CN110628692A

CN110628692A - Multi-stage microsphere based on virus-like structure for efficient cell capture

Info

Publication number: CN110628692A
Application number: CN201910841252.XA
Authority: CN
Inventors: 孙诗语; 王锐敏; 黄议达; 刘万山
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2019-12-31

Abstract

The invention discloses a multi-stage microsphere based on a virus-like structure for efficient cell capture, and relates to the field of cell biology. The multi-stage microsphere prepared by the invention is obtained by simple electrostatic interaction, covalent bond combination and nanoparticle adhesion assembly, and the surface of the multi-stage microsphere is modified by hyaluronic acid, so that the multi-stage microsphere has excellent surface hydrophilicity, biocompatibility and chemical structure stability, and in a cell capture experiment, due to the surface topological structure and chemical structure which are controllable by materials, the virus-like structure multi-stage microsphere has extremely high capture rate on unmarked cells, and has obvious progress compared with the prior art.

Description

Multi-stage microsphere based on virus-like structure for efficient cell capture

Technical Field

The invention relates to the field of cell biology, in particular to a multi-stage microsphere based on a virus-like structure for efficient cell capture.

Background

Cell capture refers to a process of separating one or more cells from a liquid by physical, chemical, biological means and the like. Cell capture is an important experimental content in biology, medical diagnosis, pathological detection and the like, and is applied to various aspects of biological experiments. For example, in immunological experiments, immune cells are specifically separated and captured for cell engineering, target cells are captured for gene programming in gene engineering, and diseased cells are captured for disease diagnosis in disease screening. Among them, capturing of tumor circulating cells for diagnosis, monitoring, analysis, etc. of cancer is attracting much attention from researchers all over the world.

The cell capture technologies that exist at present are mainly divided into pre-labeled cell capture technologies and label-free cell capture technologies. The pre-marked cell capture technology comprises a flow cytometry technology, a nano magnetic bead adsorption method and the like; label-free cell trapping includes optical tweezers technology, centrifugation technology, microfluidic technology, and the like.

Flow cytometry is a common cell analysis and sorting method in laboratories. The method comprises the steps of dyeing cells with fluorescent dye in advance, dividing a cell suspension into single-cell droplets, performing fluorescence excitation on the fluorescent dye of the cells by utilizing laser, and analyzing and sorting the cells according to the fluorescence intensity of the cells. The nano magnetic bead adsorption method needs to label cells in advance, combines specific molecules on the nano magnetic beads with the cells, separates the cells through the magnetism of the magnetic beads, and then elutes and separates the cells through pancreatin. Both the two pre-labeling cell capture methods need to pre-treat cells, the toxicity of fluorescent dye and the endocytosis of nano magnetic beads by the cells can damage the cells, and the pre-treatment time is long and the operation process is complex.

The optical tweezers technology can manipulate single cells or molecules by utilizing the mechanical effect of laser, so as to realize cell capture and separation, and the optical tweezers technology needs a large-scale precise laser emitter and a controller for support, is complex to operate and is difficult to be applied to cell capture of a large system. The microfluidic technology can adjust the size and the property of a channel, and separates and sorts cells by utilizing the physical properties of the size, the mass and the like of the cells, so that label-free cell capture is realized, but the microfluidic technology has high difficulty and the volume and the quantity of processed samples are limited.

In summary, the conventional cell capturing and separating method is complicated and expensive, and damages the cells during the capturing process, so that a simple and efficient cell capturing technology is urgently needed to be developed.

The fine ordered structure and high-level complex function of life bodies in nature are the objects of chemists and materialists in bionics. Because of its high adsorptivity to cells, natural virus particles have become important carrier materials in the fields of biological medicine and nano medicine, and have great application value in the in vivo delivery of bioactive molecules (such as chemotherapeutic drugs and therapeutic genes). However, as clinical research advances, potential hazards (e.g., immunogenicity, mutagenesis) of viral vectors are gradually exposed, and thus, the construction of advanced virus-like systems by means of material engineering has become a focus of research in the current fields of chemistry, materials and nano-research. At present, the virus-like structure is mainly used for in-situ imaging and drug delivery system construction.

Therefore, those skilled in the art are devoted to develop a multi-stage microsphere with a virus-like structure and a preparation method thereof, so that the prepared microsphere not only has high cell affinity, overcomes the size limitation that the secondary nano virus-like structure is easily phagocytized by cells in the prior art, but also has extremely high cell capture efficiency.

Disclosure of Invention

In view of the above defects in the prior art, the technical problem to be solved by the present invention is how to provide a multi-stage microsphere with a virus-like structure capable of efficiently capturing cells and a preparation method thereof, so that the prepared microsphere has high cell affinity, high biocompatibility, extremely high cell capture efficiency while overcoming the size limitation that a secondary nano virus-like structure is easily phagocytized by cells, and overcoming the defects in the prior art.

In order to achieve the aim, the invention provides a multi-stage microsphere based on a virus-like structure for efficient cell capture, wherein the multi-stage microsphere is a three-stage structure microsphere assembled by nanoparticles, nano silicon spheres and glass microspheres, and the surface of the multi-stage microsphere is modified by hyaluronic acid.

Further, the size of the nano-particles is 15nm, the size of the nano-silicon spheres is 200nm, and the glass microspheres are selected from any one of the sizes of 150, 500 and 1000 μm.

The invention also provides a preparation method of the multi-stage microspheres based on the viroid structure for high-efficiency cell capture, which comprises the following steps:

step one, adopting improvementPreparing a suspension containing nanoparticles; by usingSynthesizing nano silicon balls by the method, washing and drying for later use; carrying out surface modification treatment on the glass microspheres, washing and drying for later use;

step two, after the nano silicon spheres prepared in the step one are subjected to amination modification treatment, mixing and stirring with the suspension containing the nano particles, centrifuging, collecting, drying and calcining to obtain secondary microspheres;

step three, performing amination modification treatment on the second-stage microspheres prepared in the step two, combining the second-stage microspheres with the glass microspheres treated in the step one through EDC-NHS coupling reaction to obtain the multistage microspheres, and washing and drying the multistage microspheres;

step four, modifying hyaluronic acid to the surface of the multistage microsphere obtained in the step three through EDC-NHS coupling reaction.

Further, the specific mode of the amination modification treatment is as follows: the material was suspended in toluene (99.5%) and after addition of 3-aminopropyltriethoxysilane (APTES, 99%), refluxed at 383K for 14h more.

Further, the surface modification treatment of the glass microspheres in the first step specifically includes the following steps:

step 1, soaking the glass microspheres in a piranha solution for more than 5 hours for carboxylation treatment, and then washing and drying;

and 2, dispersing the glass microspheres carboxylated in the step 1 in dimethylformamide (DMF, 99.5%), adding a succinic anhydride solution under vigorous stirring, standing for 24 hours, washing and drying.

Further, the improvement in the first stepThe method comprises the following steps: dissolving L-arginine (99%) in a mixed solution of deionized water and octane (99%), stirring for more than 8 hours at 333K, adding tetraethyl orthosilicate (TEOS, SiO2 is more than or equal to 28.4%) and then continuing stirring for 5 hours to obtain the suspension containing the nanoparticles.

Further, the mixing reaction time of the suspension containing the nano particles and the nano silicon spheres in the second step is 20 hours.

Further, in the third step, the EDC-NHS coupling reaction time of the secondary microspheres and the glass microspheres is 20 h.

Further, in the fourth step, the EDC-NHS coupling reaction time of the hyaluronic acid and the multistage microspheres is more than 8 h.

The invention also provides an application of the multi-stage microspheres based on the virus-imitating structures, which is characterized by comprising cell capture.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) the microspheres prepared by the invention can be assembled into stable micron-sized multi-level microspheres with virus-like surface topological structures through simple electrostatic interaction, covalent bonds and crosslinking among small-sized nano particles, and the preparation method is simple and efficient;

(2) the micron-sized multistage microspheres prepared by the invention effectively overcome the size limitation that the two-stage nano virus-like structure is easily phagocytized by cells in the prior art;

(3) the multistage microsphere prepared by the invention has excellent surface hydrophilicity, biocompatibility and chemical structure stability through simple and effective surface modification;

(4) the virus-like structure multistage microsphere provided by the invention has controllable surface topological structure and chemical structure, has good affinity to cells, and shows extremely high cell capture efficiency to various cells.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram of the steps for synthesizing multi-stage microspheres with a virus-like structure and cell capture according to a preferred embodiment of the present invention;

FIG. 2 is a graph of data on the capture efficiency of MCF-7 cells by three size multi-stage microspheres (HHB-150/500/1000) prepared according to a preferred embodiment of the present invention;

FIG. 3 is a graph of data showing the efficiency of capture of MCF-7 cells by multi-stage microspheres (HHB-1000) prepared according to a preferred embodiment of the present invention;

FIG. 4 is a graph of data showing the efficiency of capture versus the effect of multistage microspheres (HHB-1000) on A549 cells prepared according to a preferred embodiment of the present invention;

FIG. 5 is a graph of data showing the efficiency of capturing SKBR-3 cells versus the effect of multistage microspheres (HHB-1000) prepared according to a preferred embodiment of the present invention;

FIG. 6 is a fluorescence microscope image of A549 cells captured by multi-stage microspheres (HHB-1000) prepared according to a preferred embodiment of the present invention, wherein FIG. a is a whole fluorescence microscope image of one microsphere captured cell, and FIG. b is a partial magnified fluorescence microscope image of FIG. a;

FIG. 7 is a fluorescence micrograph of MCF-7 cells captured by multi-stage microspheres (HHB-1000) prepared according to a preferred embodiment of the present invention, wherein FIG. a is a global fluorescence micrograph of cells captured by one of the microspheres, and FIG. b is a partially magnified fluorescence micrograph of FIG. a;

FIG. 8 is a fluorescence micrograph of MCF-7 cells captured by multi-stage microspheres (HB-1000/HHB-1000) prepared according to a preferred embodiment of the present invention, wherein FIG. a is a fluorescence micrograph of HB-1000 captured cells and FIG. b is a fluorescence micrograph of HHB-1000 captured cells;

FIG. 9 is a fluorescence micrograph of A549 cells captured by multi-stage microspheres (HHB-1000) prepared according to a preferred embodiment of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

EXAMPLE I preparation of Virus-like Structure Multi-stage microspheres

The preparation steps of the virus-like structure multistage microsphere provided by the invention are shown in figure 1, firstly, combining a nanoparticle 1 with a nano silicon sphere 2 and then calcining to obtain a secondary microsphere 3; carrying out advanced amination modification treatment on the secondary microspheres 3 to obtain secondary microspheres 4 with amino modified surfaces, and combining the secondary microspheres 4 with glass microspheres 5 according to EDC-NHS coupling reaction to obtain multistage microspheres 6; and modifying the surface of the multistage microsphere 6 with hyaluronic acid according to EDC-NHS coupling reaction to obtain the multistage microsphere 7 modified with hyaluronic acid on the surface, wherein the surface modification can enhance the surface hydrophilicity, biocompatibility and chemical structure stability of the prepared multistage microsphere. And then the cell can be used for the subsequent cell capturing operation. The specific preparation process is as follows.

1. Synthesis of multi-sized microspheres

For nanoparticles (S1), amino acid reactions are used, with modificationsThe method synthesizes the silicon dioxide nano-particles with the size of about 15 nm. 87mg of L-arginine (99%) were dissolved in a solution containing 69.5mL of deionized water and 5.23mL of octane (99%), and the mixture was stirred at 333K for 8h or more. Then 0.5mL of tetraethyl orthosilicate (TEOS, SiO2 ≥ 28.4%) was added to the previous mixture and stirred for 5 hours before the silica nanoparticles (S2) were used directly.

For nano silicon spheres (S2), the method is carried out by a common methodThe method synthesizes the silicon dioxide spheres with the size of about 200 nm. 150mL of ethanol (. gtoreq.99.7%), 11.4mL of deionized water and 7.0mL of ammonia were mixed and 8.4mL of TEOS was added to the solution with vigorous stirring at 308K. After stirring for 6 hours, nanospheres were obtained by centrifugation and washed several times with ethanol and deionized water, and the product was dried for more than 8h at 373K.

For micron sized glass microspheres (S3), glass microspheres 150/500/1000 μm in size were purchased from Sigma-Aldrich and carboxylated prior to use. The glass beads were hydroxylated by soaking in piranha solution for 5h at room temperature. The microspheres were washed with water and dried at 373K for 2 h. The hydroxylated S3 was then aminated and then dispersed in 60mL dimethylformamide (DMF, 99.5%), 5mL succinic anhydride solution (dissolved in DMF, 0.1g/mL) was added with vigorous stirring and left for 24h, the beads were washed and dried for future use.

2. Assembling multi-stage microspheres

For amination modification of the material, the material may be suspended in 90mL of toluene (99.5%), 4.8mL of 3-aminopropyltriethoxysilane (APTES, 99%) added, and the system refluxed at 383K for 14h or more to complete the amino modification.

During assembly, 100mg of aminated S2 was first dissolved in 60mL of deionized water and mixed with 10.8mL of S1 suspension and sonicated for 20 min. The mixture (S1 with negatively charged surface and S2 with positively charged surface after modification of the amination) was stirred at 333K for 20h and centrifuged at 10000rpm for 5min (note that S1 was not separated under this condition). The collected solid was dried and calcined at 823K for 5h to give secondary microspheres (HSS). Followed by amino modification of HSS.

The aminated modified HSS can be combined with carboxylated S3 by EDC-NHS coupling reaction. Carboxylated S3 was dispersed in a buffer containing excess 3- (3-dimethylaminopropyl) -1-ethylcarbodiimide hydrochloride (EDAC, 97%) and N-hydroxysuccinimide (NHS, 98%) to activate the carboxyl groups according to conventional methods, followed by addition of the aminated modified HSS and incubation at room temperature. After a reaction time of 20h, the multistage microspheres (HB) were obtained, washed with deionized water and dried for further use.

3. Hyaluronic acid functionalization

To achieve hyaluronic acid functionalized modification of the surface of the multinary microsphere, we combined the multinary microsphere with hyaluronic acid by EDC-NHS coupling using a method similar to that for synthesis of HB. The specific procedure is as follows, 50mg of hyaluronic acid are dispersed in 20mL of MES buffer (0.01M) and 7.7mg of EDAC (97%) and 6.9mg of NHS (98%) are added. The mixture was adjusted to pH 5-6 and incubated at room temperature for 30 minutes to activate the carboxyl groups. Then, 20mL of HB-containing phosphate buffer (PBS, pH 7.4, 0.1M) was added, checked and adjusted to pH 7.4-8.0 and incubated at 38 ℃ for 8h or more with slow stirring. And standing to separate out the functionalized and modified multistage microspheres (HHB), washing with deionized water, drying at normal temperature and storing for later use.

EXAMPLE II cell Capture experiment

The process of the virus-like structure multi-stage microsphere prepared by the invention when cell capture is carried out is shown in figure 1, the multi-stage microsphere 7 with the surface modified with hyaluronic acid is added into a cell suspension 8, and the mixture is shaken for a period of time, so that the cells can be captured on the microsphere surface, and a combination body 9 of the microsphere and the cells is obtained. The specific operation is as follows.

For the capture experiments, cells were digested with 0.25% Trypsin/EDTA, resuspended in phosphate buffered saline (PBS, 0.1M, pH 7.4), and counted using a hemocytometer. The cell suspension was dispensed into centrifuge tubes, and a defined amount of the multi-stage microspheres prepared in example one were added to the cell solution and the cell suspension was slowly shaken on a shaker. Thereafter, the microspheres were gently blasted with the cell suspension to remove cells that fell on the microspheres due to sedimentation. After standing, the multi-stage microspheres after cell capture can be separated.

The cell capturing effect is mainly analyzed from the aspects of cell counting statistics capturing rate and fluorescence microscopic images. Cells were stained with both DAPI (all cells were stained) and Calcein-AM (only live cells were stained).

First, the cell capturing efficiency of the multi-stage microspheres prepared by using glass microspheres of three sizes was compared.

The results of the statistical data obtained from experiments using MCF-7 cells are shown in FIG. 2. It can be seen that the cell capture efficiency of the multi-stage microspheres (HHB-1000) prepared with 1000 μm glass microspheres is significantly higher than that of the other two sizes of multi-stage microspheres. Therefore we subsequently used HHB-1000 to capture cells and compared the results with S3 (untreated 1000 μm microspheres) and S3-HA (1000 μm microspheres directly modified with hyaluronic acid). The experimental result shows that the capture rate of HHB-1000 to MCF-7 cells reaches 87.9 percent (shown in figure 3), the capture rate to A549 cells reaches 92.9 percent (shown in figure 4), the capture rate to SKBR-3 cells reaches 98.7 percent (shown in figure 5), and the capture rate is superior to S3 (the capture rate to MCF-7 cells is 2.4 percent, the capture rate to A549 cells is 2.4 percent, the capture rate to SKBR-3 cells is 0 percent, p is less than 0.005) and S3-HA (the capture rate to MCF-7 cells is 21.1 percent, the capture rate to A549 cells is 19.9 percent, the capture rate to SKBR-3 cells is 12.5 percent, and p is less than 0.001).

The data comparison shows that the multistage microspheres have great advantages in cell capture, the capture efficiency is high, and the practical application requirements are met.

Second, the binding of the multi-stage microspheres to cells was observed.

The fluorescence microscopic image of A549 captured by HHB-1000 multistage microspheres (as shown in FIG. 6) can show that the combination effect of cells and microspheres is good. Such an effect is also seen in the fluorescence microscopy images (shown in FIG. 7) of MCF-7 cells captured by HHB-1000.

Finally, the influence of hyaluronic acid modification on the efficiency of capturing cells by the multistage microspheres is comparatively analyzed.

As can be seen from the fluorescence microscopic images (shown in figure 8) obtained by respectively capturing MCF-7 cells by using the multistage microsphere HB-1000 without modified hyaluronic acid and the HHB-1000 modified with hyaluronic acid, the fluorescence intensity of the fluorescence microscopic images of the HHB-1000 group is higher, and particularly, compared with a DAPI channel image which can be dyed by all cells and a Calcein-AM channel image which can only be dyed by living cells, the number of living cells captured on the HHB-1000 is obviously greater than that of HB-1000, so that the biocompatibility of the microsphere is improved by the hyaluronic acid functionalized modification on the microsphere surface, and the method has an important significance for the cell capturing effect.

Further, Calcein-AM channel images and DAPI channel images (shown in FIG. 9) of the same field of view in the fluorescence microscopic images of A549 cells captured by HHB-1000 were subjected to cell counting, and the captured viable cell rate was calculated. The three independent calculations gave live cell rates of 96%, 91% and 92%, with an average live cell rate of 93%. According to the living cell rate, the cell capture efficiency is also corrected, so that the living cell capture efficiency of the multistage microspheres is about 82% -91% on the premise of not including captured dead cells.

The analysis of the embodiment shows that the multi-stage microspheres with the virus-like structures have high cell affinity and structural stability, overcome the size limitation that the secondary nano virus-like structures are easily phagocytized by cells, and show extremely high cell capture efficiency, particularly the cell capture efficiency aiming at living cells. And the provided preparation method can realize controllable preparation of the surface topological structure and the chemical structure of the multistage microsphere.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. The multi-stage microsphere based on the virus-like structure for efficient cell capture is characterized in that the multi-stage microsphere is a three-stage structure microsphere assembled by nanoparticles, nano silicon spheres and glass microspheres, and the surface of the multi-stage microsphere is modified with hyaluronic acid.

2. The multi-stage microsphere based on the viroid structure for high efficiency cell capture according to claim 1, wherein the size of the nanoparticle is 15nm, the size of the nano-silica sphere is 200nm, and the size of the glass microsphere is selected from any one of 150, 500 and 1000 μm.

3. The method for preparing the multi-stage microspheres based on the viroid structure for high-efficiency cell capture according to claims 1-2, wherein the method comprises the following steps:

4. The preparation method of the multi-stage microsphere based on the viroid structure for high-efficiency cell capture according to claim 3, wherein the specific mode of the amination modification treatment is as follows: the material was suspended in toluene (99.5%) and after addition of 3-aminopropyltriethoxysilane (APTES, 99%), refluxed at 383K for 14h more.

5. The method for preparing multilevel microspheres based on viroid structures for efficient cell capture according to claim 3, wherein the surface modification treatment on the glass microspheres in the first step specifically comprises the following steps:

6. The method of claim 3, wherein the improvement in the first step is a method of preparing multi-stage microspheres based on a viroid structure for efficient cell captureThe method comprises the following steps: dissolving L-arginine (99%) in a mixed solution of deionized water and octane (99%), stirring for more than 8 hours at 333K, adding tetraethyl orthosilicate (TEOS, SiO2 is more than or equal to 28.4%) and then continuing stirring for 5 hours to obtain the suspension containing the nanoparticles.

7. The method for preparing multilevel microspheres based on viroid structure for high efficiency cell capture according to claim 3, wherein the mixing reaction time of the nanoparticle-containing suspension and the nano-silica spheres in the second step is 20 h.

8. The method for preparing the multi-stage microspheres based on the viroid structure for high-efficiency cell capture according to claim 3, wherein the EDC-NHS coupling reaction time of the second-stage microspheres and the glass microspheres in the third step is 20 h.

9. The method for preparing multi-stage microspheres based on a viroid structure for high efficiency cell capture according to claim 3, wherein the EDC-NHS coupling reaction time of hyaluronic acid and the multi-stage microspheres in the fourth step is more than 8 h.

10. Use of the multi-stage microspheres based on viroid structures according to claims 1-2, wherein said use comprises cell capture.