KR20130010036A - Immobilized degradable cationic polymer for transfecting eukaryotic cells - Google Patents

Abstract

A cell transfection / cultivator comprising a solid support coated with a degradable polymer cation that is a transfection reagent is disclosed. The transfection / cultivator is conveniently stored at room temperature until use. Cell transfection can be readily accomplished by adding the subject nucleic acid and cells to be transfected to the transfection / culture medium. Cell transfection is completed in less than 1 hour using the transfection / culturer disclosed herein.

Description

Immobilized degradable cationic polymer for transfection of eukaryotic cells {IMMOBILIZED DEGRADABLE CATIONIC POLYMER FOR TRANSFECTING EUKARYOTIC CELLS}

Related application

This application claims priority to US Provisional Patent Application No. 60 / 637,344, filed December 17, 2004, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

Embodiments of the invention relate to cell transfection apparatus and methods. In particular, embodiments of the invention relate to cell transfection agents and cell incubators treated with the transfection agents. Treated cell incubators can be stored at room temperature and provide a simple and fast transfection method.

Description of related fields

Gene transfection methods can be used to introduce nucleic acids into cells and are useful for studying gene regulation and function. Particularly useful are high power assay methods that can be used to screen many pairs of DNA to identify these encoding materials by the properties of interest. Gene transfection is the transport and introduction of nucleic acids with biological function into cells, especially eukaryotic cells, in such a way that the nucleic acid retains its function in the cell. Gene transfection is widely applied to research related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy research. As gene cloning and classification in higher animals continues, researchers are attempting to discover the gene's function and identify genetic material with the desired function. These growing gene sequences require the development of systematic, high-powered approaches to characterize genetic material and analyze gene function as in other areas of research in cell and molecular biology.

Both viral and nonviral gene carriers have been used for gene delivery. Viral vectors have shown higher transfection efficiency than nonviral carriers, but the safety of viral vectors makes application difficult (Verma IM and Somia N. Nature 389 (1997), pp. 239-242; Marhsall E. Science 286 (2000), pp. 2244-2245). Non-viral transfection systems do not show the efficiency of viral vectors, but have received considerable attention because of their theoretical stability when compared to viral vectors. In addition, viral vector production is a complex and expensive process that limits the application of viral vectors in vitro. The preparation of nonviral carriers is simpler and more cost effective compared to the preparation of viral carriers, making synthetic gene carriers preferred as transfection reagents, especially in in vitro studies.

Most nonviral vectors mimic the important function of viral cell entry to overcome cellular barriers that prevent the penetration of foreign genetic material. Non-viral gene vectors based on gene carrier backbones can be classified into a) lipoplexes, b) polyplexes, and c) lipopolyplexes. Lipoplexes are usually cationic as assemblies of nucleic acid and lipid components. Gene delivery by lipoplexes is called lipofection. Polyplexes are complexes of nucleic acids and cationic polymers. Lipopolyplexes contain both lipid and polymeric components. Often such 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 cleavage synthetic peptides. Recently, bacteria and phage have also been described as shuttles for delivering nucleic acids to cells.

Most nonviral transfection reagents are synthetic cationic molecules and have been reported to "coat" nucleic acids by interacting the cationic sites on the cations with the anionic sites on the nucleic acids. Positively charged DNA-cationic molecular complexes interact with negatively charged cell membranes to promote the passage of DNA through the cell membranes by non-specific endocytosis (Schofield, Brit. Microencapsulated. Bull, 51 ( 1): 56-71 (1995)). In most conventional gene transfection protocols, cells are seeded in cell incubators 16-24 hours prior to transfection. Transfection reagents (such as cationic polymer carriers) and DNA are typically prepared in separate test tubes so that each solution is diluted in medium (fetal calf serum or antibiotic free). Then carefully add the other solution slowly and carefully to vortex the mixture while mixing. The mixture is incubated at room temperature for 15-45 minutes to form a complex between the transfection reagent and the DNA and to remove residual serum and antibiotics. Prior to transfection, the cell culture medium is removed and the cells are washed with buffer. A solution containing the DNA transfection 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. The cells are finally analyzed at one or more specific times. This is a clearly time consuming procedure, especially when the number of samples to be transfected is very large.

Some major problems exist in conventional transfection procedures. First, conventional procedures are time consuming, especially when many cell or gene samples are used for transfection testing. In addition, the results usually derived from transfection procedures are difficult to reproduce due to the number of steps required. For example, DNA-transfection reagent complex formation is affected by concentration and volume of nucleic acid and reagent, pH, temperature, type of buffer used, length and speed of vortexing, incubation time and other factors. The same reagents and procedures were followed, but different results could be obtained. Results from multi-step procedures are often affected by human or machine mistakes or other variables at each step. In addition, post-cleaning transfection of cell culture media disrupts the cells and detaches them from the surface on which they are cultured, resulting in modification and unpredictability in the final result. Because of all the factors considered, conventional transfection methods require highly skilled persons to perform transfection experiments or assays.

Researchers call for easier and more cost effective cell transfection methods, and high power cell transfection methods are needed to effectively transfect large sample numbers.

Sabatini (U.S. 2002 / 0006664A1) describes a composition containing DNA attached to a glass slide. However, this system only allows transfection with previously attached DNA. This is a major disadvantage of the system. Only by providing transfection with previously attached DNA, each investigator cannot use the desired nucleic acid.

US Patent Publication 2004 / 0138154A1, which is incorporated herein by reference, describes a cell culture / transfection apparatus in which transfection is mediated by lipid polymers. US Patent Publication 2005 / 0176132A1, incorporated herein by reference, describes a calcium salt mediated transfectable cell incubator.

US Patent Publication No. 2003 / 0215395A1, incorporated herein by reference, describes a degradable polymer that can be used for gene delivery.

As introduced above, conventional transfection is a long and technically difficult procedure. In general, three steps are required. 1) Inoculate and incubate the cells in a cell culture plate or dish until sufficient confluence is achieved. 2) Prepare the transfection reagent / nucleic acid complex. And 3) subject nucleic acid is added with the transfection reagent and further culture is performed. Two incubation periods are required and typically take two days or more to complete all steps. In contrast, embodiments of the present invention provide a simple procedure involving only one single culture step. Cell incubators previously coated with transfection reagents allow transfection by successively adding the target nucleic acid and cell culture. The transfected cells can then be cultured in the same device. Thus, the cells can be transfected and the cells can be cultured in a cell incubator without the need for further manipulation of the cells immediately after the transfection step. Transfection efficiency is comparable to normal transfection, but the time required for manipulation is reduced by more than one day. Embodiments of the present invention include transfectable cell incubators that greatly reduce the labor of transfection assays and allow for transfection with any subject nucleic acid in an easy manner by low cytotoxicity. In addition, the transfectable cell incubators of the present invention are stable upon prolonged storage at room temperature.

SUMMARY OF THE INVENTION

Embodiments of the invention relate to a device comprising a solid support coated with a transfection reagent mixture. Preferably, the transfection reagent in the coating is not complexed with biomolecules such as nucleic acids. Preferably, the solid support is polystyrene resin, epoxy resin or glass. Preferably, the coating is on the surface of the solid support. Preferably, the coating amount of the transfection reagent is about 0.1 to about 100 μg / cm ² . Preferably the transfection reagent is a polymer. More preferably, the polymer is a cationic polymer. Preferably the transfection reagent contains a degradable cationic polymer. More preferably, the degradable cationic polymer is made by linking a cationic compound or oligomer with a degradable linker. Transfection reagents may contain both degradable cationic polymers and non-degradable cationic polymers. Preferably, the ratio of non-degradable cationic polymer to degradable cationic polymer is 1: 0.5 to 1:20 (non-degradable: degradable) by weight.

In a preferred embodiment, the transfection reagent contains one or more cleavable linker molecules linking the cationic molecules in a branched arrangement with a plurality of cationic molecules, wherein the cationic molecules are selected from:

(i) a cationic compound of formula (A) or (B) or a combination thereof:

Wherein R ₁ is a hydrogen atom, alkyl of 2 to 10 carbon atoms, another Formula A, or Formula B,

R ₂ is a straight chain alkylene group of the formula-(CH ₂ ) _a- (a is an integer from 2 to 10),

R ₃ is a straight or branched chain alkylene group of the formula-(C _b H _2b )-(b is an integer from 2 to 10),

R ₄ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, another formula A, or formula B,

R ₅ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, another formula A, or formula B,

R ₆ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, formula A, or another formula B,

R ₇ is a straight or branched chain alkylene group of the formula-(C _c H _2c )-(c is an integer from 2 to 10),

R ₈ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, formula A, or another formula B);

(ii) cationic dendritic or branched polyamidoamines (PAMAM) with terminated primary or secondary amino groups;

(iii) cationic polyamino acids; or

(iv) cationic polycarbohydrates;

And the degradable linker molecule is represented by the formula:

A (Z) _d

(Wherein A is a spacer molecule having at least one degradable bond, Z is a reactive moiety that reacts with an amino group, d is an integer of at least 2, and A and Z are covalently bonded).

In a preferred embodiment, the cationic polymer or oligomer is poly (L-lysine) (PLL), polyethyleneimine (PEI), polypropyleneimine (PPI), pentaethyleneamine, N, N'-bis (2-aminoethyl) -1,3-propanediamine, N, N'-bis (2-aminopropyl) -ethylenediamine, spermine, spermidine, N- (2-aminoethyl) -1,3-propanediamine, N- (3-aminopropyl) -1,3-propanediamine, tri (2-aminoethyl) amine, 1,4-bis (3-aminopropyl) piperazine, N- (2-aminoethyl) piperazine, dendritic polya Midaamine (PAMAM), chitosan, or poly (2-dimethylamino) ethyl methacrylate (PDMAEMA).

In a preferred embodiment, the linker molecules are di- and multi-acrylates, di- and multi-acrylamides, di- and multi-isothiocyanates, di- and multi-isocyanates, di- and multi-epoxides, di And multi-aldehyde, di- and multi-acyl chlorides, di- and multi-sulfonyl chlorides, di- and multi-halides, di- and multi-anhydrides, di- and multi-maleimides, di- and multi- N-hydroxysuccinimide esters, di- and multi-carboxylic acids, or di- and multi-haloacetyl groups.

In a preferred embodiment, the linker molecule is 1,3-butanediol diacrylate, 1,4-butanediol diacrylate, 1,6-hexanediol diacrylate, 2,4-pentanediol diacrylate, 2-methyl- 2,4-pentanediol diacrylate, 2,5-dimethyl-2,5-hexanediol diacrylate, poly (ethylene glycol) diacrylate, trimethylolpropane triacrylate, pentaerythritol tetraacrylate, di (Trimethylolpropane) tetraacrylate, dipentaerythritol pentaacrylate, or polyester which has at least three acrylate or acrylamide side chain groups.

In a preferred embodiment, the polymer has a molecular weight of 500 da to 1,000,000 da. More preferably the polymer has a molecular weight of 2000 da to 200,000 da.

In a preferred embodiment, the cationic compound or oligomer has a molecular weight of 50 da to 10,000 da. In a preferred embodiment, the molecular weight of the linker molecule is between 100 da and 40,000 da.

Preferably, the solid support is a dish bottom, a multi-well plate or a continuous surface.

In some preferred embodiments, the transfection reagent is covalently associated with the nucleic acid. In another preferred embodiment, the transfection reagent is noncovalently associated with the nucleic acid.

In a preferred embodiment, the device can be stored at room temperature for at least 5 months without a significant decrease in transfection activity.

Embodiments of the invention include adding a solution containing a nucleic acid to be transfected to a device comprising a solid support coated with a transfection reagent mixture, adding eukaryotic cells to the solution, culturing the cell and nucleic acid solution. It relates to a cell transfection method comprising the step of allowing cell transfection. Preferably, the incubation time is 5 minutes to 3 hours. More preferably, the incubation time is 10 minutes to 90 minutes.

Preferably, the nucleic acid is a DNA, RNA, DNA / RNA hybrid or chemically modified nucleic acid. More preferably, the DNA is circular (plasmid), linear, fragment, or single stranded oligonucleotide (ODN). More preferably the RNA is single stranded (ribozyme) or double stranded (siRNA).

In some preferred embodiments, the cell is a mammalian cell. In some preferred embodiments, at least some of the cells undergo cell division. In some preferred embodiments, the cells are transformed cells or primary cells. In some preferred embodiments, the cells are somatic or stem cells. In some preferred embodiments, the cell is a plant cell.

Other aspects, features and advantages of the present invention will become apparent from the following detailed description of preferred embodiments.

These and other features of the invention will now be described with reference to the preferred implementation drawings, which are given for illustrative purposes but do not limit the invention.
1 shows the cell morphology of the transfected 293 cells. Transfection reagent treatments are linear polyethyleneimine (L-PEI) based polymers, lipid based polymers, degradable cationic polymers, and untreated (original 293 cells).
2 is the ratio of EGFP-positive cells.
3 shows the cell state after transfection.
4 shows the stability of transfectable cell incubators in mylar bags with O ₂ absorbent.
5 shows the stability of Mylar bag transfectable cell incubators with CO ₂ absorbent.
6 shows the stability of Mylar bag transfectable cell incubators with O ₂ and CO ₂ absorbents.
7 shows the stability of Mylar bag transfectable cell incubators.

While the described embodiments represent preferred embodiments of the invention, it will be understood that modifications may be made by those skilled in the art without departing from the spirit of the invention. Accordingly, the scope of the invention is to be determined only by the appended claims.

Embodiments of the present invention relate to transfection devices and methods that are simple, convenient and efficient compared to conventional transfection assays. The transfection device is constructed according to the method described herein by attaching the transfection reagent to the solid surface of the cell incubator. By using this device, researchers only need to attach the nucleic acid or other biomolecules and cells to be transfected to the surface of the cell incubator. There is no need to premix DNA or biomolecules with transfection reagents. This eliminates the time consuming step, which is a key requirement in conventional transfection procedures. In only about 40 minutes, the entire transfection process for 10 samples can be completed in comparison to the 2 to 5 hours or more required by the current method. This is particularly advantageous for high power transfection assays where hundreds of samples are tested simultaneously.

Compared with conventional transfection, the method described herein has several advantages. The method provides a transfection device that is very easy to store and provides a simple method for biomolecule transport or gene transfection that does not require any biomaterial / transfection reagent mixing step. The transfection procedure described herein can be completed in a very short time, such as about 5 minutes to 3 hours, providing a high power method for transfection or drug delivery, in which a large number of samples can be transfected simultaneously. .

In a preferred embodiment, the transfection reagent, which is very easily commercialized and mass produced, is easily coated on the surface of the cell incubator. Customers, such as researchers, need only add biomolecules, such as nucleic acids of interest, directly into the cell incubator to prepare the cell incubator prior to addition of the cells. The period of incubation for a period of time allows the biomolecule and the transfection reagent to form a complex that is taken up by the cell in the next step. The cells are then seeded on the surface of the cell incubator and cultured without the need to change the medium and the cells are analyzed. No medium change is necessary during the transfection process. The method described here reduces the risk of errors by dramatically reducing the number of steps involved and thus increasing the consistency and accuracy of the system.

The composition containing the transfection reagent can be attached to any suitable surface. For example, the surface may be glass, plastic (such as polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene, polycarbonate, polypropylene), silicone, metal (such as gold), nitrocellulose, methylcellulose, PTFE or Membranes, such as cellulose, paper, biomaterials (such as proteins, gelatin, agar), tissues (such as skin, endothelial tissue, bone, cartilage), or minerals (such as hydroxyapatite, graphite) . According to a preferred embodiment, the surface can be a well of a slide (glass or poly-L-lysine coated slide) or multi-well plate.

For slides such as glass slides coated with poly-L-lysine (eg, from Sigma), the transfection reagent is immobilized on the surface and dried, followed by introduction of the nucleic acid of interest or any nucleic acid introduced into the cell. Generally, nucleic acids are dropped onto glass slides in the microarray. This slide is incubated for 30 minutes at room temperature to form a nucleic acid / transfection reagent complex on the surface of the transfection device. Nucleic acid / transfection reagent complexes produce a medium for use in high power microarrays and can be used to study hundreds to thousands of nucleic acids or other biomolecules at the same time. In another embodiment, the transfection reagent can be attached to the surface of the transfection device in separate defined regions to form a microarray of the transfection reagent. In this embodiment, biomolecules, such as nucleic acids introduced into cells, are spread on the surface of the transfection device and incubated with the transfection reagent microarray. This method can be used to screen transfection reagents or other transport reagents from thousands of compounds. The results of such screening methods can be tested through computer analysis.

In other embodiments of the invention, one or more wells of a multi-well plate may be coated with one or more transfection reagents. Plates commonly used for transfection are 96-well and 384-well plates. The transfection reagent may be applied evenly to the bottom of each well of the multi-well plate. Generally, the transfection reagent is added to the plate bottom in the range of about 0.1 to 100 μg / cm ² . Moreover, the coating amount of the transfection reagent may vary depending on the type of well plate used. For example, for 6-well plates, 12-well plates or 96-well plates, the coating concentration of the transfection reagent is preferably about 0.5 to 50 μg / cm ² , more preferably about 1 to 20 μg / cm ² For 384-well plates, the coating concentration of the transfection reagent is preferably about 0.5 to about 50 μg / cm ² , more preferably about 1 to 30 μg / cm ² . In another embodiment of the invention, the 10-cm cell culture dish is coated with a transfection reagent. The transfection reagent may be applied evenly to the bottom of the dish. The transfection reagent may be added to the bottom of the dish in the range of about 0.1 to about 100 μg / cm ² , more preferably about 0.2 to about 20 μg / cm ² .

Hundreds of nucleic acids or other biomolecules can then be added to the wells, such as by multichannel pipettes or automated machines. The result of transfection is then measured using a microplate reader. Since microplate readers are commonly used in most biomaterials laboratories, the method is very convenient for analyzing transfected cells. Multi-well plates coated with transfection reagents can be widely used in most laboratories studying gene regulation, gene function, molecular therapy and signal transduction. In addition, if different kinds of transfection reagents are coated in different wells of a multi-well plate, the plates can be used to screen many transfection or delivery reagents efficiently. Recently 1,536 and 3,456 well plates have been developed, which can also be used according to the methods described herein.

In a preferred embodiment, the transfection device can be stored in a material suitable for packaging which is plastic (eg, cellophane), elastic material, thin material, Mylar®, or other polyester film material. Storage may be done with or without oxygen and / or carbon dioxide absorbents. Transfection plates prepared as described herein can be stored for at least 5 months at room temperature while retaining significant cellular transfection activity.

Transfection reagents are preferably cationic compounds capable of introducing biomolecules such as nucleic acids into cells. Preferred embodiments are cationic oligomers such as low molecular weight polyethyleneimine (PEI). More preferably, the transfection reagent is a degradable cationic polymer. Optionally, the transfection reagent comprises a cell-targeting or intracellular-targeting moiety and / or a membrane-stabilizing component as well as a delivery enhancer.

In general, transport enhancers fall into two categories. These are viral and nonviral delivery systems. As human viruses have evolved ways of overcoming barriers to transport to the nucleic acids introduced above, viruses or viral components are useful for delivering nucleic acids to cells. In addition, degradable polymers may bind or associate with viral or nonviral proteins to enhance transfection efficiency. For example, vesicular stomatitis virus G protein (VSVG) and other peptides or proteins known to those skilled in the art can be added to the polymer to improve transfection efficiency.

Another example of a viral component useful as a transport enhancer is a hemagglutination peptide (HA-peptide). This viral peptide promotes intracellular delivery of biomolecules by endosome disruption. At the acidic pH of the endosome, this protein causes the release of biomolecules and carriers into the cytoplasm.

Non-viral delivery enhancers may be polymer based or lipid based. These are usually polycations that balance the negative charge of the nucleic acid. Polycationic polymers show important expectations as nonviral gene transport enhancers, in part due to their ability to concentrate DNA plasmids of unrestricted size and stability associated with viral vectors. Examples include peptides having portions rich in basic amine acids such as oligo-lysine, oligo-arginine or combinations thereof and polyethyleneimine (PEI). These polycationic polymers facilitate transport by condensation of DNA. Branched chain polycations such as PEI and star dendrimers can mediate both DNA condensation and endosomal release (Boussif, et al. (1995) Proc. Natl. Acad. Sci USA vol. 92: 7297-7301 ). PEI is a highly branched polymer with terminal amines that are ionizable at pH 6.9 and internal amines that are ionizable at pH 3.9, and because of this configuration, can cause changes in vesicle pH, resulting in swelling of the vesicles and consequently Leads to release from endosomal capture.

Another means of enhancing delivery is to devise ligands in the transfection reagent. Such ligands should have receptors on the cells to which they are targeted. Delivery of biomolecules into cells is then initiated by receptor recognition. When a ligand binds to its specific cellular receptor, endocytosis is stimulated. Examples of ligands that have been used with various cell types to enhance biomolecular transport include galactose, transferrin, glycoprotein asialorosomucoid, adenovirus fiber, malaria circumsporozite protein, Epidermal growth factor, papilla virus capsid, fibroblast growth factor and folic acid. In the case of the folate receptor, the bound ligand is internalized through a process called potocytosis, where the receptor binds to the ligand, the surrounding membrane is blocked from the cell surface, and then the internalized material Passes cytoplasm through the vesicle membrane (Gottschalk, et al. (1994) Gene Ther 1: 185-191).

Various reagents have been used to destroy endosomes. In addition to the HA-proteins described above, incomplete viral particles have been used as endosomolystic reagents (Cotten, et al. (July 1992) Proc. Natl. Acad. Sci. USA vol. 89: pages 6094- 6098). Nonviral reagents are amphoteric or lipid based.

The release of biomolecules such as DNA into the cytoplasm of cells can be enhanced by reagents that mediate endosomal destruction, reduce degradation, or bypass all of these processes together. Chloroquine, which raises endosome pH, has been used to reduce degradation of endocytosed material by inhibiting lysosomal hydrolase (Wagner, et al. (1990) Proc Natl Acad Sci USA vol 87: 3410-3414). Branched chain polycations such as PEI and star dendrimers also promote endosomal release as introduced above.

To completely bypass endosomal degradation, subunits of toxins such as diphtheria toxin and Pseudomonas exotoxin have been used as components of chimeric proteins that can be inserted into gene / gene carrier complexes (Uherex, et al (1998) J Biol) Chem. Vol. 273: 8835-8841). These components promote the transport of nucleic acids through the endosomal membrane and retreat through the cytoplasmic net.

Once in the cytoplasm, the nucleic acid must find its path to the nucleus. Localization to the nucleus can be enhanced by the insertion of nuclear localization signals on nucleic acid-carriers. Specific amino acid sequences that function as nuclear localization signals (NLS) have been used. NLS on the cargo-carrier complex will interact with specific nuclear transport receptor proteins located in the cytosol. Once the cargo-carrier complex is associated, it is believed that the receptor protein of the complex is in multiple contact with nucleoporins, thereby transporting the complex through nuclear pores. After the cargo-carrier complex reaches its destination, it dissociates to release cargo and other ingredients.

The lower sequences from the SV40 giant T-antigen have been used to transport to the nucleus. This short sequence from the SV40 large T-antigen acts as a signal causing the transport of dissociated macromolecules into the nucleus.

Biodegradable cationic polymers typically show low cytotoxicity but also low transfection efficiency due to rapid degradation, making them somewhat less competitive for gene transfection and other carriers for other applications. These degradable cationic polymers improve transfection efficiency by combining low molecular weight cationic compounds or oligomers with degradable linkers. These linker molecules may contain biologically, physically or chemically cleavable bonds such as hydrolytic bonds, reducible bonds, peptide sequences with enzymes specific for cleavage sites, pH sensitive or ultrasonically sensitive bonds. Degradation of these polymers includes, but is not limited to, physical degradation methods such as hydrolysis, enzyme digestion and photolysis.

One of the advantages of the degradable cationic polymers described herein is that the degradation of the polymer is controllable in terms of rate and site of polymer degradation based on the type and structure of the linker.

In a preferred embodiment, the transfection reagent contains at least one degradable linker molecule that connects the cationic molecule in a branched arrangement with a plurality of cationic molecules.

Cationic oligomers such as low molecular weight polyethyleneimine (PEI), low molecular weight poly (L-lysine) (PLL), low molecular weight chitosan, and low molecular weight PAMAM dendrimers can be used to make the polymers described herein. Moreover, molecules containing amines having three or more reactive sites can be used.

Cationic oligomers can be selected from, but are not limited to:

(i) a cationic compound of formula (A) or (B) or a combination thereof:

(Wherein R ₁ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, another formula A or formula B,

R ₂ is a straight chain alkylene group of the formula-(CH ₂ ) _a- (a is an integer from 2 to 10),

R ₃ is a straight or branched chain alkylene group of the formula-(C _b H _2b )-(b is an integer from 2 to 10),

R ₄ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, another formula A, or formula B,

R ₅ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, another formula A, or formula B,

R ₆ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, formula A, or another formula B,

R ₇ is a straight or branched chain alkylene group of the formula-(C _c H _2c )-(c is an integer from 2 to 10),

R ₈ is a hydrogen atom, alkyl having 2 to 10 carbon atoms, formula A, or another formula B);

(ii) cationic dendritic or branched polyamidoamines (PAMAM) with terminated primary or secondary amino groups;

(iii) cationic polyamino acids; or

(iv) cationic polycarbohydrates.

Examples of such cationic molecules include, but are not limited to, the cationic molecules shown in Table 1.

Cationic polymers as used herein may include primary or secondary amino groups that can be bound to active ligands such as sugars, peptides, proteins and other molecules. In a preferred embodiment, lactobionic acid is combined with the cationic polymer. Galactosyl units provide useful targeting molecules towards hepatocytes due to the presence of surface galactose receptors in the cells. In another embodiment, lactose is combined with a degradable cationic polymer to introduce galactosyl units onto the polymer.

Degradable linker molecules include di- and multi-acrylates, di- and multi-methacrylates, di- and multi-acrylamides, di- and multi-isothiocyanates, di-containing at least one biodegradable spacer And multi-isocyanates, di- and multi-epoxides, di- and multi-aldehydes, di- and multi-acyl chlorides, di- and multi-sulfonyl chlorides, di- and multi-halides, di- and multi- Anhydrides, di- and multi-maleimide, di- and multi-carboxylic acids, di- and multi-α-haloacetyl acids, di- and multi-N-hydroxysuccinimide esters, but include It is not limited only. The following formula describes linkers that can be used according to preferred embodiments:

A (Z) _d

Wherein A is a spacer molecule having at least one degradable bond, Z is a reactive moiety that reacts with an amino group, d is an integer of at least 2, and A and Z are covalently bonded.

While some embodiments of reactive moieties of linker molecules are illustrated in Table 2, these examples are not intended to limit the scope of the invention. The reactive moiety is selected from acryloyl, maleimide, halide, carboxyl acyl halide, isocyanate, isothiocyanate, epoxide, aldehyde, sulfonyl chloride, and N-hydroxysuccinimide ester groups or combinations thereof It is not limited only to.

The rate of degradation of the polymer can be controlled by varying the polymer composition, feed ratio and molecular weight of the polymer. For example, if bulk alkyl groups and linkers are used, the polymer will degrade slowly. Increasing the molecular weight will also in some cases result in a decrease in degradation rate. The rate of degradation of the polymer can be controlled by adjusting the ratio of cationic polymer to linker or by varying various degradable linker molecules.

Acrylate linkers are less expensive than disulfide-containing linkers because the synthesis of disulfide-containing linkers is more difficult. The acrylate linker is hydrolyzable in aqueous solution. Thus polymers containing acrylate linkers can be degraded in various environments as long as they contain water. Thus, polymers containing acrylate linkers have broad applications compared to disulfide-linker containing polymers. In addition, although the rate of degradation of the polymer with the disulfide-linker is usually the same, the rate of degradation of the polymer synthesized by the acrylate linker may vary depending on the different acrylate linkers used.

In some embodiments, the transfection reagent can be mixed with a matrix such as a protein, peptide, polysaccharide or other polymer. Proteins are gelatin, collagen, bovine serum albumin or other proteins that can be used to attach proteins to surfaces. Polymers are hydrogels, copolymers, non-degradable or biodegradable polymers and biocompatible materials. The polysaccharide can be a compound that can form a membrane and can coat a transport reagent such as chitosan. Other reagents such as cytotoxicity reducing reagents, cell binding reagents, cell growth reagents, cell stimulating reagents or cell suppression reagents and specific cell culture compounds may also be attached to the transfection apparatus along with the transfection or delivery reagents. Transfection reagents may contain both degradable cationic polymers and non-degradable cationic polymers. The ratio of non-degradable cationic polymer to degradable cationic polymer is preferably 1: 0.5 to 1:20 (non-degradable: degradable) by weight, more preferably 1: 2 to 1:10 by weight. .

According to another embodiment, gelatin-containing gelatin and transfection reagent (eg, lipid, polymer, lipid-polymer or membrane destabilizing peptide) present in a suitable solvent such as water or twice deionized water. The transfection reagent mixture can be attached to the transfection device. In other embodiments, cell culture reagents (eg, fibronectin, collagen, salts, sugars, proteins or peptides) may also be present in the gelatin-transfection reagent mixture. This mixture is spread evenly over a surface, such as a slide or multi-well plate, to produce a transfection device containing a gelatin-transfection reagent mixture. In another embodiment, different transfection reagent-gelatin mixtures may also be dropped into separate regions of the surface of the transfection device. The product obtained is completely dried under appropriate conditions such that the gelatin-transfection reagent mixture is attached to the application site of the mixture. For example, the product obtained can be dried in a specific temperature or humidity or in a vacuum-dryer.

The concentration of transfection reagent present in the mixture depends on the transfection efficiency and the cytotoxicity of the reagent. Typically there is a balance between transfection efficiency and cytotoxicity. At the concentrations at which the transfection reagent is most effective, the concentration of the transfection reagent is at an optimal level while maintaining cytotoxicity at an acceptable level. The concentration of transfection reagent typically ranges from about 1.0 μg / ml to about 1000 μg / ml. In a preferred embodiment, the concentration is about 10 μg / ml to about 600 μg / ml. Similarly, the concentration of gelatin or other matrix depends on the experiment or assay being performed, but the concentration is usually in the range of 0.01% to 0.5% (w / v) of the transfection reagent solution.

In a preferred embodiment, the molecule introduced into the cell is a nucleic acid. Nucleic acids are DNA, RNA, DNA / RNA hybrids, peptide nucleic acids (PNA), and the like. If the DNA used is present in the vector, the vector may be any plasmid (eg, plasmid carrying green fluorescent protein (GFP) gene and / or luciferase (luc) gene) or any viral based vector (eg pLXSN). It can be of type. The DNA may be a linear fragment having a promoter sequence (such as a CMV promoter) at the 5 'end of the expressed cDNA and a poly A site at the 3' end. These gene expression elements allow the subject cDNA to be transiently expressed in mammalian cells. If the DNA is a single stranded oligodeoxynucleotide (ODN), such as antisense ODN, it can be introduced into cells to control target gene expression. In embodiments using RNA, the nucleic acid may be single stranded (antisense RAN and ribozyme) or double stranded (RNA interference, SiRNA). In most cases, RNA is modified to increase its stability and to improve function in down regulation of gene expression. In peptide nucleic acids (PNAs), the nucleic acid backbone is replaced with peptides to make the molecule more stable. The methods described herein can be used to introduce nucleic acids into cells for various purposes, such as for molecular therapy, protein function studies, or molecular mechanism studies.

Under appropriate conditions, the nucleic acid solution is added to a transfection device coated with a transfection reagent to form a nucleic acid transfection reagent complex. The nucleic acid is preferably dissolved in fetal bovine serum and antibiotic free cell culture medium such as Dulbecco's Modified Eagle's Medium (DMEM). However, any suitable cell culture medium may be used including, but not limited to, minimum essential eagle, F-12 kaigan modified medium, or RPMI 1640 medium. If the transfection reagent is evenly attached to the slide, the nucleic acid solution can be dropped to a separate location on the slide. Alternatively, the transfection reagent can be dropped to a separate location on the slide and the nucleic acid solution can simply be added to cover the entire surface of the transfection device. If the transfection reagent is attached to the bottom of the multi-well plate, the nucleic acid solution is simply added to the different wells by multi-channel pipettes, automated devices or transport methods known in the art. The resulting product (transfection apparatus coated with the transfection reagent and the desired nucleic acid) is incubated at room temperature for approximately 5 to 60 minutes, more preferably 25-30 minutes to form the nucleic acid / transfection reagent complex. . In some embodiments, for example, if different nucleic acid samples are dropped in separate locations on the slide, the DNA solution will be removed to produce a surface with nucleic acid samples bound with the transfection reagent. In another embodiment, the nucleic acid solution may be maintained on the surface. Second, cells in a suitable medium such as DMEM are plated onto the surface at an appropriate density. The product obtained (surface containing biomolecules and plated cells) is maintained under appropriate conditions to allow the nucleic acid of interest to enter the plated cells. In another embodiment, the cells are mixed with biomolecules or nucleic acids. The cell / biomolecular mixture is then added to the transfection apparatus and incubated at room temperature.

Suitable cells for use according to the methods described herein include, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells, including plant and animal cells, particularly mammalian cells. In a preferred embodiment, eukaryotic cells, such as mammalian cells (eg, humans, monkeys), are provided at appropriate densities and conditions sufficient for the introduction / introduction of the nucleic acid of interest into eukaryotic cells and for the expression of DNA or the interaction of biomolecules with cellular components. , Dog, cat, bovine or rat cells), bacteria, insect or plant cells are plated in a transfection device coated with the transfection reagent and the target nucleic acid. In certain embodiments, the cells can be selected from hematopoietic cells, neurons, pancreas cells, hepatocytes, chondrocytes, osteocytes, or myocytes. These cells may be fully differentiated cells or progenitor / stem cells.

In a preferred embodiment, the eukaryotic cells are grown in Dulbecco's modified eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) with L-glutamine and phenylsilene / streptomycin (pen / strep). It was. It will be appreciated by those skilled in the art that some cells require specific nutrients, such as growth factors and amino acids, so that certain cells must be cultured in special media. Suitable media for the cultivation of specific cell types are known to those skilled in the art. The optimal density of cells depends on the cell type and the purpose of the experiment. For example, while 70-80% confluent cells are preferred for gene transfection, the optimal conditions for oligonucleotide delivery are 30-50% confluent cells. For example, if 5 × 10 ⁴ 293 cells / well are seeded in a 96 well plate, the cells will reach 90% confluence 18-24 hours after cell inoculation. For HeLa 705 cells only 1 × 10 ⁴ cells / well are needed to achieve similar confluence rates in 96 well plates.

After the cells have been seeded onto the surface containing the nucleic acid sample / transfection reagent, the cells are incubated under optimal conditions for the cell type (eg 37 ° C., 5-10% CO ₂ ). Incubation time depends on the purpose of the experiment. Typically cells are incubated for 24 to 48 hours to allow for expression of the target gene in the case of gene transfection experiments. Analysis of intracellular trafficking of intracellular biomolecules may require several to several hours of incubation time, and cells may be observed at defined times.

The results of biomolecule delivery can be analyzed by different methods. In the case of gene transfection and antisense nucleic acid delivery, target gene expression levels can be detected by reporter genes such as green fluorescent protein (GFP) gene, luciferase gene, or beta-galactosidase gene expression. The signal of GFP can be observed directly under a microscope, the activity of luciferase can be detected with a luminometer, and blue water catalyzed by β-galactosidase is observed under a microscope or on a microplate reader. Can be measured by Those skilled in the art are familiar with how these reporters function and how they can be incorporated into gene delivery systems. Nucleic acids and their products, or other biomolecules carried in accordance with the methods described herein, and targets mediated by these biomolecules may be immunofluorescent detection or enzymatic immunocytochemistry, autoradiography, or in situ hybridization. It can be measured by various methods such as. If immunofluorescence is used to detect expression of the encoded protein, a fluorescent label antibody is used that binds the target protein (eg, added to the slide under appropriate conditions for binding of the protein to the antibody). Cells containing proteins are then identified by detecting fluorescence signals. If the transported cells can mediate gene expression, target gene expression levels can also be measured by methods such as autoradiography, in-situ hybridization and in-situ PCR. However, the method of identification depends on the nature of the delivered biomolecule, its expression, the target mediated by it, and / or the final product obtained from the delivery of the biomolecule.

[Example]

Example  One

Resolvable Cationic  Preparation of polymer

Conventional procedures for the preparation of degradable cationic polymers provided the synthesis of polymers derived from polyethyleneimine oligomers (PEI-600) with molecular weight 600 and 2,4-pentanediol diacrylate (PDODA). To the vial was added 4.32 g of PEI-600 in 25 ml of methylene chloride using a pipette or syringe. 2.09 g of PDODA was added quickly to the PEI-600 solution with stirring. The reaction mixture was stirred at rt (20 ° C.) for 4 h. This reaction mixture was then neutralized by adding 50 ml of 2M HCl. The white precipitate was centrifuged, washed with methylene chloride and dried at room temperature under reduced pressure.

Example  2

Resolvable Cationic  With polymer Transfectable  Preparation of Cell Incubator

Degradable cationic polymers were prepared as indicated in Example 1. Linear polyethyleneimine (L-PEI) based polymers and substrate based polymers were used for transfection of plasmid DNA into in vitro mammalian cells to assess transfection efficiency. For L-PEI based polymers, jet PEI (Qbiogene) transfection reagent was used. Lipofectamine 2000 (Invitrogen) and N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium salt (DOTAP; Sigma-Aldrich) were used as lipid based polymers. . The degradable cationic polymer and DOTAP were dissolved in methanol and the jet PEI and Lipofectamine 2000 were diluted with deionized water. Flat bottom 96-well cell culture plates (bottom: 0.23 cm ^{2 per} BD, BD Biosciences) were treated with these polymer solutions. The actual amount attached to the bottom is as follows: (a) degradable cationic polymer; 3 μg per well, thus 9.4 μg / cm ² , (b) jet PEI; 1 μl per well, (c) lipofectamine 2000; 0.375 μg per well, (d) DOTAP; 2 and 4 pmole per well. These plates were dried at room temperature under reduced pressure and sealed in a vacuum pack until use.

Example  3

Transfection of 293 Cells by Transfectable Cell Incubator

To 25 μl of Opti-MEM I (Invitrogen) 25 or 50 ng of pEGFP-Nl plasmid (purchased from Clontech) was added to each well and held for 25 minutes at room temperature. 293 cells 5 × 10 ⁴ were then added to 100 μl of Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% calf serum (Invitrogen) and incubated at 7.5% CO ₂ and 37 ° C. After 24 to 36 hours of incubation, transfection efficiency was assessed by observing EGFP fluorescence using an epifluorescent microscope (1 × 70, Olympus).

Transfection efficiencies are shown in Table 3. Degradable cationic polymers and jet PEI, ie L-PEI based polymers, showed high transfection efficiency.

*

Example  4

Cytotoxicity Assessment

The cytotoxicity of the described method was evaluated. The cell morphology of the transfected 293 cells as compared to Example 3 was compared by microscopic observation (FIG. 1). Cells transfected with degradable polymer showed normal morphology, similar to the original 293 cells. However, cells transfected with L-PEI based polymer (Jet PEI) and lipid based polymer (Lipofectamine 2000) were circular. We concluded that degradable cationic polymers can carry genes without damaging the cells.

Example  5

Resolvable Cationic  Optimization of the amount of polymer

Various amounts of degradable cationic polymers were attached to the cell incubator and transfection efficiency was evaluated. The degradable cationic polymer was coated on 96-well cell culture plates using the same protocol as shown in Example 2. The actual amount of polymer is as follows: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10 and 20 μg per well. Transfection was then performed as described in Example 3, and the transfected cells were incubated at 7.5% CO ₂ and 37 ° C. The amount of plasmid DNA added before cell inoculation was 0.13, 0.25, 0.50 or 1.0 μg per well. After incubation for 40 hours, the proportion and cell state of the fluorescent cells were evaluated by epifluorescence microscopy. 2 shows the proportion of EGFP-positive cells after transfection. High transfection efficiency was shown at 0.25 and 0.5 μg per well of plasmid DNA (thus 0.78 to 1.6 μg / cm ² ) and 2.5 to 5.0 μg per well of degradable cationic polymer (thus 7.8 to 16 μg / cm ² ) .

Cell states in these experiments are shown in FIG. 3. Cells transfected into the plates by L-PEI and lipid based polymers were circular and aggregated. Morphology is due to cytotoxicity. Cell states were acceptable when the amount of degradable cationic polymer attached to the bottom of the plate was 2.5 to 5.0 μg per well. In addition, all the plasmid DNA we tested provided a degradable cationic polymer and good cell state when the amount was 2.5 to 5.0 μg per well.

Example  6

Stability study

There are commercially available products with a coating on the surface of the cell incubator for specific purposes, such as to aid cell growth. Normally, the coating material is a kind of protein such as collagen or fibronectin. Because they are temperature sensitive, these cell incubators require refrigeration, which is a disadvantage, especially when bulky. For this reason, stability at room temperature is an important function.

The cell culture / transfection apparatus of the present invention was tested to study its stability after long term storage. The cell transfection apparatus was prepared as described in Example 2 and vacuum sealed in a Mylar bag (Dupont), a film with oxygen barrier material and aluminum foil, with or without oxygen and carbon dioxide absorbent. Store at 25 ° C. Thereafter, the transfection efficiency was periodically measured by plasmid DNA accompanying the luciferase gene (pCMV-LUC). The procedure for the transfectable cell incubator is as described in Example 3 except the plasmid DNA is different. The luciferase activity of the cells is determined by the Dynex MLX microtiter? Transfection efficiency was measured by measuring using a plate lighter and a luciferase assay system (Promega Corp. Madison, Wis. USA).

4, 5 and 6 show changes in transfection efficiency after 25 ° C. storage with O ₂ and / or CO ₂ absorbents in Mylar bags. There was no apparent decrease in transfection efficiency after 5 months of storage. Moreover, even when the cell incubator was stored at 25 ° C. in a Mylar bag without O ₂ and / or CO ₂ absorbents, transfection efficiency was stable and significantly higher after 5 months (FIG. 7). The cell incubator of the present invention was quite stable at room temperature. The device can be stored without using special storage conditions.

Example  7

Non-degradable Cationic  Preparation of polymer

Non-degradable polymers were prepared as follows. About 5 g of polyethyleneimine (Aldrich, product number: 408727) is dissolved in 50 ml of dichloromethane, and then dissolved in 100 ml of 2.0 M hydrogen chloride in diethyl ether (Aldrich, product number: 455180) and mixed well to dissolve the polymer hydrochloride. Formed. The polymer hydrochloride was then collected by centrifugation and washed with 150 ml of diethyl ether. This washing with diethyl ether was performed twice. The precipitate obtained after washing was dried under vacuum at room temperature for 3 hours. The dry powder was then stored at 4 ° C. with a desiccant until use.

Example  8

Resolvable Cationic  Polymer and Non-degradable Cationic  With polymer

96-well Transfection  Preparation of viable cell incubators

Degradable cationic polymers were prepared as indicated in Example 1. Non-degradable cationic polymers were prepared as indicated in Example 7. Both polymers were dissolved in methanol and mixed together to form a coating solution. The final concentration of each polymer is as follows. 40 μg / ml degradable cationic polymer, 10 μg / ml non-degradable cationic polymer. Thereafter, a flat bottom 96-well cell culture plate (0.32 cm ² per bottom well; BD Bioscience) was treated with the coating solution. In fact, 25 μl of coating solution was dispensed into each well and dried under vacuum conditions to remove methanol. Under these coating conditions, 1 μg of degradable cationic polymer was attached to each well of a 96-well plate and the density of the degradable cationic polymer was 3.1 μg / cm ² . In addition, 0.25 μg of non-degradable cationic polymer was attached to each well of a 96-well plate and the density of the non-degradable cationic polymer was 0.78 μg / cm ² . In total, 1.25 μg of polymer was attached to each well of the 96-well plate, so the density of the polymer was 3.9 μg / cm ² . The cell incubator prepared in this example was vacuum sealed in a Mylar bag with desiccant and stored at room temperature until further use.

Example  9

Resolvable Cationic  Polymer and Non-degradable Cationic  With polymer

12-well Transfectable  Preparation of Cell Incubator

Degradable cationic polymers were prepared as indicated in Example 1. Non-degradable cationic polymers were prepared as indicated in Example 7. Both polymers were dissolved in methanol and mixed together to form a coating solution. The final concentration of each polymer is as follows. 80 μg / ml degradable cationic polymer, 10 μg / ml non-degradable cationic polymer. Flat bottom 12-well cell culture plates (3.8 cm ² per well bottom; BD Bioscience) were then treated with these polymer fluids. 100 μl of the coating solution was distributed to each well and dried under vacuum conditions to remove methanol. Under these coating conditions, 8.0 μg of degradable cationic polymer was attached to each well of a 12-well plate and the density of the degradable cationic polymer was 2.1 μg / cm ² and 1.0 μg of non-degradable cationic polymer was 12-. Attachment to each well of the well plate resulted in a density of 0.26 μg / cm ² of the non-degradable cationic polymer. In total, 9.0 μg of polymer was attached to each well of a 12-well plate, so the density of the polymer was 2.4 μg / cm ² . The cell incubator prepared in this example was vacuum sealed in a Mylar bag with desiccant and stored at room temperature until further use.

Example  10

Resolvable Cationic  Polymer and Non-degradable Cationic  With polymer

6-well Transfectable  Preparation of Cell Incubator

Degradable cationic polymers were prepared as indicated in Example 1. Non-degradable cationic polymers were prepared as indicated in Example 7. Both polymers were dissolved in methanol and mixed together to form a coating solution. The final concentration of each polymer is as follows. 80 μg / ml degradable cationic polymer, 10 μg / ml non-degradable cationic polymer. Subsequently, flat bottom 6-well cell culture plates (9.6 cm ² per bottom well; BD Bioscience) were treated with these coating solutions. 200 μl of the coating solution was distributed to each well and dried under vacuum conditions to remove methanol. Under these coating conditions, 16 μg of degradable cationic polymer was attached to each well of a 6-well plate, resulting in a density of degradable cationic polymer of 1.7 μg / cm ² and 2.0 μg of non-degradable cationic polymer 6-. Attachment to each well of the well plate resulted in a density of 0.21 μg / cm ² of the non-degradable cationic polymer. In total, 18 μg of polymer was attached to each well of a 6-well plate, so the density of the polymer was 1.9 μg / cm ² . The cell incubator prepared in this example was vacuum sealed in a Mylar bag with desiccant and stored at room temperature until further use.

Example  11

Resolvable Cationic  Polymer and Non-degradable Cationic  Made of polymer

96-well Transfection  By viable cell incubator Transfection

Mammalian cells were cultured in 10 cm cell culture dishes, washed with phosphate-buffered saline and treated with trypsin solution. The trypsinized cells were then diluted in appropriate cell culture medium with serum to prepare cell suspensions. The cell density used in this example is shown in Table 4.

pEGFP-Nl plasmid was diluted in Opti-MEM and the final concentration was adjusted to 10 μg / ml. 25 μl of plasmid solution was then added to each well of a 96-well transfectable cell incubator prepared as indicated in Example 8 and maintained at room temperature for 25 minutes. Thereafter, 100 μl of the cell suspension was added to the wells and incubated at 37 ° C. with 7.5% CO ₂ . After 36-48 hours of incubation, transfection efficiency was assessed by observing EGFP fluorescence using an epifluorescence microscope (1 × 70, Olympus).

Table 4 shows the proportion of cells with EGFP fluorescence in various mammalian cell lines. The 96-well transfectable cell incubator of the present invention transfected various mammalian cell lines with high efficiency.

Example  12

Resolvable Cationic  Polymer and Non-degradable Cationic  Made of polymer

12-well Transfection  By viable cell incubator Transfection

Mammalian cells were cultured in 10 cm cell culture dishes, washed with phosphate-buffered saline and treated with trypsin solution. The trypsinized cells were then diluted in appropriate cell culture medium with serum to prepare cell suspensions. The cell density used in this example is shown in Table 4.

pEGFP-Nl plasmid was diluted in Opti-MEM and the final concentration was adjusted to 5 μg / ml. 200 μl of plasmid solution was then added to each well of a 12-well transfectable cell incubator prepared as indicated in Example 9 and held for 25 minutes at room temperature. Then 1 ml of cell suspension was added to the wells and incubated at 37 ° C. with 7.5% CO ₂ . After 36-48 hours of incubation, transfection efficiency was assessed by observing EGFP fluorescence using an epifluorescence microscope (1 × 70, Olympus).

Table 4 shows the proportion of cells with EGFP fluorescence in various mammalian cell lines. The 12-well transfectable cell incubator of the present invention transfected various mammalian cell lines with high efficiency.

Example  13

Resolvable Cationic  Polymer and Non-degradable Cationic  Made of polymer

6-well Transfection  By viable cell incubator Transfection

Mammalian cells were cultured in 10 cm cell culture dishes, washed with phosphate-buffered saline and treated with trypsin solution. The trypsinized cells were then diluted in appropriate cell culture medium with serum to prepare cell suspensions. The cell density used in this example is shown in Table 4.

pEGFP-Nl plasmid was diluted in Opti-MEM and the final concentration was adjusted to 5 μg / ml. 400 μl of plasmid solution was then added to each well of a 6-well transfectable cell incubator prepared as indicated in Example 10 and maintained at room temperature for 25 minutes. 2 ml of cell suspension were then added to the wells and incubated at 37 ° C. with 7.5% CO 2. After 36-48 hours of incubation, transfection efficiency was assessed by observing EGFP fluorescence using an epifluorescence microscope (1 × 70, Olympus).

Table 4 shows the proportion of cells with EGFP fluorescence in various mammalian cell lines. The 6-well transfectable cell incubator of the present invention transfected various mammalian cell lines with high efficiency.

Those skilled in the art will appreciate that many various modifications may be made without departing from the scope of the present invention. Accordingly, it should be clearly understood that the forms of the present invention are given by way of illustration and are not intended to limit the scope of the present invention.

Claims (1)

Various amounts of degradable cationic polymer are attached to the cell incubator, the transfection efficiency is assessed, the degradable cationic polymer is coated on the 96-well cell culture plate and transfected, and the transfected cells Was cultured at 7.5% CO ₂ and 37 ° C., and the amount of plasmid DNA was 0.13, 0.25, 0.50 or 1.0 μg per well, and after 40 hours of incubation, the proportion of fluorescent cells and the cell state were evaluated by epifluorescence microscope. And a device containing a solid support coated with a composition containing a transfection reagent not bound to a biomolecule.

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