CN111249327B - Natural mung bean-based polyphenol nano-drug carrier and application thereof - Google Patents
Natural mung bean-based polyphenol nano-drug carrier and application thereof Download PDFInfo
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Abstract
The invention relates to a polyphenol nano-drug carrier based on natural mung beans and application thereof. The polyphenol nano-drug carrier can be directly used for treating psoriasis, can also be used for loading anti-tumor drugs and treating tumors, improves the bioavailability of polyphenol substances by a simple method, improves the transmission efficiency of the drug carrier and enhances the tumor treatment effect.
Description
Technical Field
The invention relates to a polyphenol nano-drug carrier based on natural mung beans and application thereof, belonging to the technical field of nano-drug carriers.
Background
Polyphenols are a general name of compounds with a plurality of phenolic hydroxyl groups widely existing in plants, and are important metabolites in plants. Its content in plant body is second to cellulose, hemicellulose and lignin, and is widely present in some common plant foods, such as cocoa, green tea, red wine, beans, vegetables and fruits, etc. Due to the excellent antioxidant activity of polyphenol compounds, the importance of polyphenol compounds to human health is receiving more and more attention. Among them, oxidative damage is an important cause of many chronic diseases (such as cardiovascular diseases, cancer and aging), and the antioxidant function of polyphenols can play a role in preventing and treating these chronic diseases.
Polyphenols are of a wide variety and include flavonoids, tannins, ellagic acid, phenolic acids, etc., of which flavonoids and phenolic acids are the most biologically relevant class of compounds because of their important role in food and their ability to enter the circulation in the body. Research reports that after a series of metabolic processes, a large amount of low molecular weight phenolic metabolites of flavonoids enter into the internal circulation at a concentration higher than that of the parent compounds, so that neuroinflammation can be prevented or alleviated, and the development of neurodegenerative diseases can be improved. Further studies have shown that the use of gallocatechin, gallate (EGCG) or resveratrol in combination with oxaliplatin and cisplatin promotes apoptosis and inhibits proliferation of tumor cells in colorectal cancer patients, and that such enhanced cytotoxic effects are believed to be likely mediated by the autophagy pathway.
One of the major limitations of the use of flavonoids and polyphenols is their low bioavailability. The drug carrier (such as micelle, capsule, dendritic macromolecule, inorganic polyphenol nano drug carrier particle, protein, hydrogel and the like) can relieve the problems of low drug utilization rate and large toxic and side effects to a certain extent. Currently, methods of preparing microemulsions or flavonoid microcapsules, developing nanosystems or inducing enzymatic methylation, etc. have been used to enhance the absorption of flavonoids. However, these methods are complicated and costly, and most drug carriers only have a carrying function and no therapeutic function.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a polyphenol nano-drug carrier based on natural mung beans and application thereof.
The polyphenol nano-drug carrier can be directly used for treating psoriasis, can also be used for loading anti-tumor drugs and treating tumors, improves the bioavailability of flavone and polyphenol substances by a simple method, improves the transmission efficiency of the drug carrier and enhances the tumor treatment effect.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a polyphenol nano-drug carrier based on natural mung beans is prepared by decocting mung beans in water at 95-105 deg.C for 0.5-2h, centrifuging at low speed, and lyophilizing the supernatant.
According to the invention, the particle size of the polyphenol nano-drug carrier particles in the supernatant is preferably 120-200 nm, and further preferably the particle size of the polyphenol nano-drug carrier particles in the supernatant is 150-180 nm. Most preferably, the particle size of the polyphenol nano-drug carrier particles in the supernatant is 164 nm.
An application of polyphenol nano-drug carrier based on natural mung beans in treating psoriasis or loading anti-tumor drugs.
According to the invention, the polyphenol nano-drug carrier is preferably prepared by the following method:
(1) adding ultrapure water into mung bean according to the mass ratio of 1:8-12, and boiling at 95-105 ℃ for 0.5-2h to obtain a mixed solution;
(2) centrifuging the mixed solution at the rotating speed of 4000-.
According to the invention, in the step (1), the mass ratio of the mung beans to the ultrapure water is as follows: 1:10.
Preferably, in step (1), the cooking temperature is 100 ℃ and the cooking time is 1 h.
Preferably, in step (2), the centrifugation speed is 4500-5500r/min, and the centrifugation time is 4-10 min.
According to the invention, the preferable anti-tumor drug used for loading the anti-tumor drug is doxorubicin hydrochloride (DOX & HCl).
According to the invention, the specific loading method for the antitumor drug is preferably as follows:
dispersing the polyphenol nano-drug carrier with ultrapure water to obtain a resuspension solution, adding doxorubicin hydrochloride (DOX & HCl) into the resuspension solution, stirring for 8-16h to load the doxorubicin hydrochloride on the surfaces of the polyphenol nano-drug carrier particles, centrifuging, and re-dispersing in the ultrapure water to obtain the doxorubicin-loaded nano-drug dispersion solution.
According to the invention, doxorubicin hydrochloride (DOX. HCl) is preferably added in an amount such that the concentration of doxorubicin hydrochloride (DOX. HCl) in the resuspension solution is 0.1-0.5 mg/mL.
According to the invention, the particle size of the nano-drug particles loaded with adriamycin is 187 nm.
According to the invention, preferably, in order to improve the treatment effect, the polyphenol nano-drug carrier is dispersed by ultrapure water to obtain a resuspension solution, and FeCl is firstly added into the resuspension solution2·4H2O, FeCl in resuspension2·4H2The concentration of O is 3-8mg/mL, and doxorubicin hydrochloride (DOX & HCl) is added after 30 min for loading to make the final concentration of doxorubicin hydrochloride (DOX & HCl) be 0.1-0.5 mg/mL.
Adding FeCl into the heavy suspension2·4H2And O, the surfaces of the polyphenol nano-drug carrier particles are coordinated with Fe ions, and then the subsequent Fenton reaction is carried out to generate hydroxyl free radicals OH with strong oxidizing property, so that cells can be killed, and the treatment effect of the antitumor drug is greatly improved.
Preferred according to the invention, the method for psoriasis treatment is as follows: dissolving polyphenol nano-drug carrier powder in Phosphate Buffered Saline (PBS) to make the concentration of polyphenol nano-drug particles be 0.5-2.5mg/mL, and directly applying on affected parts.
Compared with the prior art, the invention has the beneficial effects that:
1. The polyphenol nano-drug carrier is obtained by a simple and direct water boiling method, has simple preparation method and low cost, can be stored for a long time after being freeze-dried into powder, and improves the bioavailability of flavone and polyphenol substances by using the simple method.
2. The polyphenol nano-drug carrier contains polyphenol and flavone components, has good antioxidant activity, so that the immune response of skin parts is adjusted, and the polyphenol nano-drug carrier can be directly used as a drug to be applied to the field of psoriasis treatment and has good treatment effect.
3. The polyphenol nano-drug carrier is used for loading anti-tumor drugs, realizes the controllable release of the anti-tumor drugs, reduces the toxic and side effects of anti-cancer drug molecules, improves the transmission efficiency of the carrier, and enhances the tumor treatment effect.
4. The polyphenol nano-drug carrier is used for loading antitumor drugs, polyphenol nano-drug carrier particles can be subjected to multifunctional modification, DOX is loaded after coordination with Fe to form a nano-drug dosage form, and OH generated by Fenton reaction is used for killing cells, so that the tumor treatment effect of DOX is promoted.
Drawings
Figure 1 is a graph of the particle size distribution of natural mung bean based polyphenol nano-drug carrier particles;
FIG. 2 is a transmission electron microscope image of polyphenol nano-drug carrier particles based on natural mung beans;
FIG. 3 is a scanning electron micrograph of polyphenol nano-drug carrier particles based on natural mung beans;
FIG. 4 is a photograph of the uptake of FITC labeled polyphenol nano-drug carrier particles by skin tissue immune cells RAW 264.7;
FIG. 5 is a graph of skin permeation results of FITC-labeled polyphenol nanoparticle, wherein a is a graph of fluorescence imaging of a mouse living body, and b is a fluorescence microscope photograph of a frozen section of a mouse skin tissue;
FIG. 6 is a graph of the effect of treatment in psoriasis model mice;
FIG. 7 is a graph of HE staining of skin tissue sections after treatment of a mouse psoriasis model is complete;
FIG. 8 is a particle size distribution diagram of the doxorubicin-loaded polyphenol nano-drug carrier;
FIG. 9 is a transmission electron microscope image of the polyphenol nano-drug carrier loaded with adriamycin;
FIG. 10 is a scanning electron micrograph of the doxorubicin-loaded polyphenol nano-drug carrier;
FIG. 11 is a particle size distribution diagram of a coordinated Fe supported doxorubicin polyphenol nano-drug carrier;
FIG. 12 is a transmission electron microscope image of a coordinated Fe-supported doxorubicin polyphenol nano-drug carrier;
FIG. 13 is a scanning electron microscope image of a coordinated Fe-supported doxorubicin polyphenol nano-drug carrier;
figure 14 is an electron paramagnetic resonance spectrum of polyphenol nanoparticle in solution;
FIG. 15 is a confocal laser photograph of detecting hydroxyl radicals in cells;
FIG. 16 is a confocal laser microscopy at 12 h after uptake of polyphenol nano-drug particles (MB-Fe-DOX NPs) by cells;
FIG. 17 is a graph of the cytotoxicity of the nano-drug carrier particles; a is the MTT profile of pure nanoparticles against MCF-7 cells, b is the MTT profile of nanoparticles against MCF-7 cells after drug loading;
FIG. 18 is a plot of the Fe ion concentration in the nano-drug particles versus the reciprocal of the relaxation time;
FIG. 19 is a magnetic resonance imaging of the nanoparticles at the tumor site in mice;
FIG. 20 is a graph of tumor size and body weight change during treatment in mice; a is the relative size of the tumor volume during treatment, b is the graph of the change in body weight of the mice during treatment;
FIG. 21 is an HE staining image of the mouse major organs after treatment was completed;
Detailed Description
The invention is further illustrated by the following examples and figures.
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Example 1
Preparing polyphenol nano-drug carrier particles based on natural mung beans:
adding 50 mL of ultrapure water into 5 g of mung beans, boiling for 1h at 100 ℃, then centrifuging for 5min at the rotating speed of 5000 r/min, removing large precipitates, reserving supernatant, and freeze-drying the supernatant for 12 h at-40 ℃ to obtain the polyphenol nano-drug carrier particles based on natural mung beans.
1) Effect of cooking time on yield of polyphenol nano-drug carrier particles
Adding 100 mL of ultrapure water into 5 g of mung beans, boiling at 100 ℃, changing boiling time to be 0.5h, 1h and 2h respectively, then centrifuging at the rotating speed of 5000 r/min for 5min to remove precipitates to obtain supernatant, freezing the supernatant at-40 ℃ for 12 h, and obtaining the yield of the polyphenol nano-drug carrier particles based on the natural mung beans under different boiling time as shown in Table 1.
TABLE 1 Effect of cooking time on the yield of polyphenol Nanoparticulate Carrier particles
| Time | Yield of polyphenol nano-drug carrier particles |
| 0.5 h | 0.37 g |
| 1 h | 0.43 g |
| 2 h | 0.46 g |
2) The obtained nano-drug carrier particles have a particle size of 164 nm (as shown in figure 1) as measured by a Malvern particle sizer, and a potential of-37.8 mV. Centrifuging the supernatant at 13000r/min, resuspending ultrapure water, dripping the resuspension solution onto a copper net containing a common carbon film, drying, performing transmission electron microscope test, wherein the test result is shown in figure 2, dripping the resuspension solution onto a silicon wafer, drying, and performing scanning electron microscope test, wherein the test result is shown in figure 3.
3) Component analysis of polyphenol nano-drug carrier particles
The BCA method, the phenol sulfate method and the ultraviolet spectrophotometer method are respectively used for detecting the components of the polyphenol nano-drug carrier particles, the detection results are shown in Table 2, and the results show that: the polyphenol nano-drug carrier particle contains 54.6% of protein, 36.0% of polysaccharide, 1.4% of polyphenol and 3.6% of flavone.
TABLE 2 Polyphenol Nanoparticulate Carrier compositions
| Composition (I) | Protein | Polysaccharides | Polyphenol | Flavone |
| Content% | 54.6 | 36.0 | 1.4 | 3.6 |
4) Uptake of FITC-labeled polyphenol nano-drug carrier by RAW264.7 cells
Seeding RAW264.7 cells in a laser confocal dish at a density of 30000 cells/well; after the cells are attached to the wall for 12 h, adding FITC marked polyphenol nano-drug carriers with equal concentration into four confocal dishes respectively, adding nano-particles for 0h, 3 h, 6h and 12 h, washing the mixture for three times by PBS, fixing the mixture by 4% paraformaldehyde, staining cell nuclei and cell membranes by Hoechst 33342 and white Germ AgglutininAlexa Fluor 633conjugate respectively, and observing the cells under a laser confocal microscope. The observation results are shown in fig. 4 (fig. 4 is a graph after ashing), and blue color of cell nuclei, red fluorescence of cell membranes and green fluorescence of FITC-labeled nanoparticles were observed under a confocal laser microscope. With the increase of time, the green fluorescence in the cells is obviously increased, which shows that the polyphenol nano-drug particles phagocytosed in the cells are gradually increased along with the time.
5) Penetration of polyphenol nano-drug carrier on mouse skin surface
To evaluate the penetration effect of polyphenol nano-drug carrier in mouse skin. First, the skin hair on the back of the mice was shaved with a razor and depilatory cream. Then, the FITC marked polyphenol nano-drug carrier particles are locally smeared on the back skin of an experimental group mouse, and the mouse smeared with phosphate buffered saline solution is used as a control group. The dorsal skin of the anesthetized mice was then imaged using a mouse fluorescence imaging system, with the results shown in fig. 5a (fig. 5a is post-ashing); the imaging result shows that the back of the mouse coated with the polyphenol nano-drug carrier particles shows obvious green fluorescence, and the back of the mouse coated with PBS has no fluorescence signal. Then, the skin tissue of the back of the mouse was cryo-sectioned, and the result is shown in fig. 5b (fig. 5b is the image after ashing), and the longitudinal section of the skin was observed under a fluorescence microscope, and green fluorescence showed that the polyphenol nano-drug carrier particles well penetrated into the skin tissue of the mouse.
6) Treatment of psoriasis model mice
Mice were divided into three groups (n =6), Control group, IMQ + PBS group, IMQ + NPs group, and the dorsal skin hair of the mice was shaved with a razor and depilatory cream. The mice in the Control group were not smeared with the IMQ cream, and the mice in the IMQ + PBS group and the IMQ + NPs group were smeared with 62.5mg of IMQ cream (5% imiquimod) on the back skin every day, and were continuously smeared for one week to induce the psoriasis model mice. In the treatment process, vaseline cream is respectively applied to the Control group mice, 20 mu L of sterile Phosphate Buffer Solution (PBS) is applied to the IMQ + PBS group mice, and 20 mu L of polyphenol nano-drug carrier solution is applied to the IMQ + NPs experimental group mice. Applied once a day for a week. The severity of skin inflammation was assessed according to the psoriasis area severity index and previously reported clinical scoring criteria. As can be seen from fig. 6, the Control group mice had no obvious scales, induration, erythema and thickening, the IMQ + PBS group mice had severe back scales, induration, erythema and thickening, while the IMQ + NPs experimental group mice showed slight scales, induration, erythema and thickening after one week of treatment, indicating that the polyphenol nano-drug particles inhibited the symptoms of psoriasis.
7) Tissue sections after treatment of psoriasis model mice
Skin hyperplasia at the affected area of psoriasis is an important criterion for the severity of psoriasis. Mice after the end of treatment were euthanized and their dorsal skin tissue sections were HE stained. The results in fig. 7 show that the skin thickening of the back of the mice in the IMQ + PBS group is significant compared with the skin thickening of the Control group, and the skin thickening of the mice in the IMQ + NPs experimental group is significantly alleviated.
Example 2
Preparation of adriamycin-loaded polyphenol nano-drug carrier
The polyphenol nano-drug carrier is resuspended by ultrapure water to obtain a resuspension solution, doxorubicin hydrochloride (DOX & HCl) with the concentration of 0.5mg/mL is added into the resuspension solution to load the doxorubicin hydrochloride on the surfaces of the polyphenol nano-drug carrier particles, then the polyphenol nano-drug carrier solution is centrifuged at the rotating speed of 13000r/min for 10 min to remove free doxorubicin hydrochloride, and the doxorubicin hydrochloride is redispersed in the ultrapure water to obtain the doxorubicin-loaded polyphenol nano-drug carrier solution.
8) The polyphenol nano-drug particle solution loaded with adriamycin has the particle size of 187 nm (as shown in figure 8) measured by a Malvern particle sizer, and the potential is-27.4 mV. Centrifuging the polyphenol nano-drug carrier solution loaded with the adriamycin at the rotating speed of 13000r/min, dripping the solution on a copper net containing a common carbon film after the ultrapure water is resuspended, airing, and carrying out a transmission electron microscope test, wherein the test result is shown in figure 9, and the polyphenol nano-drug carrier solution loaded with the adriamycin is dripped on a silicon wafer to be aired, and then carrying out a scanning electron microscope test, and the test result is shown in figure 10.
Example 3
Preparation of polyphenol nano-drug loaded with adriamycin after coordination of Fe
Resuspending the polyphenol nano-drug carrier with ultrapure water to obtain a resuspension solution, and adding FeCl into the resuspension solution2·4H2O, enabling the concentration of Fe ions in the resuspension to be 5 mg/mL; after 30min, the solution is separated at the rotating speed of 13000r/minPerforming centrifugation for 10 min, then re-dispersing the nanoparticles in ultrapure water, adding 20 mu L of doxorubicin hydrochloride (DOX & HCl) solution to make the concentration of the doxorubicin hydrochloride reach 0.3mg/mL, and loading the doxorubicin hydrochloride on the surfaces of the polyphenol nano-drug carrier particles to obtain a polyphenol nano-drug solution with coordinated Fe loaded with doxorubicin;
9) the nanoparticle solution was placed in a Malvern particle sizer to measure a particle size of 193 nm (as shown in FIG. 11), with a potential of-21.3 mV. Centrifuging the nano-drug particle solution at the rotating speed of 13000r/min, dripping the resuspended ultrapure water on a copper net containing a common carbon film for drying, and carrying out transmission electron microscope test, wherein the test result is shown in figure 12, dripping the resuspended nano-drug particles on a silicon wafer for drying and carrying out scanning electron microscope test, and the test result is shown in figure 13.
10) Detection of hydroxyl radical by electron paramagnetic resonance spectroscopy (ESR)
Fe2+Hydroxyl radicals that can be generated by reaction with hydrogen peroxide can be detected by electron paramagnetic resonance spectroscopy. Firstly, preparing 10 mmol/L hydrogen peroxide solution, 0.2 mol/L DMPO solution and polyphenol nano-drug carrier (MB-Fe-DOX NPs) solution (Fe ions 0.25 mmol/L) which coordinates Fe and loads adriamycin; then respectively adding 40 uL of the three groups of solutions into 1 mL of deionized water, and uniformly mixing; paramagnetic signals of the hydroxyl radicals are detected after 1min, the test result is shown in FIG. 14, and FIG. 14 shows that the ratio of the signal peak height to the signal peak height is 1:2:2:1, which is a characteristic peak of the hydroxyl radicals, so that the generation of the hydroxyl radicals can be judged.
11) Detection of intracellular hydroxyl radicals
Cells were seeded at 30000 cells/well density in a laser confocal dish. After the cells adhere to the wall for 12 hours, respectively adding a pure culture medium (Control group) and an adriamycin hydrochloride aqueous solution (FreeDOX group), an adriamycin-loaded polyphenol nano-drug carrier solution (MB-DOXNPs group) and a Fe-coordinated adriamycin-loaded polyphenol nano-drug carrier solution (MB-Fe-DOX NPs group) into different confocal dishes; incubating in incubator for 6 h, adding 100ul10umol/L DCFH-DA solution into the confocal dish, incubating in incubator for 20min, washing with serum-free culture medium for three times, and adding 500 ul serum-free pure culture mediumAnd observing under a laser confocal microscope. The test results are shown in FIG. 15 (FIG. 15 is a graph after ashing), and it can be seen through laser irradiation that the Control group, Free DOX group and MB-DOXNPs group show weak green fluorescence, while the MB-Fe-DOX NPs group shows obvious green fluorescence, which indicates that more hydroxyl radicals are generated in the MB-Fe-DOX NPs group because of the Fe ions and H in the cells2O2The fenton reaction occurs to generate a large number of hydroxyl radicals.
12) Cellular uptake of Nanoparticulate drug particles
Human breast cancer cell (MCF-7) cells were seeded on a confocal culture dish and cultured in 1640 medium containing 10% fetal bovine serum at 37 ℃ in a 5% carbon dioxide incubator for 12 h. After cell attachment, the cells were washed three times with PBS and the prepared MB-Fe-DOX NPs containing an equivalent doxorubicin concentration of 4 μ g/mL were added. After 12h of cell culture, the cells were fixed with 4% paraformaldehyde, followed by staining of the cell nuclei with Hoechst, cell membranes with WGA dye labeled with AF 633, and observation under a confocal laser microscope. The test results are shown in fig. 16, and by observation of a confocal microscope, a distinct red fluorescence is shown in the cell nucleus after incubation with the nanoparticles for 12h, which indicates that the released DOX enters the cell nucleus after the DOX-loaded nanoparticles are phagocytosed by the cells.
13) Cytotoxicity of polyphenol nano-drug carrier particles
a) Cell culture
Human breast cancer cell MCF-7 is cultured in 1640 liquid culture medium containing 10% fetal calf serum, and cultured in an incubator at 37 deg.C and 5% carbon dioxide.
b) Biocompatibility of polyphenol drug carriers
Cells were seeded in 96-well plates at a density of 10000 cells/well. After 12 hours of adherence, polyphenol nano-drug carriers with mass concentrations of 100 mug/mL, 200 mug/mL, 300 mug/mL, 400 mug/mL and 500 mug/mL are respectively added into five groups, and a pure culture medium is added into a control group. After 24 h incubation, 10. mu.L of 5 mg/mL MTT solution was added to each well and after 4 h incubation in the incubator, the absorbance at 570nm was measured on a microplate reader according to MTT standards. The cell viability measurement result is shown in fig. 17a, the cell viability of the polyphenol nano-drug carrier of the invention is close to one hundred percent under the dosage of 500 mug/mL, which indicates that the polyphenol nano-drug carrier has no obvious toxicity to cells, and the polyphenol nano-drug carrier is proved to have good biocompatibility.
c) Killing effect of polyphenol nano-medicine on cells
Cells were seeded in 96-well plates at a density of 10000 cells/well. After 12 h of adherence, Free DOX, MB-DOX NPs and MB-Fe-DOX NPs solutions with doxorubicin concentrations of 0.5, 1.0, 1.5, 2.0 and 4.0 mug/mL are added, and after incubation in an incubator for 24 h, the absorbance value at 570nm is measured on a microplate reader according to MTT (maximum Transmission transfer protocol) operation standards. The cell viability assay results are shown in FIG. 17b, where the killing of cells by Free DOX group was enhanced with increasing DOX concentration, the killing of cells by MB-DOXNPs group corresponding to the same concentration of doxorubicin was slightly stronger than that by Free DOX group, and finally the killing of cells by MB-Fe-DOX NPs group was strongest due to the enhancement of Fenton reaction.
14) Magnetic resonance imaging
a) Relaxation rate of MB-Fe-DOX NPs dispersion liquid magnetic resonance imaging
Firstly, quantifying the concentration of Fe ions in a nano-drug solution by ICP (inductively coupled plasma), then preparing a series of dispersions with concentration gradients, placing the dispersions in a 2 mL centrifuge tube, and fixing the scanning rotation time TE: 11ms, TR: 400ms T of the solution1And (5) signal detection. As shown in FIG. 18, 1/T1Has a linear relation with the concentration of Fe ions, and the relaxation value is 6.5 mM calculated according to the slope-1S-1The relaxation value meets the requirements of magnetic resonance imaging.
b) Magnetic resonance imaging in mouse body
The concentration of 100 mu L is 107cells/mL of mouse breast cancer cells (4T 1) are inoculated in the axilla of Balb/c mice (about 20 g), and the tumor volume reaches 200 mm3Left and right, a dose of MB-Fe-DOX NPs dispersion was injected intravenously, as shown in fig. 19, fixed scan rotation time TE: 11ms, TR: the tumor position of the mouse is scanned with time within 400ms, and the magnetic resonance imaging effect of the tumor part of the mouse can be seen at any timeThe contrast ratio is strongest at 6 h, and the imaging effect is best due to the change brought by the enrichment and metabolism of the nano-drug particles at the tumor part.
15) The examples are presented to illustrate the anti-tumor results of Balb/c mice after intravenous administration.
a) Mouse inoculation of tumor
Six-week-old Balb/c mice were randomly divided into 4 groups (n = 6) and 100. mu.L of 10 concentration was administered7cells/mL mouse mammary cancer cells (4T 1) were inoculated in the right axilla of mice (about 20 g) to achieve a tumor volume of 100 mm3On the left and right sides, intravenous administration tumor suppression treatment experiments were performed.
b) Tumor inhibition experiment
The experimental mice were divided into four groups (n = 6), PBS group, FreeDOX group, MB-DOXNPs group, MB-Fe-DOXNPs group, in which DOX was administered at a dose of 3 mg/kg. Mice were dosed with 200 μ L per mouse on days 0, 2, and 4, respectively. Tumor size and mouse body weight were measured every other day, and the mice were euthanized after continuous monitoring for 14 days. Tumor volume is used to express tumor suppression results, and it can be seen from FIG. 20a that the tumors of the PBS group mice are rapidly growing all the time, while the tumors of the FreeDOX group mice are slower than those of the PBS group, the tumor growth is partially suppressed, and the tumor suppression of the MB-DOX NPs group is enhanced, but the tumor growth can not be effectively controlled. The tumor growth of mice in the MB-Fe-DOX NPs group is obviously inhibited, which shows that the treatment effect is the best, and proves that the Fenton reaction in MB-Fe-DOX NPs drug particles is cooperated with DOX chemotherapy to play a role in enhancing the effect of inhibiting the tumor growth. Fig. 20b is a graph of the weight change of the mice during the treatment period, and the weight of the mice is not significantly changed during the treatment period, which shows that the nano-drug particles have good biocompatibility and less toxic and side effects.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (3)
1. An application of a polyphenol nano-drug carrier in preparing a preparation loaded with an anti-tumor drug,
the polyphenol nano-drug carrier is prepared by the following method:
(1) adding ultrapure water into mung bean according to the mass ratio of 1:8-12, and boiling at 95-105 ℃ for 0.5-2h to obtain a mixed solution;
(2) centrifuging the mixed solution at the rotating speed of 4500-;
the anti-tumor drug is doxorubicin hydrochloride (DOX & HCl); the specific loading method of the antitumor drug comprises the following steps:
dispersing a polyphenol nano-drug carrier with ultrapure water to obtain a resuspension solution, adding doxorubicin hydrochloride (DOX & HCl) into the resuspension solution, stirring for 8-16h to load the doxorubicin hydrochloride on the surfaces of polyphenol nano-drug carrier particles, then centrifuging to remove free doxorubicin hydrochloride, and re-dispersing the nanoparticles in the ultrapure water to obtain a doxorubicin-loaded nano-drug dispersion solution; the concentration of doxorubicin hydrochloride (DOX & HCl) in the heavy suspension is 0.1-0.5mg/mL, and the particle size of the nanoparticle loaded with doxorubicin is 187 nm.
2. The use of claim 1, wherein in step (1), the weight ratio of mung bean to ultrapure water is: 1: 10; the cooking temperature is 100 deg.C, and the cooking time is 1 h.
3. The use according to claim 1, wherein the loading method of the antitumor drug comprises the following steps: dispersing the polyphenol nano-drug carrier with ultrapure water to obtain a resuspension solution, and adding FeCl into the resuspension solution2·4H2O, FeCl in resuspension2·4H2O concentration of 3-8mg/mL, centrifuging after 0.5 h, then re-dispersing the nanoparticles in ultrapure water, and then adding doxorubicin hydrochloride (DOX. HCl) for loading to make the concentration of the doxorubicin hydrochloride (DOX. HCl) reach 0.1-0.5 mg/mL; the particle size of the obtained polyphenol nano-medicament of coordinated Fe loaded adriamycin is 193 nm.
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