CN111363762A - Method for transferring exogenous molecules into cells - Google Patents
Method for transferring exogenous molecules into cells Download PDFInfo
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- CN111363762A CN111363762A CN201811600019.4A CN201811600019A CN111363762A CN 111363762 A CN111363762 A CN 111363762A CN 201811600019 A CN201811600019 A CN 201811600019A CN 111363762 A CN111363762 A CN 111363762A
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Abstract
The present invention relates to a method for delivering exogenous molecules into cells. The method comprises the step of using a base material with a modified polydopamine layer deposited on the surface, wherein the modified polydopamine layer is a temperature-sensitive polymer graft modified polydopamine layer obtained by reacting double-bond-containing functional polydopamine with a temperature-sensitive polymer monomer. The method has the advantages of strong universality, high transmission efficiency and transfection efficiency, small cytotoxicity, high yield of modified cells and large disposable cell treatment flux.
Description
Technical Field
The invention relates to the field of biomedicine and medical instruments, in particular to a method for delivering exogenous molecules into cells.
Background
The delivery of exogenous macromolecules, such as nucleic acids, proteins and intracellular probes, into cells has important roles in basic biological research, industrial production, and clinical diagnosis and treatment. At present, the methods for delivering exogenous macromolecules into cells can be mainly divided into a carrier method and a membrane rupture method.
The carrier method is to use some substances as carriers to wrap exogenous molecules, protect the exogenous molecules from being degraded after entering cells, promote the interaction between the exogenous molecules and cell membranes, and finally protect and deliver the exogenous molecules into the cells. The carrier can be recombinant virus, vesicle, ghost cell, functional ligand and polypeptide with biological activity, or synthetic polymer and nano particle. Viral vectors deliver exogenous molecules into cells by way of viral infection, while most other vectors enter cells by way of endocytosis or membrane fusion. The most representative vectors include adenovirus, adeno-associated virus, lentivirus, polyethyleneimine, gold nanoparticles, commercial transfection reagents Lipofectamine 2000, Lipofectamine 3000, and the like.
The membrane breaking method is to enhance the permeability of the surface of a cell membrane or generate holes by utilizing a chemical membrane breaking agent (detergent and the like) or a physical external force (force, sound, light, electricity, heat and the like) so as to help exogenous molecules to enter cells. The most representative and well developed physical membrane breaking methods include electroporation and microinjection. The electroporation method is to utilize the interference of high voltage electric pulse to cell membrane to form micropores favorable for nucleic acid to enter, and the microinjection method is to introduce exogenous substances into cells directly through microinjection device.
Viral vectors are now considered to be the most efficient intracellular molecular delivery method due to their high efficiency and specificity. However, the use of viral vectors poses a great safety risk due to the strong infectivity of the virus. In addition, viral vectors are limited to the delivery of nucleic acid substances and cannot be used to deliver other exogenous molecules into cells, since only genetic material can be integrated into the viral genome for subsequent host infection experiments. This problem also exists in other support methods. Since the exogenous molecule first needs to be bound to the carrier through a certain interaction (e.g., electrostatic interaction), there is a limit to the nature of the exogenous molecule, i.e., a carrier can only be used to transfer a molecule with a specific property, which greatly reduces the versatility of the carrier method. Although the vector method is also used for the research of transferring protein or other drug molecules, most vectors are still limited to the transfer of nucleic acid substances, and vectors which are relatively versatile and can transfer a plurality of different molecules do not exist. In addition, the carrier method is limited not only in the kinds of transmissible molecules but also in the kinds of cells that can be treated. Especially for cells that are difficult to transfect, the transfection efficiency is very low. This is mainly due to the fact that the self-protection mechanism of the cells which are difficult to transfect is stronger, and the vector is difficult to enter the cells. Although the partial vector method can improve the efficiency of vector entering cells by modifying some cell-specific polypeptides, etc., the method only improves the transfection efficiency of specific cell lines, and is still limited in terms of the types of cells that can be transferred. Therefore, the vector method mainly has several disadvantages: (1) viral vectors have high transfection efficiency, but the safety problem is not negligible, and the viral vectors can only be used for delivering nucleic acid substances; (2) non-viral vectors have low versatility in delivering molecular species and cell species, and have low transfection efficiency for cells that are difficult to transfect.
On the other hand, physical membrane-breaking methods usually cause great damage to the activity of cells because they destroy the integrity of cell membranes. For example, in electroporation, the high voltage electrical pulses used to effect the entry of nucleic acid material into the interior of the cell often result in the death of a large number of cells, resulting in a substantial reduction in the number of effective cells that can ultimately be used. In the microinjection method, nucleic acid substances can be efficiently introduced into the nucleus, but it is suitable only for single cell manipulation and is not suitable for large-throughput cell processing. Other methods of cell membrane rupture using physical external forces mostly require complex instruments or devices, which makes the experimental threshold higher.
CN105420278A discloses a method for preparing cells loaded with exogenous molecules by adopting a photo-perforation mode, a substrate for preparing the cells and the cells. A photo-induced perforation kit based on a gold nanoparticle deposition film is developed, and various exogenous macromolecules are transferred into cells by utilizing the enhancement of the permeability of cell membranes of the cells adhered to the surfaces of the photo-induced perforation kit under the irradiation of laser. However, the transfection efficiency of the method is still to be improved for the cell lines which are difficult to transfect, such as primary cell mouse embryo fibroblasts, and the transfection efficiency of the method without the vector is about 53 percent. In addition, the gold nanoparticle deposition film has large surface roughness, complex surface topological structure and strong adhesion between cells and the surface, so that the modified cells are difficult to harvest, and the subsequent application is limited. However, if the cells are harvested by trypsinization, the activity of the cells will be greatly impaired, especially by fragile primary cells.
In general, the physical membrane-breaking method has major disadvantages including large loss of cell activity, low cell processing throughput, complicated required apparatus and equipment, and low cell harvesting efficiency.
Therefore, a macromolecule transmission system which has strong universality of exogenous molecules and cell types, high transmission efficiency, low cytotoxicity, capability of processing cells with large flux and high cell harvesting rate is yet to be researched.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problems in the prior art, the invention designs a method for transferring exogenous molecules into cells, which has the advantages of strong universality, high transfer efficiency and transfection efficiency, small cytotoxicity, high yield of modified cells and large disposable cell treatment flux.
Means for solving the problems
In one aspect, the present invention provides a method for delivering exogenous molecules into cells, comprising contacting a substrate having a modified polydopamine layer deposited on the surface thereof with the cells, wherein the modified polydopamine layer is obtained by reacting a double-bond-containing functional polydopamine or a functional dopamine monomer with a temperature-sensitive polymer monomer.
In one embodiment, the method comprises seeding the cells on the surface of a substrate having a modified polydopamine layer deposited on the surface, contacting the cells with an agent comprising an exogenous molecule, and illuminating the cells with a laser source in the near infrared band. Furthermore, the method also comprises the step of placing the substrate with the modified polydopamine layer deposited on the surface after the laser irradiation in an environment with the temperature lower than the temperature of the low critical solution of the temperature-sensitive polymer to promote the desorption of cells from the surface.
In another embodiment, the method further comprises the step of preparing an aqueous solution comprising dopamine and/or dopamine hydrochloride and an ethylenically unsaturated anhydride, adjusting the pH to slightly alkaline, preferably pH 8-9, and immersing the substrate in the solution to obtain a substrate having a surface deposited functional polydopamine.
In another embodiment, the method further comprises the step of dissolving dopamine and/or dopamine hydrochloride and ethylenically unsaturated anhydride in water and stirring to obtain a double bond-containing functional dopamine monomer.
In another embodiment, the ethylenically unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, citraconic anhydride, preferably the ethylenically unsaturated anhydride is selected from itaconic anhydride.
In another embodiment, the temperature-sensitive polymer monomer of the present invention at least comprises N-isopropylacrylamide, and further preferably, the temperature-sensitive polymer monomer is N-isopropylacrylamide.
In another embodiment, the substrate of the present invention may be a metal material (e.g., gold, stainless steel, titanium alloy, magnesium alloy, etc.), an inorganic non-metal material (e.g., single crystal silicon, mica, glass, etc.), an organic polymer material (e.g., polyurethane, polydimethylsiloxane, polymer electrospun fiber membrane, etc.), an experimental or instrument device (cell culture plate, microplate, micro flow channel device), etc., and preferably, the substrate is a gold plate.
In another embodiment, the exogenous molecules of the invention include one or more of a polysaccharide molecule (e.g., dextran), a protein (e.g., a gene-editing enzyme, an antibody, an antigen), a DNA (e.g., pDNA), an RNA (e.g., miRNA, siRNA), a therapeutic drug, an intracellular probe (e.g., quantum dot), a nanomaterial (e.g., a nanoparticle, a nanodevice), an aptamer, a bacterium, an artificial chromosome, an organelle (e.g., a mitochondrion), and the like.
In another embodiment, the cell of the present invention is selected from the group consisting of a cell line, preferably a cell line comprising hela cells, or a primary cell line comprising one of mouse embryonic fibroblasts, human umbilical vein endothelial cells, mouse dendritic cells.
In another technical scheme, the invention also provides application of the substrate with the modified polydopamine layer deposited on the surface in the process of transferring exogenous molecules into cells, wherein the modified polydopamine layer is obtained by reacting double-bond-containing functional polydopamine or functional dopamine monomers with temperature-sensitive polymer monomers.
In another embodiment, the present invention also provides a cell loaded with exogenous molecules prepared by the above method of the present invention.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention provides a method for transferring exogenous molecules into cells, which can greatly improve the transfer efficiency of the exogenous molecules, the cell harvest rate and the cell processing flux by preparing a specific photothermal transfer and cell release base material. Compared with the prior art of delivering exogenous molecules into cells, the invention has the following beneficial effects:
on the one hand, the method disclosed by the invention is strong in universality. Using the methods described in the present invention, it is theoretically possible to deliver into cells any substance that needs to be delivered, including, but not limited to, one or more of polysaccharide molecules (e.g., dextran), proteins (e.g., gene editing enzymes, antibodies, antigens), DNA (e.g., pDNA), RNA (e.g., 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.
On the other hand, the method of the present invention has high transfection efficiency for primary cells that are difficult to transfect. Most of the intracellular molecule transfer methods only use the conventional and easily transfected cell line such as HeLa cell as model cell to perform experiments to verify the transfer efficiency, while for the primary cells which are difficult to transfect, the molecule transfer efficiency is mostly lower, which results in poor versatility in the cell line aspect. The method of the invention is applicable to various cell lines, including conventional cell lines such as Heila cells and primary cells which are difficult to transfect, including mouse embryonic fibroblasts, human umbilical vein endothelial cells and mouse dendritic cells, and the transfection efficiency of the method reaches over 90 percent.
On the other hand, the method described in the present invention is less cytotoxic. For most of macromolecule transfer methods based on membrane rupture mechanism, the purpose is achieved by destroying the integrity of cell membranes, so the activity of the treated cells is greatly damaged; in the invention, the higher cell activity can be kept on the basis of obtaining higher transfer efficiency by regulating and controlling the power and the irradiation time of the irradiation laser.
On the other hand, the method of the present invention has a high cell yield. The invention fully utilizes the change of hydrophilicity and hydrophobicity of the temperature-sensitive polymer at different temperatures, and leads the cells to be automatically desorbed from the base material by changing the cell culture temperature so as to reduce the damage to the cell activity in cell harvesting.
In another aspect, the methods described herein allow for high throughput processing of cells. The method can treat at least tens of thousands of cells by irradiating the surface modified substrate with laser once, and can realize high-efficiency and large-scale cell treatment in a short time.
Drawings
FIG. 1 is a bar graph showing the delivery efficiency and cell activity 48h after delivery of three different sizes of exogenous molecules (dextran, bovine serum albumin, plasmid DNA encoding green fluorescent protein (pGFP)) into HeLa cells.
FIG. 2 is a cell photograph and bar graph of cells and their density before and after Hela cell release using three different substrates for dextran delivery, with a scale of 200 μm. In the figure, Chinese and English are abbreviated as Chinese meaning: Au-PDA, gold flakes depositing functional polydopamine; Au-PDA-PNIP, gold plate deposited with poly N-isopropylacrylamide graft modified polydopamine layer.
FIG. 3 is a bar graph of the transfection efficiency and cell activity 48h after transfection of plasmid DNA encoding green fluorescent protein (pGFP) into three primary cells that were difficult to transfect (mouse embryonic fibroblasts, human umbilical vein endothelial cells, mouse dendritic cells). In the figure, Chinese and English are abbreviated as Chinese meaning: mEF, mouse embryonic fibroblasts; HUVEC, human umbilical vein endothelial cells; mDC, mouse dendritic cells.
FIG. 4 shows the transfection efficiency of plasmid DNA (pGFP) encoding green fluorescent protein, the cell activity after transfection for 48h, and the detachment efficiency in Hela cells prepared by the method for preparing materials described in example 7, in which a poly-N-isopropylacrylamide graft-modified polydopamine layer was deposited. The Chinese and English abbreviations in the figures mean: Au-PDA-PNIP, gold plate deposited with poly N-isopropylacrylamide graft modified polydopamine layer.
Detailed Description
Various exemplary embodiments, features and aspects of the present invention will be described in detail below with reference to the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
Definition of
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "agent" includes any substance to be delivered into a cell. Such agents include, but are not limited to, serum-free cell culture media containing exogenous molecules.
The term "protein" is used herein to refer to a polymer of amino acid residues. The term applies to: amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, and naturally occurring amino acid polymers. The term also includes variants on traditional peptide bonds that connect the various amino acids that make up a polypeptide.
The term "monomer" means any chemical species that can be characterized by a chemical formula with a polymerizable group that can be polymerized into an oligomer or polymer to increase molecular weight.
The term "transfer" refers to the process from outside the cell to inside the cell, and the term "transfection" refers to the process from outside the cell to the nucleus, followed by translation to express the protein, and generally refers to the process of transferring nucleic acid material into the cell and successfully expressing it.
< preparation of photothermal transfer and cell-releasing substrate >
The invention firstly prepares a photo-thermal transfer and cell release substrate, and a modified poly dopamine layer is deposited on the surface of the substrate. The substrate with the modified polydopamine layer deposited on the surface can be used for transferring exogenous molecules into cells.
Dopamine (DA, chemical structure: C)6H3(OH)2-CH2-CH2-NH2CAS number: 62-31-7) as one of catechol derivatives, is a nerve conduction substance to help cells transmit pulse signals. Dopamine has recently found widespread use in biomedical and biomaterial applications due to its ubiquitous and strong adhesion to substrates. Dopamine is easily oxidized by dissolved oxygen in aqueous solution, then self-polymerization crosslinking reaction is initiated, and a closely attached polydopamine composite layer can be formed on the surface of almost any solid material. Polydopamine is a melanin-like substance, has good adhesion and outstanding light absorption performance, can absorb light from visible light to near infrared light bands, generates a photothermal effect in a matrix, and enables the local rapid and effective temperature rise of materials. However, the research of preparing a photothermal substrate for transferring exogenous molecules into cells by utilizing the photothermal effect of polydopamine is not seen at present.
The invention discovers that the base material prepared by utilizing the modified polydopamine has good photo-thermal transfer performance and cell release capacity.
In one technical scheme, the functional polydopamine is firstly deposited on the base material, and the specific method comprises the steps of preparing an aqueous solution containing dopamine and/or dopamine hydrochloride and ethylenically unsaturated anhydride, adjusting the pH value to be alkalescent, immersing the base material in the solution, and standing to obtain the base material with the functional polydopamine deposited on the surface. The substrate of the present invention, i.e., the substrate material, may be a metal material (e.g., gold, stainless steel, titanium alloy, magnesium alloy, etc.), an inorganic non-metal material (e.g., single crystal silicon, mica, glass, etc.), an organic polymer material (e.g., polyurethane, polydimethylsiloxane, polymer electrospun fiber membrane, etc.), an experimental or instrumental device (cell culture plate, microplate, microchannel device), etc. Further preferably, the substrate is a gold plate.
In another technical scheme, after the aqueous solution is prepared, stirring for 1-3h at room temperature to perform sufficient reaction; further, adjusting the pH value of the system with an alkali solution (such as tris), preferably, adjusting the pH value to 8-9, more preferably, adjusting the pH value to 8.5; further, the substrate is immersed in the solution and allowed to stand at 30-40 ℃ for 20-30 h.
In another embodiment, the ethylenically unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, citraconic anhydride, preferably, the ethylenically unsaturated anhydride is selected from itaconic anhydride. The double bond of itaconic anhydride is terminal group double bond, and the activity of the subsequent polymerization reaction is higher. The method comprises the steps of stirring aqueous solutions of itaconic anhydride and dopamine, reacting the itaconic anhydride with amino on the dopamine, opening five-membered rings of the itaconic anhydride due to nucleophilic attack to obtain acylated dopamine, immersing a substrate in the acylated dopamine, polymerizing the acylated dopamine under the weak base condition to obtain a poly-dopamine-deposited substrate with allyl groups, wherein exposed double bonds on the surface of the poly-dopamine are beneficial to subsequent grafting reaction. The specific reaction flow of itaconic anhydride and dopamine is as follows:
in another embodiment, the concentrations of dopamine or dopamine hydrochloride and ethylenically unsaturated anhydride are both in the range of 1 to 5mg/mL, preferably the concentrations of dopamine or dopamine hydrochloride and ethylenically unsaturated anhydride are the same to reduce the introduction of impurities, more preferably both 2mg/mL or 3 mg/mL. If the concentration is too high, the deposited polydopamine layer becomes thick, the accumulation of polydopamine particles is more serious, and the unevenness of the surface of the material is possibly caused; if the concentration is too low, the deposited poly-dopamine layer may be too thin and the photothermal effect is weak, and the same transfection effect cannot be obtained at the present laser intensity and irradiation time.
In one embodiment of the invention, an aqueous solution of dopamine and itaconic anhydride with a concentration of 2mg/mL is prepared, stirred for 2h at room temperature, then the pH value is adjusted to 8.5 by an alkali solution, the substrate is immersed in the above solution, and is kept stand for 24h at 37 ℃ to obtain the substrate deposited with the functional polydopamine layer containing double bonds.
And then, carrying out free radical polymerization reaction on the double bond on the functional polydopamine and a temperature-sensitive polymer monomer to generate a temperature-sensitive polymer graft modified polydopamine layer. The most important feature of temperature sensitive polymers is the presence of a critical solution temperature around which the polymer solution will undergo a discontinuous phase change. Preferably, the temperature-sensitive polymer with the low critical solution temperature is used in the invention, the polymer solution (mostly aqueous solution) is in a single-phase state below the temperature, and a phase separation phenomenon occurs above the temperature, the temperature-sensitive polymer has a certain proportion of hydrophilic groups and hydrophobic groups, the interaction between the hydrophilic groups and the hydrophobic groups and water molecules can change along with the temperature change, and the non-destructive release of the modified cells from the substrate can be promoted by utilizing the hydrophilic-hydrophobic transformation of the temperature-sensitive polymer above and below the low critical solution temperature.
Since the poly-N-isopropylacrylamide has both hydrophilic amide groups and hydrophobic isopropyl groups on the macromolecular chain, the low critical solution temperature of the macromolecular chain of the poly-N-isopropylacrylamide in an aqueous medium is about 32 ℃, and the sensitivity to temperature is very high, the temperature-sensitive polymer monomer at least comprises N-isopropylacrylamide in the invention, and more preferably, the temperature-sensitive polymer monomer is N-isopropylacrylamide.
The free radical polymerization reaction of the functional polydopamine and the temperature-sensitive polymer monomer can adopt a traditional redox free radical polymerization method, and a common initiator comprises persulfate such as ammonium persulfate, N, N, N ', N' -tetramethylethylenediamine is adopted as an initiation promoter to catalyze ammonium persulfate to generate free radicals, or ammonium persulfate and sodium bisulfite are adopted to be compounded as an initiator. The amount of initiator used can be determined autonomously by the person skilled in the art according to the reaction requirements.
The specific reaction scheme of N-isopropylacrylamide and poly-dopamine with allyl is as follows:
in one embodiment of the invention, an aqueous solution of N-isopropylacrylamide is prepared (the concentration of the N-isopropylacrylamide is 0.02-0.1g/mL, preferably 0.05g/mL, and the thickness of the obtained poly-N-isopropylacrylamide has the best temperature responsiveness), nitrogen is filled for deoxygenation, ammonium persulfate and N, N, N ', N' -tetramethylethylenediamine are added, and the substrate deposited with the functional poly-dopamine containing double bonds is soaked in the solution and stands at room temperature for 20-30h to obtain the substrate deposited with the poly-N-isopropylacrylamide graft-modified poly-dopamine layer.
In another technical scheme, the invention can also prepare a functional dopamine monomer containing double bonds, and then carry out free radical polymerization reaction on the double bonds capable of reacting on the functional dopamine monomer and the temperature-sensitive polymer monomer to generate a copolymer of dopamine and the temperature-sensitive polymer monomer. And then preparing the copolymer into an aqueous solution, adjusting the aqueous solution to be alkalescent, soaking the base material into the solution, and standing to obtain the base material with the modified polydopamine layer deposited on the surface.
In another embodiment, the functional dopamine monomer containing double bond is obtained by dissolving dopamine and/or dopamine hydrochloride and ethylenically unsaturated anhydride in water and stirring. Preferably, the ethylenically unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride and citraconic anhydride, and further preferably, the ethylenically unsaturated anhydride is selected from itaconic anhydride.
In another embodiment, the concentrations of dopamine or dopamine hydrochloride and ethylenically unsaturated anhydride are each in the range of 1 to 5mg/mL, preferably the concentrations of dopamine or dopamine hydrochloride and ethylenically unsaturated anhydride are the same to reduce the introduction of impurities. The concentration ratio of the functional dopamine monomer with double bonds to the temperature-sensitive polymer monomer is 3-6: 1 to ensure sufficient copolymerization.
In a specific embodiment of the invention, dopamine hydrochloride and itaconic anhydride are dissolved in water, and are stirred for 1-3 hours at room temperature to obtain the double-bond-containing functional dopamine monomer. And then preparing aqueous solution of N-isopropyl acrylamide and dopamine monomer containing double bonds, wherein the concentration ratio is 1:5, filling nitrogen to remove oxygen, adding azobisisobutyronitrile as an initiator into the reaction solution, and reacting for 20-30h to obtain the copolymer of dopamine and N-isopropyl acrylamide. Preparing the aqueous solution of the copolymer, adjusting the pH value of the aqueous solution to 8.5, soaking the base material in the aqueous solution, and standing the base material at the temperature of between 30 and 40 ℃ for 20 to 30 hours to obtain the base material with the modified polydopamine layer deposited on the surface.
< method for delivering exogenous molecule into cell >
The invention provides a method for transferring exogenous molecules into cells, which comprises the steps of using a substrate (namely a photothermal transfer and cell release substrate) with a modified polydopamine layer deposited on the surface, planting cells on the surface of the substrate with the modified polydopamine layer deposited on the surface, contacting the cells with a reagent containing the exogenous molecules, irradiating the cells by using a laser light source in a near infrared band, and realizing the cell transfer of the exogenous molecules by means of photoinduced perforation. The polydopamine has stronger photothermal conversion capability, and the method can reduce the laser intensity, and the laser source can use a low-intensity continuous laser source, so that the experiment cost is obviously reduced.
In one embodiment, the cell is selected from a cell line or a primary cell, preferably, the cell line comprises a hela cell, and the primary cell line comprises one of a mouse embryonic fibroblast (mEF), a Human Umbilical Vein Endothelial Cell (HUVEC), and a mouse dendritic cell (mDC). Most of the intracellular molecule transmission methods only use the conventional and easily transfected cell lines such as Heila cells to perform molecule transmission experiments, while for the primary cells which are difficult to transfect, the molecule transmission efficiency is mostly low, so that the universality of the molecule transmission system in the aspect of the cell lines is poor. The methods described in the present invention are applicable to a variety of cell lines, including primary cells that are difficult to transfect.
The method of the present invention can be used to deliver a variety of different sized substances into cells without specific requirements on the nature of the substance. For example, the exogenous molecule includes one or more of a polysaccharide molecule (e.g., dextran), a protein (e.g., a gene editing enzyme, an antibody, an antigen), a DNA (e.g., pDNA), an RNA (e.g., miRNA, siRNA), a therapeutic drug, an intracellular probe (e.g., quantum dot), a nanomaterial (e.g., nanoparticle, nanodevice), an aptamer, a bacterium, an artificial chromosome, an organelle (e.g., a mitochondrion), and the like.
In one embodiment of the invention, the photothermal and cell releasing substrate is first sterilized, the cells of interest are seeded on the substrate, and the cells are cultured for a period of time sufficient to spread the cells. Then washing cells with buffer solution, adding reagent with exogenous molecule such as serum-free cell culture medium with exogenous molecule, and irradiating with near infrared laser light source (780-3000 nm) at 1-10W/cm2Irradiating the cells on the substrate within the power density range for 0.5-10 min. And after the laser irradiation is finished for 3-5h, replacing the cell culture medium containing the exogenous molecules with a normal cell culture medium with serum, and continuing to restore the normal cultured cells. After completion of laser irradiation, the cells were incubated at 37 ℃. Further, cells may be washed with sterile phosphate buffered saline; the concentration of the exogenous molecule in the reagent can be adjusted by one skilled in the art according to the actual need.
In another technical scheme, the method further comprises the step of placing the substrate with the modified polydopamine layer deposited on the surface after laser irradiation in an environment with the temperature lower than the temperature of the temperature-sensitive polymer low critical solution to promote the cells to be detached from the surface.
When the base material with cells is placed in an environment with the temperature lower than the low critical solution temperature of the temperature-sensitive polymer, the surface of the base material grafted with the temperature-sensitive polymer is changed from hydrophobicity to hydrophilicity, and the molecular chain of the temperature-sensitive polymer swells, so that the cells on the surface are desorbed in the form of single cells or cell sheets.
In a specific embodiment of the invention, the temperature-sensitive polymer is poly-N-isopropylacrylamide, and in order to promote the harvest of cells and save the experimental cost, the substrate with the cells is placed in a refrigerator at 4 ℃ for 20-40min and then taken out, blown and beaten, and the blown and beaten cells are transferred to a new hole to realize the continuous culture.
< cells carrying exogenous molecules >
The invention further provides a cell loaded with exogenous molecules prepared by the method. The cell comprises a cell body and exogenous molecules entering the interior of the cell body, wherein the cell can be formed by the exogenous molecules directly penetrating a cell membrane and entering the cell body, or can be formed by division and proliferation of cells with the exogenous molecules inside. The method is particularly suitable for obtaining cells which are difficult to transfect and carry exogenous molecules.
Examples
The technical solution of the present invention will be further described with reference to specific examples. It should be understood that the following examples are only for illustrating and explaining the present invention and are not intended to limit the scope of the present invention.
Example 1
(1) 0.08g dopamine hydrochloride and 0.08g itaconic anhydride were dissolved in 40mL deionized water and stirred at room temperature for 2h, then the pH was adjusted to 8.5 with base solution. And immersing the gold sheet in the solution, and standing for 24h at 37 ℃ to obtain the substrate deposited with the functional polydopamine, namely Au-PDA.
(2) Preparing an aqueous solution of N-isopropylacrylamide with the concentration of 0.05g/mL, and blowing nitrogen for 30min to remove oxygen in the solution.
(3) The substrate on which the functional polydopamine layer was deposited and an aqueous solution of N-isopropylacrylamide were transferred to a glove box, and the substrate on which the functional polydopamine layer was deposited was immersed in an aqueous solution of N-isopropylacrylamide monomer, to which ammonium persulfate and N, N' -tetramethylethylenediamine were added to give final concentrations of 0.02g/mL and 0.67 μ L/mL, respectively. And reacting for 24 hours at room temperature to obtain the gold sheet with the modified poly dopamine layer deposited on the surface, namely Au-PDA-PNIP.
The content and thickness of the elements on the surface of Au-PDA and Au-PDA-PNIP are measured, and the results are shown in Table 1. The content of the surface elements is obtained by analyzing and testing X-ray photoelectron spectroscopy, and the content of each element is consistent with a theoretical value; the thickness of the sample is measured by an ellipsometer, and when the thickness of the Au surface is set to be 0nm, the thickness of the polydopamine deposition layer is 86.3nm, and the total thickness of polydopamine and poly N-isopropylacrylamide is 115.9 nm.
| Sample (I) | C(％) | N(％) | O(％) | C/N | Thickness (nm) |
| Au-PDA | 67.1±2.3 | 7.6±0.7 | 25.3±2.1 | 8.8 | 86.3±1.6 |
| Au-PDA-PNIP | 73.6±2.9 | 11.5±1.2 | 14.9±1.4 | 6.4 | 115.9±7.4 |
The content of each element is consistent with the theoretical value, and the existence of the thickness change can prove that the modified polydopamine layer is successfully deposited on the gold sheet.
(4) The substrate was sterilized with 75% alcohol, and then hela cells were seeded into the wells of a 48-well plate at a density of 5 ten thousand per well, and cultured for 12 hours to spread the cells sufficiently in the plate.
(5) The cells were washed with sterile phosphate buffer and serum-free cell culture medium with pGFP (pDNA encoding green fluorescent protein, molecular weight 3075kDa) of 1.5. mu.g per well was added.
(6) Using a laser source with a wavelength of 808nm at a wavelength of 5.1W/cm2The power density of (2) was applied to the cells in the wells for 30 s.
(7) After 4h of laser irradiation, the cell culture medium with serum was replaced to continue cell culture.
(8) After the laser irradiation is finished for 48 hours, the cell nucleus is stained by 4', 6-diamidino-2-phenylindole, and the expression condition of the green fluorescent protein is observed by a fluorescence microscope. Blue cells are stained nuclei, green cells are cells successfully expressing green fluorescent protein, and represent cells successfully transfected. The cell number of blue cells and the cell number of green cells were counted by quantifying treatment using a fluorescence microscope photograph, and the transfection efficiency was obtained by multiplying the former by 100%, and the cell activity was detected using CCK-8(cell counting kit). Transfection efficiency was 99.1% and cell viability was 95.5%, see FIG. 1.
Example 2
Steps (1) to (4) were the same as in example 1.
(5) Cells were washed with sterile phosphate buffer and serum-free cell culture medium containing dextran (molecular weight 4.4kDa) was added to give a final dextran concentration of 1mg/mL per well.
Steps (6) and (7) were the same as in example 1.
The subsequent sample treatment was carried out in three parts, namely (8-1) fluorescence microscopy for characterization of transfection and (8-2) cell release assay.
(8-1) after the laser irradiation is finished for 30min, staining the cell nucleus by using 4', 6-diamidino-2-phenylindole, and observing the condition that the dextran enters the cell by using a fluorescence microscope. Blue cells are stained nuclei and red is the color emitted by rhodamine-labeled dextran, representing successfully delivered cells. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Histogram of transfer efficiency and cell viability, with transfer efficiency 98.8% and cell viability 91.2%, see figure 1.
(8-2) after the laser irradiation is finished for 12h, placing the base material with the cells in a refrigerator at 4 ℃ for 30min, taking out, blowing, and transferring the blown cells to a new hole for culture. Before and after the cell release experiment, cells on the sample are stained by using a living cell dye, and the number of the cells before and after desorption is observed. The results are shown in FIG. 2, in which FIG. 2(a) is a fluorescence microscope result and FIG. 2(b) is a histogram of the cell density obtained by quantifying the fluorescence microscope results before and after the release.
Example 3
Steps (1) to (4) were the same as in example 1.
(5) Cells were washed with sterile phosphate buffer and serum-free cell culture medium with bovine serum albumin (molecular weight 66kDa) was added to give a final concentration of 1mg/mL of bovine serum albumin in each well.
Steps (6) and (7) were the same as in example 1.
(8) After the laser irradiation is finished for 30min, the cell nucleus is stained by 4', 6-diamidino-2-phenylindole, and the condition that bovine serum albumin enters the cell is observed by a fluorescence microscope. Blue cells are stained nuclei, and red is the color emitted by rhodamine-labeled bovine serum albumin, representing successfully delivered cells. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Histogram of transfer efficiency and cell activity, transfer efficiency was 98.0% and cell activity was 93.1%, see fig. 1.
Example 4
Steps (1) to (3) were the same as in example 1.
(4) After the substrate was sterilized with 75% alcohol, mouse embryo fibroblasts were seeded into the wells of a 48-well plate at a density of 5 ten thousand per well, and cultured for 12 hours to sufficiently spread the cells in the plate.
Step (5) was the same as in example 1.
(6) Using a laser source with a wavelength of 808nm at a wavelength of 5.1W/cm2The power density of (2) was applied to the cells in the wells for 30 s.
(7) After 4h of laser irradiation, the cell culture medium with serum was replaced to continue cell culture.
(8) After the laser irradiation is finished for 48 hours, the cell nucleus is stained by 4', 6-diamidino-2-phenylindole, and the expression condition of the green fluorescent protein is observed by a fluorescence microscope. Blue cells are stained nuclei, green cells are cells successfully expressing green fluorescent protein, and represent cells successfully transfected. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Transfection efficiency was 98.9% and cell activity was 91.8%, see fig. 3.
Example 5
Steps (1) to (3) were the same as in example 1.
(4) After the substrate was sterilized with 75% ethanol, human umbilical vein endothelial cells were seeded into the wells of a 48-well plate at a density of 5 ten thousand per well, and cultured for 12 hours to sufficiently spread the cells in the plate.
Step (5) was the same as in example 1.
(6) Using a laser source with a wavelength of 808nm at a wavelength of 5.1W/cm2The power density of (2) was applied to the cells in the wells for 30 s.
(7) After 4h of laser irradiation, the cell culture medium with serum was replaced to continue cell culture.
(8) After the laser irradiation is finished for 48 hours, the cell nucleus is stained by 4', 6-diamidino-2-phenylindole, and the expression condition of the green fluorescent protein is observed by a fluorescence microscope. Blue cells are stained nuclei, green cells are cells successfully expressing green fluorescent protein, and represent cells successfully transfected. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Transfection efficiency was 99.4% and cell activity was 96.2%, see fig. 3.
Example 6
Steps (1) to (3) were the same as in example 1.
(4) After the substrate was sterilized with 75% alcohol, mouse dendritic cells were seeded into the wells of a 48-well plate at a density of 5 ten thousand per well, and cultured for 12 hours to sufficiently spread the cells in the plate.
Step (5) was the same as in example 1.
(6) Using a laser source with a wavelength of 808nm at a wavelength of 2.3W/cm2The power density of (a) was applied for 90s to the cells in the wells.
(7) After 4h of laser irradiation, the cell culture medium with serum was replaced to continue cell culture.
(8) After the laser irradiation is finished for 48 hours, the cell nucleus is stained by 4', 6-diamidino-2-phenylindole, and the expression condition of the green fluorescent protein is observed by a fluorescence microscope. Blue cells are stained nuclei, green cells are cells successfully expressing green fluorescent protein, and represent cells successfully transfected. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Transfection efficiency was 99.2% and cell activity was 60.4%, see FIG. 3.
Example 7
(1) Dissolving 0.08g of dopamine hydrochloride and 0.08g of itaconic anhydride in 40mL of deionized water, and stirring at room temperature for 2h to obtain the dopamine monomer with double bonds.
(2) Preparing aqueous solutions of N-isopropylacrylamide and dopamine monomer with double bonds, wherein the concentrations are 0.03mg/mL and 0.15g/mL respectively, and blowing nitrogen for 30min to remove oxygen in the solutions.
(3) Adding azobisisobutyronitrile (azobisisobutyronitrile) as an initiator (the concentration is 0.0003mg/mL) into the reaction solution, and reacting for 24 hours under the protection of nitrogen to obtain the copolymer of dopamine and N-isopropylacrylamide.
(4) An aqueous solution of the copolymer was prepared at a concentration of 2mg/mL, and its pH was adjusted to 8.5 with tris.
(5) And (3) soaking the gold sheet in the solution, and standing for 24 hours at 37 ℃ to obtain the gold sheet Au-PDA-PNIP with the modified polydopamine layer deposited on the surface.
(6) The substrate was sterilized with 75% alcohol, and then hela cells were seeded into the wells of a 48-well plate at a density of 5 ten thousand per well, and cultured for 12 hours to spread the cells sufficiently in the plate.
(7) The cells were washed with sterile phosphate buffer and serum-free cell culture medium with pGFP (pDNA encoding green fluorescent protein, molecular weight 3075kDa) of 1.5. mu.g per well was added.
(8) Using a laser source with a wavelength of 808nm at a wavelength of 5.1W/cm2The power density of (2) was applied to the cells in the wells for 30 s.
(9) After 4h of laser irradiation, the cell culture medium with serum was replaced to continue cell culture.
The subsequent sample treatment was carried out in two parts, respectively (10-1) fluorescence microscopy for characterization of transfection and (10-2) cell release experiments.
(10-1) after the laser irradiation is finished for 48 hours, staining the cell nucleus by using 4', 6-diamidino-2-phenylindole, and observing the expression condition of the green fluorescent protein by using a fluorescent microscope. Blue cells are stained nuclei, green cells are cells successfully expressing green fluorescent protein, and represent cells successfully transfected. The transfer efficiency was obtained by quantitative processing using a fluorescence microscope photograph. In addition, the cell activity was measured by CCK-8 48 hours after the completion of laser irradiation. Transfection efficiency was 99.3% and cell activity was 96.6%, see fig. 4.
(10-2) after the laser irradiation is finished for 12h, placing the base material with the cells in a refrigerator at 4 ℃ for 30min, taking out, blowing, and transferring the blown cells to a new hole for culture. Before and after the cell release experiment, the cells on the sample are stained by using a living cell dye, the number of the cells before and after desorption is observed, the cell desorption efficiency is calculated, and the cell desorption efficiency is 90.6%, which is shown in figure 4.
Comparative example 1
Compared with example 1, steps (1) to (3) were omitted, gold flakes were used as the base material directly, and were sterilized with 75% ethanol, and then Hela cells were seeded into the wells of 48-well plates at a density of 5 ten thousand per well, and cultured for 12 hours to spread the cells sufficiently in the plates.
Steps (5) to (7) were the same as in example 2.
(8) And after the laser irradiation is finished for 12h, placing the substrate with the cells in a refrigerator at 4 ℃ for 30min, taking out, blowing, and transferring the blown cells to a new hole for culture. Before and after the cell release experiment, the cells on the sample are dyed by using a living cell dye, the number of the cells before and after desorption is observed, the cell desorption efficiency is calculated and is 4.5%, and the result is shown in figure 2.
Comparative example 2
Au-PDA, which was a substrate deposited with functional polydopamine obtained in the step (1) of example 1, was used as a substrate, and the gold plate was sterilized with 75% alcohol, and then hela cells were seeded into the wells of 48-well plates at a density of 5 ten thousand per well, and cultured for 12 hours to sufficiently spread the cells in the plates.
Steps (5) to (7) were the same as in example 2.
(8) And after the laser irradiation is finished for 12h, placing the substrate with the cells in a refrigerator at 4 ℃ for 30min, taking out, blowing, and transferring the blown cells to a new hole for culture. Before and after the cell release experiment, the cells on the sample are stained by using a living cell dye, the number of the cells before and after desorption is observed, the cell desorption efficiency is calculated, and the cell desorption efficiency is 24.4%, which is shown in figure 2.
Comparative example 3
pGFP (molecular weight of 3075kDa) is delivered to three primary cells (mouse embryonic fibroblast, human umbilical vein endothelial cells, mouse dendritic cells) which are difficult to transfect respectively by adding a normal cell culture medium after pGFP and Lipo2000 transfection reagents are compounded for 10min by using a commercial transfection reagent Lipo2000 according to the standard instruction of a kit.
48h after transfection, the cell nucleus was stained with 4', 6-diamidino-2-phenylindole, and the expression of green fluorescent protein was observed with a fluorescent microscope. Carrying out quantitative treatment by a fluorescence microscope photo to obtain a histogram of transfection efficiency and cell activity, wherein the transfection efficiency of mouse embryo fibroblasts is 18.9%, and the cell activity is 10.2%; the transfection efficiency of human umbilical vein endothelial cells is 12.0 percent, and the cell activity is 47.0 percent; the transfection efficiency of the mouse dendritic cells is 0.3%, and the cell activity is 66.4%. See fig. 3.
As can be seen from FIG. 1, the method of the present invention can deliver exogenous molecules of different sizes into HeLa cells, with delivery efficiencies of 95% or more, and with substantially no effect on cell activity, demonstrating that the method of the present invention has wide versatility in terms of the types of molecules that can be delivered.
As can be seen from FIG. 2, the cell density on gold plate and Au-PDA before and after cell release did not change much, while the cell density on Au-PDA-PNIP was greatly reduced after release under the same experimental conditions. The method proves that the cells after the exogenous macromolecules are delivered can be efficiently harvested from the substrate.
FIG. 3 compares the transfection efficiency of the method of the present invention with that of the commercialized transfection reagent, and it is found that the transfection efficiency of the method of the present invention can reach more than 90% for the cells difficult to transfect and maintain good activity of the cells, while the transfection efficiency of Lipo2000 for the primary cells is less than 20% under the same conditions. The method provided by the invention is proved to realize high-efficiency transfection on cells difficult to transfect and has strong universality on cell types.
As can be seen from FIG. 4, the transfection efficiency of the Au-PDA-PNIP sample prepared by deposition of the copolymer of dopamine and N-isopropylacrylamide to Hela cells, the cell activity and the cell detachment efficiency after 48h transfection all reach over 90%. The method of the invention is proved to be capable of efficiently transfecting hela cells and efficiently harvesting the cells after transferring exogenous macromolecules from the base material.
In addition to the above embodiments, the present invention also includes other embodiments, and any technical solutions formed by equivalent transformation or equivalent replacement should fall within the scope of the claims of the present invention.
Claims (10)
1. A method for transferring exogenous molecules into cells is characterized in that the method comprises the step of contacting a substrate with a modified polydopamine layer deposited on the surface with the cells, wherein the modified polydopamine layer is obtained by reacting double-bond-containing functional polydopamine or functional dopamine monomers with temperature-sensitive polymer monomers.
2. The method of claim 1, wherein said method comprises seeding said cells on a surface of a substrate having a modified polydopamine layer deposited thereon, contacting said cells with an agent comprising an exogenous molecule, and illuminating said cells with a laser source in the near infrared range.
3. The method according to claim 2, further comprising placing the substrate with the modified polydopamine layer deposited on the surface after laser irradiation in an environment at a temperature lower than the temperature of the temperature-sensitive polymer low critical solution.
4. A method according to any one of claims 1 to 3, further comprising the step of preparing an aqueous solution containing dopamine and/or dopamine hydrochloride and an ethylenically unsaturated anhydride, adjusting the pH to a weak base, preferably a pH of 8 to 9, immersing the substrate in the solution to obtain a substrate having a functional polydopamine deposited on the surface; alternatively, the method further comprises the step of dissolving the dopamine and/or dopamine hydrochloride and the ethylenically unsaturated acid anhydride in water and stirring to obtain the functional dopamine monomer with the double bond.
5. A method according to claim 4, wherein said ethylenically unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride and citraconic anhydride, and further preferably said ethylenically unsaturated anhydride is selected from itaconic anhydride.
6. The method for delivering exogenous molecules into cells according to any one of claims 1 to 5, wherein the temperature-sensitive polymer monomer at least comprises N-isopropylacrylamide, and further preferably, the temperature-sensitive polymer monomer is N-isopropylacrylamide.
7. The method for delivering exogenous molecules into cells according to any one of claims 1 to 6, wherein the substrate is a metal material (such as gold, stainless steel, titanium alloy, magnesium alloy, etc.), an inorganic non-metal material (such as monocrystalline silicon, mica, glass, etc.), an organic polymer material (such as polyurethane, polydimethylsiloxane, polymer electrospun fiber membrane, etc.), an experimental or instrument device (cell culture plate, microplate, micro-flow channel device), etc., and more preferably, the substrate is a gold plate.
8. The method of any one of claims 1-7, wherein the exogenous molecule comprises one or more of a polysaccharide molecule (e.g., dextran), a protein (e.g., a gene-editing enzyme, an antibody, an antigen), a DNA (e.g., pDNA), an RNA (e.g., miRNA, siRNA), a therapeutic drug, an intracellular probe (e.g., a quantum dot), a nanomaterial (e.g., a nanoparticle, a nanodevice), an aptamer, a bacterium, an artificial chromosome, an organelle (e.g., a mitochondrion), and the like.
9. A method according to any one of claims 1 to 8, wherein said cell is selected from the group consisting of a cell line and a primary cell, further preferably said cell line comprises HeLa cells, and said primary cell comprises one of mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells.
10. The application of the base material with the modified polydopamine layer deposited on the surface in the process of transferring exogenous molecules into cells is characterized in that the modified polydopamine layer is obtained by reacting functional polydopamine containing double bonds or functional dopamine monomers with temperature-sensitive polymer monomers.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
| CN104208749A (en) * | 2014-08-27 | 2014-12-17 | 中国科学院长春应用化学研究所 | Modified microcarrier and preparation method thereof as well as functional microcarrier |
| CN105420278A (en) * | 2015-12-09 | 2016-03-23 | 苏州大学 | Method for preparing cells carrying exogenous molecules in photoinduced perforating mode, base material for preparing cells and cells |
| CN107142281A (en) * | 2017-06-07 | 2017-09-08 | 东华大学 | The compound of polyamide-amine dendrimer and nanogold particle carries out the application process of gene transfection as non-virus carrier |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
| CN104208749A (en) * | 2014-08-27 | 2014-12-17 | 中国科学院长春应用化学研究所 | Modified microcarrier and preparation method thereof as well as functional microcarrier |
| CN105420278A (en) * | 2015-12-09 | 2016-03-23 | 苏州大学 | Method for preparing cells carrying exogenous molecules in photoinduced perforating mode, base material for preparing cells and cells |
| CN107142281A (en) * | 2017-06-07 | 2017-09-08 | 东华大学 | The compound of polyamide-amine dendrimer and nanogold particle carries out the application process of gene transfection as non-virus carrier |
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