A method for delivering exogenous molecules into cells

Abstract

This invention relates to a method for delivering exogenous molecules into cells. The method includes using a substrate with a modified polydopamine layer deposited on its surface. The modified polydopamine layer is a thermosensitive polymer-grafted polydopamine layer obtained by reacting a functional polydopamine containing double bonds with a thermosensitive polymer monomer. This method is highly versatile, has high delivery and transfection efficiency, low cytotoxicity, high modified cell yield, and high throughput for single-use cell processing.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and medical devices, and more specifically to a method for delivering exogenous molecules into cells. Background Technology

[0002] The delivery of exogenous macromolecules, such as nucleic acids, proteins, and intracellular probes, into cells plays a crucial role in basic biological research, industrial production, and clinical diagnosis and treatment. Currently, methods for delivering exogenous macromolecules into cells can be mainly divided into two types: carrier methods and cell permeation methods.

[0003] The carrier method refers to using substances as carriers to encapsulate exogenous molecules, protecting them from degradation after entering the cell and promoting their interaction with the cell membrane, ultimately escorting the exogenous molecules into the cell. These carriers can be biologically active recombinant viruses, vesicles, shadow cells, functional ligands, and peptides, or they can be synthetic polymers and nanoparticles. Viral vectors deliver exogenous molecules into cells through viral infection, while most other carriers enter cells via endocytosis or membrane fusion. The most representative carriers include adenoviruses, adenovirus-associated viruses, lentiviruses, polyethyleneimine, gold nanoparticles, and commercially available transfection reagents such as Lipofectamine 2000 and Lipofectamine 3000.

[0004] Cell membrane permeability methods refer to using chemical permeabilizing agents (such as detergents) or physical forces (force, sound, light, electricity, heat, etc.) to increase the permeability of the cell membrane surface or create pores, facilitating the entry of exogenous molecules into the cell. The most representative and mature physical permeabilization methods are electroporation and microinjection. Electroporation uses high-voltage electrical pulses to interfere with the cell membrane, creating micropores that facilitate nucleic acid entry, while microinjection directly introduces exogenous substances into the cell using a microinjection device.

[0005] Currently, viral vectors are considered the most effective intracellular molecular delivery method due to their high efficiency and specificity. However, the strong infectivity of viruses also brings significant safety risks. Furthermore, viral vectors are limited to the delivery of nucleic acid substances and cannot be used to deliver other exogenous molecules into cells, because only genetic material can be integrated into the viral genome for subsequent host infection experiments. This problem also exists in other vector methods. Since exogenous molecules must first bind to the vector through certain interactions (such as electrostatic interactions), this limits the properties of the exogenous molecules; a certain vector can only be used to deliver molecules with specific properties, greatly reducing the versatility of vector methods. Although there are studies using vector methods to deliver proteins or other drug molecules, most vectors are still limited to the delivery of nucleic acid substances; a vector with relatively strong versatility that can deliver multiple different molecules does not exist. In addition, vector methods are not only limited in the types of molecules that can be delivered, but also in the types of cells that can be processed. Especially for difficult-to-transfect cells, the transfection efficiency is very low. This is mainly because difficult-to-transfect cells have stronger self-protection mechanisms, making it difficult for vectors to enter the cells. Although some vector methods can improve the efficiency of vector entry into cells by modifying certain cell-specific peptides, this only improves the transfection efficiency of specific cell lines and is still limited in terms of the types of cells that can be transfected. Therefore, the vector method has the following main disadvantages: (1) Viral vectors have high transfection efficiency, but safety issues cannot be ignored, and they can only be used for the transfer of nucleic acid substances; (2) Non-viral vectors have low versatility in terms of the types of molecules and cell types that can be transferred, and their transfection efficiency is very low for cells that are difficult to transfect.

[0006] On the other hand, physical cell membrane disruption methods often significantly damage cell viability by disrupting the integrity of the cell membrane. For example, in electroporation, the high-voltage pulses used to allow nucleic acids to effectively enter the cell often lead to the death of a large number of cells, greatly reducing the number of usable cells. While microinjection can effectively deliver nucleic acids into the cell nucleus, it is only suitable for single-cell manipulation and not for high-throughput cell processing. Other methods that utilize physical forces to disrupt cell membranes mostly require complex instruments or devices, making the experiments more challenging.

[0007] CN105420278A discloses a method for preparing cells loaded with exogenous molecules using photoperforation, as well as the substrate and the cells used for preparation. It develops a photoperforation kit based on a gold nanoparticle-deposited membrane, utilizing the increased cell membrane permeability under laser irradiation to deliver various exogenous macromolecules into the cells. However, the transfection efficiency of this method needs improvement for difficult-to-transfect cell lines, such as primary mouse embryonic fibroblasts, where the transfection efficiency without a carrier is approximately 53%. Furthermore, the gold nanoparticle-deposited membrane has a large surface roughness and complex surface topology, resulting in strong cell adhesion and making it difficult to harvest the modified cells, limiting subsequent applications. Using trypsin digestion to harvest cells would severely damage cell viability, especially in fragile primary cells.

[0008] In summary, the main drawbacks of physical cell disruption methods include significant loss of cell viability, low cell processing throughput, complex equipment requirements, and low cell harvesting efficiency.

[0009] Therefore, a macromolecular delivery system that is versatile in terms of exogenous molecules and cell types, has high delivery efficiency, low cytotoxicity, can process cells in large quantities, and has a high cell harvest rate still needs to be studied. Summary of the Invention

[0010] The problem the invention aims to solve

[0011] To address the problems existing in the prior art, this invention designs a method for delivering exogenous molecules into cells that is highly versatile, has high delivery and transfection efficiency, low cytotoxicity, high modified cell harvest rate, and high throughput for a single cell processing step.

[0012] Solution for solving the problem

[0013] In one technical solution, the present invention provides a method for delivering exogenous molecules into cells, the method comprising contacting a substrate with a modified polydopamine layer deposited on its surface with the cells, the modified polydopamine layer being obtained by reacting a functional polydopamine containing double bonds or a functional dopamine monomer with a temperature-sensitive polymer monomer.

[0014] In one embodiment, the method includes seeding the cells on the surface of a substrate with a modified polydopamine layer deposited thereon, contacting the cells with a reagent containing exogenous molecules, and irradiating the cells with a near-infrared laser light source. Furthermore, the method further includes placing the laser-irradiated substrate with the modified polydopamine layer deposited thereon in an environment below the lower critical solution temperature of the temperature-sensitive polymer, thereby promoting cell detachment from the surface.

[0015] In another embodiment, the method further includes the step of preparing an aqueous solution containing dopamine and / or dopamine hydrochloride and olefinic unsaturated anhydride, adjusting the pH to a weakly alkaline state, preferably pH 8-9, and immersing the substrate in the solution to obtain a substrate with functional polydopamine deposited on its surface.

[0016] In another embodiment, the method further includes the step of dissolving dopamine and / or dopamine hydrochloride and olefinic unsaturated anhydride in water and stirring to obtain a functional dopamine monomer containing a double bond.

[0017] In another embodiment, the olefinic unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, and citraconic anhydride, preferably, the olefinic unsaturated anhydride is selected from itaconic anhydride.

[0018] In another embodiment, the thermosensitive polymer monomer of the present invention includes at least N-isopropylacrylamide, and more preferably, the thermosensitive polymer monomer is N-isopropylacrylamide.

[0019] In another embodiment, the substrate of the present invention may be a metallic material (such as gold, stainless steel, titanium alloy, magnesium alloy, etc.), an inorganic non-metallic material (such as monocrystalline silicon, mica, glass, etc.), an organic polymer material (such as polyurethane, polydimethylsiloxane, polymer electrospun fiber membrane, etc.), an experimental or instrumental device (cell culture plate, enzyme-labeled plate, microfluidic device), etc. Preferably, the substrate is a gold sheet.

[0020] In another embodiment, the exogenous molecules described in this invention include one or more of the following: polysaccharide molecules (such as dextran), proteins (such as gene editing enzymes, antibodies, antigens), DNA (such as pDNA), RNA (such as miRNA, siRNA), therapeutic drugs, intracellular probes (such as quantum dots), nanomaterials (such as nanoparticles, nanodevices), aptamers, bacteria, artificial chromosomes, organelles (such as mitochondria).

[0021] In another embodiment, the cells of the present invention are selected from cell lines or primary cells. Preferably, the cell line includes HeLa cells, and the primary cell line includes one of mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells.

[0022] In another technical solution, the present invention also provides the use of a substrate with a modified polydopamine layer deposited on its surface in the process of delivering exogenous molecules into cells, wherein the modified polydopamine layer is obtained by reacting a functional polydopamine containing double bonds or a functional dopamine monomer with a thermosensitive polymer monomer.

[0023] In another technical solution, the present invention also provides a cell loaded with exogenous molecules prepared by the method described above.

[0024] The effects of the invention

[0025] This invention provides a method for delivering exogenous molecules into cells. By preparing specific photothermal transfer and cell release substrates, the method can significantly improve the efficiency of exogenous molecule delivery, cell harvest rate, and cell processing throughput. Compared with existing technologies for delivering exogenous molecules into cells, the advantages of this invention are as follows:

[0026] On the one hand, the method described in this invention is highly versatile. Theoretically, using the method described in this invention, any substance that needs to be delivered into cells can be delivered, including but not limited to one or more of the following: polysaccharide molecules (such as dextran), proteins (such as gene editing enzymes, antibodies, antigens), DNA (such as pDNA), RNA (such as miRNA, siRNA), therapeutic drugs, intracellular probes (such as quantum dots), nanomaterials (such as nanoparticles, nanodevices), aptamers, bacteria, artificial chromosomes, organelles (such as mitochondria), etc.

[0027] On the other hand, the method described in this invention has high transfection efficiency for difficult-to-transfect primary cells. Most intracellular molecular delivery methods only use conventional, easily transfected cell lines such as HeLa cells as model cells to verify their delivery efficiency. However, for difficult-to-transfect primary cells, the molecular delivery efficiency is mostly low, resulting in limited versatility in cell lines. In contrast, the method described in this invention is applicable to a variety of cell lines, including conventional cell lines such as HeLa cells and difficult-to-transfect primary cells, including mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells, with transfection efficiencies exceeding 90%.

[0028] On the other hand, the method described in this invention has low cytotoxicity. Most macromolecule delivery methods based on membrane disruption mechanisms suffer significant damage to cell viability after treatment because they achieve their purpose by disrupting the integrity of the cell membrane. However, in this invention, by controlling the power and duration of the irradiated laser, high delivery efficiency can be achieved while maintaining high cell viability.

[0029] On the other hand, the method described in this invention has a high cell harvest rate. This invention fully utilizes the changes in the hydrophilicity and hydrophobicity of thermosensitive polymers at different temperatures, and by changing the cell culture temperature, cells are automatically detached from the substrate to reduce damage to cell viability during cell harvesting.

[0030] On the other hand, the method described in this invention enables high-throughput cell processing. Using the method described in this invention, at least tens of thousands of cells can be processed with a single laser irradiation on a surface-modified substrate, achieving efficient and large-scale cell processing in a short time. Attached Figure Description

[0031] Figure 1 Bar graphs showing the delivery efficiency of three different-sized exogenous molecules (dextran, bovine serum albumin, and plasmid DNA encoding green fluorescent protein (pGFP)) into HeLa cells and the cell viability 48 h after delivery.

[0032] Figure 2 Photographs and bar graphs showing cell distribution and density before and after HeLa cell release using three different substrates for dextran delivery. Scale bar is 200 μm. The abbreviations in the figures are: Au-PDA, gold flake with deposited functionalized polydopamine; Au-PDA-PNIP, gold flake with a poly(N-isopropylacrylamide) grafted polydopamine layer.

[0033] Figure 3 This image shows bar graphs illustrating the transfection efficiency of plasmid DNA encoding green fluorescent protein (pGFP) into three difficult-to-transfect primary cell types (mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells) and cell viability 48 hours after transfection. The abbreviations in the graph are as follows: mEF, mouse embryonic fibroblasts; HUVEC, human umbilical vein endothelial cells; mDC, mouse dendritic cells.

[0034] Figure 4 This study describes the preparation of gold flakes with a poly(N-isopropylacrylamide) grafted and modified polydopamine layer using the material preparation method described in Example 7. The transfection efficiency, cell viability after 48 hours of transfection, and desorption efficiency of these flakes in HeLa cells for transfecting plasmid DNA encoding green fluorescent protein (pGFP) were also investigated. The abbreviations in the figure are: Au-PDA-PNIP, gold flakes with a poly(N-isopropylacrylamide) grafted and modified polydopamine layer. Detailed Implementation

[0035] Various exemplary embodiments, features, and aspects of the present invention will now be described in detail with reference to the accompanying drawings. Numerous specific details are set forth in the following detailed description to better illustrate the invention. Those skilled in the art will understand that the invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention.

[0036] definition

[0037] Unless otherwise defined, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] The term "reagent" includes any substance to be delivered into cells. Such reagents include, but are not limited to, serum-free cell culture media containing exogenous molecules.

[0039] The term "protein" is used herein to refer to a polymer of amino acid residues. This term applies to: amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term also includes variations on conventional peptide bonds, which link multiple amino acids that make up a polypeptide.

[0040] The term "monomer" means any chemical substance that can be characterized by a chemical formula and has polymerizable groups that can be polymerized into oligomers or polymers to increase molecular weight.

[0041] The term "transfer" refers to the process from outside the cell to inside the cell, while "transfection" refers to the process from outside the cell to the cell nucleus, and then the translation and expression into proteins. Generally, it specifically refers to the process of transferring nucleic acid substances into the cell and successfully expressing them.

[0042] <Preparation of Photothermal Transfer and Cell Release Substrates>

[0043] This invention first prepares a photothermal transfer and cell release substrate, the surface of which is deposited with a modified polydopamine layer. The substrate with the modified polydopamine layer deposited on its surface, as described in this invention, can be used to deliver exogenous molecules into cells.

[0044] Dopamine (DA, chemical formula: C6H3(OH)2-CH2-CH2-NH2, CAS No.: 62-31-7), a catechol derivative, is a neurotransmitter used to help cells transmit pulse signals. Due to its universal and strong adhesion to substrates, dopamine has been widely used in biomedicine and biomaterials in recent years. Dopamine is easily oxidized by dissolved oxygen in aqueous solutions, subsequently initiating a self-polymerization and cross-linking reaction, forming a tightly adhered polydopamine composite layer on almost any solid material surface. Polydopamine is a melanin-like substance with not only good adhesion but also outstanding light absorption properties. It can absorb light in the visible to near-infrared wavelength range, generating a photothermal effect in the matrix, causing rapid and effective local heating of the material. However, there is currently no research on using the photothermal effect of polydopamine to prepare photothermal substrates for delivering exogenous molecular signals into cells.

[0045] This invention reveals that the substrate prepared using modified polydopamine has excellent photothermal transfer properties and cell release capabilities.

[0046] In one technical solution, the present invention first deposits functional polydopamine on a substrate. The specific method includes preparing an aqueous solution containing dopamine and / or dopamine hydrochloride and an olefinic unsaturated anhydride, adjusting the pH to weakly alkaline, immersing the substrate in the solution, and allowing it to stand to obtain a substrate with functional polydopamine deposited on its surface. The substrate, i.e., the base material, of the present invention can be a metallic material (such as gold, stainless steel, titanium alloy, magnesium alloy, etc.), an inorganic non-metallic material (such as monocrystalline silicon, mica, glass, etc.), an organic polymer material (such as polyurethane, polydimethylsiloxane, polymer electrospun fiber membrane, etc.), or an experimental or instrumental device (cell culture plate, ELISA plate, microfluidic device), etc. More preferably, the substrate is a gold sheet.

[0047] In another technical solution, after preparing the aqueous solution, the mixture is stirred at room temperature for 1-3 hours to allow for a full reaction; further, the pH value of the system is adjusted with an alkaline solution (such as tris(hydroxymethyl)aminomethane), preferably to 8-9, more preferably to 8.5; further, the substrate is immersed in the solution and allowed to stand at 30-40°C for 20-30 hours.

[0048] In another technical solution, the olefinic unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, and citraconic anhydride. Preferably, the olefinic unsaturated anhydride is selected from itaconic anhydride. The double bonds in itaconic anhydride are terminal double bonds, resulting in higher reactivity in subsequent polymerization reactions. When an aqueous solution of itaconic anhydride and dopamine is stirred, the itaconic anhydride reacts with the amino group on the dopamine. The five-membered ring of the itaconic anhydride is opened due to nucleophilic attack, resulting in acylated dopamine. The substrate is then immersed in the solution, and under weakly alkaline conditions, the acylated dopamine polymerizes to obtain a substrate with allyl polydopamine deposition. The exposed double bonds on the surface of the polydopamine facilitate subsequent grafting reactions. The specific reaction process of itaconic anhydride and dopamine is as follows:

[0049]

[0050] In another technical solution, the concentration range of dopamine or dopamine hydrochloride and olefinic unsaturated anhydride is 1-5 mg / mL. Preferably, the concentrations of dopamine or dopamine hydrochloride and olefinic unsaturated anhydride are the same to reduce the introduction of impurities, and more preferably, they are all 2 mg / mL or 3 mg / mL. If the concentration is too high, the deposited polydopamine layer will be thicker, and the accumulation of polydopamine particles will be more severe, which may lead to unevenness on the material surface. If the concentration is too low, the deposited polydopamine layer may be too thin, and the photothermal effect will be weak, so the same transfection effect cannot be obtained under the current laser intensity and irradiation time.

[0051] In one specific embodiment of the present invention, an aqueous solution of dopamine and itaconic anhydride, both with a concentration of 2 mg / mL, is prepared and stirred at room temperature for 2 h. Then, the pH value is adjusted to 8.5 with an alkaline solution. The substrate is immersed in the above solution and left to stand at 37°C for 24 h to obtain a substrate with a functional polydopamine layer containing double bonds deposited.

[0052] Subsequently, the double bonds on the functional polydopamine are used to undergo a free radical polymerization reaction with the thermosensitive polymer monomer to generate a thermosensitive polymer-grafted modified polydopamine layer. The most important characteristic of the thermosensitive polymer is the existence of a critical solution temperature, near which the polymer solution undergoes a discontinuous phase change. Preferably, the present invention uses a thermosensitive polymer with a low critical solution temperature. This polymer solution (mostly an aqueous solution) is in a single-phase state below this temperature, while phase separation occurs above it. The thermosensitive polymer contains a certain proportion of hydrophilic and hydrophobic groups, and their interaction with water molecules changes with temperature. Utilizing the hydrophilic-hydrophobic transformation of the thermosensitive polymer around its low critical solution temperature can promote the non-destructive release of modified cells from the substrate.

[0053] Since the poly(N-isopropylacrylamide) macromolecular chain has both hydrophilic amide groups and hydrophobic isopropyl groups, the low critical solution temperature of the poly(N-isopropylacrylamide) molecular chain in aqueous medium is around 32°C, and it is highly sensitive to temperature. Therefore, the temperature-sensitive polymer monomer in this invention includes at least N-isopropylacrylamide, and more preferably, the temperature-sensitive polymer monomer is N-isopropylacrylamide.

[0054] The free radical polymerization of functional polydopamine with temperature-sensitive polymer monomers can be carried out using traditional redox free radical polymerization methods. Commonly used initiators include persulfates such as ammonium persulfate. N,N,N',N'-tetramethylethylenediamine is used as an initiator to catalyze the generation of free radicals from ammonium persulfate, or a combination of ammonium persulfate and sodium bisulfite can be used as an initiator. The amount of initiator used can be determined by those skilled in the art based on the needs of the reaction.

[0055] The specific reaction process of N-isopropylacrylamide with allyl polydopamine is as follows:

[0056]

[0057] In one specific embodiment of the present invention, an aqueous solution of N-isopropylacrylamide (the concentration of N-isopropylacrylamide is 0.02-0.1 g / mL, preferably 0.05 g / mL, at which concentration the poly-N-isopropylacrylamide thickness has the best temperature response) is prepared, nitrogen is purged to remove oxygen, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine are added, and the substrate with the functional polydopamine containing double bonds deposited is immersed in the solution and left to stand at room temperature for 20-30 h to obtain a substrate with a poly-N-isopropylacrylamide graft-modified polydopamine layer deposited.

[0058] In another technical solution, the present invention can first prepare a functional dopamine monomer containing double bonds, and then use the reactive double bonds on it to carry out a free radical polymerization reaction with a temperature-sensitive polymer monomer to generate a copolymer of dopamine and temperature-sensitive polymer monomer. The copolymer is then prepared into an aqueous solution, adjusted to a weakly alkaline state, and the substrate is immersed in the solution. After standing, a substrate with a modified polydopamine layer deposited on its surface is obtained.

[0059] In another technical solution, the functional dopamine monomer containing a double bond is obtained by dissolving dopamine and / or dopamine hydrochloride and an olefinic unsaturated anhydride in water and stirring. Preferably, the olefinic unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, and citraconic anhydride; more preferably, the olefinic unsaturated anhydride is selected from itaconic anhydride.

[0060] In another technical solution, the concentration range of dopamine or dopamine hydrochloride and olefinic unsaturated anhydride is 1-5 mg / mL. Preferably, the concentrations of dopamine or dopamine hydrochloride and olefinic unsaturated anhydride are the same to reduce the introduction of impurities. The concentration ratio of the functional dopamine monomer with double bonds to the thermosensitive polymer monomer is 3-6:1 to ensure sufficient copolymerization reaction.

[0061] In one specific embodiment of the present invention, dopamine hydrochloride and itaconic anhydride are first dissolved in water and stirred at room temperature for 1-3 hours to obtain a functional dopamine monomer containing double bonds. Then, an aqueous solution of N-isopropylacrylamide and the dopamine monomer containing double bonds is prepared at a concentration ratio of 1:5. After nitrogen purging to remove oxygen, azobisisobutyronitrile is added to the reaction solution as an initiator, and the reaction is carried out for 20-30 hours to obtain a copolymer of dopamine and N-isopropylacrylamide. An aqueous solution of this copolymer is prepared and its pH is adjusted to 8.5. The substrate is then immersed in the above solution and allowed to stand at 30-40°C for 20-30 hours to obtain a substrate with a modified polydopamine layer deposited on its surface.

[0062] Methods for delivering exogenous molecules into cells

[0063] This invention provides a method for delivering exogenous molecules into cells. The method includes using a substrate with a modified polydopamine layer deposited on its surface (i.e., a photothermal transfer and cell release substrate); seeding cells on the surface of the substrate with the modified polydopamine layer deposited on its surface; contacting the cells with a reagent containing exogenous molecules; irradiating the cells with a near-infrared laser source; and achieving cell delivery of exogenous molecules through photoperforation. Because polydopamine has a strong photothermal conversion capability, the method described in this invention can reduce laser intensity, allowing the use of a low-intensity continuous laser source, significantly reducing experimental costs.

[0064] In one technical solution, the cells are selected from cell lines or primary cells. Preferably, the cell line includes HeLa cells, and the primary cell line includes one of mouse embryonic fibroblasts (mEF), human umbilical vein endothelial cells (HUVEC), and mouse dendritic cells (mDC). Most intracellular molecular delivery methods only use conventional, easily transfected cell lines such as HeLa cells for molecular delivery experiments. However, for difficult-to-transfect primary cells, the molecular delivery efficiency is generally low, resulting in limited versatility of the molecular delivery system in terms of cell lines. The method described in this invention is applicable to a variety of cell lines, including difficult-to-transfect primary cells.

[0065] The method described in this invention can be used to deliver various substances of different sizes into cells, and there are no special requirements on the properties of the substances. For example, the exogenous molecules include one or more of the following: polysaccharide molecules (such as dextran), proteins (such as gene editing enzymes, antibodies, antigens), DNA (such as pDNA), RNA (such as miRNA, siRNA), therapeutic drugs, intracellular probes (such as quantum dots), nanomaterials (such as nanoparticles, nanodevices), aptamers, bacteria, artificial chromosomes, organelles (such as mitochondria), etc.

[0066] In one specific embodiment of the present invention, the photothermal transfer and cell release substrate is first sterilized, and the target cells are seeded on the substrate and cultured for a period of time to allow the cells to fully spread. Then, the cells are washed with a buffer solution, and a reagent containing exogenous molecules, such as serum-free cell culture medium containing exogenous molecules, is added. A near-infrared laser light source (780–3000 nm) at 1–10 W / cm² is then used. 2Irradiate cells on the substrate for 0.5-10 minutes within the power density range. 3-5 hours after laser irradiation, replace the cell culture medium containing exogenous molecules with normal serum-containing cell culture medium to restore normal cell culture. After laser irradiation, culture the cells at 37°C. Further, cells can be washed with sterile phosphate-buffered saline solution; the concentration of exogenous molecules in the reagent can be adjusted by those skilled in the art as needed.

[0067] In another technical solution, the method of the present invention further includes placing the substrate with the modified polydopamine layer deposited on its surface after laser irradiation in an environment lower than the low critical solution temperature of the temperature-sensitive polymer, thereby promoting cell detachment from the surface.

[0068] When a substrate with cells is placed in an environment below the lower critical solution temperature of the thermosensitive polymer, the surface of the substrate grafted with the thermosensitive polymer changes from hydrophobic to hydrophilic, and the molecular chains of the thermosensitive polymer swell, causing the cells on the surface to detach in the form of single cells or cell sheets.

[0069] In one specific embodiment of the present invention, the temperature-sensitive polymer is poly(N-isopropylacrylamide). In order to promote cell harvesting and save experimental costs, the substrate containing cells is placed in a 4°C refrigerator for 20-40 minutes, then removed and blown around. The cells blown off are then transferred to new wells to continue culturing.

[0070] <Cells carrying exogenous molecules>

[0071] The present invention further provides a cell loaded with exogenous molecules prepared using the method described herein. The cell comprises a cell body and exogenous molecules entering the cell body. The cell can be formed by the exogenous molecules directly penetrating the cell membrane and entering the cell body, or it can be a cell generated through the division and proliferation of a cell containing exogenous molecules. The method described herein is particularly suitable for obtaining difficult-to-transfect cells loaded with exogenous molecules.

[0072] Example

[0073] The technical solutions of the present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain and illustrate the present invention, and are not intended to limit the scope of protection of the present invention.

[0074] Example 1

[0075] (1) Dissolve 0.08 g dopamine hydrochloride and 0.08 g itac anhydride in 40 mL of deionized water, stir at room temperature for 2 h, and then adjust the pH to 8.5 with an alkaline solution. Immerse the gold sheet in the above solution and let it stand at 37 °C for 24 h to obtain a substrate with functional polydopamine deposited on it, namely Au-PDA.

[0076] (2) Prepare an aqueous solution of N-isopropylacrylamide with a concentration of 0.05 g / mL, and purge with nitrogen for 30 min to remove oxygen from the solution.

[0077] (3) The substrate with the functional polydopamine layer deposited and the aqueous solution of N-isopropylacrylamide were transferred to a glove box. The substrate with the functional polydopamine layer deposited was then immersed in the aqueous solution of N-isopropylacrylamide monomer. Ammonium persulfate and N,N,N',N'-tetramethylethylenediamine were added to the solution to achieve final concentrations of 0.02 g / mL and 0.67 μL / mL, respectively. The reaction was carried out at room temperature for 24 h to obtain a gold sheet with a modified polydopamine layer deposited on its surface, namely Au-PDA-PNIP.

[0078] The elemental contents and thicknesses of the Au-PDA and Au-PDA-PNIP surfaces were determined, and the results are shown in Table 1. The surface elemental contents were obtained by X-ray photoelectron spectroscopy analysis, and the contents of each element were in good agreement with the theoretical values. The sample thickness was measured by an ellipsometry. With the thickness of the Au surface set to 0 nm, the thickness of the polydopamine deposition layer was 86.3 nm, and the total thickness of polydopamine and poly(N-isopropylacrylamide) was 115.9 nm.

[0079] sample 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

[0080] The fact that the content of each element matches the theoretical value and that there are variations in thickness proves that a modified polydopamine layer has been successfully deposited on the gold sheet.

[0081] (4) Disinfect the substrate with 75% alcohol, then seed HeLa cells into the wells of a 48-well plate at a density of 50,000 per well and culture for 12 hours to allow the cells to spread fully in the plate.

[0082] (5) Wash the cells with sterile phosphate buffer solution and add serum-free cell culture medium containing pGFP (pDNA encoding green fluorescent protein with a molecular weight of 3075kDa). The mass of pGFP in each well is 1.5μg.

[0083] (6) Using a laser source with a wavelength of 808nm at 5.1W / cm 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0084] (7) Four hours after laser irradiation, the cells were cultured in a cell culture medium containing serum.

[0085] (8) Forty-eight hours after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein was observed under a fluorescence microscope. Blue cells represent stained cell nuclei, and green cells represent cells that successfully expressed green fluorescent protein, indicating successfully transfected cells. Quantification was performed using fluorescence microscopy images. The number of blue cells and green cells were counted, and the transfection efficiency was obtained by dividing the latter by the former and multiplying by 100%. Cell viability was detected using a CCK-8 (cell counting kit). The transfection efficiency was 99.1%, and the cell viability was 95.5%. See [link to relevant documentation]. Figure 1 .

[0086] Example 2

[0087] Steps (1)-(4) are the same as in Example 1.

[0088] (5) Wash the cells with sterile phosphate buffer solution and add serum-free cell culture medium containing dextran (molecular weight 4.4kDa) to make the final concentration of dextran in each well 1mg / mL.

[0089] Steps (6) and (7) are the same as in Example 1.

[0090] The subsequent sample processing was carried out in three parts: (8-1) fluorescence microscopy characterization of transfection and (8-2) cell release experiment.

[0091] (8-1) Thirty minutes after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the entry of dextran into cells was observed using a fluorescence microscope. Blue cells represent stained cell nuclei, and red cells represent the color emitted by rhodamine-labeled dextran, indicating successfully delivered cells. The delivery efficiency was obtained by quantifying the fluorescence microscopy images. Additionally, cell viability was assessed using a CCK-8 assay 48 hours after laser irradiation. The delivery efficiency and cell viability are shown in the bar graph; the delivery efficiency was 98.8%, and the cell viability was 91.2%. (See [link to relevant documentation]). Figure 1 .

[0092] (8-2) Twelve hours after laser irradiation, the cell-seeded substrate was placed in a 4°C refrigerator for 30 minutes, then removed and pipetted to transfer the detached cells to new wells for culture. Before and after the cell release experiment, the cells on the sample were stained with a live cell dye to observe the cell count before and after detachment. Results are shown below. Figure 2 ,in Figure 2 (a) shows the results of fluorescence microscopy. Figure 2 (b) is a bar chart of cell density obtained by quantitative processing of fluorescence microscopy results before and after release.

[0093] Example 3

[0094] Steps (1)-(4) are the same as in Example 1.

[0095] (5) Wash the cells with sterile phosphate buffer solution and add serum-free cell culture medium containing bovine serum albumin (66 kDa) to make the final concentration of bovine serum albumin in each well 1 mg / mL.

[0096] Steps (6) and (7) are the same as in Example 1.

[0097] (8) Thirty minutes after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the entry of bovine serum albumin into cells was observed using a fluorescence microscope. Blue cells represent stained cell nuclei, and red cells represent the color emitted by rhodamine-labeled bovine serum albumin, indicating successfully delivered cells. The delivery efficiency was obtained by quantifying the fluorescence microscopy images. Additionally, cell viability was assessed using a CCK-8 assay 48 hours after laser irradiation. The delivery efficiency and cell viability are shown in the bar chart; the delivery efficiency was 98.0%, and the cell viability was 93.1%. (See [link to relevant documentation]). Figure 1 .

[0098] Example 4

[0099] Steps (1)-(3) are the same as in Example 1.

[0100] (4) Disinfect the substrate with 75% alcohol, and then seed mouse embryonic fibroblasts into the wells of a 48-well plate at a density of 50,000 per well. Culture for 12 hours to allow the cells to spread fully in the plate.

[0101] Step (5) is the same as in Example 1.

[0102] (6) Using a laser source with a wavelength of 808nm at 5.1W / cm 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0103] (7) Four hours after laser irradiation, the cells were cultured in a cell culture medium containing serum.

[0104] (8) Forty-eight hours after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein was observed under a fluorescence microscope. Blue cells represent stained cell nuclei, and green cells represent cells that successfully expressed green fluorescent protein, indicating successfully transfected cells. The transfection efficiency was obtained by quantifying the fluorescence microscope images. Additionally, cell viability was assessed using CCK-8 assays for 48 hours after laser irradiation. The transfection efficiency was 98.9%, and cell viability was 91.8%. (See [link to relevant documentation]). Figure 3 .

[0105] Example 5

[0106] Steps (1)-(3) are the same as in Example 1.

[0107] (4) Disinfect the substrate with 75% alcohol, and then seed human umbilical vein endothelial cells into the wells of a 48-well plate at a density of 50,000 per well. Culture for 12 hours to allow the cells to spread fully in the plate.

[0108] Step (5) is the same as in Example 1.

[0109] (6) Using a laser source with a wavelength of 808nm at 5.1W / cm 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0110] (7) Four hours after laser irradiation, the cells were cultured in a cell culture medium containing serum.

[0111] (8) Forty-eight hours after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein was observed under a fluorescence microscope. Blue cells represent stained cell nuclei, and green cells represent cells that successfully expressed green fluorescent protein, indicating successfully transfected cells. The transfection efficiency was obtained by quantifying the fluorescence microscope images. Additionally, cell viability was assessed using a CCK-8 assay 48 hours after laser irradiation. The transfection efficiency was 99.4%, and the cell viability was 96.2%. (See [link to relevant documentation]). Figure 3 .

[0112] Example 6

[0113] Steps (1)-(3) are the same as in Example 1.

[0114] (4) Disinfect the substrate with 75% alcohol, and then seed mouse dendritic cells into the wells of a 48-well plate at a density of 50,000 per well. Culture for 12 hours to allow the cells to spread fully in the plate.

[0115] Step (5) is the same as in Example 1.

[0116] (6) Using a laser source with a wavelength of 808nm at a speed of 2.3W / cm 2 The power density was used to irradiate the cells in the pore for 90 seconds.

[0117] (7) Four hours after laser irradiation, the cells were cultured in a cell culture medium containing serum.

[0118] (8) Forty-eight hours after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein was observed using a fluorescence microscope. Blue cells represent stained cell nuclei, and green cells represent cells that successfully expressed green fluorescent protein, indicating successfully transfected cells. The transfection efficiency was obtained by quantifying the fluorescence microscope images. Additionally, cell viability was assessed using a CCK-8 assay 48 hours after laser irradiation. The transfection efficiency was 99.2%, and cell viability was 60.4%. (See [link to relevant documentation]). Figure 3 .

[0119] Example 7

[0120] (1) Dissolve 0.08 g of dopamine hydrochloride and 0.08 g of itaconic anhydride in 40 mL of deionized water and stir at room temperature for 2 h to obtain dopamine monomer with double bonds.

[0121] (2) Prepare aqueous solutions of N-isopropylacrylamide and dopamine monomer with double bonds at concentrations of 0.03 mg / mL and 0.15 g / mL, respectively, and remove oxygen from the solution by purging with nitrogen for 30 min.

[0122] (3) Add azobisisobutyronitrile as an initiator (concentration of 0.0003 mg / mL) to the reaction solution and react for 24 h under nitrogen protection to obtain a copolymer of dopamine and N-isopropylacrylamide.

[0123] (4) Prepare an aqueous solution of the copolymer with a concentration of 2 mg / mL and adjust its pH to 8.5 with tris(hydroxymethyl)aminomethane.

[0124] (5) Immerse the gold sheet in the above solution and let it stand at 37°C for 24 hours to obtain the gold sheet Au-PDA-PNIP with a modified polydopamine layer deposited on the surface.

[0125] (6) Disinfect the substrate with 75% alcohol, then seed HeLa cells into the wells of a 48-well plate at a density of 50,000 per well and culture for 12 hours to allow the cells to spread fully in the plate.

[0126] (7) Wash the cells with sterile phosphate buffer solution and add serum-free cell culture medium containing pGFP (pDNA encoding green fluorescent protein with a molecular weight of 3075kDa). The mass of pGFP in each well is 1.5μg.

[0127] (8) Using a laser source with a wavelength of 808nm at 5.1W / cm 2 The power density was used to irradiate the cells in the pore for 30 seconds.

[0128] (9) Four hours after laser irradiation, the cells were cultured in a cell culture medium containing serum.

[0129] The subsequent sample processing was carried out in two parts: (10-1) fluorescence microscopy characterization of transfection and (10-2) cell release experiment.

[0130] (10-1) Forty-eight hours after laser irradiation, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein (GFP) was observed using a fluorescence microscope. Blue cells represent stained nuclei, and green cells represent cells that successfully expressed GFP, indicating successfully transfected cells. The transfection efficiency was obtained by quantifying the fluorescence micrographs. Additionally, cell viability was assessed using a CCK-8 assay 48 hours after laser irradiation. The transfection efficiency was 99.3%, and cell viability was 96.6%. (See [link to relevant documentation]). Figure 4 .

[0131] (10-2) Twelve hours after laser irradiation, the cell-seeded substrate was placed in a 4°C refrigerator for 30 minutes, then removed and pipetted to transfer the detached cells to new wells for culture. Before and after the cell release experiment, the cells on the sample were stained with a live cell dye to observe the cell count before and after detachment and calculate the cell detachment efficiency. The cell detachment efficiency was 90.6%. (See [reference needed]). Figure 4 .

[0132] Comparative Example 1

[0133] Compared with Example 1, steps (1)-(3) are omitted. Gold flakes are used directly as the substrate. The gold flakes are disinfected with 75% alcohol. Then, HeLa cells are seeded into the wells of a 48-well plate at a density of 50,000 per well and cultured for 12 hours to allow the cells to spread fully in the plate.

[0134] Steps (5)-(7) are the same as in Example 2.

[0135] (8) Twelve hours after laser irradiation, the cell-inoculated substrate was placed in a 4°C refrigerator for 30 minutes, then removed and pipetted to transfer the dislodged cells to new wells for culture. Before and after the cell release experiment, the cells on the sample were stained with a live cell dye to observe the cell number before and after detachment and calculate the cell detachment efficiency. The cell detachment efficiency was 4.5%. The results are shown below. Figure 2 .

[0136] Comparative Example 2

[0137] The substrate with functional polydopamine deposited, namely Au-PDA, obtained in step (1) of Example 1 was used as the substrate. The gold sheet was sterilized with 75% alcohol. Then, HeLa cells were seeded into the wells of a 48-well plate at a density of 50,000 per well and cultured for 12 hours to allow the cells to spread fully in the plate.

[0138] Steps (5)-(7) are the same as in Example 2.

[0139] (8) Twelve hours after laser irradiation, the cell-inoculated substrate was placed in a 4°C refrigerator for 30 minutes, then removed and pipetted to transfer the dislodged cells to new wells for culture. Before and after the cell release experiment, the cells on the sample were stained with a live cell dye to observe the cell count before and after detachment and calculate the cell detachment efficiency. The cell detachment efficiency was 24.4%. (See [reference needed]). Figure 2 .

[0140] Comparative Example 3

[0141] Using the commercially available transfection reagent Lipo2000, pGFP was combined with Lipo2000 transfection reagent for 10 min according to the kit's standard instructions, and then added to normal cell culture medium to transfer pGFP (molecular weight 3075 kDa) to three types of difficult-to-transfect primary cells (mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells).

[0142] Forty-eight hours after transfection, cell nuclei were stained with 4',6-diamidinyl-2-phenylindole, and the expression of green fluorescent protein was observed under a fluorescence microscope. Quantitative analysis of the fluorescence microscope images yielded bar graphs of transfection efficiency and cell viability. The transfection efficiency of mouse embryonic fibroblasts was 18.9%, and cell viability was 10.2%; the transfection efficiency of human umbilical vein endothelial cells was 12.0%, and cell viability was 47.0%; the transfection efficiency of mouse dendritic cells was 0.3%, and cell viability was 66.4%. (See also...) Figure 3 .

[0143] Depend on Figure 1 It is evident that the method described in this invention can deliver exogenous molecules of different sizes into HeLa cells with a delivery efficiency of over 95%, and has virtually no impact on cell viability, demonstrating that the method described in this invention has broad applicability in terms of the types of molecules that can be delivered.

[0144] Depend on Figure 2 It can be seen that the cell density on gold flakes and Au-PDA remained largely unchanged before and after cell release, while under the same experimental conditions, the cell density on Au-PDA-PNIP decreased significantly after release. This demonstrates that the method described in this invention can efficiently harvest cells from the substrate after delivery of exogenous macromolecules.

[0145] Figure 3Comparing the transfection efficiency of the method described in this invention with commercially available transfection reagents, it was found that the method described in this invention can achieve a transfection efficiency of over 90% for difficult-to-transfect cells while maintaining good cell viability. Under the same conditions, the transfection efficiency of Lipo2000 for primary cells is less than 20%. This demonstrates that the method described in this invention can achieve highly efficient transfection of difficult-to-transfect cells and has strong versatility for various cell types.

[0146] Depend on Figure 4 It was found that the Au-PDA-PNIP sample prepared by deposition using a copolymer of dopamine and N-isopropylacrylamide achieved a transfection efficiency of over 90% for HeLa cells, cell viability after 48 hours of transfection, and cell detachment efficiency. This demonstrates that the method described in this invention can efficiently transfect HeLa cells and efficiently harvest cells from the substrate after delivery of exogenous macromolecules.

[0147] In addition to the above embodiments, the present invention also includes other embodiments. All technical solutions formed by equivalent transformation or equivalent substitution should fall within the protection scope of the claims of the present invention.

Claims

1. A non-therapeutic method for delivering exogenous molecules into cells, characterized in that, The method includes contacting a substrate with a modified polydopamine layer deposited on its surface with the cells, wherein the modified polydopamine layer is obtained by reacting a functional polydopamine containing double bonds or a functional dopamine monomer with a temperature-sensitive polymer monomer; the method includes seeding the cells on the surface of the substrate with the modified polydopamine layer deposited on its surface, contacting the cells with a reagent containing exogenous molecules, and irradiating the cells with a near-infrared laser light source; The substrate is a metallic material, an inorganic non-metallic material, an organic polymer material, or an experimental or instrumental device; the metallic material is selected from gold, stainless steel, titanium alloy, or magnesium alloy; the inorganic non-metallic material is selected from monocrystalline silicon, mica, or glass; the organic polymer material is selected from polyurethane, polydimethylsiloxane, or polymer electrospun fiber membrane; and the experimental or instrumental device is selected from cell culture plates, enzyme-labeled plates, or microfluidic devices.

2. The non-therapeutic method for delivering exogenous molecules into cells according to claim 1, characterized in that, The method further includes placing the substrate with the modified polydopamine layer deposited on its surface after laser irradiation in an environment below the lower critical solution temperature of the temperature-sensitive polymer.

3. A non-therapeutic method for delivering exogenous molecules into cells according to claim 1 or 2, characterized in that, The method further includes the step of preparing an aqueous solution containing dopamine and / or dopamine hydrochloride and olefinic unsaturated anhydride, adjusting the pH to 8-9, and immersing the substrate in the solution to obtain a substrate with functional polydopamine deposited on the surface; or, the method further includes the step of dissolving dopamine and / or dopamine hydrochloride and olefinic unsaturated anhydride in water and stirring to obtain a functional dopamine monomer with double bonds.

4. A non-therapeutic method for delivering exogenous molecules into cells according to claim 3, characterized in that, The olefinic unsaturated anhydride is selected from one or more of itaconic anhydride, maleic anhydride, and citraconic anhydride.

5. A non-therapeutic method for delivering exogenous molecules into cells according to claim 4, characterized in that, The olefinic unsaturated anhydride is selected from itaconic anhydride.

6. A non-therapeutic method for delivering exogenous molecules into cells according to claim 1 or 2, characterized in that, The thermosensitive polymer monomer includes at least N-isopropylacrylamide.

7. A non-therapeutic method for delivering exogenous molecules into cells according to claim 6, characterized in that, The thermosensitive polymer monomer is N-isopropylacrylamide.

8. A non-therapeutic method for delivering exogenous molecules into cells according to claim 1, characterized in that, The substrate is gold flakes.

9. A non-therapeutic method for delivering exogenous molecules into cells according to claim 1 or 2, characterized in that, The exogenous molecules include one or more of the following: polysaccharide molecules, proteins, DNA, RNA, therapeutic drugs, intracellular probes, nanomaterials, aptamers, bacteria, artificial chromosomes, and organelles.

10. A non-therapeutic method for delivering exogenous molecules into cells according to claim 9, characterized in that, The polysaccharide molecule is selected from dextran; the protein is selected from gene editing enzymes, antibodies, or antigens; the DNA is selected from pDNA; the RNA is selected from miRNA or siRNA; the intracellular probe is selected from quantum dots; the nanomaterial is selected from nanoparticles or nanodevices; and the organelle is selected from mitochondria.

11. A non-therapeutic method for delivering exogenous molecules into cells according to claim 1 or 2, characterized in that, The cells were selected from cell lines and primary cells.

12. A non-therapeutic method for delivering exogenous molecules into cells according to claim 11, characterized in that, The cell line includes HeLa cells, and the primary cells include one of mouse embryonic fibroblasts, human umbilical vein endothelial cells, and mouse dendritic cells.

13. The non-therapeutic use of a substrate with a modified polydopamine layer deposited on its surface in the process of delivering exogenous molecules into cells, characterized in that, The modified polydopamine layer is obtained by reacting a functional polydopamine containing double bonds or a functional dopamine monomer with a temperature-sensitive polymer monomer; the substrate is a metallic material, an inorganic non-metallic material, an organic polymer material, or an experimental or instrumental device; the metallic material is selected from gold, stainless steel, titanium alloy, or magnesium alloy; the inorganic non-metallic material is selected from single-crystal silicon, mica, or glass; the organic polymer material is selected from polyurethane, polydimethylsiloxane, or polymer electrospun fiber membrane; the experimental or instrumental device is selected from cell culture plates, ELISA plates, or microfluidic devices; the process of delivering exogenous molecules into cells includes planting cells on the surface of the substrate on which the modified polydopamine layer is deposited, contacting the cells with a reagent containing exogenous molecules, and irradiating the cells with a near-infrared laser light source.

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