A composite material, its preparation and use

Abstract

The application provides a composite material, which comprises nanoparticles and a flexible substrate, the nanoparticles comprising one or more of carbon nanotubes, graphene, gold nanoparticles and polydopamine nanoparticles, the flexible substrate comprising one or more of thermosetting plastics such as polydimethylsiloxane and hydrogel, and the mass percentage of the nanoparticles in the composite material being 0-60 ‰. The composite material of the application is simple to prepare, has extremely strong photo-thermal conversion performance, and does not change the smooth surface of the original topological structure. Meanwhile, the composite material has universality and versatility for different cells, the delivery efficiency is close to 100%, and the modified cells can be efficiently and non-destructively released and harvested through traditional trypsin digestion, and the harvesting efficiency is more than 90%.

Description

Technical Field

[0001] This invention relates to a composite material, specifically a composite material comprising nanoparticles and a flexible substrate. This invention also relates to a method for preparing the above-mentioned composite material and its application in intracellular macromolecule delivery. Background Technology

[0002] Nanoparticles possess unique optical, electrical, and catalytic properties due to their nanoscale size. Some nanoparticles (such as metal nanoparticles like Au, metal oxide nanoparticles like iron(II,III) oxide, and polymer nanoparticles like polydopamine) have excellent surface plasmon resonance effects, enabling them to absorb large amounts of laser energy and convert it into thermal energy. Therefore, they are widely used in fields such as tumor photothermal therapy and in vivo and in vitro intracellular photothermal macromolecule delivery.

[0003] Intracellular macromolecule delivery technology, which introduces functional exogenous macromolecules to endow cells with specific properties, is crucial for numerous biomedical research projects and clinical applications. Currently, commonly used macromolecule delivery methods mainly include vector methods (including viral vectors, liposomes, or polymer vectors) and physical permeabilization methods. While viral vector methods are highly efficient, their poor safety and limitation to delivering only nucleic acids restrict their further application. During nucleic acid delivery, most viruses integrate their own DNA into the host cell, causing gene mutations in the host cell, and in severe cases, even leading to cancerous transformation of the modified cells after implantation, endangering human health. Therefore, viral vector transfection of cells is difficult to widely apply in actual clinical medicine. Liposomes and polymer vectors, on the other hand, are difficult to use on a large scale due to their low delivery efficiency and high cytotoxicity.

[0004] Compared to vector-based methods for delivering exogenous macromolecules, physical permeabilization methods are gaining increasing attention due to their high delivery efficiency, low cytotoxicity, and broad applicability, while overcoming the drawbacks of traditional viral vectors such as high infectivity and low safety. Based on the permeabilization method, physical permeabilization methods are mainly divided into mechanical permeabilization and electromagnetic / thermal permeabilization, including cell squeezing permeabilization (Reference 1), microinjection (Reference 2), electroporation (Reference 3), and photothermal permeabilization (Reference 4). Mechanical permeabilization primarily uses external force to reversibly rupture the cell membrane, thereby delivering exogenous macromolecules into the cell. Reference 1 demonstrates cell squeezing via microfluidic channels; this system can be used to prepare modified cells on a large scale, but the equipment is complex, expensive, and has low exogenous macromolecule delivery efficiency. Reference 2 discloses a microinjection device; this technology has high delivery efficiency, but it can only operate on single cells, resulting in low efficiency, and the high labor and equipment costs are not conducive to practical clinical applications. In summary, most mechanical cell disruption methods require complex instruments or devices, have high experimental thresholds, are expensive, and require too much manual skill. They do not meet the actual clinical treatment needs for obtaining large quantities of modified cells as cheaply, conveniently, quickly, efficiently, and safely as possible.

[0005] Compared to mechanical permeabilization methods, electromagnetic / thermal permeabilization methods only alter the applied electric, magnetic, optical, or thermal fields without directly interacting with cells, thus reducing cell damage. Furthermore, they are simpler to operate than mechanical methods and have gained increasing attention in recent years. Electroporation is currently the most common electromagnetic / thermal permeabilization method, offering high delivery efficiency. However, the high-voltage pulse intensity used during delivery significantly reduces cell viability, and the unsatisfactory cell adhesion and expression after harvesting limit its widespread application. Photothermal permeabilization for macromolecule delivery primarily utilizes laser irradiation of nanoparticles, leveraging their unique photothermal conversion capabilities to heat surrounding tissues or cells, causing instantaneous reversible permeation of the cell membrane. This improves cell membrane permeability, facilitating intracellular macromolecule delivery. Compared to other macromolecule delivery methods, photothermal macromolecule delivery is not only simple to operate and offers high cell throughput but also boasts strong versatility for both cells and macromolecules, making it a very promising method for live-cell macromolecule delivery. Traditional photothermal macromolecule delivery platforms use nanoparticles as photothermal agents for intracellular delivery. Although the operation is simple, it also has obvious drawbacks: 1. Since nanoparticles are easy to enter cells through endocytosis and most nanoparticles cannot be degraded through normal metabolism, they have a certain degree of cytotoxicity to cells; 2. Reducing the concentration of nanoparticles can appropriately alleviate cytotoxicity, but it will reduce photothermal efficiency, ultimately leading to a decrease in the efficiency of intracellular macromolecule delivery.

[0006] Building upon nanoparticles, to reduce nanoparticle cytotoxicity and improve intracellular macromolecule delivery efficiency, Reference 4 discloses an endogenous and exogenous macromolecule delivery platform for substrates modified with a photothermal gold nanoparticle layer. This platform enriches photothermal nanoparticles on the substrate surface through surface modification. Compared to nanoparticles, the enriched gold nanoparticle layer is not only less susceptible to endocytosis by cells, improving the cytotoxicity of photothermal materials, but also enhances photothermal efficiency, allowing for macromolecule delivery using low-intensity laser light sources, thus reducing the impact on cell viability while improving delivery efficiency. However, the surface modification of the nanoparticle layer alters the substrate surface topology, inevitably leading to side effects, making it difficult to harvest modified cells from the substrate for subsequent research and applications. To address these side effects, Reference 5 demonstrates a method combining photothermal perforation of gold nanoparticles with liposomes, successfully and efficiently transfecting difficult-to-transfect cells. However, due to the material surface topology and the ease with which cells engulf nanoparticles, it remains difficult to harvest modified cells from the substrate for subsequent research and applications. Reference 6 uses polydopamine nanoparticles as a photothermal substrate, surface-modified with the thermosensitive polymer PNIPAAM, to control cell release through temperature changes. However, the preparation temperature significantly affects the surface modification of the thermosensitive polymer, resulting in poor material reproducibility. Furthermore, the surface modification process is complex, time-consuming, and leads to a short shelf life. Additionally, the use of only one polymer brush layer results in insufficient thermosensitivity, making temperature-controlled cell release difficult and prone to cell damage. This leads to a low efficiency in harvesting viable cells, failing to truly solve the cell harvesting problem in photothermal transfection and hindering the large-scale practical application of photothermal perforation. Reference 7 uses silicon nanowires as a photothermal substrate, surface-modified with sugar-responsive phenylboronic acid. Using non-toxic natural biomolecules, such as sugars, as stimuli, cell release can be triggered under relatively mild conditions, but it still cannot efficiently harvest highly viable cells.

[0007] In summary, current photothermal intracellular macromolecule delivery platforms based on nanoparticles and nanoparticle layers still cannot meet the needs of practical applications. Therefore, further research is needed to design a macromolecule delivery system that is inexpensive, uses simple materials, is easy to operate, fast, has broad applicability to exogenous molecules and cell types, has high delivery efficiency, low cytotoxicity, can process cells in large throughputs, has high cell harvest rates, and maintains good cell viability.

[0008] Reference 1. Integr. Biol, 2014, 6, 470-475;

[0009] Reference 2. CN105420099B;

[0010] Reference 3. CN105143436B;

[0011] Reference 4. CN105420278A;

[0012] Document 5.ACS Appl.Mater.Interfaces, 2017, 9, 21593-21598;

[0013] Document 6.ACS Appl.Mater.Interfaces,2019,11,12357-12366;

[0014] Reference 7. Adv. Funct. Mater, 2019, 1906362. Summary of the Invention

[0015] The problem the invention aims to solve

[0016] To overcome the above-mentioned technical problems, the present invention provides a composite material that is inexpensive, uses simple materials, is easy and quick to operate, has strong versatility for exogenous molecules and cell types, has high delivery efficiency, low cytotoxicity, can process cells in large quantities, has a high cell harvest rate and good cell activity.

[0017] Solution for solving the problem

[0018] To address the technical problems described in this invention, the present invention provides the following technical solutions:

[0019] A composite material comprising nanoparticles and a flexible substrate, wherein the nanoparticles include one or more photothermal nanoparticles such as carbon nanotubes, graphene, gold nanoparticles and polydopamine nanoparticles, and the flexible substrate includes one or more thermosetting plastics such as polydimethylsiloxane and hydrogels, wherein the mass percentage of the nanoparticles in the composite material is 1 to 60‰.

[0020] Preferably, the nanoparticles are carbon nanotubes, and the flexible substrate is polydimethylsiloxane.

[0021] Preferably, the mass percentage of the nanoparticles in the composite material is 5 to 30‰.

[0022] Preferably, the mass percentage of the nanoparticles in the composite material is 5-20‰, more preferably 5-10‰.

[0023] On the other hand, the present invention also provides a method for preparing the above-mentioned composite material, the method comprising the following steps:

[0024] (1) Mix the nanoparticle solution and the flexible substrate or flexible substrate prepolymer thoroughly, then add it into the mold and let it stand to obtain a slurry;

[0025] (2) The obtained slurry is vacuumed and cured into a film, then removed and demolded. The obtained material is optionally processed and shaped to obtain the composite material.

[0026] Preferably, in step (1), the nanoparticle solution is a carbon nanotube solution, and the solvent of the solution is selected from water, methanol, ethanol and dimethylformamide, and the mass fraction of the carbon nanotube solution is 1-10%.

[0027] Preferably, in step (1), the flexible substrate is polydimethylsiloxane, and the flexible substrate prepolymer is a silicone rubber base and a silicone rubber curing agent with a mass ratio of 5 to 20:1.

[0028] Preferably, the method includes the following steps:

[0029] (1) Mix the carbon nanotube aqueous solution with a mass fraction of 1-10% and the polydimethylsiloxane prepolymer thoroughly, then add it evenly into the mold and let it stand for 0.5-2 hours until the slurry fills the mold;

[0030] (2) Vacuum the obtained slurry at room temperature for 0.5 to 2 hours, cure it in an oven at 60 to 80°C for 0.5 to 2 hours to form a film, then remove it and demold it. Optionally, process the obtained material into the required size to obtain the composite material.

[0031] Finally, the present invention also provides the use of the composite material in the preparation of materials for intracellular macromolecular delivery and / or cell detachment.

[0032] Preferably, the macromolecule is selected from one or more of polysaccharide molecules, proteins, DNA, RNA, intracellular probes, therapeutic drugs, aptamers, bacteria, artificial chromosomes, or organelles.

[0033] The effects of the invention

[0034] This invention discloses a nanoparticle / flexible substrate composite material (nanoparticles include, but are not limited to, carbon nanotubes, graphene, gold nanoparticles, polydopamine nanoparticles, etc.; flexible substrates include, but are not limited to, PDMS, hydrogels, etc.) that is inexpensive, easy to prepare, convenient to handle, has low cytotoxicity, and good photothermal properties. This material is simple to prepare, has extremely strong photothermal conversion performance, and can rapidly undergo photothermal conversion under low-intensity near-infrared laser light sources. The surface roughness and the number of exposed nanoparticles after film formation can be controlled by adjusting the nanoparticle concentration, ultimately obtaining a smooth surface with specific photothermal properties without altering the original topology. The composite material disclosed in this invention, taking MCNT / PDMS as an example, utilizes the superior photothermal properties of MCNT / PDMS. Irradiation with near-infrared laser enhances the permeability of cell membranes cultured on MCNT / PDMS, allowing exogenous macromolecules in the solution to enter the cells and achieving efficient intracellular macromolecule delivery. Unlike carrier methods, this invention employs a physical membrane-breaking method. By adjusting the laser intensity, it can achieve efficient delivery of various exogenous macromolecules (but not limited to dextran, proteins, pDNA, RNA, therapeutic drugs, intracellular probes, etc.) while ensuring cell viability. It can deliver cells and is universally applicable to different cell types (including but not limited to HeLa and human embryonic fibroblasts), with a delivery efficiency close to 100%. By adjusting the mass fraction of carbon nanotubes, the growth and desorption of modified cells on the MCNT / PDMS photothermal substrate can be achieved in the same way as in a culture flask. Modified cells can be efficiently and non-destructively released and harvested through conventional trypsin digestion, with a harvesting efficiency of over 90%. In addition, due to the flexible properties of MCNT / PDMS materials, it can be prepared into surfaces of any size and shape, thereby enabling high-throughput cell processing and efficient, large-volume intracellular delivery in a short time. Attached Figure Description

[0035] Figure 1 Photothermal properties of MCNT / PDMS with different MCNT contents under different laser conditions;

[0036] Figure 2 The effect of different carbon nanotube contents on the surface roughness of MCNT / PDMS;

[0037] Figure 3 Cell detachment and harvesting efficiency of MCNT / PDMS materials with different MCNT concentrations;

[0038] Figure 4 Changes in HeLa cell membrane permeability under different laser irradiation conditions (SYTOX);

[0039] Figure 5 Changes in relative cell viability of HeLa cells under different laser irradiation conditions;

[0040] Figure 6 Transfection efficiency and relative cell viability of MCNT / PDMS and Lipo2000. Detailed Implementation

[0041] The present invention provides a composite material comprising nanoparticles and a flexible substrate. The nanoparticles include one or more of carbon nanotubes, graphene, gold nanoparticles, and polydopamine nanoparticles. The flexible substrate includes one or more of thermosetting plastics such as polydimethylsiloxane (PDMS) and hydrogels. The mass percentage of the nanoparticles in the composite material is 1 to 60‰.

[0042] In a preferred embodiment, the carbon nanotubes are multi-walled carbon nanotubes (MCNTs).

[0043] In a preferred embodiment, the nanoparticles are carbon nanotubes, and the flexible substrate is polydimethylsiloxane.

[0044] The term "flexible substrate" refers to a polymer material with good elasticity, toughness, and plasticity. Due to the flexible properties of the substrate, composite materials doped with photothermal nanoparticles can be formed on any surface of a well plate or culture flask, or directly cured to obtain composite materials of specific shapes, thereby enabling photothermal macromolecule delivery in different cell culture environments.

[0045] In a preferred embodiment, the composite material consists of nanoparticles and a flexible substrate.

[0046] In a more preferred embodiment, the mass percentage of the nanoparticles in the composite material is 5-30‰, preferably 5-20‰, more preferably 5-10‰, and most preferably 10‰.

[0047] The present invention also provides a method for preparing the above-mentioned composite material, the method comprising the following steps:

[0048] (1) Mix the nanoparticle solution and the flexible substrate or flexible substrate prepolymer thoroughly, then add it into the mold and let it stand to obtain a slurry;

[0049] (2) The obtained slurry is vacuumed and cured into a film, then removed and demolded. The obtained material is optionally processed and shaped to obtain the composite material.

[0050] In a preferred embodiment, in step (1), the nanoparticle solution is a carbon nanotube solution, and the solvent of the solution is selected from water, methanol, ethanol and dimethylformamide, and the mass fraction of the carbon nanotube solution is 1-10%.

[0051] In a preferred embodiment, in step (1), the flexible substrate is polydimethylsiloxane, and the flexible substrate prepolymer is obtained by mixing a silicone rubber base and a silicone rubber curing agent in a mass ratio of 5 to 20:1. The silicone rubber base is Sylgard 184 silicone rubber base, and the silicone rubber curing agent is Sylgard 184 silicone rubber curing agent.

[0052] In a preferred embodiment, the method includes the following steps:

[0053] (1) Mix the carbon nanotube aqueous solution with a mass fraction of 1-10% and polydimethylsiloxane thoroughly, then add it evenly into the mold and let it stand for 0.5-2 hours until the slurry fills the mold;

[0054] (2) Vacuum the obtained slurry at room temperature for 0.5 to 2 hours, cure it in an oven at 60 to 80°C for 0.5 to 2 hours to form a film, then remove it and demold it. Optionally, process the obtained material into a sheet of the required size, such as a diameter or length of 0.5 to 2 cm, to obtain the composite material.

[0055] On the other hand, the present invention provides the application of the above-described composite material in the preparation of materials for the delivery and / or desorption of intracellular macromolecules.

[0056] In a preferred embodiment, the macromolecule has a mass of 0.9–500 kDa and a physical size of 1 nm–5 μm.

[0057] In a preferred embodiment, the macromolecules include, but are not limited to, polysaccharide molecules (such as dextran), proteins (such as gene editing enzymes, antibodies, antigens), DNA (such as pDNA), RNA (such as mRNA, guide RNA, miRNA, siRNA), therapeutic drugs, intracellular probes (such as quantum dots), nanomaterials (such as nanoparticles, nanodevices), aptamers, bacteria, artificial chromosomes, organelles (such as mitochondria), etc.

[0058] The present invention also provides an application of the above-mentioned composite material in intracellular macromolecule delivery and cell desorption.

[0059] Finally, the prepared MCNT / PDMS was used for intracellular macromolecule delivery and modified cell harvesting experiments. The specific experimental steps are as follows:

[0060] (1) Sterilize the MCNT / PDMS material using an autoclave, and then sterilize the cells (including but not limited to HeLa cells, human embryonic fibroblasts, etc.) at a concentration of 50,000 / cm³. 2 The cells were seeded at a density of MCNT / PDMS and cultured for 4–24 h to allow them to adhere to the sample.

[0061] (2) Wash the cells with sterile PBS and add serum-free cell culture medium containing exogenous molecules (including but not limited to plasmid DNA), wherein the final concentration of pDNA is 0.005 to 0.008 μg / mL.

[0062] (3) Using an 808nm near-infrared laser source at 1-10W / cm 2 Irradiate cells on the sample for 10–180 s within the power density range.

[0063] (4) 1-4 hours after laser irradiation, wash the cells with sterile PBS, add trypsin to digest for 0.5-5 minutes, and stop digestion with serum. Then gently blow the cells with a pipette tip until they are completely detached. Centrifuge and resuspend the detached cells to obtain the harvested cells.

[0064] The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the claims.

[0065] The MCNT aqueous solution used in this embodiment of the invention was purchased from Pioneer Nano, and the PDMS was purchased from Dow Corning.

[0066] Example 1

[0067] (1) Mix 10% MCNT aqueous solution and PDMS thoroughly according to the final carbon nanotube mass fraction of the composite material of 0-30‰, pour evenly into the mold, and let stand for 1 hour until the slurry fills the mold.

[0068] (2) Vacuum the material at room temperature for 1 hour to remove air bubbles, then cure it in an oven at 60-80°C for 1 hour. Remove the material from the oven, demold it, and cut it into round pieces with a diameter of 1.1 cm.

[0069] (3) Laser power ranged from 0.6 to 3.2 W / cm². 2 The wet solution temperature of MCNT / PDMS materials with different MCNT contents was measured using a thermal imager under laser conditions with irradiation time of 0–30 s.

[0070] The experimental results are shown in Figure 1 The results show that pure PDMS has no photothermal conversion capability, while the addition of MCNT results in 5-30‰ MCNT / PDMS materials with excellent photothermal conversion performance, providing the possibility of delivering exogenous macromolecules into cells through photothermal perforation.

[0071] Example 2

[0072] (1) Mix 10% MCNT aqueous solution and PDMS solution thoroughly according to the final carbon nanotube mass fraction of the composite material of 0-30‰, pour evenly into the mold, and let stand for 1 hour until the slurry fills the mold.

[0073] (2) Vacuum the material at room temperature for 1 hour to remove air bubbles, then cure it in an oven at 60-80°C for 1 hour. Remove the material from the oven, demold it, and cut it into round pieces with a diameter of 1.1 cm.

[0074] (3) After thorough ultrasonic cleaning with water, acetone and ethanol, dry with nitrogen for later use.

[0075] (4) Use a roughness tester to measure the surface roughness of materials with different MCNT contents.

[0076] Surface roughness of MCNT / PDMS composites with different MCNT contents, such as Figure 2 As shown, Figure 2 This indicates that MCNT / PDMS composites with an MCNT mass fraction of 0–10‰ have very low surface roughness and a completely smooth surface. However, MCNT / PDMS composites with an MCNT mass fraction of 20–30‰ exhibit increased surface roughness due to exposed MCNTs. Adjusting the MCNT content can alter the surface topology of the material.

[0077] Example 3

[0078] (1) Mix 10% MCNT aqueous solution and PDMS solution thoroughly according to the final carbon nanotube mass fraction of the composite material (0-30‰), pour evenly into the mold, and let stand for 1 hour until the slurry fills the mold. Vacuum at room temperature for 1 hour to remove air bubbles, and then cure in an oven at 60-80℃ for 1 hour. Remove from the mold and cut the material into round pieces with a diameter of 1.1 cm.

[0079] (2) Sterilize the slides using a high-temperature and high-pressure steam sterilizer. Place the slides in a 48-well plate and seed cells (including but not limited to HeLa and human embryonic fibroblasts) into the wells of the 48-well plate at a density of 10,000 to 50,000 per well.

[0080] (3) Add serum-free cell culture medium containing pGFP, and use a laser light source with a wavelength of 808 nm at 2.3 W / cm². 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0081] (4) One hour after laser irradiation, the cells were washed with sterile PBS, digested with trypsin for 0.5–5 minutes, and the digestion was stopped with serum. The cells were then gently pipetted until they were completely detached. The detached cells were centrifuged and resuspended to obtain the harvested cells. The cells on the sample were stained with DAPI dye to observe the number of cells before and after detachment and after harvesting.

[0082] Cell detachment and harvesting efficiency of MCNT / PDMS materials with different MCNT mass fractions, such as Figure 3 As shown. From Figure 3It can be seen that the cell desorption efficiency (i.e., release rate) and harvest efficiency (i.e., harvest rate) of the smooth-surfaced MCNT / PDMS (MCNT mass fraction of 0-10‰) slices can reach more than 95% and 85% respectively, which fully meets the requirements of cell harvesting.

[0083] In summary, 5-10‰ MCNT / PDMS possesses both good photothermal properties and cell detachment capabilities, making it suitable for subsequent intracellular macromolecule delivery.

[0084] Example 4

[0085] (1) Mix 10% MCNT aqueous solution and PDMS solution thoroughly according to 0-30‰ of the final carbon nanotube mass of the composite material, pour evenly into the mold, and let stand for 1 hour until the slurry fills the mold. Vacuum at room temperature for 1 hour to remove air bubbles, and then cure in an oven at 60-80℃ for 1 hour. Remove from the mold and cut the material into round pieces with a diameter of 1.1 cm.

[0086] (2) Sterilize the slides using a high-temperature and high-pressure steam sterilizer. Place the slides in a 48-well plate and seed HeLa cells into the wells of the 48-well plate at a density of 10,000 to 50,000 per well.

[0087] (3) Add serum-free cell culture medium containing SYTOX (a dye that can only pass through damaged cell membranes; the higher the fluorescence intensity, the better the cell membrane permeability), and use a laser light source with a wavelength of 808 nm at a low intensity of 0.6–3.2 W / cm². 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0088] (4) After laser irradiation for 1 to 4 hours, replace with complete culture medium to continue culturing cells.

[0089] (5) After laser irradiation for 2 to 4 hours, live cells are stained with Calcein and then fluorescence is observed using a fluorescence microscope.

[0090] (6) 48 hours after laser irradiation, the relative cell viability was detected using a CCK-8 assay kit (cell viability test kit).

[0091] Changes in HeLa cell membrane permeability (SYTOX) under different laser irradiation conditions, such as Figure 4 As shown, from Figure 4 As can be seen, pure PDMS slides have virtually no effect on cell membrane permeability, while 5–30‰ MCNT / PDMS slides have an effect on cell membrane permeability of 1.4–3.2 W / cm². 2 Both laser irradiation and light exposure significantly alter cell membrane permeability, with permeability increasing with increasing MCNT content. The activity of HeLa cells under different laser irradiation conditions is as follows: Figure 5 ,from Figure 5 It can be seen that the range is 0.6–2.3 W / cm². 2 The laser intensity can restore cells to over 80% relative cell viability. This demonstrates the feasibility of improving cell membrane permeability and delivering exogenous macromolecules by irradiating MCNT / PDMS with near-infrared laser.

[0092] In summary, a film with an MCNT content of 10‰ at a power of 2.3W / cm²... 2 When the irradiation time is 30 seconds, the obtained cells have good cell membrane permeability and cell viability. By changing the laser irradiation conditions, cells with both good viability and membrane permeability can be obtained.

[0093] Example 5

[0094] (1) Mix 10% MCNT aqueous solution and PDMS solution thoroughly to achieve a final carbon nanotube mass fraction of 10‰ in the composite material. Pour the mixture evenly into a mold and let it stand for 1 hour until the slurry covers the mold. Vacuum the mold at room temperature for 1 hour to remove air bubbles, and then cure it in an oven at 60-80℃ for 1 hour. Remove the mold and cut the material into 1.1cm diameter round pieces.

[0095] (2) Sterilize the slides using a high-temperature and high-pressure steam sterilizer. Place the slides in a 48-well plate and seed HeLa cells and human embryonic fibroblasts into the wells of the 48-well plate at a density of 10,000 to 50,000 per well.

[0096] (3) Add serum-free cell culture medium containing pGFP, and use a laser light source with a wavelength of 808 nm at 2.3 W / cm². 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0097] (4) After laser irradiation for 1 to 4 hours, replace with complete culture medium to continue culturing cells.

[0098] (5) After 48 hours of laser irradiation, the cell nuclei were stained with DAPI and the expression of green fluorescent protein was observed using a fluorescence microscope.

[0099] At 2.3W / cm 2 The transfection efficiency and relative cell viability of pGFP delivery to HeLa cells and human embryonic fibroblasts under 30s laser irradiation conditions are as follows: Figure 6 As shown in A and B, the method for delivering macromolecules into cells based on the composite material of this invention has strong versatility across cell types. Figure 6 As can be seen from B, the transfection efficiency for difficult-to-transfect cells such as human embryonic fibroblasts can reach over 93%, while maintaining good cell viability.

[0100] Comparative Example 1

[0101] The conditions for Comparative Example 1 and Example 5 were the same, the difference being that Comparative Example 1 used commercially available Lipo2000 instead of the MCNT / PDMS composite material as the transfection reagent, and the cells tested were only HeLa cells.

[0102] At 2.3W / cm 2 Transfection efficiency and relative cell viability of pGFP delivered to HeLa cells under 30s laser irradiation conditions, as shown in the figure. Figure 6 As shown in Figure A, under the same conditions, Lipo2000 has a transfection efficiency of less than 10% for primary cells, and the cells cannot maintain good viability.

Claims

1. The application of a composite material in the preparation of materials for intracellular macromolecular delivery and / or cell detachment, characterized in that, The composite material comprises nanoparticles and a flexible substrate, wherein the nanoparticles are carbon nanotubes and the flexible substrate is polydimethylsiloxane, and the mass percentage of the carbon nanotubes in the composite material is 1-10‰; the delivery of intracellular macromolecules is achieved by physical membrane disruption, allowing exogenous macromolecules in the solution to enter the cells and realize intracellular macromolecule delivery.

2. The application as described in claim 1, characterized in that, The macromolecules are selected from one or more of the following: polysaccharide molecules, proteins, DNA, RNA, intracellular probes, therapeutic drugs, aptamers, bacteria, artificial chromosomes, or organelles.

3. The application as described in claim 1 or 2, characterized in that, The mass percentage of carbon nanotubes in the composite material is 5-10‰.

4. The application as described in claim 1 or 2, characterized in that, The preparation method of the composite material includes the following steps: (1) The nanoparticle solution and flexible substrate or flexible substrate prepolymer are thoroughly mixed, then added to a mold and left to stand to obtain a slurry; (2) After vacuuming the obtained slurry, it is cured into a film, then removed and demolded. The obtained material is then optionally processed and shaped to obtain the composite material.

5. The application as described in claim 4, characterized in that, In step (1), the nanoparticle solution is a carbon nanotube solution, and the solvent of the solution is selected from water, methanol, ethanol and dimethylformamide. The mass fraction of the carbon nanotube solution is 1~10%.

6. The application as described in claim 4, characterized in that, In step (1), the flexible substrate is polydimethylsiloxane, and the flexible substrate prepolymer is a silicone rubber base and a silicone rubber curing agent with a mass ratio of 5 to 20:

1.

7. The application as described in claim 4, characterized in that, The method includes the following steps: (1) Mix the carbon nanotube aqueous solution with a mass fraction of 1~10% and the polydimethylsiloxane prepolymer thoroughly, then add it evenly into the mold and let it stand for 0.5~2h until the slurry fills the mold; (2) Vacuum the obtained slurry at room temperature for 0.5-2 hours, cure it in an oven at 60-80°C for 0.5-2 hours to form a film, then remove it and demold it. Optionally, process the obtained material into the required size to obtain the composite material.

Images