Preparation method of siRNA-carrying nanoparticles and application of siRNA-carrying nanoparticles in treatment of breast cancer

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method of nanoparticles carrying siRNA and application of the nanoparticles to treatment of breast cancer, and the preparation method comprises the following steps: firstly, carrying out hydrophobic transformation on hydrophilic doxorubicin hydrochloride molecules, and then promoting doxorubicin (DOX) molecules to generate self-aggregation by means of a solvent exchange technology to form DOX nanoparticles. The preparation method comprises the following steps: sequentially assembling amphiphilic polymaleic anhydride-octadecene-polyethylene glycol (C18-PEG), positively charged polyethyleneimine (PEI) and negatively charged Bcl-2siRNA on the surfaces of DOX nano-particles through a layer-by-layer self-assembly process driven by charge adsorption by virtue of electrostatic interaction, and finally successfully constructing the nano-particles (DOX / siRNA nano-particles) carrying siRNA. The self-delivery DOX nanoparticles are used for loading siRNA, so that the drug loading capacity can be effectively improved. After the Bcl-2 siRNA and the DOX are jointly delivered to the tumor cells, the PEI can promote the release of the Bcl-2 siRNA and silence the anti-apoptotic protein in the tumor cells in a targeted manner, so that the drug resistance of the tumor cells is effectively overcome.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a preparation method of siRNA-carrying nanoparticles and their application in the treatment of breast cancer. Background Art

[0002] Due to the heterogeneity of diseases, poor prognosis, and lack of clear molecular targets, breast cancer poses a huge challenge in clinical treatment. In addition to surgery, chemotherapy, including anthracycline drugs and taxane drugs, is the main clinical treatment method for tumors so far. For example, doxorubicin (DOX), as a widely used first-line anti-cancer drug, can produce significant toxicity to tumor cells by inducing DNA strand breaks and damage. However, the vast majority of breast cancer patients will eventually develop chemotherapy resistance, making the treatment effect unsatisfactory. Chemotherapy resistance can be induced at the level mediated by efflux pumps, involving different transmembrane and cellular carriers to regulate drug concentration and availability to cellular targets; it can also occur at the non-pump-mediated level through various modular effects, including DNA repair mechanisms, apoptosis, and survival pathways, to slow down cell death. In addition to the phenomenon of intrinsic or acquired multi-drug resistance (MDR), in most patients receiving anti-cancer drug treatment, some serious dose-related toxicities will occur. MDR reduces the accumulation of drugs in cells, increases compensatory mechanisms, and alleviates the damage caused by these drugs, ultimately leading to treatment failure and additional toxicity. Studies have found that by upregulating / downregulating genes responsible for regulating compensatory mechanisms of cell death and DNA repair, cells can be made more sensitive to the effects of chemotherapy. Currently, combining chemotherapy drugs with other treatment methods with unique mechanisms of action is becoming a promising approach for treating drug-resistant breast cancer.

[0003] Several studies have demonstrated that nucleic acids can specifically target anticancer resistance genes and restore the anticancer efficacy of the drugs used. The presence of endogenous RNA interference pathways in mammalian cells provides a powerful mechanism for the regulation of cell signaling pathways by precisely regulating gene expression. Small interfering RNA (siRNA) can "silence" the expression of specific genes with complementary sequences and is often introduced as a regulator of target gene expression and subsequent related cellular effects to induce the destruction of complementary post-transcriptional mRNA. siRNA has been found to have potential application value in regulating chemotherapy resistance genes. Dysregulation of apoptosis-mediated cell death can lead to cancer progression and reduce its sensitivity to treatment. Among them, the most studied is the Bcl-2 protein, which can regulate apoptotic cell death through the intrinsic pathway. Through in vitro and in vivo preclinical breast cancer models, Bcl-2 has been shown to be associated with melanoma and breast cancer progression and tumor metastasis. In addition, the Bcl-2 protein can protect cells from apoptosis induced by stimuli such as oxidants and viruses. Therefore, it is feasible to construct Bcl-2 siRNA using the Bcl-2 gene as a target molecule to treat drug-resistant tumors. However, siRNA has many limitations in clinical application, such as relatively large molecular weight (~13kDa) and negative charge that makes it difficult for them to diffuse through cell membranes, unstable in blood circulation, and easily degraded by nucleases. Therefore, it is urgent to design and develop a siRNA delivery system that can be transported to tumor cells and diffuse effectively to exert its effect.

[0004] In recent years, a variety of nanoparticle-mediated drug delivery systems have been developed to maintain the stability of anti-tumor drugs and improve their bioavailability. These nanodelivery systems include mesoporous silica nanoparticles, micelles, liposomes, metal organic frameworks, solid lipid nanoparticles, etc., which can solve the problems of lack of selectivity of traditional anticancer therapy drugs and low tumor cell uptake. However, nanocarriers usually have low drug loading, and some carriers will cause burden and toxicity to the body. Based on this, designing a carrier-free nanodrug delivery system that responds to and locates in the tumor microenvironment is a strategy to maximize the anti-cancer treatment effect while minimizing the side effects of existing anti-cancer drugs. In addition, in order to achieve high efficacy and low toxicity anti-tumor effects, a single chemotherapy method is often difficult to meet the needs of clinical efficacy. The use of siRNA targeting tumor resistance, combined with multiple anti-cancer mechanisms of action, is expected to sensitize the efficacy of chemotherapy drugs, thereby achieving optimized anti-breast cancer function. However, there is still a lack of reliable solutions today. Summary of the invention

[0005] The object of the present invention is to solve the disadvantages existing in the prior art, and to propose a preparation method of siRNA-loaded nanoparticles and their application in the treatment of breast cancer. This system has many advantages such as high drug loading capacity, simple process operation, responsiveness to acidic tumor microenvironment, and ability to overcome drug resistance. It has shown good therapeutic effects in both cell and animal experiments, providing new means and technologies for clinical anti-breast cancer treatment.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation method of siRNA-loaded nanoparticles, comprising the following steps:

[0008] Step 1: First, dissolve DOX in DMSO. At room temperature, while continuously stirring moderately, slowly add 1 mL of triethylamine to 10 mL of a 1 mg / mL DOX·HCl / DMSO solution, and react for 4 hours; after the reaction, hydrophilic DOX·HCl is successfully converted into hydrophobic DOX; add the 3 mg / mL DOX / DMSO solution dropwise to 5 mL of petroleum ether, and continuously stir for 5 minutes; then, through centrifugal separation means, wash three times with deionized water to finally obtain a clear DOX nanoparticle solution.

[0009] Step 2: Take 400 μL of a 0.5 mg / mL C18-PEG aqueous solution, add it to 10 mL of the DOX nanoparticle solution, then perform ultrasonic treatment on the mixed solution for 5 minutes, and incubate at room temperature for 1 hour to prepare DOX / C18P nanoparticles.

[0010] Step 3: Add the DOX / C18P nanoparticles dropwise to a 1 mg / mL PEI solution and let it stand overnight to obtain DOX / C18P / PEI nanoparticles.

[0011] Step 4: Combine DOX / C18P / PEI nanoparticles with different nitrogen-to-phosphorus ratios (N / P ratios) with Bcl-2 siRNA, and coat them on the surface of the nanoparticles through a charge adsorption-driven layer-by-layer self-assembly process to finally successfully construct DOX / siRNA nanoparticles.

[0012] Preferably, in Step 1, the particle size of the DOX nanoparticles is about 52 nm, and the potential is -17.2 ± 0.41 mV.

[0013] Preferably, in Step 2, the particle size of the DOX / C18P nanoparticles is about 53 nm, and the potential is -14.2 ± 1.19 mV.

[0014] Preferably, in step 3, after the positively charged PEI adsorbs on the surface of the DOX / C18P nanoparticles, the surface charge of the nanoparticles changes from negative to positive, and its potential is +22.3 ± 1.21 mV. At this time, the particle size of the prepared DOX / C18P / PEI nanoparticles further increases to about 55 nm.

[0015] The present invention also provides an application of the siRNA-loaded nanoparticles prepared by the above preparation method in the preparation of drugs for treating breast cancer.

[0016] 1. Test the ability of DOX nanoparticles to carry siRNA

[0017] To deeply explore the suitable conditions for the firm loading of siRNA molecules on the surface of DOX nanoparticles, purified siRNA was selected as the control sample, and different combinations of DOX / C18P / PEI nanoparticles and Bcl-2 siRNA with different nitrogen-phosphorus ratios (N / P ratios) of 1:2, 1:1, 5:2, 5:1, and 10:1 were tested. With the help of RNA agarose gel electrophoresis experiments, the presence of free siRNA after different ratio combinations was detected. The experimental results show that when the N / P ratio reaches 5:1 or more, siRNA can be fully combined with DOX / C18P / PEI nanoparticles. This conclusion is derived from the experimental phenomenon that siRNA no longer migrates in the gel at this ratio. Therefore, DOX / C18P / PEI nanoparticles and Bcl-2 siRNA react according to the N / P ratio of 5:1 to achieve the efficient and full utilization of the two raw materials. At this time, the surface charge of the prepared DOX / siRNA nanoparticles decreases to 10.2 ± 0.89 mV.

[0018] 2. Stability study

[0019] The DOX / siRNA nanoparticles were placed in 0.01 M phosphate buffer (PBS, pH 7.4), and the dynamic change of their particle size within 72 hours was tested at a constant temperature of 37 °C. The specific experimental operation process was as follows: at different time points, solution samples were taken from the above system multiple times, and the particle size of the nanoparticles was accurately measured using dynamic light scattering technology. To ensure the reliability and accuracy of the experimental results, this experiment was strictly repeated three times according to the same operation process, and finally, the average value of the three measurement results was used as the final calculation data for subsequent analysis and discussion. During the implementation process, DOX nanoparticles, DOX / C18P nanoparticles, and DOX / C18P / PEI nanoparticles were used as control groups.

[0020] 3. In vitro release performance

[0021] DOX / siRNA nanoparticles were placed in three different media with constant temperature of 37 °C and different pH values, and in vitro release kinetics tests of DOX and fluorescein-labeled small interfering ribonucleic acid (FAM-siRNA) were carried out. The media used were PBS buffer with pH 7.4, PBS buffer with pH 5.8, and PBS buffer with pH 4.3. The concentration of DOX / siRNA nanoparticles was set at 0.5 μg / mL. Once the DOX / siRNA nanoparticles were added to the release buffer, each sample was immediately transferred into an independent dialysis tube with a molecular weight cut-off (MWCO) of 14,000, and then the dialysis tube was immersed in 50 mL of buffer with corresponding different pH values, and the temperature was maintained at 37 °C. At different time intervals, 2 mL of buffer was taken from each group for the measurement of fluorescence intensity. Meanwhile, an equal volume of fresh buffer was supplemented to maintain the constant total volume of the release buffer. The fluorescence intensity was measured by using a FluoroMax 4 (Horiba JobinYvon) spectrofluorometer, so as to realize the quantitative analysis of the released DOX and FAM-siRNA. To ensure the reliability of the experimental results, this experiment was repeated three times, and the average value of the three measurement results was used as the final calculation data. Compared with neutral pH, DOX / siRNA nanoparticles are more likely to release DOX and siRNA in an acidic environment, thus reducing the damage to healthy cells and organs with neutral pH.

[0022] 4. Cell uptake test

[0023] First, normal MCF-7 cells and MCF-7 / ADR cells were seeded in 24-well plates containing DMEM medium with 10% fetal bovine serum (FBS) at a density of 5×10 4 cells per well and cultured for 24 hours. After the culture, the original medium was replaced with 1 mL of DMEM (serum-free), DOX·HCl, DOX nanoparticles, and DOX / siRNA nanoparticles, and the cells were incubated for 4 hours at a DOX concentration of 20 μg / mL. After incubation, the medium was removed, and the cells were washed three times with PBS buffer. Finally, the intracellular uptake of DOX in the cells was observed by confocal microscopy. Excitation was carried out with a laser at 488 nm, and fluorescence signals were collected in the emission wavelength range of 570 - 650 nm. DOX·HCl can be efficiently taken up by MCF-7 cells, but its internalization rate in drug-resistant MCF-7 / ADR cells decreased significantly. However, MCF-7 / ADR cells treated with DOX / siRNA nanoparticles still showed very strong red fluorescence, which confirmed the high intracellular accumulation of DOX / siRNA nanoparticles in drug-resistant cells.

[0024] Subsequently, a FACS Canto II flow cytometer was used to quantitatively determine the fluorescence uptake of DOX·HCl, DOX nanoparticles, and DOX / siRNA nanoparticles in MCF-7 cells and MCF-7 / ADR cells. The specific steps are as follows: First, MCF-7 cells and MCF-7 / ADR cells were seeded in 6-well plates at an appropriate cell concentration for cell culture, and approximately 2 mL of cell culture medium solution was added to each well. After 24 hours of cell culture, the medium was changed, and then 1 mL of DMEM (serum-free), DOX·HCl, DOX nanoparticles, and DOX / siRNA nanoparticles were added respectively to replace the medium, and the cells were incubated for 4 hours under the condition of a DOX concentration of 20 μg / mL. After the incubation, the solution in the 6-well plate was aspirated, and the cells in each well were digested with trypsin. Subsequently, the digested cells were centrifuged and washed three times with pre-cooled PBS solution to remove the substances attached to the cell surface that were not taken up by the cells. Finally, the drug-treated cells were evenly dispersed in pre-cooled PBS solution, and the fluorescence intensity of DOX in each group was detected using a flow cytometer, and the detection data were analyzed. The fluorescence intensity of MCF-7 / DOXADR cells treated with DOX / siRNA nanoparticles was significantly stronger than that of MCF-7 / DOXADR cells treated with DOX·HCl, corresponding to the observation results of the confocal laser microscopy images.

[0025] 5. Cytotoxicity test

[0026] First, take 100 μL of complete medium containing MCF-7 cells and MCF-7 / ADR, and inoculate it into a 96-well plate, with approximately 60,000 cells per well. Then incubate the cells under appropriate conditions for 24 hours. After incubation, add different substances to each group of cells: (1) DOX·HCl (20 μg / mL DOX); (2) liposome-encapsulated Bcl-2 siRNA (Lipo-siRNA), with an siRNA concentration of 50 nM; (3) a mixture of DOX·HCl (20 μg / mL DOX) and Lipo-siRNA (siRNA 50 nM); (4) DOX nanoparticles (20 μg / mL DOX); (5) a mixture of DOX nanoparticles (20 μg / mL DOX) and Lipo-siRNA (siRNA 50 nM); (6) DOX / siRNA nanoparticles (20 μg / mL DOX). Subsequently, place them in an environment of 37 °C and 5% carbon dioxide for further incubation for 48 hours and 72 hours. Then, add 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg / mL) to each well to treat the cells. After 5 hours of incubation, remove the medium and add 150 μL of DMSO to lyse the cells. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure cell viability by the MTT assay, and compare the obtained results with the control group without added drugs for comparative analysis, so as to calculate the relative cell survival percentage. The combination of DOX and Bcl-2 siRNA has a strong effect of killing tumor cells (4T1, MCF-7, MCF-7 / ADR), and is significantly better than DOX or Bcl-2 siRNA alone, indicating that the two drugs DOX and Bcl-2 siRNA have a synergistic therapeutic effect. In addition, the DOX / siRNA nanoparticles have the strongest toxicity to MCF-7 / ADR cells. Due to the size of the nanoparticles, they can bypass the action of the overexpressed efflux pump on the cell membrane and are not excreted by the cells, so that more drugs can enter their targeted sites to kill cells, thus overcoming multidrug resistance (MDR).

[0027] 6. In vivo distribution and bioimaging

[0028] BALB / c mice were purchased from Nanjing Pengsheng Biotechnology Co., Ltd. The 4T1 tumor model was constructed as follows: Inject 60 μL of PBS solution containing approximately 2×10 6 cells subcutaneously into the right hind leg of female BALB / c mice. When the tumor volume grows to 60 - 90 mm 3At this time (about the 6th day after tumor cell inoculation), it can be considered that the mice meet the conditions for subsequent animal experiments, and subsequent experimental operations can be prepared. Through intravenous injection, the DOX / siRNA nanoparticle solution was injected into the mice bearing 4T1 tumors. At the time points of 2, 6, 12, 24, and 48 hours after injection, the mice were euthanized, and the tumor tissues and major organs of the mice were removed, including the heart, liver, spleen, lungs, kidneys, etc. Subsequently, each of the obtained organs / tissues was weighed, and lysis buffer was added thereto. Then, a PowerGen homogenizer was used to homogenize the tissues to prepare a uniform tissue solution. To avoid obvious light scattering and self-quenching phenomena during subsequent fluorescence measurements, the tissue solution was diluted 10 times. At the same time, through appropriate dilution factor adjustment, the fluorescence intensities of the standard samples and the actual tissue samples were both within the linear range.

[0029] The fluorescence intensities of various organs and tissues were spectroscopically measured using a Maestro small animal fluorescence imaging system. When calculating the average fluorescence intensity of each imaged organ, the fluorescence of the tissue itself was first subtracted, and the background fluorescence of each organ when DOX / siRNA nanoparticles were not injected was subtracted, so as to carry out semi-quantitative biodistribution analysis. A laser with a wavelength of 488 nm was used for excitation, and fluorescence signals were collected in the emission wavelength range of 570 - 650 nm. All measurements were repeated three times, and the average value was used for subsequent quantitative analysis. Finally, the biodistribution of DOX / siRNA nanoparticles in different organs of the mice was calculated, and the corresponding graphs were plotted in units of percentage of the injected dose per gram of tissue (%ID g-1). After tail vein injection of DOX / siRNA nanoparticles, as time increased, the fluorescence at the tumor site gradually increased and was significantly higher than that of other organs 6 hours after administration. The size effect of the nanoparticles achieved passive targeting at the tumor site, thus enhancing the accumulation of the drug in the tumor.

[0030] The hair of the 4T1 tumor-bearing mice was removed from their head and neck to the feet. Subsequently, through intravenous injection, 200 μL of a DOX / siRNA nanoparticle solution with a concentration of 1 mg / mL was injected into the mice. At the same time, mice without any treatment were selected as the control group. At the time point of 6 hours after tail vein injection of the drug, imaging operations were performed using a Maestro small animal fluorescence imaging system. A 488 nm laser was used for excitation. After the excitation operation, in vivo spectral imaging analysis of the mice was carried out in the wavelength range of 600 - 750 nm (in 10 nm gradients).

[0031] 7. In vivo antitumor effect test

[0032] The in vivo anti-tumor effect of DOX / siRNA nanoparticles was preliminarily evaluated using 4T1 tumor-bearing mice. When the tumor volume of the mice reached approximately 70 - 90 mm 3 ³, the mice were randomly divided into 7 groups: (1) PBS, (2) DOX·HCl (1 mg / kg DOX), (3) Lipo-siRNA (10 μg siRNA), (4) DOX·HCl plus Lipo-siRNA, (5) DOX nanoparticles (1 mg / kg DOX), (6) DOX nanoparticles plus Lipo-siRNA (0.5 mg / kg DOX / 5 μg siRNA), and (7) DOX / siRNA nanoparticles (0.5 mg / kg DOX / 5 μg siRNA). Administration was via tail vein injection, with a 7-day interval between administrations, and a total of two administrations were given. The tumor size and body weight of each mouse were measured and recorded daily for a two-week period. To ensure the repeatability of the experimental results and the accuracy of the measurement data, during the experimental operation, every 6 experimental mice were divided into a group to carry out the relevant experimental operations and data collection work.

[0033] After 14 days of treatment, the tumor growth in the treatment group of mice treated with the physical mixture of DOX nanoparticles and Lipo-siRNA only increased to 1.95 ± 0.33 times, and its effect was much better than that of the treatment groups of DOX nanoparticles and Lipo-siRNA alone, which further confirmed the synergistic therapeutic effect of DOX and siRNA on the treatment of mouse tumors. The DOX / siRNA nanoparticle treatment group of mice showed the highest tumor inhibitory ability, superior to the physical mixture group of DOX / C18P nanoparticles and Bcl-2 siRNA, which was determined by the fact that DOX / siRNA nanoparticles simultaneously delivered Bcl-2 siRNA and DOX to tumor cells to jointly exert an anti-cancer effect.

[0034] By adopting the above technical solutions: First, the hydrophilic DOX·HCl molecules are subjected to hydrophobic transformation, and then, by means of solvent exchange technology, the DOX molecules are induced to self-aggregate to form DOX nanoparticles. Through electrostatic interaction, the amphiphilic C18PMH-PEG is modified on the surface of the DOX nanoparticles. Subsequently, the positively charged PEI and the negatively charged Bcl-2 siRNA are successively coated on the surface of the nanoparticles through a layer-by-layer self-assembly process driven by charge adsorption, and finally, the DOX / siRNA nanoparticles are successfully constructed. Loading siRNA with the self-delivery DOX nanoparticles can effectively improve the drug loading capacity. After the Bcl-2 siRNA and DOX are co-delivered to tumor cells, PEI can promote the release of Bcl-2 siRNA and target the silencing of anti-apoptotic proteins in tumor cells, thereby effectively overcoming the drug resistance of tumor cells. Given that the siRNA-carrying nanoparticles constructed with DOX and Bcl-2 siRNA have multiple advantages such as a non-toxic carrier, high drug loading capacity, overcoming drug resistance, and synergistic therapeutic effects, this system provides an efficient and low-toxic treatment strategy for future tumor clinical practice and is expected to solve the problems raised in the above background technology.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] In the present invention, chemotherapy drug molecules are prepared into nanoparticles through self-assembly, and then this drug nanoparticle itself is used as a carrier to deliver gene drugs for synergistic treatment. First, the hydrophilic DOX·HCl molecules are transformed into hydrophobic DOX molecules, and they are self-assembled into nanoparticles by the solvent exchange method. Subsequently, the amphiphilic C18-PEG, the positively charged PEI, and the negatively charged Bcl-2 siRNA are orderly adsorbed on the surface of the nanoparticles through hydrophobic interaction and a layer-by-layer self-assembly process mediated by electrostatic adsorption, and finally, the siRNA-carrying nanoparticles with a stable structure are constructed. This system has multiple advantages such as a high drug loading capacity, simple process operation, responsiveness to the acidic tumor microenvironment, and the ability to overcome drug resistance, and has shown good therapeutic effects in both cell and animal experiments, providing new means and technologies for clinical anti-breast cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the morphology and structure diagram of the nanoparticles prepared in the present invention; Scanning electron microscope (SEM) images of (a) DOX nanoparticles, (b) DOX / C18P nanoparticles, and (c) DOX / C18P / PEI nanoparticles. (d) The sizes and (e) Zeta potential parameters of DOX nanoparticles, DOX / C18P nanoparticles, and DOX / C18P / PEI nanoparticles.

[0038] Figure 2Electrophoretic mobility and morphology of DOX / C18P / PEI nanoparticles loaded with siRNA of the present invention at different N / P ratios; The first column is naked siRNA (N / P ratio is 0); The second to sixth columns are samples with N / P ratios of 1:2, 1:1, 5:2, 5:1, and 10:1 respectively. SEM images of DOX / siRNA nanoparticles with an N / P ratio of 5:1;

[0039] Figure 3 In vitro stability diagram of the present invention; Particle size stability study of DOX nanoparticles, DOX / C18P nanoparticles, DOX / C18P / PEI nanoparticles, and DOX / siRNA nanoparticles in PBS;

[0040] Figure 4 In vitro release diagram of the present invention; Release of (a) DOX and (b) siRNA from DOX / siRNA nanoparticles in PBS buffer (pH 7.4) and acidic PBS buffer (pH 5.8 and 4.3). Fluorescence of DOX and FAM-siRNA was measured to calculate their release amounts;

[0041] Figure 5 Cell uptake test diagram of the present invention. Subcellular localization after co-incubation of (a) DOX·HCl, (b) DOX nanoparticles, and (c) DOX / siRNA nanoparticles with MCF-7 and MCF-7 / DOXADR cells resistant to DOX for 4 hours. Blue and red represent nuclear staining and DOX fluorescence respectively. Cellular internalization levels of DOX·HCl, DOX nanoparticles, and DOX / siRNA nanoparticles in (d) MCF-7 and (e) MCF-7 / DOXADR cells were measured by flow cytometry.

[0042] Figure 6 Cytotoxicity test diagram of the present invention. Cell viability of (a, b) MCF-7 cells and (c, d) MCF-7 / ADR cells after co-incubation with different drugs for 48 hours and 72 hours.

[0043] Figure 7 In vivo distribution analysis diagram of the present invention. a) Fluorescence of DOX in major organs and tumors was measured at 2, 6, 12, 24, and 48 hours after tail vein injection. b) In vivo fluorescence images of mice 6 hours after injection of DOX / siRNA nanoparticles.

[0044] Figure 8 In vivo tumor inhibition effect diagram of the present invention. (a) Changes in tumor volume of 4T1 tumor-bearing mice over time. The DOX / siRNA nanoparticle group was set as the control group, * represents P<0.05, ** represents P<0.01. (b) Changes in body weight of 4T1 tumor-bearing mice over time. Detailed implementation mode

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus make a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the protection scope of the present invention.

[0046] Example 1

[0047] (1) Dissolve DOX·HCl in DMSO to prepare a solution with a concentration of 1 mg / mL;

[0048] (2) Mix triethylamine with DOX·HCl / DMSO and react for 4 hours;

[0049] (3) Gradually add the prepared DOX / DMSO solution (3 mg / mL) dropwise to 5 mL of petroleum ether and continuously stir for 5 minutes;

[0050] (4) Centrifuge at 13000 rpm / min for 10 min, and then wash three times with deionized water;

[0051] (5) Collect the supernatant, and place the precipitate in a vacuum drying oven to dry overnight to obtain DOX nanoparticles;

[0052] (6) Take 400 μL of C18-PEG aqueous solution with a concentration of 0.5 mg / mL and add it to 10 mL of DOX nanoparticle solution;

[0053] (7) Ultrasonically treat the mixed solution (for 5 minutes) and incubate it at room temperature for 1 hour to prepare DOX / C18P nanoparticles;

[0054] (8) Gradually add the DOX / C18P nanoparticles dropwise to the PEI solution with a concentration of 1 mg / mL and let it stand overnight to obtain DOX / C18P / PEI nanoparticles.

[0055] Example 2

[0056] (1) Dilute the DOX / C18P / PEI nanoparticles to different concentrations and add them to the Bcl-2 siRNA solution (20 mM) to prepare mixed solutions with N / P ratios of 1:2, 1:1, 5:2, 5:1, and 10:1. Then, detect the binding ability of the binding siRNA by agarose gel electrophoresis;

[0057] (2) Soak it overnight with a detergent in advance, rinse it with ultrapure water, dry it with ethanol, fill it with hydrogen peroxide (3%), let it stand at room temperature for 10 minutes, and rinse it with 0.1% DEPC water; Rinse the tools related to gel preparation with ethanol with a purity of 70%, dry them, and reserve for later use;

[0058] (3) Prepare an agarose gel: Weigh 1 g of agarose, place it in a clean conical flask (100 mL), add about 80 mL of ultrapure water, and then heat the agarose in a microwave oven to completely dissolve and homogenize it;

[0059] (4) Let the gel cool down to 60 - 70 °C, then sequentially add 18 mL of formaldehyde, 10 mL of 10x MOPS buffer solution, and 1 μL of ethidium bromide to the gel, mix them evenly, and pour the above-mentioned agarose gel;

[0060] (5) In a 500 μL microcentrifuge tube pretreated with DEPC, sequentially add: 10x MOPS buffer (8 μL), 7 μL of formaldehyde, 20 μL of formamide, and 9 μL of the sample, and mix them evenly.

[0061] (6) Place the microcentrifuge tube in a 65 °C water bath and incubate for about 15 minutes, then place it on ice for 2 minutes;

[0062] (7) Add 6 μL of loading dye to the centrifuge tube and mix evenly;

[0063] (8) Load the sample;

[0064] (9) Add 1x MOPS buffer to the electrophoresis tank and perform electrophoresis at a voltage of 7.5 V / mL;

[0065] (10) After the electrophoresis is completed, check the experimental results under an ultraviolet lamp.

[0066] Example 3

[0067] (1) Place the DOX / siRNA nanoparticles in 0.01 M PBS buffer (PBS, pH 7.4);

[0068] (2) At different time points, take out solution samples from the above system multiple times;

[0069] (3) Supplement an equal volume of fresh buffer to maintain the constant total volume of the release buffer;

[0070] (4) Use DLS to measure the particle size of the nanoparticles.

[0071] Example 4

[0072] (1) Place the DOX / siRNA nanoparticles (0.5 μg / mL) in three different culture media with constant temperature and different pH values. The culture media used are PBS buffer with a pH value of 7.4, PBS buffer with a pH value of 5.8, and PBS buffer with a pH value of 4.3;

[0073] (2) Transfer each sample into an independent dialysis tube with a molecular weight cut-off (MWCO) of 14,000. Subsequently, immerse the dialysis tube in 50 mL of buffer with the corresponding different pH values and maintain the temperature at 37 °C;

[0074] (3) Place it on a shaker at 37 °C with an oscillation speed of 100 rpm. At different time intervals after oscillation, take out 2 mL of buffer from each group. Meanwhile, supplement an equal volume of fresh buffer to maintain the constant total volume of the release buffer;

[0075] (4) Use a fluorescence spectrophotometer to measure the fluorescence intensities of DOX and FAM-siRNA.

[0076] Example 5

[0077] (1) Inoculate normal MCF-7 cells and MCF-7 / ADR cells into 24-well plates containing DMEM medium at a density of 5×10 4 cells per well and culture for 24 hours;

[0078] (2) Replace the original culture medium with 1 mL of DMEM (serum-free), DOX·HCl, DOX nanoparticles, and DOX / siRNA nanoparticles, and incubate the cells for 4 hours under the condition that the DOX concentration is 20 μg / mL;

[0079] (3) Remove the culture medium, wash the cells three times with PBS buffer, and observe the internalization of DOX in the cells through a confocal microscope. Use a laser with a wavelength of 488 nm for excitation and collect fluorescence signals in the emission wavelength range of 570 - 650 nm.

[0080] Example 6

[0081] (1) Take 100 μL of complete medium containing MCF-7 cells and MCF-7 / ADR and inoculate it into a 96-well plate. The number of cells per well is approximately 60,000. Subsequently, incubate the cells under suitable conditions for 24 hours;

[0082] (2) After incubation, different drugs were added to each group of cells: DOX·HCl (20 μg / mL DOX), Lipo-siRNA (siRNA concentration of 50 nM), a mixture of DOX·HCl (20 μg / mL DOX) and Lipo-siRNA (siRNA 50 nM), DOX nanoparticles (20 μg / mL DOX), a mixture of DOX nanoparticles (20 μg / mL DOX) and Lipo-siRNA (siRNA 50 nM), DOX / siRNA nanoparticles (20 μg / mL DOX);

[0083] (3) After 48 and 72 hours of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well to treat the cells. After 5 hours of incubation, the medium was removed, and 150 μL of DMSO was added to lyse the cells;

[0084] (4) Using an enzyme-linked immunosorbent assay (ELISA) reader, the cell viability was measured by the MTT assay, and the obtained results were compared and analyzed with the control group without drugs added to calculate the relative cell survival percentage.

[0085] Example 7

[0086] (1) 60 μL of PBS solution containing approximately 2×10 6 cells was subcutaneously injected into the right lower abdomen of female BALB / c mice;

[0087] (2) When the tumor volume grew to 60 - 90 mm 3 , the DOX nanoparticle solution was injected into the mice bearing 4T1 tumors by intravenous injection.

[0088] (3) At the time points of 2, 6, 12, 24, and 48 hours after injection, the mice were euthanized, and the tumor tissues and major organs (heart, liver, spleen, lung, kidney, etc.) of the mice were excised;

[0089] (4) Each of the obtained organs / tissues was weighed, and lysis buffer was added thereto. Then, a homogenizer was used to homogenize the tissue to prepare a uniform tissue solution;

[0090] (5) The tissue solution was diluted 10 times, and a small animal fluorescence imaging system was used to perform spectral determination of the fluorescence intensity of various organs and tissues;

[0091] (6) When calculating the average fluorescence intensity of each imaging organ, the fluorescence of the tissue itself is first deducted, and the background fluorescence of each organ when the nano-drug is not injected is subtracted, so as to carry out semi-quantitative biodistribution analysis. A laser of 488 nm is used for excitation, and the fluorescence signal is collected in the emission wavelength range of 570 - 650 nm;

[0092] (7) The hair of the 4T1 tumor-bearing mice is removed from their head and neck to the feet. Subsequently, 200 μL of a DOX nanoparticle solution with a concentration of 1 mg / mL is injected into the mice by intravenous injection;

[0093] (8) Six hours after the drug is injected via the tail vein, imaging is performed using a small animal fluorescence imaging system. A laser of 488 nm is used for excitation, and in vivo spectral imaging analysis of the mice is carried out in the wavelength range of 600 - 750 nm (in 10 nm gradients).

[0094] Example 8

[0095] (1) The 4T1 tumor-bearing mice are used to preliminarily evaluate the in vivo anti-tumor effect of the DOX / siRNA nanoparticles. When the tumor volume reaches about 70 - 90 mm 3 , the mice are randomly divided into 7 groups;

[0096] (2) The 7 groups of mice are injected with drugs via the tail vein, respectively: PBS, DOX·HCl (1 mg / kg DOX), Lipo-siRNA (10 μg siRNA), DOX·HCl plus Lipo-siRNA, DOX nanoparticles (1 mg / kg DOX), DOX nanoparticles plus Lipo-siRNA (0.5 mg / kg DOX / 5 μg siRNA), DOX / siRNA nanoparticles (0.5 mg / kg DOX / 5 μg siRNA). The drugs are administered at intervals of 7 days, and a total of two administrations are carried out;

[0097] (3) The tumor size and body weight of each mouse are measured and recorded daily, and the treatment cycle is 14 days;

[0098] (4) When the tumor volume exceeds 2000 mm 3 , euthanasia is performed on the BALB / c mice. After the treatment is over, the subcutaneous tumors of the mice are dissected and weighed for subsequent in vivo anti-tumor research.

[0099] The descriptions and practices disclosed in the present invention are easy to think about and understand for those of ordinary skill in the art. Without departing from the principle of the present invention, several improvements and refinements can also be made. Therefore, the modifications or improvements made without departing from the spirit of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A preparation method of siRNA nanoparticle carriers, characterized in that, It includes the following steps: Step 1: First, dissolve DOX in dimethyl sulfoxide (DMSO). At room temperature, while continuously stirring moderately, slowly add 1 mL of triethylamine to 10 mL of a DOX·HCl / DMSO solution with a concentration of 1 mg / mL, and react for 4 hours. After the reaction, hydrophilic DOX·HCl is successfully converted into hydrophobic DOX. Dropwise add the DOX / DMSO solution with a concentration of 3 mg / mL into 5 mL of petroleum ether, and continuously stir for 5 minutes. Subsequently, through centrifugal separation, wash three times with deionized water, and finally obtain clear DOX nanoparticles. Step 2: Take 400 μL of a C18-PEG aqueous solution with a concentration of 0.5 mg / mL, add it to 10 mL of the DOX nanoparticle solution, then perform ultrasonic treatment on the mixed solution for 5 minutes, and incubate at room temperature for 1 hour to prepare DOX / C18P nanoparticles. Step 3: Dropwise add the DOX / C18P nanoparticles into a PEI solution with a concentration of 1 mg / mL and let it stand overnight to obtain DOX / C18P / PEI nanoparticles. Step 4: Combine DOX / C18P / PEI nanoparticles with different nitrogen-phosphorus ratios with Bcl-2 siRNA, and assemble them on the surface of the nanoparticles through a charge adsorption-driven layer-by-layer self-assembly process. Finally, successfully construct siRNA-loaded nanoparticles, namely DOX / siRNA nanoparticles.

2. The preparation method of a kind of siRNA nanoparticle carrier according to claim 1, characterized in that, In Step 1, the particle size of the DOX nanoparticles is 52 nm, and the potential is -17.2 ± 0.41 mV.

3. The preparation method of a kind of siRNA nanoparticle carrier according to claim 1, characterized in that, In Step 2, the particle size of the DOX / C18P nanoparticles is 53 nm, and the potential is -14.2 ± 1.19 mV.

4. The preparation method of a kind of siRNA nanoparticle carrier according to claim 1, characterized in that, In Step 3, after the positively charged PEI adsorbs on the surface of the DOX / C18P nanoparticles, the surface charge changes from negative to positive, and its potential is +22.3 ± 1.21 mV. At this time, the particle size of the prepared DOX / C18P / PEI nanoparticles increases to 55 nm.

5. Use of the siRNA-loaded nanoparticles obtained by the preparation method according to any one of claims 1-4 in the preparation of a drug for treating breast cancer.

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