reaction) induced by the microenvironment of tumor cells. The photothermal therapy and the iron death combined tumor therapy are a novel effective method, and the defects of single photothermal therapy and iron therapy can be overcome. The key of the implementation of the technology is to construct a nano material with photo-thermal treatment and iron agent treatment, so that high iron loading capacity and low toxicity are realized while photo-thermal conversion efficiency is ensured, and the material can effectively carry therapeutic drugs, further induce the death of tumor cells, improve the treatment efficiency while the dosage is reduced, and realize the multi-means synergy.
The polyphenol compounds are also called flavonoid compounds, are compounds with a plurality of phenol groups, and have the functions of resisting oxidation, strengthening vessel walls and the like. The phenolic group of the polyphenol compound may be chelated with a metal ion to form a complex. However, the complex has the characteristics of uncontrollable appearance and size, poor biocompatibility and the like, and the application of the complex is limited. The metal-protein nano-particles are an effective strategy for constructing a stable multifunctional nano-biomaterial by utilizing the electrostatic interaction between proteins and the metal nano-particles and the good biocompatibility of the proteins.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a metal-protein nano-drug for combined tumor photothermal therapy and iron death and a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a metal-protein nanoparticle for use in therapy comprising a carrier protein having Fe complexed thereon3+Ions while passing Fe3+Ion-chelating pharmaceutically acceptable polyphenolic compounds.
In some examples, the carrier protein is selected from bovine serum albumin, human serum albumin, ovalbumin.
In some examples, the polyphenolic compound is selected from Gallic Acid (GA), Tannic Acid (TA), Ellagic Acid (EA), epigallocatechin gallate (EGCG), baicalin (Baicalein), Myricetin (Myricetin), Delphinidin (Delphinidin), and the like.
In some examples, the Fe3+The molar ratio of the ions to the polyphenol compound is (17-67): (30-60).
In some instances, therapeutic drugs that enhance iron death are also loaded.
In some examples, the therapeutic drug is selected from at least one of sorafenib, elastine, FIN56, RSL3, lanpiriropine, sulfasalazine.
In some examples, the therapeutic drug is sorafenib, Fe3+: the molar ratio of the sorafenib is 1 (0.8-5).
In a second aspect of the present invention, there is provided:
use of a metal-protein nanoparticle for the manufacture of a medicament for photothermal therapy of a tumour in combination with iron death, said metal-protein nanoparticle being as described in the first aspect of the invention.
In some examples, the tumor is selected from the group consisting of models of breast cancer, colorectal cancer, liver cancer, pancreatic cancer, ovarian cancer, squamous cell carcinoma of the tongue, melanoma, fibrosarcoma, and the like.
In a third aspect of the present invention, there is provided:
a method for preparing metal-protein nanoparticles, comprising the steps of:
s1) fully stirring the carrier protein and the ferric salt solution to ensure that Fe3+Compounding the Fe-protein complex on carrier protein to obtain Fe-protein complex;
s2) adding a polyphenol compound into the obtained Fe-protein compound, stirring and standing to obtain a crude product of the metal-protein nano-particles;
s3) purifying the crude metal-protein nano-particles to obtain the finished metal-protein nano-particles.
In some examples, the ferric salt is selected from FeCl3、Fe2(SO4)3、Fe(NO3)3FeBr3 and Fe3O4And the like.
In some examples, a therapeutic agent that enhances iron death is added to the finished metal-protein nanoparticle product and reacted sufficiently to yield metal-protein nanoparticle particles for tumor photothermal therapy in combination with iron death.
The invention has the beneficial effects that:
the inventor finds that, through experiments, different from the traditional fact that the Fenton reagent generated in the tumor photothermal treatment process can cause the oxidative degradation failure of drug molecules or the uncontrollable drug effect, after the specific iron death drug is compounded with the metal-protein nanoparticle containing the polyphenol compound and used for tumor photothermal treatment, the two drugs can unexpectedly act in a synergistic manner, and have a better killing effect on tumor cells.
According to the metal-protein nanoparticles provided by some examples of the invention, the protein can play a role in improving the stability and biocompatibility of the nanoparticles, and meanwhile, the metal-protein nanoparticles have a function of widely combining small molecules, so that a drug can be carried on related nanoparticles to realize efficient drug delivery. The polyphenol compounds and iron ions can promote the Fenton reaction under the tumor microenvironment, and the photo-thermal property of the material and the drug-induced iron death effect are combined, so that the tumor is treated in a combined manner.
Metal-protein nanoparticles, Fe, of some embodiments of the invention3+The feed ratio of protein and polyphenol compoundCan be flexibly adjusted according to the use requirement.
The particle size of the metal-protein nanoparticles of some embodiments of the present invention can be controlled by selecting different polyphenolic compounds and proteins, or by varying the reaction time.
Some embodiments of the invention provide metal-protein nanoparticles as drugs and Fe3+When the feed ratio is changed, the treatment effect is changed, and the Fe is properly improved3+When the feed amount is reduced, the good treatment effect can be achieved when the medicine concentration is low, so that the toxic and side effects are reduced.
The metal-protein nanoparticles of some embodiments of the present invention can induce the iron death of tumor cells by using drugs, and compared with other antitumor drugs, the mode of synergistic drug administration and combined photo-thermal treatment has better tumor treatment effect.
Drawings
FIG. 1 is an atomic force microscope photograph of Fe-GA @ BSA nanoparticles prepared in example 1;
FIG. 2 shows the in vitro photothermal temperature profile of Fe-GA @ BSA nanoparticles prepared in example 1;
FIG. 3 shows the results of the catalytic efficiency of the in vitro Fenton reaction of Fe-GA @ BSA nanoparticles prepared in example 1;
FIG. 4 is an atomic force microscope photograph of the Fe-GA @ BSA-SRF nanoparticles prepared in example 1;
FIG. 5 shows the changes of mitochondrial membrane potential of Fe-GA @ BSA and Fe-GA @ BSA-SRF prepared in example 1 in mouse breast cancer cell 4T1, which are observed by JC-1 detection kit through a fluorescence confocal microscope with a scale of 50 μm;
FIG. 6 is a graph of the effect of Fe-GA @ BSA and Fe-GA @ BSA-SRF prepared in example 1 on the induction of Reactive Oxygen Species (ROS) production by 4T1 in mouse breast cancer cells under different power light, as quantified by flow cytometry.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
A preparation method of metal-protein nano-drug particles Fe-GA @ BSA-SRF comprises the following steps:
(1) weighing 66mg of BSA, dissolving in ultrapure water, and stirring at room temperature for 30 minutes;
(2) 54 μ L of FeCl3Dropwise adding the solution (100mg/mL) into the BSA solution under the condition of stirring at room temperature, and stirring at room temperature for 30 min;
(3) dropwise adding 1.02mL of gallic acid solution (10mg/mL) into the Fe-BSA mixed solution under the condition of stirring, and then standing for 3 hours at room temperature in a dark place;
(4) filtering the obtained solution containing the nano particles by using an ultrafiltration tube, and washing for 2 times by using ultrapure water to obtain purified Fe-GA @ BSA nano particles; the atomic force microscope result is shown in figure 1, and the figure shows that the nano particles have uniform size and the size is about 4 nm; the in vitro photo-thermal temperature-rising curve result of the obtained nano particles is shown in figure 2, and the graph shows that under 808nm near-infrared illumination, the temperature of nano particle solutions with different concentrations is increased compared with that of pure water, and the longer the illumination time is, the higher the concentration is, and the larger the temperature-rising amplitude of the nano particles is; the catalytic efficiency of the in vitro Fenton reaction is shown in figure 3, and the graph shows that under the near-infrared illumination of 808nm and the acidic condition, Fe ions catalyze H2O2The capability of degrading methylene blue is obviously improved, namely the efficiency of Fenton reaction is enhanced.
(5) 1mL of 5mM Fe-GA @ BSA prepared in step (4) is added into 1mL of LPBS, 50 μ L of sorafenib solution (40mg/mL) is added under the condition of vigorous stirring at room temperature, ultrasonic treatment is carried out for 30 minutes by using a water bath after stirring for one hour, and then centrifugation is carried out for 5 minutes at 1000rpm to obtain Fe-GA @ BSA-SRF, wherein the atomic force microscope result is shown in FIG. 4, and the graph shows that the size of the nano-drug is uniform and is about 40 nm.
(6) The effects of the prepared Fe-GA @ BSA and Fe-GA @ BSA-SRF on photothermal combined iron treatment of mouse breast cancer cells 4T1 were studied, including killing effects on 4T1 cells, changes in mitochondrial membrane potential (see FIG. 5) and induction of Reactive Oxygen Species (ROS) production in cells (see FIG. 6). As can be seen from FIG. 5, both the Fe-GA @ BSA and Fe-GA @ BSA-SRF groups can cause significant changes in mitochondrial membrane potential, so that the green fluorescence of the JC-1 probe is significantly enhanced, the red fluorescence is significantly reduced, and the changes in mitochondrial membrane potential are the important characteristics of cell pig death. As shown in FIG. 6, Fe-GA @ BSA-SRF can induce the mouse breast cancer cell 4T1 to generate higher level of ROS under different power illumination compared with Fe-GA @ BSA, and the increased level of ROS is another important mark of cell iron death, which indicates that SRF drugs and iron death act synergistically and can achieve enhanced anti-tumor effect in combination with photothermal therapy when iron death occurs.
Example 2
The invention discloses a metal-protein nano-drug particle, which is a Fe-GA @ BSA-SRF protein-metal nano-drug particle prepared by the method, and comprises the following steps:
step (1) As in example 1
(2) 27 μ L of FeCl3Dropwise adding the solution (100mg/mL) into the BSA solution under the condition of stirring at room temperature, and stirring at room temperature for 30 min;
the steps (2) to (6) are the same as the example 1, and Fe-GA @ BSA-SRF nano-drug particles with lower unit Fe content are obtained.
Example 3
The invention discloses a metal-protein nano-drug particle, which is a Fe-TA @ OVA-SRF protein-metal nano-drug particle prepared by the method, and comprises the following steps:
(1) weighing 45mg of OVA, dissolving in ultrapure water, and stirring at room temperature for 30 minutes;
(2) 54 μ L of FeCl3Solution (100mg/mL) inDropwise adding the mixture into the OVA solution under the condition of stirring at room temperature, and stirring at room temperature for 30 min;
(3) dropwise adding 2.04mL of Tannic Acid (TA) solution (25mg/mL) into the Fe-OVA mixed solution under the condition of stirring, and then standing for 24 hours at room temperature in a dark place;
(4) filtering the obtained solution containing the nano particles by using an ultrafiltration tube, and washing for 2 times by using ultrapure water to obtain purified Fe-TA @ OVA nano particles;
(5) adding 1mL of 5mM Fe-TA @ OVA prepared in the step (4) into 1mL of PBS, adding 50 μ L of sorafenib solution (40mg/mL) under the condition of vigorous stirring at room temperature, stirring for one hour, then performing ultrasonic treatment for 30 minutes by using a water bath, and then centrifuging at 1000rpm for 5 minutes to obtain Fe-TA @ OVA-SRF;
step (6) as in example 1, the nanoparticles were obtained in which OVA was a protein and TA was a polyphenol.
Example 4
The invention discloses a metal-protein nano-drug particle, which is a Fe-GA @ BSA-RSL3 protein-metal nano-drug particle prepared by the method, and comprises the following steps:
the steps (1), (2), (3) and (4) in example 1 are reserved, and sorafenib in (5) is changed into RSL3 to obtain Fe-GA @ BSA-RSL3 nano drug particles.
Comparative example 1
The comparative example is a preparation method of metal-protein nano-drug particles, compared with the examples, the selected therapeutic drug can not induce the death of tumor cells, and the Fe-GA @ BSA-DOX protein-metal nano-drug particles prepared by the method comprise the following steps:
the steps (1), (2), (3) and (4) in the example 1 are reserved, and the sorafenib in the step (5) is changed into adriamycin to obtain Fe-GA @ BSA-DOX nano drug particles.
Comparative example 2
The comparative example is a preparation method of metal-protein nano-drug particles, compared with the examples, the selected therapeutic drug can not induce the death of tumor cells, and the Fe-GA @ BSA-CPT protein-metal nano-drug particles prepared by the method comprise the following steps:
the steps (1), (2), (3) and (4) in the example 1 are reserved, and the sorafenib in the step (5) is changed into camptothecin to obtain Fe-GA @ BSA-CPT nano-drug particles.
Comparative example 3
The comparative example is a preparation method of metal-protein nano-drug particles, compared with the examples, the selected therapeutic drugs can not induce the death of tumor cells, and the Fe-GA @ BSA-Irinotecan protein-metal nano-drug particles prepared by the method comprise the following steps:
the steps (1), (2), (3) and (4) in the example 1 are reserved, and the sorafenib in (5) is changed into Irinotecan to obtain Fe-GA @ BSA-Irinotecan nano-drug particles.
In order to confirm that a therapeutic drug capable of inducing iron death can act synergistically with the metal-protein nanoparticles prepared according to the present invention, and achieve an enhanced therapeutic effect in combination with photothermal therapy, experiments were conducted in examples 1, 4, comparative examples 1, 2 and 3, in which the drugs used in examples 1 and 4 were therapeutic drugs capable of inducing iron death, and comparative examples 1, 2 and 3 were therapeutic drugs incapable of inducing iron death. IC of each set of metal-protein nano-drug particles was calculated in 4T1 cells by MTT assay50Values, in free drug concentration as control, experimental data are shown in the following table:
as can be seen from the above table, the therapeutic drug inducing iron death acts synergistically with the metal-protein nanoparticles to significantly reduce the IC of the drug50Value, therapeutic drugs that kill tumor cells at lower concentrations without inducing iron death, after interaction with metal-protein nanoparticles, have little effect on their IC50The value is obtained. On the other hand, under near infrared illumination, each group had IC's compared to the unirradiated group50The values are all obviously reduced, which indicates that the photo-thermal combined iron death can further enhance the treatment effect.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features. The following table lists the ratio of the amounts of the components in a series of synthesis steps, and the amounts of the components of the metal-protein nano-drug particles described in the present invention can be referred to, but not limited to, the following series of combinations:
combination of | Protein (mu mol) | Fe3+(μmol) | Polyphenol (mu mol) | Medicine (mu mol) |
1 | 1 | 67 | 60 (Gallic acid) | 67 (Sorafenib) |
2 | 1 | 33 | 60 (Gallic acid) | 33 (Sorafenib) |
3 | 1 | 67 | 60 (Gallic acid) | 134 (Sorafenib) |
4 | 1 | 67 | 30 (tannic acid) | 67 (Sorafenib) |
5 | 1 | 67 | 50 (ellagic acid) | 67 (Sorafenib) |
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (1)
1. A method for preparing metal-protein nanoparticles, comprising the steps of:
s1) weighing 66mg of BSA, and dissolving in ultrapure water;
s2) adding 54 mu L of 100mg/mL FeCl3Dropwise adding the solution into the BSA solution under the condition of stirring at room temperature, and stirring at room temperature for 30 min;
s3) dropwise adding 1.02mL of 10mg/mL gallic acid solution into the Fe-BSA mixed solution under the condition of stirring, and then standing for 3 hours at room temperature in a dark place;
s4) filtering the obtained solution containing the nano-particles by using an ultrafiltration tube, and washing the solution containing the nano-particles for 2 times by using ultrapure water to obtain purified Fe-GA @ BSA nano-particles;
s5) adding 1mL of 5mM Fe-GA @ BSA to 1mL of PBS, adding 50 μ L of 40mg/mL sorafenib solution under stirring at room temperature, performing ultrasonic treatment for 30 minutes by using a water bath after stirring for one hour, and then centrifuging at 1000rpm for 5 minutes to obtain Fe-GA @ BSA-SRF.
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