CN116041470A - Cell membrane material capable of binding tumor cell CD47 with high affinity, preparation method and application thereof - Google Patents
Cell membrane material capable of binding tumor cell CD47 with high affinity, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cell membrane material with high affinity for binding tumor cells CD47, and a preparation method and application thereof, and belongs to the technical field of biology. The invention expresses the mutant SIRPalpha on the surface of a host cell membrane by means of genetic engineering, and can obtain a cell membrane material with active targeting binding capacity on a CD47 ligand on the surface of a tumor cell. The cell membrane material is utilized to carry out surface modification on the anti-tumor nano-drug, so that the drug can be delivered to the tumor part with high efficiency; meanwhile, the SIRP alpha receptor on the surface of the macrophage is blocked from being combined with the CD47 ligand by competitively combining with the CD47 ligand on the surface of the tumor cell, so that the anti-tumor activity of the macrophage is restored. Thus, the construction of this novel cell membrane material provides an innovative solution for developing cell membrane materials with targeted binding to tumor cells.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cell membrane material capable of binding tumor cells CD47 with high affinity, a preparation method and application thereof.
Background
With the development of nanotechnology, nanoparticles as drug delivery vehicles have been widely studied in the fields of diagnosis and treatment of cancer. However, most nanoparticles are recognized and cleared as foreign substances by the immune system in the body, and have poor biocompatibility, limiting the utility of these nanomaterials. Thus, there is an urgent need for a safe, effective, and easy to manufacture nano-delivery system that escapes immune system clearance, as well as enhanced tumor targeting.
The biomembrane-mediated nano-drug delivery system based on the bionic technology utilizes cell membranes as carriers, and utilizes the functions of protein and polysaccharide on the surface of the cell membranes to promote the long circulation and targeted delivery of the nuclear nanoparticles in vivo under the condition of not considering the characteristics of the inner core nano-materials, thereby being expected to realize the new breakthrough of the nano technology in tumor targeted therapy. From this, the membrane biomimetic modification nanoparticle is widely explored and studied, and how to develop an innovative membrane material is a bottleneck problem faced by current research.
In view of this, the present application is specifically proposed.
Disclosure of Invention
The invention aims to provide a cell membrane material with high affinity for binding tumor cells CD47, and a preparation method and application thereof, so as to overcome the defect of the existing cell membrane material and provide a carrier for delivering nano-drugs.
The technical conception of the invention:
the cell membrane bionic nanoparticle provides a solution for efficient drug delivery to tumor sites, but how to develop cell membrane materials with targeted binding to tumor cells is a bottleneck problem facing at present. The way of expressing specific protein capable of being targeted to bind tumor cells on the surface of cell membrane by genetic engineering provides a solution way for obtaining cell membrane material with targeted ligand binding to the surface of tumor cells.
The inventor develops a cell membrane material with specific recognition capability to tumor cells, and adopts a technical idea that specific proteins capable of combining with ligands on the surface of the tumor cells in a targeted way are expressed on the surface of the cell membrane by a genetic engineering means. Therefore, it is critical how to select specific protein variants that are effective to target binding to tumor cell surface ligands. The research literature finds that the integrin-associated protein (CD 47) is highly expressed in almost all solid tumors such as various leukemia, non-Hodgkin lymphoma, multiple myeloma, leiomyosarcoma, bladder cancer, breast cancer, colon cancer, brain cancer, liver cancer, prostate cancer and the like, and the CD47 can be combined with signal regulating protein alpha (SIRP alpha) on the surface of macrophages and send out 'do not eat me' signals, so that cancer cells escape from the attack of an immune system and participate in various biological functions such as proliferation and migration of tumor cells. Therefore, the SIRPalpha/CD 47 binding site can be used as a target for targeting tumor cell binding and a therapeutic target of tumors.
However, since sirpa acts as a transmembrane immunoglobulin, natural sirpa has a weak affinity for CD 47. In order to further improve the specific recognition capability of a target product cell membrane material on tumor cells, the inventor obtains a high-affinity SIRP alpha mutant (M-SIRP alpha) which is improved by about 5 ten thousand times compared with the natural SIRP alpha/CD 47 affinity by designing different SIRP alpha mutants, takes the high-affinity SIRP alpha as a research object, expresses the high-affinity M-SIRP alpha on the surface of a host cell membrane by a series of genetic engineering means, and then obtains the enhanced novel cell membrane material (CM-M-SIRP alpha) which actively targets and binds with a CD47 ligand on the surface of the tumor cells by adopting a cell membrane extraction technology.
The invention is realized by the following technical scheme:
in a first aspect, the present invention provides a method for preparing a cell membrane material having high affinity for binding to tumor cells CD47, comprising:
performing site-directed mutagenesis on the full-length base sequence of SIRP alpha to obtain the base sequence of mutant M-SIRP alpha;
connecting the base sequence of the fluorescent marker protein to the base sequence of the mutant M-SIRP alpha to obtain a fusion base sequence M-SIRP alpha-F;
constructing a recombinant plasmid containing a fusion base sequence M-SIRP alpha-F by using an expression plasmid;
transfecting the recombinant plasmid into a host cell, and carrying out resistance screening to obtain a recombinant cell capable of stably expressing the mutant M-SIRP alpha on the surface of a cell membrane;
after the recombinant cells are subjected to expansion culture, cell membrane materials containing mutant M-SIRP alpha are extracted.
Further, in a preferred embodiment of the invention, at least 9 amino acids of the mutant M-SIRPalpha are mutated;
preferably, the base sequence of the mutant M-SIRP alpha is shown as SEQ ID NO. 1.
Further, in a preferred embodiment of the present invention, in the above-described construction of the fusion base sequence M-SIRPalpha-F, the base sequence of the fluorescent marker protein is linked to the base sequence of the mutant M-SIRPalpha by a linker;
preferably, the base sequence of the linker is shown in SEQ ID NO. 2.
Further, in a preferred embodiment of the present invention, the fluorescent-labeled protein includes any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.
Preferably, the fluorescent marker protein is red fluorescent protein, and the base sequence of the fusion base sequence M-SIRP alpha-F is shown as SEQ ID NO. 3.
Further, in a preferred embodiment of the present invention, the above-mentioned expression plasmid in the process of constructing a recombinant plasmid includes any one of pCMV3, pCMV6, pcdna3.1 and pgl 4.50;
preferably, the fusion base sequence M-SIRPalpha-F is ligated to pCMV3 at Hind III and Xba I sites.
Further, in a preferred embodiment of the present invention, the host cells described above in the construction of the recombinant cells include any one of HEK293T, CHO, NS0, BHK and PER-C6 cells;
preferably, hygromycin B is used for resistance screening of host cells;
preferably, the concentration of hygromycin B is 380-420. Mu.g/mL.
In a second aspect, the present invention provides a cell membrane material having high affinity for binding to tumor cells CD47, prepared according to the above-described preparation method.
In a third aspect, the present invention provides an application of the cell membrane material having high affinity for binding to tumor cells CD47 in preparing a targeted tumor drug or diagnostic reagent.
In a fourth aspect, the present invention provides a targeted drug delivery system for the treatment or diagnosis of a tumor, the targeted drug delivery system comprising the cell membrane material described above, the tumor being a tumor capable of expressing CD 47.
In a fifth aspect, the present invention provides a tumor-targeted drug, which is characterized by comprising an active drug molecule and a cell membrane material encapsulated outside the active drug molecule, wherein the cell membrane material is prepared by the preparation method.
Compared with the prior art, the invention has at least the following technical effects:
the invention expresses the mutant SIRPalpha on the surface of a host cell membrane by means of genetic engineering, and can obtain a cell membrane material with active targeting binding capacity on a CD47 ligand on the surface of a tumor cell. The cell membrane material is utilized to carry out surface modification on the anti-tumor nano-drug, so that the drug can be delivered to the tumor part with high efficiency; meanwhile, the SIRP alpha receptor on the surface of the macrophage is blocked from being combined with the CD47 ligand by competitively combining with the CD47 ligand on the surface of the tumor cell, so that the anti-tumor activity of the macrophage is restored. Thus, the construction of this novel cell membrane material provides an innovative solution for developing cell membrane materials with targeted binding to tumor cells.
Compared with natural cell membrane materials, the cell membrane material of the SIRPalpha mutant with high affinity expressed on the surface has the following advantages:
1) The invention reforms the cell membrane by genetic engineering technology to obtain a cell membrane material with stronger function than the original natural cell membrane;
2) The invention has simple and convenient post-treatment, can obtain high-quality products, and can realize the amplified operation and industrialization.
Drawings
FIG. 1 shows the effect of MTT assay on HEK293T cell viability at different concentrations of hygromycin B in an embodiment of the invention;
FIG. 2Detecting the expression condition of M-SIRP alpha in a hygromycin-resistant B cell strain by using a) Western blot; b) CLSM observations of M-sirpa expression and localization in HEK 293T-M-sirpa cells (red fluorescent signal:
a protein; green fluorescent signal: WGA Alexa-Fluor 488 for labelling cell membrane dyes; blue fluorescent signal: DAPI, used to label nuclear dye, scale bar: 20 μm);
FIG. 3 shows the detection of different panel PCR products by 1% agarose gel electrophoresis;
FIG. 4 is a) CLSM observations of M-SIRPalpha expression and localization in HEK 293T-M-SIRPalpha cells of different algebra (red fluorescent signal:
a protein; green fluorescent signal: WGA Alexa-Fluor 488 for labelling cell membrane dyes; blue fluorescent signal: DAPI, used to label nuclear dye, scale bar: 10 μm); b) Detecting M-SIRP alpha expression conditions in HEK293T-M-SIRP alpha cells of different algebra in the process of Western blot generation;
FIG. 5 shows the expression of M-SIRP alpha protein on the surface of CM-M-SIRP alpha detected by Western blot;
FIG. 6 shows a) Western blot detection of CD47 expression in 4T1 cells; b) Particle size plot of CMS NPs; c) TEM images of CMS NPs (scale: 500 nm); d) CLSM observation of CMS NPs binding to 4T1 cells and e)
And (5) quantitatively analyzing fluorescence intensity. (Red fluorescent Signal: dsRED protein; green fluorescent Signal: WGA Alexa-Fluor 488 for labeling cell membrane dye; blue fluorescent Signal: DAPI for labeling cell nucleus dye; n=3; scale: 10 μm; and ". Times." represents P value)<0.01)。
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The technical scheme of the invention is as follows:
the embodiment provides a cell membrane material with high affinity for binding to tumor cells CD47, and the preparation method comprises the following steps:
step S1: site-directed mutagenesis is carried out on the full-length base sequence of SIRP alpha to obtain the base sequence of mutant M-SIRP alpha.
As a transmembrane immunoglobulin, research shows that natural SIRPalpha has weaker affinity with CD47, in order to further improve the affinity of SIRPalpha to CD47, in the step, the inventor carries out site-directed mutagenesis on the full-length base sequence of SIRPalpha, and obtains mutant M-SIRPalpha with at least 9 amino acids mutated by designing different SIRPalpha mutants, comparing and optimizing.
Preferably, the mutant M-SIRPalpha is improved by about 5 ten thousand times compared with the natural SIRPalpha/CD 47 affinity, and the base sequence is as follows:
step S2: and connecting the base sequence of the fluorescent marker protein to the base sequence of the mutant M-SIRPalpha to obtain a fusion base sequence M-SIRPalpha-F.
In order to facilitate the subsequent detection and positioning of the mutant M-SIRP alpha expressed on the surface of a host cell, the mutant M-SIRP alpha is marked by a fluorescent marking protein, and in the specific operation process, the C end of the base sequence of the M-SIRP alpha is fused with the base sequence of the fluorescent marking protein through a linker.
Preferably, the base sequence of the linker is: GGGGGTGGAGGCTCT (SEQ ID NO. 2). The linker with the flexible base sequence is favorable for separating two domains of the fusion protein, so that the fusion protein can be fully folded into respective natural conformations under the condition of no interference.
Wherein the fluorescent-labeled protein includes any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.
Preferably, the red fluorescent protein
As fluorescent marker proteins. The red fluorescent protein has the advantages of being capable of being expressed in various organisms, free of cytotoxicity and free of protein aggregation; the excitation and emission wavelength is longer, and the signal to noise ratio is higher; the fluorescent dye has high light stability and pH value stability, has high fluorescence conversion efficiency in cells, and is easier to detect; can be used for detecting deeper tissues and has good application prospect for living organism imaging.
Preferably, the base sequence of the fusion base sequence M-SIRP alpha-F is SEQ ID NO.3:
step S3: constructing recombinant plasmid pCMV3-M-SIRP alpha-F containing fusion base sequence M-SIRP alpha-F by using expression plasmid.
Further, expression plasmids include pCMV3, pCMV6, pcdna3.1, pgl4.50, and the like.
Preferably, the expression plasmid is pCMV3; the pCMV3 plasmid is used as a high copy plasmid, can be transfected into host cells by a transient transfection method, and cell strains which stably express target genes are obtained by hygromycin B screening.
Preferably, the fusion base sequence M-SIRPalpha-F is ligated to pCMV3 at Hind III and Xba I sites.
Step S4: and (3) transfecting the recombinant plasmid into a host cell, and carrying out resistance screening to obtain the recombinant cell capable of stably expressing the mutant M-SIRP alpha on the surface of a cell membrane.
Further, host cells include HEK293T, CHO, NS0, BHK and PER-C6.
Preferably, HEK293T cells are used as host cells, HEK293T is human embryo kidney cells, SV40 antigen is continuously expressed, the cells are commonly used in transfection experiments, and the transfection efficiency is high.
Further, the host cells are subjected to resistance screening using hygromycin B to obtain host cells capable of stably expressing the mutant M-sirpa.
Preferably, HEK293T cells are selected as host cells, and the lowest lethal concentration of obtaining hygromycin B to kill HEK293T cells is between 380 and 420 mug/mL, and the concentration is used as the concentration of hygromycin B in the resistance screening process, preferably the concentration of hygromycin B is 400 mug/mL.
The method comprises the steps of transfecting a linearized recombinant plasmid pCMV 3-M-SIRPalpha-F into HEK293T cells by adopting a transient transfection method, carrying out resistance screening by hygromycin B to obtain a recombinant cell HEK 293T-M-SIRPalpha of the hygromycin B, and inoculating the cell of the hygromycin B into a 96-well plate by adopting a limiting dilution method to obtain monoclonal cells.
Then, observing the expression and positioning of the M-SIRP alpha protein in the obtained monoclonal cells through a laser confocal microscope (CLSM) to obtain a cell strain HEK293T-M-SIRP alpha expressed on the surface of a cell membrane; the fusion base sequence M-SIRP alpha-F is verified to be integrated into the genome of the cell by extracting the genome of HEK293T-M-SIRP alpha cells.
Step S5: after the recombinant cells are subjected to expansion culture, cell membrane materials containing mutant M-SIRP alpha are extracted.
Through a cell subculture method, the recombinant HEK293T-M-SIRP alpha is verified to have the capability of stably expressing the M-SIRP alpha protein.
Then extracting the cell membrane by adopting the following method to obtain a cell membrane material:
1) Crushing cells by using a Dunn homogenizer to obtain homogenate;
2) Subjecting the homogenate to gradient centrifugation to obtain cell membrane material (CM-M-SIRP alpha)
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Examples
This example provides a cell membrane material with high affinity for binding to tumor cells CD47, and a method for preparing the same, comprising:
(1) Construction of recombinant plasmids:
the sequence number (NM_ 001040022.1) of the human SIRP alpha gene is searched by NCBI database, site-directed mutation is carried out on the SIRP alpha base sequence, the base sequence with 9 amino acid position mutation is obtained, the mutated SIRP alpha base sequence is shown as SEQ ID NO.1, and the mutated SIRP alpha amino acid sequence is shown as SEQ ID NO. 4. The C end of the mutated SIRP alpha base sequence is connected with red fluorescent protein through a connecting sequence GGGGGTGGAGGCTCT (SEQ ID NO. 2)
The base sequence is fused, the fused base sequence (M-SIRP alpha-F) is shown as SEQ ID NO.3, and the fused base sequence is completely synthesized by the gene. Finally, the fusion base sequence is connected to an expression plasmid pCMV3, the connection sites are HindIII and Xba I, and the constructed expression plasmid is named pCMV3-M-SIRP alpha-F.
The amino acid sequence of the SIRP alpha after mutation is shown as SEQ ID NO. 4:
(2) Hygromycin B minimum lethal concentration screening of HEK293T cells
HEK293T cells in logarithmic growth phase were collected at 5X 10 5 The cells were seeded in 6-well plates with 5% CO 2 Culturing in a 37 deg.C incubator until the cell growth density is 90%% of the medium was discarded, and fresh medium containing hygromycin B at a concentration of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600. Mu.g/mL was added to each well and culture was continued for 24 hours, and the results were repeated 3 times in parallel for each group, as shown in FIG. 1.
HEK293T viability was measured by MTT and when hygromycin B was at a concentration of 400. Mu.g/mL, HEK293T cells were completely killed, i.e., the concentration was the lowest lethal concentration of hygromycin B to kill HEK293T cells.
(3) Screening of hygromycin-resistant B cell lines
The pCMV3 vector is taken as a constitutive and stable expression plasmid, and hygromycin B can be utilized for screening to obtain a host cell strain for stably expressing target proteins. Firstly, carrying out single enzyme digestion on an expression plasmid pCMV3-M-SIRP alpha-F through an endonuclease Bgl II to obtain a linearized expression plasmid; secondly, transiently transfecting the linearized expression plasmid into HEK293T cells by Lipofectamin 3000 produced by Invitrogen company, and culturing the transfected host cells for 48 hours, and removing the culture medium; then, adding fresh culture medium with the hygromycin B concentration of 400 mug/mL for continuous culture, maintaining the resistance screening for 2 weeks, and changing the fresh culture medium every 2 days to obtain a cell strain with hygromycin B resistance; finally, inoculating the cell strain of the hygromycin B into a 96-well plate for culture by adopting a limiting dilution method, observing a red fluorescent signal of the cell by a fluorescent microscope to obtain a monoclonal cell with the red fluorescent signal, and performing expansion culture on the monoclonal cell.
(4) Western blot detection of M-SIRP alpha expression in hygromycin B cell strain
The cells from the previous step were collected, the cell surface was washed 3 times with sterile PBS buffer, and total protein was extracted by adding RTPA lysate containing the protease inhibitor Cocktail. The BCA protein concentration measuring kit is used for measuring the total protein concentration, SDS-PAGE electrophoresis separation and membrane transfer are carried out, a 5% skimmed milk powder greenhouse shaking table is closed for 1h, and a rabbit anti-human SIRP alpha monoclonal antibody (1:1000 dilution) and a human beta-actin monoclonal antibody (1:1000 dilution) are respectively added, and the temperature is 4 ℃ overnight; TBST (Tunnel boring mill) is used for washing the membrane for 3 times and 5-10 min/time, then secondary antibodies (diluted by 1:10) are respectively added, and the membrane is incubated for 1h at room temperature; TBST washes the membrane for 3 times, 5-10 min/time; development was performed using a chemiluminescent detection kit, the results of which are shown in FIG. 2 a.
As can be seen from FIG. 2a, the M-SIRP alpha protein is expressed in the hygromycin-resistant B cell line. From this, it was demonstrated that a cell line expressing M-SIRPalpha (HEK 293T-M-SIRPalpha) was obtained.
(5) Analysis of M-SIRP alpha expression site in HEK293T-M-SIRP alpha cells
Collecting the above cells in logarithmic growth phase at 1×10 4 Inoculating the cells/well into 6-well plate with cell climbing plate, 5% CO 2 Culturing in an incubator at 37 ℃ for 48 hours. Removing the culture medium, and taking 1mL of sterile PBS to clean the cell surface for 3 times; 4% paraformaldehyde fixed cells, sterile PBS and cell surface wash 3 times; then the cell membrane is stained by WGA Alexa-Fluor 488 dye for 10min, and the cell surface is washed for 3 times by sterile PBS; finally, nuclear membrane staining is carried out for 10min by utilizing DAPI, and the cell surface is washed for 3 times by using sterile PBS; the cell slide was removed, the cell surface was covered with a slide glass to which fluorescence quenching was applied, and a photograph was taken under a confocal laser microscope (CLSM), and the result is shown in fig. 2 b.
As can be seen from FIG. 2b, the M-SIRP alpha protein in HEK293T-M-SIRP alpha cells is localized to be expressed on the surface of the cell membrane. Therefore, the cell strain HEK293T-M-SIRP alpha which expresses the M-SIRP alpha protein and is positioned and expressed on the surface of a cell membrane is obtained through screening by the method.
(6) Genome extraction
Collecting the above cells in logarithmic growth phase at 1×10 7 Inoculating the cells/well into 6-well plate with cell climbing plate, 5% CO 2 Culturing in an incubator at 37 ℃ for 48 hours. Extracting genome of HEK293T-M-SIRP alpha by using a root genome extraction kit, and designing a full-length primer:
forward primer: ATGGAGCCCGCCGGCCCGGC (SEQ ID NO. 5);
reverse primer: TTACTGGAACAGGTGGTGGCGGCCCTCG (SEQ ID NO. 6);
the full length sequence of M-SIRP alpha-F was cloned (conditions: denaturation temperature: 95 ℃, denaturation time: 10s, annealing temperature: 63 ℃, annealing time: 20s, extension temperature: 72 ℃, extension time: 1.5min, number of cycles: 35), and the results are shown in FIG. 3.
From this, it was confirmed that the fusion nucleotide sequence M-SIRPa-F was integrated into the genome of the cells by extracting the genome of HEK 293T-M-SIRPa cells.
(7) Cell stability expression investigation
To examine the ability of HEK293T-M-SIRP alpha cells to stably express the M-SIRP alpha protein, the expression of the M-SIRP alpha in the cells was detected every 5 th generation by a fluorescence confocal microscope and Western blot by a cell subculture method, and the 20 th generation was detected in total.
As shown in FIG. 4, HEK293T-M-SIRP alpha cells can stably express the M-SIRP alpha protein in the 20 th generation cells after subculturing. Thus, HEK293T-M-SIRP alpha cells have the capacity of stably expressing M-SIRP alpha protein.
(8) HEK293T-M-SIRP alpha cell membrane extraction
Culturing and collecting 1×10 10 HEK293T-M-SIRP alpha cells are collected by centrifugation, 10mL of sterile PBS is used for cleaning the cell surface, the rotation speed is 500 Xg, and the cleaning is repeated for 3 times; taking 5mL of homogenate (HM buffer:0.25M sucrose, 1mM EDTA, 20mM Hepes, pH 7.4, additionally adding protease inhibitor) to resuspend cells, and crushing the cells on ice by using a Dunn homogenizer (Dounce Homogenizer) for 100 times to finally obtain homogenate; centrifuging the homogenate for 5min at 4deg.C at a rotation speed of 1,000Xg, and collecting supernatant; centrifuging the supernatant at ultra-high speed for 30min at 4deg.C and at a rotation speed of 10,000Xg, and collecting supernatant; centrifuging the supernatant at ultrahigh speed for 1h at 4 ℃ at a rotating speed of 100,000Xg, and collecting precipitate; resuspension the precipitate with sterile pre-cooled PBS, ultra-high speed centrifugation for 1h at 4deg.C at 100,000Xg, repeating the steps for 2 times, and collecting the final precipitate to obtain cell membrane material (CM-M-SIRP alpha). The content of M-SIRP alpha in the cell membrane material is detected as follows: 5460ng of the total protein was contained per 1.0 mg.
(9) Investigation of M-SIRP alpha protein in cell membrane material
Detecting the expression condition of the M-SIRP alpha protein on the surface of the CM-M-SIRP alpha by a Western blot method, and the method is the same as that of (4).
The results are shown in FIG. 5, where it can be seen that the M-SIRPalpha protein was detected in the CM-M-SIRPalpha group. Thus, the CM-M-SIRPalpha is expressed with the M-SIRPalpha protein.
(10) Investigation of cell membrane Material and tumor cell binding Condition
To examine the binding of cell membrane material to tumor cells, three negative breast cancer 4T1 cells of mice were selected as a study model.
First, the 4T1 cells were examined for CD47 expression, and Western blot analysis revealed that the 4T1 cells expressed the CD47 receptor, as shown in FIG. 6 a.
Then, the cell membrane material CM-SIRPalpha was prepared into nanoparticles (CMS NPs) by the following procedure: the obtained CM-SIRP alpha is dissolved in sterile precooled PBS solution, and is subjected to ice bath ultrasonic treatment for 3min, wherein the power is 80W. The ultrasonic sample was subjected to physical extrusion to obtain CMS NPs solution through 0.80 μm and 0.22 μm filters, and the particle size and morphology of the cell membrane nanoparticles were observed by a particle sizer and TEM, as shown in FIGS. 6b and 6c, to obtain CMS NPs with an average particle size of 164.4 nm.
Cell binding assays CMS NPs were tested for affinity to 4T1 cells, set up in the following experimental panel:
a) A PBS group;
b) CMS NPs group;
c) CD47 primary antibody + CMS NPs group (CD 47 primary antibody was added 4h before nanoparticle addition).
Taking 4T1 cells in logarithmic growth phase at 5×10 4 Inoculating the cells/wells into confocal dishes, respectively, 5% CO 2 Culturing in an incubator at 37 ℃ for 24 hours. Washing cells for 3 times by using sterile PBS, adding corresponding nanoparticle solutions according to different groups of treatment modes (quantitative analysis of protein content in the nanoparticle solutions is carried out by using a BCA method, the added protein concentration is 500 mug/mL), and gently shaking for 4 hours at room temperature; removing the supernatant, washing 3 times with sterile PBS, and staining cell membranes with WGA Alexa-Fluor 488 dye for 10 minutes; washing 3 times with sterile PBS, and performing nuclear membrane staining for 10min by using DAPI; the cells were washed 3 times with sterile PBS, photographed under a laser confocal microscope, and photographed.
As shown in fig. 6d and 6e, the binding of nanoparticles to 4T1 cells was very evident in the CMS NPs group, whereas the binding of nanoparticles to 4T1 cells was significantly reduced in the CD47 primary antibody + CMS NP group. The fluorescence intensity of the CD47 primary antibody + CMS NP group was only 12.8% of that of the CMS NPs group by fluorescent intensity quantification. Because CD47 primary antibody binds to CD47 on the surface of 4T1 cells, the binding of nanoparticles to 4T1 cells is hindered. Taken together, CMS NPs can bind efficiently to 4T1 cells via surface M-SIRPalpha.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing a cell membrane material having high affinity for binding to tumor cells CD47, comprising:
performing site-directed mutagenesis on the full-length base sequence of SIRP alpha to obtain the base sequence of mutant M-SIRP alpha;
connecting the base sequence of the fluorescent marker protein to the base sequence of the mutant M-SIRPa to obtain a fusion base sequence M-SIRPa-F;
constructing a recombinant plasmid containing the fusion base sequence M-SIRP alpha-F by using an expression plasmid;
transfecting the recombinant plasmid into a host cell, and carrying out resistance screening to obtain a recombinant cell capable of stably expressing the mutant M-SIRP alpha on the surface of a cell membrane;
and extracting cell membrane materials containing mutant M-SIRP alpha after the recombinant cells are subjected to expansion culture.
2. The method of claim 1, wherein the mutant M-sirpa has a base mutation at least 9 amino acid positions;
preferably, the base sequence of the mutant M-SIRP alpha is shown as SEQ ID NO. 1.
3. The method for preparing a cell membrane material having high affinity binding to tumor cell CD47 according to claim 1, wherein in constructing the fusion base sequence M-sirpa-F, the base sequence of the fluorescent marker protein is linked to the base sequence of the mutant M-sirpa by linker;
preferably, the base sequence of the linker is shown as SEQ ID NO. 2.
4. The method for preparing a cell membrane material having high affinity binding to tumor cells CD47 according to claim 3, wherein the fluorescent marker protein comprises any one of red fluorescent protein, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein and yellow fluorescent protein.
Preferably, the fluorescent marker protein is red fluorescent protein, and the base sequence of the fusion base sequence M-SIRP alpha-F is shown in SEQ ID NO. 3.
5. The method for preparing a cell membrane material having high affinity binding to tumor cell CD47 according to claim 1, wherein in constructing the recombinant plasmid, the expression plasmid comprises any one of pCMV3, pCMV6, pcdna3.1 and pgl 4.50;
preferably, the fusion base sequence M-SIRPalpha-F is ligated to pCMV3 at Hind III and Xba I sites.
6. The method of claim 1, wherein in constructing the recombinant cell, the host cell comprises any one of HEK293T, CHO, NS0, BHK and PER-C6 cells;
preferably, the host cells are subjected to a resistance selection using hygromycin B;
preferably, the concentration of hygromycin B is 380-420 mug/mL.
7. A cell membrane material having high affinity for binding to tumor cells CD47, prepared according to the preparation method of any one of claims 1 to 6.
8. Use of a cell membrane material having a high affinity for binding to tumor cells CD47 according to claim 7 for the preparation of a targeted tumor drug or diagnostic agent.
9. A targeted drug delivery vehicle for the treatment or diagnosis of a tumor, characterized in that it comprises nanoparticles prepared from the cell membrane material of claim 7, said tumor being a tumor expressing said CD 47.
10. A targeted tumor drug, characterized in that it comprises an active drug molecule and a cell membrane material encapsulated outside the active drug molecule, said cell membrane material being prepared according to the preparation method of any one of claims 1 to 6.
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