JP4739352B2 - Solid surface with immobilized degradable cationic polymer for transfecting eukaryotic cells - Google Patents

Description

  The present invention claims priority from US Provisional Patent Application No. 60 / 637,344 (filed December 17, 2004), which is incorporated herein by reference.

  Embodiments of the invention relate to devices and methods for cell transfection. Specifically, embodiments of the present invention relate to a cell transfection formulation and a cell culture device treated with the transfection formulation. The treated cell culture device can be stored at room temperature, providing a simple and rapid transfection method.

  Gene transfection methods can be used to introduce nucleic acids into cells and are useful for studying gene regulation and function. High throughput assays that can be used to screen large sets of DNA to identify those that encode products with interesting properties are particularly useful. Gene transfection is the delivery and introduction of biologically functional nucleic acid into a cell (particularly a eukaryotic cell) in such a way that the nucleic acid maintains its function within the cell. Gene transfection is widely applied in research related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy research. As cloning and cataloging of genes from higher organisms continues, researchers are striving to discover gene functions and identify gene products with desirable properties. This growing collection of gene sequences requires the characterization of gene products and analysis of gene function and the development of systematic and high-throughput approaches to other research areas in cell and molecular biology. The

  Both viral and non-viral gene carriers have been used for gene delivery. Viral vectors have been shown to have higher transfection efficiencies than non-viral carriers, but the safety of viral vectors has compromised their availability (Verma IM and Somia N. Nature 389 (1997). ), Pp. 239-242; Marhsall E. Science 286 (2000), pp. 2244-2245). Although non-viral transfection has never demonstrated the efficiency of viral vectors, non-viral transfection systems have received considerable attention because they are theoretically safe compared to viral vectors. In addition, the production of viral vectors is a complex and expensive process, which limits the application of viral vectors in vitro. Synthetic gene carriers are desirable (particularly for in vitro studies) as transfection reagents because the production of non-viral carriers is simple and cost effective compared to the production of viral carriers.

  Most non-viral vectors mimic the key features of viral cell entry to overcome cell barriers that attempt to prevent invasion of foreign genetic material. Non-viral gene vectors can be classified into a) lipoplexes, b) polyplexes, and c) lipopolyplexes based on the gene carrier backbone. A lipoplex is an assembly of nucleic acids and lipid components (usually cationic). Gene transfer by lipoplex is called lipofection. A polyplex is a complex of a nucleic acid and a cationic polymer. Lipopolyplex contains both a lipid component and a polymer component. Such DNA complexes are often further modified to contain a cell targeting moiety or intracellular targeting moiety and / or a membrane destabilizing component such as a viral protein or viral peptide or a membrane disruptive synthetic peptide. Is done. Recently, bacteria and phage have also been described as shuttles for introducing nucleic acids into cells.

  Most non-viral transfection reagents are synthetic cationic molecules that have been reported to “coat” nucleic acids through the interaction of cationic sites on the cation with anionic sites on the nucleic acid. The positively charged DNA-cationic molecule complex interacts with the negatively charged cell membrane to facilitate the passage of DNA through the cell membrane by nonspecific endocytosis (Schoffield, Brit. Microencapsulated. Bull, 51 ( 1): 56-71 (1995)). In conventional gene transfection protocols, cells are most often seeded on cell culture devices 16-24 hours prior to transfection. Transfection reagents (eg, cationic polymer carriers) and DNA are usually prepared in separate tubes and individual solutions are diluted in medium (containing no fetal calf serum or antibiotics). The solutions are then mixed by carefully and slowly adding one solution to the other while constantly vortexing the mixture. The mixture is incubated at room temperature for 15-45 minutes to form a complex between the transfection reagent and the DNA and remove serum and antibiotic residues. Prior to transfection, the cell culture medium is removed and the cells are washed with buffer. A 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 procedure, especially if the number of samples to be transfected is very large.

  There are several major problems with conventional transfection procedures. First, conventional procedures are time consuming, especially if there are many cells or genetic samples to be used for transfection experiments. Also, the results derived from general transfection procedures are difficult to reproduce due to the large number of steps required. For example, the formation of DNA-transfection reagent complexes can be affected by the concentration and volume of nucleic acids and reagents, pH, temperature, type of buffer used, length and speed of vortexing, incubation time, and other factors. receive. The same reagents and the same procedure can be followed, but different results may be obtained. Results derived from multi-step procedures are often affected by human or mechanical errors or other variations at each step. In addition, refreshing the cell culture medium after transfection can disrupt the cells, resulting in cell detachment from the surface where the cells are being cultured, resulting in variability and unpredictability of the final results. . Because of all the factors mentioned above, conventional transfection methods require highly skilled persons to perform transfection experiments or transfection assays.

  Researchers need easier and more cost-effective cell transfection methods, and high-throughput cell transfection methods are needed to efficiently transfect large numbers of samples.

  Sabatini (US 2002/0006664 A1) describes a DNA-containing composition deposited on a glass slide. However, this system can only transfect with pre-deposited DNA. This is a major disadvantage of this system. Since it only supports transfection with pre-deposited DNA, not all researchers can use the nucleic acid they want.

  US Patent Application Publication No. 2004/0138154 A1, which is incorporated herein by reference, describes a cell culture / transfection device in which transfection is mediated by a lipid polymer. US Patent Application Publication No. 2005 / 0176132A1, also incorporated herein by reference, describes a cell culture device capable of transfection with calcium salts.

  US Patent Application Publication No. 2003 / 0215395A1, which is incorporated herein by reference, describes degradable polymers that can be used for gene delivery.

  As mentioned above, conventional transfection is a technically difficult procedure that takes a long time. The following three steps are generally required: 1) Seed cells in cell culture plate or cell culture dish and incubate until sufficient confluence is reached; 2) Prepare transfection reagent / nucleic acid complex And 3) add the nucleic acid of interest together with the transfection reagent and perform further incubation. Two incubation periods are required and it usually takes more than 2 days to complete all steps. In contrast, embodiments of the present invention provide a simple procedure that requires only one incubation step. A cell culture device pre-coated with a transfection reagent allows transfection by successively adding the nucleic acid of interest and the cell culture. The transfected cells can then be cultured in the same device. In this way, the cells can be transfected and cultured in a cell culture device, and there is no need to further manipulate the cells immediately after the transfection step. Transfection efficiency is equivalent to normal transfection, but the time required for manipulation is reduced by more than one day. Embodiments of the present invention include a transfectable cell culture device that significantly reduces the effort of a transfection assay and allows transfection with any nucleic acid of interest in an easy manner with low cytotoxicity. It is. Moreover, the transfectable cell culture device of the present invention is stable even when stored at room temperature for a long time.

Embodiments of the invention are directed to devices that include 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 a polystyrene resin, an epoxy resin or glass. Preferably, the coating is on the surface of the solid support. Preferably, the coating amount of the transfection reagent is from about 0.1 to about 100 μg / cm ² . Preferably, the transfection agent is a polymer. More preferably, the polymer is a cationic polymer. Preferably, the transfection agent comprises a degradable cationic polymer. More preferably, the degradable cationic polymer is produced by linking cationic compounds or cationic oligomers with a degradable linker. The transfection agent may include both degradable 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.

［式中、Ｒ^１は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ^２は、式：−（ＣＨ_２）_ａ−の直鎖アルキレン基（式中、ａは２〜１０の整数である）であり；
Ｒ^３は、式：−（Ｃ_ｂＨ_２ｂ）−の直鎖または分岐鎖アルキレン基（式中、ｂは２〜１０の整数である）であり；
Ｒ^４は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ^５は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ^６は、水素原子、炭素原子数２〜１０のアルキル、式Ａ、またはもう一つの式Ｂであり；
Ｒ^７は、式：−（Ｃ_ｃＨ_２ｃ）−の直鎖または分岐鎖アルキレン基（式中、ｃは２〜１０の整数である）であり；そして
Ｒ^８は、水素原子、炭素原子数２〜１０のアルキル、式Ａ、またはもう一つの式Ｂである］；
（ｉｉ）末端１級または２級アミノ基を持つカチオン性樹状または分岐ポリアミドアミン（ＰＡＭＡＭ）；
（ｉｉｉ）カチオン性ポリアミノ酸；または
（ｉｖ）カチオン性多糖質（ｐｏｌｙｃａｒｂｏｈｙｄｒａｔｅ）；
から選択され、前記分解性リンカー分子は式：
Ａ（Ｚ）_ｄ
［式中、Ａは少なくとも一つの分解可能な結合を有するスペーサー分子であり、Ｚはアミノ基と反応する反応性残基であり、ｄは２以上の整数であって、ＡおよびＺは共有結合によって結合される］
によって表される。 In a preferred embodiment, the transfection reagent comprises a plurality of cation molecules and at least one degradable linker molecule linking the cation molecules in a branched manner, the cation molecules comprising:
(I) a cationic compound of formula (A) or (B) or a combination thereof:

[Wherein R ¹ is a hydrogen atom, an alkyl having 2 to 10 carbon atoms, another formula A or formula B;
R ² is a linear alkylene group of the formula: — (CH ₂ ) _a — (wherein a is an integer of 2 to 10);
R ³ is a linear or branched alkylene group of the formula: — (C _b H _2b ) —, where 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, an alkyl having 2 to 10 carbon atoms, Formula A, or another Formula B;
R ⁷ is a linear or branched alkylene group of the formula: — (C _c H _2c ) —, where c is an integer from 2 to 10; and R ⁸ is a hydrogen atom, a carbon atom number 2-10 alkyl, Formula A, or another Formula B];
(Ii) Cationic dendritic or branched polyamidoamines (PAMAM) with terminal primary or secondary amino groups;
(Iii) a cationic polyamino acid; or (iv) a cationic polysaccharide (polycarbohydrate);
Wherein the degradable linker molecule is of the formula:
A (Z) _d
[Wherein A is a spacer molecule having at least one decomposable bond, Z is a reactive residue that reacts with an amino group, d is an integer of 2 or more, and A and Z are covalent bonds. Combined by]
Represented by

  In a preferred embodiment, the cationic compound or cationic 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 polyamidoamine (PAMAM), chitosan Or poly (2-dimethylamino) ethyl methacrylate (PDMAEMA).

  In preferred embodiments, the linker molecules are di- and poly-acrylates, di- and poly-acrylamides, di- and poly-isothiocyanates, di- and poly-isocyanates, di- and poly-epoxides, Di- and polyvalent-aldehydes, di- and polyvalent-acyl chlorides, di- and polyvalent-sulfonyl chlorides, di- and polyvalent-halides, di- and polyvalent-anhydrides, di- and polyvalent-maleimides Di- and poly-N-hydroxysuccinimide esters, di- and poly-carboxylic acids, or di- and poly-a-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 having at least three acrylate or acrylamide side groups.

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

  In a preferred embodiment, the molecular weight of the cationic compound or cationic oligomer is 50 da to 10,000 da. In a preferred embodiment, the molecular weight of the linker molecule is 100 da to 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 agent binds the nucleic acid covalently. In other preferred embodiments, the transfection agent binds the nucleic acid non-covalently.

  In a preferred embodiment, the device can be stored at room temperature for at least 5 months without significant loss of 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 a eukaryotic cell to the solution, and the cells and It is directed to a method of cell transfection comprising the step of incubating a nucleic acid solution to cause cell transfection. Preferably, the incubation is from 5 minutes to 3 hours. More preferably, the incubation is between 10 minutes and 90 minutes.

  Preferably, the nucleic acid is 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 cell is a transformed cell or a primary cell. In some preferred embodiments, the cells are somatic cells or stem cells. In some preferred embodiments, the cell is a plant cell.

  Further aspects, features and advantages of the present invention will become apparent from the detailed description of the preferred embodiments set forth below.

  These and other features of the invention are described below with reference to the drawings of preferred embodiments, which are intended to illustrate the invention and not to limit the invention. Absent.

  While the embodiments described herein represent preferred embodiments of the present invention, it should be understood that variations will occur to those skilled in the art without departing from the spirit of the invention. Accordingly, the scope of the invention should be determined solely by the appended claims.

  Embodiments of the present invention are directed to transfection devices and transfection methods that are simple, convenient and efficient compared to conventional transfection assays. The transfection device is manufactured by affixing a transfection reagent onto the solid surface of the cell culture device according to the methods described herein. Using this device, the researcher need only add the nucleic acid or other biomolecule to be transfected and the cells to the surface of the cell culture device. There is no need to pre-mix DNA or biomolecules with the transfection reagent. This eliminates the time-consuming and critical steps necessary for conventional transfection procedures. Only about 40 minutes are required to complete all transfection steps for 10 samples, whereas current methods require more than 2-5 hours. This is particularly advantageous for high-throughput transfection assays that test hundreds of samples at once.

  The methods described herein have several advantages over conventional transfection. The methods described herein provide a transfection device that is significantly easier to store and provides a simple biomolecule delivery method or gene transfection method that does not require a biomaterial / transfection reagent mixing step. The transfection procedures described herein can be completed in a short period of time (eg, about 5 minutes to 3 hours), and high-throughput transfection methods or drug delivery that can transfect large numbers of samples at once Provide a method.

  In a preferred embodiment, the surface of a cell culture device that can be easily commercialized and mass produced is simply coated with a transfection reagent. A customer (eg, a researcher) can prepare a device prior to adding cells by simply adding a biomolecule, such as a nucleic acid of interest, directly to the surface of the cell culture device. By incubating for a predetermined time, the biomolecule and the transfection reagent can form a complex that is taken up by the cells in the next step. Next, the cells are seeded on the surface of the cell culture device, incubated (there is no need to change the medium), and the cells are analyzed. There is no need to change the medium during the transfection procedure. The methods described herein dramatically reduce the risk of error by reducing the number of stages involved, thus increasing the consistency and accuracy of the system.

  The composition containing the transfection agent can be affixed to any suitable surface. For example, the surface can be glass, plastic (eg, polytetrafluoroethylene, polyvinylidene difluoride, polystyrene, polycarbonate, polypropylene), silicon, metal (eg, gold), membrane (eg, nitrocellulose, methylcellulose, PTFE or cellulose), paper, biological It can be a material (eg protein, gelatin, agar), tissue (eg skin, endothelial tissue, bone, cartilage) or mineral (eg hydroxylapatite, graphite). According to a preferred embodiment, the surface may be a slide (glass slide or poly-L-lysine coated slide) or a well of a multiwell plate.

  In the case of slides such as glass slides coated with poly-L-lysine (eg, Sigma, Inc.), the transfection reagent is immobilized on the surface, dried and then introduced into the nucleic acid or cell of interest. The nucleic acid to be introduced is introduced. In general, nucleic acids are spotted in a microarray on the glass slide. Nucleic acid / transfection reagent complexes are formed on the surface of the transfection device by incubating the slides for 30 minutes at room temperature. This nucleic acid / transfection reagent complex provides a medium for high throughput microarrays that can be used to simultaneously examine hundreds to thousands of nucleic acids, or other biomolecules. In an alternative embodiment, the transfection reagent can be affixed to discrete discrete regions on the surface of the transfection device to form a transfection reagent microarray. In this embodiment, biomolecules such as nucleic acids to be introduced into the cells are spread on the surface of the transfection device and incubated with the transfection reagent microarray. This method can be used in screening transfection reagents or other delivery reagents from thousands of compounds. The results of such screening methods can be examined by computer analysis.

In another embodiment of the invention, one or more wells of a multi-well plate can be coated with one or more transfection reagents. Commonly used plates for transfection are 96 well plates and 384 well plates. Transfection reagents can be applied evenly to the bottom of each well in a multi-well plate. Generally, transfection reagent is applied to the bottom of the plate in the range of about 0.1 to about 100 μg / cm ² . Further, the amount of transfection reagent coating can vary depending on the type of well plate used. For example, 6-well plates, 12 when the well plates or 96-well plates, coated concentration of transfection reagent is preferably from about 0.5 to about 50 [mu] g / cm ^2, more preferably from about 1~20μg / cm ^2. 384 If the well plate, the coating concentration of the transfection reagent is preferably from about 0.5 to about 50 [mu] g / cm ^2, more preferably from about 1~30μg / cm ^2. In another embodiment of the invention, a 10 cm cell culture dish is coated with a transfection reagent. Transfection reagents can be applied evenly to the bottom of the dish. Transfection reagents can be applied 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 are then added to the wells, such as by a multi-channel pipette or automated machine. The transfection results are then determined using a microplate reader. Since microplate readers are commonly used in most biomedical laboratories, this is a very convenient method for analyzing transfected cells. Multiwell plates coated with transfection reagents can be widely used in most laboratories to study gene regulation, gene function, molecular therapy, and signal transduction. In addition, if different types of transfection reagents are coated on different wells of a multi-well plate, the plate can be used to efficiently screen many transfection or delivery reagents. Recently, 1,536 well plates and 3,456 well plates have been developed and can also be used according to the methods described herein.

  In a preferred embodiment, in a material suitable for packaging, which can be a plastic (eg cellophane), an elastomeric material, a thin metal, Mylar®, or other polyester film material. The transfection device is stored. For storage, oxygen and / or carbon dioxide absorbents may or may not be used. Transfection plates made as described herein can be stored at room temperature for at least 5 months while retaining significant cell transfection activity.

  The transfection reagent is preferably a cationic compound capable of introducing a biomolecule such as a nucleic acid into the cell. In a preferred embodiment, a cationic oligomer such as low molecular weight polyethyleneimine (PEI) is used. More preferably, the transfection agent is a degradable cationic polymer. Optionally, the transfection agent includes a cell targeting moiety or intracellular targeting moiety and / or a membrane destabilizing component in addition to the delivery enhancer.

  In general, delivery enhancers fall into two categories. They are viral carrier systems and non-viral carrier systems. Since human viruses have evolved ways to overcome the barriers to transport into the nucleus described above, the virus or viral component serves to transport nucleic acids into the cell. Also, transfection efficiency can be increased by conjugating or associating degradable polymers to viral or non-viral proteins. 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 that serves as a delivery enhancer is the hemagglutinin peptide (HA peptide). This viral peptide facilitates the transfer of biomolecules into cells by endosomal disruption. At the acidic pH of endosomes, this protein causes the release of biomolecules and carriers into the cytosol.

  Non-viral delivery enhancers can be polymer-based or lipid-based. They are generally polycations that serve to offset the negative charge of nucleic acids. Polycationic polymers are quite promising as non-viral gene delivery enhancers because, in part, they have the ability to condense DNA plasmids of any size and because of the safety concerns associated with viral vectors. Have been seen. Examples include peptides with regions rich in basic amino acids, such as oligolysine, oligoarginine or combinations thereof, and polyethyleneimine (PEI). These polycationic polymers facilitate transport by condensing DNA. Branched polycations such as PEI and starburst 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 a terminal amine that can be ionized at pH 6.9 and an internal amine that can be ionized at pH 3.9, and this configuration can cause changes in vesicle pH. , It results in swelling of the vesicles and ultimately release from capture by endosomes.

  Another means to enhance delivery is to design a ligand on the transfection reagent. This ligand must have a receptor on the targeted cell. Receptor recognition then initiates the delivery of biomolecules into the cell. When the ligand binds to its specific cellular receptor, endocytosis is stimulated. Examples of ligands that have been used with various cell types to enhance biomolecule transport are galactose, transferrin, glycoprotein asialo-olosomcoid, adenovirus fiber, malaria sporozoite protein, epidermal growth factor, human papillomavirus capsid, fiber Blast growth factor and folic acid. In the case of the folate receptor, the bound ligand is internalized by a process called protocytosis. In this process, after the receptor binds the ligand and the surrounding membrane is isolated from the cell surface, the internalized substance passes through the vesicle membrane and enters the cytoplasm (Gottschalk et al. (1994) Gene Ther 1: 185. -191).

  Various drugs have been used for endosome disruption. In addition to the HA protein described above, defective viral particles have also been used as endosome lysing agents (Cotten et al. (July 1992) Proc. Natl. Acad. Sci. USA vol. 89: 6094-6098). Non-viral agents are amphipathic or lipid based.

  Release of biomolecules such as DNA into the cytoplasm of cells can be enhanced by agents that mediate endosomal disruption, reduce degradation, or bypass this process collectively. Chloroquine, which raises endosomal pH, has been used to reduce degradation of endocytosed substances by inhibition of lysosomal hydrolase (Wagner et al. (1990) Proc Natl Acad Sci USA vol. 87: 3410-3414. ). As mentioned above, branched polycations such as PEI and starburst dendrimers also promote release from endosomes.

  To completely bypass endosomal degradation, toxin subunits such as diphtheria toxin and Pseudomonas exotoxin have been utilized as components of chimeric proteins that can be incorporated into gene / gene carrier complexes (Uherek et al. (1998). ) J Biol.Chem.vol.273: 8835-8841). These components facilitate shuttle transport of nucleic acids through the endosomal membrane and back through the endoplasmic reticulum.

  Once in the cytoplasm, the nucleic acid must reach the nucleus. Nuclear localization can be enhanced by including a nuclear localization signal on the nucleic acid-carrier. A special amino acid sequence that functions as a nuclear localization signal (NLS) is used. NLS on the cargo-carrier complex interacts with specific nuclear transport receptor proteins present in the cytosol. Once the cargo-carrier complex is assembled, it is believed that the receptor protein in the complex transports the complex through the nuclear pore by making multiple contacts with the nucleoporin. When the cargo-carrier complex reaches its destination, the cargo-carrier complex dissociates, releasing the cargo and other components.

  Subsequences derived from SV40 large T antigen are used for transport to the nucleus. This short sequence derived from the SV40 large T antigen acts as a signal that causes transport of the associated macromolecule to the nucleus.

  Biodegradable cationic polymers typically exhibit low cytotoxicity, but have low transfection efficiency due to rapid degradation, and therefore are less competitive against other carriers for gene transfer and other applications. These degradable cationic polymers improve transfection efficiency by linking low molecular weight cationic compounds or low molecular weight cationic oligomers with degradable linkers. Linker molecules are biologically, physically or chemically cleavable bonds, such as hydrolysable bonds, reducible bonds, peptide sequences with enzyme-specific cleavage sites, pH sensitive bonds, or ultrasound sensitive bonds. Etc. can be contained. Degradation of these polymers can be achieved by methods such as, but not limited to, hydrolysis, enzymatic digestion and physical degradation methods such as optical cleavage (photolysis).

  One advantage of the degradable cationic polymers described herein is that polymer degradation can be controlled with respect to polymer degradation rate and polymer degradation site based on the type and structure of the linker.

  In a preferred embodiment, the transfection reagent comprises a plurality of cationic molecules and at least one degradable linker molecule that links the cationic molecules in a branched manner.

  Cationic oligomers such as low molecular weight polyethylenimine (PEI), low molecular weight poly (L-lysine) (PLL), low molecular weight chitosan, and low molecular weight PAMAM dendrimers are used to make the polymers described herein. be able to. Furthermore, any amine-containing molecule with more than 3 reactive sites can be used.

［式中、Ｒ_１は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ_２は、式：−（ＣＨ_２）_ａ−の直鎖アルキレン基（式中、ａは２〜１０の整数である）であり；
Ｒ_３は、式：−（Ｃ_ｂＨ_２ｂ）−の直鎖アルキレン基（式中、ｂは２〜１０の整数である）であり；
Ｒ_４は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ_５は、水素原子、炭素原子数２〜１０のアルキル、もう一つの式Ａ、または式Ｂであり；
Ｒ_６は、水素原子、炭素原子数２〜１０のアルキル、式Ａ、またはもう一つの式Ｂであり；
Ｒ_７は、式：−（Ｃ_ｃＨ_２ｃ）−の直鎖または分岐鎖アルキレン基（式中、ｃは２〜１０の整数である）であり；そして
Ｒ_８は、水素原子、炭素原子数２〜１０のアルキル、式Ａ、またはもう一つの式Ｂである］；
（ｉｉ）末端１級または２級アミノ基を持つカチオン性樹状または分岐ポリアミドアミン（ＰＡＭＡＭ）；
（ｉｉｉ）カチオン性ポリアミノ酸；および
（ｉｖ）カチオン性多糖質
などから選択することができるが、これらに限定されるわけではない。 Cationic oligomers are, for example,
(I) a cationic compound of formula (A) or (B) or a combination thereof:

[Wherein R ₁ is a hydrogen atom, an alkyl having 2 to 10 carbon atoms, another formula A or formula B;
R ₂ is a linear alkylene group of the formula: — (CH ₂ ) _a — (wherein a is an integer of 2 to 10);
R ₃ is a linear alkylene group of the formula: — (C _b H _2b ) — (wherein b is an integer of 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, an alkyl having 2 to 10 carbon atoms, Formula A, or another Formula B;
R ₇ is a linear or branched alkylene group of the formula: — (C _c H _2c ) —, where c is an integer from 2 to 10; and R ₈ is a hydrogen atom, a carbon atom number 2-10 alkyl, Formula A, or another Formula B];
(Ii) Cationic dendritic or branched polyamidoamines (PAMAM) with terminal primary or secondary amino groups;
It can be selected from (iii) cationic polyamino acids; and (iv) cationic polysaccharides, but is not limited thereto.

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

  As used herein, cationic polymers can include primary amino groups or secondary amino groups that can be conjugated with active ligands (eg, saccharides, peptides, proteins, and other molecules). In a preferred embodiment, lactobionic acid is conjugated to a cationic polymer. Since galactose receptors exist on the surface of hepatocytes, the galactosyl unit becomes a useful targeting molecule targeting hepatocytes. In yet another embodiment, lactose is conjugated to a degradable cationic polymer to introduce galactosyl units on the polymer.

Degradable linking molecules include, for example, di- and polyvalent-acrylates, di- and polyvalent-methacrylates, di- and polyvalent-acrylamides, di- and polyvalent-containing at least one biodegradable spacer. Isothiocyanates, di- and polyvalent-isocyanates, di- and polyvalent-epoxides, di- and polyvalent-aldehydes, di- and polyvalent-acyl chlorides, di- and polyvalent-sulfonyl chlorides, di- and polyvalents. -Halides, di- and poly-anhydrides, di- and poly-maleimides, di- and poly-carboxylic acids, di- and poly-α-haloacetyl groups, and di- and poly-N-hydroxys There are succinimide esters. The following formula represents a linker that can be used according to a preferred embodiment:
A (Z) _d
[Wherein A is a spacer molecule having at least one decomposable bond, Z is a reactive residue that reacts with an amino group, d is an integer of 2 or more, and A and Z are covalent bonds. Combined by].

Several embodiments of reactive residues of the linker molecule are illustrated in Table 2. However, these examples do not limit the scope of the invention. The reactive residue can be selected from, but not limited to, acryloyl, maleimide, halide, carboxyl acyl halide, isocyanate, isothiocyanate, epoxide, aldehyde, sulfonyl chloride, and N-hydroxysuccinimide ester groups or combinations thereof. It is not done.

  The degradation rate of the polymer can be controlled by varying the polymer composition, feed ratio, and polymer molecular weight. For example, using linkers with bulky alkyl groups will cause the polymer to degrade slowly. There may also be a decrease in degradation rate as the molecular weight increases. The degradation rate of the polymer can be controlled by adjusting the ratio of the cationic polymer to the linker or by changing the various degradable linker molecules.

  Acrylate linkers are much less expensive than disulfide-containing linkers. This is because it is more difficult to synthesize disulfide-containing linkers. The acrylate linker can be hydrolyzable in any aqueous solution. Thus, polymers containing acrylate linkers can be degraded in a variety of environments as long as the environment contains water. Thus, a polymer containing an acrylate linker has a wide range of applications compared to a polymer containing a disulfide linker. Also, the degradation rate of polymers with disulfide linkers is usually the same, but the degradation rate of polymers synthesized using acrylate linkers can vary accordingly for different acrylate linkers used.

  In some embodiments, the transfection reagent can be mixed with a matrix (eg, a protein, peptide, polysaccharide, or other polymer). The protein can be gelatin, collagen, bovine serum albumin, or any other protein that can be used to anchor the protein to the surface. The polymers can be hydrogels, copolymers, non-degradable or biodegradable polymers and biocompatible materials. The polysaccharide can be any compound (eg, chitosan) that can form a membrane and coat the delivery reagent. Other reagents, such as cytotoxicity-reducing reagents, cell-binding reagents, cell growth reagents, cell-stimulating or cell-inhibiting reagents, and reagents for culturing specific cells, can be transferred together with transfection or delivery reagents. It can be fixed to the suction device. The transfection agent may include both degradable 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: 1: by weight. 10.

  According to another embodiment, the transfection reagent (eg lipid, polymer, lipid-polymer or membrane destabilizing peptide) and gelatin present in a suitable solvent (eg water or double deionized water). The gelatin-transfection reagent mixture containing can be affixed to the transfection device. In yet another embodiment, cell culture reagents (eg, fibronectin, collagen, salts, sugars, proteins, or peptides) may be present in the gelatin-transfection reagent mixture. The mixture is spread evenly on a surface (eg, a slide or multiwell plate), resulting in a transfection surface carrying the gelatin-transfection reagent mixture. As an alternative embodiment, different transfection reagent-gelatin mixtures may be spotted in discrete areas on the surface of the transfection device. The resulting product is thoroughly dried under appropriate conditions so that the gelatin-transfection reagent mixture is anchored to the site where the mixture was applied. For example, the resulting product can be dried at 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 cytotoxicity of the reagent. Typically, a balance is achieved between transfection efficiency and cytotoxicity. When the transfection reagent is at the most efficient concentration while maintaining cytotoxicity at an acceptable level, the concentration of the transfection reagent is at an optimal level. The concentration of transfection reagent will generally range from about 1.0 μg / ml to about 1000 μg / ml. In preferred embodiments, the concentration is from 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 generally 0.01% to 0.5% (w / v) of the transfection reagent solution. Would be in the range.

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

  A nucleic acid transfection reagent complex is formed by adding a nucleic acid solution to a transfection device coated with a transfection reagent under appropriate conditions. The nucleic acid is preferably dissolved in cell culture medium without fetal bovine serum and antibiotics, such as Dulbecco's Modified Eagle Medium (DMEM). However, any suitable cell culture medium is used, including but not limited to, for example, minimal essential Eagle's medium, F-12 Kaine's modified medium, or RPMI 1640 medium. be able to. If the transfection reagent is evenly fixed on the slide, the nucleic acid solution can be spotted at discrete locations on the slide. Alternatively, the transfection reagent may be spotted at discrete locations on the slide, in which case the nucleic acid solution can simply be added to cover the entire surface of the transfection device. If the transfection reagent is affixed to the bottom of the multi-well plate, the nucleic acid solution is added into the different wells simply by a multi-channel pipette, an automated device, or other delivery methods well known in the art. Nucleic acid / transfection reagent by incubating the resulting product (transfection device coated with transfection reagent and desired nucleic acid) at room temperature for about 5-60 minutes, more preferably 25-30 minutes A complex is formed. In some embodiments, for example when different nucleic acid samples are spotted at discrete locations on the slide, the surface carrying the nucleic acid sample complexed with the transfection reagent is removed by removing the DNA solution. can get. In other alternative embodiments, the nucleic acid solution can remain on the surface. The appropriate density of cells in an appropriate medium (eg, DMEM) is then seeded on the surface. The resulting product (the surface carrying the biomolecule and the sowed cell) is maintained under conditions that result in entry of the nucleic acid of interest into the soaked cell. In an alternative embodiment, the cells are mixed with biomolecules or nucleic acids. The cell / biomolecule mixture is then added to the transfection device and incubated at room temperature.

  Suitable cells for use in accordance with the methods described herein include prokaryotes, yeast, or higher eukaryotic cells (including plant cells and animal cells, particularly mammalian cells). In preferred embodiments, eukaryotic cells, such as mammalian cells (eg, human, monkey, dog, cat, bovine, or murine cells), bacterial cells, insect cells or plant cells, with a transfection reagent and a nucleic acid of interest. The coated transfection device is plated at a density and suitable conditions sufficient for introduction / invasion of the nucleic acid of interest into the eukaryotic cell and expression of DNA or interaction of the biomolecule with cellular components. In certain embodiments, the cells can be selected from hematopoietic cells, neuronal cells, pancreatic cells, hepatocytes, chondrocytes, bone cells, or muscle cells. The cells can be fully differentiated cells or progenitor / stem cells.

In a preferred embodiment, eukaryotic cells are grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) with L-glutamine and penicillin / streptomycin (pen / strep). One skilled in the art will appreciate that certain cells should be cultured in a special medium, as some cells require special nutrients such as growth factors and amino acids. Suitable media for culturing individual 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, 70-80% confluent cell populations are preferred for gene transfection, but for oligonucleotide delivery, the optimal conditions are 30-50% confluent cells. For example, if 5 × 10 ⁴ cells / well of 293 cells are seeded on a 96-well plate, those cells will reach 90% confluence 18-24 hours after cell seeding. In the case of HeLa 705 cells, only 1 × 10 ⁴ cells / well are required to reach a similar confluent percentage in a 96 well plate.

After seeding the cells on the surface containing the nucleic acid sample / transfection reagent, the cells are incubated under conditions optimal for that cell type (eg, 37 ° C., 5-10% CO ₂ ). The incubation time depends on the purpose of the experiment. For gene transfection experiments, cells are typically incubated for 24-48 hours in order for the cells to express the target gene. In analysis of intracellular transport of biomolecules in cells, incubation for several minutes to several hours may be required, and cells can be observed at a predetermined time point.

  The results of biomolecule delivery can be analyzed in various ways. In the case of gene transfection and antisense nucleic acid delivery, the target gene expression level can be detected by a reporter gene (such as by green fluorescent protein (GFP) gene, luciferase gene, or β-galactosidase gene expression). The signal of GFP can be observed directly under a fluorescence microscope, the activity of luciferase can be detected by a luminometer, and the blue product catalyzed by β-galactosidase can be observed under a microscope or by a microplate reader Can be determined. It is well known to those skilled in the art how these reporters function and how they can be introduced into gene delivery systems. Nucleic acids and their products, or other biomolecules delivered according to the methods described herein, and targets modulated by these biomolecules, for example, detection of immunofluorescence, or enzyme immunohistochemistry, autoradiography, Alternatively, it can be determined by various methods such as in situ hybridization. If immunofluorescence is used to detect expression of the encoded protein, a fluorescently labeled antibody that binds the target protein is used (eg, 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 is capable of modulating gene expression, target gene expression levels can also be determined by methods such as autoradiography, in situ hybridization, and in situ PCR. However, the identification method will depend on the nature of the biomolecules to be delivered, their expression products, the target regulated thereby, and / or the end product resulting from the delivery of the biomolecule.

Production of degradable cationic polymer General procedure for producing degradable cationic polymer, synthesis of polymer derived from 600 molecular weight polyethyleneimine oligomer (PEI-600) and 2,4-pentanediol diacrylate (PDODA) It describes as. 4.32 g of PEI-600 in 25 ml of methylene chloride was added to the vial using a pipette or syringe. 2.09 g PDODA was quickly added to the PEI-600 solution with stirring. The reaction mixture was stirred at room temperature (20 ° C.) for 4 hours. The 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.

Production of Transfectable Cell Culture Device Using Degradable Cationic Polymer As shown in Example 1, a degradable cationic polymer was produced. To assess transfection efficiency, plasmid DNA was transfected into mammalian cells in vitro using linear polyethyleneimine (L-PEI) -based polymers and lipid-based polymers. As the L-PEI polymer, jetPEI (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 jetPEI and Lipofectamine 2000 were diluted with deionized water. Flat bottom 96-well cell culture plates (bottom area: 0.32 cm ² per well; BD Biosciences) were treated with these polymer solutions. The actual amount adhered to the bottom was as follows: (a) Degradable cationic polymer; 3 μg per well and thus 9.4 μg / cm ² , (b) jetPEI; 1 μl per well, (c) Lipofect Amine 2000; 0.375 μg per well, (d) DOTAP; 2 and 4 pmoles per well. The plates were dried at room temperature under reduced pressure and sealed in a vacuum pack until used.

Transfection with transfectable cell culture equipment for 293 cells 25 or 50 ng of pEGFP-N1 plasmid (purchased from Clontech) in 25 μl of opt-MEM I (Invitrogen) was added to each well and kept at room temperature for 25 minutes. Next, 5 × 10 ⁴ 293 cells in 100 μl Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) containing 10% bovine serum (Invitrogen) were added and incubated at 37 ° C. under 7.5% CO ₂ . After incubation for 24-36 hours, transfection efficiency was estimated by observing EGFP fluorescence using an epifluorescence microscope (IX70, Olympus).

The transfection efficiency is shown in Table 3. Degradable cationic polymers and jetPEI (ie L-PEI based polymers) showed high transfection efficiency.

Evaluation of cytotoxicity The cytotoxicity of the described method was evaluated. The cell shape of 293 cells transfected as shown in Example 3 was compared by microscopic observation (FIG. 1). Cells transfected with the degradable polymer showed normal morphology similar to intact 293 cells. However, those transfected with L-PEI-based polymer (jetPEI) and lipid-based polymer (Lipofectamine 2000) were rounded. We conclude that degradable cationic polymers can deliver genes without damaging cells.

Optimization of the amount of degradable cationic polymer Various amounts of degradable cationic polymer were fixed on the cell culture apparatus, and transfection efficiency was evaluated. A 96-well cell culture plate was coated with a degradable cationic polymer according to the same protocol as shown in Example 2. The actual polymer amounts were as follows: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10, and 20 μg per well. Next, transfection was performed as described in Example 3, and the transfected cells were incubated at 37 ° C. under 7.5% CO ₂ . The amount of plasmid DNA added before seeding the cells was 0.13, 0.25, 0.50 or 1.0 μg per well. After 40 hours of incubation, the percentage of cells that fluoresce and the state of the cells were estimated by epifluorescence microscopy. The percentage of EGFP positive cells after transfection is shown in FIG. High transfection efficiencies are 0.25 and 0.5 μg (and thus 0.78-1.6 μg / cm ² ) of plasmid DNA per well and 2.5-5.0 μg (and thus 7.8- Assigned to degradable cationic polymers in the range of 16 μg / cm ² ).

  The state of the cells in these experiments is shown in FIG. Cells transfected with plates using L-PEI polymer and lipid polymer had a rounded shape and were aggregated. These forms were due to cytotoxicity. When the amount of degradable cationic polymer fixed to the bottom of the plate was 2.5 to 5.0 μg per well, the cell condition was acceptable. Moreover, under any plasmid DNA conditions tested by the present inventors, a good cell state was obtained with the degradable cationic polymer if the amount was 2.5 to 5.0 μg per well.

Stability testing Products with a coating on the surface of cell culture devices are sold for special purposes (eg to aid cell growth). Usually, the coating material is a kind of protein such as collagen or fibronectin. Because they are sensitive to temperature, these cell culture devices require refrigerated storage, which is a disadvantage (especially when they are bulky). Therefore, stability at room temperature is an important feature.

  The cell culture / transfection device of the present invention was tested to determine its stability after long-term storage. A cell transfection device is manufactured as described in Example 2 and is placed in a Mylar bag (Dupont Corp.), which is a film containing an oxygen barrier material and aluminum foil, with oxygen and carbon dioxide absorbent, or oxygen And vacuum sealed without carbon dioxide absorbent. Stored at 25 ° C. Next, transfection efficiency was periodically tested using plasmid DNA carrying the luciferase gene (pCMV-LUC). The procedure for the transfectable cell culture apparatus was as described in Example 3 except that the plasmid DNA was different. To determine transfection efficiency, the luciferase activity of the cells was determined using a Dynax MLX Microtiter® plate luminometer and a luciferase assay system (Promega Corp., Madison, Wis., USA).

4, 5 and 6 show the change in transfection efficiency after storage at 25 ° C. with O ₂ and / or CO ₂ absorbent in Mylar bags. There was no apparent decrease in transfection efficiency after 5 months of storage. Furthermore, even when the cell culture apparatus was kept at 25 ° C. in Mylar bags without O ₂ and / or CO ₂ absorbent, the transfection efficiency was stable after 5 months and still very high (FIG. 7). The cell culture device of the present invention is extremely stable at room temperature. This device can be stored without special storage conditions.

Preparation of non-degradable cationic polymer A non-degradable polymer was prepared as follows: After dissolving approximately 5 g of polyethyleneimine (Aldrich, product number: 408727) in 50 ml of dichloromethane, a 2.0 M hydrogen chloride-diethyl ether solution (Aldrich) was prepared. , Product number: 455180) 100 ml was added and mixed well to form the polymer hydrochloride. The polymer hydrochloride was then collected in a centrifuge and rinsed with 150 ml of diethyl ether. This rinsing with diethyl ether was performed twice. The precipitate obtained after rinsing was dried under reduced pressure conditions at room temperature for 3 hours. The dried powder was then stored at 4 ° C. with a desiccant until use.

Production of Transfectable 96-well Cell Culture Device Using Degradable Cationic Polymer and Non-degradable Cationic Polymer A degradable cationic polymer was produced as shown in Example 1. The non-degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was 40 μg / ml degradable cationic polymer and 10 μg / ml non-degradable cationic polymer. Next, flat bottom 96-well cell culture plates (bottom area: 0.32 cm ² per well; BD Biosciences) were treated with these coating solutions. In practice, 25 μl of coating solution was dispensed into each well and dried under reduced pressure to remove methanol. Under these coating conditions, 1 μg of degradable cationic polymer was affixed to each well of the 96-well plate, resulting in a density of degradable cationic polymer of 3.1 μg / cm ² . Moreover, since 0.25 μg of non-degradable cationic polymer was fixed on each well of the 96-well plate, the density of the non-degradable cationic polymer was 0.78 μg / cm ² . A total of 1.25 μg of polymer was affixed to each well of the 96-well plate, resulting in a polymer density of 3.9 μg / cm ² . The cell culture device produced in this example was vacuum sealed in a mylar bag with desiccant and stored at room temperature until further use.

Production of Transfectable 12-well Cell Culture Device Using Degradable Cationic Polymer and Non-degradable Cationic Polymer A degradable cationic polymer was produced as shown in Example 1. The non-degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was 80 μg / ml degradable cationic polymer and 10 μg / ml non-degradable cationic polymer. Next, flat bottom 12-well cell culture plates (bottom area: 3.8 cm ² per well; BD Biosciences) were treated with these polymer solutions. Methanol was removed by dispensing 100 μl of the coating solution into each well and drying under reduced pressure. Under these coating conditions, 8.0 μg of degradable cationic polymer was affixed to each well of the 12-well plate, resulting in a density of degradable cationic polymer of 2.1 μg / cm ² and 1.0 μg of non-degradable. Since the cationic polymer was anchored on each well of the 12-well plate, the density of the non-degradable cationic polymer was 0.26 μg / cm ² . A total of 9.0 μg of polymer was affixed to each well of the 12-well plate, resulting in a polymer density of 2.4 μg / cm ² . The cell culture device produced in this example was vacuum sealed in a mylar bag with desiccant and stored at room temperature until further use.

Production of Transfectable 6-well Cell Culture Device Using Degradable Cationic Polymer and Non-degradable Cationic Polymer A degradable cationic polymer was produced as shown in Example 1. The non-degradable cationic polymer was obtained as described in Example 7. Both polymers were dissolved in methanol and mixed together to make a coating solution. The final concentration of each polymer was 80 μg / ml degradable cationic polymer and 10 μg / ml non-degradable cationic polymer. Next, flat bottom 6-well cell culture plates (bottom area: 9.6 cm ² per well; BD Biosciences) were treated with these coating solutions. 200 μl of the coating solution was dispensed into each well and dried under reduced pressure conditions to remove methanol. Under these coating conditions, 16 μg of degradable cationic polymer was affixed to each well of the 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. Was fixed on each well of the 6-well plate, resulting in a non-degradable cationic polymer density of 0.21 μg / cm ² . A total of 18 μg of polymer was affixed to each well of the 6-well plate, resulting in a polymer density of 1.9 μg / cm ² . The cell culture device produced in this example was vacuum sealed in a mylar bag with desiccant and stored at room temperature until further use.

Transfected 96-well cell culture devices transfected with degradable and non-degradable cationic polymers Transfected mammalian cells are incubated in 10 cm cell culture dishes, rinsed with phosphate buffered saline, trypsin solution Was processed. Next, a cell suspension was prepared by diluting the trypsinized cells in an appropriate cell culture medium containing serum. The cell density used in this example is shown in Table 4.

The pEGFP-N1 plasmid was diluted in opti-MEM and the final concentration was adjusted to 10 μg / ml. Next, 25 μl of the plasmid solution was added to each well of a transfectable 96-well cell culture device prepared as shown in Example 8 and kept at room temperature for 25 minutes. Next, 100 μl of cell suspension was added to the wells and incubated at 37 ° C. under 7.5% CO ₂ . After incubation for 36-48 hours, transfection efficiency was estimated by observing EGFP fluorescence using an epifluorescence microscope (IX70, Olympus).

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

Transfected mammalian cells transfected with a degradable cationic polymer and non-degradable cationic polymer transfected 12-well cell culture device Incubate in 10cm cell culture dish, rinse with phosphate buffered saline, trypsin solution Was processed. Next, a cell suspension was prepared by diluting the trypsinized cells in an appropriate cell culture medium containing serum. The cell density used in this example is shown in Table 4.

The pEGFP-N1 plasmid was diluted in opti-MEM and the final concentration was adjusted to 5 μg / ml. Next, 200 μl of the plasmid solution was added to each well of a transfectable 12-well cell culture device prepared as shown in Example 9 and kept at room temperature for 25 minutes. Next, 1 ml of cell suspension was added to the wells and incubated at 37 ° C. under 7.5% CO ₂ . After incubation for 36-48 hours, transfection efficiency was estimated by observing EGFP fluorescence using an epifluorescence microscope (IX70, Olympus).

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

Transfected mammalian cells transfected with degradable cationic polymer and non-degradable cationic polymer transfected 6-well cell culture device Incubate in 10cm cell culture dish, rinse with phosphate buffered saline, trypsin solution Was processed. Next, a cell suspension was prepared by diluting the trypsinized cells in an appropriate cell culture medium containing serum. The cell density used in this example is shown in Table 4.

The pEGFP-N1 plasmid was diluted in opti-MEM and the final concentration was adjusted to 5 μg / ml. Next, 400 μl of the plasmid solution was added to each well of a transfectable 6-well cell culture device produced as shown in Example 10 and kept at room temperature for 25 minutes. Next, 2 ml of cell suspension was added to the wells and incubated at 37 ° C. under 7.5% CO ₂ . After incubation for 36-48 hours, transfection efficiency was estimated by observing EGFP fluorescence using an epifluorescence microscope (IX70, Olympus).

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

  Those skilled in the art will appreciate that many different modifications can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the above forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.

It is a figure which shows the cell shape of the transfected 293 cell. Transfection agent treatment was linear polyethyleneimine (L-PEI) -based polymer, lipid-based polymer, degradable cationic polymer, and untreated (intact 293 cells). It is a figure which shows the percentage of an EGFP positive cell. It is a figure which shows the state of the cell after transfection. FIG. 6 shows the stability of a transfectable cell culture device placed in a Mylar bag with an O ₂ absorbent. FIG. 6 shows the stability of a transfectable cell culture device placed in a mylar bag with a CO ₂ absorbent. FIG. 6 shows the stability of a transfectable cell culture device placed in a mylar bag with O ₂ and CO ₂ absorbent. It is a figure which shows the stability of the cell culture apparatus which can be transfected in the mylar bag.

Claims (29)

An apparatus for transfection of a cell comprising a solid support coated only with a composition consisting of a transfection reagent not complexed with a biomolecule,
The transfection reagent includes a plurality of cationic molecules and at least one degradable linker that links the cationic molecules in a branched manner, and the cationic molecules include:
(I) a cationic compound of formula (A) or (B) or a combination thereof:

[Wherein R ¹ is a hydrogen atom, an alkyl having 2 to 10 carbon atoms, another formula A or formula B;
R ² is a linear alkylene group of the formula: — (CH ₂ ) _a — (wherein a is an integer of 2 to 10);
R ³ is a linear alkylene group of the formula: — (C _b H _2b ) — (wherein b is an integer of 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, an alkyl having 2 to 10 carbon atoms, Formula A, or another Formula B;
R ⁷ is a linear alkylene group of the formula: — (C _c H _2c ) —, where c is an integer from 2 to 10;
R ⁸ is a hydrogen atom, an alkyl having 2 to 10 carbon atoms, Formula A, or another Formula B];
(Ii) Cationic dendritic or branched polyamidoamines (PAMAM) with terminal primary or secondary amino groups;
(Iii) a cationic polyamino acid; and
(Iv) a cationic polysaccharide;
The degradable linker molecule is selected from the group consisting of:
A (Z) _d
[Wherein A is a spacer molecule having at least one decomposable bond, Z is a reactive residue that reacts with an amino group, d is an integer of 2 or more, and A and Z are covalent bonds. Combined by]
And the device can be stored at room temperature for at least 5 months without significant loss of transfection activity.   The apparatus of claim 1, wherein the solid support is selected from the group consisting of polystyrene resin, epoxy resin and glass.   The apparatus of claim 1 wherein the coating is on the surface of a solid support. The coating amount of the transfection reagent 0. The apparatus of claim 3, wherein the apparatus is 1 to 100 μg / cm ² . The device of claim 1, wherein the transfection reagent is a cationic polymer. The device of claim 1 , wherein the transfection reagent comprises a degradable cationic polymer . The apparatus of claim 6 , wherein the degradable cationic polymer comprises a cationic compound or a cationic oligomer linked by one or more degradable linkers . The apparatus of claim 6 , wherein the transfection reagent further comprises a non-degradable cationic polymer . 9. The device of claim 8, wherein the ratio of non-degradable cationic polymer to degradable cationic polymer is from 1: 0.5 to 1:20 by weight . The cationic compound or the cationic 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 polyamidoamine (PAMAM), chitosan, and poly (2 -Selected from the group consisting of -dimethylamino) ethyl methacrylate (PDMAEMA). Device. Linker molecules are di- and poly-acrylates, di- and poly-acrylamides, di- and poly-isothiocyanates, di- and poly-isocyanates, di- and poly-epoxides, di- and polyvalents. Aldehydes, di- and poly-acyl chlorides, di- and poly-sulfonyl chlorides, di- and poly-halides, di- and poly-anhydrides, di- and poly-maleimides, di- and poly 8. The device of claim 7 , wherein the device is selected from the group consisting of: valence-N-hydroxysuccinimide ester, di- and polyvalent-carboxylic acid, and di- and polyvalent-a-haloacetyl groups . 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 penta 8. The apparatus of claim 7, selected from the group consisting of acrylates and polyesters having at least three acrylate or acrylamide side groups. The apparatus of claim 7, wherein the molecular weight of the polymer is between 500 da and 1,000,000 da . The apparatus of claim 7, wherein the molecular weight of the polymer is between 2000 da and 200,000 da . The apparatus according to claim 7, wherein the molecular weight of the cationic compound or cationic oligomer is from 50 da to 10,000 da . The apparatus of claim 7, wherein the molecular weight of the linker molecule is from 100 da to 40,000 da . The apparatus of claim 1 , wherein the solid support is a dish bottom or a multi-well plate . A method of cell transfection comprising:
  Adding to the apparatus of claim 1 a solution containing the nucleic acid to be transfected;
  Adding eukaryotic cells to the device; and
  Incubating the cell and nucleic acid solution to cause cell transfection
Including methods. 19. The method of claim 18, wherein the incubation is from 5 minutes to 3 hours. 19. The method of claim 18, wherein the incubation is from 10 minutes to 90 minutes. 19. The method of claim 18, wherein the nucleic acid is selected from the group consisting of DNA, RNA, DNA / RNA hybrids, and chemically modified nucleic acids. 24. The method of claim 21, wherein the DNA is a circular plasmid, linear, fragment or single stranded oligodeoxynucleotide. 24. The method of claim 21, wherein the RNA is a single stranded ribozyme or a double stranded siRNA. 19. The method of claim 18, wherein the cell is a mammalian cell. 19. The method of claim 18, wherein at least some of the cells undergo cell division. 19. The method of claim 18, wherein the cell is a transformed cell or a primary cell. 19. The method of claim 18, wherein the cell is a somatic cell or a stem cell. 19. The method of claim 18, wherein the cell is a plant cell. 19. The method of claim 18, wherein the solution is added after storing the device for at least 5 months.

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