CN110628692A - A multilevel microsphere based on a virus-like structure for efficient cell capture - Google Patents

Abstract

The invention discloses a multi-level microsphere based on a virus imitation structure for high-efficiency cell capture, and relates to the field of cell biology. Structural microspheres, and the surface of the multi-level microspheres is modified with hyaluronic acid. The multi-level microspheres prepared by the present invention are assembled through simple electrostatic interaction, covalent bonding and nanoparticle adhesion, and the hyaluronic acid modification on the surface of the multi-level microspheres makes the multi-level microspheres exhibit Excellent surface hydrophilicity, biocompatibility and chemical structure stability, and in the cell capture experiment, due to the controllable surface topology and chemical structure of the material, the multi-level microspheres with imitation virus structure of the present invention are effective for unlabeled cells It has a very high capture rate and has a significant improvement compared with the existing technology.

Description

A multilevel microsphere based on a virus-like structure for efficient cell capture

technical field

The invention relates to the field of cell biology, in particular to a multi-level microsphere based on a virus imitation structure for high-efficiency cell capture.

Background technique

Cell capture refers to the process of separating a variety of mixed cells in a liquid from a liquid by physical, chemical, biological and other means. As an important experimental content in biology, medical diagnosis, pathological detection, etc., cell capture is used in all aspects of biological experiments. For example, in immunology experiments, specific separation and capture of immune cells is used for cell engineering, in genetic engineering, target cells are captured for gene programming, and in disease screening, diseased cells are captured for disease diagnosis. Among them, the capture of tumor circulating cells for the diagnosis, monitoring and analysis of cancer has attracted extensive attention from researchers all over the world.

The existing cell capture technologies are mainly divided into pre-labeled cell capture technologies and unlabeled cell capture technologies. Pre-labeled cell capture techniques include flow cytometry, nano-magnetic bead adsorption, etc.; unlabeled cell capture techniques include optical tweezers, centrifugation, and microfluidics.

Flow cytometry is a common cell analysis and sorting method in the laboratory. It stains the cells with fluorescent dyes in advance, then divides the cell suspension into single-cell droplets, uses laser to excite the fluorescent dyes of the cells, and analyzes and sorts the cells according to the fluorescence intensity of the cells. The nano-magnetic bead adsorption method needs to label the cells in advance, use the specific molecules on the nano-magnetic beads to bind to the cells, and then separate them by the magnetic properties of the beads, and then separate the cells by trypsin elution. Both of these pre-labeled cell capture methods require pretreatment of the cells. The toxicity of the fluorescent dye and the endocytosis of the cells on the nano-magnetic beads will cause damage to the cells, and the pretreatment time is long and the operation process is complicated.

Optical tweezers technology can use the mechanical effect of laser to manipulate single cells or molecules, so as to realize cell capture and separation. It requires the support of large and precise laser emitters and controllers, the operation is complicated, and it is difficult to apply to large-scale cell capture. Microfluidic technology can adjust the size and properties of the channel, and use the physical properties of cells such as size, size, and quality to separate and sort cells, so as to achieve label-free cell capture. and limited quantities.

In summary, the existing cell capture and separation methods are complicated to operate, costly, and will cause damage to cells during the capture process. Therefore, we urgently need to develop a simple and efficient cell capture technology.

The fine and orderly structure and advanced complex functions of living organisms in nature have always been the objects of bionics for chemists and materials scientists. Due to its high adsorption to cells, natural virus particles have become important carrier materials in the fields of biomedicine and nanomedicine, and have great application value in the in vivo delivery of bioactive molecules (such as chemotherapy drugs and therapeutic genes). However, with the deepening of clinical research, the potential hazards of viral vectors (such as immunogenicity and induced mutations) are gradually exposed. Therefore, the construction of advanced virus imitation systems through materials engineering has become the current research field of chemistry, materials and nanotechnology. research focus. At present, the imitation virus structure is mainly used in in situ imaging and drug delivery system construction.

Therefore, those skilled in the art are committed to developing a multi-level microsphere of imitation virus structure and its preparation method, so that the prepared microsphere not only has high cell affinity, but also overcomes the secondary nano-virus imitation structure in the prior art that is easily damaged by cells. Phagocytosis is size-restricted and has extremely high cell capture efficiency.

Contents of the invention

In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is how to provide a multi-level virus imitation microsphere capable of efficiently capturing cells and a preparation method thereof, so that the prepared microsphere has high cell affinity, high biological Compatibility, while overcoming the size limitation that the secondary nano-viral structure is easy to be swallowed by cells, it has extremely high cell capture efficiency and overcomes the shortcomings of the existing technology.

In order to achieve the above object, the present invention provides a multi-level microsphere based on imitation virus structure for high-efficiency cell capture, the multi-level microsphere is a tertiary structure assembled from nanoparticles, nano-silicon spheres and glass microspheres Microspheres, and the surface of the multi-level microspheres is modified with hyaluronic acid.

Further, the size of the nanoparticles is 15nm, the size of the silicon nanospheres is 200nm, and the size of the glass microspheres is any one of 150, 500, and 1000 μm.

The present invention also provides a method for preparing multi-level microspheres based on imitation virus structure for high-efficiency cell capture as described above, said method comprising the following steps:

Step 1. Adopt improvements method to prepare a suspension containing nanoparticles; Synthesize nano-silicon spheres by method, wash and dry for later use; carry out surface modification treatment on glass microspheres, wash and dry for later use;

Step 2: After the nano-silicon spheres prepared in the step 1 are subjected to amination modification treatment, they are mixed with the suspension containing nanoparticles, collected by centrifugation and dried, and then calcined to obtain secondary microspheres;

Step 3. After the secondary microspheres prepared in the step 2 are subjected to amination modification treatment, they are combined with the glass microspheres treated in the step 1 through an EDC-NHS coupling reaction to obtain The multi-level microspheres are washed and dried;

Step 4, modifying hyaluronic acid on the surface of the multi-level microspheres obtained in the step 3 through EDC-NHS coupling reaction.

Further, the specific method of the amination modification treatment is: suspend the material in toluene (99.5%), and after adding 3-aminopropyltriethoxysilane (APTES, 99%), reflux at 383K More than 14 hours.

Further, the surface modification treatment of the glass microspheres in the step 1 specifically includes the following steps:

Step 1. Soak the glass microspheres in the piranha solution for more than 5 hours to carry out carboxylation treatment, then wash and dry;

Step 2. Disperse the carboxylated glass microspheres in step 1 in dimethylformamide (DMF, 99.5%), add succinic anhydride solution under vigorous stirring, let stand for 24 hours, wash and dry.

Further, the improvement in the step one The specific method is: after dissolving L-arginine (99%) in the mixed solution of deionized water and octane (99%), stirring at 333K for more than 8h, then adding tetraethyl orthosilicate (TEOS, SiO2 ≥28.4%) and continue stirring for 5 h to obtain the suspension containing nanoparticles.

Further, the mixing reaction time of the nanoparticle-containing suspension and the nano-silicon spheres in the second step is 20 h.

Further, the EDC-NHS coupling reaction time of the secondary microspheres and the glass microspheres in the step 3 is 20 h.

Further, the EDC-NHS coupling reaction time between the hyaluronic acid and the multi-level microspheres in the step 4 is more than 8 hours.

The present invention also provides an application of the multi-level microsphere based on the imitation virus structure as described above, characterized in that the application includes cell capture.

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

(1) The microspheres prepared by the present invention can be assembled into stable micron-scale multi-level microspheres with imitating virus surface topology through simple electrostatic interactions, covalent bonds and cross-linking between small nanoparticles, The preparation method is simple and efficient;

(2) The micron-scale multi-level microspheres prepared by the present invention effectively overcome the size limitation that the secondary nano-virus imitation structure is easily swallowed by cells in the prior art;

(3) The multi-level microspheres prepared by the present invention have excellent surface hydrophilicity, biocompatibility and chemical structure stability through simple and effective surface modification;

(4) The surface topology and chemical structure of the virus-like multi-level microspheres provided by the present invention are controllable, have good affinity to cells, and exhibit extremely high cell capture efficiency for various cells.

The idea, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention.

Description of drawings

Fig. 1 is the synthesizing steps and the schematic diagram of cell capture of the imitation virus structure multi-level microsphere of a preferred embodiment of the present invention;

Fig. 2 is three kinds of size multi-level microspheres (HHB-150/500/1000) prepared by a preferred embodiment of the present invention to the capture efficiency data figure of MCF-7 cell;

Fig. 3 is the comparison effect data diagram of the capture efficiency of MCF-7 cells by multi-stage microspheres (HHB-1000) prepared by a preferred embodiment of the present invention;

Fig. 4 is a comparison effect data diagram of the capture efficiency of A549 cells by multi-stage microspheres (HHB-1000) prepared in a preferred embodiment of the present invention;

Fig. 5 is the comparison effect data diagram of the capture efficiency of SKBR-3 cells by multi-level microspheres (HHB-1000) prepared by a preferred embodiment of the present invention;

Fig. 6 is the fluorescent microscopic image of A549 cell captured by multi-level microspheres (HHB-1000) prepared by a preferred embodiment of the present invention, wherein figure a is an overall fluorescent microscopic image of cells captured by a microsphere, and figure b is Partial magnified fluorescence microscopic image of panel a;

Figure 7 is a fluorescent microscopic image of MCF-7 cells captured by multi-stage microspheres (HHB-1000) prepared by a preferred embodiment of the present invention, wherein figure a is an overall fluorescent microscopic image of cells captured by a microsphere, Fig. b is a partially enlarged fluorescence microscopic image of Figure a;

Figure 8 is a fluorescent microscopic image of MCF-7 cells captured by multi-stage microspheres (HB-1000/HHB-1000) prepared in a preferred embodiment of the present invention, wherein Figure a is a fluorescent microscopic image of cells captured by HB-1000 Image, Figure b is a fluorescence microscopic image of cells captured by HHB-1000;

Fig. 9 is a fluorescent microscopic image of A549 cells captured by multi-level microspheres (HHB-1000) prepared in a preferred embodiment of the present invention.

Detailed ways

The following describes several preferred embodiments of the present invention with reference to the accompanying drawings, so as to make the technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned herein.

Embodiment 1. Preparation of multi-level microspheres with imitation virus structure

The preparation steps of the multi-stage microspheres with imitation virus structure provided by the present invention are shown in Figure 1. First, the nanoparticle 1 is combined with the nano-silicon sphere 2 and then calcined to obtain the secondary microsphere 3; the secondary microsphere 3 is advanced Amination modification treatment to obtain secondary microspheres 4 with amino groups on the surface, combine them with glass microspheres 5 to obtain multi-level microspheres 6 according to EDC-NHS coupling reaction; NHS coupling reaction modified the surface with hyaluronic acid to obtain multi-level microspheres 7 with hyaluronic acid modified on the surface. This surface modification can enhance the surface hydrophilicity, biocompatibility and chemical structure stability. It is then ready for subsequent cell capture operations. The specific preparation process is as follows.

1. Synthesis of Multi-sized Microspheres

For nanoparticles (S1), using amino acid reaction, modified Methods The silica nanoparticles with a size of about 15nm were synthesized. 87 mg of L-arginine (99%) was dissolved in a solution containing 69.5 mL of deionized water and 5.23 mL of octane (99%), and the mixture was stirred at 333K for more than 8 h. Then 0.5 mL of tetraethyl orthosilicate (TEOS, SiO2≥28.4%) was added into the former mixture and stirred for 5 hours to directly use the silicon dioxide nanoparticles (S2).

For nano silicon spheres (S2), through the common Methods Silica spheres with a size of about 200nm were synthesized. Mix 150 mL of ethanol (≥99.7%), 11.4 mL of deionized water and 7.0 mL of ammonia water, and add 8.4 mL of TEOS to the solution under vigorous stirring at 308K. After stirring for 6 hours, the nanospheres were obtained by centrifugation, washed with ethanol and deionized water several times, and dried at 373K for more than 8 hours.

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

2. Assembly of Hierarchical Microspheres

When modifying the material by amination, suspend the material in 90mL toluene (99.5%), add 4.8mL 3-aminopropyltriethoxysilane (APTES, 99%), and reflux the system at 383K for more than 14h. Amino modification.

During assembly, 100 mg of aminated S2 was dissolved in 60 mL of deionized water and mixed with 10.8 mL of S1 suspension and sonicated for 20 min. Then the mixture (S1 with negatively charged surface and aminated S2 with positively charged surface) was stirred at 333K for 20 h, and centrifuged at 10000 rpm for 5 min (note that S1 will not be separated under this condition). The collected solids were dried and calcined at 823 K for 5 h to obtain secondary microspheres (HSS). The HSS is then amino-modified.

Through the EDC-NHS coupling reaction, the aminated HSS can be combined with the carboxylated S3. According to the conventional method, the carboxylated S3 was dispersed in a solution containing excess 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDAC, 97%) and N-hydroxysuccinyl The carboxyl group was activated in a buffer solution of imine (NHS, 98%), and then aminated HSS was added and incubated at room temperature. After 20 h of reaction, the hierarchical microspheres (HB) were obtained, washed with deionized water and dried for further use.

3. Hyaluronic Acid Functionalization

In order to realize the functional modification of hyaluronic acid on the surface of the hierarchical microspheres, we combined the hierarchical microspheres with hyaluronic acid through EDC-NHS coupling using a method similar to the synthesis of HB. The specific method is as follows, 50 mg of hyaluronic acid was dispersed in 20 mL of MES buffer (0.01 M), and 7.7 mg of EDAC (97%) and 6.9 mg of NHS (98%) were added. The pH of the mixture was adjusted to 5-6 and incubated at room temperature for 30 minutes to activate the carboxyl groups. Then, 20 mL of HB-containing phosphate buffered saline solution (PBS, pH=7.4, 0.1 M) was added, the pH was checked and adjusted to 7.4-8.0 and incubated at 38° C. with slow stirring for more than 8 h. Afterwards, the functionalized modified hierarchical microspheres (HHB) were separated by standing, washed with deionized water, dried at room temperature and stored for future use.

Embodiment 2, cell capture experiment

The process of the multi-stage microspheres with imitation virus structure prepared by the present invention is as shown in Figure 1. The multi-stage microspheres 7 whose surface is modified with hyaluronic acid are added to the cell suspension 8, mixed and shaken for a period of time. time, the cells can be captured on the surface of the microspheres, and the combination of microspheres and cells can be obtained9. The specific operation is as follows.

During the capture experiment, the cells were first digested with 0.25% Trypsin/EDTA, then resuspended with phosphate buffer (PBS, 0.1M, pH=7.4), and counted with a hemocytometer. Divide the cell suspension into centrifuge tubes, then add a certain amount of multi-stage microspheres prepared in Example 1 into the cell solution, and shake the cell suspension slowly on a shaker. Afterwards, the microspheres were gently pipetted with the cell suspension to remove the cells that settled on the microspheres due to sedimentation. After resting, the hierarchical microspheres with captured cells can be isolated.

The cell capture effect is mainly analyzed from the perspective of cell counting statistical capture rate and fluorescence microscopic image. Cells were stained and analyzed using two dyes, DAPI (stains all cells) and Calcein-AM (stains only live cells).

First, the cell capture efficiencies of hierarchical microspheres prepared with three sizes of glass microspheres were compared.

MCF-7 cells were used for experiments, and the statistical data results are shown in Figure 2. It can be seen that the cell capture efficiency of the multi-level microspheres (HHB-1000) prepared with 1000 μm glass microspheres is significantly higher than that of the other two sizes of multi-level microspheres. Therefore, we used HHB-1000 to capture cells in the follow-up, and compared the results with S3 (untreated 1000 μm microspheres) and S3-HA (1000 μm microspheres directly modified with hyaluronic acid). The experimental results show that the capture rate of HHB-1000 on MCF-7 cells reached 87.9% (as shown in Figure 3), the capture rate on A549 cells reached 92.9% (as shown in Figure 4), and the capture rate on SKBR-3 cells The capture rate reached 98.7% (as shown in Figure 5), all better than S3 (2.4% to the capture rate of MCF-7 cells, 2.4% to the capture rate of A549 cells, and 2.4% to the capture rate of SKBR-3 cells 0%, p<0.005) and S3-HA (21.1% for MCF-7 cells, 19.9% for A549 cells, 12.5% for SKBR-3 cells, p<0.001 ).

Through data comparison, it can be seen that multi-level microspheres have great advantages in cell capture, and the capture efficiency is high, which meets the requirements of practical application.

Secondly, the combination of multi-level microspheres and cells was observed.

The fluorescent microscopic image of A549 was captured by HHB-1000 multi-level microspheres (as shown in Figure 6), and it can be seen that the cells are well combined with the microspheres. This effect can also be seen in the fluorescent microscopic images of MCF-7 cells captured by HHB-1000 (as shown in Figure 7).

Finally, the effect of hyaluronic acid modification on the cell capture efficiency of multi-stage microspheres was compared and analyzed.

It can be seen in the fluorescent microscopic images (as shown in Figure 8) that HB-1000 without modified hyaluronic acid and HHB-1000 modified with hyaluronic acid respectively captured MCF-7 cells, HHB The fluorescence intensity of the fluorescent microscopic images of the -1000 group is higher, especially comparing the DAPI channel images that can be stained for all cells and the Calcein-AM channel images that can only be stained for live cells, it can be seen that the HHB-1000 captures The number of viable cells is significantly greater than that of HB-1000, which proves that the functional modification of the surface of the microspheres with hyaluronic acid improves the biocompatibility of the microspheres, which is of great significance for the cell capture effect.

Further, cells were counted on the Calcein-AM channel image and DAPI channel image (as shown in Figure 9) in the same field of view in the fluorescent microscopic image of A549 cells captured by HHB-1000, and the captured viable cell rate was calculated. Three independent calculations obtained the living cell rate as 96%, 91% and 92%, and the average living cell rate was 93%. According to the live cell rate, we also corrected the cell capture efficiency, and obtained that the live cell capture efficiency of the multi-stage microspheres was about 82%-91% under the premise of excluding the captured dead cells.

Through the analysis of the examples, it can be seen that the multi-level microspheres of the imitation virus structure provided by the present invention have high cell affinity and structural stability, and overcome the size limitation that the secondary nano-nano virus imitation structure is easily phagocytized by cells, showing It achieves extremely high cell capture efficiency, especially for living cells. And the preparation method provided can realize the controllable preparation of the surface topological structure and chemical structure of the multi-level microsphere.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (10)

1. A multilevel microsphere based on imitation virus structure for high-efficiency cell capture, characterized in that, the multilevel microsphere is a tertiary structure microsphere assembled from nanoparticles, nano silicon spheres and glass microspheres, And the surface of the multi-level microspheres is modified with hyaluronic acid. 2. the multilevel microsphere based on imitation virus structure for efficient cell capture as claimed in claim 1, is characterized in that, the size of described nanoparticle is 15nm, and the size of described nano silicon sphere is 200nm, and described The size of the glass microspheres is any one of 150, 500, and 1000 μm. 3. A method for preparing multilevel microspheres based on imitation virus structure for high-efficiency cell capture as claimed in claims 1 to 2, characterized in that, the method comprises the following steps: Step 1. Adopt improvements method to prepare a suspension containing nanoparticles; Synthesize nano-silicon spheres by method, wash and dry for later use; carry out surface modification treatment on glass microspheres, wash and dry for later use; Step 2: After the nano-silicon spheres prepared in the step 1 are subjected to amination modification treatment, they are mixed with the suspension containing nanoparticles, collected by centrifugation and dried, and then calcined to obtain secondary microspheres; Step 3. After the secondary microspheres prepared in the step 2 are subjected to amination modification treatment, they are combined with the glass microspheres treated in the step 1 through an EDC-NHS coupling reaction to obtain The multi-level microspheres are washed and dried; Step 4, modifying hyaluronic acid on the surface of the multi-level microspheres obtained in the step 3 through EDC-NHS coupling reaction. 4. A kind of preparation method of the multilevel microspheres based on imitation virus structure that is used for efficient cell capture as claimed in claim 3, is characterized in that, the specific mode of described amination modification treatment is: suspend material in Toluene (99.5%), and after adding 3-aminopropyltriethoxysilane (APTES, 99%), reflux at 383K for more than 14h. 5. a kind of preparation method that is used for the preparation method of the multistage microsphere based on imitation virus structure for efficient cell capture as claimed in claim 3, it is characterized in that, described step 1 is carried out to the described surface of described glass microsphere The modification process specifically includes the following steps: Step 1. Soak the glass microspheres in the piranha solution for more than 5 hours to carry out carboxylation treatment, then wash and dry; Step 2. Disperse the carboxylated glass microspheres in step 1 in dimethylformamide (DMF, 99.5%), add succinic anhydride solution under vigorous stirring, let stand for 24 hours, wash and dry. 6. a kind of preparation method for the multistage microsphere based on imitation virus structure that is used for efficient cell capture as claimed in claim 3, is characterized in that, described improvement in the described step 1 The specific method is: after dissolving L-arginine (99%) in the mixed solution of deionized water and octane (99%), stirring at 333K for more than 8h, then adding tetraethyl orthosilicate (TEOS, SiO2 ≥28.4%) and continue stirring for 5 h to obtain the suspension containing nanoparticles. 7. a kind of preparation method for the multistage microsphere based on imitation virus structure that is used for efficient cell capture as claimed in claim 3, is characterized in that, the suspension that contains nanoparticle described in described step 2 and described The mixing reaction time of nano silicon spheres is 20h. 8. A kind of preparation method for the multistage microsphere based on imitation virus structure that is used for efficient cell capture as claimed in claim 3, it is characterized in that, described step 3 in described secondary microsphere and described glass microsphere The EDC-NHS coupling reaction time of the ball is 20h. 9. a kind of preparation method for the multistage microsphere based on imitation virus structure that is used for efficient cell capture as claimed in claim 3, is characterized in that, in described step 4, hyaluronic acid and described multistage microsphere EDC-NHS coupling reaction time is more than 8h. 10. An application of the multi-level microspheres based on the imitation virus structure according to claims 1-2, characterized in that the application includes cell capture.