CN112394167A - Fluorescent nano magnetic bead for capturing and identifying CTCs (biological chemical centers), and preparation method and application thereof - Google Patents
Fluorescent nano magnetic bead for capturing and identifying CTCs (biological chemical centers), and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent nano magnetic bead for capturing and identifying CTCs (biological chemical centers), and a preparation method and application thereof. The fluorescent nano magnetic bead for capturing and identifying the CTCs has a core-shell structure and comprises a magnetic bead serving as a core and a silicon dioxide layer serving as a shell, wherein fluorescent dye for capturing the CTCs through fluorescent identification is embedded in the silicon dioxide layer, the outer surface of the silicon dioxide layer is covalently connected with a polymer layer, and capturing molecules for specifically capturing the CTCs are modified on the polymer layer. The fluorescent nano magnetic beads for capturing and identifying the CTCs utilize the synergistic effect of the fluorescent dye, the anti-adhesion polymer and the double antibodies, so that the nonspecific adhesion of blood cells can be reduced, and the heterogeneous CTCs can be efficiently captured and fluorescently identified; and the preparation method is simple and has universality.
Description
Technical Field
The invention relates to a separation and identification technology of clinical CTC in medicine, in particular to an anti-adhesion fluorescent nano magnetic bead for capturing and identifying heterogeneous Circulating Tumor Cells (CTCs), a preparation method and application thereof, belonging to the field of molecular biology.
Background
Circulating Tumor Cells (CTCs) refer to malignant tumor cells that have shed from the originating tumor mass or site of metastasis into the peripheral blood of the human body. CTCs are closely related to cancer metastasis, therapeutic effects, cancer recurrence, medication guidance, and prognosis, and thus are important biomarkers for early diagnosis and treatment of cancer metastasis. The research on CTC is expected to clarify the internal mechanism of cancer metastasis, drug sensitivity and drug resistance, thereby realizing the individual effective treatment of cancer patients. However, the extremely rare number and inherent heterogeneity of CTCs in peripheral blood presents a great difficulty in efficiently capturing CTCs at high purity. In the last decade, different detection and isolation techniques have been used to isolate CTCs, such as affinity capture, microfluidics, size separation, immunomagnetic bead separation, nanostructures, etc., which rely mainly on the single biomarker EpCAM, whereas CTCs undergo epithelial-mesenchymal transition (EMT) during transfer, causing the loss or reduction of EpCAM expression, and thus techniques relying on the single antibody EpCAM may reduce the isolation of heterogeneous CTCs. The research shows that the N-cadherin receptor is highly expressed after the cancer cells are subjected to EMT, so that the N-cadherin and EpCAM antibodies are simultaneously modified on the surfaces of magnetic beads in the separation technology and are used for solving the problem of difficult heterogeneous capture of CTC.
In recent years, a series of water-soluble zwitterionic compounds have received wide attention, such as sulfobetaines and carboxybetaines. The zwitter-ionic compound has positive charges and negative charges at the same time, the surface of the formed material has strong hydration capability through electrostatic interaction, and good antifouling performance is shown. The surface modified by carboxylic betaine methyl methacrylate (CBMA) has good protein and cell resistant adsorption capacity, and the CBMA has active carboxyl functional groups, so that the surface is favorably supported for subsequent functional modification. In addition, the in-situ identification of the captured cells is also of great significance, wherein the fluorescent identification method has the advantages of simplicity and directness, so that a fluorescent layer is introduced on the surface of the magnetic beads for the fluorescent identification of the captured cells. The invention is achieved accordingly.
Disclosure of Invention
The invention mainly aims to provide fluorescent nano magnetic beads for capturing and identifying CTCs, so as to overcome the defects of the prior art.
The invention also aims to provide a preparation method of the fluorescent nano magnetic beads for capturing and identifying CTCs.
Still another object of the present invention is to provide the use of the fluorescent nanobead for capturing and identifying CTCs.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a fluorescent nano magnetic bead for capturing and identifying CTCs, which has a core-shell structure and comprises a magnetic bead serving as a core and a silicon dioxide layer serving as a shell, wherein fluorescent dye for capturing CTCs by fluorescent identification is embedded in the silicon dioxide layer, the outer surface of the silicon dioxide layer is covalently connected with a polymer layer, and capture molecules for specifically capturing CTCs are modified on the polymer layer.
In a preferred embodiment, the capture molecule is an aptamer or an antibody.
Further, the capture molecules are anti-EpCAM and anti-N-cadherin diabodies.
The embodiment of the invention also provides a preparation method of the fluorescent nano magnetic bead for capturing and identifying the CTCs, which comprises the following steps:
(1) providing a nanometer magnetic bead, forming a silicon dioxide layer on the surface of the nanometer magnetic bead, and embedding a fluorescent dye in the silicon dioxide layer;
(2) performing silanization treatment on the outer surface of the silicon dioxide layer to modify upper double bonds;
(3) covalently modifying the outer surface of the silica layer with a polymer layer;
(4) and connecting capture molecules for specifically capturing the CTCs on the polymer layer.
The embodiment of the invention also provides a capture method of the circulating tumor cells, which comprises the following steps:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the capture molecules are specifically bound to circulating tumor cells to capture the circulating tumor cells.
The embodiment of the invention also provides a fluorescence identification method of circulating tumor cells, which comprises the following steps:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the circulating tumor cells are fluorescently identified by specifically binding the capture molecules to the circulating tumor cells.
The embodiment of the invention also provides application of the fluorescent nano magnetic beads for capturing and identifying CTCs in preparation of products capable of specifically identifying and/or capturing and identifying circulating tumor cells.
Compared with the prior art, the invention has at least the following beneficial effects:
1) the method for separating and identifying CTCs based on fluorescent nano magnetic beads wrapped by the silicon dioxide layer has good cell compatibility, can keep the activity and the function of captured cancer cells to the maximum extent, and has important significance for subsequent research;
2) the fluorescent nano magnetic beads for separating and identifying CTCs provided by the invention are synthesized by introducing a fluorescent dye into a silicon dioxide shell layer through a simple embedding method, so that the magnetic beads have fluorescence performance and can directly identify captured cancer cells;
3) on the basis of modifying the specific capture molecules, the fluorescent nano magnetic beads for separating and identifying CTCs provided by the invention introduce the anti-adhesion molecules, so that the adhesion of non-target cells can be reduced on the premise of efficiently capturing target cells, and more active sites can be provided for the modification of the specific capture molecules;
4 the fluorescent nano magnetic beads for separating and identifying CTCs provided by the invention increase the capture of heterogeneous CTCs after mesenchymal transformation in blood or low expression of EpCAM by utilizing the synergistic effect of two high-expression capture molecules before and after EMT transformation of cancer cells, and reduce the loss of CTCs compared with magnetic beads only modifying anti-EpCAM single antibodies;
5) the invention also provides a method for modifying the surface of the magnetic bead.
6) The preparation method is simple and has universality.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic diagram of the application of the double-antibody modified anti-adhesion fluorescent nano magnetic beads for capturing and identifying CTCs in an exemplary embodiment of the invention.
Fig. 2 is a schematic diagram of a modification of fluorescent nanobead for capturing and identifying CTCs according to an exemplary embodiment of the present invention.
Fig. 3 is a transmission electron microscope image of a nanobead in an exemplary embodiment of the present invention.
FIG. 4 is a transmission electron microscope image of a magnetic bead wrapped by a silica layer according to an exemplary embodiment of the invention.
FIG. 5 is a confocal view of a fluorescent nano magnetic bead according to an exemplary embodiment of the invention.
FIG. 6 is a scanning electron microscope image of the interaction between fluorescent nano-magnetic beads and target cells MCF-7 according to an exemplary embodiment of the present invention.
FIG. 7 is a fluorescent identification chart of fluorescent nanobeads and target cells in an exemplary embodiment of the invention.
FIG. 8 is a graph showing the capture efficiency of fluorescent nanobeads on target cells at different concentrations in an exemplary embodiment of the invention.
FIG. 9 is a graph of the capture efficiency of fluorescent nanobeads to target cells at different incubation times in an exemplary embodiment of the invention.
FIG. 10 is a diagram illustrating the capture of cells by different types of modified magnetic beads in an exemplary embodiment of the invention.
FIG. 11 is a graph showing the study of the activity of cells after interaction with fluorescent nanobeads in an exemplary embodiment of the present invention.
FIG. 12 is a diagram of the capture of different types of cancer cells by fluorescent nano-magnetic beads modified by different capture molecules according to an exemplary embodiment of the present invention.
FIG. 13 is a diagram of the capture of a small number of MCF-7 cells by fluorescent nanobeads in an exemplary embodiment of the invention.
FIG. 14 is a diagram illustrating the capture of a small number of HeLa cells by fluorescent nano-magnetic beads according to an exemplary embodiment of the present invention.
FIG. 15 is a graph of the performance of fluorescent nanobeads for identification and enumeration of fluorescent nanobeads using a 1:1 mixture of MCF-7 cells and HeLa cells in an exemplary embodiment of the present invention.
FIG. 16 is a fluorescence plot of the identification and counting performance of fluorescent nanobeads using a 1:1 mixture of MCF-7 cells and HeLa cells simulated clinical samples in an exemplary embodiment of the invention.
FIG. 17 is a graph showing the number of CTCs detected from blood samples of healthy and patients using fluorescent nano-magnetic beads in an exemplary embodiment of the present invention.
Fig. 18 is a fluorescent image of CTCs detected from a blood sample of a cancer patient by fluorescent nanobead according to an exemplary embodiment of the present invention.
Detailed Description
The present invention will be more fully understood from the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed embodiment.
The fluorescent nano magnetic bead has a core-shell structure, and comprises a magnetic bead serving as a core and a silica layer serving as a shell, wherein fluorescent dye used for fluorescent identification and capture of CTCs is embedded in the silica layer, a polymer layer used for reducing nonspecific adhesion of blood cells is covalently connected to the outer surface of the silica layer, and capture molecules used for specific capture of CTCs are modified on the polymer layer.
In a preferred embodiment, the capture molecule is an aptamer or an antibody.
Furthermore, the capture molecules are double antibodies of anti-epithelial adhesion molecule anti-EpCAM and anti-neural cadherin anti-N-cadherin, so that the CTC with high EpCAM expression can be captured efficiently, and the CTC with low EpCAM expression or with no EpCAM expression after mesenchymal transformation can be captured efficiently.
Further, the fluorescent dye includes DiI, but is not limited thereto.
Further, the polymer forming the polymer layer includes a polymer formed of a carboxylic acid betaine methyl methacrylate (pCBMA) compound for reducing non-specific adhesion of blood cells and providing a polyvalent active functional group for modification of capture molecules.
As another aspect of the technical solution of the present invention, it relates to a method for preparing fluorescent nanobeads for capturing and identifying heterogeneous Circulating Tumor Cells (CTCs), which comprises:
(1) providing a nanometer magnetic bead, forming a silicon dioxide layer on the surface of the nanometer magnetic bead, and embedding a fluorescent dye DiI in the silicon dioxide layer;
(2) performing silanization treatment on the outer surface of the silicon dioxide layer to modify upper double bonds;
(3) covalently modifying the outer surface of the silica layer with a polymer layer;
(4) and connecting capture molecules for specifically capturing the CTCs on the polymer layer.
The invention provides a preparation method of fluorescent nano magnetic beads for efficiently capturing and identifying Circulating Tumor Cells (CTCs) with high purity, which comprises the steps of synthesizing the fluorescent nano magnetic beads with core-shell structure silica coated magnetic beads with the particle size of about 170nm, and modifying a layer of anti-adhesion polymer layer and surface functionalization of affinity molecules to the fluorescent nano magnetic beads on the outer surface of a silicon shell.
Further, the nanoscale magnetic beads are obtained through a purchase mode.
In a preferred embodiment, the step (1) specifically comprises: dispersing the nanoscale magnetic beads in a mixed solution containing tetraethyl orthosilicate, ammonia water and fluorescent dye, reacting for 2-6 hours at normal temperature under the stirring condition, thereby forming a silicon dioxide layer on the surfaces of the magnetic beads and embedding the fluorescent dye.
Further, the fluorescent dye includes DiI, but is not limited thereto.
Further, the fluorescent nano magnetic beads in the step (1) are prepared and synthesized by a method of wrapping a silicon dioxide layer, and specifically comprise: dispersing the nano magnetic beads in a solution containing tetraethyl orthosilicate (TEOS), ammonia water and a DiI dye, reacting for 2-6h at normal temperature under the stirring condition, forming a silicon dioxide layer on the surfaces of the magnetic beads through the hydrolysis action of the TEOS, embedding the DiI dye, and synthesizing the fluorescent nano magnetic beads.
In a preferred embodiment, the polymer layer is synthesized by a free radical polymerization method. Wherein, the polymer layer adopted has good cell compatibility and anti-adhesion performance.
Further, the anti-adhesion polymer layer is aimed at reducing capture of nonspecific cells by magnetic nanoparticles and providing multivalent active sites for modification of affinity molecules, and the polymer layer is mainly synthesized by using betaine methyl methacrylate (CBMA) which is a carboxylic acid with excellent anti-adhesion performance, and can provide active functional group carboxyl for modification of affinity molecules.
In a preferred embodiment, the step (3) specifically comprises: and (2) carrying out polymerization reaction on the outer surface of the double-bond modified silicon dioxide layer by using carboxylic betaine methyl methacrylate (CBMA) as a monomer and tetramethyl ethylene diamine and ammonium persulfate as initiators, controlling the reaction temperature at 30-40 ℃, and maintaining the reaction time for 30-120 min under an ultrasonic condition, so that the upper polymer layer is covalently modified on the outer surface of the silicon dioxide layer, and the polymer layer modified fluorescent nano magnetic bead is obtained.
In a preferred embodiment, the step (4) specifically comprises: and placing the fluorescent nano magnetic beads modified with the polymer layer in PBS (phosphate buffer solution) containing EDC and NHS (polyethylene glycol NHS), reacting at normal temperature, adding streptavidin for reaction, and reacting with biotin-modified capture molecules for specifically capturing CTCs to connect the capture molecules.
In a preferred embodiment, the capture molecule is an aptamer or an antibody.
Further, the affinity capture molecules for CTCs are used for realizing efficient and specific capture and identification of CTCs, and comprise antibodies of epithelial cell adhesion molecule receptors highly expressed by cancer cells before epithelial-mesenchymal transition (EMT) and antibodies of neural cadherin receptors highly expressed after EMT, such as EpCAM and N-cadherin biotinylated antibodies, and are not limited thereto.
Furthermore, the capture molecules are double antibodies of anti-epithelial adhesion molecule anti-EpCAM and anti-neural cadherin anti-N-cadherin, so that the CTC with high EpCAM expression can be captured efficiently, and the CTC with low EpCAM expression or with no EpCAM expression after mesenchymal transformation can be captured efficiently.
As a more specific embodiment, the preparation method of the present invention comprises: a silicon dioxide shell is formed on the surface of a nano magnetic bead through hydrolysis of tetraethyl orthosilicate, meanwhile, a fluorescent dye DiI is embedded, the fluorescent nano magnetic bead is synthesized, double bonds are modified on the outer surface of the silicon dioxide layer through silanization, a multifunctional linear polymer layer (pCBMA) is further modified through a free radical polymerization method, and finally amphiphilic molecules capable of being specifically recognized with heterogeneous circulating tumor cells, such as anti-epithelial adhesion molecule antibodies (anti-EpCAM) and anti-neural cadherin antibodies (anti-N-cadherin), are introduced.
In a preferred technical scheme, the method specifically comprises the following steps:
wrapping a silicon dioxide layer on the surface of a magnetic bead, embedding fluorescent dye, performing silanization treatment on the outer surface of a silicon dioxide shell to modify upper double bonds, and modifying the outer surface of the silicon shell with an anti-adhesion polymer layer by using carboxylic acid betaine methyl methacrylate, an initiator tetramethyl ethylenediamine and ammonium persulfate; and
modifying capture molecules on the anti-adhesion polymer layer.
In a preferred technical scheme, the method specifically comprises the following steps: a silica shell is formed on the surface of a nano magnetic bead through hydrolysis of tetraethyl orthosilicate, meanwhile, a fluorescent dye DiI is embedded, double bonds are modified on the outer surface of the silica layer through silanization, and a multifunctional linear polymer layer (pCBMA) is further modified through a free radical polymerization method.
In a preferred technical scheme, the method specifically comprises the following steps: the double capture molecules which can be specifically recognized with heterogeneous circulating tumor cells are introduced on the carboxyl functional groups of the polymer layer pCBMA by using a chemical condensation method.
In a more specific embodiment, a method for preparing fluorescent nanobeads for capturing and identifying CTCs may include the following steps:
the step (1) comprises the following steps: a silicon layer with a nanoscale thickness (about 25 nm) is modified on the surface of a nanoscale (about 100nm) magnetic bead, and meanwhile, a small-molecule fluorescent dye DiI is embedded in the silicon layer, so that the magnetic bead has fluorescence, and the magnetic bead can be used for directly identifying captured CTC.
The step (1) specifically comprises the following steps: adding 0.1-0.2mg of 100nm magnetic beads and 20-40 mu g of DiI dye into a water and ethanol solution with the volume ratio of 1:4-6, finally sequentially adding 0.8-1.2% of ammonia water and 0.4-0.8% of TEOS (tetraethyl orthosilicate) by volume ratio, carrying out ultrasonic treatment, stirring and reacting at normal temperature for 2-6h to obtain fluorescent Fe with the wavelength of about 170nm3O4@SiO2And (3) nanoparticles.
The step (2) comprises the following steps: fe with fluorescence of about 170nm obtained in step (1)3O4@SiO2The double bonds on the outer surface of the silicon shell of the nano-particles are modified through silanization.
The step (2) specifically comprises the following steps: the Fe with fluorescence and about 170nm obtained in the step (1)3O4@SiO2Dispersing magnetic nanoparticles in ethanol solution with a certain volume, adding 1-3% (V/V) MPS (3- (trimethoxysilyl) propyl methacrylate), ultrasonically stirring, reacting at normal temperature overnight, and cleaning with a large amount of ethanol after the reaction.
The step (3) comprises the following steps: modifying the surface of the double-bond functionalized fluorescent nano magnetic bead obtained in the step (2) with an anti-adhesion polymer layer by mainly utilizing an anti-adhesion molecule carboxylic acid betaine methyl methacrylate (CBMA) with functional groups, wherein the functional groups comprise double bonds and/or carboxyl, and performing free radical excitation polymerization on Fe3O4@SiO2The magnetic nanoparticle surface is modified with an anti-adhesion polymerization linear compound pCBMA, and the pCBMA not only has excellent anti-adhesion performance and reduces the interference of blood cells, but also can provide more modification sites for the modification of antibodies.
The step (3) specifically comprises the following steps: dispersing the double-bond functionalized fluorescent nano magnetic beads obtained in the step (2) into a certain amount of deionized water, adding 0.05-0.1% (w/v) of tetramethylethylenediamine, 0.1-0.15% (w/v) of ammonium persulfate and 0.5-5% (w/v) of CBMA monomer, and carrying out ultrasonic reaction for 0.5-2h at 30-40 ℃. After the reaction is finished, the reaction solution is separated and cleaned by a magnetic frame.
The step (4) comprises the following steps: and (3) modifying the fluorescent nano magnetic beads of the pCBMA in the step (3), and simultaneously modifying anti-EpCAM and anti-N-cadherin double antibodies on the functional group carboxyl of the pCBMA linear polymer by a chemical condensation method. The fluorescent nano magnetic bead can efficiently capture the CTC with high EpCAM expression, and can also efficiently capture the CTC with low EpCAM expression and high N-cadherin expression after EMT conversion, thereby reducing the loss of heterogeneous CTC.
The step (4) specifically comprises the following steps: dispersing the fluorescent nano magnetic beads modified by pCBMA in the step (3) in PBS solution containing 0.05-0.2M EDC and 0.01-0.05M NHS of coupling reagent with activated carboxyl, carrying out ultrasonic treatment, and reacting at normal temperature for 20-90 min. After the reaction, the mixture was washed with PBS, then 0.00005 to 0.0001% (w/v) SA was added thereto, stirred, reacted overnight at room temperature, and washed. Then two antibodies of biotin modified anti-EpCAM and anti-N-cadherin are added for reaction for 1-24 h. Storing in a refrigerator at 4 ℃ in the dark for later use.
In a more preferred embodiment, step (1) comprises: adding 0.1-0.2mg of 100nm magnetic beads and 20-40 mu g of DiI dye into a water and ethanol solution with the volume ratio of 1:4-6, finally sequentially adding 0.8-1.2% of ammonia water and 0.4-0.8% of TEOS (tetraethyl orthosilicate) by volume ratio, carrying out ultrasonic treatment, stirring and reacting at normal temperature for 2-6h to obtain fluorescent Fe with the wavelength of about 170nm3O4@SiO2And (3) nanoparticles.
In a more preferred embodiment, step (2) comprises: the Fe with fluorescence and about 170nm obtained in the step (1)3O4@SiO2Dispersing magnetic nanoparticles in ethanol solution with a certain volume, adding 1-3% (V/V) MPS (3- (trimethoxysilyl) propyl methacrylate), ultrasonically stirring, reacting at normal temperature overnight, and cleaning with a large amount of ethanol after the reaction.
In a more preferred embodiment, step (3) comprises: dispersing the double-bond functionalized fluorescent nano magnetic beads obtained in the step (2) into a certain amount of deionized water, adding 0.05-0.1% (w/v) of tetramethylethylenediamine, 0.1-0.15% (w/v) of ammonium persulfate and 0.5-5% (w/v) of CBMA monomer, and carrying out ultrasonic reaction for 0.5-2h at 30-40 ℃. After the reaction is finished, the reaction solution is separated and cleaned by a magnetic frame.
In a more preferred embodiment, step (4) comprises: dispersing the fluorescent nano magnetic beads modified by pCBMA in the step (3) in PBS solution containing 0.05-0.2M EDC and 0.01-0.05M NHS of coupling reagent with activated carboxyl, carrying out ultrasonic treatment, and reacting at normal temperature for 20-90 min. After the reaction, the mixture was washed with PBS, then 0.00005 to 0.0001% (w/v) SA was added thereto, stirred, reacted overnight at room temperature, and washed. Then two antibodies of biotin modified anti-EpCAM and anti-N-cadherin are added for reaction for 1-24 h. Storing in a refrigerator at 4 ℃ in the dark for later use.
Compared with the prior art, the fluorescent nano magnetic beads for CTC separation and identification disclosed by the invention utilize the anti-adhesion molecules CBMA to form a polymer layer on the outer surface of the silicon shell, so that the non-specific adhesion of blood cells can be greatly reduced, and multivalent active sites are provided for modification of capture molecules. In addition, the capture molecules modified by the polymer layer are anti-EpCAM and anti-N-cadherin double antibodies, and the synergistic effect of the double antibodies is utilized, so that the CTC with high EpCAM expression can be efficiently captured, and the CTC with low EpCAM expression or after mesenchymal transformation without EpCAM expression can also be efficiently captured. Meanwhile, the fluorescent dye embedded in the silicon shell enables the magnetic beads to have fluorescence performance, and captured cells can be directly identified through fluorescence imaging. The method provides a new capture substrate for high-purity and high-activity CTC in clinic.
The method has simple and convenient process and low cost. Firstly, a silicon dioxide shell is formed on the surface of a nano magnetic bead through hydrolysis of tetraethyl orthosilicate, meanwhile, a fluorescent dye DiI is embedded, fluorescent nano magnetic beads are synthesized, the fluorescent nano magnetic beads can be used for directly identifying captured cells, then double bonds are modified on the outer surface of a silicon dioxide layer through silanization, a multifunctional linear polymer layer (pCBMA) is further modified through a free radical polymerization method, adhesion of blood cells can be reduced, multivalent active sites are provided for modification of affinity molecules such as antibodies for specifically identifying CTCs, and finally double capture molecules such as anti-epithelial adhesion molecule antibodies (anti-EpCAM) and anti-neural cadherin antibodies (anti-N-cadherin) capable of specifically identifying heterogeneous circulating tumor cells are introduced through carboxyl functional groups on the polymer. By utilizing the synergistic effect of the double antibodies, the CTC with high EpCAM expression can be efficiently captured, and the CTC with low EpCAM expression or with no EpCAM expression after mesenchymal transformation can also be efficiently captured.
Another aspect of the embodiments of the present invention provides fluorescent nanobeads for capturing and identifying CTCs, prepared by the above method.
The invention also provides an operation process of the fluorescent nano magnetic bead for capturing and identifying the CTCs.
In another aspect of the embodiments of the present invention, a fluorescent nano magnetic bead with different affinity molecules can be replaced for capturing tumor cells with different phenotypes.
Another aspect of the embodiments of the present invention also provides the use of the fluorescent nanobead for capturing and identifying CTCs.
In another aspect, the present invention relates to a reagent for capturing and identifying heterogeneous Circulating Tumor Cells (CTCs), comprising fluorescent nanobeads for capturing and identifying CTCs, wherein the fluorescent nanobeads are provided with capture molecules for specifically capturing the heterogeneous circulating tumor cells.
Further, the agent further comprises a pharmaceutically acceptable carrier.
As another aspect of the technical solution of the present invention, it relates to a method for capturing circulating tumor cells, the method comprising:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the capture molecules are specifically bound to circulating tumor cells to capture the circulating tumor cells.
In another aspect, the present invention relates to a method for the fluorescent identification of circulating tumor cells, comprising:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the circulating tumor cells are fluorescently identified by specifically binding the capture molecules to the circulating tumor cells.
As another aspect of the technical scheme of the invention, the invention relates to the application of the fluorescent nano magnetic beads for capturing and identifying CTCs in the preparation of products capable of specifically recognizing and/or capturing and identifying circulating tumor cells.
As another aspect of the technical scheme of the invention, the invention relates to the application of the reagent for capturing and identifying the circulating tumor cells, which is related to the line, in the preparation of products capable of specifically recognizing and/or capturing the circulating tumor cells.
Further, the application comprises the step of counting and identifying the CTCs in the clinical patient blood sample by using the fluorescent nano magnetic beads for capturing and identifying the CTCs.
The prepared anti-adhesion fluorescent nano magnetic bead modified by the double antibodies can be used for counting and identifying CTCs in clinical patient blood samples. The invention relates to a technology for separating and identifying clinical medical CTCs, in particular to a preparation method and application of anti-adhesion fluorescent nano magnetic beads for capturing and identifying heterogeneous Circulating Tumor Cells (CTCs), and belongs to the field of molecular biology. The fluorescent nano magnetic beads for separating and identifying CTCs form a polymer layer on the outer surface of a silicon shell by using the anti-adhesion molecules CBMA, so that the non-specific adhesion of blood cells can be greatly reduced, and multivalent active sites are provided for modification of capture molecules. In addition, the capture molecules modified by the polymer layer are anti-EpCAM and anti-N-cadherin diabodies, and by utilizing the synergistic effect of the diabodies, the CTCs with high EpCAM expression can be efficiently captured, and the CTCs with low EpCAM expression or after mesenchymal transformation without EpCAM expression can also be efficiently captured. Meanwhile, the fluorescent dye embedded in the silicon shell enables the magnetic beads to have fluorescence performance, and captured cells can be directly identified through fluorescence imaging. The method provides a new capture substrate for high-purity and high-activity CTCs clinically and efficiently.
One aspect of the invention includes: fluorescent molecules are introduced on the surfaces of the magnetic beads during synthesis, so that the magnetic beads can directly identify cancer cells, anti-adhesion molecules are introduced to reduce non-specific adhesion of the magnetic beads to blood cells, and CTCs affinity capture molecules are utilized to realize efficient and high-specificity capture of the CTCs.
Another aspect of the invention includes: the synergistic effect of two high-expression affinity capture molecules before and after the EMT transformation of the cancer cells is utilized to increase the capture of heterogeneous CTCs after the mesenchymal transformation in blood or the low expression of EpCAM, and compared with magnetic beads only modifying anti-EpCAM single antibodies, the loss of CTCs is reduced.
The fluorescent nano magnetic beads for capturing and identifying the CTCs provided by the invention have good cell compatibility, are simple and economical to prepare, and can realize the fluorescent identification of the captured CTCs.
The technical solutions of the present invention will be described in further detail below with reference to several preferred embodiments and accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The conditions used in the following examples may be further adjusted as necessary, and the conditions used in the conventional experiments are not generally indicated.
Example 1
The preparation method of the fluorescent nano magnetic beads for capturing and identifying the CTCs in the embodiment specifically comprises the following steps:
a) adding 0.1-0.2mg of 100nm magnetic beads and 20-40 mu g of DiI dye into a water-ethanol solution with the volume ratio of 1:4-6, and finally sequentially adding 0.8-1.2% of ammonia water and 0.4-0.8% of TEOS (tetraethyl orthosilicate)Tetraethyl ester), ultrasonic treatment, stirring, reacting for 2-6h at normal temperature, and separating and cleaning by a magnetic frame to obtain Fe with fluorescence of about 170nm3O4@SiO2And (3) nanoparticles.
b) B, mixing the Fe with fluorescence of about 170nm obtained in the step a3O4@SiO2Dispersing magnetic nanoparticles in ethanol solution with a certain volume, adding 1-3% (V/V) MPS (3- (trimethoxysilyl) propyl methacrylate), ultrasonically stirring, reacting at normal temperature overnight, and cleaning with a large amount of ethanol after the reaction. Then dispersing the magnetic beads in a certain amount of deionized water, adding 0.05-0.1% (w/v) tetramethylethylenediamine, 0.1-0.15% (w/v) ammonium persulfate and 0.5-5% (w/v) CBMA monomer, and carrying out ultrasonic reaction at 30-40 ℃ for 0.5-2 h. After the reaction is finished, the reaction solution is separated and cleaned by a magnetic frame.
c) The method comprises the following steps: and d, dispersing the fluorescent nano magnetic beads modified by pCBMA in the step b into PBS (phosphate buffer solution) containing 0.05-0.2M EDC and 0.01-0.05M NHS of a coupling reagent with activated carboxyl, carrying out ultrasonic treatment, and reacting at normal temperature for 20-90 min. After the reaction is finished, washing by PBS, then adding 0.00005-0.0001% (w/v) of SA, stirring, reacting overnight at normal temperature, washing, and adding biotin-modified anti-EpCAM and anti-N-cadherin diabody.
And (3) placing the magnetic nanoparticles in a BSA solution with the concentration of 0.1-2% (w/v) for reaction for 10-60min to block the NHS groups which are not completely reacted. Obtaining fluorescent nano magnetic beads (F-MNPs) modified by the double antibodies, and storing the fluorescent nano magnetic beads (F-MNPs) in a refrigerator at 4 ℃ in a dark place for later use.
The modified flow charts are shown in FIGS. 1 and 2. Fig. 3 is a transmission electron microscope image of the nano magnetic bead in the embodiment, fig. 4 is a transmission electron microscope image of the magnetic bead wrapped by the silica layer, fig. 5 is a laser confocal image of the fluorescent nano magnetic bead, and fig. 6 and 7 are a scanning electron microscope image and a fluorescence identification image of the effect of the fluorescent nano magnetic bead and the target cell MCF-7.
Example 2
First, to determine the optimal amount of functionalized fluorescent nanomagnetic particles to use to capture cells, 0.5mL was applied at a density of 2 × 105The cell sap/mL and the anti-EpCAM modified fluorescent nano magnetic beads with different concentrations which are pre-dispersed in 0.5mL of PBS solutionMixing, culturing in a 37 ℃ cell culture box, collecting the captured cells with the aid of a magnetic frame, washing with PBS for three times, counting, and finally calculating the capture efficiency of the functionalized magnetic nanoparticles with different usage amounts. FIG. 8 is a graph showing the efficiency of the capture behavior of MCF-7 cells by different fluorescent nano-magnetic bead concentrations, and it can be seen from FIG. 8 that the capture of target cells is maximized at a concentration of 0.1 mg/mL.
Example 3
To determine the optimal incubation time for the capture of cells by functionalized fluorescent nanomagnetic particles, 0.5mL was applied at a density of 2 × 105Respectively mixing the/mL cell sap with anti-EpCAM modified fluorescent nano magnetic beads with different concentrations which are pre-dispersed in 0.5mL PBS solution, putting the mixture into a 37 ℃ cell culture box for culture, collecting the captured cells with the assistance of a magnetic frame, washing the cells with PBS for three times, counting, and finally calculating the capture efficiency under different incubation times. FIG. 9 is a graph showing the efficiency of the capture behavior of MCF-7 cells at different incubation times, and it can be seen from FIG. 9 that the capture efficiency of the cells reaches the maximum value at 20 min.
Example 4
The capture behavior of different modified magnetic beads on cancer cells is examined by taking an EpCAM positive breast cancer cell strain MCF-7 as a model cell. Digesting and stripping MCF-7 cells which are cultured for two days and have good growth state by using 0.25 percent of pancreatin, then discarding the trypsin liquid, adding fresh culture solution to blow and beat the cells uniformly, counting the cells, and adjusting the cell suspension to 105and/mL. And (3) incubating the modified nano magnetic beads and 1mL of prepared cell suspension in a cell incubator for 20min, washing 3-5 times by using PBS, and counting. FIG. 10 is a graph showing the capture behavior of different types of magnetic beads on MCF-7 cells. The experimental result shows that the magnetic beads without modifying the anti-adhesion molecules have certain adhesion behavior to cancer cells, the magnetic beads modified by the anti-adhesion molecules have good anti-adhesion performance, and the antibody modified nano magnetic beads have good selectivity and capture efficiency to the cancer cells.
Example 5
The effect of functionalized magnetic nanoparticles on the activity of captured cells was analyzed using AM (green fluorescence, live cells) and PI (red fluorescence, dead cells) dual fluorescence staining. In order to avoid the interference caused by the autofluorescence of the magnetic nanoparticles, the magnetic nanoparticles used in the synthesis process are not embedded with DiI. Incubating fluorescent double-antibody functionalized magnetic beads with MCF-7, collecting captured cells under the assistance of an external magnetic field, incubating the captured cells with dead and live double-fluorescent dyes in a PBS solution for 20min, cleaning, observing and analyzing an experimental result by using a fluorescence microscope, and finally counting the survival rate of the cells. FIG. 11 is the activity data and fluorescence map of the cells after the interaction of fluorescent nano-magnetic beads and MCF-7 cells. The experimental result shows that the fluorescent nano magnetic bead has good biocompatibility and can keep the activity of cells.
Example 6
Testing the specificity of both antibodies
The inventor selects a cell strain MCF-7 which is EpCAM positive and N-cadherin negative, a cell strain HeLa which is EpCAM negative and N-cadherin positive, and a cell strain CCRM-CEM which is EpCAM negative and N-cadherin negative to carry out cell capture experiments. The results of the evaluation of the capture specificity of the antibodies are summarized in FIG. 12. The experimental result shows that the antibody modified nano magnetic beads have good selectivity on different types of cancer cells.
Example 7
Dispersing 10, 20, 50, 100 and 200 DiO pre-stained MCF-7 or HeLa cells into 1mL of PBS solution respectively, then incubating with 0.1mg of double-antibody functionalized fluorescent nano magnetic beads, separating and collecting the captured cells by using a magnetic frame, washing for three times, and counting the captured cells by using a fluorescence microscope.
10, 20, 50, 100 and 200 DiO pre-stained MCF-7 or HeLa cells were dispersed in 0.5mL of whole blood solution, respectively, and then incubated with 0.1mg of diabody-functionalized fluorescent nanobead dispersed in 0.5mL of DMEM, and the captured cells were collected and washed three times using a magnetic rack, and counted using a fluorescence microscope.
MCF-7 and HeLa cells pre-stained with DiO were mixed in a ratio of 1:1, 10, 20, 50, 100 and 200 mixed cells were dispersed in 0.5mL of a whole blood solution, respectively, and then incubated with 0.1mg of diabody-functionalized fluorescent nanobeads dispersed in 0.5mL of DMEM, and captured cells were collected by a magnetic frame and washed three times, and the captured cells were counted by a fluorescence microscope. Meanwhile, mixing unstained MCF-7 and HeLa cells according to a ratio of 1:1, dispersing 10, 20, 50, 100 and 200 mixed cells into 0.5mL of whole blood solution respectively, then incubating with 0.1mg of double-antibody functionalized fluorescent nano magnetic beads dispersed in 0.5mL of DMEM, collecting and washing captured cells three times through a magnetic frame, and counting the captured cells by using a fluorescent microscope.
The results are shown in fig. 13, 14, 15 and 16, respectively, fig. 13 is a graph of the capture of fluorescent nanobeads to a small number of MCF-7 cells, fig. 14 is a graph of the capture of fluorescent nanobeads to a small number of HeLa cells, fig. 15 is a graph of the identification and counting performance of fluorescent nanobeads using a simulated clinical sample in which MCF-7 cells and HeLa cells are mixed at 1:1, and fig. 16 is a fluorescence graph of the identification and counting performance of fluorescent nanobeads using a simulated clinical sample in which MCF-7 cells and HeLa cells are mixed at 1: 1. The experiment result shows that the fluorescent nano magnetic bead has good sensitivity.
Example 8
A patient sample (1 mL) was collected using an EDTA anticoagulant vacuum blood collection tube and centrifuged to separate Peripheral Blood Mononuclear Cells (PBMCs) including CTCs. Human leukocyte separation fluid (Ficoll-Paque) is added into a SepMateTM-15 centrifuge tube in advance, and then blood diluted by sterile PBS with the same volume is added, so that the interface between the two is kept clear as much as possible. The cells were then centrifuged at 2000rpm for 20 minutes, after which time the cells were observed to be bottommost. Transferring the solution above the red blood cells into a new centrifuge tube, adding a certain amount of PBS solution, mixing uniformly, centrifuging again at 1500rpm for 20 minutes, taking out the supernatant, adding a certain amount of PBS again to disperse the obtained cells, centrifuging again, and then incubating with 0.1mg of magnetic beads in 1mL of PBS solution. After washing, the cells were fixed with 4% paraformaldehyde for 30min, then blocked with blocking solution for one hour, then immunostained with 0.3% Triton X-100, 2% BSA, Alexa Fluor 488-labeled anti-CD45, and Alexa Fluor 647-labeled anti-Pan-Keratin (PanCK) in PBS buffer, stained in a refrigerator at 4 ℃ for 8 hours in the dark, stained with Hoechst 33342, washed with PBS, and finally observed for experimental results using confocal laser. The fluorescence image showed that the cells of F-MNPs +/PanCK +/CD45-/Hoechst 33342+ were considered to be CTCs, and the cells of F-MNPs-/PanCK-/CD45+/Hoechst 33342+ were WBCs. The results are shown in fig. 17 and 18, respectively, fig. 17 is a graph of the number of CTCs detected by fluorescent nanobeads from blood samples of healthy persons and patients, and fig. 18 is a fluorescent image of CTCs detected by fluorescent nanobeads from blood samples of cancer patients.
In summary, according to the technical scheme of the invention, the double-antibody modified anti-adhesion polymer fluorescent nano magnetic bead with good cell compatibility is constructed, and the fluorescent nano magnetic bead has high capture efficiency, capture specificity and sensitivity, and identification capability, and is simple in preparation method.
In addition, the present inventors have also conducted experiments with other materials and conditions, etc. listed in the present specification by way of the above examples, and have also made fluorescent nanobeads with good cell compatibility, which have higher cell capture specificity and sensitivity.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.
Claims (15)
1. The fluorescent nano magnetic bead is characterized by having a core-shell structure and comprising a magnetic bead serving as a core and a silicon dioxide layer serving as a shell, wherein fluorescent dye used for fluorescent identification and capture of CTCs is embedded in the silicon dioxide layer, the outer surface of the silicon dioxide layer is covalently connected with a polymer layer, and capture molecules used for specific capture of CTCs are modified on the polymer layer.
2. The fluorescent nanobead for capturing and identifying CTCs according to claim 1, wherein: the capture molecule is an aptamer or an antibody; preferably, the capture molecules are anti-EpCAM and anti-N-cadherin diabodies.
3. The fluorescent nanobead for capturing and identifying CTCs according to claim 1, wherein: the fluorescent dye comprises DiI.
4. The fluorescent nanobead for capturing and identifying CTCs according to claim 1, wherein: the compound forming the polymer layer includes carboxylic acid betaine methyl methacrylate.
5. A preparation method of fluorescent nano magnetic beads for capturing and identifying CTCs is characterized by comprising the following steps:
(1) providing a nanometer magnetic bead, forming a silicon dioxide layer on the surface of the nanometer magnetic bead, and embedding fluorescent dye in the silicon dioxide layer;
(2) performing silanization treatment on the outer surface of the silicon dioxide layer to modify upper double bonds;
(3) covalently modifying the outer surface of the silica layer with a polymer layer;
(4) and connecting capture molecules for specifically capturing the CTCs on the polymer layer.
6. The preparation method according to claim 5, wherein the step (1) specifically comprises: dispersing the nanoscale magnetic beads in a mixed solution containing tetraethyl orthosilicate, ammonia water and fluorescent dye, reacting for 2-6 hours at normal temperature under the stirring condition, thereby forming a silicon dioxide layer on the surfaces of the magnetic beads and embedding the fluorescent dye.
7. The production method according to claim 5 or 6, characterized in that: the fluorescent dye comprises DiI.
8. The method of claim 5, wherein: the polymer layer is synthesized by a free radical polymerization method.
9. The method according to claim 8, wherein the step (3) specifically comprises: and (2) carrying out polymerization reaction for 30-120 min at 30-40 ℃ on the outer surface of the double-bond-modified silicon dioxide layer by taking carboxylic acid betaine methyl methacrylate as a monomer and tetramethyl ethylenediamine and ammonium persulfate as initiators, so as to covalently modify the upper polymer layer on the outer surface of the silicon dioxide layer, and thus obtaining the polymer layer-modified fluorescent nano magnetic bead.
10. The method according to claim 8, wherein the step (4) specifically comprises: and placing the fluorescent nano magnetic beads modified with the polymer layer in a PBS (phosphate buffer solution) solution containing EDC and NHS, reacting at normal temperature, adding streptavidin for reaction, and reacting with biotin-modified capture molecules for specifically capturing CTCs to connect the capture molecules.
11. The production method according to claim 5 or 10, characterized in that: the capture molecule is an aptamer or an antibody; preferably, the capture molecules are anti-EpCAM and anti-N-cadherin diabodies.
12. A method of capturing circulating tumor cells, the method comprising:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the capture molecules are specifically bound to circulating tumor cells to capture the circulating tumor cells.
13. A method for the fluorescent identification of circulating tumor cells, comprising:
contacting the circulating tumor cells with fluorescent nanobeads for capture and identification of CTCs; and
the circulating tumor cells are fluorescently identified by specifically binding the capture molecules to the circulating tumor cells.
14. Use of the fluorescent nanobead for capturing and identifying CTCs according to any one of claims 1 to 4 for the preparation of a product capable of specifically recognizing and/or capturing and identifying circulating tumor cells.
15. The use according to claim 14, comprising the step of counting and identifying CTCs in a clinical patient's blood sample using said fluorescent nanobead for capturing and identifying CTCs.
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| CN114081968A (en) * | 2021-11-24 | 2022-02-25 | 致慧医疗科技(上海)有限公司 | Charge reversal type nano material and preparation method and application thereof |
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