Preparation method of core-shell nanoparticles with photo-thermal-phase change dual functions and intracellular delivery application of core-shell nanoparticles

Abstract

The invention relates to a preparation method of core-shell nanoparticles with photo-thermal-phase change dual functions and intracellular delivery application of the core-shell nanoparticles, and belongs to the field of nano materials and cell delivery. The invention provides a photothermal-phase change bifunctional core-shell nanoparticle applied to intracellular delivery of biomacromolecules, the nanoparticle is of a core-shell structure, a high-molecular polymer is taken as a shell, liquid fluorocarbon and a photothermal material are taken as a core, the nanoparticle is prepared by an ultrasonic emulsification method, and the preparation method is simple. The boiling point of the liquid fluorocarbon is low, and the photo-thermal material has good photo-thermal conversion performance, so that the core-shell nanoparticles can quickly generate photo-thermal steam microbubbles under laser induction. Under the synergistic effect of laser and core-shell nanoparticles, generated steam microbubbles are induced to generate micropores in plant cell walls and cell membranes or animal cell membranes, so that the physical barrier of the cell walls and the cell membranes is overcome, and the efficiency of delivering exogenous functional biomacromolecules into cells is remarkably improved.

Description

Technical Field

[0001] The present invention relates to a preparation method of a photothermal-phase change dual-functional core-shell nanoparticle and its intracellular delivery application, belonging to the fields of nanomaterials and cell delivery. Background Art

[0002] Intracellular delivery refers to the process by which exogenous substances enter plant or animal cells through cell walls and cell membranes and carry out molecular exchange within the cells. This process is of great significance for the normal functions, growth, development of cells, and their response to environmental changes. However, due to the physical barrier effects of cell walls and cell membranes, exogenous biomolecules often have difficulty in efficiently entering the interior of cells. Traditional cell delivery methods, such as Agrobacterium-mediated transformation, particle bombardment, and electroporation, although achieving some success to a certain extent, still have limitations such as low efficiency, complex operation, and high cost. Therefore, developing new delivery systems to overcome these challenges has become a research hotspot in the field of cell biotechnology.

[0003] As a novel delivery carrier, nanoparticles exhibit advantages such as high transformation efficiency, genetic stability, and simplicity of operation due to their small size, high surface area, and controllability, and can effectively penetrate cell walls and cell membranes to deliver functional biomolecules into cells. However, how to improve the penetrability of nanoparticles and achieve efficient molecular delivery remains the main technical problem currently faced. Laser-induced optical poration is an emerging cell membrane penetration technology that uses pulsed lasers to generate temporary micropores on cell membranes, enabling exogenous substances to enter cells through these micropores. Compared with traditional physical methods, laser-induced optical poration technology has higher spatial precision, lower risk of cell damage, and can achieve precise delivery in specific regions or specific cell populations. Therefore, combining laser-induced optical poration technology with nanotechnology and applying it in cell delivery can overcome the physical barriers of cell walls and cell membranes, significantly improve the delivery efficiency of exogenous substances, and simultaneously reduce potential damage to cells.

[0004] Liquid fluorocarbons and photothermal materials are materials with unique properties. Liquid fluorocarbons have excellent chemical stability and low boiling points, and are widely used in ultrasound contrast agents, coolants, and drug delivery systems. Photothermal materials, on the other hand, with their excellent photothermal conversion performance, good biocompatibility, and tunable surface functionalization characteristics, have been widely used in medical imaging and targeted drug delivery. In recent years, the development of nanotechnology has made the combination of these two types of substances have broader application prospects, especially in the field of laser-induced cell delivery. The boiling point of liquid fluorocarbons is only a few dozen degrees, which can provide a low-boiling environment for photothermal materials and effectively improve the efficiency of optical perforation. Therefore, developing a photothermal nanoparticle for intracellular delivery of biomacromolecules, providing its preparation method and its application in laser-induced optical perforation-mediated intracellular delivery, has important research significance and application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a photothermal-phase change dual-functional core-shell nanoparticle and its intracellular delivery application. The photothermal-phase change dual-functional core-shell nanoparticle is prepared by an ultrasonic emulsification method. The preparation method is simple and can generate steam microbubbles under laser induction to achieve cell perforation, thereby delivering functional biomacromolecules into the cell interior.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The first technical problem to be solved by the present invention is to provide a photothermal-phase change dual-functional core-shell nanoparticle. The photothermal-phase change dual-functional core-shell nanoparticle has a core-shell structure, with a polymer as the outer shell and liquid fluorocarbon and photothermal material as the inner core.

[0008] The polymer is selected from any one of polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), chitosan (CS), and sodium alginate (SA); the liquid fluorocarbon is selected from any one of perfluoropentane (PFP) and perfluorohexane (PFH); the photothermal material is any one or more of iron oxide, gold, silver, Prussian blue, carbon nanotubes, and Mxene photothermal materials.

[0009] The mass ratio of the polymer, the volume of the liquid fluorocarbon, and the mass of the photothermal material is 100 mg: 400 μL: (1 - 10) mg, preferably 100 mg: 400 μL: 7 mg.

[0010] The hydrated particle size of the photothermal-phase change dual-functional core-shell nanoparticle is 200 - 900 nm, preferably 500 - 600 nm. This nanoparticle can generate steam microbubbles under laser induction and instantaneously perforate the cell wall and cell membrane in one step.

[0011] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned photothermal-phase change dual-functional core-shell nanoparticles, comprising the following steps:

[0012] Step (1): Dissolve the polymer in an organic solvent to form a polymer solution, then disperse the photothermal material in the polymer solution by water bath sonication to form a mixed solution, and then add liquid fluorocarbon and gently shake to obtain a homogeneous mixture;

[0013] Step (2): Add an emulsifier to the homogeneous mixture, ultrasonically emulsify it with an ultrasonic cell disruptor, volatilize the solvent overnight in a fume hood, wash it with ultrapure water, and centrifuge to obtain nanoparticles;

[0014] Step (3): Under pulsed laser irradiation, test its ability to generate vapor microbubbles.

[0015] In step (1), the organic solvent is one of dichloromethane and chloroform.

[0016] The amount of the organic solvent has no influence on the structure and properties of the nanoparticles. Generally, the volume ratio of the organic solvent to the mass of the polymer is 1 mL: 40 mg.

[0017] In step (2), the emulsifier is a 2% by mass polyethylene glycol-124 solution, the ultrasonic power of the ultrasonic cell disruptor is 40%, it is turned on for 3 seconds and turned off for 7 seconds, and the ultrasonic time is 30 minutes. The centrifugation speed is 4000 - 10000 rpm.

[0018] In step (3), when testing the ability of different nanoparticles to generate vapor microbubbles, the pulsed laser energy and the nanoparticle concentration need to be kept the same.

[0019] The third technical problem to be solved by the present invention is to point out that the above-mentioned photothermal-phase change dual-functional core-shell nanoparticles are applied to the field of intracellular delivery of cells.

[0020] The above-mentioned photothermal-phase change dual-functional core-shell nanoparticles are used for intracellular delivery of functional biomacromolecules to plant cells or animal cells.

[0021] Furthermore: The above-mentioned photothermal-phase change dual-functional core-shell nanoparticles are used for intracellular delivery of functional biomacromolecules to plant cells or animal cells, and are characterized in that the efficiency of delivering FITC-Dextran 10 (10 kDa) into cells can reach up to 85.7%, and the net acquisition rate can reach 50.3%

[0022] Furthermore, the method for using the photothermal-phase change dual-functional core-shell nanoparticles in intracellular delivery of functional biomacromolecules to plant cells or animal cells is as follows: Pulse laser scanning is performed on the photothermal-phase change dual-functional core-shell nanoparticles. Due to the photothermal conversion characteristics of the nanoparticles and the low boiling point characteristics of liquid fluorocarbon, vapor microbubbles are rapidly generated under pulse laser scanning and quickly rupture to generate physical forces acting on the plant cell wall and cell membrane or animal cell membrane, resulting in instantaneous perforations. Furthermore, the functional biomacromolecules quickly pass through the pores to reach the inside of the cells and play their roles.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The photothermal-phase change dual-functional core-shell nanoparticles of the present invention have the performance of converting the light energy of pulsed laser into heat energy under pulsed laser scanning. The heat generation instantaneously causes the surrounding liquid to volatilize to generate vapor microbubbles. Furthermore, the vapor microbubbles rupture, achieving a perforation effect on the cell wall and cell membrane, and promoting the entry of biomacromolecules into the cells.

[0025] (2) In the photothermal-phase change dual-functional core-shell nanoparticles of the present invention, the low-boiling-point liquid fluorocarbon phase, due to its boiling point of only dozens of degrees, reduces the heat required for generating unit vapor bubbles, improves the light perforation efficiency, and furthermore, the low-boiling-point liquid fluorocarbon plays a buffering role and reduces the damage to cells.

[0026] (3) The photothermal-phase change dual-functional core-shell nanoparticles of the present invention, in synergistic action with laser, can effectively overcome the physical barriers formed by the cell wall and cell membrane and improve the efficiency of intracellular delivery of functional biomacromolecules to cells.

[0027] (4) The photothermal-phase change dual-functional core-shell nanoparticles of the present invention have a simple preparation method, low cost, and great application potential. Description of the Drawings

[0028] Figure 1 It is a preparation flow chart of the photothermal-phase change dual-functional core-shell nanoparticles for intracellular delivery of biomacromolecules to cells.

[0029] Figure 2 It is for PLA / PFH / Fe 3 O 4 TEM images (a) and enlarged images (b) of the core-shell nanoparticles.

[0030] Figure 3 It is for the PLA / PFH / Fe 3 O 4 Confocal laser scanning microscopy images of the core-shell nanoparticles prepared in Example 1 delivering FITC-Dextran 10 (10 kDa) to onion cells under laser induction with different energy densities.

[0031] Figure 4 Scanning confocal microscopy images of FITC-Dextran 10 (10 kDa) delivery to onion cells induced by laser with different energy densities without nanoparticle mediation. Detailed implementation manners

[0032] The technical solutions of the present invention will be further elaborated through the following examples, aiming to better understand the content of the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0033] Example 1

[0034] Preparation method of the photothermal-phase change bifunctional core-shell nanoparticles ( Figure 1 ), and its steps are mainly as follows:

[0035] Step (1): Dissolve the high molecular polymer polylactic acid (PLA) in dichloromethane solvent, stir at room temperature until completely dissolved, then add iron oxide to the PLA solution. Under ice-water bath, ultrasonically crush for 6 minutes, with an ultrasonic power of 40%, 3 seconds on and 7 seconds off, to make the iron oxide nanoparticles evenly dispersed in the PLA solution. Add perfluorohexane to the above solution and gently shake evenly to make the mass ratio of polylactic acid, the volume of perfluorohexane, and the mass of iron oxide be 100 mg: 400 μL: 7 mg

[0036] Step (2): Add 2% polyvinyl alcohol-124 (PVA-124) by mass to the mixture, ultrasonically crush for 30 minutes, with an ultrasonic power of 40%, 3 seconds on and 7 seconds off. After emulsification is completed, volatilize the solvent overnight in a fume hood, centrifuge (4000 rpm, 20 minutes), take the supernatant and continue to centrifuge (10000 rpm, 20 minutes). The precipitate is the PLA / PFH / Fe 3 O 4 core-shell nanoparticles with a hydrated size of 500 - 600 nm, disperse them into deionized water, and store them at 4 °C for standby.

[0037] Step (3): Use a 9.4 J / cm 2 pulsed laser to scan the PLA / PFH / Fe with a concentration of 0.1 mg / ml 3 O 4 core-shell nanoparticles, and on average 15 vapor microbubbles can be generated under one pulsed laser.

[0038] For the PLA / PFH / Fe prepared in this example 3 O4 The core-shell nanoparticles were characterized. They were composed of PLA / PFH / Fe 3 O 4 As shown by the TEM of the core-shell nanoparticles ( Figure 2 ), the PLA / PFH / Fe 3 O 4 nanoparticles had a core-shell structure. Since the PLA shell encapsulated PFH and Fe 3 O 4 , the lipid center dispersed the Fe 3 O 4 nanoparticles with a darker color. Surrounding them was PFH with a lighter color, and the outermost layer was the PLA film layer. Since there was a small amount of Fe 3 O 4 on the PLA film, its color was also darker. This indicated that the PLA / PFH / Fe 3 O 4 core-shell nanoparticles were successfully synthesized. The hydrodynamic diameter of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles was detected using a laser particle size analyzer. By centrifugation, the hydrodynamic diameter of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles was 200 - 900 nm, preferably 500 - 600 nm. The hydrodynamic diameter of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles was tested continuously for 3 weeks. The particle size of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles changed little in deionized water, indicating good in vitro stability of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles.

[0039] Comparative Example 1

[0040] A preparation method of the core-shell nanoparticles with photothermal-phase change dual functions. Its steps were basically the same as those in Example 1, except that in step (1), the mass ratio of polylactic acid, the volume of perfluorohexane, and the mass of iron oxide was 100 mg: 400 μL: 1 mg; the test showed that 2 vapor microbubbles could be generated under an average of one pulsed laser in step (4).

[0041] Comparative Example 2

[0042] A preparation method of the core-shell nanoparticles with photothermal-phase change dual functions. Its steps were basically the same as those in Example 1, except that in step (1), the mass ratio of polylactic acid, the volume of perfluorohexane, and the mass of iron oxide was 100 mg: 400 μL: 5 mg; the test showed that 8 vapor microbubbles could be generated under an average of one pulsed laser in step (4).

[0043] Comparative Example 3

[0044] The preparation method of the photothermal-phase change dual-functional core-shell nanoparticles is basically the same as that of Example 1, except that in step (1), the mass ratio of polylactic acid, the volume of perfluorohexane, and the mass of iron oxide is 100 mg: 400 μL: 10 mg; tests show that about 15 vapor microbubbles can be generated under an average of one pulsed laser in step (4).

[0045] Comparing Example 1 with Comparative Examples 1, 2, and 3, it can be seen that the ability of the PLA / PFH / Fe 3 O 4 core-shell nanoparticles to generate vapor microbubbles under laser induction is higher than that of Comparative Examples 1 and 2, and is similar to that of Comparative Example 3. Because the content of the photothermal material iron oxide will affect the photothermal conversion ability of the nanoparticles, with the increase of the iron oxide content, the ability of the laser-induced nanoparticles to generate vapor microbubbles increases. However, the encapsulation ability of the nanoparticles is limited. When the addition amount of iron oxide reaches a certain value, the excess iron oxide will not be encapsulated inside the nanoparticles. Therefore, its ability to generate vapor microbubbles will not continue to increase.

[0046] Example 2

[0047] Investigate the relationship between the photothermal conversion performance of the photothermal-phase change dual-functional core-shell nanoparticles and the laser energy density and the concentration of the core-shell nanoparticles.

[0048] Test method: Add PLA / PFH / Fe 3 O 4 core-shell nanoparticles with different concentrations (0.5, 0.1, 0.05, 0.01 mg / mL) into a 96-well cell culture microplate, and the final volume is 200 μL. Use a pulsed laser to scan the PLA / PFH / Fe 3 O 4 core-shell photothermal nanoparticles, adjust their laser energy density (9.4, 7.21, 5.87, 4.68 J / cm 2 ), and observe and photograph the number of vapor microbubbles generated by the PLA / PFH / Fe 3 O 4 core-shell nanoparticles under laser induction under an inverted dark-field microscope.

[0049] Experimental results: Under the pulsed laser scan, the PLA / PFH / Fe 3 O 4 core-shell nanoparticles can absorb laser energy, convert it into heat, and manifest it in the form of vapor microbubbles. With the decrease of the laser energy density, the number of vapor microbubbles generated by the core-shell nanoparticles gradually decreases because the PLA / PFH / Fe 3 O 4The laser energy absorbed per unit area by the core-shell nanoparticles decreases. As the concentration of the core-shell nanoparticles decreases, the number of vapor microbubbles generated also decreases because, under the condition of the same laser energy density, the number of PLA / PFH / Fe 3 O 4 core-shell nanoparticles decreases. That is, the generation of vapor microbubbles by the absorption of laser energy by the core-shell nanoparticles is related to the laser energy density and the concentration of the core-shell nanoparticles.

[0050] Example 3

[0051] To investigate the adsorption ability of the core-shell nanoparticles with photothermal-phase change dual functions on the cell surface, Rhodamine B was introduced to prepare PLA-RB / PFH / Fe 3 O 4 core-shell nanoparticles with fluorescent dye. The preparation method is as follows:

[0052] Step (1): Dissolve 80 mg of polylactic acid (PLA) and 6.462 mg of diisopropylethylamine in 0.16 mL of dichloromethane solvent. Separately, dissolve 2 mg of Rhodamine B in 0.4 mL of dichloromethane solvent. Mix the two solutions and stir at room temperature. After reacting for 18 hours in the dark, precipitate 4 times in cold diethyl ether to remove the unreacted fluorescent dye, and obtain Rhodamine B-labeled polylactic acid (PLA-RB).

[0053] Step (2): Dissolve 30 mg of PLA-RB in 0.75 mL of dichloromethane. Add 5.6 mg of iron oxide to the solution. Under an ice-water bath, ultrasonically crush for 6 minutes with an ultrasonic power of 40%, 3 seconds on and 7 seconds off to uniformly disperse the iron oxide nanoparticles in the PLA-RB solution. Add 120 μL of perfluorohexane to the above solution and gently shake evenly.

[0054] Step (3): Add a 2% (mass fraction) polyvinyl alcohol (PVA) solution to the mixture. Under an ice-water bath, ultrasonically crush for 30 minutes with an ultrasonic power of 40%, 3 seconds on and 7 seconds off. After emulsification is completed, volatilize the solvent overnight in a fume hood and observe its fluorescence staining under a confocal microscope.

[0055] Step (4): Centrifuge (8000 rpm, 20 minutes) to remove the precipitate. Take the supernatant and continue to centrifuge (14000 rpm, 20 minutes). The precipitate is the PLA-RB / PFH / Fe 3 O 4 core-shell nanoparticles with a hydrated size of about 471 nm. Disperse them in deionized water and store at 4 °C for later use.

[0056] Example 4

[0057] Examine the adsorption ability of the photothermal-phase change dual-functional core-shell nanoparticles on the cell surface, taking onion cells as an example.

[0058] Experimental grouping: Sample 1: Control group (Cell), Sample 2: Group of cells incubated with core-shell nanoparticles (Cell+PLA / PFH / Fe 3 O 4 ), Sample 3: Group of cells incubated with fluorescently labeled core-shell nanoparticles (Cell+PLA-RB / PFH / Fe 3 O 4 ), Sample 4: Cells rinsed once after incubation with fluorescently labeled core-shell nanoparticles (Cell+PLA-RB / PFH / Fe 3 O 4 -1), Sample 5: Cells rinsed twice after incubation with fluorescently labeled core-shell nanoparticles (Cell+PLA-RB / PFH / Fe 3 O 4 -2).

[0059] Experimental method: Take fresh onion inner epidermal cells as model cells, cut the cell samples to a size of 0.6 cm×1.2 cm, place them on a glass slide. Add 70 μL of 3% sucrose solution to Sample 1, add 70 μL of 1 mg / mL PLA / PFH / Fe 3 O 4 to Sample 2, and add 70 μL of PLA-RB / PFH / Fe 3 O 4 to Samples 3-5, then cover with a cover slip. After incubation for 20 minutes, use a 1 mL pipette to aspirate the sucrose solution and rinse Samples 4 and 5 1 and 2 times respectively. Transfer the samples to a confocal microscope and observe the adsorption of core-shell nanoparticles on their surfaces under a 561 nm excitation light. Next, scan the onion samples with a pulsed laser (2.68 J / cm 2 ), transfer the samples to a confocal microscope, observe the fluorescence of core-shell nanoparticles on their surfaces under a 561 nm excitation light, and measure the average fluorescence intensity on the surface before and after scanning the laser.

[0060] Experimental results: Before and after laser scanning, there was no obvious fluorescence in the control group and the group of cells incubated with core-shell nanoparticles. Fluorescence could be observed on the cell walls in the group of cells incubated with fluorescently labeled core-shell nanoparticles before and after rinsing. Moreover, as the number of rinsing times increased, the average fluorescence intensity on the cell surface decreased, which was because some fluorescently labeled core-shell nanoparticles were not adsorbed on the cell surface and were rinsed away. Obvious pores could be observed on the cell surface after laser scanning, which confirmed that laser-induced photoporation occurred in the core-shell nanoparticles, and the average fluorescence intensity decreased. It was very likely that the core-shell nanoparticles absorbed energy and evaporated and disappeared in the form of vapor bubbles, or the core-shell nanoparticles internalized into the cell interior after the pores were opened on the onion surface.

[0061] Example 5

[0062] Application of a photothermal-phase change dual-functional core-shell nanoparticle in the field of intracellular delivery of cells, taking onion cells as an example.

[0063] Test method: Take fresh onion inner epidermal cells as model cells, cut the cell sample to a size of 0.6 cm × 1.2 cm, place it on a glass slide, add 70 μL of 1 mg / mL PLA / PFH / Fe 3 O 4 core-shell nanoparticles and incubate for 20 minutes. Then use a 1 mL pipette to aspirate 3% sucrose solution to wash away the unadsorbed PLA / PFH / Fe 3 O 4 core-shell nanoparticles, and retain 70 μL of 3% sucrose solution. Then add 20 μL of 3 mg / mL fluorescein isothiocyanate-dextran (FITC-Dextran, FD10) to the onion sample, cover with a coverslip. Transfer the sample to under a pulsed laser, adjust different laser energy densities, and scan half of the sample. After the laser scanning is completed, add propidium iodide (PI) to stain dead cell nuclei, rinse off the excess fluorescent dye, and transfer it to a confocal microscope to observe the delivery results and take pictures.

[0064] To explore the effect of pulsed laser on the efficiency of delivering biological macromolecules by PLA / PFH / Fe 3 O 4 core-shell nanoparticles in cells, laser energy densities of 9.4, 7.21, 5.85, 4.68, 2.68, 2.05, 1.67, 1.33 J / cm 2 were used. The hydrated particle size of the core-shell nanoparticles used was approximately 500 nm.

[0065] Experimental results: In the experimental group with the addition of PLA / PFH / Fe 3 O 4 core-shell nanoparticles ( Figure 3 ), the delivery efficiency and mortality decreased with the decrease of the pulsed laser energy density, which indicates that a higher laser energy density is beneficial for the evaporation of PLA / PFH / Fe 3 O 4 core-shell nanoparticles to generate vapor microbubbles, perforate the cell wall, and promote the delivery of FD-10 into onion cells. The highest net acquisition rate was 50.3%, and at this time, the laser energy density was 7.21 J / cm 2 . Without the addition of core-shell nanoparticles ( Figure 4) Only relying on the action of laser, at high energy density, there will be a low delivery efficiency. It can be seen that the core-shell nanoparticles absorb laser energy and convert it into heat, which is manifested in the form of vapor microbubbles, further perforating the cell wall and cell membrane surfaces, and playing a greater role in the delivery process.

[0066] In summary, the present invention provides a core-shell nanoparticle with good in vitro stability and dual functions of photothermal conversion and phase change for intracellular delivery of biomacromolecules in cells, which has good photothermal conversion performance. Among them, the photothermal material absorbs pulsed laser and converts it into heat under pulsed laser. The surrounding liquid absorbs heat and generates vapor microbubbles, and the low-boiling liquid fluorocarbon improves the efficiency of generating vapor bubbles and reduces the damage to cells at high temperatures. On the other hand, a polymer with good biocompatibility is selected to reduce the cytotoxicity of the nanoparticles. The present invention is applied to intracellular delivery of cells. By laser induction, the core-shell nanoparticle with dual functions of photothermal conversion and phase change generates vapor microbubbles. The moment the vapor microbubbles burst, they can perforate the plant cell wall and cell membrane or animal cell membrane, and push the functional biomacromolecules into the cell interior, effectively solving the physical barriers of the cell wall and cell membrane and improving the efficiency of intracellular delivery of functional biomacromolecules in cells.

Claims

1. Core-shell nanoparticles with dual functions of photothermal and phase change, characterized by: The nanoparticles are of a core-shell structure, with a high molecular polymer as an outer shell and liquid fluorocarbon and photothermal material as an inner core.

2. The core-shell nanoparticles with photothermal-phase change dual functions according to claim 1, characterized in that: The high molecular polymer is selected from any one of polylactic acid (PLA), poly(lactide-glycolide) (PLGA), chitosan (CS), and sodium alginate (SA); the liquid fluorocarbon is selected from any one of perfluoropentane (PFP) and perfluorohexane (PFH); the photothermal material is any one or more of ferroferric oxide, gold, silver, Prussian blue, carbon nanotubes, and Mxene photothermal materials.

3. The core-shell nanoparticles with photothermal-phase change dual functions according to claim 1, characterized in that: The mass ratio of the high molecular polymer, the volume of the liquid fluorocarbon and the mass ratio of the photothermal material is 100 mg:400 μL:(1-10) mg, preferably 100 mg:400 μL:7 mg.

4. The core-shell nanoparticles with photothermal-phase change dual functions according to claim 1, characterized in that: The hydrated particle size of the core-shell nanoparticles is 200-900 nm, preferably 500-600 nm.

5. A method for preparing core-shell nanoparticles with photothermal-phase change dual functions according to claim 1, characterized in that: The following steps are involved: Step (1), dissolving a high molecular weight polymer in an organic solvent to form a polymer solution, then dispersing the photothermal material in a polylactic acid solution by water bath ultrasound to form a mixed solution, then adding liquid fluorocarbon, and gently shaking to obtain a uniform mixture; Step (2), adding an emulsifier to the uniform mixture, ultrasonically emulsifying the mixture using an ultrasonic cell disruptor, evaporating the solvent in a fume hood overnight, washing with ultrapure water, and centrifuging to obtain nanoparticles; Step (3), testing its ability to generate steam microbubbles under pulsed laser irradiation.

6. The method for preparing core-shell nanoparticles with photothermal-phase change dual functions according to claim 5, characterized in that: In step (1), the organic solvent is one of dichloromethane and chloroform.

7. The method for preparing core-shell nanoparticles with photothermal-phase change dual functions according to claim 5, characterized in that: In step (2), the emulsifier is a 2% by mass polyethylene glycol-124 solution, the ultrasonic cell disruptor has an ultrasonic power of 40%, is on for 3 seconds and off for 7 seconds, and the centrifugal speed is 4000-10000 rpm.

8. The method for preparing core-shell nanoparticles with photothermal-phase change dual functions according to claim 5, characterized in that: In step (3), the ability of different nanoparticles to generate steam microbubbles is tested, and the pulse laser energy and nanoparticle concentration must be kept the same.

9. The photothermal-phase change dual-functional core-shell nanoparticles according to any one of claims 1 to 4 are used in the field of intracellular delivery.

10. The photothermal-phase change dual-functional core-shell nanoparticles according to claim 9 are applied to the field of intracellular delivery: characterized in that: The photothermal-phase change dual-functional core-shell nanoparticles are used to deliver functional biomacromolecules into plant cells or animal cells; Further: The method of using the core-shell nanoparticles with dual photothermal and phase change functions for delivering functional biomacromolecules into plant cells or animal cells is as follows: pulse laser scanning is performed on the core-shell nanoparticles with dual photothermal and phase change functions. Due to the photothermal conversion characteristics of the nanoparticles and the low boiling point characteristics of liquid fluorocarbon, nanobubbles are rapidly generated under pulse laser scanning, and they quickly rupture to generate physical forces acting on the plant cell wall and cell membrane or animal cell membrane, resulting in instantaneous perforation, so that the functional biomacromolecules quickly pass through the pores to reach the interior of the cell and exert their effects.

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