CN110564669A - auxiliary platform for transferring exogenous molecules to cells and preparation method and application thereof - Google Patents
auxiliary platform for transferring exogenous molecules to cells and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title description 9
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 40
- 239000010703 silicon Substances 0.000 claims abstract description 40
- 239000002070 nanowire Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 25
- 229920000642 polymer Polymers 0.000 claims abstract description 20
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical group OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000758 substrate Substances 0.000 claims description 19
- 239000003999 initiator Substances 0.000 claims description 16
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- 229920001577 copolymer Polymers 0.000 claims description 9
- CZDWJVSOQOMYGC-UHFFFAOYSA-N 4-borono-2-fluorobenzoic acid Chemical compound OB(O)C1=CC=C(C(O)=O)C(F)=C1 CZDWJVSOQOMYGC-UHFFFAOYSA-N 0.000 claims description 7
- 238000002716 delivery method Methods 0.000 claims description 7
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- KWNPRVWFJOSGMZ-UHFFFAOYSA-N 2-boronobenzoic acid Chemical compound OB(O)C1=CC=CC=C1C(O)=O KWNPRVWFJOSGMZ-UHFFFAOYSA-N 0.000 claims description 2
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- DBVFWZMQJQMJCB-UHFFFAOYSA-N 3-boronobenzoic acid Chemical compound OB(O)C1=CC=CC(C(O)=O)=C1 DBVFWZMQJQMJCB-UHFFFAOYSA-N 0.000 claims description 2
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- 238000001035 drying Methods 0.000 description 4
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 229910021589 Copper(I) bromide Inorganic materials 0.000 description 2
- 239000007821 HATU Substances 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
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- 150000001875 compounds Chemical class 0.000 description 1
- QTMDXZNDVAMKGV-UHFFFAOYSA-L copper(II) bromide Substances [Cu+2].[Br-].[Br-] QTMDXZNDVAMKGV-UHFFFAOYSA-L 0.000 description 1
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- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- Life Sciences & Earth Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention relates to an intracellular and extracellular source macromolecule transfer platform which comprises a silicon nanowire array and a polymer brush formed on the surface of the silicon nanowire array, wherein the polymer brush is provided with a phenylboronic acid group. This patent has decorated the discernment molecule on the surface and has been used for catching suspension cell, then adopts the supplementary light thermal perforation's of surface technique to improve the transfer efficiency to the cell to solved the problem that present most supplementary transfer techniques of surface are only applicable to adherent cell, realized the high efficiency macromolecule transfer to suspension cell.
Description
Technical Field
the invention particularly relates to an auxiliary platform for transferring exogenous molecules to cells, and a preparation method and application thereof.
Background
Suspension cells are widely distributed throughout cell lines, such as hematopoietic cells, circulating blood cells, macrophages, and lymphocytes. The delivery of exogenous macromolecules (e.g., proteins, nucleic acids, etc.) into suspension cells is the key to understanding the development of various disease treatment protocols, and has important roles in the fields of cell therapy, regenerative medicine, disease diagnosis, and drug screening. Suspension cells are more difficult to transfect than adherent cells. The existing methods for delivering exogenous macromolecules into suspended cells mainly comprise biochemical methods and physical methods.
the biochemical method adopts some vectors and the like to assist exogenous macromolecules to enter cells, and can be mainly divided into two types of viral vectors and non-viral vectors. At present, viral vectors are the most advanced nucleic acid vectors for clinical application due to their high efficiency and specificity. However, viral vectors also present some serious challenges, such as: immunogenicity, safety, and complexity of preparation, which are also reasons why viral vectors have been difficult to obtain FDA approval. Due to the limitations of viral vectors, researchers have developed a variety of non-viral vectors, such as cationic polymers, liposomes, inorganic nanoparticles, and cell-penetrating peptides. However, the transfection efficiency of these vectors is relatively low. In addition, most non-viral vectors are used for nucleic acid transfection, and few vectors are used for protein and carbohydrate delivery. And one vector may be suitable for delivery of only certain species with specific labels, but not others without such labels, and thus such vectors are less versatile for delivery of macromolecules. Meanwhile, for suspension cells which are difficult to transfect, the self-protection mechanism is stronger, so that the transfer efficiency is very low.
The physical method is a method for promoting exogenous macromolecules to enter cells based on cell membrane breakage. The most typical and currently used method for suspension cell delivery is electroporation. Electroporation is the transient increase of the permeability of cell membranes by the action of high-intensity electric fields, which promotes the entry of exogenous macromolecules in the surrounding medium into the interior of cells, but the high pulse intensity of electroporation causes massive death of suspended cells.
1. A method for preparing cells loaded with exogenous molecules by adopting a photo-induced perforation mode, a base material for preparing the cells and the cells. Publication No.: CN 105420278A.
The gold nanoparticles are deposited on the surface of the gold sheet, and macromolecular transmission to various adherent cells is realized by utilizing the photothermal effect of the gold nanoparticles, but the gold nanoparticles have no effect on exogenous macromolecular transmission of suspended cells.
2. An optimized electroporation transfection method for suspension cells. Publication No.: CN 103981218A.
The invention belongs to the field of mammalian cell genetic engineering, and particularly relates to a method for transfecting suspension cells by using an optimized electroporation technology. However, the transfection efficiency is still to be improved, and there is a problem that the electroporation technique is much harmful to the cell activity.
3. A method for pretreating a cell culture plate for suspension cell liposome transfection. Publication No.: CN 102719480A.
the invention discloses a method for pretreating a cell culture plate for suspension cell liposome transfection, which adopts a surface-assisted liposome transfection method and still belongs to the field of a carrier method. In particular, in the case of suspended lymphocytes, the positive charge content on the cell membrane surface is significantly increased, thereby increasing the difficulty of transfecting nucleic acid into lymphocytes using a vector such as cationic liposome.
Therefore, development of a universal, simple and efficient surface-assisted delivery system capable of delivering macromolecules to adherent cells and delivering exogenous molecules to suspension cells without damage is yet to be researched.
Disclosure of Invention
the invention aims to solve the technical problem of providing an auxiliary platform, a preparation method and application thereof. And various exogenous molecules are efficiently transferred into cells by virtue of the photothermal effect, so that the adherent cells and the suspension cells are efficiently transferred. Further, the modified cells can be harvested by utilizing the sugar response, and the damage to the cells is small.
In order to solve the technical problems, the invention adopts the following technical scheme:
The invention provides an auxiliary platform which comprises a silicon nanowire array and a polymer brush formed on the surface of the silicon nanowire array, wherein the polymer brush is provided with a phenylboronic acid group.
since sialic acid is present on most cell membrane surfaces and is overexpressed on the cell membrane surfaces of most cancer cells, T cells, B cells, and natural killer cells, phenylboronic acid can bind to sialic acid, and the phenylboronic acid ester bond can be dissociated by the action of fructose molecules, and the sugar response is more cell-friendly than temperature response. Therefore, the polymer brush with the phenylboronic acid group and capable of capturing suspended cells is modified on the photothermal effect substrate silicon nanowire array, exogenous macromolecule transfer to various suspended cells is achieved, and by adding fructose molecules, cells on the surface of the auxiliary platform can be harvested without damage for subsequent research and application.
The surface assistance of the invention ensures that the contact between macromolecules such as DNA and the like and suspended cells is tighter, which is beneficial to improving the transfer efficiency, and the surface-assisted silicon nanowire array has the photothermal effect, utilizes the photothermal perforation to promote cell membranes to become more permeable, is beneficial to enabling exogenous macromolecules to enter the interior of the cells through the cell membranes, and can better maintain the activity of the cells.
Preferably, the thickness of the polymer brush is 10-30 nm. The number of cells trapped on the surface increases with the thickness of the polymer brush when the thickness of the polymer brush is less than 20nm, and remains substantially unchanged when the thickness reaches 20nm, so that the optimal thickness of the polymer brush is about 20nm for the combined consideration of cost and performance.
Preferably, the length of the silicon nanowire array is 5-35 μm. It was found experimentally that the photothermal effect increases with longer lengths of silicon nanowires, but the compatibility with cells becomes poor because long silicon nanowires are not suitable for cell growth. Further preferably, the length of the silicon nanowire is 10 to 28 μm, and more preferably 10 to 18 μm.
According to one embodiment, the auxiliary platform is prepared by modifying the silicon nanowire array by an initiator, then grafting a copolymer, and finally modifying a modifier containing a phenylboronic acid group.
Preferably, the initiator is 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, or the initiator is formed by modifying a silane coupling agent 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane on the silicon nanowire array, and then modifying 2-bromoisobutyryl bromide or 2-chloroisobutyryl chloride.
Preferably, the monomers forming the copolymer are hydroxyethyl methacrylate and 2-aminoethyl methacrylate.
Preferably, the modifier is one or more of 4-carboxy-3 fluorobenzeneboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid and 2-carboxyphenylboronic acid.
according to one embodiment, the auxiliary platform further comprises an exogenous molecule loaded on the silicon nanowire array and/or the polymer brush, so that the concentration of the exogenous molecule can be increased, and the transfer efficiency can be improved. The auxiliary platform can be directly used as an excellent platform for in vitro transfection of cells, and further used for cell therapy of diseases.
In the present invention, the foreign molecule may be a desired substance such as DNA, RNA, polypeptide, protein, saccharide, drug, etc.
The auxiliary platform is used for supporting the near infrared light at a speed of 1-10W/cm2When the illumination intensity irradiates for 10-50 s, the temperature of the auxiliary platform can be increased, so that the cell membrane permeability of the cell can be increased, exogenous molecules can conveniently enter the cell, and the transfer efficiency is improved.
the auxiliary platform is not only suitable for adherent cells, but also suitable for suspension cells.
The second aspect of the present invention provides a method for preparing the auxiliary platform, including the following steps:
(1) Reacting the silicon nanowire array with an initiator solution to obtain a silicon nanowire array modified with an initiator;
(2) In the presence of an initiating system, reacting the silicon nanowire array modified with the initiating agent with a monomer to obtain a substrate with a copolymer grafted on the surface;
(3) In the presence of a condensation reagent, reacting the base material with a modifier solution to prepare the auxiliary platform;
(4) selectively loading exogenous molecules on the auxiliary platform.
According to a specific embodiment, the preparation method comprises the following steps:
(1) the volume ratio is 2-3: 1, cleaning the silicon wafer by using a mixed solution of sulfuric acid and hydrogen peroxide, and drying by using nitrogen; preparing a mixed aqueous solution containing hydrofluoric acid and silver nitrate, wherein the feeding volume ratio of water to hydrofluoric acid is 2-4: 1, and the concentration of the silver nitrate is 8-9 g/L; adding the mixed aqueous solution into a cleaned silicon wafer, reacting for 5-30 min at 45-55 ℃, and then cleaning and drying to obtain a silicon nanowire array;
(2) The volume ratio is 2-3: 1, cleaning a silicon nanowire array by using a mixed solution of sulfuric acid and hydrogen peroxide, then putting the cleaned silicon nanowire array into an anhydrous toluene solution of 1-3 wt% of 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, standing at room temperature for 20-30 h, and then cleaning to obtain a silicon nanowire array modified with an initiator;
(3) Adding 2-aminoethyl methacrylate and hydroxyethyl methacrylate into anhydrous methanol and deionized water to prepare a mixed solution, wherein the concentration of the 2-aminoethyl methacrylate is 0.1-0.2 mol/L, the concentration of the hydroxyethyl methacrylate is 0.6-0.7 mol/L, and then sequentially adding CuBr2uniformly mixing bipyridine and ascorbic acid, adding the mixture into a silicon nanowire array modified with an initiator, reacting at room temperature for 10-60 min, and cleaning to remove residual monomers and physically adsorbed polymers to obtain a substrate with a copolymer brush grafted on the surface;
(4) preparing 4-carboxyl-3 fluorobenzeneboronic acid anhydrous N, N-dimethylformamide solution with the concentration of 4-6 mg/mL, adding O- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate and N, N-diisopropylethylamine, controlling the final concentrations to be 3-4 mg/mL and 2-3 mu L/mL respectively, blowing nitrogen for 20-40 min, then moving into a glove box, adding the mixed liquid into a base material, and reacting at room temperature for 10-15 h to obtain the auxiliary platform.
Further, the auxiliary platform is placed into a solution containing exogenous molecules, and the auxiliary platform loaded with the exogenous molecules is obtained by standing.
The third aspect of the present invention provides an in vitro cell delivery method of the auxiliary platform, comprising the following steps:
(1) planting cells on the auxiliary platform;
(2) Adding a serum-free culture medium for cell culture, wherein when an adopted auxiliary platform is loaded with exogenous molecules, the serum-free culture medium is added with or does not contain the exogenous molecules; when the adopted auxiliary platform is not loaded with exogenous molecules, the serum-free culture medium is added with the exogenous molecules;
(3) Near infrared light is adopted to be 1-10W/cm2irradiating for 10-50 s at the illumination intensity to obtain the cell containing the exogenous molecule.
In a fourth aspect of the present invention, there is provided a method for releasing cells by the delivery method, wherein the serum-free cell culture medium is replaced with a fructose solution 1 to 10 hours after the irradiation is completed, and the cells are collected.
In a fifth aspect, the invention provides a cell prepared by the platform aid, or a cell prepared by the delivery method, or a cell collected by the release method.
due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
This patent has modified the discernment molecule that can catch suspension cell on the surface, has improved the capture rate to suspension cell to adopt the technique of the supplementary light and heat perforation of surface can improve the transfer efficiency to the cell, thereby solved the problem that present most of supplementary transfer techniques of surface only are applicable to adherent cell, realized the high efficiency macromolecule transfer to suspension cell.
Drawings
FIG. 1 is a representation of example 1, wherein FIG. 1(a) is a top SEM representation of SN-PHB prepared in example 1, FIG. 1(b) is a cross-sectional SEM representation of SN-PHB prepared in example 1, and FIG. 1(c) is a single TEM representation of SN-PHB prepared in example 1;
FIG. 2 is a graph of water contact angle data for SiNWAs, SN-Br, SN-PHA and SN-PHB prepared in example 1;
FIG. 3 is the experimental results of example 1, in which FIG. 3(a) is a graph showing the transfection efficiency of the sample flow data in example 1 for the plasmid DNA (pGFP) encoded by green fluorescent protein into Ramos cells, and FIG. 3 (b) is a graph showing the results of the sample flow data in example 1 for the characterization of the mean fluorescence intensity of pGFP after transfection into Ramos cells;
FIG. 4 is a graph showing the density statistics of Ramos cells released by fructose and sample capture in example 1;
FIG. 5 is a graph showing the results of the proliferation potency of the sample tested in example 1 using CCK-8;
FIG. 6 is the results of the experiment of example 2, wherein FIG. 6(a) is a graph of the flow data of the samples of example 2 characterizing the transfection efficiency of pGFP into T cells, and FIG. 6(b) is a graph of the results of the flow data of the samples of example 2 characterizing the mean fluorescence intensity of pGFP after transfection into T cells;
FIG. 7 is a graph showing the results of the proliferation potency of the sample tested in example 2 using CCK-8.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are not indicated by the manufacturer and are commercially available.
example 1:
(1) Preparation of SiNWAs: the silicon wafer was cleaned with a mixed solution of sulfuric acid/hydrogen peroxide (7:3 by volume) and blown dry with nitrogen. Preparing a mixed aqueous solution containing hydrofluoric acid and silver nitrate (wherein the mixed aqueous solution contains 90mL of deionized water, 30mL of hydrofluoric acid and 1.02g of silver nitrate), adding the mixed aqueous solution into a cleaned silicon wafer, reacting for 10min at 50 ℃, cleaning with 25% dilute nitric acid solution and deionized water, and drying in a vacuum drying oven to obtain the SiNWAs.
(2) modification initiator on SiNWAs: the reaction solution is prepared by using sulfuric acid: and (3) putting the SiNWAs cleaned by the hydrogen peroxide mixed solution (7:3 volume ratio) into an anhydrous toluene solution of 2% 2-bromo-2-methylpropionate (3-trimethoxysilyl) propyl ester, standing at room temperature for 24 hours, and cleaning by using anhydrous toluene to obtain the SiNWAs modified with the initiator, wherein the SiNWAs are marked as SN-Br.
(3) Surface grafting of poly (hydroxyethyl methacrylate (HEMA) -co-2-Aminoethyl Methacrylate (AMA)) by electron transfer activated regenerative atom transfer radical polymerization (ARGET-ATRP) method: preparing a polymerization reaction liquid: AMA and HEMA at a molar ratio of 20:80, 2.2 mmole AMA and 8.8 mmole HEMA were dissolved in a mixed solution of 7mL of anhydrous methanol and 7mL of deionized water, and then 7. mu. moles of CuBr were added in that order2Uniformly mixing 44 mu mol of bipyridyl (bpy) and 98 mu mol of ascorbic acid (Vc), adding the mixture into SN-Br, reacting at room temperature for 30min, alternately cleaning residual monomers and physically adhered polymers on the surface by using anhydrous methanol and deionized water to obtain a substrate with the surface grafted with AMA and HEMA copolymer brush, and marking the substrate as SN-PHA.
(4) Surface post-modification of 4-carboxy-3-fluorobenzeneboronic acid: preparing a 5mg/mL solution of 4-carboxy-3-fluorobenzeneboronic acid (CFPBA) in anhydrous N, N-dimethylformamide, and then adding O- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA) to control the final concentrations to be 3.8mg/mL and 2.58 mu L/mL respectively. Then blowing nitrogen for 30min, moving the mixture into a glove box, adding the mixed liquid into SN-PHA, and reacting for 12h at room temperature to obtain SN-PHB; the SEM picture of the SN-PHB and the TEM picture of a single SN-PHB are shown in FIG. 1.
The water contact angle data for SiNWAs, SN-Br, SN-PHA, and SN-PHB are shown in FIG. 2.
Table 1 shows XPS characterization during sample preparation.
TABLE 1
(5) SN-PHB substrates were sterilized with 75% ethanol, rinsed with sterile PBS, and then placed in PBS containing 1.5 μ g pGFP for 4h to preload the substrate with pGFP.
(6) then, the human B-lymphocytoma cells Ramos are planted on the surface of the base material at the density of 10 ten thousand per hole, and the cells are cultured for 4 hours to be fully adhered on the surface of the base material.
(7) The non-adherent cells were washed with sterile PBS along with some proteins, salts, etc., and then serum free medium containing pGFP, the mass of pGFP in each well was 1.5. mu.g.
(8) using a laser source with a wavelength of 808nm at 2.3W/cm2was irradiated for 30s, the plate was then returned to the cell incubator for 4h, and the following samples were processed in two parts: the flow cytometry in (9-1) was used to characterize pGFP transfection and proliferation potency of Ramos cells released in (9-2) and the cell release by fluorescence microscopy, respectively:
(9-1) replacing the culture solution with a sterile 60mM fructose solution, shaking at 120rpm for 20min, lightly blowing and collecting released cells by using a gun head, centrifugally suspending the cells into a new culture medium, placing the new culture medium in a new culture hole for continuous culture for 48h, collecting the Ramos cells in sterile PBS after 48h, and performing flow cytometry test by using the Ramos cells without any treatment as a control group to characterize the transfection efficiency of the Ramos cells; the results are shown in FIG. 3, in which FIG. 3(a) is a graph of sample flow data characterizing the transfection efficiency of pGFP into Ramos cells and (b) is a graph of sample flow data characterizing the mean fluorescence intensity of pGFP after transfection into Ramos cells. As can be seen from the flow data diagram, the transfection efficiency of the SN-PHB material reaches about 85 percent after the SN-PHB material is irradiated by near infrared light, and the SN-PHB material can efficiently transfect the exogenous macromolecule pGFP to suspension cells.
(9-2) the culture solution is changed into a sterile 60mM fructose solution, the mixture is shaken at 120rpm for 20min, then the released cells are collected by slightly blowing with a gun head and then centrifugally resuspended into a new culture medium, the culture is continued in a new culture hole, and the cells which are continuously cultured are tested for the proliferation capacity by adopting CCK-8. Before and after the cell release experiment, cells on the sample were stained with nuclear dye 4',6-diamidino-2-phenylindole (DAPI), and the number of cells before and after detachment was observed. The test results are shown in fig. 4 and 5. FIG. 4 is a density statistic of Ramos cells released by fructose and sample capture. Si-PHB is the PHB polymer brush modified on the silicon chip substrate, SiNWAs is the silicon nanowire array prepared from the silicon chip, and SN-PHB is the PHB polymer brush modified on the SiNWAs. As can be seen from the data diagram, on the planar silicon chip and the PHB-modified Si-PHB substrate, Ramos cells captured by the substrate are very few, while the SiNWs and SN-PHB substrate can enhance the capturing of Ramos due to the topological structure of SiNWAs and the affinity of phenylboronic acid groups in PHB to Ramos cells. And after the addition of the fructose solution, the Ramos cells captured on the SN-PHB surface are released basically in full. It is proved that the material prepared by the invention can capture the suspended Ramos cells, and the captured cells can be harvested from the substrate through sugar responsiveness and used in subsequent characterization research and application. Fig. 5 shows the result of the proliferation potency of Ramos cells tested by CCK-8, and it can be seen from the figure that Ramos cells delivered by laser irradiation can maintain good cell proliferation activity using the material of the present invention.
Example 2:
(1) Preparation of SiNWAs: the silicon wafer was cleaned with a mixed solution of sulfuric acid/hydrogen peroxide (7:3 by volume) and blown dry with nitrogen. Preparing a mixed aqueous solution containing hydrofluoric acid and silver nitrate (wherein the mixed aqueous solution contains 90mL of deionized water, 30mL of hydrofluoric acid and 1.02g of silver nitrate), adding the mixed aqueous solution into a cleaned silicon wafer, reacting for 10min at 50 ℃, cleaning with 25% dilute nitric acid solution and deionized water, and drying in a vacuum drying oven to obtain the SiNWAs.
(2) Modification initiator on SiNWAs: the reaction solution is prepared by using sulfuric acid: and (3) putting the SiNWAs cleaned by the hydrogen peroxide mixed solution (7:3 volume ratio) into an anhydrous toluene solution of 2% 2-bromo-2-methylpropionate (3-trimethoxysilyl) propyl ester, standing at room temperature for 24 hours, and cleaning by using anhydrous toluene to obtain the SiNWAs modified with the initiator, wherein the SiNWAs are marked as SN-Br.
(3) surface grafting of poly (hydroxyethyl methacrylate (HEMA) -co-using ARGET-ATRPpoly-2-Aminoethyl Methacrylate (AMA)): preparing a polymerization reaction liquid: AMA and HEMA at a molar ratio of 20:80, 2.2 mmole AMA and 8.8 mmole HEMA were dissolved in a mixed solution of 7mL of anhydrous methanol and 7mL of deionized water, and then 7. mu. moles of CuBr were added in that order2uniformly mixing 44 mu mol of bipyridyl (bpy) and 98 mu mol of ascorbic acid (Vc), adding the mixture into SN-Br, reacting at room temperature for 30min, alternately cleaning residual monomers and physically adhered polymers on the surface by using anhydrous methanol and deionized water to obtain a substrate with the surface grafted with AMA and HEMA copolymer brush, and marking the substrate as SN-PHA.
(4) Surface post-modification of 4-carboxy-3-fluorobenzeneboronic acid: preparing a 5mg/mL solution of 4-carboxy-3-fluorobenzeneboronic acid (CFPBA) in anhydrous N, N-dimethylformamide, and then adding O- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA) to control the final concentrations to be 3.8mg/mL and 2.58 mu L/mL respectively. And then blowing nitrogen for 30min, moving the mixture into a glove box, adding the mixed liquid into SN-PHA, and reacting for 12h at room temperature to obtain SN-PHB.
(5) SN-PHB substrates were sterilized with 75% ethanol, rinsed with sterile PBS, and then placed in PBS containing 1.5 μ g pGFP for 4h to preload the substrate with pGFP.
(6) the T cells are planted on the surface of the substrate at the density of 10 ten thousand per hole, and the cells are cultured for 4 hours to be fully adhered on the surface of the substrate.
(7) non-adherent T cells were washed with sterile PBS along with some proteins, salts, etc., and then serum-free medium containing pGFP, the mass of pGFP in each well was 1.5. mu.g.
(8) Using a laser source with a wavelength of 808nm at 2.3W/cm2Was irradiated for 30s, the plate was then returned to the cell incubator for 4h, and the following samples were processed in two parts: the flow cell count in (9-1) characterizes pGFP transfection and the proliferative capacity of the released T cells in (9-2), respectively.
(9-1) the culture solution was changed to a sterile 60mM fructose solution, shaken at 120rpm for 20min, then gently blowing and beating the collected and released cells by using a gun head, then centrifugally suspending the cells into a new culture medium, placing the cells into a new culture hole for further culture for 48 hours, collecting the T cells in sterile PBS after 48 hours, taking the T cells without any treatment as a negative control group, and adopting a commercial Lipo2000 transfection reagent to transfect pGFP into the T cells as a positive control (the Lipo2000 transfects pGFP into the T cells: planting the T cells in a 48-hole plate at the density of 10 ten thousand per hole, adding the Lipo2000 and the pGFP after compounding for 10min after 4h, adding the Lipo2000 and the pGFP into the planted T cells, replacing a culture medium containing the Lipo2000 and pGFP compound with a fresh culture medium containing serum after 6h of transfection, and continuously culturing for 48h), and carrying out flow cytometry test on the three samples to characterize the transfection efficiency; the results are shown in FIG. 6, which is a graph of the results of (a) sample flow data characterizing pGFP transfection efficiency into T cells and (b) sample flow data characterizing the mean fluorescence intensity of pGFP after transfection into T cells. As can be seen from the flow data diagram, the efficiency of the commercial transfection reagent Lipo2000 for transfecting pGFP by T cells is almost zero, and the transfection efficiency of the material SN-PHB in the invention reaches more than 80% after near infrared light irradiation, which proves that the invention can efficiently transfect exogenous macromolecule pGFP by suspended T cells which are difficult to transfect.
(9-2) the culture solution is changed into a sterile 60mM fructose solution, the mixture is shaken at 120rpm for 20min, then the released cells are collected by slightly blowing with a gun head and then centrifugally resuspended into a new culture medium, the culture is continued in a new culture hole, and the cells which are continuously cultured are tested for the proliferation capacity by adopting CCK-8. The test results are shown in fig. 7, and it can be seen from fig. 7 that the T cells delivered by laser irradiation can maintain good cell proliferation activity using the material of the present invention.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. An auxiliary platform, comprising: the polymer brush comprises a silicon nanowire array and a polymer brush formed on the surface of the silicon nanowire array, wherein the polymer brush is provided with a phenylboronic acid group.
2. The assistance platform according to claim 1, wherein: the thickness of the polymer brush is 10-30 nm.
3. the assistance platform according to claim 1, wherein: the length of the silicon nanowire array is 5-35 mu m.
4. the assistance platform according to claim 1, wherein: the auxiliary platform is prepared by modifying the silicon nanowire array through an initiator, then grafting a copolymer and finally modifying a modifier containing a phenylboronic acid group.
5. The assistance platform according to claim 4, wherein: the initiator is 2-bromo-2-methylpropanoic acid (3-trimethoxysilyl) propyl ester, or the initiator is formed by modifying a silane coupling agent 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane on the silicon nanowire array, and then modifying 2-bromo isobutyryl bromide or 2-chloro isobutyryl chloride; monomers for forming the copolymer are hydroxyethyl methacrylate and 2-aminoethyl methacrylate; the modifier is one or more of 4-carboxyl-3 fluorobenzeneboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid and 2-carboxyphenylboronic acid.
6. The assistance platform according to claim 1, wherein: the auxiliary platform also comprises exogenous molecules loaded on the silicon nanowire array and/or the polymer brush.
7. A method of preparing an assistive platform of any of claims 1 to 6, wherein: the method comprises the following steps:
(1) Reacting the silicon nanowire array with an initiator solution to obtain a silicon nanowire array modified with an initiator;
(2) In the presence of an initiating system, reacting the silicon nanowire array modified with the initiating agent with a monomer to obtain a substrate with a copolymer grafted on the surface;
(3) In the presence of a condensation reagent, reacting the base material with a modifier solution to prepare the auxiliary platform;
(4) selectively loading exogenous molecules on the auxiliary platform.
8. an in vitro cell delivery method of the helper platform of any one of claims 1 to 6, wherein: the method comprises the following steps:
(1) Planting cells on the auxiliary platform;
(2) Adding a serum-free culture medium for cell culture, wherein when an adopted auxiliary platform is loaded with exogenous molecules, the serum-free culture medium is added with or does not contain the exogenous molecules; when the adopted auxiliary platform is not loaded with exogenous molecules, the serum-free culture medium is added with the exogenous molecules;
(3) Near infrared light is adopted to be 1-10W/cm2Irradiating for 10-50 s at the illumination intensity to obtain the cell containing the exogenous molecule.
9. A method of releasing cells by the delivery method of claim 8, comprising: and (3) replacing the serum-free cell culture medium with a fructose solution 1-10 hours after the irradiation is finished, and collecting cells.
10. A cell produced with the aid of the platform-assisted method of any one of claims 1 to 6, or produced by the delivery method of claim 8, or collected by the delivery method of claim 9.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160024122A1 (en) * | 2012-01-18 | 2016-01-28 | Wisconsin Alumni Research Foundation | Bioreversible boronates for delivery of molecules into cells |
| CN105504301A (en) * | 2015-12-07 | 2016-04-20 | 复旦大学 | Dendritic macromolecule-copolymer cell capturing material as well as preparation method and application thereof |
| US20170274098A1 (en) * | 2016-03-25 | 2017-09-28 | Institute For Basic Science | Gene delivery system using polymer network |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN105504301A (en) * | 2015-12-07 | 2016-04-20 | 复旦大学 | Dendritic macromolecule-copolymer cell capturing material as well as preparation method and application thereof |
| US20170274098A1 (en) * | 2016-03-25 | 2017-09-28 | Institute For Basic Science | Gene delivery system using polymer network |
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| CN114917415A (en) * | 2022-03-21 | 2022-08-19 | 四川大学 | Degradable composite membrane for heart occluder and preparation method and application thereof |
| CN114917415B (en) * | 2022-03-21 | 2023-01-06 | 四川大学 | Degradable composite membrane for heart occluder and preparation method and application thereof |
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