CN112957469A - PH-responsive magnetic nano core-shell drug-loading system and construction method and application thereof - Google Patents
PH-responsive magnetic nano core-shell drug-loading system and construction method and application thereof Download PDFInfo
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- CN112957469A CN112957469A CN202110226722.9A CN202110226722A CN112957469A CN 112957469 A CN112957469 A CN 112957469A CN 202110226722 A CN202110226722 A CN 202110226722A CN 112957469 A CN112957469 A CN 112957469A
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention provides a pH-responsive magnetic nano core-shell drug-loading system and a construction method and application thereof. The pH-responsive magnetic nano core-shell drug delivery system provided by the invention has magnetic targeting property, an external magnetic field can be used for causing the movement of magnetic nanoparticles, when a drug-containing carrier reaches cancer cell tissues, the acidic micro-area environment (pH is 5.8) enables a capping agent 'gatekeeper' to degrade and break bonds, and 'gate' is opened, so that drugs are released to kill tumor cells, the cytotoxicity and positioning conditions of materials are monitored through a series of cell experiments, and the increase of apoptosis amount is found along with the increment of drug concentration, so that the novel pH-responsive synergistic drug controlled release system has potential application value for the treatment of cancers.
Description
Technical Field
The invention belongs to the technical field of nano treatment, and particularly relates to a pH-responsive magnetic nano core-shell drug loading system and a construction method and application thereof.
Background
In recent years, many anticancer drugs have been studied and emerged. Despite our progress, cancer has not been completely cured to date. Many anticancer drugs target specific molecules on cancer cells and can also enter normal cells and produce adverse effects. The development of effective drug carriers or administration routes is very important for improving the targeting of drugs. Currently, various types of nanoparticles have been reported as potential drug delivery or diagnostic drugs, including liposomal nanoparticles, polymeric and metallic nanoparticles, and other inorganic nanoparticles. Biocompatible rice particles with diameters from a few nanometers to 250 nanometers are considered to have great potential in cancer drug delivery. Meanwhile, since nanoparticles can retain vascular leakage of tumor vessels in solid tumors due to their relatively large size, many of the currently under-developed nanocarrier cancer therapies rely on an Enhanced Permeability and Retention (EPR) effect to passively accumulate and kill cancer cells in the tumor microenvironment.
In the nano materials for diagnosis and treatment, superparamagnetic ferroferric oxide microspheres (SPION) are paid the attention of researchers of antitumor drugs, and the tiny size property enables the microspheres to be accumulated at the position of a pathological change part by fully utilizing the effects of permeation enhancement and retention, and can realize the fixed-point delivery under the action of an external magnetic field, thereby achieving the aim of targeted drug delivery. As is well known, the carrier of the antitumor drug has certain toxic and side effects on human cells, and zirconium dioxide (ZrO)2) Widely used in the oral medicine industry and benefited from better biocompatibility, thus utilizing zirconium dioxide structureThe built mesoporous drug delivery system has low biological toxicity, and the rich mesoporous structure of the mesoporous drug delivery system improves the loading rate of anticancer drugs. Bismuth oxide (Bi) widely used in various industries2O3) Not only are less toxic, but also protect cytotoxic drugs from premature release, and they themselves may also release bi at their destination3+Exert cytotoxic effect. Therefore, the development of a simple and rapid strategy for combining a pH response and a magnetic response drug delivery system is expected to further promote the application of the drug delivery system in the biomedical field.
Disclosure of Invention
The invention aims to provide a pH-responsive magnetic nano core-shell drug delivery system, which is characterized in that a zirconia mesoporous shell layer is modified on the surface of a magnetic core, a drug such as an anticancer drug Daunomycin (DNM) is loaded in a pore channel of the mesoporous shell layer, nano bismuth oxide is used as a pH-responsive gating blocking pore channel, the system has magnetic targeting property, and the drug can be released in an acidic environment in vivo.
The second purpose of the invention is to provide a construction method of the pH-responsive magnetic nano core-shell drug carrier system.
The third purpose of the invention is to provide the application of the pH-responsive magnetic nano core-shell drug carrier system.
For the purpose, the invention is realized by the following technical schemes:
a pH-responsive magnetic nano core-shell drug-loading system is characterized in that a mesoporous shell layer is modified on the surface of a magnetic inner core to form a core-shell structure, an anti-tumor drug is electrostatically adsorbed in a pore channel of the mesoporous shell layer, and then a pH-sensitive substance (such as nano bismuth oxide) is electrostatically adsorbed in the pore channel to be used as a pH-responsive gating plugging pore channel.
The invention takes nano ferroferric oxide as a magnetic core, zirconium dioxide as a mesoporous shell and Bi2O3Blocking the pores of the mesoporous shell, therefore, a preferred embodiment of the present invention is Bi2O3Plugged mesoporous ZrO2The system is based on the characteristics and properties of cancer cells, and takes zirconium dioxide coated ferroferric oxide as a biological materialFe3O4@ZrO2And in a pH-sensitive system-Bi2O3Nano particle pair of biological material Fe loaded with anticancer drug3O4@ZrO2The obtained drug-carrying system is used for carrying anticancer drugs. Due to ZrO2Presence of a coating layer, nano Fe3O4Is protected by a magnetic core, and the surface of the magnetite particles is suitable for uniformly coating mesoporous ZrO2. The present invention is directed to the use of Bi2O3The oxide is a 'gatekeeper', the targeted antitumor drug system is controlled by the acid-base environment in vivo, and Bi is in the acid environment2O3The drug channel is controlled to be opened to release the drug. Under the action of an external magnetic field, the drug-loaded nano particles enter tumor cells and are influenced by the acidic microenvironment of the tumor cells, and the blocking substances are dissolved to release the drugs. Therefore, in order to kill cancer cells, the pH sensitive drug-loaded nanoparticles can enter an acidic microenvironment of the tumor cells in a targeted manner under the action of an external magnetic field, so that the blockage can be dissolved and the drug can be released.
The drug-carrying system is a targeted anti-tumor drug system, and the anti-tumor drug is anthracycline antibiotic.
Further, the anthracycline is doxorubicin, daunomycin, idarubicin, mitoxantrone, or epirubicin.
On the other hand, the construction method of the pH-responsive magnetic nano drug-carrying system comprises the following steps:
(1) the superparamagnetic ferroferric oxide nano particles are prepared by a chemical coprecipitation method. The method specifically comprises the following operations: first, 2.7g of FeCl was weighed3·6H2O and 1.20g FeCl2·4H2O, adding 200mL of deoxidized deionized water into a 500mL round-bottom flask in a nitrogen environment, heating to 80 ℃ in an oil bath kettle, reacting for 10min at a constant temperature, then quickly adding 12mL of 25% ammonia water, testing the pH range by using a pH test paper, adjusting the pH value to 10-11, immediately turning the liquid black, and increasing the stirring speed to 4.0 krp/min; stopping reaction after 30min, cooling to room temperature, performing adsorption separation by using magnetism, and alternately washing with ethanol and deionized water to obtain magnetic nanoparticlesThree sub-times, 40mL of deionized water was added and the mixture was sealed, and 1mL of the sample was taken from the mixture in a test tube and the concentration was determined by differential method.
(2)Fe3O4@ZrO2The preparation of (1): and (2) ultrasonically dispersing the ferroferric oxide synthesized in the last step uniformly, calculating and sucking 200mg of turbid liquid into a beaker, cleaning the beaker with deionized water for 3 times, dispersing the nanoparticles into a 500mL three-neck flask with 140mL of absolute ethanol, adding 60mL of deionized water, stirring vigorously at room temperature, quickly adding 2mL of 25% concentrated ammonia water into the three-neck flask, reacting for 20min, dropwise adding 0.06mL of 0.1mol/L zirconium oxychloride solution, standing and separating black nanoparticles by using magnetic adsorption after reacting for 24 hours, and alternately washing the flask with ethanol and distilled water for 3 times to remove redundant reactants. Dispersing the washed nano-microspheres in a mixed solution of 70mL of ethanol and 70mL of water containing 350mg of cetyltrimethylammonium bromide (CTAB), adding 2mL of 25% ammonia water, reacting for 20min under vigorous stirring at room temperature after uniform ultrasonic dispersion, then dropwise adding 1mL of 0.1mol/L zirconium oxychloride solution, reacting for 24h, separating out magnetic nanoparticles under the action of an external magnetic field after the reaction is finished, and alternately washing for 3 times by using ethanol and water to obtain Fe3O4@ZrO2And (4) nano microspheres.
(3) Preparation of MMZr: fe containing CTAB template agent in the last step3O4@ZrO2The nano particles are ultrasonically dispersed in 200mL calcium nitrate-absolute ethyl alcohol (10mg/mL), the nano particles are transferred into a 500mL three-neck flask, the stirring speed is 4.0krp/min, the flask is placed in an oil bath kettle at 80 ℃ for heating and refluxing for 6h, the flask is kept to be at room temperature, then the magnetic nano particles are washed for 2-3 times by deionized water under the magnetic action to ensure that CTAB is completely washed away, and the flask is sealed by 20mL deionized water to obtain the MMZr (Fe without template agent CTAB) with a core-shell structure3O4@ZrO2) And (4) nano microspheres.
(4) Adding Bi after adding the antitumor drug and successfully loading the drug to obtain MMZr @ X2O3The blocked microsphere containing the medicine MMZr @ Bi is obtained2O3-X, wherein X represents an antineoplastic drug; the method specifically comprises the following operations: adding antitumor drug into MMZr nanometer microsphere to obtain medicine containing preparationMicrospheres; then 5mg of nano Bi is added into the cleaned nano microspheres2O3Dispersing evenly, and plugging for 12h at room temperature in dark. After the reaction is finished, adsorbing and standing the carrier, washing the surface for 2-3 times by using PBS (phosphate buffer solution) with the pH value of 7.40, and adding excessive Bi in the test tube2O3Is removed.
After systemic administration, larger particles with diameters greater than 200nm are usually sequestered by the spleen due to mechanical filtration and eventually removed by the phagocytic system, resulting in a reduction in blood circulation time. On the other hand, smaller particles less than 10nm in diameter are rapidly removed by extravasation and renal clearance. Particles from 10-100nm are the best choice for intravenous injection and exhibit the longest blood circulation time. The aperture of the mesoporous synthesized by the system is between 2 nanometers and 50 nanometers.
Compared with the prior art, the invention has the following beneficial effects:
the pH-responsive magnetic nano core-shell drug delivery system provided by the invention has magnetic targeting property, and can use an external magnetic field to cause the movement of magnetic nanoparticles, wherein the pH response is that the pH value of tumor cells is lower than that of normal cells, when a drug-containing carrier reaches cancer cell tissues, the acidic micro-area environment (pH is 5.8) enables a capping agent ' gate keeper ' to degrade and break bonds, and the gate ' is opened, so that drugs are released to kill the tumor cells, the cytotoxicity and positioning conditions of the materials are monitored through a series of cell experiments, and the increase of apoptosis is found along with the increase of drug concentration, so that the novel pH-responsive synergistic drug controlled-release system has potential application value for the treatment of cancers.
Drawings
FIG. 1 shows MMZr @ Bi2O3-a scheme for the preparation of DNM nanoparticles;
FIG. 2 is a mechanism diagram of the controlled release of pH-responsive drug-loaded nanosystems to kill cancer cells under the action of an external magnetic field;
FIG. 3 shows MMZr @ Bi2O3-electron microscopy of DNM nanoparticles;
FIG. 4 is Fe3O4、Fe3O4@ZrO2-Zeta potential map of CTAB, MMZr nanoparticles;
FIG. 5 shows DNM, MMZr @ Bi2O3、MMZr@Bi2O3-ultraviolet spectrum of DNM nanoparticles;
FIG. 6 shows DNM, MMZr and MMZr @ Bi2O3-infrared spectrum of DNM nanoparticles;
FIG. 7 is a diagram showing the magnetic dispersion effect of the magnetic nano-carrier according to the present invention;
FIG. 8 is a graph of the release rate of the non-blocking (A) and blocking (B) nanocarriers stimulated at different pH conditions;
FIG. 9 shows the concentration of different empty carriers MMZr @ Bi in the blood compatibility experiment2O3A photograph of hemolysis mixed with a suspension of red blood cells;
FIG. 10 shows the concentrations of nanoparticles MMZr and MMZr @ Bi2O3、Bi2O3、DNM、MMZr@Bi2O3-effect of DNM on cell survival of human normal cells Hacat, tumor cells a549, MCF-7;
FIG. 11 is a photograph of the magnetic field area and the opposite magnetic field-free area in the petri dish at 0h and 24h in the magnetic targeting test;
FIG. 12 is a Prussian blue staining photograph of different tumor cells by the magnetic nano-carrier system of the present invention;
FIG. 13 cellular uptake of magnetic nanocarriers of the invention by tumor cells A549, MCF-7;
FIG. 14(A) is the photo of the change of scratches at the same positions of 0h and 24h under the optical microscope in the cell migration test, and (B) is the comparison of the migration areas of the drug-loaded nanoparticles and the blank group in the cell migration test;
FIG. 15 shows the same DNM, MMZr @ Bi2O3-cell growth cycle map at DNM concentration;
FIG. 16 shows MMZr @ Bi at different concentrations for fluorescence microscopy2O3-a map of apoptosis detected under DNM conditions;
FIG. 17 shows MMZr @ Bi at different concentrations in a flow cytometer2O3-a map of apoptosis quantitatively detected under DNM conditions;
FIG. 18 is a graph of mitochondrial membrane potential detection at different dosing concentrations detected under a flow cytometer;
figure 19 is a flow cytometer autophagy plot at different dosing concentrations.
Detailed Description
In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
The medicines involved in the invention are abbreviated as follows:
DNM: daunomycin, FBS: fetal bovine serum, MTT: 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt and CTAB (cetyl trimethyl ammonium bromide).
The construction method of the pH responsive magnetic nano drug-carrying system of the antitumor drug comprises the following steps:
step 1: the superparamagnetic ferroferric oxide nano particle is prepared by a chemical coprecipitation method, and 2.7g FeCl is weighed firstly3·6H2O and 1.20g FeCl2·4H2O, adding 200mL of deoxidized deionized water into a 500mL round-bottom flask in a nitrogen environment, heating to 80 ℃ in an oil bath kettle, reacting for 10min at a constant temperature, then quickly adding 12mL of 25% ammonia water, testing the pH range by using a pH test paper, adjusting the pH value to 10-11, immediately turning the liquid black, and increasing the stirring speed to 4.0 krp/min; and stopping the reaction after reacting for 30min, cooling to room temperature, performing adsorption separation by using magnetism, alternately washing the prepared magnetic nanoparticles by using ethanol and deionized water for three times, finally adding 40mL of deionized water for sealing, taking 1mL of sample from the solution in a test tube, and measuring the concentration of the sample by using a differential method.
And step 3: preparing a nano-carrier of MMZr, namely preparing Fe containing CTAB template in the step 23O4@ZrO2The nanoparticles of (1) were ultrasonically dispersed in 200mL of calcium nitrate-anhydrous ethanol (10mg/mL), transferred to a 500mL three-necked flask, stirred at 4.0krp/min, the flask was placed in an oil bath at 80 ℃ and heated under reflux for 6h, left to stand at room temperature and then the magnetic nanoparticles were washed with deionized water 2-3 times under magnetic action to ensure complete washing of CTAB, sealed with 20mL of deionized water, and 1mL of the solution was taken out of the tube and the concentration thereof was determined by differential method.
And 4, step 4: firstly, 5mg of the nano-carrier in the step 3 is taken out to be placed in a test tube, and 4mL of DNM solution with the concentration of 1.0mg/mL is added. Slightly shaking, magnetically adsorbing and standing, sucking 0.2mL of supernatant as a sample for 0h, placing the test tube in a constant temperature shaking table (150rp/min, 30 ℃) and shaking in the dark, immobilizing for 24h and 48h, taking 0.5mL of supernatant, measuring the concentration of the supernatant by using an ultraviolet spectrophotometer, and calculating the immobilized amount. And recovering the supernatant after immobilization for 48h, and washing the nanospheres by using PBS (phosphate buffer solution) with the pH value of 7.40 to obtain the drug-immobilized magnetic nanospheres.
And 5: adding 5mg of nano Bi into the cleaned nano microspheres2O3Dispersing evenly, and plugging for 12h at room temperature in dark. After the reaction is finished, adsorbing and standing the carrier, washing the surface for 2-3 times by using PBS (phosphate buffer solution) with the pH value of 7.40, and adding excessive Bi in the test tube2O3Is removed.
Example 1 for MMZr @ Bi2O3Preparation and characterization of the-DNM nanosystems
MMZr@Bi2O3The preparation flow of the-DNM nano system is shown in figure 1The mechanism of antitumor study is shown in FIG. 2, the system adopts coprecipitation method to synthesize magnetic core, and soft template method to construct mesoporous Fe3O4@ZrO2With nano Bi2O3The nano-carrier immobilized with the DNM drug for gated plugging responds to release of the drug in a low-pH environment of tumor cells.
Scanning electron microscope (TEM) and zeta potential analyzer results As shown in FIGS. 3 and 4, the size distribution and surface charge of the nanoparticles were obtained, demonstrating ZrO2Has been successfully coated with Fe3O4And CTAB has been removed clean.
The ultraviolet absorption spectrum result is shown in fig. 5, DNM has an absorption peak at 480nm, the pure carrier has no absorption peak, and the drug-loaded nanoparticles have an ultraviolet absorption peak at 480nm, which proves that the drug is immobilized.
The results of infrared absorption spectroscopy are shown in FIG. 6, in which C-N and N-H stretches in DNM have characteristic absorption peaks of 1616cm-1，Fe3O4@ZrO2Middle 602cm-1Has a characteristic absorption peak of 570cm of Zr-O bond and Fe-O bond-1And (5) vibrating. In addition, at MMZr @ Bi2O3A characteristic peak 1411cm for daunomycin was also observed in DNM-1Prove ZrO2Has been successfully coated with Fe3O4And the drug has been successfully loaded.
FIG. 7 shows the measurement results of the magnetic response effect of the nano system. The magnetic response performance of the carrier is good after the zirconium oxide is wrapped. And the carrier is not separated in a short time under the condition of no magnetic field, so that the dispersibility of the magnetic nano-microspheres is proved to be better.
Example 2 sustained release test of drug-loaded nanocarriers under different pH conditions
In order to verify the drug release condition of the nano-carrier compound under different pH stimulation conditions, the non-blocked and non-blocked nano-particles after drug loading are respectively dissolved in the same volume and different concentrations, and the sustained release rate is calculated after ultraviolet measurement of supernatant after sustained release in 0h,1h,2h,4h,6h,8h,12h and 24h, and the result is shown in figure 8. The drug release is more along with the enhancement of the acidic environment, which shows that the drug-loaded magnetic fluid shows pH responsiveness, and the characteristic is very important for the intelligent targeted therapy of cancer cells.
Experimental example 3 cell experiment
Cell culture
Cell lines A549 cells (human lung cancer cells) and MCF-7 (human breast cancer cells) were obtained from the university of Guangdong pharmacy institute of medicine, and Hacat cells (human normal skin cells) were obtained from the university of Guangdong pharmacy institute of college. DMEM medium containing 10% FBS and 1% penicillin-streptomycin is used as growth environment, and the growth environment is 5% CO at 37 DEG C2Culturing in a cell culture box under the condition of optimal humidity, changing a culture medium for 2-3 days, carrying out passage once, and finally selecting the cells in the logarithmic growth phase with good conditions for experiment.
Blood compatibility test
Deionized water and physiological saline are respectively used as positive control and negative control, 2.5mL of 2% erythrocyte suspension is respectively added into a carrier with the final concentration of 3mg/mL, the OD value is measured, the hemolysis rate is calculated, and the phenomenon of hemolysis under beating is observed. The results are shown in fig. 9, which shows that the supernatant of the test tube added with the magnetic fluid is light yellow, while the test tube added with the ultrapure water has hemolysis, and the hemolysis rate of the magnetic fluid is less than 5% of the hemolysis rate of the medical material according to the hemolysis rate, thus proving that the carrier can be safely used for medical treatment.
Cell viability assay-MTT assay
Determination of A549 cells in free DNM, MMZr @ Bi Using the MTT assay2O3、Bi2O3、DNM、MMZr@Bi2O3Cell viability in the presence of DNM. Tumor cells A549, MCF-7 cells and human normal skin cells Hacat with good logarithmic phase growth condition are taken, respectively digested into single cell suspension by 0.25% pancreatin, and after cell counting is carried out on a blood counting cell plate, the cell density is adjusted to be 2 multiplied by 10 by using a complete culture medium4cell/ml, inoculated into 96-well plates at 100. mu.L/well, at 37 ℃ with 5% CO2Culturing for 24h in an incubator; after the cells are attached to the wall, the culture medium is removed, and each group is added with the mixture containing free DNM, MMZr and MMZr @ Bi into each hole2O3、Bi2O3、DNM、MMZr@Bi2O3100. mu.L of culture Medium for DNM samples, set at 5 concentrationsAnd 5 multiple wells are arranged for each concentration, and the culture is carried out in an incubator. And 4h before the culture is finished, sucking out the culture medium containing the medicine in the plate, adding 100 mu L of culture medium containing 10% MTT, continuing to culture for 4h, removing the supernatant after the culture is finished, adding 150 mu L of DMSO into each hole, slightly shaking for reaction for 10min to fully dissolve crystal particles, and measuring the OD value at the position of 490nm wavelength on an enzyme-labeling instrument.
As shown in FIG. 10, in cancer cells, the pure carrier MMZr was not too toxic and the nanoparticles Bi were2O3And MMZr @ Bi2O3Has certain cytotoxicity, and simultaneously MMZr @ Bi2O3The cytotoxicity of DNM was stronger than that of pure DNM, indicating MMZr @ Bi2O3-DNM has a synergistic anti-cancer effect; meanwhile, compared with a pure drug DNM drug loading system MMZr @ Bi under the same concentration condition2O3The DNM is more harmful to normal cells, which indicates that the MMZr @ Bi synthesized by the invention2O3The DNM nano-particles have a targeting effect and stimulate the release of pH response drugs under the acidic condition of tumor cells.
Magnetic targeting study
A549 cells are inoculated in a 60mm culture dish for culture and adherence, and 1.5 mu g of nano-particle MMZr @ Bi is added2O3-DNM. For magnetic targeting studies, a magnet (about 4T) was placed under one side of the dish. After incubation for 24h, photographing and recording the magnetic field area and the opposite magnetic field-free area in the culture dish. The result is shown in fig. 11, the cell morphology of the medicated group magnetic targeting region changes, and obvious magnetic nanoparticle aggregation can be seen, and the cells in the magnetic targeting region shrink while the blank group cells have no obvious change in the magnetic region and the nonmagnetic region, which indicates that the magnetic nanoparticles can be locally enriched under the action of the external magnetic field, thereby achieving the purpose of controllable passive targeted therapy.
Prussian blue staining
The uptake of the magnetic nanoparticles in the cells can be verified by a Prussian blue staining experiment, A549 cells and MCF-7 cells are inoculated in a 6-well plate, a blank control group is added with a normal culture medium after the cells are cultured for 24 hours and adhered, and an experiment group is added with MMZr @ Bi with the concentration of 2 mug/mL2O3The culture mediumCulturing for 12 h. The supernatant was removed, washed three times with PBS, fixed with 4% paraformaldehyde solution at 4 ℃ for 30min, and the fixing solution was washed off again with PBS. Then 5% potassium (II) ferrocyanide trihydrate solution and 5% HCl are added to each well in a 1:1 ratio, incubated at 37 ℃ for 1h, counterstained with neutral red stain for 30s, washed three times with PBS, and recorded by optical microscopy. The results are shown in FIG. 12, where blue particles were present in the cells of the experimental group. This demonstrates MMZr @ Bi2O3Can well enter cells.
Flow cytometer test cell uptake
The cultured cells were observed under a microscope, and the cells that grew well and were in the logarithmic growth phase were selected for experiments, and adherent cells were digested with 0.25% trypsin, and resuspended with complete culture medium after digestion was completed. After counting, cells were seeded into 12-well plates at 8000-10000 cells per well. Adding a drug-containing complete culture solution with five drug concentration gradients near the IC50 value after the cells are fully paved by 80%, continuously culturing for 24h, washing away the nanoparticles which are not completely taken up by PBS, washing the digested cells for three times by PBS, putting the cells into a flow tube, and analyzing by a flow cytometer. The flow cytometry analyzer is used for obtaining a fluorescence map as shown in fig. 13, and then flowjo software is used for obtaining the fluorescence map according to the flow map, and the result shows that as the drug concentration of the drug-loaded nanoparticles increases, the flow fluorescence map moves to the right, the fluorescence intensity is enhanced compared with a blank group, and the drug can be absorbed by tumor cells and the cytotoxicity is stronger as the drug concentration increases.
Cell scratch test-cell migration
A549 cells were plated at 5X 10 per well5The density of individual cells was seeded in the medium in 6-well plates and after overnight culture, the supernatant was removed. The monolayer of cells at the bottom of the 6-well plate was streaked with a 10 μ L sterile white tip and cells detached during the streaking were washed away with PBS. With a catalyst containing MMZr @ Bi2O3The medium of DNM was incubated for 48 hours and the change of the scratch at the same position was recorded by optical microscopy for 0h and 24 h. As shown in FIG. 14, the cell migration area was decreased in the dosed group compared to the blank group as time passed, and MMZr @ Bi was observed2O3DNM shows an inhibitory effect on cell migration.
Cell cycle assay
Collecting cells growing in logarithmic phase, digesting with pancreatin for 1-2min, blowing into cell suspension with pipette gun, counting with blood counting plate at 1.0 × 106Cells per well were added to a six well plate and cells were incubated at 37 ℃ with 5% CO2After culturing for 24h, the 6-well plate was taken out, the medium was aspirated, washed 2 times with sterile PBS, and 1.5mM DNM, MMZr @ Bi were added2O3DNM, blank control plus fresh medium. Continuously culturing the six-hole plate for 24 h; removing the residue containing MMZr @ Bi2O3-DNM nanoparticle medium, discarding medium, washing several times with PBS, adding ETDA-containing pancreatin for digestion for 2min, removing pancreatin, adding the previously collected cell culture fluid, gently blowing and beating until all cells fall off, collecting these suspensions in a centrifuge tube for routine 5min, then sucking centrifuged supernatant, adding 1mL cold 70% ethanol, gently blowing and beating and mixing well, placing in a refrigerator at 4 ℃ for 24h, then taking out for centrifugation, sucking cell supernatant, adding 1mL cold PBS for resuspension, then centrifuging again, sucking supernatant, flicking the bottom of a test tube with fingers to disperse well, finally adding 0.5mL PI staining solution per tube to stain it, allowing it to be resuspended with cells, incubating at 37 ℃ in dark for 30min, and performing flow detection.
As shown in FIG. 15, blank cell group G0/G1The phase ratio was 38.46%, the S phase ratio was 32.13%, G2The ratio of the/M phase was 29.41%, DNM in cell group G0/G1The phase ratio was 44.45%, the S phase ratio was 29.11%, G2The ratio of the/M period is 26.44%, MMZr @ Bi2O3DNM in cell group G0/G1The phase ratio is 50.8%, the S phase ratio is 25.76%, G2The ratio of the/M period was 23.44%, compared to the blank control, MMZr @ Bi2O3DNM predominantly arrested A549 cells at G0/G1And has a stronger retarding effect than DNM.
Fluorescence microscope and flow cytometer for measuring cell apoptosis
Cells growing in log phase are digested with pancreatin for 1-2min, gently blown with a pipette to form a suspension, counted on a hemocytometer at 1.0X 106Cell density per well was seeded in 6-well plates and cells were incubated at 37 ℃ with 5% CO2After culturing for 24h, taking out a 6-hole plate, sucking out the culture medium, washing for 2 times by sterile PBS, adding the drug-loaded nano microspheres with different concentrations for culturing for 24h, sucking out the culture medium, washing for three times by PBS, adding 0.5ml of Hoechst 33258 staining solution in each air, and culturing at 37 ℃ by 5% CO2Culturing in a cell culture box for 20-30min, discarding staining solution, and washing with PBS or culture solution for 2-3 times to perform fluorescence detection.
Collecting cells growing in logarithmic phase, digesting with pancreatin for 1-2min, blowing into cell suspension with pipette gun, counting with blood counting plate at 1.0 × 106Each well was added to a six well plate and cells were incubated at 37 ℃ with 5% CO2After the culture is carried out for 24h conventionally in the incubator, the 6-hole plate is taken out, the culture medium is sucked out, the sterile PBS is washed for 2 times, and the MMZr @ Bi is respectively added2O3DNM, adding fresh culture medium to the blank control group, and culturing for 24 h; sucking the cell culture solution into a centrifuge tube by using a pipette gun, cleaning the centrifuge tube for 2 times by using PBS (phosphate buffer solution), adding pancreatin containing EDTA (ethylene diamine tetraacetic acid) for digesting for 1min, sucking the pancreatin, adding the culture solution collected before, blowing and beating the cells lightly, collecting the cells into the centrifuge tube completely, cleaning the centrifuge tube by using the PBS, centrifuging the supernatant, adding 195 mu l of Annexin V-FITC binding solution, 5 mu l of Annexin V-FITC and 10ul of PI into each centrifuge tube in sequence, mixing the mixture lightly, incubating the mixture for 20min in a dark place at room temperature, then placing the mixture in an ice bath in a dark place, and resuspending the mixture for 3 times in the incubating process so as to improve the dyeing effect, and performing flow detection immediately.
As shown in FIG. 16, MMZr @ Bi at various concentrations2O3Effect of DNM on cells. After 24 hours of drug treatment, the nucleus of the control group is complete, the chromatin is distributed uniformly, and light blue weak fluorescence is presented. And in MMZr @ Bi2O3In the DNM group, with the increasing concentration of the drug, the blue bright spots are more and more, and the staining fluorescence of a large number of cells in the cells is observed plusThe typical apoptosis phenomenon that strong and nuclear chromatin condenses and deviates to a crescent shape on one side is that more and more apoptotic cells are available, which indicates that the drug is dose-dependent.
For quantitative evaluation by MMZr @ Bi2O3DNM-induced apoptosis, we performed flow assays using Annexin V/PI double staining. As shown in fig. 17, it is known that the apoptosis rate reached 42%, the number of early and late withers increased, and the mechanical damage of the cells was less as the drug concentration increased.
Mitochondrial membrane potential detection
The tumor cells (A549) were dispersed in 6-well cell culture plates at a density of 2.5X 105After the cells cultured overnight attached to the cell culture medium, the compound was added to the cell culture medium in a concentration gradient and allowed to act on the cells. After 48h of treatment, the digested monolayer cells were covered with an appropriate amount of pancreatin, the washed cells were collected, and the cells were resuspended in serum-free medium. Then, JC-1 staining solution was mixed into the cell suspension, and the mixture was placed at 37 ℃ with 5% CO2The incubator is incubated for 30min in the dark. Finally, the cells were washed twice with staining buffer, the buffer suspended the cells and passed through a cell screen, and the samples were analyzed using a flow cytometer. JC-1 is an ideal fluorescent probe widely applied to the detection of membrane potential (delta psi). When the membrane potential is higher, JC-1 is gathered in a mitochondrial matrix to form a polymer, and red fluorescence can be generated; at lower membrane potentials, JC-1 cannot aggregate to the mitochondrial matrix, and is monomeric, producing green fluorescence. The relative proportion of red and green fluorescence is most commonly used to measure the magnitude of the membrane potential. Finally, the results obtained were analyzed using flowjo7.6.1 software.
As shown in fig. 18, the mitochondrial membrane potential after nanoparticle treatment was lower than that of the untreated blank group and the positive control group, and the higher the drug concentration was, the more significant the decrease in the mitochondrial membrane potential was. And the cells are deviated to the right, JC-1 is changed from a polymer to a monomer, and the drug-loaded nano particles are proved to be capable of reducing the mitochondrial membrane potential of tumor cells so as to induce the death of the cells. From this it was preliminarily speculated that the nanoparticles initiate apoptosis in the mitochondrial pathway.
Autophagy study
Currently, staining cells with acid-labeled MDCs is another rapid method for assessing autophagy activity. As can be seen from the flow cytometer detection results in FIG. 19, MMZr @ Bi2O3The fluorescence intensity of the DNM-treated A549 cells is increased compared with that of the control group, which indicates that the nanoparticles can induce autophagy of the A549 cells and cause cell death.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A pH-responsive magnetic nano core-shell drug loading system is characterized by comprising a magnetic core, a mesoporous shell and a pH-responsive gate, wherein the mesoporous shell is modified on the surface of the magnetic core, and after anti-tumor drugs are loaded in channels of the mesoporous shell, the mesopores are plugged by the pH-sensitive substance serving as the responsive gate.
2. The pH-responsive magnetic nano core-shell drug delivery system of claim 1, wherein the magnetic core is a superparamagnetic ferroferric oxide nanoparticle.
3. The pH-responsive magnetic nano core-shell drug delivery system according to claim 2, wherein the mesoporous shell layer is made of zirconium dioxide.
4. The pH-responsive magnetic nano core-shell drug delivery system of claim 3, wherein the pH-sensitive substance is Bi2O3Nanoparticles.
5. The pH-responsive magnetic nano core-shell drug delivery system according to claim 1, wherein the anti-tumor drug is an anthracycline.
6. The pH-responsive magnetic nano core-shell drug delivery system of claim 5, wherein the anthracycline is doxorubicin, daunorubicin, idarubicin, mitoxantrone, or epirubicin.
7. The preparation method of the pH-responsive magnetic nano core-shell drug-loading system according to claim 4, characterized by comprising the following steps:
(1)Fe3O4@ZrO2carrying out ultrasonic dispersion on superparamagnetic nano ferroferric oxide uniformly, washing with deionized water, continuously dispersing with absolute ethyl alcohol, violently stirring at room temperature, adding 25% concentrated ammonia water, dropwise adding 0.1mol/L zirconium oxychloride solution into the solution, reacting for 24 hours, standing and separating black nano microspheres by using magnetic adsorption, and alternately washing with ethyl alcohol and distilled water to remove redundant reactants; dispersing the washed nano-microspheres in a mixed solution of ethanol and water which contains CTAB and is mixed in equal proportion, continuously adding 25% ammonia water, reacting under vigorous stirring at room temperature after uniform ultrasonic dispersion, then dropwise adding 0.1mol/L zirconium oxychloride solution, reacting for 24 hours, separating out magnetic nanoparticles under the action of an external magnetic field after the reaction is finished, and washing with ethanol and water alternately to obtain Fe3O4@ZrO2Nano-microspheres;
(2) preparing MMZr, namely preparing Fe containing CTAB template agent in the last step3O4@ZrO2Ultrasonically dispersing the nano particles in 10mg/mL calcium nitrate-absolute ethyl alcohol, stirring at the rotating speed of 4.0krp/min, placing the nano particles in an oil bath kettle at the temperature of 80 ℃, heating and refluxing for 6h, standing to room temperature, washing the magnetic nano particles with deionized water under the magnetic action to wash CTAB, and sealing with 20mL deionized water to obtain the MMZr nano microspheres with the core-shell structure;
(3) preparation of plugged microspheres containing drug MMZr @ Bi2O3Adding an anti-tumor drug X into the MMZr nano microspheres to obtain drug-containing microspheres; then adding nanometer Bi into the cleaned nanometer microspheres2O3Dispersing uniformly, blocking for 12h in the dark at room temperature, adsorbing and standing the carrier after the reaction is finished, and buffering by PBS (phosphate buffer solution) with pH value of 7.40Cleaning the surface with the solution to remove excess Bi in the test tube2O3Removing to obtain the product.
8. The preparation method according to claim 7, wherein in the step (1), the superparamagnetic ferroferric oxide nanoparticles are prepared by a chemical coprecipitation method, and the preparation method comprises the following steps: firstly, FeCl is weighed3·6H2O and FeCl2·4H2O, adding deoxidized deionized water into a round-bottom flask in a nitrogen environment, heating to 80 ℃ in an oil bath kettle, reacting at a constant temperature, rapidly adding 25% ammonia water, testing the pH range by using a pH test paper, adjusting the pH value to 10-11, immediately turning the liquid into black, and stirring for reaction; stopping reaction after 30min of reaction, cooling to room temperature, performing adsorption separation by using magnetism, alternately washing the prepared magnetic nanoparticles by using ethanol and deionized water, and finally adding deionized water for sealing.
9. The preparation method according to claim 7, wherein in the step (3), the antitumor agent is doxorubicin, daunomycin, idarubicin, mitoxantrone or epirubicin.
10. The use of the pH-responsive magnetic nano core-shell drug delivery system of any one of claims 1 to 6 in the preparation of a medicament for the treatment of a tumor.
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