HK1088637B - Solid surface for biomolecule delivery and high-throughput assay - Google Patents

Description

Solid surface for biomolecule delivery and high throughput assay methods

Technical Field

The present invention relates to methods for introducing biomolecules, such as nucleic acids, into cells by culturing the cells on a solid surface coated with transfection reagents and biomolecules for routine and high throughput transfection assays. The invention also relates to methods of use and methods of production of the transfectable surface for use in the experimental methods.

Background

Gene transfection methods can be used to introduce nucleic acids into cells, and can be used to study gene regulation and gene function. High throughput assays that can be used to screen large batches of DNA to identify encoded products with properties of interest are particularly useful. Gene transfection is the delivery and introduction of biologically functional nucleic acids into cells, such as eukaryotic cells, in such a way that the function of the nucleic acid is retained in the cell. Gene transfection is widely used in studies involving gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy. As the cloning and cataloging of genes from higher organisms continues, researchers have attempted to discover the function of the genes and identify gene products with desirable properties. The growing collection of gene sequences requires the development of systematic, high-throughput approaches to the identification of gene products and analysis of gene function, as well as other fields of research in cellular and molecular biology.

Both viral and non-viral gene vectors have been used in gene delivery. Viral vectors exhibit higher transfection efficiencies than non-viral vectors, but the safety of viral vectors hampers their applicability (Verma I.M and Somia N.Nature 389(1997), pp.239-242; Marhsall E.science 286(2000), pp.2244-2245). Although non-viral transfection systems do not show the efficiency of viral vectors, the former are of great interest because of their theoretical safety profile compared to viral vectors. In addition, the preparation of viral vectors is a complex and expensive process, which limits the in vitro applications of viral vectors. The preparation of non-viral vectors is simpler and less costly than the preparation of viral vectors, making synthetic gene vectors a transfection agent needed for in vitro studies.

Most non-viral vectors mimic important features of viral entry into cells to overcome cellular barriers aimed at preventing infiltration of foreign genetic material. Non-viral gene vectors based on gene vector backbones can be classified as a) lipid complexes (lipoplex), b) polyplex (polyplex), and c) lipid polyplex (lipoplex). Lipoplexes are collections of nucleic acids with a lipid component, which is usually a cation. Gene transfer via the lipid complex is called lipofection. Polyplexes are nucleic acid complexes with cationic polymers. The lipoplex contains a lipid component and a polymer component. Typically the above-mentioned DNA complexes are further modified to contain cell-targeting or intracellular targeting moieties and/or membrane destabilizing components, such as viral proteins or peptides or membrane-disrupting (membrane-disrupting) synthetic peptides. Recently, bacteria and bacteriophages have also been referred to as shuttle mechanisms for transferring nucleic acids into cells.

Most non-viral transfection agents are synthetic cationic molecules and are reported to "coat" nucleic acids by interaction between cationic sites on the cations and anionic sites on the nucleic acids. The positively charged DNA-cationic molecule complex interacts with the negatively charged cell membrane to facilitate the passage of DNA across the cell membrane by nonspecific endocytosis. (Schofield, Brit. Microencapsed. Bull, 51 (1): 56-71 (1995)). In most conventional gene transfection protocols, cells are seeded on cell culture equipment for 16-24 hours prior to transfection. Transfection reagents (e.g., cationic polymer carriers) and DNA are typically prepared in separate tubes, and each respective solution is diluted in culture medium (without fetal bovine serum or antibiotics). The two solutions were then mixed by the following steps: one solution was carefully and slowly added to the other while the mixture was constantly stirred. The mixture is incubated at room temperature for 15-45 minutes to form transfection reagent-DNA complexes and to remove residual serum and antibiotics. Prior to transfection, the cell culture medium was removed and the cells were washed with buffer. The solution containing the DNA-transfection reagent complex is added to the cells and the cells are incubated for about 3-4 hours. The medium containing the transfection reagent is then replaced with fresh medium. Finally, the cells are analyzed at one or more specific time points. This is clearly a time-consuming process, especially when the number of samples to be transfected is very large.

There are several major problems with conventional transfection methods. First, conventional methods are time consuming, especially when the number of cells or gene samples to be used in a transfection experiment is large. Furthermore, the results obtained are difficult to repeat, since the conventional transfection methods require a large number of steps. For example, in the production of DNA-transfection reagent complexes, the concentration and volume of nucleic acids and reagents, pH, temperature, the type of buffer used, the length and speed of agitation, incubation time, and other factors will all affect the formation of the complexes. Although the same reagents and procedures may be used, different results may be obtained. The results obtained with multi-step methods are often affected by human or mechanical errors or other variations in the steps. Furthermore, renewing the cell culture medium after transfection disturbs the cells and may cause them to detach from the surface on which they are cultured, leading to variable and unpredictable end results. For all the reasons mentioned above, conventional transfection methods require highly skilled technicians to perform the transfection experiments or experimental procedures.

Researchers need simpler, less costly cell transfection methods and require high throughput cell transfection methods to efficiently transfect large numbers of samples.

Disclosure of Invention

The invention provides a method for introducing a biomolecule into a eukaryotic cell, comprising the steps of: (a) coating a solid surface with a biomolecule delivery agent (delivery reagent), (b) adding biomolecules to be introduced into eukaryotic cells to the solid surface, (c) seeding the solid surface with cells at a sufficiently high density under suitable conditions to introduce the biomolecules into the eukaryotic cells. According to embodiments of the invention, the surface may be selected from the group consisting of a vial (flash), a dish, a multi-well plate, a slide, and an implantation device. The biomolecule delivery agent or transfection agent may be selected from polymers, lipids, lipid-polymers and/or combinations and/or derivatives thereof containing a cell targeting or intracellular targeting moiety and/or membrane destabilizing component and one or more delivery enhancers (enhancer).

According to embodiments of the invention, the biomolecule delivery agent may be attached to the surface by: i.e. uniformly coating the agent on the surface or dispensing (spot) the biomolecule delivery agent on discrete areas of the surface. The solid surface coated with the biomolecule delivery agent may further contain a matrix agent (matrix reagent) selected from the group consisting of proteins, peptides, polysaccharides and polymers. The protein may be selected from gelatin, bovine serum albumin, and extracellular matrix components, such as, but not limited to, collagen, laminin, and fibronectin. The polymer may be selected from hydrogels, biodegradable polymers, and biocompatible materials.

According to an embodiment of the present invention, the biomolecule delivery reagent coated to the solid surface may further comprise a cell culture agent selected from the group consisting of a cytoreductive agent (cytoreductive agent), a cell binding/adhesion agent, a cell growth agent, a cell stimulation agent and a cell inhibition agent.

The biomolecules may be selected from nucleotides, proteins, peptides, sugars, polysaccharides and organic compounds. Preferred biomolecules are selected from the group consisting of DNA, RNA and DNA/RNA hybrids. The nucleotides may be circular (plasmid), linear or single stranded oligodeoxynucleotides. The RNA may be single-stranded (ribozyme) or double-stranded (siRNA) RNA.

The solid surface used according to the method of the invention may be selected from, but is not limited to, a slide or a multiwell plate.

Eukaryotic cells used in accordance with embodiments of the present invention can be, but are not limited to, mammalian cells. The mammalian cell can be a dividing cell or a non-dividing cell. The mammalian cell may be a transformed cell or a primary cell. The mammalian cell may be a somatic cell or a stem cell. The eukaryotic cell may be a plant cell, a yeast cell, or an insect cell.

The invention provides a high-throughput drug screening experiment method, which comprises the following steps: (a) attaching a delivery agent to a solid surface, (b) attaching a biomolecule to be introduced into eukaryotic cells to the delivery agent, (c) seeding the cells at a sufficiently high density on a surface containing the delivery agent and the biomolecule under suitable conditions to introduce the biomolecule into the eukaryotic cells, and (d) detecting the eukaryotic cells to which the biomolecule has been delivered.

Biomolecules may be selected from, but are not limited to, nucleotides, proteins, peptides, sugars, polysaccharides, and organic compounds, and nucleotides may be selected from, but are not limited to, DNA, RNA, and DNA/RNA hybrids. The DNA may be circular (plasmid), linear or single stranded Oligodeoxynucleotides (ODN). The RNA may be single-stranded (ribozyme) or double-stranded (siRNA) RNA.

The eukaryotic cell is preferably a mammalian cell. The mammalian cell can be a dividing cell or a non-dividing cell, and the cell can be a transformed cell or a primary cell. The mammalian cell may be a somatic cell or a stem cell. Eukaryotic cells may be selected from, but are not limited to, plant cells, bacterial cells, and insect cells. Detection of cells that have been delivered with biomolecules can be carried out by detecting the biomolecules themselves, their products, their target molecules, products catalyzed or regulated by the biomolecules.

Drawings

FIG. 1 is a schematic representation of a transfection assay using a transfectable cell culture device or slide.

Figure 2 illustrates the effect of using a transfectable surface coated with various cationic polymer-gelatin transfection mixtures on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of linear PEI, NDT-CP-B-1 and NDT-CP-1 are shown in the graph. The amount of Superfect was 15, 7.5 and 3.75. mu.g/well, respectively. The transfection efficiency of GFP gene was about 30-35%, and NDT-CP-1 showed the highest efficiency.

Figure 3 illustrates the effect of using a transfectable surface coated with various cationic polymer-gelatin transfection mixtures on luciferase reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of linear PEI, NDT-CP-B-1 and NDT-CP-1 were 8, 4, 2 and 1. mu.g/well, respectively. The amount of Superfect was 15, 7.5 and 3.75. mu.g/well. The luciferase activity of all samples was higher than 5X 10⁷RLU/mg protein.

Figure 4 illustrates the effect of using a transfectable surface coated with various cationic lipid-polymer-gelatin transfection mixtures on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of lipid-polymer shown are 8, 4 and 2 μ g/well. In the tested lipid-polymer transfection agent, the transfection efficiency of GFP can reach 20-25%.

Figure 5 illustrates the effect of using a transfectable surface coated with various cationic lipid-polymer-gelatin transfection mixtures on luciferase reporter gene transfection in 923 cells in a 96-well plate cell culture device system. NDT-LP-2 can have luciferase activity greater than 10 in lipid-polymer-gelatin transfection mixture systems⁶RLU/mg protein.

Figure 6 illustrates the effect of using a transfectable surface coated with cationic lipid-gelatin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. Transfection efficiencies mediated by lipofectamine 2000-gelatin transfection mixtures were up to 30% in 96-well plate systems.

Figure 7 illustrates the effect of using a transfectable surface coated with cationic lipid-gelatin transfection mixture on luciferase reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The luciferase activity of lipofectamine reagent 2000 can reach up to about 10 in a lipid-gelatin transfection mixture system⁷RLU/mg protein.

Figure 8 illustrates the effect of using a transfectable surface coated with cationic polymer-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of NDT-CP-1 and NDT-CP-B-1 are shown in the figure. The amount of Superfect was 15 and 7.5. mu.g/well, respectively. The efficiency of GFP transfection mediated by the cationic polymer-laminin system is up to 50%.

Figure 9 illustrates the effect of using a transfectable surface coated with cationic polymer-laminin transfection mixture on luciferase reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of NDT-CP-1 and NDT-CP-B-1 are shown in the figure. The amount of Superfect was 15, 7.5 and 3.75. mu.g/well, respectively. Luciferase activity of cationic polymer-laminin system up to 10⁸RLU/mg protein.

Figure 10 illustrates the effect of using a transfectable surface coated with cationic lipid-polymer-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of lipid-polymer shown are 8, 4 and 2 μ g/well. GFP transfection mediated by the lipid-polymer-laminin transfection mixture system was up to 45% efficient.

Figure 11 illustrates fluorescence in 923 cells in a 96-well plate cell culture device system using a transfectable surface coated with a cationic lipid-polymer-laminin transfection mixtureEffect of transfection of the enzyme reporter gene. The amount of lipid-polymer is shown in the figure. Luciferase activity of the lipopolymer-laminin transfection mixture system can reach 8 x 10⁷RLU/mg protein.

Figure 12 illustrates the effect of using a transfectable surface coated with cationic lipid-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. Transfection efficiency mediated by lipofectamine-laminin transfection mixture system was up to 45%.

Figure 13 illustrates the effect of using a transfectable surface coated with cationic lipid-laminin transfection mixture on luciferase reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The luciferase activity of the lipofectamine reagent 2000-laminin transfection mixture system can reach up to 1.5 multiplied by 10⁷RLU/mg protein.

Figure 14 illustrates the effect of using a transfectable surface coated with cationic polymer-collagen transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of NDT-CP-1 and NDT-CP-B-1 are shown in the figure. The amount of Superfect was 15 and 7.5. mu.g/well, respectively. The efficiency of GFP transfection mediated by the cationic polymer-collagen transfection mixture system was up to 40%.

Figure 15 illustrates the effect of using a transfectable surface coated with cationic lipid-polymer-collagen transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of lipid-polymer shown are 8, 4 and 2 μ g/well. Transfection efficiency mediated by the cationic lipid-polymer-collagen transfection mixture system was up to 35%.

Figure 16 illustrates the effect of using a transfectable surface coated with cationic lipid-collagen transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The efficiency of transfection mediated by the cationic lipid-collagen transfection mixture system was up to 35% similar to that mediated by the lipofectamine 2000-gelatin transfection mixture system.

Figure 17 illustrates the effect of using a transfectable surface coated with cationic polymer-gelatin-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of NDT-CP-1 and NDT-CP-B-1 are shown in the figure. The amount of Superfect was 15 and 7.5. mu.g/well, respectively. The efficiency of GFP transfection mediated by the cationic polymer-gelatin-laminin transfection mixture system was up to 42%.

Figure 18 illustrates the effect of using a transfectable surface coated with cationic lipid-polymer-gelatin-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The amounts of lipid-polymer shown are 8, 4 and 2 μ g/well. The efficiency of GFP transfection mediated by the cationic lipid-polymer-gelatin-laminin transfection mixture system was up to 40% maximum.

Figure 19 illustrates the effect of using a transfectable surface coated with cationic lipid-gelatin-laminin transfection mixture on GFP reporter gene transfection in 923 cells in a 96-well plate cell culture device system. The transfection efficiency mediated by the cationic lipid-gelatin-laminin transfection mixture system can reach up to 30%.

Figure 20 illustrates the GFP reporter gene transfection assay using a transfectable slide system drop-coated with cationic polymer (NDT-CP-1) -gelatin or cationic lipid (lipofectamine reagent 2000) -gelatin transfection mixture in 923 cells. The transfectable slide was immersed in the GFP plasmid solution. Although the entire slide was covered with the GFP plasmid solution, only the cells at the spot coated with the transfection mixture showed GFP signal. This indicates that the transfection reagent was well attached to the slide without spreading. The above results indicate that the present technology can be used in transfection array (array) applications, and that the technology can screen thousands of target genes or gene drugs in cell-based transfection assay methods for genomic function studies or gene drug development (antisense ODN or siRNA).

Figure 21 illustrates the GFP reporter transfection assay in 923 cells using a transfectable slide system coated with cationic polymer (NDT-CP-1) -gelatin or cationic lipid (lipofectamine reagent 2000) -gelatin transfection mixture. Transfectable slides were loaded with GFP plasmid DNA by drop-coating 1-4. mu.l of GFP plasmid (20. mu.g/ml) solution and allowed to air dry (airdry). The slide was then placed on the bottom of a six-well plate, followed by seeding of 293 cells. The GFP signal was analyzed by fluorescence microscopy. Only the areas spotted with GFP plasmid DNA showed green fluorescent signal, indicating that the plasmid DNA was well attached to the spotted areas with transfection mixture on the glass surface. The technology can be used in transfection array applications, and can screen thousands of target genes or gene drugs in cell-based transfection assay methods for genomic function studies (cDNA library screening) or gene drug development (antisense ODN or siRNA).

Figure 22 illustrates the GFP reporter gene transfection assay using a transfectable slide system drop-coated with cationic polymer (NDT-CP-1) -laminin or cationic lipid (lipofectamine reagent 2000) -laminin transfection mixture in 923 cells. The transfectable slide was immersed in the GFP plasmid solution. Although the entire slide was covered with the GFP plasmid solution, only the cells at the spot coated with the transfection mixture showed GFP signal. This indicates that the transfection reagent was well attached to the slide without spreading. The above results indicate that the present technology can be used in transfection array applications, which can screen thousands of target genes or gene drugs in cell-based transfection assay methods for genomic function studies or gene drug development (antisense ODN or siRNA).

Figure 23 illustrates the GFP reporter gene transfection assay method in 923 cells using a transfectable slide system coated with cationic polymer (NDT-CP-1) -laminin or cationic lipid (lipofectamine reagent 2000) -laminin transfection mixture. Transfectable slides were loaded with GFP plasmid DNA by dispensing 1-4. mu.l of GFP plasmid (20. mu.g/ml) solution and allowed to air dry. The slide was then placed on the bottom of a six-well plate, followed by seeding of 293 cells. The GFP signal was analyzed by fluorescence microscopy. Only the areas spotted with GFP plasmid DNA showed green fluorescent signal, indicating that the plasmid DNA was well attached to the glass surface in the areas spotted with transfection mixture. The technology can be used in transfection array applications, and can screen thousands of target genes or gene drugs in cell-based transfection assay methods for genomic function studies (cDNA library screening) or gene drug development (antisense ODN or siRNA).

Figure 24 illustrates the effect of transfection of antisense ODN into Hela705 Luc cells using transfection reagent-laminin mixtures in a 96-well cell culture device. The results show that the transfectable surface consisting of the cationic polymer-laminin transfection mixture, the lipid-polymer-laminin mixture, or the lipid-laminin transfection mixture exhibits significant target RNA blockade, indicating that both plasmids and oligonucleotides can be successfully delivered into mammalian cells by the above protocol.

Figure 25 illustrates the effect of using transfection reagent-laminin mixture on siRNA delivery to 293 cells in a 96-well cell culture device. Compared to the non-siRNA control group, the transfectable surface consisting of the cationic polymer-laminin transfection mixture, the lipid-polymer-laminin mixture, or the lipid-laminin transfection mixture showed significant target RNA blockade, indicating that both plasmid and siRNA can be successfully delivered into mammalian cells by the above protocol.

FIG. 26 illustrates typical results for Tat peptide signal in Hela cells. In fig. 26A, Tat is coated on a solid surface with a gelatin matrix. FIG. 26B shows a non-Tat control. In the Tat-coated group, about 60-80% of the cells exhibited FITC signaling. This indicates that both nucleic acids and peptides can be delivered into cells according to embodiments of the invention.

FIG. 27 illustrates the effect of targeting molecules (transfer) on gene transfer mediated by cationic Polymer (PLL) -laminin transfection mixture in HepG2 cells using transfectable surface technology in a 96-well cell culture device system. The results show that the introduction of targeting molecules (transfer) in PLL-laminin based transfectable surface systems can significantly improve transfection efficiency.

FIG. 28 illustrates the effect of membrane interference peptides (VSVG peptides) on gene transfer mediated by cationic Polymer (PLL) -laminin transfection mixtures in 293 cells using transfectable surface technology in a 96-well cell culture device system. The results show that the introduction of a membrane interfering peptide (VSVG peptide) in a PLL-laminin based transfectable surface system can significantly improve transfection efficiency.

Figure 29 illustrates the effect of delivering GFP reporter gene to HUV-EC cells using transfection reagent-laminin mixture in a 96-well cell culture device. The GFP transfection efficiency of NDT-CP-1 was about 20%, while that of Superfect was about 15%. Lipofectamine reagents show very low transfection efficiencies (< 1.0%). This indicates that not only transformed cell lines, such as 293, Hela, HepG2, can be transfected, but also primary cells can be transfected with the cationic polymer-laminin transfection mixture system.

Figure 30 illustrates the effect of transfection reagent-laminin mixture on luciferase reporter gene delivery to HUV-EC cells in a 96-well cell culture apparatus. The transfection efficiencies of NDT-CP-1 and Superfect were 1.26X 10, respectively⁷And 8.26X 10⁶RLU/mg protein. Lipofectamine reagent 2000 exhibits lower efficiency compared to the cationic polymer-laminin mixture system. This further confirms that primary cells can be transfected with different genes according to embodiments of the invention.

Figure 31 illustrates the cell survival fraction of 293 cells in 96-well plates coated with transfection reagent-gelatin after transfection. All samples showed high survival scores (> 65%). This indicates that the cytotoxicity of the transfection reagent used according to the method of the invention is acceptable.

FIG. 32 illustrates the effect of introducing a cytoreductive agent (glutamine) on a transfection reagent-laminin based transfectable surface on the enhancement of cytotoxicity in 293 cells. "none" indicates no glutamine in the sample and "G" indicates glutamine in the sample. The results show that glutamine (cytoreductive agent) significantly improved transfection cytotoxicity compared to the glutamine-free group, indicating that cytoreductive agent is an excellent candidate agent for reducing cytotoxicity caused by transfection agents and transfection methods.

FIG. 33 illustrates the effect of the introduction of cell adhesion and cell stimulating agents (laminin) on the enhanced cytotoxicity in 293 cells on a transfectable surface based on transfection reagent-gelatin. "L" indicates the presence of laminin in the sample, and "none" indicates the absence of laminin in the sample. The results indicate that cell adhesion agents, such as laminin, significantly increase cytotoxicity caused by transfection agents and transfection methods in transfectable surface technology systems.

FIG. 34 illustrates the assessment of the shelf life (shelf-life) of transfectable surfaces in stability studies. The results show that there is no significant difference in transfection performance of the as-manufactured (flash-made) transfectable surface compared to the transfectable surface after 9 days of treatment at 37 ℃, indicating that the transfectable surface can have a shelf life of 1.5 years when stored at 4 ℃.

FIG. 35 is a table illustrating NDT synthetic polymer and lipid-polymer structures.

Detailed Description

The present invention describes a novel transfection apparatus and method which is simple, convenient and effective compared to conventional transfection assays. According to the method of the invention, the transfection device is manufactured by attaching a transfection reagent to the solid surface of the cell culture device. By using the above described device, researchers need only add nucleic acids or other biomolecule carrier systems to the surface of the cell culture device, without the need to pre-mix the DNA or biomolecules with transfection reagents. This eliminates the critical time-consuming steps required for conventional transfection methods. Scientists need only about 40 minutes to complete the entire transfection process for 10 samples, compared to 2-5 hours or more for the current methods. This is particularly advantageous for high throughput transfection assays that test hundreds of samples simultaneously.

The novel methods described herein have several advantages over conventional transfection. The transfection device provided by the present invention is very easy to store and does not require a biomaterial/transfection agent mixing step in the simple biomolecule delivery or gene transfection methods provided by the present invention. The transfection method of the present invention can be accomplished in a short time, e.g., about 40 minutes, and in the high throughput transfection or drug delivery methods provided, a large number of samples can be transfected simultaneously.

The present invention describes a novel method and apparatus for gene delivery that overcomes the problems common to the conventional experimental approaches described above. The transfection reagent is simply coated on the surface of the cell culture device, and thus can be easily commercialized and mass-produced. The customer, e.g., a researcher, need only add biomolecules, e.g., nucleic acids of interest, directly to the surface of the cell culture device to prepare the device prior to transfection. The cells are then seeded on the surface of the cell culture device and incubated without changing the media, and then analyzed. Medium replacement during transfection is not necessary. The method of the present invention significantly reduces the risk of error by reducing the number of steps involved, thereby improving the consistency and accuracy of the system.

According to the methods of the invention, transfection reagents are attached to the surface of a slide, multiwell plate, or other surface to form a transfection device. By using the above described device one need only add DNA or other biomolecules to the surface and allow the transfection reagent to form complexes with the DNA or biomolecules. The reaction takes place for about 30 minutes, and the cells are then seeded and incubated on the surface under conditions suitable for the introduction of the biomolecule into the cells.

Any surface suitable for attaching a mixture containing nucleic acids/biomolecules to its surface may be used. For example, the surface can be glass, plastic (e.g., polytetrafluoroethylene, polyvinylidene fluoride, polystyrene, polycarbonate, polypropylene), silicon, metal (e.g., gold), membrane (e.g., nitrocellulose, methyl nitrocellulose, PTFE, or cellulose), paper, biological material (e.g., protein, gelatin, agar), tissue (e.g., skin, endothelial tissue, bone, cartilage), mineral (e.g., hydroxyapatite, graphite). According to a preferred embodiment, the surface may be a glass slide (glass or poly-L-lysine coated glass slide) or a well of a multiwell plate.

For a slide, e.g., a slide coated with poly-L-lysine (e.g., Sigma, Inc.), a transfection reagent is attached to the surface and dried, and then nucleic acids of interest or nucleic acids, proteins, peptides, or small molecule drugs to be introduced into the cells are introduced. The slides were incubated at room temperature for 30 minutes to form biomolecule/transfection reagent complexes on the surface of the transfection device. The biomolecule/transfection reagent complex creates a medium that can be used in high throughput microarrays and can be used to study hundreds to thousands of nucleic acids, proteins, peptides, and other small molecule drugs simultaneously. In another embodiment, the transfection reagents or drug delivery reagents may be attached to a discontinuous, defined area of the surface of the transfection device to form a microarray of transfection reagents or drug delivery reagents. In the above embodiments, the molecules to be introduced into the cells, such as nucleic acids, are coated on the surface of the transfection device along with a transfection or delivery agent. The method can be used to screen transfection or other delivery agents from thousands of compounds. The results of the screening method can be verified by computer analysis.

In another embodiment of the invention, one or more wells of a multi-well plate may be coated with a transfection or drug delivery agent. Common plates used in transfection and drug screening are 96-well and 384-well plates. Transfection or biomolecule delivery agents may be uniformly coated on the bottom of the plate. Hundreds of nucleic acids, proteins, peptides or other biomolecules are then added to the wells by, for example, a multi-channel pipette or an automated machine. The transfection results were then determined by using a microplate reader. This is a very convenient way of analyzing transfected cells, since microplate readers are commonly used in most biomedical laboratories. Multi-well plates coated with transfection or biomolecule delivery agents are widely used in most laboratories to study gene regulation, gene function, molecular therapy and signal transduction, and drug screening. Also, if different kinds of transfection agents are coated on different wells of a multi-well plate, the plate can be used more effectively for screening multiple transfection agents or delivery agents. Recently, 1,536 and 3,456 well plates have been developed, which plates can also be used according to the methods of the present invention.

The transfection or delivery agent is preferably a cationic compound that can introduce biomolecules, such as nucleic acids, proteins, peptides, sugars, polysaccharides, organic compounds, and other biomolecules into the cell. Preferred embodiments employ cationic oligomers such as low molecular weight Polyethylenimine (PEI), low molecular weight poly (L-lysine) (PLL), low molecular weight chitosan or low molecular weight dendrimers (dendrimers). According to its modular composition, reagents can be divided into: a lipid, a polymer, a lipid-polymer and/or combinations and/or derivatives thereof, said agent comprising a cell targeting or intracellular targeting moiety and/or a membrane destabilizing component and a delivery enhancing agent.

According to one embodiment, the delivery agent may be mixed with a matrix, such as a protein, peptide, polysaccharide, or other polymer. The protein may be gelatin, collagen, bovine serum albumin, or any other protein that may be used to attach the protein to a surface. The polymers may be hydrogels, copolymers, non-degradable or biodegradable polymers and biocompatible materials. The polysaccharide can be any compound capable of forming a film and coating the delivery agent, such as chitosan. Other agents, such as cytotoxicity reducing agents, cell binding agents, cell growth agents, cell stimulating or cell inhibiting agents, and compounds for culturing specific cells may also be applied to the transfection device along with the transfection or delivery agent.

According to another embodiment, a gelatin-transfection reagent mixture containing a transfection reagent (e.g., a lipid, polymer, lipid-polymer, or membrane destabilizing peptide) and gelatin in a suitable solvent, such as water or secondary deionized water, may be attached to the transfection device. In another embodiment, the cell culture agent may also be present in a gelatin-transfection reagent mixture. The mixture is uniformly applied to surfaces such as slides and multi-well plates, thereby creating a transfection surface containing a gelatin-transfection reagent mixture. In another embodiment, different transfection reagent-gelatin mixtures may also be spotted on discrete areas of the surface of the transfection device. The resulting product is completely dried under suitable conditions so that the gelatin-transfection reagent mixture adheres to the site of the coating mixture. For example, the resulting product may be dried at a particular temperature or humidity or in a vacuum dryer.

The concentration of transfection reagent in the mixture depends on the transfection efficiency and the cytotoxicity of the reagent. There is generally a balance between transfection efficiency and cytotoxicity. The concentration that is most effective for the transfection agent while maintaining cytotoxicity at acceptable levels is the optimal level for the concentration of the transfection agent. The concentration of transfection reagent is generally from about 1.0. mu.g/ml to about 1000. mu.g/ml. In a preferred embodiment, the concentration is from about 40. mu.g/ml to about 600. mu.g/ml. Similarly, the concentration of gelatin or other matrix depends on the experiment or experimental method to be performed, but is typically 0.01% to 0.5% of the transfection reagent. According to the embodiment shown in the examples, the gelatin concentration is about 0.2% of the transfection agent.

The molecules to be introduced into the cell may be nucleic acids, proteins, peptides, Peptide Nucleic Acids (PNAs) and other biomolecules. The nucleic acid may be DNA, RNA, and DNA/hybrids, etc. If the DNA used is present in a vector, the vector may be of any type, such as a plasmid (e.g., pCMV-GFP, pCMV-luc) or a virus-based vector (e.g., pLXSN). The DNA may also be a linear fragment and have a promoter sequence (e.g.CMV promoter) at the 5 'end of the cDNA to be expressed and a poly A site at the 3' end. The above gene expression elements allow the cDNA of interest to be transiently expressed in mammalian cells. If the DNA is a single stranded Oligodeoxynucleotide (ODN), such as an antisense ODN, it can be introduced into a cell to modulate target gene expression. In embodiments using RNA, the nucleic acid may be a single-stranded (antisense RNA and ribozyme) or double-stranded (RNA interference, SiRNA) RNA. In most cases, the RNA is modified to increase its stability and enhance its role in the down-regulation of gene expression. In Peptide Nucleic Acids (PNA), the nucleic acid backbone is replaced with a peptide, making the molecule more stable. In particular embodiments, the methods of the invention can introduce proteins, peptides, and other molecules into cells for a variety of purposes, such as molecular therapy, protein function studies, or molecular function studies.

Under appropriate conditions, biomolecules are added to the transfection device coated with transfection or delivery agents to form biomolecule/delivery agent complexes. The biomolecule is preferably dissolved in fetal calf serum and antibiotic free cell culture Medium, such as Dulbecco's Modified Eagle Medium (DMEM). If the transfection or delivery agent is uniformly attached to the slide, the biomolecules can be dispensed at discrete locations on the slide. Alternatively, the transfection or delivery reagent may be dispensed at discrete locations on the slide, and the biomolecules may simply be added to cover the entire surface of the transfection device. If the transfection or delivery reagent is attached to the bottom of a multiwell plate, the biomolecules can be simply added to the different wells by multi-channel pipettes, automated equipment, or other methods. The resulting product (transfection device coated with transfection or delivery agent and biomolecules) was incubated at room temperature for about 25 minutes to form biomolecule/transfection agent (delivery agent) complexes. In some cases, for example, where different types of biomolecules are spotted on discrete locations of a slide, the DNA solution will be removed to produce a surface containing biomolecules complexed with transfection reagents. In other cases, the biomolecule solution may remain on the surface. The surface is then coated with cells of moderate density in a suitable medium. The resulting product (containing the biomolecule and the surface of the coated cells) is maintained under conditions that allow the biomolecule to enter the coated cells.

Cells suitable for use in accordance with the methods of the present invention include prokaryotes, yeast, or higher eukaryotic cells, including plant cells and animal cells, particularly mammalian cells. Eukaryotic cells, such as mammalian cells (e.g., human, monkey, dog, cat, bovine, or murine cells), bacterial, insect, or plant cells are coated onto a transfection device coated with a transfection or delivery agent and biomolecules at a sufficiently high density under suitable conditions to introduce/introduce the biomolecules into the eukaryotic cells and express DNA or to interact with cellular components. In particular embodiments, the cell may be selected from hematopoietic cells, neuronal cells, pancreatic cells, hepatic cells, chondrocytes, osteocytes, or myocytes. The cells may be fully differentiated cells or progenitor/stem cells.

In a preferred embodiment, eukaryotic cells are grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat-inactivated Fetal Bovine Serum (FBS) and L-glutamine and penicillin/streptomycin (pen/strep). One skilled in the art will appreciate that certain cells should be cultured in specialized media because certain cells require special nutrients, such as growth factors and amino acids. The optimal density of cells depends on the type of cell and the purpose of the experiment. For example, for gene transfection, the preferred population is 70-80% confluent cells (confluent cells), but for oligonucleotide delivery, the best situation is 30-50% confluent cells. In the exemplary embodiment, if at 5 × 10⁴Individual 293 cells/well were seeded onto 96-well plates and cells reached 90% confluence 18-24 hours after cell seeding. For Hela705 cells, only 1X 10 cells were required⁴A similar percentage of confluence can be achieved in 96-well plates per cell per well.

After seeding cells on a surface containing biomolecules/delivery agents, the cells are seeded under optimal conditions for the cell type (e.g., 37 ℃, 5-10% CO)₂) And (5) culturing the cells. The incubation time depends on the purpose of the experiment. Generally, in gene transfection experiments, for the expression of target genes in cells, cultured cells for 24 ~ 48 hours. In analyzing intracellular trafficking of biomolecules in cells, incubation may be required for minutes to hours, and the cells may be observed at a specified time point.

The results of biomolecule delivery can be analyzed by different methods. In the case of gene transfection and antisense nucleic acid delivery, the expression level of the target gene can be detected by a reporter gene, such as Green Fluorescent Protein (GFP) gene, luciferase gene, or β -galactosidase gene expression. The GFP signal can be observed directly under the microscope, the luciferase activity can be detected by luminometer, and the β -galactosidase-catalyzed blue product can be observed under the microscope or measured by a microplate reader. Those skilled in the art are familiar with how the above reporter genes function and how they are introduced into gene delivery systems. Nucleic acids and their products, proteins, peptides or other biomolecules delivered according to the methods of the invention, as well as targets regulated by such biomolecules, can be determined by a variety of methods, such as detection of immunofluorescence or enzymatic immunocytochemistry, autoradiography, or in situ hybridization. If immunofluorescence is used to detect expression of the encoded protein, a fluorescently labeled antibody that binds to the target protein is used (e.g., added to the slide under conditions suitable for binding of the antibody to the protein). The cells containing the protein are then identified by detecting the fluorescent signal. If the delivered molecule modulates gene expression, the expression level of the target gene can also be determined by methods such as autoradiography, in situ hybridization, and in situ PCR. However, the identification method depends on the properties of the delivered biomolecule, the expression product of the biomolecule, the target regulated by the product and/or the final product produced by biomolecule delivery.

Examples

Transfection agent

Branched PEI_25k(M_wPolyethyleneimine to 25KDa) and linear PEI_25k(M_w25KDa) from Polysciences Inc (wallington, pa). Superfect^(TM)(Valencia, Calif.) solutions were used as provided by the manufacturer. Transfection reagent LipofectAMINE^TMPurchased from Life Techonologies (gaithersburg, maryland) and used as supplied by the manufacturer. NDT-CP-B-1 (degradable) and NDT-CP-1 (non-degradable) are made from PEI₆₀₀(M_w600Da) synthetic linker different polymeric transfection agents. NDT-LP-1 is a lipid-polymer containing both polymer and lipid structures on the same molecule. The structure of the NDT polymeric transfection reagent is shown in fig. 35.

Preparation of transfectable surfaces

Transfection surfaces prepared from gelatin-based transfection mixtures

Preparation of 0.2% gelatin

Type B gelatin powder, i.e. 225Bloom (Sigma, cat # G-9391), was dissolved in sterile MiliQ water by gently stirring the solution in a water bath at 60 ℃ for 15 minutes. The 0.2% gelatin solution was then cooled at room temperature and the solution was filtered through a 0.45 μm porous acetate membrane (CA) while still warm (-37-40 ℃). A 100ml solution was prepared and the filtered gelatin solution was stored in 50ml aliquots at 4 ℃.

Preparation of transfection mixtures with gelatin

All transfection reagents were diluted in 0.2% gelatin solution. Linear PEI_25kAnd the concentration of the specially synthesized polymer sample is 320.0-40.0 mug/ml, and the branched PEI_25kThe concentration of (b) is 160.0 mu g/ml to 20.0 mu g/ml. The concentration of Superfect is 600.0-75.0 mu g/ml, and the concentration of lipofectamine reagent is 200.0-25.0 mu g/ml. The concentrations of the synthetic polymer and the lipid-polymer (NDT) were 320.0. mu.g/ml to 40.0. mu.g/ml.

Production of transfectable surfaces with 96-well plates and transfection reagent-gelatin mixtures

To each well of a 96-well plate 25. mu.l of transfection/gelatin solution was added. The plate was shaken for several seconds to ensure that the entire bottom surface was covered with transfection/gelatin solution. The plates were then air dried in a tissue culture hood for several hours (about 5-6 hours). The dried plates were stored at 4 ℃ until use (FIGS. 2-7).

Production of transfectable slide surfaces by drop coating of transfection reagent-gelatin mixture

1-4. mu.l of transfection/gelatin solution was drop coated on the PLL-coated slide. The slides were placed in a clean hood for about 1 hour until completely dry. The dried slides were stored at 4 ℃ until use (FIGS. 20, 21).

Transfection surfaces prepared from laminin-based transfection mixtures

Laminin (Sigma, cat # L2020) was diluted in PBS until the final concentration was 40.0. mu.g/ml and stored at 4 ℃.

Preparation of transfection mixtures with laminin

All transfection reagents were diluted in 40.0. mu.g/ml laminin solution. Linear PEI_25kAnd the concentration of the specially synthesized polymer sample is 320.0-40.0 mug/ml, and the branched PEI_25kThe concentration of (b) is 160.0 mu g/ml to 20.0 mu g/ml. The concentration of Superfect is 600.0-75.0 mu g/ml, and the concentration of lipofectamine reagent is 200.0-25.0 mu g/ml. The concentrations of the synthetic polymer and the lipid-polymer (NDT) were 320.0. mu.g/ml to 40.0. mu.g/ml.

Production of transfectable surfaces with 96-well plates and transfection reagent-laminin mixtures

To each well of a 96-well plate 25. mu.l of transfection/laminin solution was added. The plate was shaken for several seconds to ensure that the entire bottom surface was covered with transfection/gelatin solution. The plates were then air dried in a tissue culture hood for several hours (about 5-6 hours). The dried plates were stored at 4 ℃ until use (FIGS. 8-13).

Production of transfectable slide surfaces by drop-coating of transfection reagent-laminin mixture

1-4. mu.l of transfection/gelatin solution was drop coated on the PLL-coated slide. The slides were placed in a clean hood for about 1 hour and allowed to dry completely. The dried slides were stored at 4 ℃ until use (FIGS. 22-23).

Transfection surfaces prepared from collagen-based transfection mixtures

Collagen (Sigma, cat # C8919) was diluted in PBS until a final concentration of 120.0. mu.g/ml and stored at 4 ℃.

Preparation of transfection mixtures with laminin

All transfection reagents were diluted in 120.0. mu.g/ml collagen solution. Linear PEI_25kAnd concentration of specially synthesized polymer sample320.0-40.0. mu.g/ml, and branched PEI_25kThe concentration of (b) is 160.0 mu g/ml to 20.0 mu g/ml. The concentration of Superfect is 600.0-75.0 mu g/ml, and the concentration of lipofectamine reagent is 200.0-25.0 mu g/ml. The concentrations of the synthetic polymer and the lipid-polymer (NDT) were 320.0. mu.g/ml to 40.0. mu.g/ml.

Production of transfectable surfaces with 96-well plates and transfection reagent-collagen mixtures

25 μ l of transfection/collagen solution was added to each well of the 96-well plate. The plate was shaken for several seconds to ensure that the entire bottom surface was covered with transfection/gelatin solution. The plates were then dried in a tissue culture hood for several hours (about 5-6 hours). The dried plates were stored at 4 ℃ until use (FIGS. 14-16).

Transfection surfaces prepared from gelatin/laminin-based transfection mixtures

Laminin (Sigma, catalog No. L2020) was diluted in 0.2% gelatin until the final concentration was 40.0 μ g/ml and stored at 4 ℃.

Preparation of transfection mixtures with gelatin and laminin

All transfection reagents were diluted in 40.0 μ g/ml laminin/0.2% gelatin solution. Linear PEI_25kAnd the concentration of the specially synthesized polymer sample is 320.0-40.0 mug/ml, and the branched PEI_25kThe concentration of (b) is 160.0 mu g/ml to 20.0 mu g/ml. The concentration of Superfect is 600.0-75.0 mu g/ml, and the concentration of lipofectamine reagent is 200.0-25.0 mu g/ml. The concentrations of the synthetic polymer and the lipid-polymer (NDT) were 320.0. mu.g/ml to 40.0. mu.g/ml.

Production of transfectable surfaces with 96-well plates and transfection agent-gelatin-laminin mixtures

To each well of a 96-well plate, 25. mu.l of transfection/gelatin/laminin solution was added. The plate was shaken for several seconds to ensure that the entire bottom surface was covered with transfection/gelatin solution. The plates were then dried in a tissue culture hood for several hours (about 5-6 hours). The dried plates were stored at 4 ℃ until use (FIGS. 17-19).

Preparation of plasmid DNA

Plasmids pCMV-GFP and pCMV-luc were constructed according to standard DNA recombination protocols. Expression of the Green Fluorescent Protein (GFP) and firefly luciferase gene cDNA is controlled by the human Cytomegalovirus (CMV) promoter, and transcripts are stabilized by the gene expression enhancer, the chicken β -globin intron. The Plasmid was amplified in DH 5. alpha. E.coli and purified using Qiagen Plasmid Max Preparation Kit (Qiagen Plasmid Max Preparation Kit) according to the manufacturer's instructions. The quantity and quality of the purified plasmid DNA was checked by spectrophotometric analysis at 260 and 280nm and by electrophoresis in a 0.8% agarose gel. The purified plasmid DNA was dissolved in sterile double distilled water and stored at-20 ℃.

Preparation of DNA solution with DMEM

pCMV-GFP or pCMV-luc plasmid was diluted in DMEM until the final concentration was 10. mu.g/ml. Thirty (30) μ l of DNA solution was added to the transfectable surface of a 96-well plate or slide and incubated at room temperature for 20-30 minutes.

Preparation of antisense oligonucleotides

Luciferase 705 reporter systems were developed by Dr.Kole of University of Northern Carolina (Kang SH et al, Biochemistry 1998; 37 (18): 6235-9). In the above system, human β -globin with a mutation at 705 was inserted into the sequence between luciferase cdnas. Introducing the plasmid into Hela cells to obtain stable gene expression; the cell line was designated Hela luc 705. Usually the cells show low luciferase activity, because the gene product with mis-splicing (luciferase) does not show activity. However, an antisense oligonucleotide that binds to the 705 sequence will block the wrong splice site and produce a luciferase protein with biological activity. Luciferase 705 was used as a functional model to evaluate the efficiency of antisense oligonucleotide delivery. Higher luciferase activity indicates higher efficiency of antisense delivery.

In this study, an 18 nucleotide 2' -O-phosphorothioate methyl ester oligonucleotide was synthesized in combination with the luc705 sequence. The sequence is CCUCUUACCUCAGUUACA. The antisense oligonucleotides were diluted in optimal MEM to a final concentration of 0.6. mu. mol/L. Mu.l of antisense oligonucleotide was added to the transfectable surface of each well and incubated at room temperature for 25 minutes (FIG. 24).

Preparation of siRNA

sirnas are double-stranded RNA fragments containing 21-25 base pairs (bp) that bind to and destroy target mrnas and cause down-regulation of gene expression levels. In the experiment, luciferase plasmids and siRNA synthetic cassettes (cassettes) targeting the luciferase gene were prepared in optimal MEM and added to the transfectable plates described above and incubated for 25 minutes. The amount of luciferase plasmid was 0.5. mu.g/well and the siRNA synthesis cassette was about 0.5. mu.g/well (FIG. 25).

Tat peptide delivery

Biotin-labeled Tat-peptides were prepared at various concentrations (50, 25, 12.5 and 6.3 μ g/well) with 0.2% gelatin solution and coated on 96-well plates, respectively. The plates were dried by placing in a cell culture hood for several hours. At 1.5X 10⁴Wells Hela cells were seeded on peptide-coated plates and incubated at 37 ℃ for 4 hours. Cells were fixed with 0.2% glutaraldehyde/PBS for 5 minutes and then treated with 10% methanol. After blocking the cells with 10% serum at 37 ℃ for 30 minutes, they were incubated with streptavidin-FIFC at 37 ℃ for 30 minutes. Cells were washed with PBS and the fluorescence signal was observed under a fluorescence microscope. If the peptide is successfully delivered into the cell, the biotin-conjugated peptide can specifically bind to streptavidin-FIFC and enable the peptide to generate a fluorescent signal. A stronger FITC signal in the cells indicates more peptide was transported into the cells (fig. 26).

Effect of targeting moieties in transfection mixtures on transfectable surface System mediated Gene transfer

Preparation of transferrin conjugated poly-L-lysine

Transferrin can be absorbed by hepatocytes in the transferrin reporter-mediated endocytic pathway. Transferrin has been reported to have been successfully used as a cell targeting molecule that can improve gene delivery efficiency in hepatocytes (Wagner E, Ogris M, Zanner W.adv Drug Deliv Rev 1998; 30 (1-3): 97-113).

Targeting molecule (transferrin) pairs in laminin-based transfection mixture systems Effect of cationic polymeric transfection Agents mediated Gene transfer

In the assay, 25. mu.L of poly-L-lysine conjugated to transferrin (PLL-T) and 40.0. mu.g/ml laminin were coated on 96-well plates. poly-L-lysine (PLL) was used as a control. The PLL-T and PLL concentrations were 320.0 μ g/ml to 40.0 μ g/ml. After air drying, 25. mu.l of luciferase plasmid solution (20.0. mu.g/ml, polymer/DNA ratio 16: 1-2: 1) was added to the plate and incubated at room temperature for 25 minutes. The dried plates were ready for use in the relevant experiments (fig. 27).

Use of membrane-destabilizing components in transfection mixtures for transfectable surface system-mediated gene transfer Influence of

VSVG is a viral envelope protein with membrane destabilizing properties that cause membrane fusion and disrupt cell membranes. VSVG has been used as a gene transfection enhancer, which can significantly enhance cationic Polymer (PLL) -mediated gene transfection. Peptides from the cell fusion region of the VSVG protein have been synthesized. The peptide has the sequence RRRQGTWLNPGFPPQSCGYATVTDARRR, and has amino acid arginine at the C terminal and the N terminal respectively to improve the solubility of the peptide.

VSVG peptide and poly-L-lysine were diluted in 40.0. mu.g/ml laminin. The VSVG peptide concentration was 1.0mg/ml, while the PLL concentration was 640. mu.g/ml to 160.0. mu.g/ml. In the control group, only PLL was diluted in laminin solution at the same concentration. 25 μ l of PLL or PLL + VSVG peptide solution was added to a 96-well plate and air dried. The amounts of PLL were 16.0, 8.0 and 4.0. mu.g/well, respectively (FIG. 28).

Gene transfer mediated by cytoreductive agent in transfection mixture to transfectable surface systemMoving shadow Sound box

Glutamine is a cytoreductive agent that protects cells from ammonia-induced cytotoxicity (Nakamura E and Hagen S J. am J Physiol rather lever Physiol 283G 1264-1275, (2002)). Since almost all cationic polymers or cationic lipids contain amino groups, the addition of glutamine to the transfection mixture in the preparation of the transfectable surface has a role in protecting cells from transfection cytotoxicity. Glutamine (100mmol/L) and various transfection reagents (NDT-CP-B-1, NDT-CP-1, Superfect and Lipofectamine reagent 2000) were diluted in 0.2% gelatin solution, 25. mu.l of the glutamine/transfection reagent/gelatin containing solution was added to a 96-well plate and allowed to air dry. Then 25. mu.l of GFP plasmid solution in optimal MEM (20.0. mu.g/ml) was added to each well and incubated for 25 minutes at room temperature. The dried plate was ready for use (fig. 32).

Effect of cell Adhesives in transfection mixtures on transfectable surface System-mediated Gene transfer Sound box

Laminin is a matrix that supports cell growth and differentiation. Laminin is a commonly used cell adhesion agent. In this experiment, transfection reagents NDT-CP-B-1, NDT-CP-1, NDT-LP-2, Superfect and lipofectamine reagent 2000 were diluted with 0.2% gelatin or a mixture of 0.2% gelatin and 40.0. mu.g/ml laminin, and 25. mu.l of the solution was added to a 96-well plate. The amount of NDT polymer used in each well was 8, 4 or 2. mu.g, the amount of Superfect was 15, 7.5 and 3.8. mu.g/well, and the amount of lipofectamine reagent 2000 was 2.5, 1.25 and 0.63. mu.g/well, respectively. After air drying, 25. mu.l of GFP plasmid in optimal MEM (20. mu.g/ml) was added to each well and incubated at room temperature for 25 minutes, then at 5X 10⁴One well 293 cells were seeded onto the plate. The cells were then incubated at 37 ℃ for 24 hours. Transfection efficiency and cytotoxicity were analyzed by fluorescence microscopy and MTT assay (fig. 33).

Cell culture

HEK 293T cells were maintained in DMEM (Gibco) containing 10% fetal bovine serum, 100 units/ml penicillin and 100. mu.g/ml streptomycin. In the above medium, the doubling time of the cells is about 20 hours, and the cells are separated every 3 to 4 days to avoid excessive confluency.

The Hela705 cell line was taken from human cervical carcinoma Hela cells after introduction of the firefly luciferase gene with the mutant β -globin intron (mutation at position 705), which resulted in luciferase protein mutation due to incorrect splicing. However, mutated introns can be corrected by specific antisense oligonucleotides after they block the wrong splice site (Kang SH et al, Biochemistry 1998; 37 (18): 6235-9). The cell lines were maintained in DMEM (Gibco) containing 10% fetal bovine serum, 100 units/ml penicillin and 100. mu.g/ml streptomycin. Hygromycin at 200. mu.g/ml was added to the medium to maintain the luc-705 plasmid. In this medium, the doubling time of the cells is about 20 hours, and the cells are separated every 3-4 days to avoid excessive confluency.

The human hepatoma cell line HepG2 was maintained in α -MEM medium (Gibco) containing 10% fetal bovine serum, 100 units/ml penicillin and 100. mu.g/ml streptomycin. In the above medium, the doubling time of the cells is about 20 hours, and the cells are separated every 3 to 4 days to avoid excessive confluency.

HUV-EC cell lines, human primary endothelial cells, were grown and maintained in EBM medium (Cambrex Corp.) containing 10% fetal bovine serum, 100 units/ml penicillin and 100. mu.g/ml streptomycin and different types of growth factors according to the manufacturer's instructions (FIGS. 29-30).

Cells were prepared and seeded onto transfectable surfaces

Cells were harvested from a 10cm dish immediately prior to transfection in a tissue culture hood as follows:

a. the medium was removed, the cells were rinsed with 2ml of PBS, the solution was spread onto the plate, and then the solution was immediately removed.

b. 0.5ml of trypsin-EDTA was added to the cells and spread evenly on the plate, and immediately thereafter the trypsin-EDTA was removed.

c. The cells were placed in the hood for 3-5 minutes. The plate is then agitated to detach the cells from the surface of the plate.

d. Six (6) ml of 37 ℃ whole medium was added to the cell plate and the solution was pipetted up and down 12-15 times with a 10ml pipette until a single cell suspension was obtained while avoiding excessive foam generation. An additional 14ml of medium was added and the cells were suspended completely in the solution.

e. The number of cells was determined in a hemocytometer.

f. Diluting HEK 293T, HepG2 cells in a sterile basin to a final concentration of 4-5 × 10⁵Cells/well, and 100. mu.l (4-5X 10) of each well of a 96-well plate was seeded⁴A cell). The optimal concentration of Hela705 and HUV-EC cells is 1-2X 10⁵Cells/ml.

GFP reporter gene transfection experimental method

The Green Fluorescent Protein (GFP) gene was used for initial screening. After transfection, the GFP signal in the cells was observed under a fluorescence microscope (Olympus, filter 520 nm). The cells were photographed with a 10X objective. The percentage of cells with GFP signal in transfected cultures was determined from the counts of the three fields to obtain the optimal amount of cationic polymer.

Luciferase assay

Luciferase activity measurements were performed using a chemiluminescence Assay according to the manufacturer's instructions (Luciferase Assay System; Madison, Wis., USA). Briefly, 30 hours after gene transfer, cells were rinsed twice with PBS and then lysed with lysis buffer (1% Triton X-100, 100mM K) at room temperature₃PO₄2mM dithiothreitol, 10% glycerol and 2mM EDTA, pH7.8) for 15 minutes. Then in a photometer at room temperatureMu.l aliquots of cell lysate were mixed with 50. mu.l luciferase assay reagent using a syringe. Light emission was measured three times over a 10 second period and expressed as RLU (relative light units). Relative Light Units (RLU) were determined by BSA protein assay (pierce, Rockford, Illinois) and normalized to the protein content of each sample. All experiments were performed in triplicate.

Cytotoxicity test method-MTT test method

Cytotoxicity of transfection reagents on mammalian cells was assessed using the 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) method. Forty (40) hours after transfection, 10. mu.l of MTT solution (5.0mg/ml, Sigma) was added to each well and incubated for 3 hours. The medium was then removed and 200. mu.l DMSO was added to dissolve the formazanCrystals (formalzancrytal). The absorbance of the solution was measured at 570 nm. The viability of the cells was calculated using the following equation: viability (%) { absorbance }_{570 (sample)}Absorbance_{570 (control)}} × 100 (FIG. 31).

Stability study

LPEI, BPEI, NDT-CP-B-1, NDT-CP-1, Superfect and lipofectamine reagents in 0.2% gelatin were coated in 96-well plates as described above. After air drying, the plates were incubated at 37 ℃ for 9 days. And (5) standby. Luciferase plasmid (20.0. mu.g/ml in optimal MEM) was added to the plate (previously and thereafter incubated at 37 ℃) at 25. mu.l/well and incubated for 25 minutes. Inoculation of 5X 10 in plates⁴293 cells and incubated at 37 ℃ for 48 hours. The gene transfection efficiency of the transfectable surface of the freshly fabricated plate was compared to that of 9 days of incubation at 37 ℃ (FIG. 34).

While the invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention. All references cited herein are incorporated by reference.

Claims (22)

1. A method for introducing a biomolecule into eukaryotic cells in vitro comprising the steps of: (a) providing a solid surface at least partially coated with a transfection reagent comprising an oligoethyleneimine (PEI) linked with a degradable linker, (b) adding biomolecules to be introduced into eukaryotic cells to the solid surface, and (c) seeding the solid surface with cells at a sufficiently high density under suitable conditions to introduce the biomolecules into the eukaryotic cells, wherein the biomolecules are not pre-mixed with the transfection reagent.

2. The method of claim 1, wherein the surface is selected from the group consisting of a bottle, a dish, a multi-well plate, a slide, and an implantation device.

3. The method of claim 1, wherein the transfection reagent further comprises an additive selected from the group consisting of a cellular targeting moiety, an intracellular targeting moiety, a membrane destabilizing component, and one or more transfection enhancing agents.

4. The method of claim 1, wherein the transfection agent is attached to the surface by: namely, the transfection agent is uniformly coated on the surface or the transfection agent is dripped on discontinuous areas of the surface.

5. The method of claim 1, wherein the transfection reagent further comprises a matrix agent selected from the group consisting of proteins, glycoproteins, peptides, polysaccharides, polymers, and mixtures thereof.

6. The method of claim 5, wherein the protein is selected from the group consisting of gelatin, collagen, laminin, fibronectin, and bovine serum albumin, or a mixture thereof.

7. The method of claim 5, wherein the polymer is selected from the group consisting of a hydrogel, a biodegradable polymer, and a biocompatible material.

8. The method of claim 1, wherein the transfection reagent further comprises a cell culture agent selected from the group consisting of cytoreductive agents, cell binding agents, cell growth agents, cell stimulating agents, and cytostatic agents.

9. The method of claim 1, wherein the eukaryotic cell is a mammalian cell.

10. The method of claim 9, wherein the mammalian cell is a dividing cell or a non-dividing cell.

11. The method of claim 9, wherein the mammalian cell is a transformed cell or a primary cell.

12. The method of claim 9, wherein the mammalian cell is a somatic cell or a stem cell.

13. The method of claim 1, wherein the cell is a plant cell or an insect cell.

14. The method of claim 4, wherein the transfection reagent is attached to the surface by means of a manual or automated mechanical device.

15. The method of claim 1, wherein said biomolecule is selected from the group consisting of DNA, RNA, and DNA/RNA hybrids.

16. The method of claim 1, wherein the biomolecule is selected from the group consisting of a linear oligodeoxynucleotide, a plasmid, and a single stranded Oligodeoxynucleotide (ODN).

17. The method of claim 1, wherein the biomolecule is selected from the group consisting of single-stranded RNA or double-stranded RNA.

18. A method of detecting whether a biomolecule can enter a cell, comprising the steps of: (a) introducing a biomolecule into the cell by the method of any one of the preceding claims, and (b) detecting whether the biomolecule has been delivered into the cell.

19. The method of claim 18, wherein the biomolecule is selected from the group consisting of nucleotides, proteins, peptides, sugars, polysaccharides, and organic compounds.

20. The method of claim 18, wherein the cell is selected from the group consisting of a plant cell, an insect cell, and a bacterial cell.

21. The method of claim 18, wherein the detecting is performed by detecting the biomolecule, biomolecule product, biomolecule activity, activity of a biomolecule product, target molecule, biomolecule catalyzed product, or biomolecule regulated product.

22. The method of claim 1, wherein the degradable linker is 1, 6-hexanediol diacrylate.