An engineered exosome targeting breast cancer drug-resistant cells, preparation method and application

Abstract

The present application relates to the biomedical field, and relates to an engineered exosome targeting breast cancer drug-resistant cells, a preparation method and application. The engineered exosome targeting breast cancer drug-resistant cells is cloned from N terminal to C terminal in sequence with the first 133 amino acids of CD63, the RBD domain of spike and FLAG-tag, and they are constructed in a PCDH-puro plasmid. First, CD63(N)-RBD-FLAG is stably expressed in HEK293T cells, then the culture supernatant is extracted, and the exosome is obtained through gradient ultracentrifugation. The present application provides an engineered exosome, which has high expression of CD63(N)-RBD-FLAG and high targeting property based on high expression of ACE2 to drug-resistant cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an engineered exosome targeting drug-resistant breast cancer cells, its preparation method, and its application. In particular, it relates to a modified exosome fused with CD63(N)-SPIKE(RBD)-FLAG that targets drug-resistant breast cancer cells that highly express ACE2. Background Technology

[0002] Breast cancer is the most common malignant disease in women, and chemotherapy is the main treatment strategy for patients with locally advanced or metastatic breast cancer. However, a large number of patients gradually lose response to chemotherapy drugs, and cancer cells develop resistance to various anticancer drugs, which is a key reason why advanced breast cancer is difficult to treat. When cells are attacked by drugs, some specific proteome reprogramming occurs, which is different from that of parent cells. Many specific proteins are also expressed in drug-resistant cells. Therefore, identifying or targeting these proteins can be a treatment option for drug-resistant patients. In our previous studies, we found that angiotensin-converting enzyme 2 (ACE2) expression is upregulated in breast cancer cells after stimulation by chemotherapy drugs, and ACE2 is stably and highly expressed in drug-resistant cells. ACE2 is a key functional receptor for SARS-CoV-2 to enter host cells. ACE2 has become a focus of global attention. SARS-CoV-2 is a single-stranded RNA virus with relatively simple genetic material and a rapid mutation rate. The spike protein expressed on its envelope is a key protein for recognizing the host. The RBD domain of the spike recognizes and binds to the host ACE2, which is the first step in viral infection of the host. In view of the high ACE2 expression characteristic of drug-resistant cells, we envision constructing an ACE2-specific targeted drug delivery system to enhance the treatment effect for drug-resistant patients.

[0003] Extracellular vesicles, especially exosomes, are key carriers secreted by cells to perform intercellular communication and substance delivery functions. Exosomes are small vesicles with a diameter of 30-150 nm. Almost all cell types can produce and release exosomes. Exosomes are naturally present in body fluids, have a topological structure similar to cells, and contain DNA, RNA, proteins, lipids, and small molecule metabolites. Exosome formation involves the invagination of the cell membrane to form multivesicular bodies (MVBs). These MVBs contain intraluminal vesicles (ILVs), which are exosome precursors. The MVBs fuse with the plasma membrane, releasing the exosomes into the extracellular space. Exosomes can serve as a biocompatible drug delivery system, but they lack specific targeting capabilities.

[0004] Constructing engineered exosomes involves modifying the proteins contained within them to achieve specific targeting capabilities. Exosomes contain many membrane proteins, such as various four-transmembrane proteins (CD9, CD63, CD37, CD81, CD82), heat shock proteins (HSPs), tumor susceptibility gene 101 protein (Tsg101), and ALG-2 interacting protein X (Alix), all of which are commonly expressed antigens on the exosome surface. Modifying the expression of these proteins can enhance the targeting ability of exosomes. Therefore, is it possible to construct engineered exosomes that target drug-resistant cells to improve the therapeutic effect on drug-resistant patients? Summary of the Invention

[0005] To address the technical challenges in the background and overcome the shortcomings of existing technologies, this invention provides an engineered exosome targeting drug-resistant breast cancer cells, its preparation method, and its application, which can solve the problem of difficult treatment for drug-resistant breast cancer patients.

[0006] To achieve the above objectives, the present invention provides an engineered exosome that targets drug-resistant breast cancer cells, wherein the engineered exosome fused with the protein CD63(N)-SPIKE(RBD)-FLAG.

[0007] Specifically, the engineered exosomes targeting drug-resistant breast cancer cells sequentially clone the first 133 amino acids of CD63, the RBD domain of spike, and the FLAG-tag from the N-terminus to the C-terminus, and construct them in the PCDH-puro plasmid, named CD63(N)-RBD-FLAG.

[0008] Furthermore, the plasmid construction scheme adopted homologous recombination, and two pairs of primers were designed to clone CD63(N) and SPIKE(RBD) by PCR, respectively. The primers are shown in the table below:

[0009]

[0010] The PCR system is as follows:

[0011]

[0012] The PCR procedure is as follows:

[0013] Preheating: 98℃, 30s; Denaturation: 98℃, 10s; Annealing: 55℃, 10s; Extension: 72℃, 30s; Denaturation-annealing extension...

[0014] Repeat the process 30 times; store at 4°C.

[0015] PCDH-puro plasmid was linearized by single enzyme digestion, and the digestion system was incubated at 37°C for 1 hour.

[0016] 1ng 1μl 2μl Up to 20 μl 20μl

[0017] The linearized plasmid vector and PCR product were purified and recovered by agarose gel electrophoresis, and then ligated for homologous recombination in the following system at 50°C for 15 min:

[0018]

[0019] This invention also provides a method for preparing the above-mentioned engineered exosomes targeting drug-resistant breast cancer cells, the preparation method comprising the following steps:

[0020] 1) Stable expression of CD63(N)-RBD-FLAG in HEK293T cells;

[0021] 2) Extract the culture supernatant and obtain exosomes by gradient ultracentrifugation.

[0022] This invention also provides the application of the engineered exosomes that target drug-resistant breast cancer cells in the preparation of exosome-enhanced drugs for breast cancer targeting.

[0023] This invention also provides the application of the above-mentioned engineered exosomes targeting drug-resistant breast cancer cells in the preparation of drugs that encapsulate therapeutic drugs to kill drug-resistant breast cancer cells.

[0024] The present invention also provides the application of the above-mentioned engineered exosomes targeting drug-resistant breast cancer cells in the preparation of drugs that inhibit breast cancer cell growth and ACE2-specific targeted drug delivery systems.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention provides an engineered exosome that highly expresses CD63(N)-RBD-FLAG and has high targeting ability against drug-resistant cells based on high ACE2 expression.

[0027] 2. Construct an engineered exosome that integrates expression of CD63 and spike to target drug-resistant breast cancer cells that highly express ACE2, thereby improving the treatment outcome for drug-resistant patients.

[0028] 3. A modified exosome comprising expressing a fusion-expressed modified membrane protein, such that the exosome specifically targets drug-resistant breast cancer cells. Attached Figure Description

[0029] Figure 1 Western blotting analysis of plasmid overexpression in cells and exosomes

[0030] Figure 2 Compare the exosome uptake of drug-resistant and drug-sensitive breast cancer cells.

[0031] Figure 3 Compare exosome uptake before and after T47D epirubicin stimulation.

[0032] Figure 4 Compare the uptake of different exosomes in drug-resistant cells.

[0033] Figure 5 Compare the exosome uptake of drug-resistant cells before and after ACE2 knockdown.

[0034] Figure 6 A schematic diagram of modified fusion-expressing CD63(N)-SPIKE(RBD)-FLAG exosomes targeting drug-resistant breast cancer cells that highly express ACE2. Detailed Implementation

[0035] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0036] The plasmid construction scheme of this invention adopts homologous recombination, and two pairs of primers are designed to clone CD63(N) and SPIKE(RBD) by PCR, respectively. The primers are shown in the table below:

[0037]

[0038] The PCR system is as follows:

[0039]

[0040] The PCR procedure is as follows:

[0041] Preheating: 98℃, 30s; denaturation: 98℃, 10s; annealing: 55℃, 10s; extension: 72℃, 30s; repeat denaturation, annealing and extension cycles 30 times; store at 4℃.

[0042] PCDH-puro plasmid linearization by single enzyme digestion, the enzyme digestion system is as follows (37℃, 1h):

[0043] 1ng 1μl 2μl Up to 20 μl 20μl

[0044] The linearized plasmid vector and PCR products were purified and recovered by agarose gel electrophoresis, and then ligated by homologous recombination in the following system (50℃, 15 min):

[0045]

[0046] Transformation of recombinant products:

[0047] DH5α chemocompetent cells were thawed on ice. 10 μl of the recombinant product was added to 100 μl of competent cells, mixed, and incubated on ice for 30 min. After heat shock in a 42°C water bath for 45 s, the cells were immediately cooled on ice for 3 min. 1 ml of LB liquid medium was added, and the cells were shaken at 37°C for 1 h (200 rpm). After centrifugation at 5000 rpm for 5 min, 900 μl of supernatant was discarded. The cells were resuspended in the remaining medium and gently spread on LB solid medium plates containing ampicillin resistance using a sterile spreader. The cells were incubated upside down in a 37°C incubator for 16 h.

[0048] Plasmid extraction:

[0049] Pick a single colony from the plate and inoculate it into 20 ml of LB liquid medium. Amplify at 37°C and 200 rpm. After 16 h, extract the plasmid using a plasmid extraction kit and set aside for later use.

[0050] Example 1 Virus Packaging

[0051] HEK293T cells in good growth condition, in the logarithmic growth phase, and with a confluence of 60%-80%, were used for plasmid transfection and virus packaging. The required amount of plasmid is shown in the table below:

[0052]

[0053] Each group of plasmids was transfected into HEK293T cells with PEI (polyethyleneimine). After 48 hours, the supernatant was collected, and the precipitate was removed to obtain the virus solution.

[0054] HEK293T cells were infected with a 1:1 mixture of virus and cell culture medium. After 48 hours, cells stably expressing the virus were selected using puromycin (puro) to select for expression. The culture supernatant from stably expressing cells was continuously collected for exosome extraction.

[0055] Exosomes were obtained by extracting the culture supernatant and then using the following gradient ultracentrifugation method:

[0056] Centrifuge at 3000g for 10 minutes at 4℃ and collect the supernatant.

[0057] Centrifuge at 10,000g for 30 minutes at 4℃ and collect the supernatant.

[0058] Centrifuge at 100,000g, 4℃ for 70 min, discard the supernatant, retain the precipitate, and resuspend in a large amount of PBS.

[0059] Centrifuge at 100,000g, 4℃ for 70 min, discard the supernatant, retain the precipitate, and resuspend in 100μl PBS.

[0060] Example 2: Exosome uptake

[0061] PKH26(sigma) was used to stain exosomes.

[0062] Solution A: Add a certain amount of exosomes to 250 μl Diluentc;

[0063] Solution B: 250 μl Diluentc plus 1 μl PKH26.

[0064] Mix solution A and solution B and let stand for 5 minutes. Add 1 ml of pure serum and stop for 1 minute. Centrifuge at 100,000 g for 70 minutes. Resuspend the cells in culture medium and apply to the cells. After a certain period of time, detect the amount of fluorescence in the cells, which is the amount of exosomes taken up by the cells.

[0065] The exosomes obtained above were quantified: protein concentration was determined by BSA protein quantification method, and exosomes with the same total protein content were subsequently added. The exosomes extracted by this method were found to be rich in CD63(N)-RBD-FLAG, such as... Figure 1 As shown.

[0066] Results of breast cancer drug resistance cell induction assay: The human breast cancer cell line MDA-MB-468 was induced to become a drug-resistant cell line MDA-MB-468 / EPR through gradient chemotherapeutic drug induction. The ACE2 expression level in the drug-resistant cells was as follows: Figure 2 As shown in Figure A.

[0067] When equal amounts of the above exosomes were added to MDA-MB-468 sensitive and resistant cells, the resistant cells took up significantly more exosomes rich in CD63(N)-RBD-FLAG than the sensitive cells. Figure 2 As shown in B.

[0068] Example 3: Epirubicin-stimulated T47D cells to take up exosomes

[0069] T47D cells with good growth and a confluence of about 50% were pre-stimulated with 0.1 μM epirubicin for 24 h. Exosomes were stained with PKH67 (Bio-Lepto). The staining method was to dilute the staining solution and diluent at a ratio of 1:10 to prepare a stock solution. The stock solution was then diluted 25 times and added to serum-free culture medium. The exosomes to be stained were then added and stained at 4°C for 30 min. The stained exosomes were then added to the pre-stimulated T47D cells, and the uptake was observed after a certain period of time.

[0070] The results show that the exosomes provided by this invention can better target drug-resistant cells, enabling drug-resistant cells to take up more exosomes, which provides a basis for encapsulating therapeutic drugs in exosomes to kill drug-resistant cells.

[0071] In breast cancer cells T47D, ACE2 stress response is upregulated by the chemotherapy drug epirubicin, such as... Figure 3As shown in Figure A, T47D cells significantly uptake more of the aforementioned engineered exosomes rich in CD63(N)-RBD-FLAG after epirubicin stimulation, such as... Figure 3 As shown in B. The exosomes provided by this invention exhibit high targeting specificity for cells that highly express ACE2.

[0072] The engineered exosomes rich in CD63(N)-RBD-FLAG provided by this invention were taken up by more exosomes in drug-resistant cells MDA-MB-468 / EPR compared to control exosomes, such as... Figure 4 As shown.

[0073] The exosomes provided by this invention exhibit stronger targeting of drug-resistant cells compared to unmodified exosomes. Furthermore, knocking down ACE2 in drug-resistant cells MDA-MB-468 / EPR significantly reduces exosome uptake, such as... Figure 5 As shown, this indicates that the high targeting ability of these exosomes to drug-resistant cells depends on the high expression of ACE2.

Claims

1. An engineered exosome targeting drug-resistant breast cancer cells, characterized in that, The engineered exosome fusion expression protein CD63(N)-SPIKE(RBD)-FLAG targeting drug-resistant breast cancer cells; The engineered exosomes targeting drug-resistant breast cancer cells sequentially cloned the first 133 amino acids of CD63, the RBD domain of spike, and the FLAG-tag from the N-terminus to the C-terminus, and constructed them in the PCDH-puro plasmid, named CD63(N)-RBD-FLAG.

2. The engineered exosome targeting drug-resistant breast cancer cells according to claim 1, characterized in that, The plasmid construction method adopted homologous recombination. Two pairs of primers were designed to clone CD63(N) and SPIKE(RBD) by PCR, respectively. The primers are as follows: 。 3. A method for preparing engineered exosomes targeting drug-resistant breast cancer cells according to any one of claims 1 to 2, characterized in that, The preparation method includes the following steps: 1) Stable expression of CD63(N)-RBD-FLAG in HEK293T cells; 2) Extract the culture supernatant and obtain exosomes by gradient ultracentrifugation.

4. The use of an engineered exosome targeting drug-resistant breast cancer cells according to any one of claims 1 to 2 in the preparation of exosome-enhanced drugs for breast cancer targeting.

5. The use of an engineered exosome targeting drug-resistant breast cancer cells according to any one of claims 1 to 2 in the preparation of a drug for killing drug-resistant breast cancer cells by encapsulating exosomes with therapeutic agents.

6. The use of an engineered exosome targeting drug-resistant breast cancer cells according to any one of claims 1 to 2 in the preparation of an exosome-based drug for inhibiting breast cancer cell growth.

7. The use of an engineered exosome targeting drug-resistant breast cancer cells according to any one of claims 1 to 2 in the design of an exosome-specific targeted drug delivery system for ACE2.

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