CN109718381B

CN109718381B - Subcellular targeting nano-drug delivery system

Info

Publication number: CN109718381B
Application number: CN201910177112.7A
Authority: CN
Inventors: 姜玮; 王磊; 丁胜勇; 徐晓文
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-03-08
Filing date: 2019-03-08
Publication date: 2020-10-16
Anticipated expiration: 2039-03-08
Also published as: CN109718381A

Abstract

The disclosure belongs to the technical field of nano-drug delivery systems, and particularly relates to a subcellular targeting nano-drug delivery system. Aiming at the problem that an anti-tumor drug cooperative delivery system in the prior art is difficult to realize subcellular targeting, the disclosure provides a detachable MSN cooperative delivery nano-carrier for subcellular targeted drug delivery, the carrier is of a core-shell-hook structure, and the MSN is used as a core material for carrying PTX; polymerized polyphenol (EGCg) as a shell material for use in encapsulating drugs in MSN (MSN @ EGCg) and providing a modification site for the hooks; aptamer AS1411 ligated double stranded DNA (dsDNA-Apt) was used AS a hook to bind nucleolin and carry Dox. The nano-carrier can respectively deliver Dox and PTX to cell nucleus and cytoplasm, improves the intracellular targeting capability of drug delivery, is applied to the development of antitumor drugs, and has considerable prospect.

Description

Subcellular targeting nano-drug delivery system

Technical Field

The present disclosure belongs to the technical field of nano-drug delivery systems, and particularly relates to a sub-cell targeted nano-drug delivery system with a core-shell-hook structure.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Although chemotherapy is a classical means of cancer treatment, its effectiveness is often limited by the non-selective delivery of free chemotherapeutic agents. These drugs enter normal and cancer cells indiscriminately by simple diffusion, inevitably resulting in reduced chemotherapeutic efficacy and serious side effects. In order to solve these problems, researchers in the field have found that nanoparticles have unique size and good characteristics of passing through tumor blood vessels, and can be used as nanoparticle-mediated drug delivery (nanocarrier) for targeting tumor cell delivery systems. In addition, specific ligand-modified nanocarriers are capable of binding to corresponding biomarkers on the cell membrane and following the movement of these markers are directed into cells and even subcellular compartments. Thus, a range of targeted nanocarriers were developed for efficient drug delivery. Among them, the subcellular targeting nanocarriers are more favorable for drug accumulation in subcellular action compartments and mediate cell function damage and apoptosis more directly and effectively, and are promising approaches for improving chemotherapeutic effects.

Recent studies have shown that nanocarriers carrying two or more drugs (co-delivery nanocarriers) can synergistically damage tumor cells from different sites of action and reduce dose-dependent side effects by reducing the amount of drug used. The inventor finds that the current nano-carrier for synergistic delivery can only target tumor cells at a cellular level, and does not realize targeted delivery at a subcellular level. As a practical site for DNA replication, the nucleus is a subcellular compartment in which many chemotherapeutic drugs act. For example, the classical chemotherapeutic drug doxorubicin (Dox) induces tumor cell apoptosis by inserting DNA into the nucleus or inhibiting topoisomerase II. In addition, if drugs whose action site is the nucleus are released in the cytoplasm, these drugs are rapidly degraded by cytoplasmic acids and enzymes during diffusion, so that the efficacy of the drugs is lowered. The inventors believe that the development of a nucleus-targeted synergistic delivery nanocarrier may be effective in reducing the above-mentioned drug degradation and further enhancing the therapeutic efficacy. However, successful construction of nuclear targeted cooperative delivery nanocarriers is hampered by biological barriers. In order to enter the nucleus, a component in the nanocarrier of less than 9nm must pass through the nuclear pore complex (4-10nm) in the nuclear membrane, but nanoparticles of this size are rapidly eliminated during cycling. Conversely, larger nanoparticles that are resistant to elimination cannot pass through the nuclear membrane due to size, nor can they act as a component of entry into the nucleus. Therefore, to simultaneously satisfy these requirements, it is necessary to construct a cooperative delivery nanocarrier in which intracellular components can be detached.

In the past decades, Mesoporous Silica Nanoparticles (MSNs) have been widely used in nanocarriers, and have advantages of large mesoporous volume, high biocompatibility, and easy surface modification. Various drugs such as Dox, Paclitaxel (PTX) were shown to be able to be loaded in the mesopores of MSN. To avoid early drug leakage, various gating materials such as gold nanoparticles, polymers, proteins, and nucleic acids have been used to block the mesopores of MSNs. Among these materials, polymerized polyphenols are gradually attracting attention due to structural changes in acid-base and redox environments. Polyphenols can polymerize to a reversible coating on metallic or non-metallic surfaces in alkaline and oxidative environments, which can be used to encapsulate drugs in MSNs. In addition, the resulting polyphenolic coating is capable of adsorbing amino compounds, including amino-modified DNA, and specific DNA can be used as targeting ligands and drug carriers. In an acidic environment, the polyphenol coating can be depolymerized by glutathione and release adsorbed DNA and drug within the mesopores. By utilizing the concentration difference of protons and glutathione inside and outside the tumor cells, the polyphenol is expected to become a good material for modifying the MSN surface.

Disclosure of Invention

In the context of the above-mentioned studies, the present disclosure constructs a synergistic delivery nanocarrier with detachable components induced by depolymerization of polymerized polyphenols for subcellular targeted drug delivery. The nano-carrier has a core-shell-hook structure, and adopts MSN as a core material for carrying PTX; polymerized polyphenol (epigallocatechin-3-gallate, EGCg) as shell material is used in encapsulating drugs in MSN (MSN @ EGCg) and providing modification sites for hooks; aptamer AS1411 ligated double stranded DNA (dsDNA-Apt) was used AS a hook to bind nucleolin and carry Dox. The nanocarrier delivery system first recognizes and binds nucleolin overexpressed on the cancer cell membrane through surface hooks and guides the nanocarrier into cells. Subsequently, the shell is depolymerized by glutathione in the weak acid environment of the cancer cells. Finally, the nucleus remains in the cytoplasm and releases PTX, while nucleolin-bound hooks target the nucleus and release Dox under nucleolin guidance. Fluorescence of coumarin 6(a surrogate for PTX) and Dox occurred in the cytoplasm and nucleus, respectively, of cancer cells by cellular fluorescence imaging, suggesting that nanocarriers can deliver PTX and Dox to the respective subcellular action compartments, respectively. Compared with the non-subcellular targeting nano-carrier, the nano-carrier has 19.1 percent and 54 percent increase of cytotoxicity on MCF-7 and MCF-7/ADR cells respectively under the total drug concentration of 4.5 mu M, and has 20.4 percent reduction of cytotoxicity on normal liver cells. The nano-carrier provided by the disclosure provides an effective strategy for subcellular targeting drug delivery, can realize delivery of adriamycin to cell nucleus and delivery of paclitaxel to cytoplasm, and is a potential tumor treatment method.

In a first aspect of the present disclosure, an MSN @ EGCg nanocarrier is provided, which has a core-shell structure, mesoporous silica particles (MSN) are used as a core material for loading a drug a, and polymerized polyphenol composed of EGCg is used as a shell material for encapsulating pores of mesoporous silica.

The research disclosed by the disclosure shows that by adopting EGCg as a shell material, the formed polymerized polyphenol coating can be kept stable in a neutral environment, MSN early-stage drug leakage is avoided, glutathione can be fully depolymerized in a weak acid environment, and the gating effect in tumor cells is realized.

In a second aspect of the present disclosure, a preparation method of the above MSN @ EGCg nanocarrier is provided, the preparation method comprising the steps of: synthesizing a mesoporous silicon dioxide material from a silicon material under an alkaline condition, adding the mesoporous silicon dioxide material into a buffer solution, and adding EGCg to prepare MSN @ EGCg.

Preferably, the preparation method of the mesoporous silica material is as follows: adding an alkaline solution into a CTAB solution, uniformly stirring, heating to a certain temperature, reacting for a period of time, then adding Tetraethoxysilane (TEOS) into a reaction system at a certain speed, keeping the certain temperature for continuing reacting for a period of time after a white precipitate is observed to be generated, slowly cooling to room temperature, washing to obtain a precipitate, drying the precipitate, and calcining for a period of time to obtain the mesoporous silica material.

Further preferably, the alkaline solution is a sodium hydroxide solution.

Preferably, the CTAB solution is added into the alkaline solution and then uniformly stirred, and the solution is heated to 70-90 ℃ to continue to react for 25-35 min.

The mesoporous silica material formed under the reaction condition can have a good hole structure, is beneficial to effective encapsulation of subsequent polymerized polyphenol, and can realize effective release of the medicine after the encapsulation is removed.

More preferably, the adding speed of the Tetraethoxysilane (TEOS) is 2-3 mL, and the adding is finished within 8-12 min.

The silicon material is added into the reaction system at the speed, which is beneficial to obtaining the mesoporous silicon dioxide material with uniform shape and stable performance.

Preferably, after the formation of white precipitate is observed, the reaction is continued for 1.5 to 2.5 hours at 70 to 90 ℃ to stabilize the precipitate.

Preferably, the dried white solid is calcined at 520-570 ℃ for 5-7h to obtain a product.

The mesoporous silicon dioxide material is obtained by calcining at the temperature, which is beneficial to completely removing residual water and surfactant in the material and forming uniform holes.

Preferably, the step of adding EGCg to prepare MSN @ EGCg is as follows: suspending the prepared MSN in a BICINE buffer solution, and adding EGCg under stirring; after the reaction solution was further uniformly stirred for a while, 6. mu.L of 12mM acetic acid was added to the reaction solution, and immediately centrifuged at 8000rpm for 2 minutes to obtain a white solid for use.

Further preferably, after 5-7h of reaction with EGCg, acetic acid is added and centrifuged to obtain a white solid.

Further preferably, the mass ratio of the MSN to the EGCg is 0.8-1.2: 0.6-1.

The core-shell structure obtained by the mass ratio reaction can meet the encapsulation requirement of core materials, reduce the early leakage of the drug in the transfer process of an organism, and on the other hand, the shell structure can react and decompose with glutathione in time in the environment in cancer cells to realize the release of the drug.

In a third aspect of the present disclosure, a MSN @ EGCg-dsDNA-Apt nanocarrier is provided, wherein the nanocarrier has a core-shell-hook structure, and double-stranded DNA (dsDNA-Apt) connected with an aptamer AS1411 is used AS a hook structure to modify the surface of the nanocarrier of the first aspect, so AS to bind nucleolin and carry a drug B.

In a fourth aspect of the present disclosure, there is provided a method for preparing the above MSN @ EGCg-dsDNA-Apt nanocarrier, wherein the method further comprises the following steps after the steps of the second aspect are completed: putting the dsDNA-Apt and the MSN @ EGCg into a BICINE buffer solution together, and incubating for a period of time at a certain temperature to obtain a white product, namely the MSN @ EGCg-dsDNA-Apt.

Preferably, the incubation temperature is 36-38 ℃, and the incubation time is 2-4 h.

In a fifth aspect of the disclosure, an application of the MSN @ EGCg nanocarrier of the first aspect and/or the MSN @ EGCg-dsDNA-Apt nanocarrier of the third aspect in preparing an anti-tumor drug is provided.

In a sixth aspect of the present disclosure, an antitumor drug is provided, wherein the MSN @ EGCg nanocarrier of the first aspect and/or the MSN @ EGCg-dsDNA-Apt nanocarrier of the third aspect is used for loading the drug.

Preferably, the drug A is a drug with an action site in cytoplasm.

Further preferably, the drug a is Paclitaxel (PTX).

Preferably, the drug B is a drug with an action site in a cell nucleus.

Further preferably, the drug B is a doxorubicin (Dox).

Advantageous effects of the disclosure

1. Most of the nano-carriers which are cooperatively delivered in the prior art can only achieve the target cancer cells at the cellular level, and the target on the subcellular structure is difficult to achieve. The MSN @ EGCg-dsDNA-Apt nano carrier provided by the disclosure adopts mesoporous silicon dioxide to load a medicine, coats polymerized polyphenol to encapsulate the medicine, and is also provided with double-stranded DNA (dsDNA-Apt) connected with an aptamer AS1411 on the surface, the medicine is loaded by adopting the structure, cancer cells can be identified through a hook structure, the polymerized polyphenol shell material is decomposed in an acidic cell environment after the medicine enters the inside of the cells, so that the medicine loaded in the mesoporous silicon dioxide material is released in cytoplasm, and on the other hand, the hook structure is reduced in size and enters the cell nucleus under the affinity action of the aptamer and nucleolin, so that the cell nucleus targeting of the medicine is realized. The nano-carrier in the disclosure realizes the respective delivery of cytoplasm and nucleus while realizing the synergistic delivery of the drug.

2. The drug carrier in the disclosure adopts mesoporous silica to load PTX, and adopts double-stranded DNA (dsDNA-Apt) connected with aptamer AS1411 to load Dox. Research proves that compared with a non-subcellular targeting nano-carrier, the nano-carrier has 19.1% and 54% increased cytotoxicity on MCF-7 and MCF-7/ADR cells respectively under the total drug concentration of 4.5 mu M, and has 20.4% reduced cytotoxicity on HL-7702 cells, so that the nano-carrier can effectively increase the inhibition effect on cancer cells and reduce the toxicity on normal cells.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a schematic diagram of the application of the MSN @ EGCg-dsDNA-Apt nano-carrier in subcellular targeted drug delivery.

FIG. 2 is a TEM image of the MSN synthesized in example 1;

wherein, fig. 2(a) is a TEM image of MSN at 50000x magnification; FIG. 2(b) is a TEM image of MSN at 200000 magnification.

FIG. 3 is a graph of HR-TEM characterization of the MSN @ EGCg-dsDNA-Apt nanocarrier synthesis process of example 1:

FIG. 3(a) is MSN;

fig. 3(b) MSN @ EGCg with 2h incubation time;

fig. 3(c) MSN @ EGCg with incubation time of 6 h;

FIG. 3(d) is MSN @ EGCg-dsDNA-Apt with DNA concentration of 5. mu.M;

FIG. 3(e) shows MSN @ EGCg-dsDNA-Apt at a DNA concentration of 20. mu.M.

FIG. 4 is a graph showing the fluorescence intensity plotted against the number of insertions of Dox in example 1;

FIG. 5 is a standard curve (a) of the concentration of PTX determined by HPLC method in example 1 and a graph of the in vitro release of PTX in MSN-PTX @ EGCg;

wherein, FIG. 5(a) is a standard curve chart of the concentration of PTX measured by HPLC;

FIG. 5(b) is a graph of PTX release profile in MSN-PTX @ EGCg at different pH conditions;

FIG. 6 is a graph showing the fluorescence intensity of different cells after 4 hours of incubation of MSN-C6@ EGCg-Apt-Dox with free C6 and Dox at 37 ℃ measured by the flow cytometry in example 1;

wherein, FIGS. 6(a) and 6(b) are the fluorescence intensities of C6 and Dox in HL-7702 cells, respectively;

FIGS. 6(C) and 6(d) are C6 and Dox fluorescence intensities in MCF-7 cells;

FIGS. 6(e) and 6(f) are C6 and Dox fluorescence intensities in MCF-7/ADR cells.

FIG. 7 is a photograph of fluorescence of cells after 6 hours of co-incubation with MSN-C6@ EGCg-dsDNA-Apt-Cy 5-Dox;

wherein, FIGS. 7(a), 7(f) and 7(k) are Brightfield diagrams of HL-7702, MCF-7 and MCF-7/ADR, respectively;

FIGS. 7(b), 7(g) and 7(l) are fluorescence diagrams of Hoechst 33342 of HL-7702, MCF-7 and MCF-7/ADR, respectively;

FIGS. 7(c), 7(h) and 7(m) are fluorescence diagrams of Cy5 for HL-7702, MCF-7 and MCF-7/ADR, respectively;

FIGS. 7(d), 7(i) and 7(n) are the Dox fluorescence maps of HL-7702, MCF-7 and MCF-7/ADR, respectively;

FIGS. 7(e), 7(j) and 7(o) are fluorescence diagrams of C6 of HL-7702, MCF-7 and MCF-7/ADR, respectively.

FIG. 8 is a graph showing the measurement of cell activity of various cells after incubation with a sample at 37 ℃ for 24 hours by the MTT method in example 1;

wherein FIG. 8(a) is a chart of HL-7702 cell activity; FIG. 8(b) is a MCF-7 cell activity diagram; FIG. 8(c) is a MCF-7/ADR cell activity diagram.

Detailed Description

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 disclosure 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 disclosure. 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.

As described in the background art, the nano-carrier cooperatively delivered in the prior art can only achieve the target at the cellular level, and the effective site of the drug actually acting in the cell nucleus is difficult to be reached efficiently. In order to solve the technical problems, the present disclosure provides an MSN @ EGCg-dsDNA-Apt nanocarrier, which can simultaneously achieve targeting of cancer cells and targeting of subcellular structures, and respective delivery of cell nucleus and cytoplasm, effectively reduce toxicity to normal cells, and improve therapeutic effect.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

1.1 instruments and reagents

The oligonucleotides in this example were synthesized and purified by Shanghai Biotechnology Ltd, the sequences of which are listed in Table 1. Tetraethoxysilane (TEOS) is available from shanghai national medicine limited. Cetyl trimethylammonium nitrate (CTAB) and epigallocatechin-3-gallate were obtained from Solebao bioscience and technology, Inc. Coumarin 6 and glutathione were purchased from alatin biochemical technologies, inc. Doxorubicin is available from huafeng group ltd. Paclitaxel was supplied by Chenxin pharmaceuticals, Inc. MTT was purchased from Sigma-Aldrich. All solutions were prepared with ultra pure water (>18.25 M.OMEGA.). Other chemicals used in the experiment were all analytically pure and no further purification was required during use.

Fluorescence spectroscopy was performed on a Hitachi F-7000 fluorescence spectrophotometer. The UV absorption spectrum scan was performed using Hitachi U-2910 UV-Vis spectrometer. The transmission electron microscope image was observed and recorded using a JEM-1011 transmission electron microscope. High resolution tem images were observed and recorded using a JEM2100 tem. The cell fluorescence images were observed and recorded using a carl zeiss Axio Observer. MTT experiments were measured using a TECAN Infinite M200 microplate reader. Flow cytometry experiments were performed on a Beckman Coulter Flow Cytometer.

Table 1: oligonucleotide sequences used in this work

1.2 Synthesis of MSN

0.5g CTAB and 240mL of water are placed in an eggplant-shaped bottle and stirred at a constant speed for 2 minutes. Then 1.75mL of 2M sodium hydroxide solution was added slowly. After stirring was continued for 5 minutes, the solution was heated to 80 ℃ and the reaction was continued for 30 minutes. Subsequently, 2.5mL of TEOS was added dropwise over 10 minutes, and generation of a white precipitate was observed. The reaction was continued for 2 hours while maintaining 80 ℃ to stabilize the precipitate, and then the reaction solution was slowly cooled to room temperature. The precipitate was collected by centrifugation (14000rpm) and the resulting precipitate was washed with water and ethanol (3 minutes x 3 times). Finally, the white solid obtained was dried in vacuo and the product obtained was calcined in a muffle furnace at 550 ℃ for 6 hours.

1.3 preparation of MSN @ EGCg

1mg of MSN was ultrasonically suspended in 2mL of BICINE buffer (0.6M sodium chloride, 0.1M N, BICINE, pH 7.8), and then 0.8mg of EGCg was added with stirring. After the reaction solution was stirred at a constant speed for 6 hours, 6. mu.L of acetic acid (12mM) was added, and the mixture was immediately centrifuged at 8000rpm for 2 minutes.

1.4 characterization of dsDNA-Apt

Purchased dsDNA-Apt and dsDNA-Apt-Cy5 were dissolved using TE buffer (0.1mM EDTA,1mM Tris, pH 8.0). The oligonucleotide chain was then annealed at 100 ℃ for 5 minutes and allowed to cool to room temperature. The assembly of oligonucleotides was characterized using gel electrophoresis experiments. Electrophoresis experiments (36mA,30 min, TAE buffer) were performed using 8% polyacrylamide gels. After completion of the electrophoresis, the gel was stained with ethidium bromide for 10 minutes. Gels were imaged using an Alpha Inotech Alpha Imager.

1.5 preparation of MSN @ EGCg-dsDNA-Apt and MSN @ EGCg-dsDNA-Apt-Cy5

The annealed dsDNA-Apt or ds-DNA-Apt-Cy5 was co-incubated with MSN @ EGCg in BICINE buffer at 37 ℃ for 3 hours to complete the DNA adsorption process. The obtained product was then collected by centrifuging the reaction solution and washed 3 times with water to remove BICINE buffer. Finally, the supernatant was discarded and the white product was suspended in PBS buffer (pH 7.4).

1.6 characterization of MSN, MSN @ EGCg and MSN @ EGCg-dsDNA-Apt

The size and shape of MSN were observed by TEM, and the mesoporous structure of MSN, MSN @ EGCg and MSN @ EGCg-dsDNA-Apt were observed by HR-TEM. To examine the effect of different adsorption times (2 h, 6 h) and different DNA concentrations (5. mu.M, 20. mu.M) on the synthesis, each sample was diluted to 0.5mg/mL with PBS buffer (pH 7.4).

1.7 drug carrying study

5mg of paclitaxel and 15mg of MSN were ultrasonically dissolved or suspended in 2mL of dichloromethane, and then stirred open in a water bath at 37 ℃ for 4 hours until 0.5mL of solution remained. Subsequently, the solution was centrifuged at 8000rpm for 6 minutes, and the resulting precipitate was washed with 0.5mL of dichloromethane. All upper layer liquids were combined and diluted to 500mL with methanol. Paclitaxel concentration was measured using high performance liquid chromatography (HPLC, Agilent-1200, US) chromatography (acetonitrile: water ═ 50:50, 292 nm). The carrying efficiency (EF%) of paclitaxel was calculated by the following formula:

wherein W_{total PTX}，W_{free PTX}And W_{nanoparticles}Representing the total amount of paclitaxel dissolved in dichloromethane, the amount of free paclitaxel remaining and the mass of MSN, respectively.

Dox-carried studies were performed in PBS buffer (pH 7.4). The method for synthesizing EGCg-coated drug-loaded MSN is the same as the method for synthesizing MSN @ EGCg. To measure the number of inserted Dox in each dsDNA-Apt, dsDNA-Apt solutions of 0.9. mu.M, 1.0. mu.M, 1.1. mu.M, 1.2. mu.M, 1.3. mu.M, 1.4. mu.M and 1.5. mu.M, respectivelyIncubate with 9. mu.M doxorubicin solution for 3 hours. The solution (F-7000) was then measured using an F-7000 fluorescence spectrometer (λ ex 476nm, λ em 550-750 nm)_n) And blank (F)₀) The fluorescence intensity of (2). The obtained result was used as fluorescence intensity (F)_n-F₀) -curve of the Dox number.

1.8 in vitro PTX Release study

In vitro PTX release studies were performed using the dialysis bag (MWCO ═ 3500) method, since PTX molecules were able to pass through the dialysis bag and MSN was not. To confirm the release rate of PTX, three samples were dispersed in PBS (pH 7.4, 0.5% twain80), PBS (pH 6.0, 0.5% twain80) and PBS (pH 6.0,10mM GSH, 0.5% twain80), respectively. Each sample was taken out by 1mL, filled in a dialysis bag, and then immersed in 15mL of the corresponding dispersion solvent. All samples were then placed in a constant temperature constant speed shaker and shaken at 100rpm for 48 hours at 37 ℃. 0.1mL of external solvent was removed at predetermined time points and the concentration of PTX was determined by HPLC. After each removal, 0.1mL of fresh solvent was added accordingly. The HPLC method for PTX measurement is the same as described previously.

1.9 cell lines and cell cultures

Human normal liver cells (HL-7702 cells) and human breast cancer cells (MCF-7 cells) were obtained from a cell bank collected from a type of culture of the Chinese academy of sciences. Drug-tolerant human breast cancer cells (MCF-7/ADR cells) were obtained from the group of professor Zhao Yun Xue, university, Shandong. All cells were cultured using RPMI 1640 cell culture medium and 10% bovine embryonic serum (FBS), 37 ℃ and 5% carbon dioxide environment.

1.10 drug accumulation Studies of cells

PTX has a weak fluorescence emission capacity, and therefore intracellular drug accumulation was analyzed by fluorescence using coumarin 6 instead of PTX. HL-7702, MCF-7 and MCF-7/ADR cells were placed in 6-well plates and incubated for 24 hours. It is appropriate to ensure the state and density of the cells. The original culture medium was then aspirated, and free Dox and C6, and MSN-C6@ EGCg-dsDNA-Apt-Dox (Dox 5.0. mu.M, C62.5. mu.M) were added to the plates, respectively. After 4 hours of co-incubation, the sample cells were washed with cold PBS and finally the fluorescence intensity of each sample was measured using a flow cytometer. Ensure that the temperature of the sample is maintained at 0 ℃ throughout the measurement.

1.11 cellular fluorescence imaging Studies

HL-7702, MCF-7 and MCF-7/ADR cells were placed in a confocal cell and incubated for 24 hours. The condition of the cells is ensured to be appropriate so that the number of cells is just evenly distributed at the bottom of the culture tank. The original medium was then discarded and 150. mu.L of MSN-C6@ EGCg-dsDNA-Apt-Cy5-Dox (Dox 5.0. mu.M, C62.5. mu.M) was added. The samples were stained with Hoechst 33342 stain for 30 minutes and washed with cold PBS and finally visualized using a fluorescence microscope.

1.12 in vitro cytotoxicity Studies

HL-7702, MCF-7 and MCF-7/ADR cells were plated in 96-well plates, approximately 4000 cells were seeded per well and incubated for 24 hours. Then, the original medium was discarded, and different concentrations (0.15. mu.M, 0.75. mu.M, 1.5. mu.M, 3.0. mu.M, 4.5. mu.M, 6.0. mu.M drug total) of free Dox, free PTX, free Dox and PTX, MSN @ EGCg-dsDNA-Apt, MSN-PTX @ EGCg-dsDNA-Apt, MSN @ EGCg-dsDNA-Apt-Dox, MSN-PTX @ EGCg-dsDNA-Apt-Dox, and a mixture of MSN-PTX @ EGCg-dsDNA-Apt and MSN-Dox @ EGCg-dsDNA-Apt were added, respectively, as expressed by MSN- (PTX + Dox) @ Cg-dsDNA-Apt. The incubation was continued for 24 hours, then washed 3 times with cold PBS and MTT solution (100. mu.L, 0.5mg/mL, RPMI 1640) was added, respectively. After 4 hours of co-incubation, the solution from each well was discarded and 150. mu.L DMSO was added. The absorbance at 570nm was measured after shaking for 5 minutes in a microplate reader. All experiments were repeated 3 times. Cell activity (%) was calculated using the following formula:

wherein A is_sample，A_controlAnd A_blankRepresenting the absorbance at 570nm of the sample, control and blank, respectively.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

SEQUENCE LISTING

<110> Shandong university

<120> subcellular targeting nano-drug delivery system

<130>2010

<160>2

<170>PatentIn version 3.3

<210>1

<211>59

<212>DNA

<213> Artificial sequence

<400>1

ccccgccccc tgt-NH2tgggggc ggggtttttt tttggtggtg gtggttgtgg tggtggtgg 59

<210>2

<211>59

<212>DNA

<213> Artificial sequence

<400>2

ccccgccccc tgt-NH2tgggggc ggggttttt-Cy5t tttggtggtg gtggttgtgg tggtggtgg 59

Claims

1. The MSN @ EGCg-dsDNA-Apt nano carrier is characterized by having a core-shell-hook structure, and modifying the surface of the MSN @ EGCg nano carrier by using double-stranded DNA (namely dsDNA-Apt) connected with an aptamer AS1411 AS a hook structure, wherein the double-stranded DNA is used for combining nucleolin and carrying a medicament B; the MSN @ EGCg nano-carrier is of a core-shell structure, mesoporous silica particles are used as a core material for loading a drug A, and polymerized polyphenol consisting of EGCg is used as a shell material for packaging holes of the mesoporous silica; the drug A is a drug with an action site in cytoplasm; the medicine B is a medicine with an action site in a cell nucleus; the drug A is paclitaxel; the medicine B is adriamycin.

2. The method of claim 1, wherein the method of making the MSN @ EGCg-dsDNA-Apt nanocarrier comprises the steps of: synthesizing a mesoporous silicon dioxide material from a silicon material under an alkaline condition, adding the mesoporous silicon dioxide material into a buffer solution, and adding EGCg to prepare MSN @ EGCg.

3. The method for preparing the MSN @ EGCg-dsDNA-Apt nanocarrier of claim 2, wherein the mesoporous silica material is prepared by the following steps: adding an alkaline solution into a CTAB solution, uniformly stirring, heating to 70-90 ℃, continuously reacting for 25-35 min, then adding ethyl orthosilicate into a reaction system at a certain speed, keeping 70-90 ℃ for continuously reacting for 1.5-2.5 h after white precipitate is observed to be generated, slowly cooling to room temperature, washing to obtain precipitate, drying the precipitate, and calcining for a period of time to obtain the mesoporous silica material.

4. The method of claim 3, wherein the alkaline solution is sodium hydroxide solution.

5. The method for preparing the MSN @ EGCg-dsDNA-Apt nanocarrier of claim 3, wherein the amount of tetraethoxysilane added is 2-3 mL and is completed within 8-12 min.

6. The method for preparing the MSN @ EGCg-dsDNA-Apt nanocarrier of claim 2, wherein the step of adding EGCg to prepare MSN @ EGCg comprises the following steps: suspending the prepared MSN in a BICINE buffer solution, and adding EGCg under stirring; and (3) continuously and uniformly stirring the reaction solution for a period of time, adding acetic acid into the reaction solution, and centrifuging to obtain a white solid for later use.

7. The method for preparing the MSN @ EGCg-dsDNA-Apt nano-carrier of claim 6, wherein after the EGCg is added and reacted for 1.5-2.5 h, half volume of the reaction solution is taken out, added with acetic acid and centrifuged to obtain a white solid, and the operation is repeated after the rest reaction solution is stirred for a while.

8. The method of claim 6, wherein the weight ratio of MSN @ EGCg-dsDNA-Apt nanocarrier to EGCg is 0.8-1.2: 0.6-1.

9. The method for preparing the MSN @ EGCg-dsDNA-Apt nanocarrier according to any one of claims 2 to 8, wherein the method for preparing further comprises the following steps after the completion of the steps of any one of claims 2 to 8: and putting the dsDNA-Apt and the MSN @ EGCg into a BICINE buffer solution together, and incubating for 2-4 h at 36-38 ℃ to obtain a white product, namely the MSN @ EGCg-dsDNA-Apt.

10. The use of the MSN @ EGCg-dsDNA-Apt nanocarrier of claim 1 in the preparation of an anti-tumor medicament.

11. An anti-tumor drug, wherein the MSN @ EGCg-dsDNA-Apt nanocarrier of claim 1 is used for loading the drug.