CN116656618A

CN116656618A - Exosomes of PARP1/2 siRNA and application of exosomes in breast cancer treatment

Info

Publication number: CN116656618A
Application number: CN202310684409.9A
Authority: CN
Inventors: 李伟; 梁展文; 陈凯; 吴梦瑶; 徐彩华; 龚斐然; 何康; 徐梦丹
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-08-29

Abstract

The invention belongs to the field of medicine, and particularly relates to an exosome of PARP1/2 siRNA and application thereof in breast cancer treatment. The engineering exosome contains PARP1/2 siRNA, and recombinant fusion CD63-CXCL12 protein is expressed on the engineering exosome envelope. The invention constructs a targeted drug delivery system specific to breast cancer tissues, which can specifically inhibit PARP in breast cancer cells and avoid the influence on normal cells.

Description

Exosomes of PARP1/2 siRNA and application of exosomes in breast cancer treatment

Technical Field

The invention belongs to the field of medicine, and particularly relates to an exosome of PARP1/2 siRNA and application thereof in breast cancer treatment.

Background

Breast cancer is the most common malignancy in women worldwide, and GLOBOCAN 2020 worldwide cancer patient statistics show that 1929 ten thousand new cancers worldwide, of which breast cancer first exceeds lung cancer, are the most common cancers. In female malignant tumors, the incidence and mortality of breast cancer are all the first, and the future is expected to be continuously rising. Despite significant advances and efficacy in surgery and adjuvant therapy (chemotherapy, endocrine and targeted therapy) in early breast cancer, up to 30% of patients experience recurrence or death, while about 5% -10% of patients are advanced breast cancer at the time of first diagnosis. The treatment of advanced refractory breast cancer is still based on palliative chemotherapy.

DNA damage occurs continuously in cells due to exogenous or endogenous stimuli of the cells. Cells thus evolve a variety of mutually coordinated DNA damage repair pathways to ensure the integrity of genetic material. Common DNA damage repair methods can be divided into two categories: one class is nucleic acid excision repair (nucleotide excision repair, NER), base Excision Repair (BER) and mismatch repair (MMR) for repairing DNA single strand lesions (dnaingle-strand damage); another class is the repair of DNA double-strand Damage (DSB) by Non-homologous end joining (Non-homologous end joining, NHEJ) and homologous recombination repair (Homologous recombination, HR). BRCA1 and BRCA2 are important genes involved in homologous recombination repair (Homologous recombination, HR). People with genetic defects of BRCA1 and BRCA2 are more prone to develop tumors, but the tumors are more sensitive to anti-tumor treatment due to the defects of DNA damage repair mechanisms.

Poly (ADP-ribose polymerase, PARP) is an important DNA base repair enzyme, acting mainly through Base Excision Repair (BER), PARP1/2 belongs to the DNA dependent PARP in the PARP family, and activated PARP1/2 accounts for more than 90% of the total enzyme activity in cells. When the cell DNA is subjected to SSB by external stimulus or endogenous factors, PARP1/2 can be combined to damaged DNA and catalyze self and H1 and other histones to generate PAR, PARP1 and the histones after PAR can recruit related repair proteins to the damaged DNA on one hand, and can loose chromosomes on the other hand so as to provide space positions for the repair proteins.

Based on the important role of PARP1/2 in DNA single strand damage repair, PARP inhibitors have been developed as sensitizers for radiotherapy and chemotherapy drugs as early as the 70 s of the 20 th century. In 2005, the proposed synthetic lethal theory provides an important theoretical basis for applying PARP inhibitors alone to patients with HR repair defects, and the theory proposes: PARP inhibitors can inhibit DNA single strand damage repair by inhibiting the BER pathway when DNA single strand damage occurs. When a DNA damage that is not repaired in time encounters an ongoing replication fork, a replication-dependent DNA double strand damage is created. In normal cells, replication-dependent DNA double-stranded lesions can be repaired by the HR repair pathway, whereas in breast cancer cells with defects in HR repair caused by BRCA1/2 mutations, double-stranded lesions of DNA cannot be repaired in time, resulting in cell death.

Although PARP inhibitors have significant therapeutic effects on BRCA1/2 mutated breast cancers, the occurrence of severe toxic responses has prevented their further clinical development. Because PARP inhibitors act systemically, the lack of selectivity makes them undesirable for a number of systems, including the blood system: including thrombocytopenia, anemia, and even rare but fatal, acute Myeloid Leukemia (AML), etc., it is therefore particularly important that tumor tissue specifically inhibit PARP expression.

siRNA is a double stranded molecule, typically 19-21 base pairs, capable of modulating expression of a particular gene by cleaving homologous mRNA. The invention can reduce the expression of PARP1/2 in breast cancer cells by delivering the siRNA targeting PARP1/2 into the breast cancer cells, thereby achieving the purpose of treating breast cancer. Despite the high potential of siRNA technology in tumor treatment, clinical applications have been limited to date. On the one hand, siRNAs are large or negatively charged molecules that cannot penetrate the lipid bilayer of the cell membrane by passive diffusion. On the other hand, naked siRNA is vulnerable to enzymatic degradation and clearance in the in vivo environment, making it difficult for siRNA to reach tumor sites to function. Thus, there is an urgent need for a vector capable of safely and efficiently delivering siRNA to breast cancer to achieve its effect.

Exosomes (exosomes) are extracellular vesicles of about 40-160nm in diameter. The protein, lipid, nucleic acid and the like can be selectively packaged and released outside cells, and released to exosomes outside the cells, can be captured by cells residing in a microenvironment, and can also be carried to distant tissues and organs along with biological fluid to carry and transfer important signal molecules, so that a brand-new information transfer system between cells is formed. In order to maximize the delivery efficiency and overcome the obstacle of the siRNA delivery process, thereby improving the synthetic lethality of the HR repair defect breast cancer caused by PARP1/2 siRNA, and the exosome-based siRNA delivery system has obvious advantages. The engineered design allows the surface modification of specific molecules to achieve breast cancer specific targeting, which has the following advantages: (1) good biocompatibility and low toxicity; (2) The siRNA is prevented from being damaged by in vivo environment, and the circulation time of the siRNA in blood is prolonged; (3) siRNA can be specifically targeted to breast cancer to reduce systemic side effects.

However, in the prior art, there are few reports on PARP1/2 siRNA exosomes capable of specifically recognizing breast cancer cells.

Disclosure of Invention

Although targeted inhibition of PARP1/2 can be achieved by exosome delivery of PARP1/2 siRNA, if exosomes are not capable of specifically recognizing breast cancer cells, such exosomes are also capable of inhibiting expression of PARP1/2 in normal cells, thereby potentially producing toxic side effects on normal cells, and therefore exosomes delivering PARP1/2 siRNA are required to have breast cancer cell specificity. CXCL12 is also called as matrix cell-derived factor-1 (Stromal cell derived factor-1, SDF-1), is a protein with the size of 8-12kDa, belongs to chemotactic cytokine family, CXCR4 is a specific receptor of CXCL12, is a G protein coupled receptor with a 7-time membrane penetrating structure composed of 352 amino acids, is highly expressed on the surface of breast cancer cells, can activate various intracellular cell signaling pathways after being combined with CXCL12, including mTOR, PI3K/AKT, NF-kappaB, JAK/STAT and the like, and participates in regulating the outflow of intracellular calcium ions so as to promote the migration and growth of cells.

293T cells are a cell line with very low immunogenicity and paracrine properties.

The invention expresses CD63-CXCL12 recombinant protein in 293T cells to obtain the breast cancer targeted nanoscale exosome, and the exosome can perform the treatment effect only on breast cancer cells by targeting and delivering PARP1/2 siRNA to the breast cancer cells, thereby avoiding toxic and side effects on normal cells.

Specifically, the technical scheme of the invention is as follows:

in a first aspect the invention discloses an engineered exosome for breast cancer treatment, said engineered exosome comprising a PARP1/2 siRNA.

Preferably, the engineered exosome envelope expresses a recombinant CD63-CXCL12 fusion protein.

The second aspect of the invention discloses the application of the engineering exosome in preparing medicaments for treating breast cancer.

The third aspect of the invention discloses a method for preparing an engineered exosome for breast cancer treatment, comprising the following steps:

s1: extracting a targeting exosome expressing recombinant protein CD63-CXCL 12;

s2: loading PARP1/2 siRNA into targeted exosomes in S1 results in engineered exosomes for breast cancer treatment.

Preferably, in S2, the nucleotide sequence of the PARP1 gene siRNA is as set forth in SEQ ID NO: 5-9; the nucleotide sequence of the PARP2 gene siRNA is shown as SEQ ID NO: 10-14.

Preferably, the S1 includes:

s11: preparing a CD63-CXCL12 fusion protein;

s12: constructing an expression vector pLentai-PuroR-CMV-CD63-CXCL12-EGFR;

s13: extracting recombinant plasmid pLentai-PuroR-CMV-CD63-CXCL12-EGFR;

s14: transfecting recombinant plasmid pLentai-Puror-CMV-CD63-CXCL12-EGFR into cells, and extracting the targeting exosomes expressing recombinant protein CD63-CXCL 12.

More preferably, the nucleotide sequence of the CD63-CXCL12 fusion gene is as set forth in SEQ ID NO:21, said CD63-CXCL12 fusion gene is translated into said CD63-CXCL12 fusion protein.

More preferably, in S14, the cell is a 293T cell.

The invention also discloses a medicine for treating breast cancer, which comprises the engineering exosome or the engineering exosome prepared by the method.

The invention also discloses the application of the engineering exosome prepared by the method in preparation of the breast cancer resistant medicine.

Breast cancer cells with BRCA1/2 mutations, which have the defect of homologous recombination repair mechanisms, are sensitive to inhibitors of poly ADP-ribose diphosphate polymerase (polyADP-ribose polymerase, PARP), and can be treated with PARP inhibitors. PARP inhibitors have serious adverse effects, however, because PARP inhibitors act systemically and lack selectivity characteristics that cause adverse effects on a number of systems including the blood system: including thrombocytopenia, anemia, and even rare but fatal Acute Myeloid Leukemia (AML), etc., it is therefore particularly important to specifically inhibit PARP expression in tumor tissues. The invention obtains breast cancer targeted exosomes by expressing CD63-CXCL12 recombinant proteins in 293T cells; loading PARP1/2 siRNA into the exosome by electroporation to obtain an engineered exosome; the engineered exosomes targeted delivery of PARP1/2 siRNA to breast cancer to kill tumor cells. The breast cancer tissue specific targeted drug delivery system constructed by the invention can specifically inhibit PARP in breast cancer cells, thereby exerting the therapeutic effect only aiming at the breast cancer cells and avoiding the toxic and side effects on normal cells.

Compared with the prior art, the invention has the following advantages:

the currently clinically adopted oral PARP inhibitor does not have the capability of specifically identifying tumor cells, so that the PARP inhibitor has serious toxic and side effects. The invention constructs a targeted drug delivery system specific to breast cancer tissues, which can specifically inhibit PARP in breast cancer cells and avoid the influence on normal cells.

Drawings

FIG. 1 shows the result of enzyme digestion verification. * Nc=no cleavage control, 1 kb=1 kb dnamarker, e=xmai-BamHI cleavage.

Fig. 2 is a schematic diagram of flow cytometry detection of fluorescence intensity statistical analysis of empty vector and recombinant plasmid EGFP, P <0.01.

FIG. 3 is a schematic diagram showing the expression of CD63 and CXCL12 in 293T cells.

FIG. 4 is a diagram showing statistical analysis of red fluorescence intensity of normal cells and breast cancer cells incubated by the engineering exosomes,

**P<0.01。

FIG. 5 is a schematic representation of engineered exosome-mediated down-regulation of PARP1/2 expression in breast cancer cells.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the scope of the examples.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The reagents and materials used in the present invention are commercially available.

Example 1

The embodiment discloses an engineering exosome for breast cancer treatment, which specifically comprises the following steps:

1. the method comprises the following steps:

construction of 1.1CD63-CLCL12 fusion Gene

1.1.1 design and Synthesis of primers

Based on the gene sequence of the fusion protein CD63-CXCL12 (SEQ ID NO: 21), the PCR primers were designed by themselves as follows: p1:5'-CCACCGCCATGGTGGCGCCCGGGA-3' (SEQ ID NO: 1) (CCCGGG is XmaI recognition site)

P2：5’-AGTGGCTACGAGGTGATGT-3’(SEQ ID NO：2)

P3：5’-ACGACCT-3’TGGCGTTCAT-3’(SEQ ID NO：3)

P4:5'-AGGTTCAAGATGTGAGGATCCGC-3' (SEQ ID NO: 4) (GGATCC is a BamHI recognition site)

Amplification of 1.1.2CD63, CXCL12 Gene fragment

The 293T cell cDNA is used as a template, the P1 and the P2 of the CD63 gene are used as primers, and the CD63 gene is amplified, and the reaction system is as follows:

the PCR reaction conditions were: pre-denaturation at 98℃for 5min, denaturation at 98℃for 30s, annealing at 61℃for 1min, extension at 72℃for 2min,35cycle, extension at 72℃for 10min, and preservation at 4 ℃.

The 293T cDNA is used as a template, P3 and P4 of CXCL12 sequences are used as primers, and the CXCL12 sequences are amplified, and the reaction system is as follows:

Purification of 1.1.3PCR product

And (3) after the amplified PCR product is subjected to 1% agarose gel electrophoresis analysis, cutting off a target band, and purifying by a DNA gel recovery kit to obtain a purified PCR product.

Amplification of 1.1.4CD63-CXCL12 fusion Gene

The fusion gene CD63-CXCL12 is amplified by using a method of overlap, extension and splice PCR (SOE-PCR) by taking PCR recovery products of CD63 and CXCL12 as templates and P1 and P4 as primers, and the reaction system is as follows:

the PCR reaction conditions were: pre-denaturation at 98℃for 8min, denaturation at 98℃for 30s, annealing at 64℃for 1min, extension at 72℃for 2min,42cycle, extension at 72℃for 10min, and preservation at 4 ℃.

After the amplified PCR product is analyzed by 1% agarose gel electrophoresis, a target band is cut off, and the PCR product is purified by a DNA gel recovery and purification kit.

1.2 construction of the expression vector pLentai-Puror-CMV-CD63-CXCL12-EGFR

1.2.1pLentai-Puror-CMV-EGFR vector cleavage step

The vector pLentai-PuroR-CMV-EGFR was digested with BamHI and XmaI to prepare a reaction system:

after being evenly mixed, the mixture is incubated in a water bath kettle at 37 ℃ for 2 hours; 1.5% agarose gel electrophoresis, it can be seen that 7700bp and 848bp bands are obtained, and 7700bp vector bands are recovered. (10XNEBufferr 3.1 from New Englandbiolabs cat# B6003S) 1.2.2T4 ligation step

Establishing T4 connection system

After mixing, the mixture was left to join overnight at 16 ℃. (10XT4 connection Buffer from Thermo Scientific cargo number B69)

1.2.3 conversion of ligation products

(1) Taking out competent TOP10 E.coli cells in a refrigerator at-80deg.C, placing on an ice box of an ultra-clean workbench, adding 5 μl of the connection product when the cells are melted into ice-water mixture, gently blowing to mix the connection product, and ice-bathing for 30min;

(2) Water bath at 42 ℃ for 90s and ice bath for 4min;

(3) Adding 800 μl of culture medium, and shake culturing at 37deg.C for 4 hr;

(4) Mu.l of the culture product was pipetted onto a solid culture plate with Amp resistance (10. Mu.g/ml) and incubated overnight at 37 ℃.

1.3 extraction of recombinant plasmid pLentai-Puror-CMV-CD63-CXCL12-EGFR

Plasmid extraction was performed using a plasmid miniprep kit (Labselect, inc.).

(1) Taking 1 to 5ml of bacterial liquid, centrifuging at 12000rpm for 1 minute, absorbing and discarding the supernatant, then adding 200 mu l of solution P1, adding 200 mu l of solution P2 after fully mixing, gently turning up and down for 6 to 8 times until the solution becomes clear and viscous, immediately adding 250 mu l of solution P3, and gently turning up and down for 6 to 8 times. Followed by centrifugation at 12000rpm for 10 minutes.

(2) Transferring the supernatant to an adsorption column at 12000rpm, and centrifuging for 1 minute; the waste liquid was discarded, 700. Mu.l of Washing buffer was added thereto, and the mixture was centrifuged at 12000rpm for 1 minute, and the mixture was repeated twice. Placing the adsorption column into a recovery tube, centrifuging at 12000rpm for 2 minutes, discarding the waste liquid, standing at room temperature until the residual liquid in the adsorption column is dried. The column was transferred to a fresh EP tube, 20. Mu.l of eluent was added dropwise, and the mixture was allowed to stand at room temperature for 5 minutes at 12000rpm and centrifuged for 2 minutes. The solution was collected and the Thermo NanoDrop-2000 was used to measure plasmid concentration and stored at-20 ℃.

1.4293T cell transfection recombinant plasmid pLentai-Puror-CMV-CD63-CXCL12-EGFR

Cell transfection was performed using the Thermo Fisher Scientific company lipofectamine kit, as follows:

(1) Experiments were performed using 293T cells in the logarithmic growth phase. After cell digestion and counting, the cells were used in a 2X 10 per well ⁵ Is plated in 6-well plates, cultured overnight, and transfected after cells adhere.

(2) Preparing a solution 1: MEM medium (250. Mu.l) +lipo3000 (3.75. Mu.l). Preparing a solution 2: MEM medium (250. Mu.l) +plasmid (5. Mu.g) +P3000. Solutions 1 and 2 were left at room temperature for 15 minutes, respectively, followed by mixing and incubation for 5 minutes.

(3) After the cells were rinsed with PBS, the mixture of (2) was added, and the cells were cultured for 6 hours and changed to DMEM complete medium.

1.5 identification of expression of CD63-CXCL12 fusion proteins in 293T cells by Western blot.

(1) Protein cleavage: firstly, preparing a lysate: 200. Mu.l of RIPA lysate was mixed with 4. Mu.l of protease inhibitor, 4. Mu.l of phosphatase inhibitor and 2. Mu.l of PMSF on ice and prepared for use. To the cell pellet or exosome pellet, 200. Mu.l of lysate was added, followed by incubation on ice for 20 minutes, and after 12000 Xg, centrifugation was performed at 4℃for 5 minutes, and the supernatant was collected. Protein concentration was determined using BCA assay.

(2) Protein denaturation: SDS-PAGE protein loading buffer (5X) was added to the supernatant followed by a water bath at 100deg.C for 10 minutes to allow complete denaturation of the protein.

(3) Electrophoresis: an 8% SDS-PAGE gel was prepared, run at 80V constant pressure for 30 minutes after loading, then run at 120V constant pressure until bromophenol blue was 0.5cm from the lower edge of the gel.

(4) Transferring to membrane, taking out SDS-PAGE gel, fixing in the order of power cathode, sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper, sponge and power anode, placing in a transferring tank, transferring to membrane for 90 min under constant pressure of 100V.

(5) The PVDF membrane was removed and placed in a blocking solution containing 5% nonfat dry milk formulated with TBST and blocked for 1 hour at room temperature.

(6) Incubating primary antibodies: the PVDF membrane was removed and blocked with a blocking solution according to 1: the primary antibody was diluted in a ratio of 1000, the membrane was placed in the primary antibody solution, gently shaken on a shaker, and incubated overnight at 4 ℃.

(7) Incubating a secondary antibody: the PVDF membrane was removed, washed 3 times with TBST, and then the secondary antibody was incubated at room temperature, and after 1 hour, washed 3 times with TBST.

(8) Color development: preparing ECL luminous liquid: reinforcing liquid: stabilizing solution = 1:1. ECL luminescent droplets were applied to the protein-binding side of PVDF membranes. After the excessive luminous liquid is sucked by filter paper, the film is pressed into a sheet by an X-ray film, and then the film is sequentially put into a developing solution and a fixing solution, washed by water, photographed and counted.

1.6 extraction of Targeted exosomes expressing the recombinant protein CD63-CXCL12

The old 293T cell culture broth was aspirated, rinsed 3 times with PBS, and in order to ensure that the collected culture supernatant was free from the influence of foreign body confounding factors, the culture medium was replaced with DMEM medium containing 10% of FBS from which the foreign body was removed (ultracentrifuge removal of the foreign body), and the culture broth was collected after a conventional culture for 36 hours. 50ml of the culture was centrifuged at 300 Xg at 4℃for 10 minutes, and the pellet was discarded. Then, 2000 Xg was centrifuged at 4℃for 10 minutes, and the precipitate was discarded. Then 10000 Xg, centrifuged at 4℃for 30 minutes, and the precipitate was discarded. Followed by centrifugation at 100000 Xg at 4℃for 70 minutes, at which time the pellet was the exosome. The supernatant was pipetted off, followed by a 200. Mu.l PBS resuspension, followed by 100000 Xg, centrifugation at 4℃for 70 minutes, the supernatant was pipetted off, and 50. Mu.l PBS resuspension was added. Placing in a refrigerator at-80deg.C for preservation.

1.7PARP1/2 siRNA sequence and targeting site

siRNA targeting PARP1/2 sites were designed and synthesized as follows:

1.8 construction of engineered exosomes by electroporation of PARP1/2 siRNA into the targeted exosomes

Purified targeted exosomes and PARP1/2 siRNA were mixed in electroporation buffer, electroporated in electroporation cuvette using Bio-Rad gene pulserXcell electroporation system at 350V, followed by incubation of the mixture at 37 ℃ for 30min to restore exosome envelope.

1.9 observation of exosomes by transmission electron microscopy

Mu.l of the exosome suspension was added dropwise to the Formvar-carbon film coated copper-loaded mesh, allowed to stand for 1 minute and then air-dried. The Formvar membrane side was rinsed 3 times with PBS and the copper mesh side was kept dry. Fixation was performed for 20 minutes at room temperature using 1% glutaraldehyde. Using ddH ₂ After O rinse twice, stain with 2% uranyl acetate for 15 minutes. Along with itAfter 10 minutes of rinsing with methylcellulose-UA, the mixture was air-dried. Observed under a transmission electron microscope and photographed.

1.10 identification of particle size of exosomes

With ddH ₂ O dilutes exosome suspension, and the exosome particle concentration is controlled to be 1x10 ⁷ Ml to 1x10 ⁹ Between/ml, the number and size of examples in the samples were determined using a ZetaView PMX110 instrument at 405nm laser, and the exosome size was analyzed using Nanoparticle tracer analysis (Nanoparticle TrackingAnalysis, NTA) software.

1.11 exosome tracing and targeting identification

(1) Exosome staining

The exosome content was determined by BCA assay, 500. Mu.g exosome was mixed with 4. Mu.g/ml of Dil red dye in equal volume and incubated at 4℃for 2 hours in the absence of light. After subsequently re-suspending the exosomes with 1ml of PBS, 100000×g, centrifuged at 4 ℃ for 70 min, the supernatant was pipetted off and re-suspended with 50 μl of PBS, which is red fluorescent-labeled exosomes (Dil-exosomes). Placing in a refrigerator at-80deg.C, and storing in dark place.

(2) Exosome and cell incubation

And (3) incubating the exosomes with the Dil fluorescent markers by adopting different cell lines, and observing the uptake condition of the breast cancer to the engineered exosomes compared with other cell lines in the same time. The following operations were all performed in the dark. Will be 5x10 ⁵ Individual cells were seeded in 6-well plates with cell slide placed thereon, 50. Mu.g of Dil-Exosome was added to the culture solution after 12 hours, the culture solution was pipetted off after 24 hours, followed by rinsing 3 times with PBS and 1ml of 4% paraformaldehyde was added thereto for fixation for 15 minutes at room temperature. After aspiration of the fixative, 1ml of 0.1% Triton X-100 was added and the cells incubated for 5 minutes followed by 3 washes with PBS. Finally, the sealing piece liquid sealing piece of the anti-fluorescence quenching agent of the premixed DAPI is used for shading for 15 minutes. Exosome phagocytosis was observed under a fluorescence microscope. And detecting exosome phagocytosis by using a flow cytometer.

1.12 detection of PARP1/2 expression levels in breast cancer cells by real-time fluorescent quantitative PCR experiments

The primers required for the experiment were synthesized by Shanghai Biotechnology Co. The primer sequences involved in this section are shown in the following table:

experiments were performed using SYBR Green PCR Master Mix from Roche, the system is as follows:

the reaction system is placed in a LightCycler 96 real-time fluorescence quantitative PCR instrument. PCR amplification of cDNA was performed in a two-step method.

The conditions are as follows: step 1, pre-denaturation: setting 1 cycle at 95 ℃ for 30 seconds; step 2, PCR reaction: at 95 ℃,5 seconds, 60 ℃,30 seconds, 40 cycles are set. 3 duplicate wells were set for each sample. Using GAPDH as an internal reference, the Ct value (the number of cycles required for the fluorescence intensity to reach a set threshold) of each sample was then measured, and the relative expression level of the target gene was 2 ^-△△CT And (3) calculating:

Δct (experimental group) =ct (gene of interest, experimental group) -CT (reference gene, experimental group)

Δct (control) =ct (target gene, control) -CT (reference gene, control)

ΔΔct= Δct (experimental group) - Δct (control group)

2. Results

2.1 identification of recombinant plasmid pLentai-PuroR-CMV-CD63-CXCL12-EGFR

The recombinant plasmid pLentai-PuroR-CMV-CD63-CXCL12-EGFR was digested with XmaI and BamHI, and as a result, two approximately 9000 and 1300bp size bands, namely a pLentai-PuroR-CMV-EGFR linear fragment and a CD63-CXCL12 fusion gene fragment, were seen in FIG. 1, and the recombinant expression plasmid pLentai-PuroR-CMV-CD63-CXCL12-EGFR was successfully constructed.

2.2 transfection of recombinant plasmid pLentai-Puror-CMV-CD63-CXCL12-EGFR into 293T cells

The recombinant plasmid was transfected into 293T cells, and the 293T cells expressed EGFP to emit green fluorescence, and the fluorescence intensity was measured using a flow cytometer, and the results are shown in FIG. 2. Indicating that the engineering plasmid can express the integrated recombinant CD63-CXCL12 gene in cells.

2.3 verification of the fusion Gene CD63-CXCL12

The expression of CD63 and CXCL12 in 293T cells transfected with the empty vector was low, while the expression of CD63 and CXCL12 in 293T cells transfected with the engineering plasmid was high, indicating that the recombinant plasmid was able to successfully express the integrated CD63 and CXCL12 in 293T cells, and the results are shown in FIG. 3.

2.4 extraction, preparation and identification of engineered exosomes

The culture solution of 293T cells transfected with engineering plasmids is collected, exosomes in the culture solution are extracted by an ultracentrifugation method, PARP1/2 siRNA is delivered into the exosomes by electroporation, the exosomes are constructed as engineering exosomes, and the structure of the exosomes is observed under an electron microscope, and has no obvious difference from the structure of the general exosomes, so that the modified engineering exosomes do not change the structural properties of the exosomes. Particle size of the engineered exosomes was detected by NTA. The particle sizes of the engineering exosomes are not obviously different, which indicates that the structural properties of the modified engineering exosomes are not changed.

2.5 determination of engineered exosome targeting

To verify the targeting of the engineered exosomes, the engineered exosomes were labeled with red dye Dil and used to incubate human normal breast cells (HS 578 BST) and breast cancer cells (MDA-MB-231), red fluorescence was measured by flow cytometry, more red fluorescence was found in the breast cancer cells, indicating that the engineered exosomes tended to be enriched in tumor cells rather than normal tissue cells, indicating that the engineered exosomes had targeting to breast cancer, and the results are shown in fig. 4.

2.6 engineering exosomes to down-regulate PARP1/2 expression in breast cancer cells

The expression of PARP1/2 in breast cancer cells transfected with normal exosomes (control) and engineered exosomes (CD 63-CXCL 12) was detected by a real-time fluorescent quantitative PCR method and the results are shown in FIG. 5.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. An engineered exosome for use in the treatment of breast cancer, wherein the engineered exosome comprises PARP1/2 siRNA.

2. The engineered exosome of claim 1, wherein the engineered exosome envelope expresses a recombinant CD63-CXCL12 fusion protein.

3. Use of an engineered exosome according to claim 1 in the manufacture of a medicament for breast cancer treatment.

4. A method of preparing an engineered exosome for breast cancer treatment, comprising:

s1: extracting a targeting exosome expressing recombinant protein CD63-CXCL 12;

5. The method of claim 4, wherein in S2, the nucleotide sequence of the PARP1 gene siRNA is as set forth in SEQ ID NO: 5-9; the nucleotide sequence of the PARP2 gene siRNA is shown as SEQ ID NO: 10-14.

6. The method of claim 4, wherein S1 comprises:

s11: preparing a CD63-CXCL12 fusion protein;

s12: constructing an expression vector pLentai-PuroR-CMV-CD63-CXCL12-EGFR;

s13: extracting recombinant plasmid pLentai-PuroR-CMV-CD63-CXCL12-EGFR;

7. The method of claim 6, wherein the nucleotide sequence of the CD63-CXCL12 fusion gene is set forth in SEQ ID NO:21, said CD63-CXCL12 fusion gene is translated into said CD63-CXCL12 fusion protein.

8. The method of claim 6, wherein in S14, the cell is a 293T cell.

9. A medicament for treating breast cancer, comprising the engineered exosome of claims 1-2 or the engineered exosome prepared by the method of claims 4-8.

10. Use of an engineered exosome prepared according to the method of claims 4-8 in the preparation of an anti-breast cancer drug.