CN112915209A

CN112915209A - Composite material and preparation method and application thereof

Info

Publication number: CN112915209A
Application number: CN201911241426.5A
Authority: CN
Inventors: 陈红; 王蕾; 唐鹤鸣
Original assignee: Bmd Biotechnology Suzhou Co ltd
Current assignee: Bmd Biotechnology Suzhou Co ltd
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2021-06-08

Abstract

The invention provides a composite material which comprises nano particles and a flexible base material, wherein the nano particles comprise one or more of carbon nano tubes, graphene, gold nano particles and polydopamine nano particles, the flexible base material comprises one or more of thermosetting plastics such as polydimethylsiloxane and hydrogel, and the mass percentage of the nano particles in the composite material is 0-60 per thousand. The composite material is easy 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 universality on different cells, the delivery efficiency is close to 100%, the modified cells can be efficiently released and harvested without damage through the traditional pancreatin digestion, and the harvesting efficiency is over 90%.

Description

Composite material and preparation method and application thereof

Technical Field

The invention relates to a composite material, in particular to a composite material containing nano particles and a flexible base material, and also relates to a preparation method of the composite material and application of the composite material in intracellular macromolecule delivery.

Background

The nano-particles have unique optical, electric and catalytic properties due to the nano-scale size, and some nano-particles (for example, metal nano-particles such as Au, metal oxide nano-particles such as ferroferric oxide and polymer nano-particles such as polydopamine) have excellent surface plasmon resonance effect, can absorb laser energy in a large amount and convert the laser energy into heat energy, and are widely applied to the fields of tumor photothermal therapy, in-vivo and in-vitro intracellular photothermal macromolecule delivery and the like.

Intracellular macromolecule delivery technology can impart specific properties to cells by introducing functional exogenous macromolecules, and is the key to the development and clinical application of numerous biomedical research works. The currently used methods for delivering macromolecules mainly comprise a carrier method (including viral vectors, liposome or polymer carriers) and a physical membrane breaking method. Among them, the viral vector method has high efficiency, but its safety is poor and only nucleic acid can be delivered, which limits its further application. When delivering nucleic acid, most viruses integrate the DNA of the virus into host cells, so that host cell gene mutation is caused, and serious and even the modified cells become cancerous after being implanted into a human body, thereby endangering human health. Therefore, it is difficult to widely use the virus vector method for transfecting cells in actual clinical medicine. Liposomes and polymeric carriers are difficult to use on a large scale due to low delivery efficiency and strong cytotoxicity.

Compared with the technology of delivering the exogenous macromolecule by a carrier method, the technology of delivering the exogenous macromolecule by a physical membrane rupture method is more and more concerned by people because the delivery efficiency is high, the cytotoxicity is low, the universality is strong, and the defects of strong infectivity, low safety and the like of the traditional virus carrier are overcome. The physical membrane rupture methods are mainly classified into a mechanical membrane rupture method and an electromagnetic/thermal membrane rupture method according to the difference of membrane rupture methods, and include a plurality of directions such as a cell extrusion membrane rupture method (document 1), a microinjection method (document 2), an electroporation method (document 3), a photothermal membrane rupture method (document 4), and the like. The mechanical membrane breaking method is mainly characterized in that the cell membrane is reversibly broken under the action of external force on the cell, so that exogenous macromolecules are delivered into the cell. Document 1 shows that the cell is extruded through the microfluidic channel, and the system can prepare modified cells on a large scale, but the equipment is complex, the price is high, and the delivery efficiency of exogenous macromolecules is low. Document 2 discloses a microinjection apparatus that is efficient in delivery, but can only operate on single cells, and is inefficient, and the labor and equipment costs are high, which is not favorable for practical clinical applications. In summary, most of the mechanical membrane-breaking methods require complicated instruments or devices, have high experimental threshold, expensive experimental price and high manual requirements, and do not meet the requirements of cheaply, conveniently, quickly, efficiently and safely obtaining a large amount of modified cells as much as possible in actual clinical treatment.

Compared with a mechanical membrane breaking method, the electromagnetic/thermal membrane breaking method only changes external electric, magnetic, optical and thermal fields without direct interaction with cells, reduces cell damage, is simpler and easier to operate compared with the mechanical method, and has attracted more attention in recent years. Electroporation is currently the most common of electromagnetic/thermal membrane disruption methods, and its delivery efficiency is high. However, the high-voltage electric pulse used in the delivery process has high intensity, so that the activity of the cells is greatly reduced, and the popularization and the application of the high-voltage electric pulse are limited due to the non-ideal adherence effect and expression condition of the cells after harvesting. The photothermal membrane-breaking macromolecule delivery method mainly utilizes laser to irradiate the nanoparticles, utilizes the unique photothermal conversion capacity of the nanoparticles to heat surrounding tissues or cells, and enables cell membranes to be subjected to instantaneous reversible perforation, so that the permeability of the cell membranes is improved to carry out intracellular macromolecule delivery. Compared with other macromolecule delivery methods, the photothermal macromolecule delivery method is simple to operate, high in cell flux, and high in universality of cells and macromolecules, and is a very promising living cell macromolecule delivery means. The traditional photo-thermal macromolecule delivery platform carries out intracellular delivery by taking nano particles as photo-thermal agents, and has obvious defects although the operation is simple: 1. the nanoparticles are easy to enter cells through endocytosis, and most nanoparticles cannot be degraded through normal metabolism, so the nanoparticles have certain cytotoxicity to the cells; 2. reducing the nanoparticle concentration may suitably slow down cytotoxicity, but may result in a decrease in photothermal efficiency, ultimately leading to a decrease in the efficiency of intracellular macromolecule delivery.

On the basis of nanoparticles, in order to reduce the cytotoxicity of nanoparticles and improve the delivery efficiency of macromolecules in cells, document 4 discloses an intracellular and extracellular source macromolecule delivery platform with a gold nanoparticle layer with a photo-thermal effect modified on the surface of a substrate. The photo-thermal nano particles are enriched on the surface of the base material through surface modification, compared with nano particles, the enriched gold nano particle layer is not easy to be endocytosed by cells, the cytotoxicity of the photo-thermal material is improved, the photo-thermal efficiency is improved, a low-intensity laser light source can be used for macromolecule delivery, and the influence on the cell activity is reduced while the delivery efficiency is improved. However, the substrate surface topology changes due to the surface modification of the nanoparticle layer, which brings unavoidable side effects, and results in difficulty in harvesting modified cells from the substrate for subsequent research and application. In order to solve the side effects caused by surface modification of the nanoparticle layer, document 5 shows a method of combining photothermal perforation of gold nanoparticles with liposomes, so as to successfully and efficiently transfect cells which are difficult to transfect. However, due to the surface topology of the material and the tendency of the cells to phagocytose nanoparticles, it is difficult to harvest the modified cells from the substrate for subsequent research and application. Document 6 discloses that polydopamine nanoparticles are used as a photo-thermal base material, a temperature-sensitive polymer PNIPAAM is modified on the surface, and cell release is controlled by temperature change. But the preparation temperature has great influence on the surface modification temperature-sensitive polymer, so that the material repeatability is poor, the surface modification operation of the material is complex, the time consumption is long, and the quality guarantee period of the prepared material is short. In addition, due to the fact that only one layer of polymer brush is modified, temperature sensitivity is insufficient, temperature control cell release is not easy to operate, cells are easy to damage and the like, the efficiency of really harvesting effective cells with good activity is low, the problem of cell harvesting in photothermal transfection cannot be really solved, and the photothermal perforation method is difficult to be applied in large-scale practice. Document 7 uses silicon nanowires as photo-thermal substrates, sugar-responsive phenylboronic acids are surface-modified, and non-toxic natural biomolecules, such as sugars, are used as stimuli, which can trigger cell release under milder conditions, but still cannot efficiently harvest highly active cells.

In summary, current photothermal intracellular macromolecule delivery platforms based on nanoparticle particles and nanoparticle layers still fail to meet practical application requirements. Therefore, the design of a macromolecule transmission system which is cheap, simple in material, convenient and fast to operate, strong in universality of exogenous molecules and cell types, high in transmission efficiency, low in cytotoxicity, capable of processing cells with large flux, high in cell harvesting rate and good in cell activity is yet to be researched.

Document 1, Integr. biol,2014,6, 470-475;

document 2.CN 105420099B;

document 3.CN 105143436B;

document 4.CN 105420278A;

ACS appl. Mater. interfaces,2017,9, 21593-;

document 6.ACS appl. Mater. interfaces,2019,11, 12357-12366;

document 7.adv.funct.mater,2019,1906362.

Disclosure of Invention

Problems to be solved by the invention

In order to overcome the technical problems, the invention provides the composite material which is cheap, simple in material, convenient and quick to operate, strong in universality of exogenous molecules and cell types, high in transfer efficiency, small in cytotoxicity, capable of treating cells with large flux, high in cell harvesting rate and good in cell activity.

Means for solving the problems

In order to solve the technical problems, the invention provides the following technical scheme:

the composite material comprises nanoparticles and a flexible base material, wherein the nanoparticles comprise one or more of photothermal nanoparticles such as carbon nanotubes, graphene, gold nanoparticles and polydopamine nanoparticles, the flexible base material comprises one or more of thermosetting plastics such as polydimethylsiloxane and hydrogel, and the mass percentage of the nanoparticles in the composite material is 1-60 per thousand.

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

Preferably, the mass percentage of the nanoparticles in the composite material is 5-30 per mill.

Preferably, the mass percentage of the nanoparticles in the composite material is 5-20 per mill, and preferably 5-10 per mill.

On the other hand, the invention also provides a preparation method of the composite material, which comprises the following steps:

(1) fully mixing the nanoparticle solution and the flexible base material or the flexible base material prepolymer, then adding the mixture into a mold, and standing to obtain slurry;

(2) and (3) vacuumizing the obtained slurry, curing to form a film, taking out the film, demolding, and optionally processing and molding the obtained material to obtain the composite material.

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

Preferably, in the step (1), the flexible base material is polydimethylsiloxane, and the prepolymer of the flexible base material is a silicone rubber base and a silicone rubber curing agent in a mass ratio of 5-20: 1

Preferably, the method comprises the steps of:

(1) fully mixing 1-10% by mass of a carbon nanotube aqueous solution and a polydimethylsiloxane prepolymer, uniformly adding the mixture into a mold, and standing for 0.5-2 hours until the mold is fully paved with slurry;

(2) and vacuumizing the obtained slurry at room temperature for 0.5-2h, curing in an oven at the temperature of 60-80 ℃ for 0.5-2h to form a film, taking out the film, demolding, and optionally processing the obtained material into a required size to obtain the composite material.

Finally, the invention also provides the application of the composite material in preparing materials for delivering intracellular macromolecular substances and/or desorbing cells.

Preferably, the macromolecular substance is selected from one or more of a polysaccharide molecule, a protein, DNA, RNA, an intracellular probe, a therapeutic drug, an aptamer, a bacterium, an artificial chromosome, or an organelle.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention discloses a nano particle/flexible base material composite material (nano particles include but are not limited to carbon nano tubes, graphene, gold nano particles, polydopamine nano particles and the like, and flexible base materials include but are not limited to PDMS, hydrogel and the like) which is cheap in material, easy to prepare, convenient to operate, low in cytotoxicity and good in photo-thermal performance. The material is easy to prepare, has extremely strong photo-thermal conversion performance, and can rapidly perform photo-thermal conversion under a low-intensity near-infrared laser light source; the surface roughness of the material and the quantity of the nano particles exposed on the surface after film formation can be controlled by adjusting the concentration of the nano particles, and finally, a smooth surface with specific photo-thermal performance without changing the original topological structure is obtained. The composite material disclosed by the invention takes MCNT/PDMS as an example, and due to the good photo-thermal effect of the MCNT/PDMS, the permeability of cell membranes of cells cultured on the MCNT/PDMS can be enhanced by irradiating near-infrared laser, so that exogenous macromolecules in a solution can enter the cells, and the efficient intracellular macromolecule transfer is realized; different from a carrier method, the method disclosed by the invention adopts a physical membrane breaking method, can realize high-efficiency delivery of various exogenous macromolecular substances (but not limited to dextran, protein, pDNA, RNA, therapeutic drugs, intracellular probes and the like) on the premise of ensuring cell activity by adjusting laser intensity, has universality and universality (including but not limited to Hela, human embryo fibroblasts and the like) on different cells, and has delivery efficiency close to 100%; by adjusting the mass fraction of the carbon nano tubes, the modified cells grow and desorb on the MCNT/PDMS photo-thermal base material like in a culture bottle, the modified cells can be released and harvested efficiently and nondestructively by traditional pancreatin digestion, and the harvesting efficiency is over 90 percent; in addition, due to the flexible characteristic of the MCNT/PDMS material, the surface with any size and shape can be prepared, so that high-flux treatment on cells is realized, and efficient and large-amount intracellular delivery can be realized in a short time.

Drawings

FIG. 1 shows the photo-thermal properties of MCNT/PDMS with different MCNT contents under different laser conditions;

FIG. 2 the effect of different carbon nanotube contents on the surface roughness of MCNT/PDMS;

FIG. 3 cell detachment and harvesting efficiency of MCNT/PDMS material at different MCNT concentrations;

FIG. 4 shows the cell membrane permeability change (SYTOX) of Hela under different laser irradiation conditions;

FIG. 5 shows the relative cell viability of Hela cells under different laser irradiation conditions;

FIG. 6 transfection efficiency and relative cell activity of MCNT/PDMS and Lipo 2000.

Detailed Description

The invention provides a composite material which comprises nano particles and a flexible base material, wherein the nano particles comprise one or more of carbon nano tubes, graphene, gold nano particles, polydopamine nano particles and the like, the flexible base material comprises one or more of thermosetting plastics such as Polydimethylsiloxane (PDMS) and the like, hydrogel and the like, and the mass percentage of the nano particles in the composite material is 1-60 per mill.

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

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

The term "flexible substrate" refers to a polymeric material that possesses good elasticity, toughness, and plasticity. Due to the flexible characteristic of the base material, the composite material doped with photo-thermal nano particles can be formed on any surface of a pore plate or a culture bottle, or the composite material with a specific shape can be obtained by direct curing and molding, so that photo-thermal macromolecule delivery can be carried out in different cell culture environments.

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

In a more preferred embodiment, the mass percentage of the nanoparticles in the composite material is 5 to 30%, preferably 5 to 20%, more preferably 5 to 10%, and most preferably 10%.

The invention also provides a preparation method of the composite material, which comprises the following steps:

In a preferred embodiment, in the step (1), the nanoparticle solution is a carbon nanotube solution, 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%.

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

In a preferred embodiment, the method comprises the steps of:

(1) fully mixing 1-10% by mass of a carbon nanotube aqueous solution and polydimethylsiloxane, uniformly adding the mixture into a mold, and standing for 0.5-2 hours until the mold is fully paved with slurry;

(2) and vacuumizing the obtained slurry at room temperature for 0.5-2h, curing in an oven at the temperature of 60-80 ℃ for 0.5-2h to form a film, taking out the film, demolding, and optionally processing the obtained material into a sheet with a required size, such as a diameter or a length of 0.5-2 cm to obtain the composite material.

In another aspect, the present invention provides the use of a composite material as described above in the preparation of a material for delivery and/or desorption of macromolecular substances within cells.

In a preferred embodiment, the macromolecules have a mass of 0.9 to 500kDa and a physical size of 1nm to 5 μm.

In a preferred embodiment, the macromolecular species include, but are not limited to, polysaccharide molecules (e.g., dextran), proteins (e.g., gene editing enzymes, antibodies, antigens), DNA (e.g., pDNA), RNA (e.g., mRNA, guide RNA, miRNA, siRNA), therapeutic drugs, intracellular probes (e.g., quantum dots), nanomaterials (e.g., nanoparticles, nanodevices), aptamers, bacteria, artificial chromosomes, organelles (e.g., mitochondria), and the like.

The invention also provides an application of the composite material in intracellular macromolecule delivery and cell desorption.

Finally, the prepared MCNT/PDMS is used for intracellular macromolecule transfer and modified cell harvest experiments. The specific experimental steps are as follows:

(1) sterilizing MCNT/PDMS material with sterilizing pot, and sterilizing cells (including but not limited to Hela cells, human embryo fibroblasts, etc.) at 5 ten thousand/cm²The cell density is planted in MCNT/PDMS, and the cell is cultured for 4-24 h to adhere to the wall of the sample.

(2) The cells were washed with sterile PBS and serum-free cell culture medium with exogenous molecules (exogenous molecules including but not limited to plasmid DNA) was added, with pDNA at a final concentration of 0.005-0.008. mu.g/mL.

(3) Using a laser source with 808nm near-infrared band at 1-10W/cm²Irradiating the cells on the sample within the power density range for 10-180 s.

(4) And after laser irradiation is finished for 1-4 h, cleaning the cells by using sterile PBS, adding pancreatin for digestion for 0.5-5 min, slightly blowing and beating the serum by using a gun head until the cells completely fall off after the serum is stopped digestion, and centrifuging and resuspending the blown cells to obtain the harvested cells.

The following examples are for illustrative purposes only and do not limit the scope of the claims.

The MCNT aqueous solution used in the examples of the present invention was purchased from pioneer nanometer, and PDMS was purchased from Dow Corning.

Example 1

(1) Fully mixing MCNT aqueous solution with the mass fraction of 10% and PDMS according to the mass fraction of 0-30 per mill of the final carbon nano tube of the composite material, uniformly pouring the mixture into a mold, and standing for 1 hour until the mold is fully paved with slurry.

(2) Vacuumizing for 1h at room temperature to remove bubbles, and then curing for 1h in an oven at the temperature of 60-80 ℃. Taking out, demolding, and cutting into 1.1cm diameter circular slice.

(3) Respectively at the laser power of 0.6-3.2W/cm²And measuring the solution temperature of the MCNT/PDMS material with different MCNT contents in a wet state by using a thermal imager under the laser condition with the irradiation time of 0-30 s.

The experimental result is shown in fig. 1, which indicates that pure PDMS has no photo-thermal conversion capability, and after MCNT is added, 5-30% o of MCNT/PDMS material has good photo-thermal conversion performance, thereby providing possibility for delivering exogenous macromolecules into cells through photo-thermal perforation.

Example 2

(1) Fully mixing MCNT aqueous solution with the mass fraction of 10% and PDMS solution according to the mass fraction of 0-30 per mill of the final carbon nano tube of the composite material, uniformly pouring the mixture into a mold, and standing for 1 hour until the mold is fully paved with slurry.

(3) Ultrasonic cleaning with water, acetone and ethanol, and blow-drying with nitrogen.

(4) The surface roughness of the materials with different MCNT contents was measured by a roughness meter.

The surface roughness of MCNT/PDMS composite materials with different MCNT contents is shown in figure 2, and figure 2 shows that the MCNT/PDMS composite materials with MCNT mass fraction of 0-10 per mill have small roughness and completely smooth surfaces. And the surface of the MCNT/PDMS composite material with the MCNT mass fraction of 20-30 per mill is exposed, so that the roughness is increased. The surface topology of the material can be changed by adjusting the MCNT content on the surface.

Example 3

(1) Fully mixing MCNT aqueous solution with the mass fraction of 10% and PDMS solution according to the mass fraction of 0-30 per mill of the final carbon nano tube of the composite material, uniformly pouring the mixture into a mold, and standing for 1 hour until the mold is fully paved with slurry. Vacuumizing for 1h at room temperature to remove bubbles, and then curing for 1h in an oven at the temperature of 60-80 ℃. Taking out, demolding, and cutting into 1.1cm diameter circular slice.

(2) Sterilizing the slices with high-temperature high-pressure steam sterilizer. The slide is paved in a 48-well plate, and cells (including but not limited to Hela and human embryo fibroblasts) are planted in the holes of the 48-well plate at the density of 1-5 ten thousand per hole.

(3) Adding serum-free cell culture medium with pGFP, and irradiating with laser light source with wavelength of 808nm at 2.3W/cm²The power density of (2) was applied to the cells in the wells for 30 s.

(4) And after the laser irradiation is finished for 1h, cleaning the cells by using sterile PBS, adding pancreatin for digestion for 0.5-5 min, slightly blowing and beating the cells by using a gun head after the serum is stopped from being digested until the cells completely fall off, and centrifuging and resuspending the blown cells to obtain the harvested cells. Cells on the sample were stained with DAPI dye and the number of cells before and after detachment and after harvest was observed.

The cell detachment and harvesting efficiencies of MCNT/PDMS materials of different MCNT mass fractions are shown in fig. 3. As can be seen from FIG. 3, the desorption efficiency (i.e., the release rate) and the harvesting efficiency (i.e., the harvesting rate) of the MCNT/PDMS (the MCNT mass fraction is 0-10 ‰) sheet cells with smooth surfaces can respectively reach more than 95% and 85%, and the requirements of cell harvesting are completely met.

In conclusion, 5-10% o of MCNT/PDMS has good photothermal property and cell desorption capability, and can be used for subsequent intracellular macromolecule delivery.

Example 4

(1) Fully mixing MCNT aqueous solution with the mass fraction of 10% and PDMS solution according to 0-30 per mill of the final mass of the carbon nano tube of the composite material, uniformly pouring the mixture into a mould, and standing for 1h until the mould is fully paved with slurry. Vacuumizing for 1h at room temperature to remove bubbles, and then curing for 1h in an oven at the temperature of 60-80 ℃. Taking out, demolding, and cutting into 1.1cm diameter circular slice.

(2) Sterilizing the slices with high-temperature high-pressure steam sterilizer. And (3) paving the slices in a 48-hole plate, and planting the Hela cells in the holes of the 48-hole plate at the density of 1-5 ten thousand per hole.

(3) Adding serum-free cell culture medium with SYTOX (a dye capable of only passing broken cell membrane, the higher the fluorescence intensity, the better the cell membrane permeability), and using laser light source with wavelength of 808nm at low intensity of 0.6 to3.2W/cm²The power density of (2) was applied to the cells in the wells for 30 s.

(4) And after the laser irradiation is finished for 1-4 h, replacing the medium with a complete medium to continue culturing the cells.

(5) After laser irradiation for 2-4 h, staining the living cells with Calcein (Calcein), and observing the fluorescence condition with a fluorescence microscope.

(6) After 48 hours of laser irradiation, CCK-8 kit (cell activity test kit) is used for detecting the relative cell activity of cells

The cell membrane permeability change (SYTOX) of Hela under different laser irradiation conditions is shown in FIG. 4. it can be seen from FIG. 4 that pure PDMS sheet does not substantially change the cell membrane permeability, while MCNT/PDMS is 5-30 ‰ MCNT/PDMS at 1.4-3.2W/cm²The cell membrane permeability is obviously changed by illumination, and the cell membrane permeability is increased along with the increase of the MCNT content. The activity of Hela cells under different laser irradiation conditions is shown in FIG. 5, and 0.6-2.3W/cm can be seen from FIG. 5²The laser intensity can make the cell possess more than 80% of relative cell activity. This demonstrates the feasibility of improving cell membrane permeability and delivering exogenous macromolecules by irradiating MCNT/PDMS with a near-infrared laser.

In summary, the power of the tablet with MCNT content of 10 ‰ is 2.3W/cm²And the irradiation time was 30 seconds, the obtained cell membrane permeability and cell activity were both good. By changing the laser irradiation conditions, cells with good activity and membrane permeability can be obtained.

Example 5

(1) Fully mixing MCNT aqueous solution with the mass fraction of 10% and PDMS solution according to the mass fraction of 10 per mill of the final carbon nano tube of the composite material, uniformly pouring the mixture into a mold, and standing for 1h until the mold is fully paved with slurry. Vacuumizing for 1h at room temperature to remove bubbles, and then curing for 1h in an oven at the temperature of 60-80 ℃. Taking out, demolding, and cutting into 1.1cm diameter circular slice.

(2) Sterilizing the slices with high-temperature high-pressure steam sterilizer. And (3) paving the slices in a 48-hole plate, and planting Hela cells and human embryo fibroblasts into the holes of the 48-hole plate at the density of 1-5 ten thousand per hole.

(3) Addition of plasmid containing pGFPSerum cell culture medium, using laser light source with wavelength of 808nm at 2.3W/cm²The power density of (2) was applied to the cells in the wells for 30 s.

(5) After 48h of laser irradiation, cell nuclei were stained with DAPI, and green fluorescent protein expression was observed with a fluorescence microscope.

At 2.3W/cm²And the transfection efficiency and relative cell activity for delivering pGFP to Hela cells and human embryonic fibroblasts under the 30s laser irradiation conditions are shown in fig. 6a and B, respectively. It can be seen that the method for delivering macromolecular substances into cells based on the composite material of the present invention has strong versatility for cell types, and it can be seen from B in fig. 6 that the transfection efficiency of cells which are difficult to transfect, such as human embryonic fibroblasts, can also reach more than 93%, and the good activity of the cells is maintained.

Comparative example 1

The conditions of comparative example 1 and example 5 were the same, except that in comparative example 1, the MCNT/PDMS composite was replaced with commercial Lipo2000 as a transfection reagent, and the cells tested were Hela cells only.

At 2.3W/cm²Transfection efficiency and relative cell activity for delivering pGFP to Hela cells under 30s laser irradiation are shown in FIG. 6A. As can be seen from the figure, under the same conditions, the transfection efficiency of Lipo2000 to primary cells was less than 10%, and the cells could not maintain good activity.

Claims

1. The composite material is characterized by comprising nano particles and a flexible base material, wherein the nano particles comprise one or more of carbon nano tubes, graphene, gold nano particles and polydopamine nano particles, the flexible base material comprises one or more of polydimethylsiloxane and hydrogel, and the mass percentage of the nano particles in the composite material is 1-60 per thousand.

2. The composite of claim 1, wherein the nanoparticles are carbon nanotubes and the flexible substrate is polydimethylsiloxane.

3. The composite material of claim 1, wherein the nanoparticles are present in the composite material in an amount of 5 to 30% by weight.

4. A composite material according to any one of claims 1 to 3, wherein the nanoparticles are present in the composite material in a mass percentage of 5 to 20%, preferably 5 to 10%.

5.A method of making a composite material according to any one of claims 1 to 4, the method comprising the steps of:

6. The method according to claim 5, wherein in the step (1), the nanoparticle solution is a carbon nanotube solution, 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%.

7. The preparation method of the composite material according to claim 5, wherein in the step (1), the flexible base material is polydimethylsiloxane, and the prepolymer of the flexible base material is a silicone rubber base and a silicone rubber curing agent in a mass ratio of 5-20: 1.

8. A method of preparing a composite material according to claim 5, comprising the steps of:

9. Use of a composite material according to any one of claims 1 to 4 in the manufacture of a material for delivery of macromolecular substances and/or detachment of cells within cells.

10. Use according to claim 9, wherein the macromolecular substance is selected from one or more of a polysaccharide molecule, a protein, DNA, RNA, an intracellular probe, a therapeutic drug, an aptamer, a bacterium, an artificial chromosome, or an organelle.