CN114569731B - Molecular beacon modified nano-carrier and application thereof in preparation of anti-tumor products - Google Patents

Abstract

The invention relates to a molecular beacon modified nanometer carrier and its application in the preparation of anti-tumor products. The molecular beacon in the nanocarrier can be used for silencing, imaging and drug delivery of MDR1mRNA in drug-resistant cancer cells, so as to realize drug-resistant cell targeting and inhibit drug efflux; in addition, the molecular beacon in the nanocarrier and MDR1mRNA Hybridization enables in situ imaging of MDR1 mRNA; and detachment of the molecular beacon causes drug release inside the nanocarrier. The above-mentioned nanocarriers have important application prospects in the imaging and drug delivery of drug-resistant tumor cells, which can significantly inhibit the drug resistance of tumor cells, enhance the delivery efficiency of chemotherapy drugs and the inhibitory effect of drug-resistant tumors.

Description

A nano-carrier modified by a molecular beacon and its application in the preparation of anti-tumor products

technical field

The invention belongs to the technical field of anti-tumor nano preparations, and in particular relates to a molecular beacon-modified nano-carrier and its application in the preparation of anti-tumor products.

Background technique

The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

In chemotherapy, the key to ensuring the effect of chemotherapy is to maintain the effective concentration of chemotherapy drugs in cancer cells. Maintenance of intracellular drug concentrations is generally limited by two critical factors. One is drug delivery efficiency, which is affected by the water solubility, stability, non-specific delivery and penetration efficiency of some chemotherapy drugs, which inevitably leads to insufficient drug concentration in cancer cells; the other is drug resistance after chemotherapy, usually It is caused by the reduction of intracellular drug accumulation caused by the overexpression of ABC transporters, especially P-glycoprotein (P-glycoprotein, P-gp), which is encoded by MDR1 mRNA in drug-resistant cancer cells and has drug pumping function. Due to the influence of these two factors, the drug concentration in drug-resistant cancer cells is not enough to reach the lethal threshold, which ultimately leads to limited therapeutic effect. Therefore, there is an urgent need to develop novel drug delivery systems for efficient drug delivery and to overcome drug resistance to ensure therapeutically effective intracellular drug concentrations.

The rapid development of nanomaterials has brought opportunities to exploit innovative drug delivery systems to bypass delivery barriers. Mesoporous silica nanoparticles (MSNs) are porous nanomaterials that have attracted increasing attention in biomedical applications. Thanks to their inherent characteristics of large pore volume, adjustable pore size, easy surface modification, high cell uptake efficiency, and high biocompatibility, MSNs show great advantages in the preparation of nanocarriers with high drug loading capacity and delivery efficiency. At present, a variety of MSN-based nanocarriers have been reported. For example, Andre E. Nel's group developed a lipid-coated mesoporous silicon nanocarrier for the delivery of chemotherapeutic drugs for pancreatic cancer. These carriers can effectively deliver drugs into cancer cells, but in drug-resistant cancer cells, the drugs released from the nanocarriers are still exposed to P-gp and are excreted from the cells, reducing the intracellular drug concentration. Therefore, how to overcome drug resistance in delivery remains a great challenge. To address this issue, utilizing the abundant active sites on the surface of MSNs and the designability of functional nucleic acid molecules (such as antisense oligonucleotides, siRNAs, or aptamers), rationally construct integrated nanocarriers that can effectively deliver drugs and overcome Drug resistance has application potential. At the same time, the ability to visualize intracellular events in real time is another critical factor in evaluating the drug release and gene silencing capabilities of nanocarriers, but has been barely explored.

Contents of the invention

Based on the above technical background, the present invention constructs a multifunctional molecular beacon (Molecular beacon, MB) modified mesoporous silicon nanocarrier for MDR1 mRNA silencing, imaging and drug delivery in drug-resistant cancer cells. The nanocarriers (Dox@MSN-AD-MBs) are modified by modifying quencher-labeled anchor DNA (AD) on the surface of MSN, encapsulating doxorubicin (Dox), and then using FAM-labeled molecular beacons Constructed by blocking. After uptake by drug-resistant cancer cells, MB on the surface of nanocarriers will hybridize with MDR1 mRNA, dehybridize with AD and fall off from the carrier at the same time. This hybridization event can achieve a triple effect: first, silence MDR1 mRNA and downregulate P-glycoprotein (P-gp) expression levels; second, restore FAM fluorescence and in situ imaging of MDR1 mRNA; third, open blocked pores and release Dox, produces cytotoxicity. The results of qRT-PCR and Western blot showed that the expression of MDR1 mRNA and P-gp in HepG2/ADR and MCF-7/ADR cells decreased after co-incubation with MSN-AD-MBs. Fluorescence imaging experiments enabled visualization of intracellular hybridization events of MB and MDR1 mRNA and monitoring of MDR1 mRNA expression levels. The ability of nanocarriers to inhibit drug-resistant cancer cells was investigated by cytotoxicity test. Compared with free Dox, Dox@MSN-AD-MBs showed higher inhibitory potency against HepG2/ADR cells and MCF-7/ADR cells. The present invention provides a new idea for the development of multifunctional nanocarriers, and has application potential in inhibiting drug resistance of cancer cells, and specifically provides the following technical solutions:

The first aspect of the present invention provides a molecular beacon modified nanocarrier, the main body of the nanocarrier is a porous carrier, the porous carrier is loaded with chemotherapeutic drugs, and the surface of the porous carrier is modified with molecular beacons and anchored DNA The hybrid strand; the molecular beacon blocks the through hole on the surface of the porous carrier, and the anchor DNA is fixed on the surface of the porous carrier through chemical bonds.

Preferably, the porous carrier is mesoporous silicon nanoparticle (MSN), which is a hollow sphere with through holes on the surface, the cavity of the hollow sphere is loaded with the chemotherapeutic drug, and the particle size of the hollow sphere is 120- 150nm, the size of the through hole is 2-3nm.

Further, the above-mentioned mesoporous silicon nanoparticles are synthesized by a sol-gel method, and the specific steps are as follows: slowly add an aqueous solution of cetyltrimethylammonium bromide (CTAB) into the lye and heat up to 75-85°C, and then Slowly add tetraethyl orthosilicate (TEOS) and maintain 75-85° C. for 0.5-3 hours to react to obtain a white precipitate which is the mesoporous silicon nanocarrier.

It should be clear that the chemotherapeutic drug is a small molecule chemical entity with anti-tumor activity, and is not limited to the therapeutic mechanism or type of the drug, as long as it can be loaded into the cavity of the porous carrier and released through the through hole. Applicable to the above-mentioned nanocarriers. In one embodiment of the verification of the present invention, the chemotherapeutic drug is doxorubicin (Dox). Further, in the aforementioned mesoporous silicon nanocarrier loaded with doxorubicin, the doxorubicin is loaded into the cavity of the mesoporous silicon nanoparticle by diffusion.

In addition, in the design of the molecular beacon and anchor DNA hybrid chain described in the first aspect, the molecular beacon is modified with a fluorescent dye, the anchor DNA is modified with a quencher group, and the two nucleotide chains are hybridized. When the above-mentioned nano-carriers do not show fluorescence, when the nano-carriers are taken up by cells and tumor cells, the molecular beacons have high affinity with the targets in the tumor cells, detach from the surface of the carrier and perform tumor cell imaging by fluorescence.

Based on the above design, the target of the molecular beacon can be adjusted according to the type of tumor cells and the purpose of drug design. In the preferred mode of the present invention, the nanocarrier is applied to the treatment of drug-resistant tumors, and the target of the molecular beacon It is a drug resistance-related gene, a specific example is MDR1, and the aptamer chain has a high affinity with MDR1 mRNA, and can silence MDR1 mRNA in drug-resistant tumor cells and perform imaging.

Further, the 5' end of the anchor DNA is connected to the surface of the nanocarrier through a chemical bond, and the 3' end is marked with a quenching group; further, the chemical bond is an amide bond, and the 5' end of the anchor DNA It has amino modification, and is connected to the carboxyl group on the surface of the nanocarrier through amide condensation; the 5' end of the aptamer chain is labeled with a fluorescent group; further, the quenching group is BHQ-1, and the fluorescent The group is FAM.

In a specific implementation of the above preferred technical solution, the sequence of the molecular beacon is as follows:

FAM-AGGTCGGTAAGCTTCAAGATCCATCCCGACCTCGCGAATGATTAGGTCGATAAGCTACAGGAGGCTACATGACCTCGCGAATGATT;

The sequence of the anchor DNA is as follows:

_H2N -AATCATTCGCG-BHQ1.

The nano-carrier modified by the molecular beacon described in the above scheme is prepared as follows: the mesoporous silicon nanoparticles are synthesized by the sol-gel method, and amino groups are connected on the surface of the mesoporous silicon nanoparticles to obtain aminated silica (MSN- NH ₂ ), carboxylate MSN-NH ₂ to obtain carboxylated mesoporous silicon nanoparticles (MSN-COOH); modify the aminated anchor DNA on the surface of MSN-COOH to generate anchor DNA modified by amide condensation reaction MSN (MSN-AD); Dissolve Dox in the buffer solution, add MSN-AD and stir the reaction in a dark environment to load the drug; continue to add molecular beacons to the drug-loaded MSN-AD buffer to complete the hybridization The molecular marker-modified nanocarrier (Dox@MSN-AD-MBs) can be obtained.

Further, in the above preparation method, the method of connecting amino groups on the surface of mesoporous silicon particles is as follows: add 3-aminopropyltriethoxysilane (APTES) to the toluene solution of MSN, heat up to 110-120°C and react for 1- After 3 hours, the solid part was obtained. After washing the solid part, it was dispersed in the methanolic hydrochloric acid solution and refluxed for 14-18 hours to obtain the MSN-NH ₂ .

Further, in the above preparation method, the method of carboxylation of MSN-NH ₂ is as follows: add MSN-NH ₂ and succinic anhydride to N,N-dimethylformamide (DMF), and react at room temperature for 7-9 hours , you can get MSN-COOH.

Further, in the above preparation method, the preparation method of the MSN-AD is as follows: add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC ) and N-hydroxysulfosuccinimide (Sulfo-NHS), react for 12 to 17 minutes to activate the carboxyl group; add PBS and aminated anchor DNA, and continue to react for 24 hours to generate MSN-AD.

Further, the stirring reaction time in the dark environment is 10-14 hours.

Further, in the preparation method, molecular beacons are added to the buffer of MSN-AD loaded with drugs and reacted for 4-8 hours.

The second aspect of the present invention provides the use of the molecular beacon-modified nanocarrier described in the first aspect as an anti-tumor active ingredient.

In the second aspect above, the application as an anti-tumor active ingredient includes but is not limited to any of the following:

(1) administering the molecular beacon-modified nanocarrier described in the first aspect to an individual in need of treatment;

(2) using the nano-carrier modified by the molecular beacon described in the first aspect for the preparation of anti-tumor products;

(3) Using the molecular beacon-modified nanocarrier described in the first aspect as an anti-tumor model agent.

In the application of the above-mentioned aspect (1), the "individual in need of treatment" means an individual in need of treatment for a tumor-related disease, preferably, a drug-resistant tumor; the "treatment" includes remission of the disease, Inhibition, improvement and cure; In addition, the "individual" means any animal, preferably a mammal, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or spirits Long animals, and most preferably humans.

In the application of the above (2) aspect, the anti-tumor products include but not limited to anti-tumor drugs, health care products with anti-tumor effects, special medical food, and medical devices used in anti-tumor treatment.

In the application of the above-mentioned aspect (3), the "anti-tumor model drug" includes drug preparations involved in in vivo and in vitro models for screening anti-tumor drugs and evaluating their efficacy. Based on the design of the first aspect of the present invention, the molecular beacon can hybridize with MDR1 mRNA after detaching from the nanocarrier, and at the same time realize in situ imaging of tumor cells. Play the situation to observe.

The third aspect of the present invention provides a pharmaceutical composition, which includes the molecular beacon-modified nanocarrier described in the first aspect.

Preferably, the above pharmaceutical composition includes other active ingredients and/or pharmaceutically necessary excipients.

Further, the other active ingredients include but are not limited to one or more of antineoplastic drugs, anti-inflammatory drugs, immunomodulatory drugs, analgesic drugs, and hemostatic drugs.

The fourth aspect of the present invention provides a drug for treating drug-resistant tumors, in which the molecular beacon-modified nanocarrier described in the first aspect is used as an active ingredient.

The beneficial effects of the above one or more technical solutions are:

In a solution provided by the present invention, a molecular beacon containing the antisense sequence of MDR1 mRNA is used as the gate of MSN to construct a multifunctional mesoporous silicon nanocarrier for silencing and imaging of MDR1 mRNA in drug-resistant cancer cells and drug delivery. By imaging intracellular MDR1 mRNA, the nanocarrier can be used to distinguish drug-resistant cancer cells from non-drug-resistant cancer cells. As the antisense sequence of MDR1 mRNA, MB can effectively silence MDR1 mRNA, inhibit its translation and down-regulate the expression of P-gp, thereby reducing drug efflux and used to combat drug resistance. Compared with free Dox, Dox@MSN-AD-MBs showed significantly enhanced inhibitory potency against HepG2/ADR cells and MCF-7/ADR cells. The IC ₅₀ values of free Dox and Dox@MSN-AD-MBs on HepG2/ADR cells were 0.47 μM and 1.2 μM, respectively, and the IC ₅₀ values on MCF-7/ADR cells were 2.9 μM and >5.0 μM, respectively. By integrating the functions of cancer marker imaging, gene silencing and drug delivery, this nanocarrier provides a new idea for the development of multifunctional drug delivery system, which has potential applications in the diagnosis and treatment of cancer and the suppression of drug resistance.

Description of drawings

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention.

Fig. 1 is the principle of the silencing, imaging and drug delivery of MDR1mRNA in drug-resistant cancer cells using the mesoporous silicon nanocarrier modified by the molecular beacon described in the present invention;

Fig. 2 is non-denaturing PAGE verification principle feasibility in embodiment 1;

Lane M: Marker; Lane 1: MDR1 mRNA target sequence (500nM); Lane 2: MB (500nM); Lane 3: Anchor DNA (500nM); Lane 4: MB (500nM) + anchor DNA (500nM); Lane 5: Anchored DNA-MB hybrid (500nM) + MDR1 mRNA target sequence (500nM);

Fig. 3 is the construction and characterization of MSN-AD-MBs described in embodiment 1;

(A) TEM image of MSN; (B) hydrated particle size distribution curves of MSN, MSN-AD and MSN-AD-MBs; (C) BET nitrogen adsorption-desorption curve of MSN; (D) pore size distribution curve of MSN; (E) Surface Zeta potential of MSN, MSN-NH ₂ , MSN-COOH, MSN-AD and _MSN -AD-MBs; (F) FT- IR spectrum;

Fig. 4 is the photo of MSN-COOH (left) and MSN-AD (right) described in embodiment 1;

Fig. 5 is the modification amount of MB described in embodiment 1 and the loading amount result of Dox;

(A) Fluorescence signal intensity-concentration linear relationship of FAM-labeled MB; (B) Fluorescence signal intensity-concentration linear relationship of Dox; (C) Fluorescence emission spectra of the solution before and after FAM-labeled MB modification; (D) Fluorescence emission spectra of the solution before and after Dox loading Fluorescence emission spectrum;

Fig. 6 is the stability of nano-carrier and Dox release investigation result in vitro;

(A) MSN-AD-MBs in PBS buffer system and PBS buffer system containing glutathione, DNase I, MDR1 mRNA target sequence FAM fluorescence signal recovery rate-time relationship diagram; (B) Dox@MSN-AD - Dox release rate-time curves of MBs co-incubated with different concentrations of MDR1mRNA;

Fig. 7 is the relative expression level of MDR1 mRNA in qRT-PCR analysis HepG2 cell, HepG2/ADR cell, MCF-7 cell and MCF-7/ADR cell described in embodiment 1;

Figure 8 is a confocal fluorescence imaging picture of HepG2 cells, HepG2/ADR cells, MCF-7 cells and MCF-7/ADR cells after MSN-AD-MBs treatment described in Example 1; the scale bar is 20 μm;

Figure 9 is the confocal fluorescence imaging images of different tumor cells treated with free Dox and Dox@MSN-AD-MBs;

Among them, (A) is HepG2/ADR cells; (B) is MCF-7/ADR cells; the scale bar is 20 μm;

Figure 10 is the relative expression level of MDR1 mRNA in tumor cells after qRT-PCR analysis of different concentrations of MSN-AD-MBs (in MB concentration) to treat tumor cells;

Among them, (A) is HepG2/ADR cell; (B) is MCF-7/ADR cell;

Figure 11 is the MTT method analysis MSN-AD-MBs, the survival rate of tumor cells after free Dox and Dox@MSN-AD-MBs (in terms of Dox concentration) treatment;

Among them, (A) are HepG2/ADR cells treated with different concentrations of the above drugs; (B) are MCF-7/ADR cells treated with different concentrations of the above drugs; (C) are HepG2/ADR cells treated with the same concentrations of the above drugs. ADR cells; (D) is the survival rate of MCF-7/ADR cells treated with the same concentration of the above drugs; the Dox concentrations used in HepG2/ADR cells and MCF-7/ADR cells were 1.3 μM and 4.0 μM, respectively.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used here is only for describing specific embodiments, and is not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof.

In order to enable those skilled in the art to understand the technical solution of the present invention more clearly, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

Example 1

Reagents and Instruments

The nucleic acids used in this example (see Table 1 and Table 2 for specific sequences) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Tetraethoxysilane (TEOS) was purchased from Sinopharm Group Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), cetyltrimethylammonium (CTAB) and succinic anhydride were purchased from Suleibao Biotechnology Co., Ltd. (Beijing, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) was purchased from BBI Bioscience Co., Ltd. (Shanghai, China). 3-Aminopropyl-triethoxysilane (APTES) was purchased from Mecox Lane Bioscience Co., Ltd. (Beijing, China). Doxorubicin (Dox) was purchased from Guangdong Huafeng Pharmaceutical Co., Ltd. (Guangdong, China). Glutathione (GSH) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Beijing, China). DNase I was purchased from NEB Beijing Company (Beijing, China). Trypsin, RPMI 1640, DMEM and PBS buffers were purchased from Biological Industries Ltd. (Israel). Tetramethylazolazolium blue (MTT) was purchased from Sigma-Aldrich Chemical Reagents, Inc. (NJ, USA). The reagents used in this work were of analytical grade, and the aqueous solutions and buffers were prepared with ultrapure water.

Table 1 Nucleic acid sequence used in this embodiment

Note: The green part in MB represents the MDR1 mRNA recognition sequence, the underlined part represents the stem of the hairpin, and FAM represents the FAM fluorophore label; the underlined part in Ctrl-MB represents the stem of the hairpin; the H in the anchor DNA ₂ N stands for amino modification, BHQ-1 stands for BHQ-1 quencher label.

Table 2 The qRT-PCR primer sequences used in this embodiment

Transmission electron microscopy (TEM) images were taken with a JEM2100 microscope (Japan). The Brunauer-Emmett-Teller (BET) nitrogen adsorption-desorption isotherm was drawn using ASAP2020 (USA). Dynamic light scattering (DLS) and surface zeta potential data of nanoparticles were measured using Malvern Nano ZSAnalyzer (UK). Fourier transform infrared (FT-IR) spectra were recorded using a Tensor II spectrometer (Germany). A F-7000 Hitachi fluorescence emission spectrometer (Japan) was used to measure the fluorescence signal intensity and collect fluorescence emission spectra. Hitachi U-2910 spectrometer (Japan) was used to collect UV-vis absorption spectrum and measure absorbance. Fluorescent imaging pictures were taken with a Leica TCS SP8 confocal laser scanning microscope (CLSM) (Germany). MTT experimental data were measured by Tecan multimode microplate reader (Switzerland).

Gel electrophoresis experiment

The binding feasibility of molecular beacon (MB) to anchor DNA and the ability of MB to recognize the target sequence of MDR1 mRNA were verified by polyacrylamide gel electrophoresis (PAGE). First, prepare a 12% PAGE gel, take acrylamide solution (7.5mL, 40%), 5×TBE buffer (Tris 88mM, boric acid 88mM, EDTA 2.0mM, pH 8.3, 5.0mL), N,N,N Mix ',N'-tetramethylethylenediamine (18 μL), ammonium persulfate solution (0.1 g mL ^-1 , 180 μL) and DEPC water (12.5 mL) to make a gel. After the sample was injected into the gel, it was electrophoresed in 1×TBE buffer at 15° C. for 1 h at a constant current of 25 mA. After electrophoresis, the gel was taken out and stained in SYBR Gold (1/100,000) solution in the dark for 40 min. Finally, the stained gel was placed on the Bio-RAD gel imaging system for imaging.

Synthesis of Aminated Mesoporous Silicon Nanoparticles

MSNs were synthesized using the classic sol-gel method. The specific steps are: dissolving CTAB (0.5 g) in deionized water (240 mL), and slowly adding NaOH solution (2.0 M, 1.8 mL) under stirring. Then the temperature was raised to 80° C., TEOS (4.0 mL) was added slowly, and the reaction was maintained at 80° C. for 2 h, resulting in a white precipitate. After cooling to room temperature, the white precipitate was separated by centrifugation (12000 rpm, 6 min), washed with ethanol and water, and dried in an oven. The linkage of the amino group is carried out by post-modification method. Specifically, the dried solid (400 mg) was suspended in toluene (40 mL), APTES (80 μL) was added under stirring conditions, and the temperature was raised to 110° C. for 2 h to obtain aminated silica. After the above solid was separated by centrifugation and washed with ethanol, it was dispersed in methanolic hydrochloric acid solution (37.4% hydrochloric acid 2.0mL, methanol 160mL) and refluxed for 16h to remove the internal CTAB template to obtain aminated mesoporous silicon nanoparticles (MSN -NH ₂ ). The obtained MSN-NH ₂ was washed with ethanol and dried in an oven for use.

Preparation of MSN-AD-MBs

Before modifying DNA, carboxylate MSN- _NH2 . Carboxylation is obtained by reaction of anhydrides with amino groups. Specifically, MSN-NH ₂ (50mg) and succinic anhydride (500mg) were added to N,N-dimethylformamide (DMF, 15mL) and reacted at room temperature for 8 hours to obtain carboxylated mesoporous silicon nano Granules (MSN-COOH). MSN-COOH was centrifuged and washed with water, then resuspended in MES buffer (MES 100mM, pH 6.0, 5.0mL) for subsequent experiments.

Aminated anchor DNA was modified on the surface of MSN-COOH by amide condensation reaction. Take the above MSN-COOH (5.0mg, 0.5mL) suspension, add EDC (15.0mg) and Sulfo-NHS (25.0mg) under stirring condition, react for 15min, and activate the carboxyl group. Add PBS (1.0mL, pH 7.4) and aminated anchor DNA (30μM, 450μL) and continue to react for 24h to generate anchor DNA-modified MSN (MSN-AD). MSN-AD was separated by centrifugation, washed with water and dispersed in PBS buffer.

The preparation of MSN-AD-MBs is carried out on the basis of MSN-AD. The above MSN-AD (22 μL) suspension was diluted to 1.0 mL with PBS, MB (100 μM, 10 μL) was added under stirring, and the reaction was continued for 6 h to complete hybridization to prepare nanocarriers (MSN-AD-MBs). MSN-AD-MBs were separated by centrifugation and washed with PBS buffer. The supernatant and washing liquid were combined, and the reduction of MB in PBS buffer during the modification process was calculated by the fluorescence method, which was the amount of MB modified on the MSN surface.

Preparation of Dox@MSN-AD-MBs

Dox (1.0 mg) was dissolved in PBS buffer (1.0 mL), MSN-AD (200 μL) was added under stirring, and the reaction was stirred for 12 h in the dark to fully load Dox, and then MB (100 μM, 45 μL) was added. Continue to react for 6h to obtain drug-loaded nanocarriers (Dox@MSN-AD-MBs). Stop the reaction, separate the prepared Dox@MSN-AD-MBs by centrifugation, wash with PBS buffer, and suspend Dox@MSN-AD-MBs in PBS buffer (1.0 mL) for use. The carrying amount of Dox was calculated by fluorescence method, and the amount of Dox loaded in MSN was determined by the decrease of Dox before and after loading.

Study on Dox release in vitro

The in vitro release of Dox was investigated by the semi-permeable membrane bag diffusion method. In order to verify the specificity of drug-loaded nanocarriers releasing Dox, in this example, different concentrations (0, 1.0, 2.0 and 4.0 μM) of the MDR1 mRNA target sequence were mixed with Dox@MSN-AD-MBs and mixed at 100 rpm at 37°C. The speed oscillates for 24h. During the shaking process, samples were taken at predetermined time points (0.5, 1, 2, 3, 4, 5, 6.5, 8, 12 and 24 h), 0.1 mL each time, and the Dox in the supernatant was quantitatively analyzed after centrifugation. PBS buffer (0.1 mL) was added after each sampling to ensure that the total volume remained constant. Dox was quantified by a fluorometric method. Specifically, first measure the fluorescence signal intensity of different concentrations of Dox solutions, draw a standard curve of fluorescence signal intensity and Dox concentration, and then quantify the Dox concentration according to the standard curve.

Study on the Stability of Nanocarriers

In order to investigate the stability of nanocarriers, in this example, MSN-AD-MBs were co-incubated with MDR1 mRNA target sequence (100nM), GSH (15mM), and DNase I (3.5mU mL ^-1 ). Samples were taken at predetermined time points (10, 20, 30, 40, 50 and 60 min), and the fluorescence signal intensity of the FAM group was measured after centrifugation. After quantifying FAM, the fluorescence recovery rate of FAM was calculated and compared with the blank group.

Cells and Cell Culture

HepG2 cells were purchased from the Chinese Academy of Sciences, HepG2/ADR cells were purchased from Aiyan Biotechnology Company, MCF-7 cells and MCF-7/ADR cells were purchased from Dr. Liu Zhenzhen, School of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University and School of Pharmacy, Qilu Medical College, Shandong University, respectively. Kindly provided by Professor Han Xiuzhen. HepG2 cells and HepG2/ADR cells were cultured with DMEM medium containing 10% fetal bovine serum and 1.0% antibiotics, MCF-7 cells and MCF-7/ADR cells were cultured with RPMI 1640 medium containing 10% FBS and 1.0% antibiotics . All cells were cultured at 37 °C in an incubator with 5.0% _CO2 .

Cell fluorescence imaging experiment

HepG2 cells, HepG2/ADR cells, MCF-7 cells, and MCF-7/ADR cells were inoculated into confocal culture dishes and incubated for 24 h, and then added with MSN-AD-MBs (50 μg mL ^-1 , 150 μL) Fresh culture medium was incubated for another 4 h, and imaged under a fluorescent microscope.

Intracellular Dox accumulation analysis

HepG2/ADR and MCF-7/ADR cells were inoculated into confocal culture dishes respectively, and after incubation for 24 hours, the medium was discarded. Then the cells were washed with PBS buffer, and the cells were divided into two groups, and the two groups of cells were co-incubated with the medium containing free Dox and Dox@MSN-AD-MBs (both containing 1.0 μM Dox) for 4 h, and placed in Imaging was performed under a fluorescence microscope.

MDR1 mRNA expression level analysis

The relative expression levels of MDR1 mRNA in different cells were analyzed by qRT-PCR. HepG2 cells, MCF-7 cells, HepG2/ADR cells and MCF-7/ADR cells were seeded in six-well plates and cultured with different concentrations of MSN-AD-MBs (containing 0, 50, 100 and 200 nM of MBs) After 48 hours of medium culture (HepG2 cells and MCF-7 cells were cultured with culture medium), the total RNA of the cells was extracted with Trizol reagent. GAPDH was selected as an internal reference for qRT-PCR experiments, and the cycle program was 95°C for 3min and 60°C for 30s (45 cycles). The relative expression level of MDR1 mRNA was calculated by the 2 ^-ΔΔCt method.

Western blot analysis

HepG2/ADR cells and MCF-7/ADR cells were seeded in six-well plates, incubated with different concentrations of MSN-AD-MBs (containing 0, 50, 100 and 200 nM MB) for 48 h, and then washed with warm saline Wash the cells 3 times and remove the medium. Next, RIPA lysis buffer containing protease and phosphatase inhibitors is added to extract the total protein of the cells. The extracted protein was added to the protein sample loading buffer, heated to 100°C for 5 minutes to denature the protein, and stored in a -20°C refrigerator after aliquoting to avoid repeated freezing and thawing. The expression level of P-glycoprotein (P-gp) was characterized according to the western blot analysis manual. Specific steps are as follows:

First, make 8.0% separating gel and 4.0% stacking gel, the method is: take deionized water (7.05mL), Tris hydrochloric acid buffer (1.5M, pH 8.8, 3.75mL), sodium lauryl sulfate solution (10 %, 150μL), acrylamide solution (30%, 4.95mL), N,N,N',N'-tetramethylethylenediamine (30μL), ammonium persulfate solution (0.1g mL ^-1 , 75μL) To make the separating gel, after adding the above mixed solution to the glass plate, carefully add 0.1% sodium lauryl sulfate solution to ensure the smooth surface of the gel. After standing for 1 hour, the 0.1% sodium dodecyl sulfate solution was removed, and then the laminated gel was prepared. Deionized water (5.4mL), Tris hydrochloric acid buffer (1.5M, pH 6.8, 2.5mL), sodium lauryl sulfate solution (10%, 100μL), acrylamide solution (30%, 2.0mL), N, Mix N,N',N'-tetramethylethylenediamine (20 μL) and ammonium persulfate solution (0.1 g mL ^-1 , 50 μL) to prepare a separating gel. After pouring the gel into the glass plate, insert the comb and let it stand for 1 hour to complete the gel making.

Then, add the above-mentioned total protein extract (loading amount: 30 μg per lane), and perform electrophoresis. The initial voltage is 80V. After the blue band of the loading buffer enters the separation gel, increase the voltage to 120V and continue the electrophoresis until the blue band completely runs out, and the electrophoresis ends. After electrophoresis, the protein was transferred to PVDF membrane (Millipore Immobilon-P, 0.45 μm) in transfer buffer. After the membrane transfer, place the PVDF membrane in 5% skimmed milk dissolved in TBST buffer (10mM Tris, 150mM NaCl, 0.05% Tween-20, pH 7.2-7.5), and incubate gently at room temperature for 1h to seal Nonspecific protein binding sites on membranes. After washing the PVDF membrane with TBST buffer three times, the PVDF membrane at the corresponding protein position was cut, and placed in the corresponding primary antibody incubation solution, and incubated overnight at 4°C. The next day, the PVDF membrane was washed three times with TBST, and incubated with horseradish peroxide-labeled secondary antibody at room temperature for 2 h with gentle shaking.

Finally, after washing 3 times with TBST, the PVDF membrane was placed in an imager for imaging.

Cytotoxicity study

First, HepG2/ADR cells and MCF-7/ADR cells were inoculated into 96-well plates, and after incubation for 24 hours, the medium was discarded. Different concentrations of free Dox, MSN-AD-MBs and Dox@MSN-AD-MBs (Dox concentrations of HepG2/ADR cells were 0.1, 0.5, 1.0, 1.25 and 1.5 μM, Dox of MCF-7/ADR cells Concentrations of 0.1, 1.0, 2.0, 3.0 and 5.0 μM, MSN-AD-MBs added in the same amount as Dox@MSN-AD-MBs) were incubated in fresh medium for 48h. After discarding the medium, the cells were washed with PBS, and MTT solution (0.5 mg mL ^-1 , 100 μL) was added to each well. After further incubation for 4 h, the supernatant was removed and DMSO (100 μL per well) was added. To dissolve the formazan produced. After shaking for 5 min, measure the absorbance value of each well at 570 nm with a microplate reader. Cell viability (%) was calculated using the following formula:

Where A _sample , A _control and A _blank represent the absorbance of the sample, control and blank.

In order to further verify the role of MB in enhancing the inhibitory efficiency of drug-resistant cancer cells, this example treated HepG2/ADR cells with nanocarriers loaded with MB and ctrl MB (MB containing 8 MDR1 mRNA mismatched bases) and MCF-7/ADR cells, and examine cell viability. The pretreatment method of the cells was the same as the above-mentioned cytotoxicity investigation method. After washing the cells with PBS buffer, HepG2/ADR cells and MCF-7/ADR cells were treated with the same concentration of free Dox, Dox@MSN-AD-ctrl MBs, respectively. and Dox@MSN-AD-MBs (the Dox concentration of HepG2/ADR cells was 1.3 μM, and the Dox concentration of MCF-7/ADR cells was 4.0 μM) for 48 h, and after the same treatment, the microplate reader was used to measure each The OD value of each hole at 570nm. The cell viability calculation formula is the same as above.

Design rationale for multifunctional molecular beacon-modified mesoporous silicon nanocarriers for MDR1 mRNA silencing, imaging, and drug delivery in drug-resistant cancer cells

The principle of multifunctional molecular beacon (MB)-modified mesoporous silicon nanocarriers for MDR1 mRNA silencing, imaging and drug delivery in drug-resistant cancer cells is shown in Figure 1. First, the small-molecule chemotherapeutic drug Dox was loaded into the cavity of carboxylated mesoporous silicon nanoparticles (MSN-COOH) by diffusion to form Dox-loaded MSN-COOH (Dox@MSN-COOH). Then, Dox@MSN-AD was constructed by modifying amino group and quencher (BHQ-1) double-labeled anchor DNA (AD) on the surface of Dox@MSN-COOH by amide condensation. On this basis, drug-loaded nanocarriers (Dox@MSN-AD-MBs) were prepared by linking FAM-labeled MBs via complementary base pairing. MB is responsible for recognizing MDR1 mRNA and blocking MSNs. When Dox@MSN-AD-MBs were taken up by drug-resistant cancer cells, the MB on the surface hybridized with MDR1 mRNA, unhybridized with AD, and fell off from the carrier. The hybridization event between MB and MDR1 mRNA has a triple effect: first, the hybridization event between MB and MDR1 mRNA can be used to suppress drug resistance by silencing MDR1 mRNA and down-regulating the expression level of P-gp; secondly, the hybridization event between MB and MDR1 mRNA can make The distance between the FAM fluorophore and the BHQ-1 quencher restores fluorescence for in situ imaging of MDR1 mRNA; finally, the separation of MB can also unblock the MSN, open the pores of MSN, release Dox, and exert cytotoxicity. The multifunctional MB-modified mesoporous silicon nanocarrier constructed in this chapter has two advantages for cancer marker sensing and drug delivery. First, by loading Dox and modifying MB, the nanocarrier combines the imaging and drug delivery functions of MDR1 mRNA in drug-resistant cancer cells, which helps to inhibit cancer cells while analyzing the expression information of cancer markers in situ in real time; Second, by integrating MB with MDR1 mRNA silencing effect, the nanocarrier down-regulates the expression levels of MDR1 mRNA and P-gp, thereby inhibiting the drug resistance of drug-resistant cancer cells, effectively enhancing the delivery efficiency of chemotherapeutic drugs and Inhibitory potency of drug-resistant cancer cells.

Gel Electrophoresis Characterization

Non-denaturing PAGE was used to verify the feasibility of hybridization of MB to the anchor DNA and the binding ability of MB to the target sequence of MDR1 mRNA. As shown in Figure 2, lanes 1-3 represent the MDR1 mRNA target sequence band, MB band and anchor DNA band, respectively. After mixing anchor DNA and MB, a clear band appeared in lane 4, which moved slower than MB alone (lane 2) and anchor DNA (lane 3), indicating that the anchor DNA hybridized to MB Bands, indicating that the anchor DNA can hybridize to MB. Lane 5 is the electrophoresis result after the anchor DNA-MB hybrid and the MDR1 mRNA target sequence are mixed, and the anchor DNA-MB hybrid band (lane 4) disappears in the lane, indicating that when the MDR1 mRNA target sequence exists, the MDR1 mRNA target sequence Can compete with MB to form MDR1mRNA-MB hybrid; at the same time, the anchor DNA band appears (lane 3), indicating that the combination of MDR1 mRNA and MB can dissociate MB from the anchor DNA. PAGE results showed that the interaction between the designed nucleic acid sequences met the design requirements.

Construction and characterization of MSN-AD-MBs

TEM images (Fig. 3A) showed that the synthesized MSNs were porous spherical particles with an average particle size of about 138.4 nm. The average hydrated particle size of MSN was further measured by DLS, which was about 146.9nm (Figure 3B), which was slightly larger than the average particle size observed under the electron microscope, because the particle size measured by DLS method was that the nanoparticles were solvated in aqueous solution The particle size of the formed hydrated ions will be slightly larger than the MSN particle size directly observed under TEM. Studies have shown that MSN within the particle size range of 135-200nm has less cytotoxicity and is easily taken up by cells, which has advantages in biomedical applications. In addition, the present embodiment has carried out BET nitrogen adsorption-desorption test, and the BET adsorption-desorption isotherm of MSN (Fig. 3C) is the type IV isotherm, shows that MSN has mesoporous structure, and its average pore diameter is further calculated as 2.3nm (Fig. 3D ).

Next, this example characterizes the preparation process of nanocarriers. The average hydrated particle sizes of MSN, MSN-AD and MSN-AD-MBs were determined by DLS method. As shown, the average hydrated particle size of MSN-AD (155.5 nm) and MSN-AD-MBs (163.2 nm) was larger than that of MSN, which also indicated the successful modification of anchoring DNA and MBs. In order to further prove this point, in this example, the surface Zeta potential values of the structures appearing during the preparation of MSN-AD-MBs were measured ( FIG. 3E ). MSN presents a negative potential due to the abundant silicon hydroxyl groups on the surface; the surface Zeata potential of MSN-NH ₂ presents a positive potential due to the easy binding of amino groups to protons; after carboxylation and DNA modification, due to the negative charge of carboxyl and nucleic acid itself, MSN-COOH and MSN-AD-MBs surface Zeta potential value is negative. The above shows that the change of Zeta potential value provides evidence for the success of carboxylation and DNA modification. Next, in this example, MSN-NH ₂ , MSN-COOH and MSN-AD-MBs were verified by FT-IR. As shown in Figure 3F, the FT-IR spectrum of MSN-NH ₂ has an absorption peak at 1503 cm ^-1 , which is the bending vibration peak of the NH bond and is the characteristic absorption peak of the amino group, confirming the existence of the amino group; the MSN-COOH The characteristic absorption peak of the amide bond (1556cm ^-1 , NH vibration peak) and the characteristic absorption peak of the carboxyl group (1720cm ^-1 , the stretching peak of the carbonyl group in the carboxyl group) appear in the spectrum, indicating the successful connection of the carboxyl group; in the spectrum of MSN-AD-MBs In the figure, the peak at 1720cm ^-1 disappears, and only the characteristic absorption peak of amide bond (1557cm ^-1 ) remains, which is because the carboxyl group and amino group form an amide bond, which further proves the successful preparation of MSN-AD-MBs.

In addition, after modifying the anchor DNA on MSN-COOH, this example took pictures of MSN-COOH and MSN-AD respectively. As shown in Figure 4, the colors of MSN-COOH and MSN-AD are white and pink, respectively, and pink is the color of the anchored DNA labeled by BHQ-1. This color change visually proves the successful modification of the anchored DNA.

Modified amount of MB and loaded amount of Dox

MB modification and Dox loading were calculated by subtraction method. Standard curves were first drawn for FAM-labeled MB and Dox, respectively. As shown in Figure 5A, within the concentration range of 40-250nM, the FAM fluorescence signal intensity (F _FAM ) has a good linear relationship with the MB concentration (C _MBs ), and the linear equation is F _FAM =29.2C _MBs +45.1(R ² = 0.999); within the concentration range of 0.1-20 μg mL ^-1 , Dox fluorescence signal intensity (F _Dox ) and Dox concentration (C _Dox ) showed a good linear relationship (Fig. 5B), and the linear equation was F _Dox ＝10.9C _Dox -1.18 (R ² =0.997). Then the fluorescent signal intensities of MB before and after modification and Dox before and after loading were measured (Figure 5C and Figure 5D), and the concentrations of MB and Dox were converted according to the working curve. The reduction of MB before and after modification and the reduction of Dox before and after loading were MB modification and Dox loading, which were 4.2 μmol g ^-1 and 100.9 mg g ^-1 , respectively.

Study on the Stability of Nanocarriers and the Release of Dox in Vitro

Since intracellular enzymes and reducing substances often lead to non-specific degradation or destruction of nanocarriers, resulting in false positive signals for imaging or non-specific release of drugs, this example examines the stability of MSN-AD-MBs in different environments . Use PBS buffer, PBS buffer containing DNase I and GSH to incubate MSN-AD-MBs, simulate the intracellular environment, intracellular enzyme environment and reducing environment, respectively, measure the recovery rate of FAM fluorescence signal and pass with positive samples (with MDR1mRNA The medium incubation of the target sequence incubation) was compared to assess the stability of the vector. As shown in Figure 6A, after incubation with PBS buffer and PBS buffer containing DNase I and GSH for 60 min, the recovery rate of fluorescence signal of MSN-AD-MBs was not significantly different from that of negative samples, which was significantly lower than that of positive samples with the same incubation conditions , indicating that MSN-AD-MBs have good tolerance to intracellular enzymes and reducing substances, and the carrier has good stability. This is because the steric hindrance of MSN can effectively prevent the degradation of nucleic acid modified on the surface of MSN by interfering substances in the environment such as nucleases, thereby ensuring the structural integrity and stability of the nanocarrier.

The in vitro release of Dox@MSN-AD-MBs was investigated using a semipermeable membrane bag method, the principle of which is that the semipermeable membrane bag allows Dox to pass through but not MSN. By testing the Dox fluorescence signal intensity at different time points and converting it to the Dox concentration according to the standard curve, this example draws the Dox release rate-time curve incubated with different concentrations of the MDR1 mRNA target sequence. As shown in Figure 6B, in the absence of MDR1 mRNA, the release rate of Dox within 24 h was 12.6%, which was significantly lower than that in the presence of the MDR1 mRNA target sequence. The release rate of Dox at the same time point increased with the concentration of MDR1 mRNA target sequence. After incubation with 4 μM MDR1 mRNA target sequence for 24 h, the release rate of Dox reached 83.2%. These data indicated that the nanocarriers could release the carried drug Dox in response to the MDR1 mRNA target sequence, and the release rate of Dox increased with the concentration of the MDR1 mRNA target sequence, which was consistent with the design principle.

Cell Fluorescence Imaging Studies

In order to verify the feasibility of MSN-AD-MBs for intracellular MDR1 mRNA imaging, this embodiment selected two groups of cell lines with significant differences in MDR1 mRNA expression levels for fluorescence imaging experiments, respectively HepG2 cells (human liver cancer cell line ) and HepG2/ADR cells (human Dox-resistant liver cancer cell line), MCF-7 cells (human breast cancer cells) and MCF-7/ADR cells (human Dox-resistant breast cancer cell line). First, the present embodiment investigated the relative expression levels of MDR1 mRNA in two groups of cells by qRT-PCR method. As shown in Figure 7, the qRT-PCR results showed that the relative expression levels of MDR1 mRNA in drug-resistant cell lines HepG2/ADR cells and MCF-7/ADR cells were significantly higher than those in non-drug-resistant cell lines HepG2 cells and MCF-7 cells, Consistent with literature reports.

Then, in this example, the fluorescence imaging method was used to investigate the application of nanocarriers for intracellular MDR1 mRNA imaging. As shown in Figure 8, after incubation with MSN-AD-MBs for 4 h, drug-resistant cells HepG2/ADR cells and MCF-7/ADR cells showed strong FAM fluorescence signals under confocal laser scanning microscopy (CLSM), significantly Higher than the non-drug-resistant cell lines HepG2 cells and MCF-7 cells treated in the same way. The results of fluorescence imaging were consistent with those detected by qRT-PCR, indicating that MSN-AD-MBs can image MDR1 mRNA in living cells and be used to distinguish drug-resistant cancer cells from non-drug-resistant cancer cells. The reason is that after the nanocarriers are taken up by the cells, under the action of MDR1 mRNA, MB falls off from the nanocarriers, away from the quencher group to restore fluorescence, thereby indicating MDR1 mRNA.

As mentioned in the Introduction, P-gp overexpression in drug-resistant cancer cells is one of the main obstacles leading to insufficient intracellular drug accumulation. Theoretically, after Dox@MSN-AD-MBs are taken up by drug-resistant cancer cells, due to the silencing effect of MB on MDR1 mRNA, the expression level of P-gp in the cells is down-regulated, which can reduce the efflux of Dox and increase the intracellular accumulation. In order to prove this point, this example investigated the accumulation of Dox in drug-resistant cancer cells co-incubated with the medium containing free Dox and Dox@MSN-AD-MBs by fluorescence imaging experiments, and compared with the blank cells without Dox For comparison. As shown in Figure 9, no Dox fluorescence was observed in the control cells, indicating that the cells themselves did not produce interference signals. The Dox fluorescence signal in HepG2/ADR cells treated with Dox@MSN-AD-MBs (Fig. 9A) was significantly stronger than that of HepG2/ADR cells treated with free Dox at the same concentration, indicating that the drug-loaded nanocarriers can effectively enhance the effect of Dox on drug-resistant cancer. The accumulation in cells is consistent with the experimental principle. Similarly, MCF-7/ADR cells treated with Dox@MSN-AD-MBs (Fig. 9B) were significantly stronger than MCF-7/ADR cells treated with free Dox at the same concentration, which further proved the above conclusion. The above results indicate that nanocarriers have potential applications in improving drug delivery efficiency.

MDR1 mRNA silencing investigation

Studies have shown that under the premise of effective delivery into cells, antisense oligonucleotides can silence the expression of corresponding mRNA by hybridizing with mRNA and inhibit the translation process. The nanocarrier proposed by the present invention integrates the MB containing the antisense sequence of MDR1 mRNA, therefore, this example assumes that the nanocarrier can be used to silence MDR1 mRNA and down-regulate P-gp expression. In order to verify this point, the present embodiment measured the relative expression levels of MDR1 mRNA and P-gp in HepG2/ADR cells and MCF-7/ADR cells before and after MSN-AD-MBs treatment by qRT-PCR method and Western blot method respectively . As shown in Figure 10A and Figure 10B, after incubation with MSN-AD-MBs for 48h, the relative expression levels of MDR1 mRNA in HepG2/ADR cells and MCF-7/ADR cells decreased, and the degree of decline increased with that of MSN-AD-MBs Increased concentration and enhanced, showing a concentration-dependent relationship. Western blot experiments showed that after incubation with MSN-AD-MBs for 48 hours, the expression level of P-gp in HepG2/ADR cells and MCF-7/ADR cells also decreased, and the degree of decrease also increased with the concentration of MSN-AD-MBs increase (Figure 10C and Figure 10D). The results of qRT-PCR and Western blot experiments showed that MSN-AD-MBs can effectively silence MDR1mRNA in drug-resistant cancer cells and correspondingly down-regulate the expression level of P-gp, which is consistent with the hypothesis, indicating that the nanocarriers are effective in combating drug resistance in cancer cells Has application potential.

Cytotoxicity

In order to investigate the ability of Dox@MSN-AD-MBs to fight drug resistance and enhance the inhibitory effect of cancer cells, this example measured the survival rate of HepG2/ADR cells and MCF-7/ADR cells treated with different conditions by MTT method to investigate Cytotoxicity of nanocarriers against drug-resistant cells. On this basis, this embodiment calculates the half maximal inhibitory concentration (Half maximalinhibitory concentration, IC ₅₀ ) of Dox to evaluate the efficacy of Dox in inhibiting drug-resistant cancer cells in different delivery methods. First, the present embodiment measured the cell viability of HepG2/ADR cells and MCF-7/ADR cells after incubation with different concentrations of MSN-AD-MBs, free Dox and Dox@MSN-AD-MBs for 24h (Fig. 11A and Figure 11B). Among them, the survival rate of HepG2/ADR cells and MCF-7/ADR cells treated with MSN-AD-MBs was higher than 90%, indicating that the nanocarriers have good safety. At the same Dox concentration, the cell survival rates of HepG2/ADR cells and MCF-7/ADR cells treated with Dox@MSN-AD-MBs were significantly lower than those of the same kind of cells treated with the same concentration of free Dox. When Dox concentrations were 1.5 μM and 5.0 μM, the cell viability of HepG2/ADR cells and MCF-7/ADR cells treated with Dox@MSN-AD-MBs was significantly higher than that of HepG2/ADR cells and MCF-7/ADR cells treated with free Dox. ADR cells were 40.6% and 73.1% lower, respectively. The calculated IC ₅₀ values of Dox@MSN-AD-MBs and free Dox on HepG2/ADR cells were 0.47 μM and 1.2 μM, respectively, and the IC ₅₀ values on MCF-7/ADR cells were 2.9 μM and >5.0 μM, respectively. The above results showed that, at the same Dox concentration, Dox@MSN-AD-MBs exhibited higher inhibitory efficacy on HepG2/ADR cells and MCF-7/ADR cells, indicating that nanocarriers can effectively enhance the inhibition of drugs against drug-resistant cancer cells efficacy. The reason why nanocarriers enhance the efficacy of drugs to inhibit drug-resistant cancer cells is that nanocarriers down-regulate the expression level of P-gp by silencing MDR1 mRNA, thereby reducing the efflux effect of drug-resistant cancer cells on Dox and increasing the concentration of intracellular Dox.

Finally, in order to further verify the effect of MB containing MDR1 mRNA antisense sequence on drug-resistant cancer, this example designed ctrl MB containing 8 MDR1 mRNA mismatched bases as a control, and measured the effect of MB containing free Dox, Dox HepG2/ADR cells co-incubated with @MSN-AD-ctrl MBs and Dox@MSN-AD-MBs (for HepG2/ADR cells and MCF-7/ADR cells, the Dox concentrations were 1.3 μM and 4.0 μM, respectively) and cell viability of MCF-7/ADR cells. As shown in Figure 11C, compared with free Dox-treated and Dox@MSN-AD-ctrl MBs-treated HepG2/ADR cells, Dox@MSN-AD-MBs-treated HepG2/ADR cells showed significantly higher The low cell survival rate indicated that the nanocarriers could fight drug resistance and enhance the anticancer potency of chemotherapeutic drugs only when the MB containing the antisense sequence of MDR1 mRNA was present. Interestingly, the cell survival rate of HepG2/ADR cells treated with Dox@MSN-AD-ctrl MBs was higher than that of HepG2/ADR cells treated with free Dox, which may be because Dox@MSN-AD-ctrl MBs not only Not having the effect of silencing MDR1 mRNA to combat drug resistance, also hindered drug release from nanocarriers, further reducing intracellular Dox concentration. Similar results also appeared in MCF-7/ADR cells under the same treatment conditions (Fig. 11D), which also indicated that only when the MB containing the antisense sequence of MDR1 mRNA was present, the carrier could suppress drug resistance and enhance drug inhibitory efficacy . The results of the cytotoxicity experiment verified the necessity of MB containing the antisense sequence of MDR1mRNA to suppress drug resistance, which was consistent with the design principle.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong University

<120> A Molecular Beacon Modified Nanocarrier and Its Application in the Preparation of Antitumor Products

<130> 2010

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<170> PatentIn version 3.3

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Claims (12)

1. A nano-carrier modified by a molecular beacon, characterized in that, the main body of the nano-carrier is a porous carrier, and the chemotherapeutic drug is loaded in the porous carrier, and the surface of the porous carrier is modified with molecular beacons and anchored DNA. hybridized chain; the molecular beacon blocks the through-holes on the surface of the porous carrier, and the anchor DNA is fixed on the surface of the porous carrier through chemical bonds; The porous carrier is a mesoporous silicon nanoparticle, which is a hollow sphere with through holes on the surface. The cavity of the hollow sphere is loaded with the chemotherapeutic drug. The particle diameter of the hollow sphere is 120-150 nm. The through hole The size is 2~3nm; The chemotherapeutic drug is adriamycin; In the above mesoporous silicon nanocarrier loaded with doxorubicin, the doxorubicin is loaded into the cavity of the mesoporous silicon nanoparticle by diffusion; The molecular beacon MDR1 mRNA has a high affinity and can silence MDR1 mRNA in drug-resistant tumor cells; The 5' end of the anchor DNA is connected to the surface of the nanocarrier through a chemical bond, and the 3' end is marked with a quenching group; The chemical bond is an amide bond, the 5' end of the anchor DNA has an amino modification, and is connected to the carboxyl group on the surface of the nanocarrier through amide condensation; the 5' end of the molecular beacon is marked with a fluorescent group; The quenching group is BHQ-1, and the fluorescent group is FAM; The sequence of the molecular beacon is as follows: FAM-AGGTCGGTAAGCTTCAAGATCCATCCCGACCTCGCGAATGATTAG GTCGATAAGCTACAGGAGGCTACATGACCTCGCGAATGATT; The sequence of the anchor DNA is as follows: _H2N -AATCATTCGCG-BHQ1. 2. The nano-carrier modified by molecular beacons as claimed in claim 1, wherein the above-mentioned mesoporous silicon nanoparticles are synthesized by a sol-gel method, and the steps are as follows: cetyltrimethylammonium bromide Slowly add the aqueous solution of lye into the lye and raise the temperature to 75-85°C, then slowly add tetraethyl orthosilicate and maintain the temperature at 75-85°C for 0.5-3h to obtain white precipitates which are mesoporous silicon nanoparticles. 3. The nano-carrier modified by molecular beacon as claimed in any one of claims 1-2, characterized in that, the nano-carrier modified by molecular beacon, its preparation method is as follows: adopt sol-gel method to synthesize mesoporous silicon Nanoparticles, connect amino groups on the surface of mesoporous silicon nanoparticles to obtain aminated silica, and carboxylate MSN- _NH2 to obtain carboxylated mesoporous silicon nanoparticles; modify the aminated anchor DNA by amide condensation reaction Generate anchored DNA-modified MSN on the surface of MSN-COOH; dissolve Dox in the buffer solution, add MSN-AD and stir the reaction in a dark environment to load the drug; continue to add to the buffer of the loaded MSN-AD After the molecular beacon reaction is completed and the hybridization is completed, the nano-carrier modified by the molecular marker can be obtained. 4. The nano-carrier modified by molecular beacons as claimed in claim 3, characterized in that, in the preparation method, the method of connecting amino groups on the surface of mesoporous silicon nanoparticles is as follows: add 3-aminopropane to the toluene solution of MSN Triethoxysilane, heated to 110-120 ° C for 1-3 hours to obtain a solid part, the solid part was washed and dispersed in methanolic hydrochloric acid solution and refluxed for 14-18 hours to obtain the MSN-NH ₂ ; In the preparation method, the method of carboxylation of MSN- _NH2 is as follows: add MSN- _NH2 and succinic anhydride to N,N-dimethylformamide, and react at room temperature for 7-9 hours to obtain MSN -COOH; In the preparation method, the preparation method of the MSN-AD is as follows: add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxyl to the suspension of MSN-COOH Thiosuccinimide reacts for 12-17 minutes to activate the carboxyl group; add PBS and aminated anchor DNA and continue to react for 24 hours to generate MSN-AD; The stirring reaction time in the dark environment is 10～14h; In the preparation method, molecular beacons are added to the buffer solution of the MSN-AD loaded with drugs and reacted for 4-8 hours. 5. The application of the nano-carrier modified by the molecular beacon according to any one of claims 1-4 in the preparation of anti-tumor products. 6. The application of the molecular beacon modified nanocarrier as an anti-tumor active ingredient according to claim 5, wherein the anti-tumor product is an anti-tumor active ingredient. 7. The application of the molecular beacon modified nanocarrier as an anti-tumor active ingredient according to claim 5, wherein the anti-tumor product is an anti-tumor drug. 8. The application of the molecular beacon modified nanocarrier as an anti-tumor active ingredient according to claim 5, wherein the anti-tumor product is a medical device used in the anti-tumor treatment process. 9. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the molecular beacon-modified nanocarrier according to any one of claims 1-4. 10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition comprises other active ingredients and/or pharmaceutically necessary adjuvants. 11. pharmaceutical composition as claimed in claim 10, is characterized in that, The other active ingredients include but are not limited to one or more of antineoplastic drugs, anti-inflammatory drugs, immunomodulatory drugs, analgesic drugs, and hemostatic drugs. 12. A drug for treating drug-resistant tumors, characterized in that, in the drug, the molecular beacon-modified nanocarrier according to any one of claims 1-4 is used as an active ingredient.

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