CN106701828A - Method for increasing probability of nanoprobe puncturing through cytomembrane - Google Patents

Abstract

The invention is a method for increasing the probability of nanoprobes penetrating cell membranes, which relates to the field of cell biology and cell transfection. By spreading a layer of rat tail collagen type I protein on the cell membrane surface, the fluidity of the cell membrane surface is inhibited to achieve The effect of greatly increasing the probability of nanoprobes penetrating cell membranes. The method for attaching the rat tail collagen type Ⅰ protein to the surface of the cell membrane is as follows: step 1: prepare HEPES solution; step 2: prepare the solution of rat tail collagen type Ⅰ protein; step 3: take the target cell culture solution and remove the culture medium; step 4: immerse the target cells obtained in step 3 with the mixed solution obtained in step 2, and let it stand for a period of time; step 5: remove the remaining rat tail collagen type Ⅰ solution, and obtain cells with rat tail collagen type Ⅰ protein Target cells; step 6: the target cells obtained in step 5 and attached with rat tail collagen type I protein were placed in culture medium and cultured for at least 24 hours.

Description

A method to increase the probability of nanoprobes penetrating cell membranes

technical field

The invention relates to the fields of cell biology and cell transfection. A layer of rat tail collagen type I protein is prepared on the cell membrane surface, and a method for increasing the probability of nanometer probes penetrating the cell membrane is invented.

Background technique

Cell transfection methods are divided into biological transfection (lentiviral transfection and adenoviral transfection), chemical transfection (liposome transfection) and physical transfection (electroporation, microinjection and nanoprobe technology), each Each method has its own advantages and disadvantages. The methods commonly used at present are biological transfection and chemical transfection methods, but the transfected cells have the risk of canceration, and the cytotoxicity is increased, and the transfection efficiency is very low. The probability of successful transfection of pluripotent stem cells does not exceed 0.1%. . The physical transfection method is safe and reliable, and it is also an important method for a large number of scientists to study cell transfection in recent years. In the physical transfection method, electroporation can transfect cells with high throughput, but high voltage will cause a large number of cell death, and the transfection process is uncontrollable; microinjection can transfect cells in time and space. Controlled transfection, but direct transfection of adherent cells is difficult. At present, more researchers are beginning to pay attention to the cell transfection method based on nanoprobe technology. The method can realize direct single-cell transfection of adherent cells, with controllable time and space, and can observe the biological characteristics of transfected cells in situ. But the difficulty lies in the low penetration rate of commercial nanoprobes to the cell membrane, which will directly affect the transfection efficiency of cells. Therefore, there is a need to increase the probability of nanoprobes penetrating cell membranes.

Contents of the invention

Aiming at the deficiency of the background technology, the present invention invents a method for increasing the probability of the nano-probe penetrating the cell membrane.

The invention mainly spreads a layer of rat tail collagen type I protein on the surface of the cell membrane to inhibit the fluidity of the cell membrane surface and achieve the purpose of increasing the probability of the nanometer probe penetrating the cell membrane. Therefore, the technical solution of the present invention is a method for increasing the probability of nano-probes penetrating the cell membrane. The method attaches rat tail collagen type I protein to the surface of the cell membrane, thereby increasing the probability of the nano-probe penetrating the cell membrane.

Further, a method for attaching rat tail collagen type I protein to the cell membrane surface is as follows:

Step 1: Prepare HEPES (4-hydroxyethylpiperazineethanesulfonic acid) solution;

Step 1.1: Dissolve HEPES powder in sterilized triple-distilled water to obtain a HEPES solution, and dissolve 1.2g of HEPES powder per 100ml of sterilized triple-distilled water;

Step 1.2: Filter the HEPES solution obtained in step 1.1 with a filter with a pore size of 0.2uM, and place the filtered solution in a sealed refrigerator;

Step 2: preparing a solution of 0.2 mg/ml rat tail collagen type I protein;

The rat tail collagen type I solution was mixed with the HEPES solution obtained in step 1, and the concentration of the rat tail collagen type I protein in the mixed solution was 0.2 mg/ml;

Step 3: Take the target cell culture solution, remove the culture medium in the cell culture solution, and obtain the target cells;

Step 4: immerse the target cells obtained in step 3 with the mixed solution obtained in step 2, and let it stand for a period of time, so that the rat tail collagen type I protein in the mixed solution is attached to the cell surface;

Step 5: remove the remaining rat tail collagen type Ⅰ solution to obtain the target cells attached with rat tail collagen type Ⅰ protein;

Step 6: The target cells obtained in step 5 and attached with rat tail collagen type I protein are placed in culture medium and cultured for at least 24 hours.

In the present invention, a layer of rat tail collagen type I protein is spread on the surface of the cell membrane to inhibit the fluidity of the cell membrane surface, thereby achieving the effect of greatly increasing the probability of the nanometer probe penetrating the cell membrane.

Description of drawings

Fig. 1 is the human fibroblast in the petri dish and the individual fibroblast topography figure that atomic force microscope obtains;

Figure 2 is a schematic diagram of the spreading of rat tail collagen on the cell membrane surface;

Figure 3 is a force curve diagram of the interaction between the probe and the cell;

Fig. 4 is nanoprobe micrograph;

Figure 5 is a schematic diagram of probe and single cell interaction;

Fig. 6 is a schematic diagram of the force curve of the needle tip penetrating the cell membrane.

Specific method steps

1. Culture method of human fibroblasts (Fibroblasts referred to as "FB")

Fibroblasts are the main cell source of induced pluripotent stem cells, so this experiment takes human fibroblasts as an example. Fibroblasts were cultured in a 35mm diameter petri dish, the culture medium was DMEM/HIGH GIUCOSE and 10% fetal bovine serum (FBS), and the cells were cultured in an incubator at 37°C and 5% CO ₂ for at least 24 hours.

2. Preparation of Rat Tail Collagen Type Ⅰ

1), preparation of 0.05mol/ml HEPES (4-hydroxyethylpiperazineethanesulfonic acid) solution

① Weigh 1.2g of HEPES powder with an electronic balance;

②In the aseptic operation workbench, dissolve 1.2g of HEPES powder in 100ml of sterilized three-distilled water;

③Filt the above solution with a disposable 0.2uM filter to remove the HEPES powder, put the filtered solution into a culture bottle, seal the bottle mouth with a parafilm and place it in a 4°C refrigerator for later use;

2) Preparation of 0.2 mg/ml rat tail collagen type Ⅰ protein solution

①In the aseptic operation workbench, take 1ml HEPES (0.05mol/ml) solution into 1.5ml sterilized EP tube;

②Add 40ul rat tail collagen type I solution (5mg/ml) to the above solution, mix well and set aside.

Drug source

Note: HEPES powder: biosharp

Rat tail collagen type Ⅰ: Shengyou Biotechnology Co., Ltd.

3. Spreading and verification of mouse tail collagen type Ⅰ protein on the cell membrane surface

1) Spreading of mouse tail collagen type Ⅰ protein on the cell membrane surface

The formation of rat tail collagen type I protein on the surface of the cell membrane is divided into the following five steps (Figure 2):

1. Fibroblasts were cultured in a 35mm diameter petri dish, the culture medium was DMEM/HIGH GIUCOSE (medium (high sugar) containing sodium pyruvate) and 10% fetal bovine serum (FBS), and the cells were cultured at 37°C. Incubate for at least 24 hours in a 5% CO ₂ incubator;

2. Absorb the culture solution in the culture dish in the aseptic operation workbench;

3. Add an appropriate amount of the prepared mixed solution to the culture dish in step 2 in the aseptic operation workbench, subject to submerging the cells, and then let it stand for 5-10 minutes;

4. Suck away the remaining solution in the culture dish in the aseptic operation workbench. At this time, there will be a layer of rat tail collagen type I protein on the surface of the cell membrane. Figure 2 is the thickened curve on the surface of the cell membrane;

5. Add culture medium (DMEM/HIGH GIUCOSE and 10% fetal bovine serum (FBS)) to it, and cultivate for at least 24 hours.

2) Verification of rat tail collagen type Ⅰ protein on the cell membrane surface

Rat tail collagen type Ⅰ protein is a kind of protein molecule. When rat tail collagen type Ⅰ protein is spread on the surface of the cell membrane, the protein molecules on the surface will increase a lot compared to cells without rat tail collagen type Ⅰ protein. At this time, If the probe is used to interact with the cell, the probability of adhesion will increase accordingly, which can be reflected from the force curve of the probe-cell interaction. Figure 3 is the force curve of the interaction between the cell and the atomic force microscope probe. The two curves are the needle entry curve and the needle return curve. When the protein molecules on the surface of the cell membrane increase, one end of the protein molecule is more likely to adhere to the probe. , when the probe returns to the needle, the needle will split the protein molecule attached to the needle tip, which is reflected in the force curve, that is, there is an obvious force jump when the needle is returned (Figure 3 AB section), this phenomenon is called adhesion phenomenon , the corresponding jump force is called the adhesion force. In the experiment, the probability of probe adhesion before and after adding rat tail collagen type Ⅰ protein can be compared to prove whether the cell membrane surface has been covered with rat tail collagen type Ⅰ protein.

Table 1 shows the comparison of the probability of probe adhesion before and after adding rat tail collagen type Ⅰ protein. At this time, the needle speed of the probe was 2 μm/s, and the force was 1 nN. Three independent experiments were performed on the cells without rat tail collagen type Ⅰ protein and the cells with rat tail collagen type Ⅰ protein. There are 10 cells in each group of experiments, and each cell is repeated 5 times, and the probability of adhesion in the force curve is finally calculated. When no rat tail collagen type Ⅰ protein was added, the probability of sticking back to the needle curve was only 15.2%, and the probability of sticking to the back needle curve increased by 60.00% after adding rat tail collagen type Ⅰ protein, which proved that the surface of the cell membrane was attached A layer of rat tail collagen type I protein.

Table 1 Probability of probe adhesion before and after adding rat tail collagen type Ⅰ protein

4. Nanoprobe penetration experiment based on atomic force microscope

The experiment was divided into two groups, one group was not added rat tail collagen type Ⅰ protein, and the other group was added rat tail collagen type Ⅰ protein. Each group of experiments was carried out 3 times independently, with 10 cells in each experiment, and each cell was repeated 5 times. The tip used in the experiment is a commercial pyramid-shaped silicon nitride probe, as shown in Figure 4, with an elastic modulus of 0.01N/m-0.3N/m, and Figure 5 is a diagram of the interaction between the probe and a single cell in the optical microscope in the experiment , the needle tip is aimed at the nuclear region of the target cell. The loading condition of the experimental setup is 1nN, and the needle speed is 2μm/s.

Figure 6 is the force curve of the probe penetrating the cell membrane. The probe penetrating the cell membrane is roughly divided into three processes: first, the probe gradually approaches the cell, as shown in section AB of the figure; when the probe contacts the cell, the probe begins to exert pressure on the cell , as shown in section BC; when the needle tip continues to exert pressure on the cells, the force curve jumps, which means that the needle tip has penetrated the cell membrane, as shown in section CD. By counting the probability of a jump in the force curve before and after adding rat tail collagen type Ⅰ protein on the cell membrane surface, the effect of rat tail collagen type Ⅰ protein on the cell membrane surface on the cell membrane penetration rate can be obtained, as shown in Table 2.

Experimental results

Table 2 shows the probability of the probe penetrating the cell membrane after adding rat tail collagen type Ⅰ protein on the surface of the cell membrane. Under the same experimental conditions, when no rat tail collagen type Ⅰ protein was added, the probability of the probe penetrating the cell membrane was only 18.15%, and after adding the rat tail collagen type Ⅰ protein, the probability of the probe penetrating the cell membrane increased to 40.2% %. The experimental results proved that after adding rat tail collagen type Ⅰ protein on the surface of the cell membrane, the penetration rate of the probe to the cell membrane was significantly increased. This method has important practical application value for the physical transfection of single cells.

Table 2 The probability of the probe penetrating the cell membrane before and after adding rat tail collagen type Ⅰ protein

Claims (2)

1. a kind of method for increasing nano-probe penetration cell film probability, the type albumen of Collagen type-I I is attached to cell by the method Film surface, so as to improve the probability that nano-probe penetrates the cell film.

2. it is a kind of the type albumen of Collagen type-I I is attached to surface of cell membrane method be：

Step 1：Prepare HEPES solution；

Step 1.1：HEPES powder is dissolved in the tri-distilled water of sterilizing, HEPES solution is obtained, per 100ml in the tri-distilled water of sterilizing Dissolving 1.2gHEPES powder；

Step 1.2：The HEPES solution obtained with the filtering step 1.1 that aperture is 0.2uM, the solution after filtering is close Envelope refrigeration is placed；

Step 2：Prepare the solution of the type albumen of 0.2mg/ml Collagen type-Is I；

The HEPES solution that the type solution of rat tail collagen protein I is obtained with step 1 is mixed, and makes the rat-tail glue in mixed solution The concentration of former I type albumen is 0.2mg/ml；

Step 3：Target cell culture solution is taken, the nutrient solution in removal cell culture solution obtains target cell；

Step 4：The target cell that the mixed solution immersion step 3 obtained with step 2 is obtained, stands a period of time, makes mixing molten The type albumen of Collagen type-I I in liquid is attached to cell surface；

Step 5：The type solution of remaining rat tail collagen protein I is removed, acquisition is attached with the target cell of the type albumen of Collagen type-I I；

Step 6：The target cell for being attached with the type albumen of Collagen type-I I that step 5 is obtained is placed in nutrient solution and cultivates at least 24 Individual hour.