阅读说明：本技术 一种靶向BIRC6基因的siRNA核酸脂质体及其制备方法与用途 (siRNA nucleic acid liposome targeting BIRC6 gene and preparation method and application thereof ) 是由 谭亚南 关新元 李咏梅 李珊珊 罗敏 于 2021-08-05 设计创作，主要内容包括：本发明公开了一种靶向BIRC6基因的siRNA核酸脂质体及其制备方法和用途,其中,所述靶向BIRC6基因的siRNA核酸脂质体包括脂质体组分和BIRC6抑制剂；所述脂质体组分包括DOTAP、Chol、DPPC和DSPE-PEG2000；所述BIRC6抑制剂为靶向BIRC6的siRNA,所述靶向BIRC6的siRNA序列为GUUUCAAAGCAGGAUGAUGdTdT。本发明提供的siRNA核酸脂质体表面带正电性,不易被核酸酶降解,能够使负载的siRNA进入细胞靶向性沉默BIRC6基因,并能有效抑制三阴性乳腺癌细胞的增殖和肿瘤形成,达到治疗三阴性乳腺癌的目的。本发明siRNA核酸脂质体具备良好的细胞摄取和溶酶体逃逸能力,具有BIRC6靶向性,有利于基因药物在体内的靶向运输,实现基因药物体内长循环和对BIRC6高表达肿瘤的靶向沉默效果。(The invention discloses a siRNA nucleic acid liposome targeting a BIRC6 gene and a preparation method and application thereof, wherein the siRNA nucleic acid liposome targeting the BIRC6 gene comprises a liposome component and a BIRC6 inhibitor; the liposome component comprises DOTAP, Chol, DPPC and DSPE-PEG 2000; the BIRC6 inhibitor is siRNA targeting BIRC6, and the siRNA sequence of the BIRC6 is GUUUCAAGCAGGAUGAUGdTdT. The siRNA nucleic acid liposome provided by the invention has positive electricity on the surface, is not easily degraded by nuclease, can enable the loaded siRNA to enter a cell targeting silencing BIRC6 gene, and can effectively inhibit the proliferation and tumor formation of triple negative breast cancer cells, thereby achieving the purpose of treating triple negative breast cancer. The siRNA nucleic acid liposome has good cell uptake and lysosome escape capacity, has the BIRC6 targeting property, is beneficial to the targeted transportation of gene drugs in vivo, and realizes the long circulation in gene drug objects and the targeted silencing effect on BIRC6 high-expression tumors.)

1. An siRNA nucleic acid liposome targeting BIRC6 gene, which is characterized by comprising a liposome component and a BIRC6 inhibitor; the liposome component comprises DOTAP, Chol, DPPC and DSPE-PEG 2000; the BIRC6 inhibitor is siRNA targeting BIRC6, and the siRNA sequence of the BRIC6 is GUUUCAAGCAGGAUGAUGdTdT.

2. The siRNA nucleic acid liposome targeting BIRC6 gene in claim 1, wherein the liposome component comprises, by weight, 70 parts of DPPC, 0-20 parts of DOTAP, 5 parts of Chol and 5 parts of DSPE-PEG 2000.

3. The siRNA nucleic acid liposome targeting BIRC6 gene of claim 1, wherein the mass ratio of the liposome components and the BIRC6 inhibitor is 10-100: 1.

4. The siRNA nucleic acid liposome targeting BIRC6 gene of claim 1, wherein the mass ratio of the liposome components and the BIRC6 inhibitor is 60: 1.

5. A method for preparing siRNA nucleic acid liposome targeting BIRC6 gene as described in any one of claims 1-4, comprising the steps of:

dissolving DPPC, DOTAP, Chol and DSPE-PEG2000 in ethanol, and heating in water bath at 60 deg.C to obtain ethanol phase solution;

rapidly dispersing the ethanol phase solution into an F68 aqueous solution, stirring at the rotating speed of 400rpm for 8min, and cooling the prepared solution at room temperature to obtain a PEG modified cationic liposome solution;

preparing siRNA targeting BIRC6 into an siRNA solution with the concentration of 2 mu M by DEPC water, mixing the siRNA solution with the PEG modified cationic liposome solution according to a set mass ratio, whirling for 30s, and standing for 30min at room temperature to obtain the siRNA nucleic acid liposome targeting BIRC6 gene.

6. Use of the siRNA nucleic acid liposome targeting BIRC6 gene as described in any one of claims 1-5 in the preparation of a medicament for treating or preventing diseases caused by abnormal expression of BIRC6 gene.

7. Use according to claim 6, wherein the disease is triple negative breast cancer.

8. A liposome preparation comprising the siRNA nucleic acid liposome targeting BIRC6 gene of any one of claims 1-5 and a pharmaceutically acceptable carrier.

Technical Field

The invention relates to the technical field of drug delivery, in particular to siRNA nucleic acid liposome targeting BIRC6 gene and a preparation method and application thereof.

Background

Triple-negative breast cancer (TNBC) is a type of breast cancer in which ER and PgR expression are deleted and HER2 is overexpressed or gene amplification is deleted, and accounts for 12% -17% of all breast cancers. TNBC is biologically more aggressive, with a higher recurrence rate in patients and a worse 5 year prognosis compared to other subtypes. Although the PARP inhibitor, PD-L1 antibody atezolizumab, is currently approved for TNBC patients with BRCA mutations, positive for PD-L1 expression, respectively, targeted therapy against TNBC is still in the early stages and chemotherapy remains the standard treatment. TNBC patients, despite having a certain response rate to chemotherapy, still have 60% -70% of patients who are not susceptible to chemotherapy. Therefore, there is an urgent need to identify more molecular targets that can be used for TNBC therapy and to develop corresponding targeted therapeutic approaches.

BIRC6 is a very large (near 530kDa) and less studied member of the Inhibitor of Apoptosis Protein (IAP) family. Our previous studies found that BIRC6 is highly expressed in triple negative breast cancer, and that overexpression can promote proliferation of triple negative breast cancer cells, and is associated with short prognosis survival time of triple negative breast cancer patients, revealing that silencing BIRC6 expression is expected to be useful in triple negative breast cancer treatment.

At present, there are no inhibitors that silence BIRC6 expression clinically. Gene therapy using siRNA can down-regulate the expression of abnormal genes in cancer cells and show greater potential in cancer therapy. However, unmodified siRNA is easily degraded by nuclease, and has poor cellular uptake efficiency, and clinical application is limited. At present, siRNA silencing BIRC6 gene has not been studied in the treatment drugs such as triple negative breast cancer.

Accordingly, the prior art is yet to be improved and developed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a siRNA nucleic acid liposome targeting BIRC6 gene and a preparation method and application thereof.

The technical scheme of the invention is as follows:

an siRNA nucleic acid liposome targeting BIRC6 gene, which comprises liposome components and a BIRC6 inhibitor; the liposome component comprises DOTAP, Chol, DPPC and DSPE-PEG 2000; the BIRC6 inhibitor is siRNA targeting BIRC6, and the siRNA sequence of the BRIC6 is GUUUCAAGCAGGAUGAUGdTdT.

The siRNA nucleic acid liposome targeting the BIRC6 gene comprises 70 parts of DPPC, 0-20 parts of DOTAP, 5 parts of Chol and 5 parts of DSPE-PEG2000 in parts by weight.

The siRNA nucleic acid liposome targeting the BIRC6 gene is characterized in that the mass ratio of the liposome component to the BIRC6 inhibitor is 10-100: 1.

The siRNA nucleic acid liposome targeting the BIRC6 gene is characterized in that the mass ratio of the liposome component to the BIRC6 inhibitor is 60: 1.

A method for preparing siRNA nucleic acid liposome targeting BIRC6 gene comprises the following steps:

dissolving DPPC, DOTAP, Chol and DSPE-PEG2000 in ethanol, and heating in water bath at 60 deg.C to obtain ethanol phase solution;

rapidly dispersing the ethanol phase solution into an F68 aqueous solution, stirring at the rotating speed of 400rpm for 8min, and cooling the prepared solution at room temperature to obtain a PEG modified cationic liposome solution;

preparing siRNA targeting BIRC6 into an siRNA solution with the concentration of 2 mu M by DEPC water, mixing the siRNA solution with the PEG modified cationic liposome solution according to a set mass ratio, whirling for 30s, and standing for 30min at room temperature to obtain the siRNA nucleic acid liposome targeting BIRC6 gene.

An application of siRNA nucleic acid liposome targeting BIRC6 gene in preparing a medicament for treating or preventing diseases caused by abnormal expression of BIRC6 gene.

The use of (a), wherein the disease is triple negative breast cancer.

A liposome preparation, wherein the siRNA nucleic acid liposome targeting BIRC6 gene and a pharmaceutically acceptable carrier are contained.

Has the advantages that: the invention provides a siRNA nucleic acid liposome targeting BIRC6 gene, which is formed by combining a cationic liposome component and a BIRC6 inhibitor and then forming the siRNA nucleic acid liposome in an electrostatic adsorption mode. The cationic liposome has positive charges, and can be tightly combined with siRNA with negative charges through electrostatic action to form a compound of the liposome and nucleic acid, so that escape of an endo-lysosome is promoted, the nucleic acid is protected from degradation by nuclease, and guarantee is provided for further drug effect exertion of nucleic acid drugs; on the other hand, the PEG phospholipid is introduced, so that the phagocytosis of the nanoparticles by macrophages of a reticuloendothelial system can be reduced, the in vivo long circulation is realized, the retention and accumulation time of a nucleic acid delivery system in tumor tissues is prolonged, the toxic and side effects are reduced, and the high-efficiency and low-toxicity treatment target is realized. The siRNA nucleic acid liposome has positive electricity on the surface, is not easily degraded by nuclease, can enable the loaded siRNA to enter a cell targeting silent BIRC6 gene, and can effectively inhibit the proliferation and tumor formation of triple negative breast cancer cells, thereby achieving the purpose of treating triple negative breast cancer; the siRNA nucleic acid liposome targeting the BIRC6 gene is beneficial to targeted transportation of a gene medicament in vivo, can improve the treatment efficiency of triple negative breast cancer, and has important significance for the targeted treatment of BIRC6 high-expression tumors by the gene medicament.

Drawings

FIG. 1 shows the preparation process of siRNA nucleic acid liposome targeting BIRC6 gene.

FIG. 2 shows the particle size and surface potential of pCLNs containing DOTAP in different mass ratios.

FIG. 3 shows gel electrophoresis block diagram of siRNA nucleic acid liposomes.

FIG. 4 shows the particle size and potential of siRNA nucleic acid liposomes.

The results shown in fig. 5, a to C, show that siRNA nucleic acid liposomes can efficiently deliver siRNA targeting BIRC6 to triple negative breast cancer cells in vitro and in vivo, a shows that after MDA-MB-468 cells were incubated with free Cy3-siRNA or pCLNs/Cy3-siRNA for a specified time, intracellular fluorescence intensity of Cy3-siRNA was measured with a flow cytometer; b shows that after MDA-MB-468 cells are incubated with pCLNs/Cy3-siRNA for a specified time, the intracellular localization and lysosome escape of liposomes are observed by laser confocal scanning microscopy; c shows that after intravenous injection of free Cy3-siRNA or pCLNs/Cy3-siRNA into MDA-MB-468 xenograft tumor mice, their in vivo distribution was observed.

The results shown in a to G in fig. 6 show that siRNA nucleic acid liposome can significantly inhibit proliferation and tumor formation of triple negative breast cancer cells, a shows protein expression of BIRC6 in MDA-MB-468 cells treated with nucleic acid liposome (left) and relative cell proliferation rate (right); b shows the apoptosis ratio of MDA-MB-468 cells after being treated by nucleic acid liposome and EGFR inhibitor gefitinib; C-E shows representative pictures of MDA-MB-468 xenografts treated with nucleic acid liposomes and the EGFR inhibitor gefitinib (C), tumor volume (D) and tumor weight (E); f shows immunohistochemical analysis of MDA-MB-468 xenograft tumors treated with nucleic acid liposomes and an EGFR inhibitor gefitinib; g shows the mean fluorescence intensity of Ki67 and clear caspase3 staining.

Detailed Description

The invention provides a siRNA nucleic acid liposome targeting BIRC6 gene and a preparation method and application thereof, and a person skilled in the art can realize the siRNA nucleic acid liposome by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The experimental procedures, in which specific conditions are not specified in the examples below, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1

Preparation of PEG modified cationic liposome

The PEG-modified cationic liposome provided in this example 1 is prepared by the following steps:

according to the weight portion, 70 portions of thermosensitive lipid DPPC (dipalmitoylphosphatidylcholine), 0 portion of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine), 5 portions of Chol (cholesterol) and 5 portions of DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) are dissolved in ethanol solution and heated in water bath at 60 ℃. 2mL of an aqueous solution of F68 (1mg/mL) was heated to 60 ℃. The ethanol phase solution was rapidly dispersed into an aqueous solution of F68 at 60 ℃ and stirred at 400rpm for 8 min. And cooling the prepared solution at room temperature to obtain the PEG modified cationic liposome (pCLNs).

Example 2

Preparation of PEG modified cationic liposome

The PEG-modified cationic liposome provided in this example 2 is prepared by the following steps:

according to the weight portion, 70 portions of thermosensitive lipid DPPC (dipalmitoylphosphatidylcholine), 5 portions of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine), 5 portions of Chol (cholesterol) and 5 portions of DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) are dissolved in ethanol solution and heated in water bath at 60 ℃. 2mL of an aqueous solution of F68 (1mg/mL) was heated to 60 ℃. The ethanol phase solution was rapidly dispersed into an aqueous solution of F68 at 60 ℃ and stirred at 400rpm for 8 min. And cooling the prepared solution at room temperature to obtain the PEG modified cationic liposome (pCLNs).

Example 3

Preparation of PEG modified cationic liposome

The PEG-modified cationic liposome provided in this example 3 is prepared by the following steps:

according to the parts by weight, 70 parts of thermosensitive lipid DPPC (dipalmitoylphosphatidylcholine), 10 parts of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine), 5 parts of Chol (cholesterol) and 5 parts of DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) are dissolved in an ethanol solution and heated in a water bath at 60 ℃. 2mL of an aqueous solution of F68 (1mg/mL) was heated to 60 ℃. The ethanol phase solution was rapidly dispersed into an aqueous solution of F68 at 60 ℃ and stirred at 400rpm for 8 min. And cooling the prepared solution at room temperature to obtain the PEG modified cationic liposome (pCLNs).

Example 4

Preparation of PEG modified cationic liposome

The PEG-modified cationic liposome provided in this example 4 is prepared by the following steps:

according to the parts by weight, 70 parts of thermosensitive lipid DPPC (dipalmitoylphosphatidylcholine), 20 parts of DOTAP ((2, 3-dioleoyl-propyl) -trimethylamine), 5 parts of Chol (cholesterol) and 5 parts of DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) are dissolved in an ethanol solution and heated in a water bath at 60 ℃. 2mL of an aqueous solution of F68 (1mg/mL) was heated to 60 ℃. The ethanol phase solution was rapidly dispersed into an aqueous solution of F68 at 60 ℃ and stirred at 400rpm for 8 min. And cooling the prepared solution at room temperature to obtain the PEG modified cationic liposome (pCLNs).

The particle size and surface potential of pCLNs containing DOTAP with different mass ratios are respectively measured by a particle size and surface potential meter. As a result, as shown in FIG. 2, when the weight part of the DOTAP component was 0 part, the particle diameter of pCLNs was 65.47. + -. 1.0nm, and the surface potential was-11.4. + -. 1.89 mV. As the proportion of the DOTAP component in parts by weight was gradually increased to 20 parts, the particle diameter of pCLNs was 100.2. + -. 11.2nm and the surface potential was 40.6. + -. 4.41 mV. The results show that as the proportion of the DOTAP component increases, the particle size of the pCLNs increases, but still remains around 100nm, and can be preferentially aggregated to tumor tissues through an "enhanced penetration and retention" (EPR) effect during in vivo transport; the surface charge is reversed from negative charge to positive charge and is more than 30mV, which is beneficial to stabilizing the composite siRNA with negative charge and protecting the siRNA from degradation of nuclease.

Example 5

1) Preparation of PEG modified cationic liposome

The preparation method of the PEG-modified cationic liposome provided in this example 5 is the same as that in example 4 and is not set forth herein.

2) Preparation of a composite siRNA liposome:

the composite siRNA liposome provided in this example 5 was prepared by the following steps:

the siRNA of the BIRC6 gene is prepared into siRNA solution with the concentration of 2 mu M by DEPC water, and the siRNA sequence of the BIRC6 gene is preferably GUUUCAAGCAGGAUGAUGdTdT. The siRNA solution and the PEG-modified cationic liposome pCLNs solution were slowly mixed at a set mass ratio (mass of pCLNs: siRNA mass 10: 1), vortexed for 30s, and then allowed to stand at room temperature for 30min to obtain a pCLNs/siRNA complex (as shown in fig. 1).

Example 6

1) Preparation of PEG modified cationic liposome

The preparation method of the PEG-modified cationic liposome provided in this example 6 is the same as that in example 4 and is not set forth herein.

2) Preparation of a composite siRNA liposome:

the composite siRNA liposome provided in this example 6 is prepared by the following steps:

the siRNA of the BIRC6 gene is prepared into siRNA solution with the concentration of 2 mu M by DEPC water, and the siRNA sequence of the BIRC6 gene is preferably GUUUCAAGCAGGAUGAUGdTdT. The siRNA solution and the PEG-modified cationic liposome pCLNs solution were slowly mixed at a set mass ratio (mass of pCLNs: siRNA mass 20: 1), vortexed for 30s, and then allowed to stand at room temperature for 30min to obtain a pCLNs/siRNA complex (as shown in fig. 1).

Example 7

1) Preparation of PEG modified cationic liposome

The preparation method of the PEG-modified cationic liposome provided in this example 7 is the same as that in example 4 and is not set forth herein.

2) Preparation of a composite siRNA liposome:

the complex siRNA liposome provided in this example 7 was prepared by the following steps:

the siRNA of the BIRC6 gene is prepared into siRNA solution with the concentration of 2 mu M by DEPC water, and the siRNA sequence of the BIRC6 gene is preferably GUUUCAAGCAGGAUGAUGdTdT. The siRNA solution and the PEG-modified cationic liposome pCLNs solution were slowly mixed at a set mass ratio (pCLNs mass: siRNA mass: 40: 1), vortexed for 30s, and then allowed to stand at room temperature for 30min to obtain a pCLNs/siRNA complex (as shown in fig. 1).

Example 8

1) Preparation of PEG modified cationic liposome

The preparation method of the PEG-modified cationic liposome provided in this example 8 is the same as that in example 4 and is not set forth herein.

2) Preparation of a composite siRNA liposome:

the complex siRNA liposome provided in this example 8 is prepared by the following steps:

the siRNA of the BIRC6 gene is prepared into siRNA solution with the concentration of 2 mu M by DEPC water, and the siRNA sequence of the BIRC6 gene is preferably GUUUCAAGCAGGAUGAUGdTdT. The siRNA solution and the PEG-modified cationic liposome pCLNs solution were slowly mixed at a set mass ratio (pCLNs mass: siRNA mass 60: 1), vortexed for 30s, and then allowed to stand at room temperature for 30min to obtain a pCLNs/siRNA complex (as shown in fig. 1).

Example 9

1) Preparation of PEG modified cationic liposome

The preparation method of the PEG-modified cationic liposome provided in this example 9 is the same as that in example 4 and is not set forth herein.

2) Preparation of a composite siRNA liposome:

the complex siRNA liposome provided in this example 9 is prepared by the following steps:

the siRNA of the BIRC6 gene is prepared into siRNA solution with the concentration of 2 mu M by DEPC water, and the siRNA sequence of the BIRC6 gene is preferably GUUUCAAGCAGGAUGAUGdTdT. The siRNA solution and the PEG-modified cationic liposome pCLNs solution were slowly mixed at a set mass ratio (pCLNs mass: siRNA mass: 100: 1), vortexed for 30s, and then allowed to stand at room temperature for 30min to obtain a pCLNs/siRNA complex (as shown in fig. 1).

To test the optimal mass ratio of liposome components to siRNA in the pCLNs/siRNA complexes obtained in examples 5-9 of the present invention, we performed gel electrophoresis blocking experiments. First, a 1% agarose gel was prepared and ethidium bromide (final concentration 50. mu.g/100 mL) was added. Mixing the pCLNs/siRNA compound with 5 multiplied loading buffer solution, adding the mixture into a gel hole, and setting electrophoresis parameters: 100V, 30 min. After the electrophoresis is finished, the gel is taken out, observed in a gel imaging system and photographed. The results show that the mobility of siRNA through the gel is reduced with the increase of pCLNs mass, when the mass ratio of pCLNs to siRNA is 60:1, the siRNA band is completely blocked in the gel hole (as shown in FIG. 3), and the liposome is completely sealed with siRNA to form stable complex. The proportion is selected as the preferable compounding proportion of siRNA of the BIRC6 gene and PEG modified cationic liposome pCLNs.

3) The physical and chemical properties of the siRNA liposome are studied:

cationic liposomes pCLNs and pCLNs/siRNA complexes were prepared according to the methods of examples 4 and 9, the particle size and surface potential of the corresponding liposomes were determined using a microparticle size and surface potential meter, and the spatial structure of the liposomes was studied using transmission and scanning electron microscopy. The results show that pCLNs and pCLNs/siRNA complexes exhibit the conventional spherical morphology and good size uniformity of liposomes (as shown in FIG. 4A). When pCLNs were mixed with siRNA, the particle size of the liposomes changed from 100.2. + -. 11.2nm to 155.3. + -. 1.6nm and the zeta potential changed from 40.6. + -. 4.41mV to 18.9. + -. 1.31mV (as shown in FIG. 4B), indicating that the pCLNs/siRNA complex was successfully prepared.

4) Cell uptake and tumor targeting ability studies of siRNA liposomes:

siRNA liposomes were prepared according to the method in example 9, using Cy 3-labeled siRNA. Triple negative breast cancer cells MDA-MB-468 were inoculated into 24-well plates or 35mm glass-bottom dishes and incubated overnight, and pCLNs/Cy3-siRNA complexes were added for 1h, 4h and 12h of incubation, with a final siRNA concentration of 100 nM. To assess siRNA uptake by cells, cells were treated with trypsin and harvested and analyzed for fluorescence by flow cytometry. The results show that the proportion of fluorescent cells increases with increasing incubation time of pCLNs/Cy3-siRNA and remains high after 12h of incubation (as shown in FIG. 5A). In order to achieve efficient cytoplasmic gene silencing, escape of siRNA liposomes from lysosomes after entry into the cell is essential. To study the escape of siRNA from lysosome, 0.5mL Lysotracker Blue (labeled lysosome) reagent was added to cells before observation, incubated at 37 ℃ for 30min, the medium was discarded, PBS was washed, 4% paraformaldehyde was fixed and mounted, and the distribution of the complex in cells was observed by confocal laser microscopy and a fluorescent photograph was taken. As shown in FIG. 5B, after 4h incubation, red fluorescence (Cy3-siRNA) and blue fluorescence (LysoTracker) co-localized in MDA-MB-468 cells, indicating that Cy3-siRNA localized in lysosomes. After 12h incubation, the red fluorescence separated from the blue fluorescence, suggesting that Cy3-siRNA had escaped from the lysosome into the cytoplasm. The results show that the prepared siRNA liposome has good cell uptake and lysosome escape capacity.

Further, we evaluated whether pCLNs coated siRNA could reach the tumor site through the blood and be stable in circulation. Firstly, MDA-MB-468 cells are implanted into a mammary fat pad of a female nude mouse, and an in-situ triple negative breast cancer model is established. When the tumor volume reaches 100mm³In this case, pCLNs/Cy3-siRNA or free Cy3-siRNA was injected intravenously at a siRNA dose of 20. mu.g. At the indicated times after injection, mice were imaged using a Maestro in vivo imaging system. The results showed that the fluorescence intensity in the tumors of the group injected with pCLNs/Cy3-siRNA gradually increased until the fluorescence remained high after 36h of injection, while there was no fluorescence in the tumors of the group injected with Cy3-siRNA (as shown in FIG. 5C). These results indicate that the prepared siRNA nucleic acid liposomes can be efficiently delivered to tumor sites after injection and are stable in circulation.

5) In vitro therapeutic effect study of siRNA liposomes on triple negative breast cancer cells:

the pCLNs/siBIRC6 complex was prepared by selecting liposome fractions and siRNA targeting BIRC6 according to the method of example 9, and the expression level of BIRC6 was examined by immunoblotting after incubating the complex with MDA-MB-468 cells for 48 h. The results show that pCLNs/siBIRC6 enables efficient knock-down of BIRC6 (as shown on the left of FIG. 6A). Subsequently, MDA-MB-468 cells are inoculated in a 96-well plate, after adherence, corresponding liposome is added, and after incubation for 72h, the proliferation rate of the cells is detected through an MTT experiment. The results show that pCLNs/siBIRC6 can significantly inhibit cell growth (as shown on the right of FIG. 5A). Further, the corresponding liposome or the EGFR inhibitor gefitinib is added into MDA-MB-468 cells, and after incubation for 72h, the apoptosis ratio of the cells is detected by flow cytometry. The results show that the pCLNs/siBIRC6 complex significantly increased the level of apoptosis compared to gefitinib treatment (as shown in figure 6B). The results show that the prepared siRNA liposome can effectively inhibit the growth of triple negative breast cancer cells in vitro.

6) In vivo efficacy study of siRNA liposomes on triple negative breast cancer cells:

we evaluated the antitumor effect of pCLNs/siBIRC6 complex in an orthotopic nude mouse model seeded with MDA-MB-468 cells. Specifically, MDA-MB-468 cells are injected into a mammary fat pad of a female nude mouse with the age of 4-5 weeks, and when the tumor volume reaches-100 mm³At the time, the mice were randomly divided into 4 treatment groups. gefitinib is intragastrically administered at 100mg/kg daily; pCLNs/siBIRC6 (20. mu.g of siBIRC6 per injection) or an equivalent of pCLNs/siNC was injected intravenously, repeatedly 1 time per week for 6 weeks. Mouse tumor volume and weight were monitored. The results showed that pCLNs/siBIRC6 gene therapy was more effective in inhibiting tumor growth (as shown in figure 6C) and had a 91.58% reduction in tumor volume (as shown in figure 6D) compared to the gefitinib-treated group. Furthermore, the tumor weight of the pCLNs/siBIRC 6-treated group was also lower than that of the gefitinib-treated group (as shown in FIG. 6E). Subsequently, tumor tissues were removed for immunohistochemical staining and FIGS. 6F-G show that pCLNs/siBIRC6 treatment was more effective than gefitinib in blocking cell proliferation (Ki67) and inducing apoptosis (cleared caspase 3). These results show that the prepared siRNA nucleic acid liposome can effectively inhibit the tumor formation of triple negative breast cancer cells.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.