Two-dimensional nano material and light synergistically enhanced intracellular delivery method

Abstract

The invention discloses a two-dimensional nano material and light synergistically enhanced intracellular delivery method for cells. According to the method, efficient intracellular delivery of cells can be synergistically realized under the mediation of exogenous pulse laser by using a two-dimensional material. A two-dimensional material is synthesized through an HF etching method, after the two-dimensional material and cells are co-incubated for a period of time, model molecules (fluorescent macromolecules, protein, nucleic acid and the like) are added, after exogenous pulse laser is used for treatment, the two-dimensional material absorbs heat to enable surrounding water to be vaporized to generate bubbles, the bubbles are continuously expanded through continuous irradiation of the exogenous pulse laser, and therefore the two-dimensional material is obtained. When a certain degree is reached, mechanical force generated by rupture enables cell membranes and cell wall opening biological functional macromolecules (protein, nucleic acid and the like) to enter cells through free diffusion, and exogenous functional macromolecules are integrated with genetic materials in the cells, so that long-term stable expression is realized. By adjusting the laser energy density and various parameters (material concentration, material size, laser energy density and the like), high delivery efficiency is guaranteed, and cells also have high activity. The characterization of the material by various test means shows that the characterization is successful, and meanwhile, efficient intracellular delivery of cells is also realized.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and uses MXene and light to synergistically enhance intracellular delivery of cells. Background Art

[0002] In recent years, due to population growth and the intensification of the greenhouse effect globally, the "high-temperature stress" caused by global warming poses a serious threat to world food security, and plant genetic engineering has gradually attracted people's attention. Traditional genetic engineering can be divided into physical methods and biological methods. Among them, biological methods represented by Agrobacterium-mediated transformation and physical methods represented by gene gun and electroporation-mediated transformation are widely used in plant genetic engineering. However, these systems have limitations such as species dependence, tissue damage, low transformation efficiency, and high cost. In recent years, gene delivery methods based on nanotechnology have been developed for genetic transformation. Gene engineering mediated by nanomaterials will be beneficial to promoting plant genetic engineering in modern agriculture. Currently, excellent transformation efficiency, good biocompatibility, sufficient protection for exogenous nucleic acids, and regeneration potential have been achieved in some cells of animals and protoplasts of plants, such as Arabidopsis thaliana and tobacco. However, its wide application faces many challenges. There are still some drawbacks, especially when selecting different objects, different nanocarriers need to be designed. Common difficult-to-find carriers include carbon-based nanocarriers (carbon quantum dots, carbon nanotubes), metal oxide nanocarriers (nano-ferroferric oxide, nano-titanium dioxide, nano-cerium dioxide, etc.), and inorganic oxides (mesoporous silica). Based on this, we propose a technology of photoporation mediated by a photothermal nanomaterial. The photothermal material releases heat, causing the surrounding water vapor to vaporize and generate bubbles. When the bubbles expand to a certain extent and burst, the mechanical force generated makes pores in the cell membrane and cell wall, and biofunctional macromolecules (proteins, nucleic acids, etc.) enter the cell. The exogenous functional macromolecules can achieve long-term and stable expression by integrating with the cell genetic material. By adjusting relevant parameters, while ensuring high delivery efficiency, the cells also have high activity. Summary of the Invention

[0003] The object of the present invention is to provide a method for intracellular delivery of cells, which can effectively improve the delivery efficiency while maintaining a high survival rate of cells, and integrate exogenous genetic material into cells to achieve long-term and stable expression, aiming at the problems of low transfection efficiency and long experimental period in the prior art.

[0004] To solve the above technical problems, the present invention provides the following technical solutions:

[0005] MXene is prepared by using the hydrofluoric acid etching method, and the prepared MXene is used as a two-dimensional material to synergistically cooperate with light to obtain the best delivery effect by regulating multiple parameters.

[0006] One of the technical problems solved by the present invention is the low delivery efficiency. The cell membrane and cell wall effectively block most external interferences as cell barriers. To achieve high delivery efficiency without damaging the cell membrane and cell wall, the present invention uses the optical perforation technique. By controlling multiple parameters such as the pulsed laser energy density and the concentration of MXene, while achieving high delivery efficiency, the integrity of the cell membrane and cell wall is maintained, that is, the cell survival rate is high, so as to achieve a high net acquisition rate. The method includes the following steps:

[0007] Add diluted hydrochloric acid into a polytetrafluoroethylene reaction kettle, then add 2 g of lithium fluoride into the diluted hydrochloric acid solution and react for 10 min. Immediately add 1 g of aluminum carbide to the solution in multiple portions, with a sampling time interval of 40 s each time. After all additions are completed, seal with a sealing film and react in a water bath at 35 °C for 24 h. After the reaction is completed, aliquot the suspension of the container into centrifuge tubes, centrifuge (3500 r, 5 min), and repeat multiple times until the pH of the supernatant is ≥6. Take the black liquid in the centrifuge tube, ultrasonicate in an ice-water bath, and after completion, centrifuge (3500 r 5 min). After centrifugation, take the supernatant and place it in a petri dish for freeze-drying. In the above steps, the concentration of the diluted hydrochloric acid is 9 mol / L.

[0008] Compared with the prior art, the present invention has the following remarkable advantages:

[0009] The present invention utilizes the two-dimensional photothermal nanomaterial MXene synthesized by the hydrofluoric acid etching method with good biocompatibility. Due to its excellent photothermal conversion performance, biocompatibility, and a large number of active groups on its surface (including hydroxyl groups (-OH), carboxyl groups (-COOH), and amino groups (-NH2)), it can adhere well to the cell membrane and cell wall. At the same time, under the irradiation of an external 532 nm pulsed laser, it can be observed through a microscope that the MXene solution can generate nanobubbles. The mechanical force generated by the continuous endothermic expansion and final rupture of the nanobubbles perforates the cell wall and cell membrane of the inner epidermis of the onion. The exogenous FD10 can penetrate into the cells. Then, PI is added to perform live-dead staining on the treated cells. After washing away the excess substances, finally, it can be observed through a confocal microscope that while achieving a relatively high delivery efficiency, a relatively high survival rate can still be maintained. Compared with the Agrobacterium transfection method and the optical perforation method, this process is completed instantaneously, with an extremely fast processing speed and a lower and safer pulsed laser energy density required.

[0010] Beneficial effects

[0011] (1) The method for synergistically enhancing intracellular delivery of cells by a two-dimensional nanomaterial and light in the present invention controls the concentration of the nanomaterial, the size of the nanomaterial, and the pulsed laser energy density, while maintaining a relatively high delivery efficiency and a relatively high cell survival rate;

[0012] (2) A method for intracellular delivery of two-dimensional nanomaterials synergistically enhanced by light, which uses MXene to significantly improve the delivery efficiency while ensuring high biocompatibility;

[0013] (3) A method for intracellular delivery of two-dimensional nanomaterials synergistically enhanced by light has excellent delivery effects;

[0014] (4) A method for intracellular delivery of two-dimensional nanomaterials synergistically enhanced by light has a high processing throughput and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0016] Wherein:

[0017] Figure 1 is the TEM image of MXene.

[0018] Figure 2 is the photothermal performance characterization of MXene. The temperature rise and fall of MXene under the irradiation of 808 nm infrared laser.

[0019] Figure 3 is the experimental result taken by a laser confocal microscope. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following will make a detailed description of the specific embodiments of the present invention in combination with the embodiments.

[0021] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0022] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that may be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it a separate or alternative embodiment that mutually excludes other embodiments.

[0023] Example 1

[0024] Preparation of a two-dimensional nanomaterial, the steps are as follows:

[0025] (1) Take 30 ml of HCl with a concentration of 11.7 mol / L, prepare 9 mol / L HCl, then weigh 2 g of LiF and dissolve it in the prepared HCl, and stir magnetically for a period of time. Weigh 1 g of Ti3AlC2 and add it to HF and stir magnetically for 24 h.

[0026] (2) The next day, dispense the suspension of the container into centrifuge tubes, centrifuge (3500 r, 5 min), repeat many times until the pH of the supernatant is ≥ 6 (the supernatant turns black). Prepare a Buchner funnel and start suction filtration (ensure that there is always water in the funnel, rinse the remaining liquid that has not been poured out with a wash bottle, repeat many times until the funnel is full of water).

[0027] (3) Take out the black solid on the filter paper, ultrasonicate it in an ice-water bath. After the ultrasonication ends, centrifuge (3500 r 5 min). After the centrifugation ends, take the supernatant and place it in a petri dish, freeze-dry it, and take out the solid after three days to calculate the yield.

[0028] (4) Treat the freeze-dried powder with different ultrasonication times to obtain solutions with different particle sizes. The potential of the solution is measured to be -40 mV by a Malvern nanoparticle size analyzer. The synthesis of the material is confirmed by observing the particle size and thickness and other microscopic morphologies of MXene by using a field emission scanning electron microscope and an atomic force microscope.

[0029] Example 2

[0030] By using a Fourier transform infrared spectrometer (VERTEX 80V, Germany) to test the MXene solid powder prepared in Example 1, the generation of hydroxyl groups can be observed at 3200 - 3600 cm-1; by using a field emission scanning electron microscope (JSM-7600F, Japan) and an atomic force microscope (Dimesion Edge, Germany), the size and surface morphology of the MXene prepared in Example 1 can be observed. Use a Malvern nanoparticle size analyzer (BeNano90 ZETA, China) to measure the surface potential of MXene before and after the reaction in Example 1; it can be proved that the MXene in Example 1 is successfully synthesized, as Figure 1 shown.

[0031] Example 3

[0032] The MXene solution in Example 1 was irradiated with an 808 nm laser generator (LR-MFJ-808 / 5000 mW, China), and at the same time, an infrared camera (Testo, Germany) was used to record the temperature rise. The MXene solution in Example 1 was irradiated with a pulsed laser (MW-QZ-532 / 20 μJ, China), and an inverted research microscope (Olympus IX73, China) was used to observe the microbubble effect, demonstrating that the MXene in Example 1 has good photothermal properties, as Figure 2 shown.

[0033] Example 4

[0034] (1) Take a fresh onion, peel off the outermost two layers of onion scales, and use a scalpel and forceps to take the inner epidermal cells of the outermost layer of the remaining onion scales, and place them on a clean glass slide.

[0035] (2) Then, 50 μL of 0.33 M sucrose solution was dropped on the freshly taken onion epidermal cells and covered with a coverslip, and incubated for 3 min.

[0036] (3) After completion, scrape off the remaining onion scales on one side with a pointed forceps, and then add 50 μL of MXene to incubate with the onion epidermal cells for 30 min.

[0037] (4) After incubation, add 10 μL of 3 mg / mL FD10 and then treat with laser.

[0038] (5) After treatment, add 10 μL of 1 mg / mL PI (Propidium Iodide), incubate for 5 min, and then rinse off the excess FD10 and PI with 0.33 M sucrose.

[0039] (6) After cleaning, place the sample under a confocal microscope for observation, photographing and recording, as Figure 3 shown.

[0040] Comparative Example 1

[0041] A method for intracellular delivery of cells synergistically enhanced by a two-dimensional nanomaterial and light, the implementation steps of which are basically the same as those in Example 4, except that in step (3), MXene treated with ultrasonic times of 10 min, 30 min, 60 min, 90 min, and 120 min respectively was used, and a series of experimental samples were finally obtained. After observation and counting by a confocal microscope, the final net acquisition rates were 31.8%, 36.88%, 55.02%, 52.49%, and 53.78% respectively.

[0042] Comparing Example 4 with Comparative Example 1, it can be seen that the ultrasonic treatment time of MXene in Example 1 is longer than that in Comparative Example 1, and the final net acquisition rate of Example 1 is higher than that of Comparative Example 1. Because too long ultrasonic time will result in too small MXene size, so the microbubbles generated under pulsed laser irradiation will also become smaller, and the mechanical force generated when the microbubbles burst will also become smaller, resulting in a decrease in delivery efficiency and a decrease in the final net acquisition rate.

[0043] Comparative Example 2

[0044] A method for enhancing intracellular delivery of cells by two-dimensional nanomaterials in cooperation with light, the implementation steps of which are basically the same as those of Example 4, except that in step (3), MXene treated with concentrations of 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL are used respectively, and a series of experimental samples are finally obtained. After observation and counting by a confocal microscope, the final net acquisition rates are 14.96%, 17.56%, 39.61%, 31.54%, and 26.55% respectively.

[0045] Comparing Example 4 with Comparative Example 2, it can be seen that the concentration time of MXene in Example 1 is higher than that in Comparative Example 1, and the final net acquisition rate of Example 1 is higher than that of Comparative Example 1. Because too high a concentration will result in stronger toxicity of MXene, so the number of microbubbles generated under pulsed laser irradiation will also increase, and the mechanical force generated when the microbubbles burst will also increase, resulting in a decrease in cell survival rate and a decrease in the final net acquisition rate.

[0046] Comparative Example 3

[0047] A method for enhancing intracellular delivery of cells by two-dimensional nanomaterials in cooperation with light, the implementation steps of which are basically the same as those of Example 4, except that in step (3), MXene treated with laser energy densities of 5.32 J / cm 2 , 8.21 J / cm 2 , 10.7 J / cm 2 , 18.7 J / cm 2 , 28.85 J / cm 2 are used respectively, and a series of experimental samples are finally obtained. After observation and counting by a confocal microscope, the final net acquisition rates are 38.03%, 46.95%, 55.02%, 42.25%, and 48.77% respectively.

[0048] Comparing Example 4 with Comparative Example 3, it can be seen that the pulsed laser energy density in Example 1 is higher than that in Comparative Example 1, and the final net acquisition rate of Example 1 is higher than that in Comparative Example 1. Because the pulsed laser energy density will cause the microbubble effect to become stronger, the microbubbles generated under pulsed laser irradiation will also become larger, and the mechanical force generated when the microbubbles burst will also become larger, resulting in a decrease in cell survival rate and a decrease in the final net acquisition rate.

[0049] Example 5

[0050] The experimental samples in Example 4 were designed with the control groups as Cell, Cell+FD, Cell+FD+MXene, Cell+FD+Laser, and the experimental group as Cell+FD+MXene+Laser. The experimental results were observed through a confocal microscope. At an excitation wavelength of 488 nm, the cells filled with green in the field of view were positive cells, and at an excitation wavelength of 561 nm, the cells with orange-red fluorescence emitted by the cell nuclei were dead cells. By counting with ImageJ, the final results could be obtained from the statistical data, proving that the cell delivery efficiency of the experimental group was much higher than that of the control group, as Figure 3 shown.

[0051] Example 6

[0052] Using the experimental group samples in Example 5, the optimal concentration for achieving the highest delivery effect was explored. The freeze-dried powder in Example 1 was dissolved in deionized water and ultrasonically homogenized at room temperature to obtain concentrations of 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL,

[0053] 5 mg / mL, 10 mg / mL, etc. The MXene with different concentrations was experimented according to the steps of Example 4. Finally, the best delivery effect was obtained at a concentration of 2.5 mg / mL;

[0054] Using the experimental group samples in Example 5, the optimal energy density for achieving the highest delivery effect was explored. The freeze-dried powder in Example 1 was dissolved in deionized water, and the pulsed laser energy density was adjusted to obtain 2.67 J / cm 2 , 5.32 J / cm 2 , 10.7 J / cm 2 , 18.7 J / cm 2 , 28.85 J / cm 2 , and the MXene with a concentration of 2.5 mg / mL was experimented according to the steps of Example 4. Finally, the best delivery effect was obtained at a concentration of 2.5 mg / mL and an energy density of 10.7 J / cm 2 ;

[0055] Using the experimental group samples in Example 5, explore the best delivery effect at the optimal MXene sonication time. Dissolve the freeze-dried powder in Example 1 in deionized water, and adjust the MXene sonication time to 10 min, 30 min, 60 min, 90 min, and 120 min respectively, and control the energy density to be 10.7 J / cm 2 , conduct experiments on MXene with a concentration of 2.5 mg / mL according to the steps in Example 4. Finally, at a concentration of 2.5 mg / mL and an energy density of 10.7 J / cm 2 , the best delivery effect is achieved when the MXene sonication time is 60 min;

[0056] It is determined that when obtaining the optimal delivery efficiency and survival rate, the MXene concentration is 2.5 mg / mL, the MXene sonication time is 60 min, and the laser energy density is 10.7 J / cm 2 .

[0057] The present invention provides a method for synergistically enhancing intracellular delivery of cells by two-dimensional nanomaterials and light. The two-dimensional material MXene used is synthesized by hydrofluoric acid etching. The light source used in the experiment is a pulsed laser of 532 nm. The inner epidermis cells of onions are used as the experimental object. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A method for intracellular delivery of two-dimensional nanomaterials synergistically enhanced by light, characterized in that: A two-dimensional photothermal material was synthesized, which synergistically opened pores in the cell membrane and cell wall under the action of an exogenous pulsed laser, achieving high-throughput, safe, and efficient intracellular delivery of exogenous functional macromolecules. Finally, ImageJ was used to count the cells to calculate the delivery efficiency and cell survival rate, and determine the optimal parameters.

2. The method for intracellular delivery of a two-dimensional nanomaterial synergistically enhanced with light according to claim 1, characterized in that: This includes selecting MXene as a two-dimensional material. Compared with other two-dimensional materials, the surface of MXene contains a large amount of hydroxyl (-OH), carboxyl (-COOH) and amino (-NH2) groups, which can adhere to the cell wall to improve the final delivery efficiency. This method includes but is not limited to the two-dimensional material MXene. The same effect can be achieved by using other two-dimensional photothermal materials through the same operation.

3. The method for intracellular delivery of a two-dimensional nanomaterial synergistically enhanced with light according to claim 2, characterized in that: Including the synthesis of the two-dimensional material MXene by HF etching method, take 30ml of 11.7mol / L HCl to dilute and prepare 9mol / L HCl. Then weigh 2g LiF and dissolve it in the diluted HCl, stir it magnetically for a period of time, weigh 1g Ti3AlC2 and add it to HF and stir it magnetically for 24h. The next day, the suspension in the container is divided into centrifuge tubes and centrifuged (3500r / min, 5min), repeatedly until the pH of the supernatant is ≥6. Take out the black liquid in the centrifuge tube and perform ice-water bath ultrasound. After the ultrasound, centrifuge it. After the end, take the supernatant into a culture dish and freeze-dry it.

4. The method for intracellular delivery of two-dimensional nanomaterials and light synergistically enhanced according to claim 1, characterized in that: The external pulse laser used is a 532nm pulse laser.

5. The method for intracellular delivery of two-dimensional nanomaterials and light synergistically enhanced according to claim 1, characterized in that: The cells used include onion inner epidermal cells as experimental objects. Onion inner epidermal cells can be regarded as single-layer cells, and nanoparticles can adhere to the cells to the greatest extent. Most importantly, onion inner epidermal cells have no autofluorescence and are large in size, which is convenient for subsequent observation of results.

6. The method according to claim 1, wherein the specific steps are as follows: (1) The method comprises taking freshly purchased Hexi No. 2 red onions, taking onion epidermal cells, and then dropping 50 μL of 0.33 M sucrose solution on the onion epidermal cells just taken out and covering them with a cover glass, and incubating for 3 minutes. After the incubation, the remaining onion scales on one side are scraped off with pointed tweezers; (2) Then, 50 μL of MXene was added and incubated with the onion epidermal cells for 30 min; (3) After incubation, add 10 μL of 3 mg / mL FD10 and treat with laser; (4) After completion, add 10 μL 1 mg / mL PI (Propidium Iodide), incubate for 5 min, and then rinse off excess FD10 and PI with 0.33 M sucrose. (5) After washing, the sample was placed under a confocal microscope for observation, photography and recording. Cells filled with green fluorescence in the field of view were successfully delivered cells and recorded as positive cells, while cells with red fluorescence in the nucleus were recorded as dead cells. The delivery efficiency and survival rate were calculated by counting cells using ImageJ software.

7. The method for intracellular delivery of a two-dimensional nanomaterial synergistically enhanced with light according to claim 6, characterized in that: The sucrose solution comprises dissolving sucrose in deionized water and stirring it uniformly at room temperature. The FD10 solution comprises dissolving FD10 in deionized water and stirring it uniformly at room temperature. The PI solution comprises dissolving PI in deionized water and stirring it uniformly at room temperature. The MXene solution comprises dissolving the freeze-dried powder of claim 3 in deionized water and stirring it uniformly at room temperature.

8. The method for intracellular delivery of a two-dimensional nanomaterial synergistically enhanced with light as described in claim 1, characterized in that: The exogenous functional macromolecule used was FD10 (FITC-Dextran 10 kda) as a model molecule.

9. The method for intracellular delivery of two-dimensional nanomaterials and light synergistically enhanced according to claim 6, characterized in that: The efficiency of FD10 delivered to onion inner epidermal cells could reach up to 63.97%, the survival rate could reach up to 86%, and the net gain rate could reach up to 55.02%.

10. The method for intracellular delivery of a two-dimensional nanomaterial synergistically enhanced with light as claimed in claim 1, characterized in that: This method is applicable to animal and plant cells, and the same effect can be achieved through the same operation.

Images