Bionic magnetic bead for capturing circulating tumor cells in vivo, preparation method and application
Targeted bionic magnetic beads are constructed through genetic engineering and synthetic biology technology, which solves the problem of low capture efficiency in vitro CTCs, and achieves efficient and highly specific CTCs capture and isolation, which improves the sensitivity of early cancer detection.
Patent Information
- Application Number
- CN202411654709.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-03-04
AI Technical Summary
The prior art methods for capturing circulating tumor cells (CTCs) in vitro have problems such as low detection throughput, insufficient capture, easy equipment blockage and lack of specificity, which is difficult to meet the needs of early cancer detection.
Through genetic engineering and synthetic biology technology, bionic magnetic beads targeting epithelial and interstitial phenotype CTCs are constructed, and magnetic bodies are naturally synthesized by microorganisms and stable expression of fusion protease enzyme cleavage sites to combine specific antibodies and proteins to achieve efficient capture and isolation of CTCs.
It realizes efficient capture of heterogeneous CTCs in the flowing blood of the human body, improves the capture rate and sensitivity, and through the use of specific protease cleavage sites, it can be separated gently and orderly to avoid damage.
Smart Images
Figure CN119552851A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of immunomagnetic bead preparation, and in particular to a bionic magnetic bead for capturing circulating tumor cells in vivo, a preparation method and an application thereof. Background Art
[0002] Capturing and analyzing circulating tumor cells (CTCs) is of great significance for tumor research. It can reduce the tumor burden of tumor patients, delay tumor metastasis, detect tumors early, monitor tumor recurrence, evaluate drug efficacy, and guide treatment. The capture mechanisms of CTCs are divided into the following categories: based on physical properties (size, charge, density, elasticity, etc.), based on affinity recognition, and based on various nanostructures. At present, there are many methods for capturing and separating CTCs in vitro. In vitro blood testing is the most common method, but the blood samples tested are small, accounting for only 0.15% of the total blood. At the same time, the proportion of CTCs in blood samples is extremely small (<3%), resulting in a significantly insufficient amount of CTCs captured in vitro and low detection sensitivity, which is difficult to meet the needs of early cancer detection.
[0003] In recent years, a variety of methods for capturing and enriching CTCs in vivo have been developed, which to some extent solve the problem of low throughput of CTCs detection. Among these methods, label-free enrichment methods based on physical characteristics can enrich many subtypes of CTCs and increase the amount of CTCs captured, but this method still has problems such as easy clogging of filter membranes or micropores, loss of small CTCs, the need for pretreatment steps such as centrifugation and dilution, and lack of sufficient specificity in isolated cells; CTCs capture methods based on biological affinity strategies have strong specificity, but there are few methods for capturing CTCs in flowing blood, and there is a limitation that heterogeneous CTCs cannot be captured. Summary of the invention
[0004] In view of this, the purpose of this application is to provide a biomimetic magnetic bead for capturing circulating tumor cells in vivo, a preparation method and an application. The preparation method of the biomimetic magnetic bead is based on genetic engineering and synthetic biology technology, and uses naturally synthesized magnetosomes of microorganisms and eukaryotic cell membranes that stably express fusion protease cleavage sites, connecting peptides, and CTCs epithelial phenotype markers (such as EpCAM, CK, E-Cadherin) and CTCs mesenchymal phenotype markers (such as Vimentin, N-Cadherin, Twist) as raw materials to construct magnetic beads targeting mesenchymal phenotype / epithelial phenotype CTCs. The prepared biomimetic magnetic beads can capture heterogeneous CTCs by affinity adsorption with different targeting properties, and the captured CTCs are gently and orderly separated from the biomimetic magnetic beads through the specific protease cleavage sites on the biomimetic magnetic beads, thereby releasing CTCs without loss.
[0005] In order to achieve or at least partially achieve the above objectives, the present application provides the following technical solutions: In a first aspect, the present application provides a biomimetic magnetic bead for capturing circulating tumor cells in vivo, wherein the biomimetic magnetic bead comprises at least one of a first biomimetic magnetic bead targeting epithelial phenotype circulating tumor cells and a second biomimetic magnetic bead targeting mesenchymal phenotype circulating tumor cells; wherein, The first biomimetic magnetic bead is formed by a first targeted cell membrane coated with magnetic particles, and the first targeted cell membrane comprises an anti-EpCAM antibody having an amino acid sequence as shown in SEQ ID NO: 1; The second bionic magnetic beads are formed by second targeted cell membrane-coated magnetic particles, and the second targeted cell membrane comprises a NEFL protein having an amino acid sequence as shown in SEQ ID NO:2.
[0006] In a second aspect, the present application provides an anti-EpCAM antibody gene recombinant plasmid, which includes a CMV promoter, a signal peptide gene sequence, an anti-EpCAM antibody gene fragment, a TEV protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and an eGFP green fluorescent protein gene sequence connected in sequence; wherein the nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5, the signal peptide gene sequence is shown in SEQ ID NO:6, the nucleotide sequence of the anti-EpCAM antibody gene fragment is shown in SEQ ID NO:7, the TEV protease cleavage site gene sequence is shown in SEQ ID NO:8, the first connexin gene sequence is shown in SEQ ID NO:9, the transmembrane protein gene sequence is shown in SEQ ID NO:10, the second connexin gene sequence is shown in SEQ ID NO:11, and the eGFP green fluorescent protein gene sequence is shown in SEQ ID NO:12.
[0007] In a third aspect, the present application provides a NEFL protein gene recombinant plasmid, which comprises a CMV promoter, a signal peptide gene sequence, a NEFL protein gene fragment, an HRV 3C protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and a MCHERRY fluorescent protein gene sequence connected in sequence; wherein the nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5, the signal peptide gene sequence is shown in SEQ ID NO:6, the nucleotide sequence of the NEFL protein gene fragment is shown in SEQ ID NO:13, the HRV 3C protease cleavage site gene sequence is shown in SEQ ID NO:14, the first connexin gene sequence is shown in SEQ ID NO:9, the transmembrane protein gene sequence is shown in SEQ ID NO:10, the second connexin gene sequence is shown in SEQ ID NO:11, and the MCHERRY red fluorescent protein gene sequence is shown in SEQ ID NO:15.
[0008] In a fourth aspect, the present application provides an engineered cell, wherein the engineered cell comprises an anti-EpCAM antibody gene recombinant plasmid or a NEFL protein gene recombinant plasmid.
[0009] In a fifth aspect, the present application provides a method for preparing biomimetic magnetic beads, the preparation method comprising any one of (A) to (C): (A) A method for preparing a first biomimetic magnetic bead targeting epithelial phenotype circulating tumor cells, comprising the steps of: (1) Transforming the anti-EpCAM antibody gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express anti-EpCAM antibodies; collecting and lysing the engineered cells that highly express anti-EpCAM antibodies, and isolating and obtaining a suspension of cell membranes targeting epithelial phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) The cell membrane suspension of the epithelial phenotype circulating tumor cells obtained in (1) is mixed with the magnetic microparticles obtained in (2). The first biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells were obtained by mixing the two. (B) A method for preparing a second biomimetic magnetic bead targeting mesenchymal phenotype circulating tumor cells, comprising the steps of: (1) Transforming the NEFL protein gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express NEFL protein; collecting and lysing the engineered cells that highly express NEFL protein, and isolating and obtaining a suspension of cell membranes targeting mesenchymal phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) mixing the suspension of cell membranes of circulating tumor cells with mesenchymal phenotype obtained in (1) with the magnetosomes obtained in (2) Mixing is performed to obtain second biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells; (C) A method for preparing a third biomimetic magnetic bead that simultaneously targets epithelial phenotype circulating tumor cells and mesenchymal phenotype circulating tumor cells, comprising the steps of: The first biomimetic magnetic beads prepared in (A) and the second biomimetic magnetic beads prepared in (B) are mixed.
[0010] In a sixth aspect, the present application provides the use of the aforementioned bionic magnetic beads to capture heterogeneous circulating tumor cells in vivo.
[0011] Compared with the prior art, the technical solution of this application has at least the following beneficial effects: 1. This application uses genetic engineering to obtain a large number of eukaryotic cells that express anti-EpCAM antibodies and NEFL proteins that bind to Vimentin, providing a basis for large-scale batch production of targeted bionic magnetic beads while avoiding the shortcomings of targeted antibody inactivation and low coupling efficiency caused by traditional chemical coupling methods.
[0012] 2. The bionic magnetic beads provided in this application can achieve a higher capture rate of circulating tumor cells in human blood.
[0013] 3. The specific protease cleavage sites on the biomimetic magnetic beads of the present application achieve gentle and orderly separation of captured CTCs from the biomimetic magnetic beads, thereby releasing CTCs without loss.
[0014] 4. The microvesicles and magnetosomes used in this application are both biogenic materials with the advantages of low immunogenicity and good biocompatibility. The magnetic bead assembly steps are relatively simple and can be easily applied to large-scale and efficient engineering production. 5. The magnetosomes used in this application are natural fermentation products of microorganisms, and have the advantages of low production cost, stable structure, and simple purification. They also have superparamagnetism and high coercivity, which makes the adsorption of CTCs stable and not easy to fall off when combined with microfluidic technology for in vivo detection, thereby improving the sensitivity of in vivo detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a schematic diagram of the construction structure of the anti-EpCAM antibody gene recombinant plasmid provided in the examples of the present application.
[0016] Figure 2 This is a schematic diagram of the construction structure of the NEFL protein gene recombinant plasmid provided in the examples of the present application.
[0017] Figure 3 Transmission electron microscopy images of the suspension of biomimetic magnetic beads provided in the embodiments of the present application; wherein A is a transmission electron microscopy image of the suspension M1 of biomimetic magnetic beads prepared for targeting epithelial phenotype CTCs, and B is a transmission electron microscopy image of the suspension M2 of biomimetic magnetic beads prepared for targeting mesenchymal phenotype CTCs.
[0018] Figure 4 The results of anti-EpCAM antibody / NEFL protein expression in THP-1 engineered cells, targeted cell membranes, and biomimetic magnetic beads were detected by western blot; A is the anti-EpCAM antibody expression result, and B is the NEFL protein expression result. DETAILED DESCRIPTION
[0019] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0020] Those skilled in the art will appreciate that, unless otherwise stated, the terms "said", "the", "the aforementioned", and "above" used in the text of the present application may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.
[0021] In the text of this application, the terms "first", "second" and the like are used to distinguish between identical or similar items with substantially the same functions and effects. This is only for the purpose of clearly describing the technical solutions of the embodiments of this application, and shall not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
[0022] Those skilled in the art will appreciate that the experimental methods in the examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
[0023] In the text of this application, the term "EpCAM" refers to epithelial cell adhesion molecule, also known as CD326, which is a single-channel type I plasma membrane glycoprotein; the term "Vimentin" is an intermediate filament protein, mainly expressed in mesenchymal cells; the term "NEFL" refers to light peptide neurofilament protein, which can bind to "Vimentin"; the terms "TEV protease" and "HRV 3C protease" are both commonly used tool enzymes used to remove affinity tags on fusion proteins, and have strong site specificity, among which "TEV protease" is the full name of tobacco etch virus protease, and the recognition site is "ENLYFQ↓(G / S)"; "HRV 3C protease" is a recombinant enzyme, and the recognition site is "LE(V / A / T)LFQ↓GP".
[0024] In the text of this application, the term "magnetic particles" refers to the magnetic material inside the magnetic bead structure, and its types include but are not limited to at least one of the following types: Fe3O4, Fe3S4, γ-Fe2O3, ferrite, etc. When an external magnetic field acts on the magnetic beads, the "magnetic particles" inside the magnetic beads will respond to the magnetic field, thereby making the magnetic beads magnetic. By utilizing the magnetic properties of the "magnetic particles", the magnetic beads can be controlled by an external magnetic field, including separation, mixing, and transportation. The term "magnetosome" is a cell organelle unique to magnetotactic bacteria. They are magnetic nanoparticles composed of magnetite crystals coated with biofilms, and their main components are Fe3O4 and Fe3S4. Therefore, "magnetosomes" can be used as a type of magnetic particles.
[0025] The embodiment of the present application is based on genetic engineering and synthetic biology technology, and uses naturally synthesized magnetosomes of microorganisms and eukaryotic cell membranes that stably express fusion protease cleavage sites, connecting peptides and anti-EpCAM antibodies and NEFL proteins that bind to Vimentin as raw materials to construct magnetic beads targeting epithelial phenotype and mesenchymal phenotype CTCs. The prepared biomimetic magnetic beads can use different targeting and affinity adsorption to capture heterogeneous CTCs. The captured CTCs are gently and orderly separated from the biomimetic magnetic beads through specific protease cleavage sites on the biomimetic magnetic beads, thereby releasing CTCs without loss. The embodiment of the present application uses synthetic biology to produce targeted antibodies on cell membranes, avoiding the multi-step, unstable coupling efficiency and high antibody use cost of traditional chemical coupling methods. At the same time, the naturally synthesized magnetosomes are single magnetic domain nanoparticles with uniform size, superparamagnetism and good biocompatibility, and have high yield and simple preparation methods. The CTCs recovered by the biomimetic magnetic beads prepared in the examples of the present application have high integrity and activity, and can realize the classification and collection of CTCs mesenchymal phenotype / epithelial phenotype subtypes, and can be used for subsequent bioinformatics analysis, culture and drug sensitivity experiments, etc.
[0026] Based on this, an embodiment of the present application provides a bionic magnetic bead for capturing circulating tumor cells in vivo, the bionic magnetic beads comprising at least one of a first bionic magnetic bead targeting epithelial phenotype circulating tumor cells and a second bionic magnetic bead targeting mesenchymal phenotype circulating tumor cells; wherein the first bionic magnetic bead is formed by a first targeted cell membrane-coated magnetic particle, and the first targeted cell membrane comprises an anti-EpCAM antibody having an amino acid sequence as shown in SEQ ID NO:1; the second bionic magnetic bead is formed by a second targeted cell membrane-coated magnetic particle, and the second targeted cell membrane comprises an amino acid sequence as shown in SEQ ID NO:2 as a NEFL protein.
[0027] There are multiple combinations of the above-mentioned biomimetic magnetic beads for capturing circulating tumor cells in vivo. The first combination is a first biomimetic magnetic bead that only contains epithelial phenotype CTCs. The first biomimetic magnetic bead can bind to the epithelial phenotype marker of CTCs, namely, epithelial cell adhesion molecule (EpCAM), through the anti-EpCAM antibody expressed on the first targeted cell membrane, thereby achieving the purpose of capturing epithelial phenotype CTCs; the second combination is a second biomimetic magnetic bead that only contains a mesenchymal phenotype CTCs. The second biomimetic magnetic bead binds to the mesenchymal phenotype marker of CTCs, namely, Vimentin, through the NEFL protein expressed on the second targeted cell membrane, thereby achieving the purpose of capturing mesenchymal phenotype CTCs; the third combination is a combination of the above-mentioned first biomimetic magnetic bead and the second biomimetic magnetic bead, thereby achieving the purpose of simultaneously capturing epithelial phenotype CTCs and mesenchymal phenotype CTCs. To achieve the capture and separation extraction of heterogeneous CTCs (epithelial phenotype CTCs and mesenchymal phenotype CTCs), the first combination and the second combination can be used to extract epithelial phenotype CTCs and mesenchymal phenotype CTCs respectively, or the third combination, that is, the mixed magnetic beads of the first bionic magnetic beads and the second bionic magnetic beads, can be used to simultaneously extract epithelial phenotype CTCs and mesenchymal phenotype CTCs, so as to achieve the purpose of capturing heterogeneous CTCs. The capture and separation of the two phenotypes of CTCs do not interfere with each other in the same sample system.
[0028] In certain embodiments, when the biomimetic magnetic beads include first biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells, the first targeted cell membrane further includes a TEV protease cleavage site with an amino acid sequence as shown in SEQ ID NO: 3; when the biomimetic magnetic beads include second biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells, the second targeted cell membrane further includes an HRV 3C protease cleavage site with an amino acid sequence as shown in SEQ ID NO: 4. Through the specific protease cleavage sites expressed on the targeted cell membranes of the biomimetic magnetic beads, the captured epithelial phenotype CTCs and mesenchymal phenotype CTCs can be removed by tobacco etch virus (TEV) protease and recombinantly expressed human 3C (HRV 3C) protease, respectively, to achieve separation between CTCs and biomimetic magnetic beads.
[0029] In certain embodiments, the magnetic particles are magnetosomes.
[0030] Based on this, the present application embodiment provides an anti-EpCAM antibody gene recombinant plasmid, such as Figure 1 As shown, the anti-EpCAM antibody gene recombinant plasmid includes a CMV promoter, a signal peptide gene sequence, an anti-EpCAM antibody gene fragment, a TEV protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and an eGFP green fluorescent protein gene sequence connected in sequence; wherein the nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5, the signal peptide gene sequence is shown in SEQ ID NO:6, the nucleotide sequence of the anti-EpCAM antibody gene fragment is shown in SEQ ID NO:7, the TEV protease cleavage site gene sequence is shown in SEQ ID NO:8, the first connexin gene sequence is shown in SEQ ID NO:9, the transmembrane protein gene sequence is shown in SEQ ID NO:10, the second connexin gene sequence is shown in SEQ ID NO:11, and the eGFP green fluorescent protein gene sequence is shown in SEQ ID NO:12.
[0031] Based on this, the present application embodiment provides a NEFL protein gene recombinant plasmid, such as Figure 2 As shown, the NEFL protein gene recombinant plasmid includes a CMV promoter, a signal peptide gene sequence, a NEFL protein gene fragment, an HRV3C protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and a MCHERRY fluorescent protein gene sequence connected in sequence; wherein the nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5, the signal peptide gene sequence is shown in SEQ ID NO:6, the nucleotide sequence of the NEFL protein gene fragment is shown in SEQ ID NO:13, the HRV 3C protease cleavage site gene sequence is shown in SEQ ID NO:14, the first connexin gene sequence is shown in SEQ ID NO:9, the transmembrane protein gene sequence is shown in SEQ ID NO:10, the second connexin gene sequence is shown in SEQ ID NO:11, and the MCHERRY red fluorescent protein gene sequence is shown in SEQ ID NO:15.
[0032] The above-mentioned anti-EpCAM antibody gene recombinant plasmid and NEFL protein gene recombinant plasmid can be used to prepare engineered cells respectively. The cell membrane of the engineered cells is the targeted cell membrane used to prepare the above-mentioned bionic magnetic beads. The preparation of bionic magnetic beads is achieved by coating magnetic particles with the targeted cell membrane.
[0033] Based on this, an embodiment of the present application provides an engineered cell, wherein the engineered cell includes an anti-EpCAM antibody gene recombinant plasmid or a NEFL protein gene recombinant plasmid.
[0034] The preparation principle of the above-mentioned engineered cells is as follows: the exogenous gene, that is, the gene sequence of the above-mentioned recombinant plasmid, is effectively inserted into the genome of the human cell line using a lentiviral vector, thereby forming a recombinant cell line with the function of expressing the exogenous gene, that is, the engineered cell.
[0035] Based on this, the present application embodiment provides a method for preparing biomimetic magnetic beads, the preparation method comprising at least one of (A) to (C): (A) A method for preparing a first biomimetic magnetic bead targeting epithelial phenotype circulating tumor cells, comprising the steps of: (1) Transforming the anti-EpCAM antibody gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express anti-EpCAM antibodies; collecting and lysing the engineered cells that highly express anti-EpCAM antibodies, and isolating and obtaining a suspension of cell membranes targeting epithelial phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) mixing the suspension of cell membranes of circulating tumor cells with epithelial phenotype obtained in (1) with the magnetosomes obtained in (2) Perform hybrid ultrasound to obtain the first biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells; (B) A method for preparing a second biomimetic magnetic bead targeting mesenchymal phenotype circulating tumor cells, comprising the steps of: (1) Transforming the NEFL protein gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express NEFL protein; collecting and lysing the engineered cells that highly express NEFL protein, and isolating and obtaining a suspension of cell membranes targeting mesenchymal phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) mixing the suspension of cell membranes of circulating tumor cells with mesenchymal phenotype obtained in (1) with the magnetosomes obtained in (2) Performing hybrid ultrasound to obtain a second biomimetic magnetic bead targeting mesenchymal phenotype circulating tumor cells; (C) A method for preparing a third magnetic bead that simultaneously targets epithelial phenotype circulating tumor cells and mesenchymal phenotype circulating tumor cells, comprising the steps of: The first biomimetic magnetic beads prepared in (A) and the second biomimetic magnetic beads prepared in (B) are mixed.
[0036] The above-mentioned preparation methods include three types. The type of magnetic beads to be prepared is determined according to the type of CTCs to be captured and separated, so as to select the corresponding preparation method. When the biomimetic magnetic beads are first biomimetic magnetic beads, method (A) is used for preparation; when the biomimetic magnetic beads are second biomimetic magnetic beads, method (B) is used for preparation; when the biomimetic magnetic beads include both the first biomimetic magnetic beads and the second biomimetic magnetic beads, method (C) is used for preparation, that is, the first biomimetic magnetic beads and the second biomimetic magnetic beads are prepared separately and then mixed to form the third biomimetic magnetic beads.
[0037] Based on this, the embodiments of the present application provide the use of the aforementioned bionic magnetic beads to capture heterogeneous circulating tumor cells in vivo.
[0038] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.
[0039] Preparation of biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells This embodiment provides a method for preparing biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells, and the specific steps are as follows: (1) Preparation of suspension targeting epithelial phenotype circulating tumor cells: Genetic engineering was used to obtain stably transformed engineered cells through lentiviral vector infection technology. The recombinant plasmid (anti-EpCAM antibody gene recombinant plasmid) fused with anti-EpCAM antibody gene fragment, TEV protease cleavage site gene sequence, membrane localization signal peptide gene sequence and eGFP green fluorescent protein gene sequence was cloned and inserted into the human monocytic THP-1 genome. The stably transformed THP-1 cells were proliferated to obtain a large number of engineered cells that stably expressed anti-EpCAM monoclonal antibodies on the cell membrane. The engineered cells were collected, washed and resuspended with 1× PBS buffer, and the detergent sodium deoxycholate was added to a final concentration of 30 mM. The cells were treated with low-frequency ultrasound (ultrasound parameters were set as follows: power of 30 W, 2 seconds per ultrasound, 2 seconds between two ultrasounds, and ultrasound treatment time of 5 minutes). The suspension after ultrasound treatment was collected and centrifuged at 4°C and 10,000 g for 30 minutes. The supernatant was collected to remove large cell debris and organelles in the precipitate. The collected supernatant was centrifuged at 4 Centrifuge at 100,000 g for 40 minutes at ℃, remove the supernatant, rinse the precipitate 2-3 times with 1× PBS buffer, resuspend the precipitate, and enrich the suspension of cell membranes targeting epithelial phenotype circulating tumor cells; (2) Preparation of magnetosomes: The cultured magnetotactic bacteria suspension was centrifuged at 8000 g for 5 minutes, the magnetotactic bacteria cells were collected, and the cells were washed twice with 1× PBS buffer, and the cells were resuspended at a ratio of cell wet weight (g): buffer volume (mL) = 1:20, and the magnetotactic bacteria were disrupted by ultrasound (ultrasonic parameters were 200 W, each ultrasound for 3 seconds, and an interval of 5 seconds between two ultrasounds). After ultrasound disruption for 30 minutes, the disrupted magnetotactic bacteria cell suspension was placed on a magnetic rack to adsorb the magnetosomes, and the liquid portion was discarded to remove cell debris. The separated magnetosomes were rinsed with 1× PBS to obtain a magnetosome suspension, and then the magnetosome suspension was treated with low-frequency ultrasound at an ultrasound parameter of 20 W, each ultrasound for 2 seconds, and an interval of 3 seconds between two ultrasounds, for a total of 6 minutes. The magnetic rack adsorption, rinsing and ultrasound treatment were repeated 8 to 10 times, and the OD280 nm was measured until it was less than 0.05. The suspension was freeze-dried overnight to obtain purified magnetosomes. (3) Preparation of biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells: Take the suspension of cell membranes of epithelial phenotype circulating tumor cells collected in step (1) and mix it evenly with the magnetic bodies prepared in step (2) at a ratio of 3:1 (w / w); perform low-frequency ultrasonic treatment (ultrasonic parameters are 20 W, each ultrasonic treatment is 2 seconds, and the interval between two ultrasonic treatments is 3 seconds) for 10 minutes to obtain a biomimetic magnetic bead suspension, place the biomimetic magnetic bead suspension after ultrasonic treatment on a magnetic rack, adsorb the biomimetic magnetic beads, discard the supernatant, rinse the magnetic beads with 1× PBS, repeat this step 3 to 5 times, and finally obtain a suspension M1 of biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells.
[0040] Preparation of biomimetic magnetic beads targeting circulating tumor cells with mesenchymal phenotype This embodiment provides a method for preparing biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells, and the specific steps are as follows: (1) Preparation of suspension targeting the cell membrane of circulating tumor cells with mesenchymal phenotype: Genetic engineering was used to obtain stably transformed engineered cells through lentiviral vector infection technology. The recombinant plasmid fused with NEFL protein gene fragment, HRV 3C protease cleavage site gene sequence, membrane localization signal peptide gene sequence and MCHERRY red fluorescent protein gene sequence (NEFL protein gene recombinant plasmid) was cloned and inserted into the human monocytic THP-1 genome. The stably transformed THP-1 cells were proliferated to obtain a large number of engineered cells that stably expressed NEFL protein on the cell membrane. The engineered cells were collected, washed and resuspended with 1× PBS buffer, and the detergent sodium deoxycholate was added to a final concentration of 30 mM. The cells were treated with low-frequency ultrasound (ultrasound parameters were set as follows: power of 30 W, 2 seconds per ultrasound, 2 seconds between two ultrasounds, and ultrasound treatment time of 5 minutes). The suspension after ultrasound treatment was collected and centrifuged at 4°C and 10,000 g for 30 minutes. The supernatant was collected to remove large cell debris and organelles in the precipitate. The collected supernatant was centrifuged at 4 The mixture was centrifuged at 100,000 g for 40 minutes at ℃, the supernatant was removed, the precipitate was rinsed 2-3 times with 1× PBS buffer, and the precipitate was resuspended to enrich the suspension of cell membranes targeting mesenchymal phenotype circulating tumor cells; (2) Preparation of magnetosomes: The cultured magnetotactic bacteria suspension was centrifuged at 8000 g for 5 minutes, the magnetotactic bacteria cells were collected, and the cells were washed twice with 1× PBS buffer, and the cells were resuspended at a ratio of cell wet weight (g): buffer volume (mL) = 1:20, and the magnetotactic bacteria were disrupted by ultrasound (ultrasonic parameters were 200 W, each ultrasound for 3 seconds, and an interval of 5 seconds between two ultrasounds). After ultrasound disruption for 30 minutes, the disrupted magnetotactic bacteria cell suspension was placed on a magnetic rack to adsorb the magnetosomes, and the liquid portion was discarded to remove cell debris. The separated magnetosomes were rinsed with 1× PBS to obtain a magnetosome suspension, and then the magnetosome suspension was treated with low-frequency ultrasound at an ultrasound parameter of 20 W, each ultrasound for 2 seconds, and an interval of 3 seconds between two ultrasounds, for a total of 6 minutes. The magnetic rack adsorption, rinsing and ultrasound treatment were repeated 8 to 10 times, and the OD280 nm was measured until it was less than 0.05. The suspension was freeze-dried overnight to obtain purified magnetosomes. (3) Preparation of biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells: Take the cell membrane suspension of circulating tumor cells targeting mesenchymal phenotype collected in step (1) and mix it evenly with the magnetic bodies prepared in step (2) at a ratio of 3:1 (w / w); perform low-frequency ultrasonic treatment (ultrasonic parameters are 20 W, each ultrasonic treatment is 2 seconds, and the interval between two ultrasonic treatments is 3 seconds) for 10 minutes to obtain a biomimetic magnetic bead suspension, place the biomimetic magnetic bead suspension after ultrasonic treatment on a magnetic rack, adsorb the biomimetic magnetic beads, discard the supernatant, rinse the magnetic beads with 1× PBS, repeat this step 3 to 5 times, and finally obtain a suspension M2 of biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells.
[0041] Preparation of hybrid biomimetic magnetic beads targeting circulating tumor cells with epithelial and mesenchymal phenotypes A suspension M1 of biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells and a suspension M2 of biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells were prepared respectively according to the aforementioned method, and M1 and M2 were mixed to obtain a suspension M3 of mixed biomimetic magnetic beads targeting epithelial phenotype and mesenchymal phenotype circulating tumor cells.
[0042] Figure 3 3 are transmission electron micrographs of suspensions of biomimetic magnetic beads prepared in the examples, wherein A is a transmission electron micrograph of suspension M1 of biomimetic magnetic beads targeting epithelial phenotype CTCs, and B is a transmission electron micrograph of suspension M2 of biomimetic magnetic beads targeting mesenchymal phenotype CTCs. It can be seen from the figures that the magnetic cores of the magnetic beads in suspensions M1 and M2 are crystals of 30 to 50 nm, and the cell membranes coated thereon are uniform in size and morphology and well dispersed.
[0043] Figure 4 The results of western blot detection of anti-EpCAM antibody-eGFP fusion protein / NEFL-MCHERRY fusion protein expression in THP-1 engineered cells, targeted cell membranes, and biomimetic magnetic beads, wherein A is the expression result of anti-EpCAM antibody-eGFP fusion protein, and B is the expression result of NEFL-MCHERRY fusion protein. It can be seen from the figure that both the anti-EpCAM antibody-eGFP fusion protein for epithelial phenotype CTCs and the NEFL-MCHERRY fusion protein for capturing mesenchymal phenotype CTCs can be efficiently expressed in the corresponding THP-1 engineered cells, targeted cell membranes, and biomimetic magnetic beads.
[0044] CTCs capture experiment The bionic magnetic beads targeting epithelial phenotype CTCs were used to capture MCF-7 cells (EpCAM high expression), and the bionic magnetic beads targeting mesenchymal phenotype CTCs were used to capture MDA-MB-231 cells (Vimentin high expression).
[0045] (1) Epithelial phenotype CTCs capture experiment 1000, 5000, and 10000 Hoechst 33342 pre-stained MCF-7 (EpCAM high expression) cells were added to a simulated blood system containing 10 mL of fresh whole blood (rubber tube inner diameter 0.8 / 1.6 / 3.2 mm, length 80 cm, sequentially connected to a micro pump and centrifuge tube to form a closed circulation system), and the rubber tube was injected with a suspension of biomimetic magnetic beads M1 and placed under a magnetic field; the micro pump was turned on and the flow rate was controlled to 10 cm / s. After circulating for 30 minutes, the magnetic field was removed and the magnetic beads were taken out. After washing with PBS three times, the isolated CTCs were stained with PI, and the active MCF-7 cells (Hoechst 33342+PI-) were counted by flow cytometry, and the capture efficiency was calculated using the following formula: CTCs capture positive rate = captured active CTCs / total CTCs The results of the epithelial phenotype CTCs capture experiment are shown in Table 1.
[0046] Table 1 From the results in Table 1, it can be seen that the biomimetic magnetic bead suspension M1 has a higher capture efficiency for epithelial phenotype CTCs.
[0047] (2) Mesenchymal phenotype CTCs capture experiment 1000, 5000, and 10000 Hoechst 33342 pre-stained MDA-MB-231 (Vimentin high expression) cells were added to a simulated blood system containing 10 mL of fresh whole blood (rubber tube inner diameter 0.8 / 1.6 / 3.2 mm, length 80 cm, sequentially connected to a micro pump and a centrifuge tube to form a closed circulation system), and the rubber tube was injected with a suspension of biomimetic magnetic beads M2 and placed under a magnetic field. The micro pump was turned on and the flow rate was controlled to 10 cm / s. After circulating for 30 minutes, the magnetic field was removed and the magnetic beads were taken out. After washing with PBS three times, the isolated CTCs were stained with PI, and the active MDA-MB-231 cells (Hoechst33342+PI-) were counted by flow cytometry, and the capture efficiency was calculated using the following formula: CTCs capture positive rate = captured active CTCs / total CTCs The results of the mesenchymal phenotype CTCs capture experiment are shown in Table 2.
[0048] Table 2 From the results in Table 2, it can be seen that the biomimetic magnetic bead suspension M2 also has a high capture efficiency for mesenchymal phenotype CTCs.
[0049] In summary, the bionic magnetic beads provided in the embodiments of the present application can capture epithelial phenotype CTCs and mesenchymal phenotype CTCs, have good selectivity and specificity, high sensitivity, and can effectively separate magnetic beads and CTCs, and can achieve simultaneous classification and collection of epithelial phenotype CTCs and mesenchymal phenotype CTCs.
[0050] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.
Claims
1. A biomimetic magnetic bead for capturing circulating tumor cells in vivo, characterized in that: The bionic magnetic beads include at least one of a first bionic magnetic bead targeting epithelial phenotype circulating tumor cells and a second bionic magnetic bead targeting mesenchymal phenotype circulating tumor cells; wherein, The first biomimetic magnetic bead is formed by a first targeted cell membrane coated with magnetic particles, and the first targeted cell membrane comprises an anti-EpCAM antibody having an amino acid sequence as shown in SEQ ID NO: 1; The second bionic magnetic beads are formed by second targeted cell membrane-coated magnetic particles, and the second targeted cell membrane comprises a NEFL protein having an amino acid sequence as shown in SEQ ID NO:
2.
2. The bionic magnetic bead according to claim 1, characterized in that: When the bionic magnetic beads include first bionic magnetic beads targeting epithelial phenotype circulating tumor cells, the first targeting cell membrane also includes a TEV protease cleavage site whose amino acid sequence is shown in SEQ ID NO:3; when the bionic magnetic beads include second bionic magnetic beads targeting mesenchymal phenotype circulating tumor cells, the second targeting cell membrane also includes an HRV 3C protease cleavage site whose amino acid sequence is shown in SEQ ID NO:
4.
3. The bionic magnetic bead according to claim 1, characterized in that: The magnetic particles are magnetosomes.
4. An anti-EpCAM antibody gene recombinant plasmid, characterized in that: The anti-EpCAM antibody gene recombinant plasmid includes a CMV promoter, a signal peptide gene sequence, an anti-EpCAM antibody gene fragment, a TEV protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and an eGFP green fluorescent protein gene sequence connected in sequence; wherein, The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5; The signal peptide gene sequence is shown in SEQ ID NO:6; The nucleotide sequence of the anti-EpCAM antibody gene fragment is shown in SEQ ID NO: 7; The TEV protease cleavage site gene sequence is shown in SEQ ID NO: 8; The first connexin gene sequence is shown in SEQ ID NO:9; The transmembrane protein gene sequence is shown in SEQ ID NO: 10; The second connexin gene sequence is shown in SEQ ID NO: 11; The eGFP green fluorescent protein gene sequence is shown in SEQ ID NO:
12.
5. A NEFL protein gene recombinant plasmid, characterized in that: The NEFL protein gene recombinant plasmid includes a CMV promoter, a signal peptide gene sequence, a NEFL protein gene fragment, an HRV 3C protease cleavage site gene sequence, a first connexin gene sequence, a transmembrane protein gene sequence, a second connexin gene sequence and a MCHERRY red fluorescent protein gene sequence connected in sequence; wherein, The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:5; The signal peptide gene sequence is shown in SEQ ID NO:6; The nucleotide sequence of the NEFL protein gene fragment is shown in SEQ ID NO: 13; The HRV 3C protease cleavage site gene sequence is shown in SEQ ID NO: 14; The first connexin gene sequence is shown in SEQ ID NO:9; The transmembrane protein gene sequence is shown in SEQ ID NO: 10; The second connexin gene sequence is shown in SEQ ID NO: 11; The MCHERRY red fluorescent protein gene sequence is shown in SEQ ID NO:
15.
6. An engineered cell, characterized in that: The engineered cells include an anti-EpCAM antibody gene recombinant plasmid or a NEFL protein gene recombinant plasmid.
7. A method for preparing biomimetic magnetic beads, characterized in that: The preparation method comprises at least one of (A) to (C): (A) A method for preparing a first biomimetic magnetic bead targeting epithelial phenotype circulating tumor cells, comprising the steps of: (1) Transforming the anti-EpCAM antibody gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express anti-EpCAM antibodies; The engineered cells that highly express anti-EpCAM antibodies are collected and lysed to isolate and obtain a suspension of cell membranes targeting epithelial phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) mixing the suspension of cell membranes of circulating tumor cells with epithelial phenotype obtained in (1) with the magnetosomes obtained in (2) The mixture is mixed to obtain the first biomimetic magnetic beads targeting epithelial phenotype circulating tumor cells; (B) A method for preparing a second biomimetic magnetic bead targeting mesenchymal phenotype circulating tumor cells, comprising the steps of: (1) Transforming the NEFL protein gene recombinant plasmid into human monocytic THP-1 cells, culturing and obtaining engineered cells that highly express NEFL protein; collecting and lysing the engineered cells that highly express NEFL protein, and isolating and obtaining a suspension of cell membranes targeting mesenchymal phenotype circulating tumor cells; (2) Cultivating and collecting magnetotactic bacteria, disrupting them by ultrasound to obtain a cell suspension, and magnetically adsorbing the magnetosomes in the cell suspension; (3) mixing the suspension of cell membranes of circulating tumor cells with mesenchymal phenotype obtained in (1) with the magnetosomes obtained in (2) Mixing is performed to obtain second biomimetic magnetic beads targeting mesenchymal phenotype circulating tumor cells; (C) A method for preparing a third magnetic bead that simultaneously targets epithelial phenotype circulating tumor cells and mesenchymal phenotype circulating tumor cells, comprising the steps of: The first biomimetic magnetic beads prepared in (A) and the second biomimetic magnetic beads prepared in (B) are mixed.
8. Use of the bionic magnetic beads according to any one of claims 1 to 3 in capturing heterogeneous circulating tumor cells in vivo.