CN113201493B

CN113201493B - Preparation method of erythrocyte clusters with ideal morphology

Info

Publication number: CN113201493B
Application number: CN202110392824.8A
Authority: CN
Inventors: 刘威; 彭伟
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2022-07-05
Anticipated expiration: 2041-04-13
Also published as: CN113201493A

Abstract

The invention discloses a preparation method of a red blood cell cluster with an ideal morphology, and belongs to the technical field of biological medicines. The invention realizes the controllable regulation of the morphology of the red blood cells by controlling the modification amount of DSPE-PEG-X molecules on the surfaces of the red blood cells, thereby controllably preparing the red blood cells with enough deformation capacity and further preparing the red blood cell clusters with ideal morphology on the basis of the red blood cells. The invention realizes the controllable preparation of the erythrocyte clusters with ideal morphology, and the prepared erythrocyte clusters can enrich target rare cells with high efficiency and high specificity.

Description

Preparation method of erythrocyte clusters with ideal morphology

Technical Field

The invention belongs to the technical field of biological medicines, relates to optimization of a red blood cell cluster preparation method, and particularly relates to a preparation method of a red blood cell cluster with an ideal morphology.

Background

Both patents CN111826349A, CN111826351A have disclosed the use of red blood cell cluster materials for specific capture and enrichment of Circulating Tumor Cells (CTCs) in peripheral blood. Based on the principle that intermolecular force exists between folic acid (or antibody) modified erythrocyte membrane protein and the surface modified microspheres, erythrocytes can be adsorbed on the surface modified microspheres; and because the negative charges on the surfaces of the red blood cells cause electrostatic repulsion among the red blood cells, the red blood cells are possibly difficult to form a full surface layer adsorption state on the surfaces of the microspheres, and the electrostatic repulsion among the red blood cells can be greatly weakened through electrostatic adsorption of cationic polymer polybrene molecules on the surfaces of the red blood cells in advance, so that full surface layer adsorption of the red blood cells on the surfaces of the microspheres is realized. The erythrocyte clusters formed after folic acid (or antibody) modified erythrocytes are tightly adsorbed on the surfaces of the microspheres can well avoid nonspecific adsorption with leukocytes while CTCs are targeted, so that high-purity CTCs are finally obtained by capture.

In fact, even though folic acid (or antibody) -modified erythrocytes are adsorbed by polybrene in advance to reduce repulsive force between erythrocytes, there is a possibility that erythrocyte clusters with a surface covered with compact erythrocytes cannot be obtained, that is, the conventional erythrocyte cluster preparation technology cannot stably and controllably prepare ideal erythrocyte clusters, and further optimization and improvement of the preparation technology are urgently needed.

Disclosure of Invention

The invention aims to solve the problems in the existing red blood cell cluster preparation technology and provides a preparation method of red blood cell clusters with ideal morphology. The ideal morphology of the erythrocyte clusters means that the surfaces of the core microspheres are adsorbed and covered by erythrocytes as much as possible, the surfaces of the core microspheres exposed by the formed erythrocyte clusters are quite dispersed, and the adsorbed erythrocytes are close enough to each other and are not beneficial to nonspecific adsorption of white blood cells on the surfaces of the core microspheres; the free CTCs can be captured by specific binding with antigen-antibody on the surface of the red blood cells.

The present invention first analyzes the principle of red blood cell cluster formation. According to the research results of the predecessors, carboxyl or amino on the surface of the core microsphere can form hydrogen bond acting force with protein molecules, so that the protein is adsorbed. And membrane protein molecules are distributed on the surface of the erythrocyte membrane, so that the core microspheres realize the adsorption of the erythrocyte by adsorbing the membrane protein on the surface of the erythrocyte membrane. For a less fresh blood cell sample, the membrane surface protein content may be attenuated, which is detrimental to the adsorption of the core microspheres to the aged red blood cells, and thus the red blood cells extracted from an stale blood sample cannot be prepared into a complete red blood cell cluster. In view of this, red blood cell clusters prepared each time by using red blood cells extracted from fresh blood are in many cases certainly able to obtain more ideal red blood cell clusters, but there are still cases of failure, thus explaining that the freshness of red blood cells is not the most critical influencing factor.

After many experiments, the relatively ideal red cell clusters can be prepared even if stale red cells exist according to the existing red cell cluster preparation method. Under the condition of not considering the freshness of the red blood cells, the shapes of the red blood cells obtained after DSPE-PEG-FA modification is added each time are slightly different, smooth and round spherical red blood cells are obtained in part of experiments, the spherical red blood cells with sharp spines on the surfaces are obtained in part of experiments, meanwhile, most of red blood cell clusters prepared by the red blood cells are not ideal, most of the red blood cell clusters prepared by the red blood cells are ideal red blood cell clusters, and therefore, the correlation of the shapes of the red blood cells after DSPE-PEG-FA modification on the ideal degree of the finally prepared red blood cell clusters is proved.

The modification of folic acid on the surface of red blood cells is realized by the hydrophobic acting force of a DSPE segment of DSPE-PEG-X type molecules in cell membranes, and the principle is shown in figure 1. And certain steric hindrance exists between the modified PEG chain segments in the DSPE-PEG-FA, so when the modification amount of the DSPE-PEG-FA is large, the shape of the erythrocyte is changed due to the steric repulsion between the PEG chain segments, and the erythrocyte is changed into a spherical shape from the original disc shape. It is therefore assumed that if the modification amount of DSPE-PEG-FA is small, the change degree of the morphology of the red blood cells should be small, so the invention firstly explores the influence of the modification amount of DSPE-PEG-X on the surface of the red blood cells on the morphology of the red blood cells. In the research, when the DSPE-PEG-X modification amount on the surface of the erythrocyte membrane is small, the steric hindrance generated between DSPE-PEG-X molecules is small, and only the surface of the erythrocyte generates convex deformation; when the modification amount of DSPE-PEG-X on the surface of the erythrocyte is larger, larger steric hindrance exists between PEG chain segments, so that the shape of the erythrocyte is converted into a ball shape with sharp spines; when the amount of DSPE-PEG-X modification is further increased, the pricks become smaller and the overall shape of the red blood cells becomes more rounded, as shown in FIG. 2. Further experiments show that the red blood cells with small DSPE-PEG-X modification amount have better deformation capability, while the spherical thorn-shaped red blood cells with large modification amount have poorer deformation capability, as shown in FIG. 3.

In the process of preparing the red blood cell clusters, the red blood cell clusters need to be separated from redundant red blood cells in a blowing suspension mode, so that the redundant red blood cells are separated out, and pure red blood cell clusters are obtained. For the red blood cells with better deformability, when the red blood cells are close to the surface modified microspheres, the red blood cells are in contact with the microspheres in a larger area in a deformation mode, so that the generated adsorption force is larger, the impact force of the blowing and suspending operation hardly causes the red blood cells to be separated from the surfaces of the microspheres, and finally the obtained red blood cell clusters with the surfaces tightly adsorbing the red blood cell layers are obtained. However, the rigidity of the red blood cells with poor deformability is relatively high, so that when the red blood cells are close to the surface-modified microspheres, the red blood cells are difficult to contact with each other in a large area due to deformation, and thus the adsorption force between the red blood cells and the surface-modified microspheres is relatively weak, and the impact force generated by the suspension operation easily causes the adsorbed red blood cells to peel off from the surfaces of the red blood cell clusters, so that it is difficult to finally obtain the red blood cell clusters with the surfaces tightly adsorbing the red blood cell layers.

From the existing experimental results, excessive DSPE-PEG-X modification can cause too strong erythrocyte rigidity, and further cause reduction of the adsorption quantity on the surface of the microsphere. Fig. 4 shows the results of preparing clusters from five morphologies of red blood cells and the same amount of microspheres, and it can be seen from the graph that the red color of the prepared red blood cell clusters changes regularly with the change of the morphology of the red blood cells, the shade of the red color represents the difference of the adsorption amount of the red blood cells, and the adsorption amount of the red blood cells of the sample with the darker color is larger. From left to right, sample 1 has the largest amount of DSPE-PEG-X modification, and the more spherical red blood cells are, the greater the rigidity, the least amount of red blood cells are adsorbed onto the prepared red blood cell clusters, and thus the lightest apparent color of the red blood cell clusters. The apparent color of the red blood cell clusters of

samples

2 and 3 gradually deepens with the gradually decreasing amount of the modified DSPE-PEG-X, which indicates that the red blood cell deformability is stronger with the decreasing amount of the modified DSPE-PEG-X, so that the red blood cell adsorption amount of the red blood cell clusters gradually increases. The apparent color of

samples

3 and 4 did not differ much as the amount of modification by DSPE-PEG-X was further decreased, indicating that the red blood cell deformability did not change much even though the amount of modification by DSPE-PEG-X was further decreased, and finally the amount of red blood cells adsorbed by the red blood cell clusters did not change much. The sample 5 is a red blood cell cluster prepared by using original red blood cells without modification of DSPE-PEG-X molecules, the surface of the red blood cells is only modified with polybrene, and the original red blood cells without modification of the DSPE-PEG-X molecules have the strongest deformability, so that the ideal red blood cell cluster can be obtained, and the apparent color of the red blood cell cluster is not different from that of the sample 3 and the sample 4.

As can be concluded from the results of the above exploratory experiments, the key to preparing the ideal clusters is to maintain sufficient deformability of the red blood cells using appropriate surface modification methods. Particularly for the erythrocyte with the surface modified with DSPE-PEG-X molecule, the modification amount of the molecule on the surface of the erythrocyte needs to be controlled within a reasonable range, and the ideal erythrocyte cluster can be finally obtained.

Based on the above, the purpose of the invention is realized by the following technical scheme:

a method for preparing erythrocyte cluster with ideal shape and appearance includes such steps as controlling the modification amount of DSPE-PEG-X molecules on the surface of erythrocyte to regulate the shape and shape of erythrocyte, and preparing erythrocyte with enough deformation capacityThe red cell clusters with ideal morphology are prepared. The preparation method of the red blood cell cluster can refer to Chinese patents CN111826349A (a red blood cell cluster for enriching circulating tumor cells based on a size filtration method) and CN111826351A (a magnetic red blood cell cluster for enriching circulating tumor cells based on a magnetic separation method). In the preparation method, the dosage relation between the erythrocyte and the DSPE-PEG-X is preferably 5X 10⁷The number of red blood cells is less than 0.04mg DSPE-PEG-X.

Further, the preparation method of the red blood cell clusters with ideal morphology comprises the following steps:

(1) modification of the surface of erythrocytes with folate or RGD or tumor cell-targeting antibodies according to method A or B

A. Modification of surfaces of erythrocytes with folic acid or RGD: adding DSPE-PEG-FA or DSPE-PEG-RGD into the erythrocyte dispersion, incubating, centrifuging, and washing to obtain erythrocyte with surface modified folic acid or RGD. Wherein the dosage proportion relationship of erythrocytes and DSPE-PEG-FA or DSPE-PEG-RGD is every 5 × 10⁷Adding DSPE-PEG-FA or DSPE-PEG-RGD below 0.04mg into red blood cells.

B. Antibody on erythrocyte surface modification: adding DSPE-PEG-biotin into the erythrocyte dispersion liquid, incubating, centrifuging and washing to obtain biotin-modified erythrocytes; adding biotin-modified erythrocytes into an SA (streptavidin) solution, uniformly mixing, incubating, and centrifuging and washing to obtain SA-modified erythrocytes; and adding the red blood cells modified by the SA into a biotinylated antibody solution, uniformly mixing, incubating, and centrifugally washing to obtain the red blood cells modified by the antibody. Wherein the dosage proportion relation of the erythrocyte, the DSPE-PEG-biotin, the SA and the biotinylated antibody is 5 multiplied by 10⁷10-100 μ g of DSPE-PEG-biotin with red blood cells less than 0.04mg and 0.5-5 μ g of biotinylated antibody with SA.

(2) Modification of polybrene on surface of erythrocyte

And (2) adding the red blood cells with the surfaces modified with folic acid, RGD or the antibody in the step (1) into polybrene solution, mixing uniformly, incubating, centrifuging and washing to obtain the red blood cells with the surfaces modified with polybrene and folic acid, RGD or the antibody, and dispersing the red blood cells with PBS for later use. Wherein the red blood cells are surface-modified with folic acid, RGD or antibodyThe proportional relation between the cell and the polybrene is 5 multiplied by 10⁷1-5 mg of polybrene per erythrocyte.

(3) Preparation of red blood cell clusters

And (3) uniformly mixing the magnetic microspheres with the surface modified with carboxyl or amino groups and the red blood cells obtained in the step (2), centrifuging to ensure that the red blood cells and the microspheres are close to each other at the bottom of a centrifugal tube, incubating, finally carrying out magnetic separation and washing on the mixture by using a magnetic frame for resuspension, washing and removing excessive free red blood cells, and finally obtaining magnetic red blood cell clusters, namely the red blood cell clusters with ideal morphology.

Further, in the method A of the step (1), the red blood cell dispersion liquid is obtained by dispersing red blood cells by using a PBS solution; adding DSPE-PEG-FA or DSPE-PEG-RGD into the erythrocyte dispersion liquid, wherein the PBS solution containing the DSPE-PEG-FA or DSPE-PEG-RGD is added; the molecular weight of the DSPE-PEG-FA or DSPE-PEG-RGD is 2000-10000; the incubation is static incubation at 4-37 ℃ for 0.5-30 min.

Further, the method A of the step (1) is as follows: based on the red blood cell content of the red blood cell dispersion, 5X 10 cells were taken out for each fixed amount⁷Quantitatively adding 0-0.04 mg of PBS solution containing DSPE-PEG-FA or DSPE-PEG-RGD with the molecular weight of 2000-10000 into red blood cells, standing and incubating for 30min at 4 ℃, and centrifuging and washing for three times by PBS to obtain the red blood cells with the surfaces modified with folic acid or RGD.

Further, in the method B of the step (1), the red blood cell dispersion is obtained by dispersing red blood cells with a PBS solution; adding DSPE-PEG-biotin into the erythrocyte dispersion liquid, namely adding PBS solution containing the DSPE-PEG-biotin; the molecular weight of the DSPE-PEG-biotin is 2000-10000; the SA solution is a PBS solution of SA, and the biotinylated antibody solution is a PBS solution of a biotinylated antibody; the first incubation is standing incubation at 4-37 ℃ for 0.5-30 min; the second and third incubations are incubation for 30 min-2 h at 4-37 ℃.

Further, the method B of the step (1) is as follows: based on the red blood cell content of the red blood cell dispersion, 5X 10 cells were taken out for each fixed amount⁷Quantitatively adding 0-0.04 mg of DSPE with molecular weight of 2000-10000 into red blood cellsA PBS solution of PEG-biotin, standing and incubating for 30min at 4 ℃, and centrifuging and washing three times by PBS to obtain biotin-modified red blood cells; uniformly mixing biotin-modified erythrocytes with 50-1000 mu L of SA solution with the concentration of 100 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by PBS to obtain SA-modified erythrocytes; and finally, uniformly mixing the SA modified erythrocyte with 100-1000 mu L of biotinylated antibody solution with the concentration of 5 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by PBS to obtain the erythrocyte of the surface modified tumor cell targeted antibody.

Further, in the step (2), the polybrene solution is a PBS solution of polybrene; the solution used for dispersing the red blood cells is PBS; the incubation is performed for standing incubation for 0.5-30 min at 4-37 ℃.

Further, the step (2) is: step (1) is carried out according to the proportion of 5 multiplied by 10⁷Mixing the erythrocyte with folic acid, RGD or antibody surface modified obtained by the dosage of the erythrocyte with 50-500 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifugally washing for three times by PBS to obtain the erythrocyte with polybrene surface modified and FA, RGD or antibody, and dispersing the erythrocyte into the PBS solution for later use.

Further, in the step (3), the number of the red blood cells is 50-500 times of the number of the magnetic microspheres, and the centrifugation condition is 200-600 g for 1-10 min; the incubation is performed for 0.5-30 min at 4-37 ℃.

Further, the step (3) is: washing the magnetic microspheres with the surface modified with carboxyl or amino for three times by PBS (phosphate buffer solution), adding the red blood cells obtained in the step (2) with the number more than 100 times of that of the microspheres, uniformly mixing the red blood cells and the red blood cells, centrifuging the mixture in a centrifuge at a centrifugal speed of 400g for 1min to ensure that the red blood cells and the microspheres are close to each other, centrifuging, standing and incubating the mixture at 4 ℃ for 30min, blowing and suspending the mixture at the bottom of the centrifuge tube, magnetically separating and washing the mixture by a magnetic frame, and washing and removing excessive free red blood cells to obtain magnetic red blood cell clusters, namely the red blood cell clusters with ideal morphology.

Biomimetic materials of erythrocyte clusters have been used for specific capture and enrichment of CTCs in peripheral blood, and the density of surface erythrocyte wrapping determines the purity of the CTCs obtained by final enrichment. The prior art has unstable factors and is difficult to ensure that the erythrocyte clusters with compact surfaces for adsorbing erythrocytes are obtained in each preparation. The invention provides a technical scheme capable of stably preparing ideal red blood cell clusters, the obtained ideal red blood cell clusters can recognize and capture tumor cells with high specificity through surface modifiers of the ideal red blood cell clusters, and a large number of red blood cells wrapped by the cluster surfaces can avoid non-specific adsorption of other unrelated cells, so that target tumor cells can be efficiently enriched with high specificity.

Drawings

FIG. 1 is a schematic diagram of the surface modification of DSPE-PEG-FA on erythrocytes.

FIG. 2 is a graph showing the effect of DSPE-PEG-X modification on the morphology of erythrocytes.

FIG. 3 is a graph comparing the effect of different DSPE-PEG-X modifications on the surface of erythrocytes on deformability, wherein the erythrocytes shown in FIG. 3(a) correspond to the erythrocytes shown in FIG. 2(a), and the erythrocytes shown in FIG. 3(b) correspond to the erythrocytes shown in FIG. 2 (e).

Fig. 4 is a color chart of red cell clusters solutions prepared from red blood cells of different morphologies, and the red cell clusters in samples 1 to 5 were red cell clusters prepared in comparative example 2, comparative example 1, example 3, example 2, and example 1, respectively.

FIG. 5 is a bright field plot of red blood cell clusters prepared from the raw red blood cells of FIG. 2(a) without modification of the DSPE-PEG-X molecule.

FIG. 6 is a bright field image of red blood cell clusters prepared from the raw red blood cells of FIG. 2(b) modified with DSPE-PEG-FA molecules.

FIG. 7 is a bright field image of red blood cell clusters prepared from the raw red blood cells of FIG. 2(c) modified with DSPE-PEG-biotin molecules.

FIG. 8 is a bright field plot of red blood cell clusters prepared from the raw red blood cells of FIG. 2(d) modified with DSPE-PEG-FA molecules.

FIG. 9 is a bright field image of red blood cell clusters prepared from the raw red blood cells of FIG. 2(e) modified with DSPE-PEG-RGD molecules.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 erythrocyte clusters prepared from primitive erythrocytes without modification of the DSPE-PEG-X molecule in FIG. 2(a)

Separating whole blood with percoll separating medium to obtain pure red blood cell, washing red blood cell with PBS solution for three times, dispersing red blood cell with PBS solution, and counting the red blood cell content in red blood cell dispersing liquid with cell counting plate. The morphology of the original erythrocytes without modification of DSPE-PEG-X is shown in FIG. 2 (a).

Polybrene modification: quantitatively taking out 0.5X 10 based on the content of red blood cells in the red blood cell dispersion⁸Adding 100 μ L polybrene PBS solution with concentration of 10mg/mL into each erythrocyte, standing and incubating for 30min at 4 deg.C, centrifuging and washing with PBS for three times to obtain surface-modified polybrene erythrocyte, and dispersing into PBS solution for use.

Preparing red cell clusters: washing magnetic microspheres (5-6 μm in particle size, magnetic polystyrene microspheres, Sedrin Sijie chromatographic technology development center) with surface modified carboxyl groups with PBS for three times, adding 100 times of modified red blood cells counted by the microspheres, uniformly mixing the two, centrifuging the mixture in a centrifuge at a centrifugation speed of 400g for 1min to ensure that the red blood cells and the microspheres are close to each other, centrifuging the mixture, standing the mixture at 4 ℃ for incubation for 30min, blowing and suspending the mixture at the bottom of the centrifuge tube, performing magnetic separation and washing on the mixture by using a magnetic frame, washing and removing excessive free red blood cells, and finally obtaining the magnetic red blood cell clusters.

The bright field photograph of the red cell clusters is shown in FIG. 5. As can be seen, the red cell clusters prepared from the red blood cells in FIG. 2(a) are relatively ideal. From the optical microscope photo of the erythrocyte clusters, the surfaces of most erythrocyte clusters are fully covered with a layer of erythrocytes, the exposed surfaces of the cluster core microspheres are too dispersed, thus being not beneficial to nonspecific adsorption of leukocytes by the core microspheres, and the erythrocyte layer with compact surfaces is stacked to be beneficial to the rejection effect of the erythrocyte clusters on the leukocytes, thereby realizing the capture of high-purity CTCs.

Example 2 erythrocyte clusters prepared from Primary erythrocytes modified with DSPE-PEG-FA molecule in FIG. 2(b)

Separating whole blood with percoll separating medium to obtain pure red blood cell, washing red blood cell with PBS solution for three times, dispersing red blood cell with PBS solution, and counting the red blood cell content in red blood cell dispersing liquid with cell counting plate.

Folic acid modification: quantitatively taking out 2X 10 based on the content of red blood cells in the red blood cell dispersion⁸Quantitatively adding 0.02mg of DSPE-PEG-FA solution with molecular weight of 5000 into the red blood cells, standing and incubating at 4 deg.C for 30min, and centrifuging and washing with PBS for three times to obtain folic acid modified red blood cells, wherein the appearance is shown in FIG. 2 (b).

Modification of polybrene: mixing the erythrocyte modified with folic acid with 300 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifuging and washing for three times by PBS to obtain the erythrocyte modified with surface polybrene and folic acid, and dispersing the erythrocyte modified with surface polybrene and folic acid into the PBS solution for later use.

Preparing red cell clusters: washing the magnetic microspheres with the surface modified carboxyl groups for three times by PBS, adding modified red blood cells with the number being 100 times of the counted number of the microspheres, centrifuging the modified red blood cells and the modified red blood cells for 1min at a centrifuging speed of 400g after the two are uniformly mixed, centrifuging the red blood cells and the microspheres to the bottom of a centrifuge tube to ensure that the red blood cells and the microspheres are close to each other, standing and incubating the mixture at 4 ℃ for 30min after centrifugation, blowing and suspending the mixture at the bottom of the centrifuge tube, performing magnetic separation and washing on the mixture by a magnetic frame, washing and removing excessive free red blood cells, and finally obtaining magnetic red blood cell clusters.

The bright field photograph of the red cell clusters is shown in FIG. 6. As can be seen, the red cell clusters prepared from the red blood cells in FIG. 2(b) are relatively ideal. From the optical microscope photo of the erythrocyte clusters, the surfaces of most erythrocyte clusters are fully covered with a layer of erythrocytes, the exposed surfaces of the cluster core microspheres are too dispersed, thus being not beneficial to nonspecific adsorption of leukocytes by the core microspheres, and the erythrocyte layer with compact surfaces is stacked to be beneficial to the rejection effect of the erythrocyte clusters on the leukocytes, thereby realizing the capture of high-purity CTCs.

Example 3 erythrocyte clusters prepared from Primary erythrocytes modified with DSPE-PEG-biotin molecule in FIG. 2(c)

EpCAM antibody modification: quantitatively taking out 1.5X 10 based on the content of red blood cells in the red blood cell dispersion⁸Quantitatively adding 0.12mg of DSPE-PEG-biotin PBS solution with molecular weight of 10000 into red blood cells, standing and incubating for 30min at 4 ℃, and centrifuging and washing for three times by PBS to obtain the red blood cells of surface modified biotin; uniformly mixing biotin-modified erythrocytes with 200 mu L of SA solution with the concentration of 100 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifuging and washing for three times by PBS to obtain erythrocytes with surface-modified SA; and finally, uniformly mixing the SA modified erythrocyte with 500 mu L of biotinylated EpCAM antibody solution with the concentration of 5 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by PBS to obtain the erythrocyte with the surface modified EpCAM antibody, wherein the appearance of the erythrocyte is shown in figure 2 (c).

Modification of polybrene: mixing the red blood cells modified with the EpCAM antibody with 200 mu L of polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifuging and washing for three times by PBS to obtain the red blood cells of the surface modified polybrene and the EpCAM antibody, and dispersing the red blood cells into the PBS solution for later use.

The bright field photograph of the red cell clusters is shown in FIG. 7. As can be seen, the red cell clusters prepared from the red blood cells in FIG. 2(c) are relatively ideal. From the optical microscope photo of the red blood cell clusters, the surfaces of most red blood cell clusters are fully covered with a layer of red blood cells, the exposed surfaces of the cluster core microspheres are too dispersed, so that the non-specific adsorption of white blood cells by the core microspheres is not facilitated, the red blood cell layer with a tight surface is stacked, the rejection effect of the red blood cell clusters on the white blood cells is facilitated, and the high-purity CTCs can be captured.

Comparative example 1 erythrocyte clusters prepared from DSPE-PEG-FA-modified erythrocytes in FIG. 2(d)

Folic acid modification: quantitatively taking out 1X 10 based on the content of red blood cells in the red blood cell dispersion⁸Quantitatively adding 0.4mg of DSPE-PEG-FA solution with molecular weight of 2000 into the red blood cells, standing and incubating at 4 deg.C for 30min, and centrifuging and washing with PBS for three times to obtain folic acid modified red blood cells, wherein the appearance is shown in FIG. 2 (d).

Modification of polybrene: mixing the erythrocyte modified with folic acid with 200 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifuging and washing for three times by PBS to obtain the erythrocyte modified with surface polybrene and folic acid, and dispersing the erythrocyte modified with surface polybrene and folic acid into the PBS solution for later use.

The bright field photograph of the red cell clusters is shown in FIG. 8. As can be seen, the red blood cell clusters produced by the red blood cells in FIG. 2(d) are not ideal and have many defects. From the optical microscope photo of the erythrocyte clusters, the surface of a larger part of erythrocyte clusters has obvious gaps, namely, modified erythrocytes are not adsorbed on the part of the surface where the cluster core microspheres are located, and the exposed surface of the core microspheres has obvious nonspecific adsorption effect on leukocytes, which has great adverse effect on the purity of the captured CTCs.

Comparative example 2 erythrocyte clusters prepared from DSPE-PEG-RGD-modified erythrocytes shown in fig. 2(e)

RGD modification: quantitatively taking out 1X 10 based on the content of red blood cells in the red blood cell dispersion⁸Quantitatively adding 0.4mg of DSPE-PEG-RGD PBS solution with molecular weight of 2000 into red blood cells, standing and incubating at 4 deg.C for 30min, and centrifuging and washing with PBS for three times to obtain folic acid modified red blood cells, the appearance of which is shown in FIG. 2 (e).

Modification of polybrene: mixing red blood cells modified with RGD with 200 μ L polybrene PBS solution with concentration of 10mg/mL, standing and incubating for 30min at 4 deg.C, centrifuging and washing with PBS for three times to obtain surface-modified polybrene and RGD red blood cells, and dispersing into PBS solution for use.

Preparing red cell clusters: washing the magnetic microspheres with the surface modified with carboxyl groups for three times by using PBS (phosphate buffer solution), adding modified red blood cells with the number more than 100 times of the counted number of the microspheres, uniformly mixing the magnetic microspheres and the modified red blood cells, centrifuging the mixture in a centrifuge at a centrifugal speed of 400g for 1min to ensure that the red blood cells and the microspheres are close to each other to the bottom of a centrifuge tube, centrifuging, standing and incubating the mixture at 4 ℃ for 30min, blowing the mixture at the bottom of the centrifuge tube, magnetically separating and washing the mixture by using a magnetic frame, washing and removing excessive free red blood cells, and finally obtaining the magnetic red blood cell clusters.

The bright field photograph of the red cell clusters is shown in FIG. 9. As can be seen, the red blood cell clusters produced by the red blood cells in FIG. 2(e) are not ideal and have many defects. From the optical microscope photo of the erythrocyte clusters, the surface of a larger part of erythrocyte clusters has obvious gaps, namely, modified erythrocytes are not adsorbed on the part of the surface where the cluster core microspheres are located, and the exposed surface of the core microspheres has obvious nonspecific adsorption effect on leukocytes, which has great adverse effect on the purity of the captured CTCs.

The examples provided above are merely illustrative of the method of the present invention and are not intended to limit the remainder of the disclosure in any way. Other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A preparation method of red blood cell clusters with ideal morphology is characterized by comprising the following steps: the preparation method realizes the controllable regulation of the morphology of the red blood cells by controlling the modification quantity of DSPE-PEG-X molecules on the surfaces of the red blood cells, so that the red blood cells with enough deformation capacity can be controllably prepared, and the red blood cell clusters with ideal morphology are prepared on the basis of the red blood cells; wherein, X represents folic acid, RGD or biotin; the dosage relationship between the red blood cells and the DSPE-PEG-X is 5 multiplied by 10⁷DSPE-PEG-X with the dose of less than 0.04mg per erythrocyte;

the method for preparing the red blood cell clusters with ideal morphology comprises the following steps:

modification of polybrene on the surface of erythrocytes: adding red blood cells with the surface modified by DSPE-PEG-X into polybrene solution, mixing uniformly, incubating, centrifuging, washing to obtain red blood cells with the surface modified by polybrene and DSPE-PEG-X, and dispersing the red blood cells for later use; wherein the dosage proportion relation of the erythrocytes and polybrene in the erythrocytes for modifying the DSPE-PEG-X is 5 multiplied by 10⁷1-5 mg of polybrene per erythrocyte;

preparing red cell clusters: uniformly mixing the magnetic microspheres with the surface modified with carboxyl or amino, the surface modified polybrene and the red blood cells of DSPE-PEG-X, centrifuging, incubating, resuspending the mixture, and carrying out magnetic separation and washing on the mixture by using a magnetic frame to obtain the red blood cell clusters with ideal morphology.

2. A preparation method of red blood cell clusters with ideal morphology is characterized by comprising the following steps: the method comprises the following steps:

A. Modification of surfaces of erythrocytes with folic acid or RGD: adding DSPE-PEG-FA or DSPE-PEG-RGD into the erythrocyte dispersion, incubating, centrifuging, and washing to obtain erythrocytes with folic acid or RGD modified on the surface; wherein the dosage proportion relationship of red blood cells and DSPE-PEG-FA or DSPE-PEG-RGD is 5 × 10⁷Adding DSPE-PEG-FA or DSPE-PEG-RGD below 0.04mg into red blood cells;

B. antibody on erythrocyte surface modification: adding DSPE-PEG-biotin into the erythrocyte dispersion liquid, incubating, centrifuging and washing to obtain biotin-modified erythrocytes; adding biotin-modified erythrocytes into the SA solution, uniformly mixing, incubating, and centrifuging and washing to obtain SA-modified erythrocytes; adding the red blood cells modified by the SA into a biotinylated antibody solution, uniformly mixing, incubating, and centrifuging and washing to obtain red blood cells modified by the antibody; wherein the dosage proportion relation of the erythrocyte, the DSPE-PEG-biotin, the SA and the biotinylated antibody is 5 multiplied by 10⁷10-100 μ g of DSPE-PEG-biotin with red blood cells less than 0.04mg and 0.5-5 μ g of biotinylated antibody of SA;

(2) modification of polybrene on surface of erythrocyte

Adding the red blood cells with the surfaces modified with folic acid, RGD or antibodies in the step (1) into polybrene solution, mixing uniformly, incubating, centrifuging and washing to obtain the red blood cells with the surfaces modified with polybrene and folic acid, RGD or antibodies, and dispersing the red blood cells for later use; wherein the dosage proportion relation of erythrocytes and polybrene in erythrocytes with folic acid, RGD or antibody modified on the surface is 5 × 10⁷1-5 mg of polybrene per erythrocyte;

(3) preparation of red blood cell clusters

And (3) uniformly mixing the magnetic microspheres with the surface modified with carboxyl or amino groups with the red blood cells obtained in the step (2), centrifuging, incubating, re-suspending the mixture, and carrying out magnetic separation and washing on the mixture by using a magnetic frame to obtain the red blood cell clusters with ideal morphology.

3. The method for preparing the red blood cell clusters with ideal morphology according to claim 2, wherein: in the method A in the step (1), the molecular weight of the DSPE-PEG-FA or the DSPE-PEG-RGD is 2000-10000, and the incubation is static incubation for 0.5-30 min at 4-37 ℃.

4. The method according to claim 2, wherein the method comprises: the method A in the step (1) comprises the following steps: based on the red blood cell content of the red blood cell dispersion, 5X 10 cells were taken out for each fixed amount⁷Quantitatively adding 0-0.04 mg of PBS solution containing DSPE-PEG-FA or DSPE-PEG-RGD with the molecular weight of 2000-10000 into red blood cells, standing and incubating for 30min at 4 ℃, and centrifuging and washing for three times by PBS to obtain the red blood cells with the surfaces modified with folic acid or RGD.

5. The method according to claim 2, wherein the method comprises: in the method B in the step (1), the molecular weight of the DSPE-PEG-biotin is 2000-10000, the first incubation is static incubation at 4-37 ℃ for 0.5-30 min, and the second and third incubations are static incubation at 4-37 ℃ for 30 min-2 h.

6. The method according to claim 2, wherein the method comprises: the method B in the step (1) comprises the following steps: based on the red blood cell content of the red blood cell dispersion, 5X 10 cells were taken out for each fixed amount⁷Quantitatively adding 0-0.04 mg of PBS solution containing DSPE-PEG-biotin with the molecular weight of 2000-10000 into red blood cells, standing and incubating for 30min at 4 ℃, and centrifugally washing for three times by using PBS to obtain the biotin-modified red blood cells; uniformly mixing biotin-modified erythrocytes with 50-1000 mu L of SA solution with the concentration of 100 mu g/mL, and standing and incubating at 37 DEG CCentrifuging and washing for three times by PBS (phosphate buffer solution) for 30min to obtain SA modified erythrocytes; and finally, uniformly mixing the SA modified red blood cells with 100-1000 mu L of biotinylated antibody solution with the concentration of 5 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by using PBS (phosphate buffer solution) to obtain the red blood cells of the surface modified tumor cell targeted antibody.

7. The method according to claim 2, wherein the method comprises: the incubation in the step (2) is static incubation at 4-37 ℃ for 0.5-30 min.

8. The method according to claim 2, wherein the method comprises: the step (2) is as follows: step (1) is carried out according to the proportion of 5 multiplied by 10⁷Mixing the erythrocyte with folic acid, RGD or antibody surface modified obtained by the dosage of the erythrocyte with 50-500 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifugally washing for three times by PBS to obtain the erythrocyte with polybrene surface modified and FA, RGD or antibody, and dispersing the erythrocyte into the PBS solution for later use.

9. The method according to claim 2, wherein the method comprises: in the step (3), the number of the red blood cells is 50-500 times of the number of the magnetic microspheres, the centrifugation condition is 200-600 g, the centrifugation is 1-10 min, and the incubation is static incubation for 0.5-30 min at 4-37 ℃.

10. The method according to claim 2, wherein the method comprises: the step (3) is as follows: washing the magnetic microspheres with the surface modified with carboxyl or amino for three times by PBS, adding the erythrocyte obtained in the step (2) with the number more than 100 times of that of the microspheres, uniformly mixing the magnetic microspheres and the erythrocyte, centrifuging the mixture in a centrifuge at the centrifugal speed of 400g for 1min, standing and incubating the mixture at 4 ℃ for 30min, blowing and suspending the mixture at the bottom of the centrifuge tube, and carrying out magnetic separation and washing on the mixture by a magnetic frame to obtain the erythrocyte clusters with ideal shapes.