KR100667171B1 - PAMAM Derivatives for Delivery of Materials into Cells and Method for Transferring Desired Materials into Cells using The Derivatives - Google Patents

Abstract

The present invention provides a PAMAM derivative for intracellular material transfer in which all or part of the surface primary amine of the PAMAM dendrimer is grafted with at least one material selected from the group of arginine, lysine, and guanidine. The PAMAM derivatives according to the present invention efficiently carry out the transport of required substances into cells, and in particular, can function as a vector for delivering various genetic materials related to intracellular expression.

PAMAM, dendrimer, vector

Description

PAMAM Derivatives for Delivery of Materials into Cells and Method for Transferring Desired Materials into Cells using The Derivatives}

¹ H NMR Spectra (300 MHz, D ₂ O) for PAMAM, PAMAM-Arg, PAMAM-Lys

Figure 2 is a result of performing agarose gel electrophoresis of the polyplex (polymer / DNA complex) according to the present invention by changing the charge ratio [A: PAMAM-Arg, B: PAMAM-Lys]

FIG. 3A shows DNA complex formation assay results of polymers using picogreen reagents at various charge ratios [●: PEI, ▲: PAMAM, ■: PAMAM-Arg, ▼: PAMAM-Lys, Δ: PAMAM-OH]

3B shows zeta potential measurement results of polymer / DNA complexes at various charge ratios (▲: PAMAM, ■: PAMAM-Arg, ▼: PAMAM-Lys).

Figure 4 (a) (b) is a cytotoxicity test for 293 cell line and HepG2 cell line using the PAMAM derivatives (PAMAM-Arg, PAMAM-Lys) according to the present invention [(a): 293 cells, (b ): HepG2 cells, ●: PEI, ▲: PAMAM, ■: PAMAM-Arg, ▼: PAMAM-Lys]

Figure 4c is a result of the cytotoxicity test for HepG2 cell line using the PAMAM derivative (PAMAM-Gu) according to the present invention

5 is a transfection efficiency measurement result for 293 cell line and HepG2 cell line using the PAMAM derivative according to the present invention [▲: PAMAM, ■: PAMAM-Arg, ▼: PAMAM-Lys]

6 is a transfection efficiency measurement results for Neuro 2A cell line using the PAMAM derivative according to the present invention

7 is a transfection efficiency measurement results for primary rat aortic vascular smooth muscle cells using PAMAM derivatives according to the present invention

8 is a transfection efficiency measurement results for the C2C12 cell line using the PAMAM derivative according to the present invention

9 is a comparative test result of gene transfer efficiency in HepG2 cells using PAMAM derivatives according to the present invention

Figure 10 is a PAMAM derivative gene transfer animal experimental photograph according to the present invention

11 is a result of observing the difference of intracellular penetration capacity of PAMAM derivatives according to the present invention by fluorescence microscope

The present invention relates to a vector capable of delivering a desired substance into a cell. More particularly, the present invention relates to a PAMAM derivative for intracellular substance delivery that has improved transfection efficiency without cytotoxicity based on PAMAM dendrimer.

The need for effective, reliable and safe gene (RNA, DNA) delivery technologies continues to grow with the development of gene therapy. In the past decades, a lot of research and development has been conducted to find an efficient way to deliver therapeutic genes into cells for the purpose of gene therapy in humans. Numerous clinical trials reported to date have been associated with viral vector systems (retroviruses, adenoviruses) that provide effective transduction and high levels of gene expression. However, there are still some stability issues that need to be addressed, such as short-term and long-term risks such as endogenous toxicity, host immune response and the possibility of activation of oncogenes due to the inserted gene. For this reason, there is a growing demand for alternatives to viral vectors, namely non-viral vector systems, and various techniques have emerged that are considered safer and more desirable for gene delivery and clinical gene therapy. Non-viral vector systems usually use plasmid DNA alone or various types of DNA complexes such as cationic liposomes and polyionic polymers. Inefficiencies and cytotoxicity associated with currently used synthetic nonviral systems must be considered in vivo use. As a result, only very few nonviral vectors have reached clinical trials.

Many synthetic and natural cationic polymers have been introduced in nonviral vector systems and have been tested for applicability in gene therapy. Although some polymers have shown promise early in the test, their use in intracellular gene transfer has been limited by unexpected properties such as low transfection efficiency and inherent cytotoxicity in cells. Nevertheless, polyionic dendrimers still attract the attention of many scientists because of their well-defined structures and their ability to easily control surface functionality in terms of biomedical applications. To date, polyamidoamine (PAMAM) dendrimers and polyethyleneimine (PEI) dendrimers have been tested for their availability, and for PEI, relatively high in vitro with some expected results in the cells. Transfection efficiency was shown.

As mentioned above, one of the major problems with nonviral gene delivery systems compared to viral vectors is their low efficiency. In order to solve this problem, a method of linking or conjugating a cell specific ligand and an oligopeptide such as a TAT-derived peptide or an oligo arginine derivative has been attempted. Recently, some basic peptides, known as protein transduction sites (PTDs) or membrane transfer signals (MTSs), have been identified, characterized and introduced into a variety of therapeutic methods for drug, protein, oligonucleotide and plasmid DNA delivery. Interestingly, such sequences are known to contain amino acid residues usually charged with cations such as arginine, lysine. The actual mechanism is not clear and there is debate as to whether entry into the cell membrane is by endocrine pathway or non-endocytic pathway or direct membrane permeation, but a large number of groups have reported increased intracellular delivery. have. Most experiments for delivery have been carried out by covalently binding nucleic acids to signal peptides, and some experiments have been carried out by the formation of simple complexes by electrostatic attraction, but so far no satisfactory efficiency and safety It does not provide.

The present invention has been proposed to solve the problems of the prior art as described above, and an object of the present invention is to efficiently carry out the transport of the required material into cells, and in particular, to transfer various genetic materials related to intracellular expression. To provide a novel PAMAM derivative that can greatly contribute to the expression of the desired gene.

In another aspect, the present invention provides a method for delivering a desired substance including a gene into the cell using the PAMAM derivative.

In order to achieve the above object, the present invention provides a PAMAM derivative for intracellular substance transfer in which all or part of the surface primary amine of the PAMAM dendrimer is grafted with at least one substance selected from the group of arginine, lysine, and guanidine. do.

The present invention provides a PAMAM derivative for gene delivery, wherein the intracellular delivery material is one of a substance selected from the group consisting of small molecules, peptides, proteins, oligonucleotides, and plasmids.

The present invention provides a gene transfer PAMAM derivative in which all or part of the surface primary amine of the PAMAM dendrimer is preferably grafted with arginine alone.

The present invention provides a gene transfer PAMAM derivative in which all or part of the surface primary amine of the PAMAM dendrimer is preferably grafted with lysine.

The present invention provides a gene transfer PAMAM derivative in which all or part of the surface primary amine of the PAMAM dendrimer is preferably grafted with guanidine.

The present invention provides a gene delivery PAMAM derivative, characterized in that the PAMAM dendrimer is generation 10 or less, preferably generation 3 to generation 6.

The present invention provides a PAMAM derivative capable of performing intracellular selective mass transfer by using the properties of each of the PAMAM dendrimers having a large difference in intracellular permeability.

In addition, the present invention is a substance intended to be delivered to the cell and PAMAM derivatives for intracellular material shearing in which all or part of the surface primary amine of the PAMAM dendrimer is grafted with at least one substance selected from the group of arginine, lysine, and guanidine. It provides a method for delivering a substance into cells by binding.

Hereinafter, the content of the present invention in more detail as follows.

The present invention provides a novel polymer vector for efficiently delivering the required substances in the cell, and in particular, based on the PAMAM dendrimer, the primary amine group on the surface is replaced with other cationic compounds to improve the transfer efficiency of genes and the like. It provides an elevated PAMAM derivative.

According to the present invention, cationic compounds to be grafted at the position of the primary amine group present on the surface of the existing PAMAM include lysine, arginine or guanidine, which may be grafted as a single substance, and two or more components are competitively grafted. Although it may be open, the form used independently is good.

These cationic compounds may be grafted over the entire primary amine groups present on the surface of PAMAM, and some of them may be grafted, for example, about 50 to 90%.

The PAMAM which can be used in the present invention is not particularly limited, for example, generation (G) 10 or less, preferably G3 to G6.

PAMAM derivatives according to the present invention can be prepared by the following process. The protected cationic compound may be obtained through an amino acid coupling reaction to PAMAM, followed by removal of the protecting group used in the reaction, precipitation in an organic solvent, and washing with water. The obtained product is powdered through lyophilization. When arginine is used as the cationic compound in the above reaction, 4 equivalents of HOBt, HBTU, Fmoc-Arg (pbf) -OH and 7.3 equivalents of DIPEA can be carried out by mixing in anhydrous DMF solution for 4 hours at room temperature. In this case, a two-step process is required to remove the Fmoc and pbf used as protecting groups. Even when cationic compounds are used with lysine or guanidine, the overall process is the same as in the example of arginine, but the specific protecting group may be different (for example, the pbf protecting group may be Boc for lysine. Deprotectors may be used other than those used for arginine, eg TFA alone). As described above, the protecting group of the cationic compound required in the manufacturing process is different, but the same may be used depending on the conditions. In this case, two or more cationic compounds may be grafted to the terminal of the PAMAM by a single process.

The PAMAM derivative prepared by the above process may be applied as a use of a vector for delivering a predetermined substance into a cell. The PAMAM derivative according to the present invention has a positive charge in an aqueous solution, and thus it is possible to form a complex (polyplex, polyplex) by negatively charged materials and electrostatic attraction in the aqueous solution. This property suggests that the PAMAM derivatives of the present invention can be used as vectors for intracellular delivery. For example, for gene therapy, DNA must be expressed inside the cell (cell nucleus) as a therapeutic protein (cell transformation, transfection), but DNA alone is difficult to penetrate into the cell. Cell transformation is difficult on its own. The reason is that since the cell membrane is generally negatively charged, it is difficult for DNA with negative charge to enter the cell due to electrical repulsion. Accordingly, the PAMAM derivative according to the present invention forms a polyplex with oligonucleotides including negatively charged DNA, which is neutral or cationic as a whole, thereby facilitating penetration into cells.

The oligonucleotides include single or double chain DNA or RNA and plasmids. The oligonucleotides may be DNA or plasmids comprising RNAs encoding genes of any protein, RNA and RNAs are mRNAs, tRNAs, rRNAs as well as antisense RNAs and ribozymes complementary to the target DNA, RNA sequence of any cell. (Rybozyme). Genes encoding the protein include genes encoding polypeptides associated with the treatment or diagnosis of a disease. The polypeptide encoded by the gene preferably includes various hormones, tissue-adhesive antigens, cell adhesion proteins, cytokines, various antibodies, cellular receptors, intracellular or extracellular enzymes and fragments thereof. In addition, the oligonucleotides preferably include gene expression regulators, for example, transcriptional promoters, enhancers, silencers, operators, terminators, attenuators, and other expression regulators.

In addition to the oligonucleotide, the substance that can be delivered into the cell using the PAMAM derivative according to the present invention may be, for example, one substance selected from the group of low molecular weight compounds, peptides, and proteins. Examples of low molecular weight compounds include 5-fluorouracil, ibuprofen, paclitaxel, curcumin, chloramphenicol, actinomycin, doxorubicin, and the like. Peptides include p53 peptide, p16 peptide, p21 peptide, Smac peptide, VHL tumor suppressor peptide, Merlin, MAK19 and the like. Examples of proteins include α-interferon, β-interferon, erythropoietin, insulin, interleukin-2, human growth factor (HGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and the like.

The PAMAM derivative according to the present invention is a method of delivering a biofunctional or drug molecule such as the low molecule, peptide or protein other than the gene as follows.

1) Host-Guest

The above method includes all methods of inserting a low molecular weight (guest) into the space generated inside the PAMAM derivative host according to the present invention and transferring the same into cells, for example, drug molecules that are difficult to dissolve in an aqueous solution. Water-soluble PAMAM derivatives are mixed in an organic solvent which is dissolved in each other, and then a complex is formed by dissolving the remaining precipitate in an aqueous solution after evaporating the organic solvent. Alternatively, the complex may be prepared by dissolving both in an organic solvent mixed with water and then dispersing it in an aqueous solution. Through this method, drug molecules with extremely low solubility in aqueous solution can be dissolved in aqueous solution with high solubility, thereby enabling biochemical or medicinal applications. The size of the drug molecule to be inserted is limited by the internal space of the PAMAM derivative, and the concentration, the type of organic solvent used as the solvent, the pH of the aqueous solution, and the composition may be changed depending on the properties of the drug.

2) chemical bonding method

This method is a method of chemically binding a biofunctional molecule or the like to the surface of the PAMAM derivative according to the present invention is delivered to the cell in the form of a kind of prodrug (prodrug). The method can be largely divided into the following two types.

① stable binding in vivo

The binding method includes all the methods of bioconjugating a biofunctional molecule through a PAMAM derivative according to the present invention and a stable amide bond or -C-N- bond in vivo. First, since the surface of the PAMAM derivative is exposed to primary amines and guanidine functional groups having different pKa values, buffer conditions for binding using only primary amines are necessary. For example, in order to bind the nucleophilic reaction of the primary amine of the PAMAM derivative, the binding site functional group of the transfer material is chemically treated with a functional group activated by a leaving group such as -COOH functional group or various halogen molecules. It must be modified and bound via a direct reaction between the PAMAM derivative and the delivery material or through a coupling agent.

② biodegradable bonds

The present method is similar to the above ① method, but includes all the methods of replacing the bonds with biodegradable bonds rather than in vivo stable bonds. In this manner, for example, ester bonds degraded through hydrolysis, ortho-esters degraded at low pH in an endosome environment, acetal, hydrazone bonds, disulfide bonds degraded at intracellular reduction potential, and the like can be used. .

Hereinafter, the content of the present invention will be described in detail through examples. However, the following examples are merely illustrative for the purpose of describing the present invention and they should not be used in any sense to limit the scope of the present invention.

<Example>

(1) Experimental process

1) material

PAMAM G4 (Starburst), 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT), piperidine, N, N-dimethylformamide (DMF) , N, N-diisopropylethylamine (DIPEA), and BSA were purchased from Sigma-Aldrich Korea. N-hydroxybenzotriazole (HOBt) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium (HBTU) from Anaspec (San Jose, Calif.) Was purchased and Fmoc-L-Arg (pbf) -OH was purchased from Nova Biochem (San Diego, Calif.). Luciferase assay kit was purchased from Promega (Madison, WI). Picogreen reagent was purchased from Molecular Probes. Fetal bovine serum (FBS), grace serum (FCS), 100 × antibiotic-antifungal and Dulbecco's modified Eagle's medium (DMEM) were purchased from GIBCO (Gettysburg, MD). DMEM / F-12 was purchased from Cambrex (Walkersville, MD). Collagenase type II, eletasses, soybean trypsin inhibitors were purchased from Washington (Lakewood, NJ).

2) Synthesis of PAMAM-Arg and PAMAM-Lys

Amino acid coupling with PAMAM was performed by mixing 4 equivalents of HOBt, HBTU, Fmoc-Arg (pbf) -OH and 7.3 equivalents of DIPEA in anhydrous DMF solution for 4 hours at room temperature. The resultant was precipitated in ethyl ether and washed with excess ether. Fmoc groups of dendrimers bound to Fmoc-Arg (pbf) were removed by adding 30% piperidine to DMF (v / v). After 1 hour of deprotection the mixture was precipitated in ethyl ether and washed with excess ether. In order to deprotect the pbf group of arginine (treatment at room temperature for 6 hours), trifluoroacetic acid: triisopropylsilane: H ₂ O in a volume fraction of 95: 2.5: 2.5 was used. The final product was precipitated in ethyl ether and washed with excess ether. The product was dissolved in water and dialyzed against pure water overnight at 4 ° C. The product was then lyophilized and recovered as a white powder.

The entire process of PAMAM-lysine was also performed in the same manner as described above. However, Fmoc-Lys (Boc) -OH was used, and 90% TFA was used to remove the BOC group (1 hour at room temperature). The yield of the product was usually over 99% and the ¹ H NMR spectra (300 MHz, D ₂ O) of the polymer is shown in FIG.

3) Synthesis of PAMAM-Gu

Instead of bioconjugating arginine directly to the PAMAM dendrimer, the primary amine on the surface of the PAMAM dendrimer was converted directly to the guanidine functional group, focusing on the fact that arginine had a guanidine functional group. Potassium carbonate and 1H-pyrazole-1-carboxamidine in an amount corresponding to a 5-fold molar ratio of PAMAM dendrimer primary amine were dissolved in water, mixed with PAMAM dendrimer solution dissolved in water, and reacted at room temperature for 20 hours. Thereafter, the dialysis membrane of MWCO 1000 was dialyzed for 2 days and then purified by lyophilization.

As a result of 300 MHz ¹ H NMR, 55-61% of the PAMAM dendrimer surface primary amine was found to be converted to a guanidine functional group.

4) Plasmid Preparation

The results of gene transfection were observed using the firefly luciferase gene as the reporter gene. A fortineus pyralis luciferase expression plasmid (pCN-Luci) having a cell nuclear direct signal composed of 21 amino acids derived from SV40 large T antigen was constructed by subcloning into pCN. pCN vectors are known to be better expressed than vectors with only CMV promoter. Plasmid DNA was transferred to E.Coli TOP10 recipient cells and highly purified plasmids were isolated from Qiagen (Valencia, CA. USA) using the Plasmid Purification Mega Kit according to the manufacturer's instructions. The plasmid was precipitated in isopropanol, washed with 70% ethanol and resuspended in distilled water. DNA was stored at -20 ° C until use.

5) Agarose gel electrophoresis experiment

Complexes were formed by varying the charge ratio between the polymer and the pCN-Luciferase (pCN-Luci) plasmid for 30 minutes at room temperature in HEPES buffer (25 mM, pH 7.4, 10 mM MgCl ₂ ). Each sample was analyzed by electrophoresis on 0.7% agarose gel and stained by incubating for 1 hour in a buffer containing EtBr (0.5 μg / ml) at 37 ° C.

6) PicoGreen Essay for Polyplex Formation

Polyplexes of plasmid DNA (1.0 μg) and dendrimer were prepared in 200 μl of HEPES-buffered saline (HBS, 25 mM HEPES, 150 mM NaCl, pH 7.4) with varying charge ratios in the range of 0.5 to 30, and the mixture at room temperature. Incubate for 30 minutes. Then, 200 μl of picogreen reagent diluted in TE buffer (10 mM Tris, 1 mM EDTA, pH7.5) was added and incubated for 2 more minutes. Before measuring the fluorescence intensity with a spectrofluorometer (JASCO FP-750) the polyplexes were diluted to a total of 2 ml of TE buffer. The excitation (λ _ex ) and emission (λ _em ) wavelengths were 480 nm and 520 nm, respectively.

7) Zeta Potential and Particle Size Measurement

Zeta potential values and polyplex sizes were measured using a Malvern Zetasizer 3000HAs system (Malvern Instruments, Worcestershire, UK) using PCS 1.61 software. Polyplexes were formed in HBS (10 mM HEPES, 1 mM NaCl, pH 7.4) for zetapotent experiments and with a final concentration of 5 μg / ml plasmid DNA in water, respectively, for size determination.

8) Cell culture

Human fetal kidney 293 cells and neuro 2A (murine neuroblastoma) cells were cultured in DMEM containing 10% FBS. Human liver carcinoma HepG2 cells were cultured in MEM containing 10% FBS. Primary rat aortic vascular smooth muscle cells were cultured in DMEM / F12 containing 10% FBS. Cells were maintained on a plastic tissue dish (Falcon) at 37 ° C. in a humid environment containing 5% CO ₂ /95% air. All media contained 1 × antibiotic-antifungal agent.

9) Primary Rat Aortic Tube Smooth Muscle Cell Preparation

Cells were isolated from rat aortic vascular smooth muscle in primary culture. In summary, the enzyme mixture containing 1 mg / ml collagenase type II, 0.25 mg / ml elastase, 1 mg / ml soybean trypsin inhibitor and 2 mg / ml BSA prepared in DMEM / F12 was preheated at 37 ° C. , And incubated with the aorta obtained from 6-week-old SD rats and cut small pieces (Charles River, Japan). Enzyme treatment was carried out for 30 to 45 minutes with stirring at 37 ℃. The resulting cell suspension was centrifuged at 1000 rpm for 5 minutes at 4 ° C., and the resulting cell pellet was washed with DMEM / F12 containing 10% FCS, 100 mg / ml penicillin and 0.1 mg / ml streptomycin. Cells were then maintained in DMEM / F12 medium containing 10% FBS and 1 ′ antibiotic-antifungal agents.

10) In vitro Cytotoxicity Test

Colorimetric MTT assay was performed for cytotoxicity assay. In summary, 293 and HepG2 cells were dispensed in 96 well plates at a concentration of 1 × 10 ⁴ cells / well and incubated in 100 μl medium for 1 day before incubation with polymer. Cells were treated with PEI 25kDa, PAMAM G4, PAMAM-Arg and PAMAM-Lys for 2 days, and then 26 μl MTT stock solution (2 mg / ml) was added to each well and incubated for 4 hours. The medium was removed, 150 μl DMSO was added and the absorbance was measured at 570 nm using a microplate reader.

11) Transfection Experiments and Essays

Dispense 293 (5 × 10 ⁴ cells / well), HepG2 (5 × 10 ⁴ cells / well), and Neuro2A (1 × 10 ⁵ cells / well) cells into a 24-well plate And incubated in 600 μl of medium containing 10% FBS for 1 day before transfection. Primary rat vascular smooth muscle cells were dispensed with 4 × 10 ⁴ cells / well and incubated for 2 days prior to transfection. Polyplexes of plasmid DNA and dendrimers were prepared in 150 μl of FBS free medium under various charge ratios, and the mixture was incubated at room temperature for 30 minutes. For conditions without FBS, the medium was replaced with 600 μl of FBS free medium before transfection. After 4 hours of polyplex treatment, the medium was replaced with 600 μl medium containing 10% FBS. Cells were incubated for two more days before the assay. After removing the culture medium, the cells were washed with DPBS and digested for 30 minutes at room temperature using 150 μl reporter digestion buffer (Promega). Luciferase activity was measured using an LB9507 luminometer (Berthold, Germany). Protein content was measured using a Micro BCA assay reagent kit (Pierce, Rockford, IL).

12) Gene transfer animal experiment of PAMAM derivative

Complexes of plasmid / PAMAM-R derivatives containing the LacZ gene were injected into the brains of normal rats and then examined for gene transfer in vivo via X-Gal staining.

13) Intracellular Penetration Ability of PAMAM-Arg and PAMAM-Lys

After incubating approximately 30000 HeLa cell lines in 1.8 mL of DMEM medium, 20 μl of PAMAM-Arg and PAMAM-Lys labeled with FITC were mixed, and after 48 hours, the difference in intracellular infiltration capacity of both PAMAM derivatives was observed by fluorescence microscopy. It was.

(2) Experiment result

1) Synthesis and Characterization of PAMAM-Arg and PAMAM-Lys

Arginine-rich peptides can be provided as effective gene transfer vectors. It has also been found that oligoarginine (n = 6-9) is a membrane transport signal, and many chimeric peptides have been developed over the years. Recently, arginine dendrimers have been reported by Kasai et al. According to this, 8 or 16 arginine residues were introduce | transduced into each primary amino group of the backbone of a polyL-lysine dendrimer, and the polyl-lysine dendrimer itself and the angiogenesis inhibitory ability of cancer cells were compared. Recently, Okuda et al. Reported that high transfection efficiency can be obtained by replacing the terminal lysines of dendritic polymer-L-lysine with arginine. The present invention is available by grafting arginine residues on dendritic surfaces of PAMAM dendrimers (generation 4), which are commercially available at low prices, moderately small in molecular weight and suitably containing tertiary amines known to contribute to the endosomal buffering effect, The possibility of a strong transfection agent was investigated. Arginine was conjugated to the surface primary amine of PAMAM dendrimer and placed at each end of PAMAM to determine the effect of the introduction of arginine residues, a major unit of cell-penetrating peptides, on gene transfer efficiency. The purpose of designing and synthesizing dendrimers is that the spatially oriented extra arginine residues after complexing with DNA contribute to promoting uptake by the cell and consequently to increase transfection efficiency.

As shown in FIG. 1, the ¹ H NMR spectra of the polymer are as follows.

¹ H NMR (300 MHz, D2O) PAMAM: δ2.44 (-NCH ₂ C H ₂ CO-of PAMAM Unit, 248H), 2.63 (-CONHCH ₂ C H ₂ N- of PAMAM Unit, 120H and -NC H ₂ C H ₂ N- of PAMAM unit, 4H), 2.72 (-CONHCH ₂ C H ₂ NH ₂ -of PAMAM unit, 128H), 2.83 (-NC H ₂ CH ₂ CO- of PAMAM unit, 248H), 3.25 (- CONHC H ₂ CH ₂ N- of PAMAM unit, 248H). PAMAM-Arg: δ 1.68 (-HCCH ₂ C H ₂ CH ₂ NH- of arginine, 116H), 1.93 (-HCC H ₂ CH ₂ CH ₂ NH- of arginine, 116H), 2.63 (-NCH ₂ C H ₂ CO- of PAMAM unit, 248H), 2.83 (-CONHCH ₂ C H ₂ N- of PAMAM unit and -NC H ₂ C H ₂ N- of PAMAM unit), 2.96 (-CONHCH ₂ C H ₂ NH2- of PAMAM unit ), 3.13 (-NC H ₂ CH ₂ CO-of PAMAM unit), 3.25 (-HCCH ₂ CH ₂ C H ₂ NH- of arginine), 3.46 (-CONHC H ₂ CH ₂ N- of PAMAM unit and -CONHCH ₂ C H ₂ NHCO- of PAMAM unit), 4.01 ( -H CCH ₂ CH ₂ CH ₂ NH- of arginine, 58H). PAMAM-Lys: δ 1.45 (-CHCH ₂ C H ₂ CH ₂ CH ₂ NH ₂ -of lysine, 120H), 1.73 (-CHCH ₂ C H ₂ CH ₂ NH ₂ -of lysine, 120H), 1.89 (-CHC H ₂ CH2CH2NH2- of lysine, 120H), 2.53 (-NCH2C H 2 CO- of PAMAM _{unit, 248H), 2.81 (-CONHCH 2} C H 2 N- of PAMAM unit, and -NC H ₂ C H ₂ N- of PAMAM unit), 3.02 (-CONHCH ₂ C H ₂ NH ₂ of PAMAM unit, -NC H ₂ CH ₂ CO- of PAMAM unit and -CHCH ₂ CH ₂ CH ₂ H ₂ NH ₂ -of lysine), 3.39 (-CONHC) H ₂ N- CH ₂ of PAMAM unit and -CONHCH ₂ C H ₂ NHCO- of PAMAM _{unit), 3.92 (-C H CH 2} CH 2 CH 2 CH 2 NH 2 - of lysine, 60H).

The number of arginine and lysine residues added was calculated from ¹ H NMR data. Approximately 58 arginine molecules were bound to one PAMAM dendrimer containing 64 surface primary amines, which also matches well in the preparation of PAMAM-Lys (60 lysine per PAMAM). PAMAM-Lys was prepared and compared with the control reagent to determine the possible role for the increase of polymer MW and charge density.

2) Analysis of Complex Formation by Agarose Gel Electrophoresis and PicoGreen Reagent Assays

To assess the formation of dendrimer / DNA complexes, agarose gel electrophoresis on polyplexes was performed at different charge ratios (FIG. 2). Plasmid DNA was shown to be completely retarded at PAMAM-Arg or PAMAM-Lys and charge ratio 2. In addition, picogreen reagents have been used to evaluate polyplex formation at various charge ratios for more accurate analysis of complex formation. A commonly used method for analyzing polymer / DNA complex morphology is the EtBr exclusion assay. However, the method has some problems, which are highly dependent on the concentration of EtBr used and the sensitivity is lowered by 10 to 15 times compared to the positive control. The present invention provides a new method using phycogreen reagent as a more sensitive probe as a method for quantifying the degree of complex formation with DNA. This method has been found to be very reproducible and very sensitive to the observation of complex formation. The polymer was self-assembled with plasmid DNA by incubating for 30 minutes at room temperature before adding a buffer containing the appropriate amount of picogreen reagent. Plasmid DNA that does not form a complex is exposed to picogreen reagents, and the resulting fluorescence increases with the degree of complex formation. As shown in FIG. 3A, the inhibition of fluorescence resulting from dendrimer and precomplex formation increased with increasing charge ratio and reached flat zero near charge ratio 4. This is because the plasmid DNA was efficiently aggregated into particles and could not react with the phycogreen reagent to which the DNA was added. Interestingly, pure PAMAM was observed to form a complex completely at a lower charge ratio 2 than at least another polymer requiring a charge ratio of 4. These results indicate that pure PAMAM is more effective in forming complete complexes, and that PAMAM-Arg and PAMAM-Lys can effectively form condensed complexes as PEI and PAMAM at charge ratio 4. The surface of pure PAMAM is considered to be covered with primary amines, except for the higher pKa values compared to the alpha amines of lysine or arginine and secondary or tertiary amines of PEI, respectively, which are included in the calculation of the charge ratio. As a control, a PAMAM-OH polymer that did not form a diionic complex with DNA was also tested and the relative fluorescence was observed to remain around 100% at all weight ratios tested.

3) Zeta Potential and Size Measurement of Polyplexes with Plasmid DNA

From the agarose gel delay and picogreen assay results, it was estimated that the modified dendrimers, PAMAM-Arg and PAMAM-Lys, can form compact polyplexes. The surface charge of the composite was measured and shown in FIG. 3B. Consistent with the previous properties obtained from the picogreen assay curve based on the charge ratio in FIG. 3A, the unmodified PAMAM was 20 mV at a charge ratio of 2 compared to other complexes including DNA and modified PAMAM-Arg and PAMAM-Lys. The two modified PAMAMs were found to show less than 10 mV at the same charge ratio. In addition, the zeta potential values measured for all the polyplexes showed an equivalent value of around 20 to 25 mV at a charge ratio of 4 or more.

The formation of complexes at the nanometer level is generally considered to be one of the important factors in polyplex mediated gene delivery. The average particle size of the polyplexes was investigated by dynamic laser light scattering. As shown in Table 1, it can be seen that the polymer effectively condenses DNA into nanometer-sized particles having a size of 185-250 nm under optimized transfection conditions. Interestingly, the average size of the polyplex consisting of PAMAM and DNA was 245 nm. This was slightly larger compared to that of PAMAM-Arg / DNA, PAMAM-Lys / DNA and PEI / DNA with sizes around 200 nm. Referring to the results in Table 1 based on the previous picogreen reagent assay, the charge densities per modified dendrimer showed 16% and 25% increase for PAMAM-Arg and PAMAM-Lys, respectively, when compared to pure PAMAM. This means that the modified polymer can also completely compensate for the phosphate anions of the plasmid DNA and form mature polyplexes such as pure PAMAM or PEI. Thus, at charge ratio 6 determined to be sufficient for formation of the complete complex, PAMAM-Arg and PAMAM-Lys were able to form complexes that exposed a large number of excess surface arginine or lysine residues. In addition, no visible precipitation due to size increase was detected even when 150 μg / ml DNA was combined with PAMAM-Arg in pure or 5% glucose solution.

TABLE 1

PEI PAMAM PAMAM-Arg PAMAM-Lys MW (Da) 25000 ^a 14215 ^a 23321 ^b 21895 ^b (+) Count / Polymer 581 64 122 124 (+) Number / 1 µg 1.3990 × 10 ¹⁶ 2.7104 × 10 ¹⁵ 3.1493 × 10 ¹⁵ 3.4094 × 10 ¹⁵ Average size (nm) 208.5 ± 1.5 246.4 ± 1.5 200.2 ± 2.6 185.1 ± 0.5

^a molecular weight provided by the manufacturer

^b Molecular weight determined by ¹ HNMR analysis of each polymer

^c Mean size and mean ± standard deviation measured by dynamic light scattering experiments were given as n = 3. The charge ratio (N / P) of the mock-up was 7.8 for PEI, 6.0 for PAMAM, PAMAM-Arg and PAMAM-Lys.

4) Cytotoxicity

Toxicity of cationic polymers has been reported to be a function of cell membrane and polymer interaction and / or cell uptake efficiency. As shown in Fig. 4 (a) (b) was compared the cytotoxicity of the reagent against 293 and HepG2 cells. Each cell was incubated for 48 hours with increasing polymer content in the presence of serum. From the results, PEI was highly toxic to both cells, and PAMAM was much less toxic. PAMAM-Arg and PAMAM-Lys both showed slightly increased toxicity compared to pure PAMAM. This is believed to be due to the increased charge density and the molecular weight of each modified polymer. However, the toxicity of PAMAM-Arg and PAMAM-Lys were both much lower than those of PEI.

4C shows cytotoxicity results after 4 hours of culture as a cytotoxicity test result of PAMAM-GU on HepG2 cells. According to this, as in the case of the above PAMAM-Arg and PAMAM-Lys, PEI, a gene carrier, is very toxic, whereas PAMAM derivatives were significantly lower in cytotoxicity similar to PAMAM itself.

5) Transfection Efficiency for Cell Lines

First, transfection efficiency of PAMAM-Arg and PAMAM-Lys was measured. For the experiment, 293 cells were first selected because the cells are generally sensitive to nonviral transfection reagents. As shown in FIG. 5A, the transfection efficiencies of PAMAM, PAMAM-Arg and PAMAM-Lys exhibited levels of efficiency with or without serum or substantially equivalent to PEI. Whether conventionally grafted arginine contributes to gene delivery capacity has not been directly observed. To characterize this, HepG2 cells were selected for the next step. This is because the transfection efficiency of pure PAMAM (G4) is usually 10 times less than PEI. 5B shows experimental results of HepG2 cells in the presence or absence of serum under optimum conditions for each polymer. According to this, PAMAM-Arg according to the present invention has advantages over pure PAMAM, and has been shown to be comparable to PEI. It can also be seen from the results of PAMAM-Lys that the increased molecular weight and charge increased the transfection ability by grafting basic amino acids to PAMAM. However, the efficiency of transfection was not as great as that of pure PAMAM. To elucidate this, transfection was performed again for PAMAM-Arg, PAMAM-Lys and PAMAM with different charge ratios. Charge ratio was carried out over 1-12, according to Figure 5D it can be seen that PAMAM-Arg is significantly improved compared to PAMAM-Lys and pure PAMAM. In addition, Neuro 2A cells were additionally tested for gene transfer applications in neurons. Two different concentrations of DNA (0.2 and 1.0 μg / well) were used to compare the transfection efficiency and cytotoxicity of each complex, and the results were more apparent. As shown in FIG. 6A, PAMAM-Arg showed significantly higher expression levels than other reagents. The transfection efficiency of PAMAM-Arg was more than 2 orders of magnitude larger than lipofectamine or pure PAMAM, and more than 1 orders of magnitude compared to PEI. Cytotoxicity experiments were performed on polyplexes and the results are shown in FIG. 6B. Strong cytotoxicity was observed in lipofectamine / DNA at the concentration of DNA used (67% and 53% survival for 0.2 and 1.0 μg DNA / well, respectively). On the other hand, other polymer / DNA complexes showed negligible toxicity at both concentrations. From the above results, it can be confirmed that the PAMAM derivative according to the present invention is excellent in transfection efficiency as in the examples of HepG2 and Neuro 2A cells without cytotoxicity.

Next, an experiment to measure transfection efficiency with respect to PAMAM-Gu was performed. To this end, gene transfer efficiencies in C2C12 cells and HepG2 cells were examined according to the presence or absence of serum in culture medium.

FIG. 8 is a comparison result of gene transfer efficiency in C2C12 cells, and FIG. 9 is a result of comparison experiment of gene delivery efficiency in HepG2 cells, and each ratio represents a mass ratio of DNA and polymer. As can be seen from the diagram, the PAMAM derivatives grafted with guanidine functional groups can be confirmed that the gene transfer efficiency is improved by 10 to 100 times or more compared with the conventional PAMAM.

6) Transfection Efficiency for Primary Rat Vascular Smooth Muscle Cells

From the previous experiment, additional transfection experiments were performed on primary rat vascular smooth muscle cells. As shown in FIG. 7A, PAMAM-Arg was shown to significantly improve transfection efficiency compared to pure PAMAM and PAMAM-Lys, and the efficiency was comparable to that of PEI. DNA administration dependence of luciferase expression with the vector was tested and the results are shown in FIG. 7B. In the absence of serum, expression increased with the amount of DNA introduced. However, in the presence of serum, the expression level remained low despite the increase in DNA dose. From these results, it was confirmed that PAMAM-Arg mediated transfection in the cells was disturbed in the presence of serum.

7) Result of gene transfer animal experiment of PAMAM derivative

As shown in FIG. 10, X-Gal staining occurred in the surrounding area along the injection direction, and as a result, it was shown that external genes could be delivered and expressed in biological tissues of the brain, which are known to be very difficult.

8) Intracellular Penetration Ability of PAMAM-Arg and PAMAM-Lys

The results of observing the difference in intracellular penetration ability of both PAMAM derivatives by fluorescence microscope are shown in FIG. 11. The photo above is a general optical photo and the photo below is a fluorescence micrograph. In the case of PAMAM-Lys, it can be seen that the polymer is concentrated in the cell membrane and the cytoplasmic part, and fluorescence appears around the cell, whereas PAMAM-Arg penetrates into the cell nucleus and shows fluorescence. The experimental results showed that PAMAM-Lys and PAMAM-Arg showed a large difference in intracellular permeability due to the difference in the surface grafted amino acid groups. Expect.

Therefore, PAMAM-Arg is a growth factor that is involved in the growth and differentiation of cells and foreign genes, such as therapeutic DNA, which enable the expression of desired proteins through processes such as transcription and translation. And its receptors, various transcription factors to control the expression of the target genes and their inhibitors, and drugs that act primarily on DNA, such as anticancer agents, also need to be delivered into the cell nucleus, so they are also used for delivery of these drugs. Can be.

On the other hand, PAMAM-Lys is suitable for delivery of substances to be delivered to the cytoplasm, for example, substances that inhibit the target mRNA present in the cytoplasm, such as siRNA, and many drugs that act on various enzymes or organelles in the cytoplasm. Can be.

According to the present invention, it is possible to efficiently carry out the transport of the required material into the cell, has no cytotoxicity, and in particular, functions as a vector for delivering various genetic materials related to intracellular expression, thereby greatly contributing to the expression of a desired gene.

Claims (11)

A PAMAM derivative for intracellular material transfer in which all or part of the surface primary amines of the PAMAM dendrimers of generations 3 to 5 are grafted with at least one substance selected from the group of arginine, lysine, and guanidine. The intracellular substance delivery PAMAM derivative according to claim 1, which is used as a vector for delivering a substance selected from the group of low molecular substances, peptides, proteins, oligonucleotides, and plasmids. The PAMAM derivative for intracellular material transfer according to claim 1, wherein all or part of the surface primary amine of the PAMAM dendrimer is grafted with arginine alone. The PAMAM derivative according to claim 1, wherein all or part of the surface primary amine of the PAMAM dendrimer is grafted with lysine. The PAMAM derivative for intracellular material transfer according to claim 1, wherein all or part of the surface primary amine of the PAMAM dendrimer is grafted with guanidine. delete delete 4. The PAMAM derivative for intracellular material delivery according to claim 3, wherein the delivery material is a material to be penetrated into the cell nucleus. 5. The PAMAM derivative for intracellular material delivery according to claim 4, wherein the delivery material is a material to be penetrated into the cell membrane or cytoplasm. All or part of the surface primary amines of the PAMAM dendrimers from generation 3 to generation 6 are intended to be delivered intracellularly with PAMAM derivatives for intracellular mass shearing, which have been grafted with at least one substance selected from the group of arginine, lysine, and guanidine. A method of binding a substance and delivering the substance into a cell. The method of claim 10, wherein the substance to be delivered into the cell is one substance selected from the group of low molecular weight compounds, peptides, proteins, oligonucleotides, and plasmids.

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