COMPOSITE MATERIAL AND PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

The present disclosure provides a composite material. The composite material comprises nanoparticles and a flexible substrate, the nanoparticles comprise one or more of carbon nanotubes, graphene, gold nanoparticles, and polydopamine nanoparticles, the flexible substrate comprises one or more of thermosetting plastics such as polydimethylsiloxane and a hydrogel, and the mass percentage of the nanoparticles in the composite material is 0 to 60‰. The composite material of the present disclosure is easy to prepare, has extremely strong photothermal conversion performance, and does not change the smooth surface of an original topological structure. Meanwhile, the composite material has universality and versatility for different cells, the delivery efficiency is close to 100%, and modified cells may be efficiently and non-destructively released and harvested by means of traditional trysinization, and the harvesting efficiency is 90% or more.

Claims

1.-8. (canceled)

9. A method for intracellular macromolecular substance delivery and/or cell detachment which comprises utilizing a composite material, wherein the composite material comprises nanoparticles and a flexible substrate, the nanoparticles comprise one or more of carbon nanotubes, graphene, gold nanoparticles, and polydopamine nanoparticles, the flexible substrate comprises one or more of polydimethylsiloxane and a hydrogel, and a mass percentage of the nanoparticles in the composite material is 1 to 60‰; wherein the method comprises irradiating the composite material and cells cultured thereon with near-infrared laser.

10. The method according to claim 9, wherein the macromolecular substance is selected from one or more of polysaccharide molecules, proteins, DNA, RNA, intracellular probes, therapeutic drugs, aptamers, bacteria, artificial chromosomes, or organelles.

11. The method according to claim 9, wherein the nanoparticles are carbon nanotubes, and the flexible substrate is polydimethylsiloxane.

12. The method according to claim 9, wherein the mass percentage of the nanoparticles in the composite material is 5 to 30‰.

13. The method according to claim 9, wherein the mass percentage of the nanoparticles in the composite material is 5 to 20‰.

14. The method according to claim 9, wherein the mass percentage of the nanoparticles in the composite material is 5 to 10‰.

15. The method according to claim 9, wherein a preparation method for the composite material comprises: (1) thoroughly mixing a nanoparticle solution and a flexible substrate or a flexible substrate prepolymer, and then adding a mixture into a mold and allowing to stand to obtain a slurry; and (2) vacuumizing the obtained slurry and curing to form a film, then taking out the mold and releasing the film from the mold, and optionally forming the resulting material to obtain said composite material.

16. The method according to claim 15, wherein in step (1), the nanoparticle solution is a carbon nanotube solution, and a solvent of the solution is selected from the group consisting of water, methanol, ethanol, and dimethylformamide, and a mass fraction of the carbon nanotube solution is 1 to 10%.

17. The method according to claim 15, wherein in step (1), the flexible substrate is polydimethylsiloxane, and the flexible substrate prepolymer is a composition of a silicone rubber base and a silicone rubber curing agent in a mass ratio of 5 to 20:1.

18. The method according to claim 11, wherein a preparation method for the composite material comprises: (1) thoroughly mixing a carbon nanotube aqueous solution with a mass fraction of 1 to 10% and a polydimethylsiloxane prepolymer, and then adding a mixture into a mold uniformly and allowing to stand for 0.5 to 2 hours until the mold is fully covered by the slurry; and (2) vacuumizing the obtained slurry at room temperature for 0.5 to 2 hours, and curing in an oven at 60 to 80° C. for 0.5 to 2 hours to form a film, then taking out the mold and releasing the film from the mold, and optionally processing the resulting material into a desired dimension to obtain said composite material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 Photothermal properties of MCNT/PDMS with different contents of MCNT under different laser conditions;

[0033] FIG. 2 effect of different contents of carbon nanotubes on surface roughness of MCNT/PDMS;

[0034] FIG. 3 cell detachment and harvesting efficiency of MCNT/PDMS materials with different MCNT concentrations;

[0035] FIG. 4 changes of Hela cell membrane permeability under different laser irradiation conditions (SYTOX);

[0036] FIG. 5 changes of relative cell viability of Hela cells under different laser irradiation conditions;

[0037] FIG. 6 transfection efficiency and relative cell viability of MCNT/PDMS and Lipo2000.

DETAILED DESCRIPTION

[0038] The present disclosure provides a composite material, wherein the composite material comprises nanoparticles and a flexible substrate, the nanoparticles comprise one or more of carbon nanotubes, graphene, gold nanoparticles, polydopamine nanoparticles, and the like, the flexible substrate comprises one or more of thermosetting plastics such as polydimethylsiloxane (PDMS) and a hydrogel, and a mass percentage of the nanoparticles in the composite material is 1 to 60‰.

[0039] In a preferred embodiment, the carbon nanotubes are multi-walled carbon nanotubes (MCNT).

[0040] In a preferred embodiment, the nanoparticles are carbon nanotubes, and the flexible substrate is polydimethylsiloxane.

[0041] The term “flexible substrate” refers to a polymer material with good elasticity, toughness, and plasticity. Due to the flexible characteristic of the substrate, a composite material doped with the photothermal nanoparticles may be formed on any surface of a well plate or a culture flask, or a composite material with a specific shape may be obtained by direct curing molding, so that macromolecule delivery may be carried out in different cell culture environments via the photoporation effect.

[0042] In a preferred embodiment, the composite material is a composition of nanoparticles and a flexible substrate.

[0043] 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‰.

[0044] The present disclosure also provides a preparation method for the above-described composite material, comprising:

[0045] (1) thoroughly mixing a nanoparticle solution and a flexible substrate or a flexible substrate prepolymer, and then adding a mixture into a mold and allowing to stand to obtain a slurry; and

[0046] (2) vacuumizing the obtained slurry and curing to form a film, then taking out the mold and releasing the film from the mold, and optionally forming the resulting material to obtain said composite material.

[0047] In a preferred embodiment, in step (1), the nanoparticle solution is a carbon nanotube solution, and a solvent of the solution is selected from the group consisting of water, methanol, ethanol, and dimethylformamide, and a mass fraction of the carbon nanotube solution is 1 to 10%.

[0048] 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.

[0049] In a preferred embodiment, the method comprises:

[0050] (1) thoroughly mixing a carbon nanotube aqueous solution with a mass fraction of 1 to 10% and a polydimethylsiloxane prepolymer, and then adding a mixture into a mold uniformly and allowing to stand for 0.5 to 2 hours until the mold is fully covered by the slurry; and

[0051] (2) vacuumizing the obtained slurry at room temperature for 0.5 to 2 hours, and curing in an oven at 60 to 80° C. for 0.5 to 2 hours to form a film, then taking out the mold and releasing the film from the mold, and optionally processing the resulting material into a disc with a desired dimension such as 0.5 to 2 cm in diameter or length to obtain said composite material.

[0052] On the other hand, the present disclosure provides a use of the above-described composite material for preparing a material for intracellular macromolecular substance delivery and/or cell detachment.

[0053] In a preferred embodiment, the mass of the above-described macromolecular substance is 0.9 to 500 kDa, and the physical dimension is 1 nm to 5 μm.

[0054] In a preferred embodiment, the macromolecular substance includes, 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 and nanodevices), aptamers, bacteria, artificial chromosomes, or organelles (e.g., mitochondria), and the like.

[0055] The present disclosure further provides a use of the composite material in intracellular macromolecule delivery and cell detachment.

[0056] Finally, the prepared MCNT/PDMS is used for intracellular macromolecule delivery and harvesting of modified cells. The specific experimental method comprises:

[0057] the MCNT/PDMS material is sterilized by a sterilizing pot, cells (including but not limited to Hela cells, human embryonic fibroblast, and the like) are seeded on the MCNT/PDMS at a density of 50,000/cm.sup.2, and the cells are cultured for 4 to 24 hours to enable the cells to adhere to the wall of the sample.

[0058] The cells are washed with sterile PBS, and a serum-free cell culture medium with exogenous molecules (including but not limited to plasmid DNA) is added at a final pDNA concentration of 0.005 to 0.008 μg/mL.

[0059] The cells on the sample are irradiated with a laser source in the near infrared wavelength of 808 nm at a power density of 1 to 10 W/cm.sup.2 for 10 to 180 seconds.

[0060] 1 to 4 hours after laser irradiation, the cells are washed with sterile PBS and trypsin is added to digest for 0.5 to 5 minutes. After the serum stopped digestion, the cells are gently blown off with a gun head until the cells completely fall off. The cells which are blown off are centrifuged and resuspended to obtain harvested cells.

[0061] The following Examples are for illustrative purposes only and do not limit the scope of the claims.

[0062] The aqueous solution of MCNT used in Examples of the present disclosure was purchased from Pioneer Nano and PDMS was purchased from Dow Corning.

Example 1

[0063] (1) After thoroughly mixing the MCNT aqueous solution with the mass fraction of 10% and the PDMS according to the final carbon nanotube mass fraction of the composite material of 0 to 30‰, the mixture was uniformly poured into a mold and allowed to stand for 1 hour until the mold was fully covered by the slurry.

[0064] (2) The bubbles were removed by vacuumizing at room temperature for 1 hour, and then the slurry was cured in an oven at 60 to 80° C. for 1 hour. After the mold being taken out, the resultant was released from the mold, and the resulting material was cut into discs with the diameter of 1.1 cm.

[0065] (3) The solution temperature of MCNT/PDMS materials with different MCNT contents in wet state was measured by thermal imager under the laser power of 0.6 to 3.2 W/cm.sup.2 and the irradiation time of 0 to 30 seconds, respectively.

[0066] The experimental results were shown in FIG. 1, which showed that pure PDMS has no photothermal conversion ability, while after adding MCNT, 5 to 30‰MCNT/PDMS materials have good photothermal conversion performance, which provides the possibility of delivering exogenous macromolecules into cells via photoporation.

Example 2

[0067] (1) After thoroughly mixing the MCNT aqueous solution with the mass fraction of 10% and the PDMS according to the final carbon nanotube mass fraction of the composite material of 0 to 30‰, the mixture was uniformly poured into a mold and allowed to stand for 1 hour until the mold was fully covered by the slurry.

[0068] (2) The bubbles were removed by vacuumizing at room temperature for 1 hour, and then the slurry was cured in an oven at 60 to 80° C. for 1 hour. After the mold being taken out, the resultant was released from the mold, and the resulting material was cut into discs with the diameter of 1.1 cm.

[0069] (3) The disc was thoroughly ultrasonically cleaned with water, acetone and ethanol, and dried with nitrogen for later.

[0070] (4) The surface roughness of the materials with different MCNT contents was measured by a roughness meter

[0071] The surface roughness of MCNT/PDMS composite materials with different MCNT contents was shown in FIG. 2. FIG. 2 showed that the surface roughness of MCNT/PDMS composite materials with a mass fraction of MCNT of 0 to 10‰ was very small, and the surface was completely smooth. However, the surface roughness of MCNT/PDMS composite material with a mass fraction of MCNT of 20 to 30‰ was increased due to the exposure of MCNT on the surface, leading to the increase in the roughness. The topological structure of the material surface might be changed by adjusting the MCNT content on the surface.

Example 3

[0072] (1) After thoroughly mixing the MCNT aqueous solution with the mass fraction of 10% and the PDMS according to the final carbon nanotube mass fraction of the composite material of 0 to 30‰, the mixture was uniformly poured into a mold and allowed to stand for 1 hour until the mold was fully covered by the slurry. The bubbles were removed by vacuumizing at room temperature for 1 hour, and then the slurry was cured in an oven at 60 to 80° C. for 1 hour. After the mold being taken out, the resultant was released from the mold, and the resulting material was cut into discs with the diameter of 1.1 cm.

[0073] (2) A sterilizing pot with high-temperature and high-pressure steam was used to sterilize the disc. The disc was laid on the 48-well plate, and the cells (including but not limited to Hela, human embryonic fibroblast) were seeded into the wells of a 48-well plate at a density of 1 to 50,000/well.

[0074] (3) The serum-free cell culture medium with pGFP was added, and the cells in the wells were irradiated with a laser light source with a wavelength of 808 nm at a power density of 2.3 W/cm.sup.2 for 30 seconds.

[0075] (4) 1 hour after laser irradiation, the cells were washed with sterile PBS and trypsin was added to digest for 0.5 to 5 minutes. After the serum stopped digestion, the cells were gently blown off with a gun head until the cells completely fell off. The cells which were blown off were centrifuged and resuspended to obtain harvested cells. The cells on the sample were stained with DAPI dye, and the number of cells before and after detachment and after harvesting was observed.

[0076] The cell detachment and harvesting efficiency of MCNT/PDMS materials with different MCNT mass fractions were shown in FIG. 3. It can be seen from FIG. 3 that the cell detachment efficiency (release rate) and harvesting efficiency (harvesting rate) of MCNT/PDMS (the mass fraction of MCNT was 0 to 10‰) sheets with smooth surfaces reached up to 95% and 85% respectively, which fully met the requirements of cell harvesting.

[0077] To sum up, 5 to 10‰ MCNT/PDMS has both good photothermal performance and cell detachment ability, which may be used for subsequent intracellular macromolecule delivery.

Example 4

[0078] (1) After thoroughly mixing the MCNT aqueous solution with the mass fraction of 10% and the PDMS according to the final carbon nanotube mass fraction of the composite material of 0 to 30‰, the mixture was uniformly poured into a mold and allowed to stand for 1 hour until the mold was fully covered by the slurry. The bubbles were removed by vacuumizing at room temperature for 1 hour, and then the slurry was cured in an oven at 60 to 80° C. for 1 hour. After the mold being taken out, the resultant was released from the mold, and the resulting material was cut into discs with the diameter of 1.1 cm.

[0079] (2) A sterilizing pot with high-temperature and high-pressure steam was used to sterilize the disc. The disc was laid on the 48-well plate, and Hela cells were seeded into the wells of a 48-well plate at a density of 1 to 50,000/well.

[0080] (3) The serum-free cell culture medium with SYTOX (a dye that may only pass through the damaged cell membrane, and the higher the fluorescence intensity, the better the cell membrane permeability) was added, and the cells in the wells were irradiated with a laser source with a wavelength of 808 nm at a power density of low intensity of 0.6 to 3.2 W/cm.sup.2 for 30 seconds.

[0081] (4) 1 to 4 hours after laser irradiation, the medium was changed to complete medium to continue cell culture.

[0082] (5) 2 to 4 hours after laser irradiation, the living cells were stained with Calcein, and then the fluorescence was observed by a fluorescence microscope.

[0083] (6) 48 hours after laser irradiation, the relative cell viability was detected by CCK-8 kit (cell viability test kit).

[0084] The changes of Hela cell membrane permeability (SYTOX) under different laser irradiation conditions were shown in FIG. 4. It can be seen from FIG. 4 that pure PDMS did not change the permeability of cell membrane, but 5 to 30‰ MCNT/PDMS might change the permeability of cell membrane obviously at 1.4 to 3.2 W/cm.sup.2, and the cell permeability increased with the increase of MCNT content. The viability of Hela cells under different lase irradiation conditions was shown in FIG. 5. It can be seen from FIG. 5 that the relative cellular viability of cells may be 80% or more with the laser intensity of 0.6 to 2.3 W/cm.sup.2. This demonstrated the feasibility of improving cell membrane permeability and delivering exogenous macromolecules by irradiating MCNT/PDMS with near-infrared laser irradiation.

[0085] To sum up, when the power was 2.3 W/cm.sup.2 and the irradiation time was 30 seconds, the cell membrane permeability and cell viability of the disc with MCNT content of 10‰ were better. By changing the laser irradiation conditions, cells excellent in both viability and membrane permeability might be obtained.

Example 5

[0086] (1) After thoroughly mixing the MCNT aqueous solution with the mass fraction of 10% and the PDMS according to the final carbon nanotube mass fraction of the composite material of 10‰, the mixture was uniformly poured into a mold and allowed to stand for 1 hour until the mold was fully covered by the slurry. The bubbles were removed by vacuumizing at room temperature for 1 hour, and then the slurry was cured in an oven at 60 to 80° C. for 1 hour. After the mold being taken out, the resultant was released from the mold, and the resulting material was cut into discs with the diameter of 1.1 cm.

[0087] (2) A sterilizing pot with high-temperature and high-pressure steam was used to sterilize the disc. The disc was laid on the 48-well plate, and Hela cells and human embryonic fibroblast cells were seeded into the wells of a 48-well plate at a density of 1 to 50,000/well.

[0088] (3) The serum-free cell culture medium with pGFP was added, and the cells in the wells were irradiated with a laser light source with a wavelength of 808 nm at a power density of 2.3 W/cm.sup.2 for 30 seconds.

[0089] (4) 1 to 4 hours after laser irradiation, the medium was changed to complete medium to continue cell culture.

[0090] (5) 48 hours after laser irradiation, the nuclei were stained with DAPI, and then the expression of green fluorescent protein was observed by the fluorescence microscope.

[0091] The transfection efficiency and relative cell viability of pGFP delivered to Hela cells and human embryonic fibroblasts under a laser irradiation condition with 2.3 W/cm.sup.2 and 30 seconds were shown in FIG. 6 A and B, respectively. It can thus be seen that the method for delivering macromolecular substances into cells based on the composite material of the present disclosure is highly versatile for cell types, and it can be seen from B in FIG. 6 that the transfection efficiency for difficult-to-transfect cells such as human embryonic fibroblasts might reach 93% or more, and the good viability of the cells was maintained.

Comparative Example 1

[0092] The conditions of Comparative Example 1 and Example 5 are the same, except that commercial Lipo2000 was used as transfection reagent in Comparative Example 1 instead of MCNT/PDMS composite material, and only Hela cells were tested.

[0093] The transfection efficiency and relative cell viability of pGFP delivered to Hela cells under a laser irradiation condition with 2.3 W/cm.sup.2 and 30 seconds were shown in FIG. 6A. As can be seen from that figure, under the same conditions, the transfection efficiency of Lipo2000 to the primary cell was less than 10%, and the good activity of cells was not maintained.