WO2012134135A2 - Spermine copolymer and gene therapy using the spermine copolymer as a nucleic-acid carrier - Google Patents

Abstract

The present invention relates to a spermine copolymer and to gene therapy using the spermine copolymer as a nucleic-acid carrier. The spermine copolymer of the present invention is formed by copolymerizing spermine (SPE) using a linker compound that contains two or more hydroxyl groups, and thus may exhibit superior binding capacity in therapeutic nucleic acids such as DNA, effectively protect DNA from nucleases, and exhibit physico-chemical properties appropriate for use as a nucleic-acid carrier. Further, the hydroxyl groups of the linker compound may prevent the access of other blood proteins and the resulting aggregation thereof to achieve the improved stability of the nucleic-acid carrier in the blood, thereby exhibiting extremely low in vitro and in vivo cytotoxicity and extremely high transduction efficiency. Therefore, the spermine copolymer of the present invention may be effectively used as a nucleic acid carrier for gene therapy.

Description

Spermine copolymer and gene therapy using it as nucleic acid carrier

The present invention relates to spermine copolymers and gene therapy using them as nucleic acid carriers.

Gene therapy is the treatment of diseases by delivering the therapeutic nucleic acid to the desired organs in the body to allow the expression of new proteins in cells, rather than treating the symptoms of the disease, rather than treating the cause of the disease. . Gene therapy can have good selectivity over treatment with common drugs and can be applied over long periods of time to improve the rate and rate of treatment of diseases that are difficult to control with other therapies. However, since therapeutic nucleic acids such as DNA are vulnerable to hydrolysis by enzymes in vivo and have low efficiency of influx into cells, nucleic acids that enable high expression efficiency by safely delivering therapeutic nucleic acids to desired target cells for effective gene therapy. It is essential to develop a gene carrier.

Nucleic acid carriers must be low or free of toxicity and must be able to deliver nucleic acids to the cells of interest selectively and effectively. Such nucleic acid carriers can be broadly divided into viral and non-viral. Until recently, viral vectors with high transduction efficiency were used as nucleic acid carriers in clinical trials. However, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are not only complicated in their manufacturing process, but can also be immunogenic, potentially infectious, prone to inflammation, and nonspecific DNA. Due to the safety issues such as the insertion of the limited size of the acceptable DNA and the human body is limited to apply. As a result, non-viral vectors have been in the spotlight as a substitute for viral vectors.

Non-viral vectors have the advantage of being repeatedly administered with a minimum of immune response, specific delivery to specific cells, excellent safety and storage stability, and easy mass production. Examples of such nonviral vectors include cationic liposomes, N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), alkylammonium (alkylammonium), cationic cholesterol derivatives, gramicidin, and the like. However, conventional liposome-based nucleic acid carriers have low blood stability and low price competitiveness.

Recently, the cationic polymer in the non-viral vector has attracted a lot of attention because it can form a complex through the negatively charged DNA and ionic bonds. Such cationic polymers include poly-L-lysine (PLL), poly (4-hydroxy-L-proline ester), polyethyleneimine (PEI), poly [α- (4-aminobutyl) -L-glycolic acid], Polyamidoamine dendrimers, poly [N, N '-(dimethylamino) ethyl] methacrylate (PDMAEMA), and the like, which protect DNA from enzymatic degradation by compressing therapeutic nucleic acids such as DNA to form nanoparticles. It quickly penetrates into the cell and helps it escape from the endosome. Most non-viral vectors have advantages such as biodegradability, low toxicity, non-immunogenicity, and ease of use, compared to viral vectors, but have problems such as relatively low transduction efficiency and limited particle size.

In particular, most cationic polymers used as non-viral vectors show high transduction efficiency in vitro, where serum concentrations are low, but in vivo environments, cationic polymer / nucleic acid complexes are affected by various factors present in serum. There is a problem that the transduction efficiency of is severely inhibited and the influx of nucleic acids is not smooth. This is because excessive positive charges occur on the surface of the cationic polymer / nucleic acid complex in vivo, causing nonspecific interaction with plasma proteins and blood components. Therefore, the transduction efficiency of the cationic polymer is significantly reduced in vivo in which a large amount of serum is present, but not in an environment in which serum-free medium or serum is present in a very low concentration, and when applied in vivo, the lung Therapeutic application of the cationic polymer is inevitably limited because it can cause aggregation, accumulation in the liver and spleen, and further opsonization and removal by the reticuloendothelial system. Polyethyleneimine (PEI), which is widely studied as a non-viral vector, also has a significant decrease in transduction efficiency in vivo, and has problems such as low expression effect of nucleic acid due to high cytotoxicity and low blood compatibility.

Therefore, there is an urgent need for the development of nucleic acid carriers that enhance transduction efficiency while maintaining the advantages of existing non-viral vectors.

Accordingly, the present inventors have diligently researched to develop nucleic acid carriers having low cytotoxicity and high transduction efficiency. As a result, the hydrophilic nucleic acid carriers are known to have low transduction efficiency and low cell uptake efficiency. A spermine copolymer obtained by copolymerizing two or more spermines (SPEs) using a linker compound containing two or more hydroxyl groups shows high binding ability to therapeutic nucleic acids such as DNA and effectively protects the therapeutic nucleic acids from nucleases. In addition to exhibiting suitable physicochemical properties for use as a nucleic acid carrier, the hydroxyl group of the linker enhances the stability of the nucleic acid carrier in the blood by preventing access to and subsequent aggregation of other blood proteins, thereby in vitro and in vivo. Very low cytotoxicity and significantly improved transduction efficiency. The present invention was completed by confirming that the spermine copolymer can be usefully used as a nucleic acid carrier for gene therapy.

It is an object of the present invention to provide a spermine copolymer with markedly improved transduction efficiency without showing cytotoxicity as a nucleic acid carrier.

Another object of the present invention is to provide a method for preparing the spermine copolymer.

It is another object of the present invention to provide a nucleic acid delivery complex in which a therapeutic nucleic acid is bound to the spermine copolymer.

Another object of the present invention to provide a pharmaceutical composition for gene therapy containing the nucleic acid delivery complex as an active ingredient.

The spermine copolymer of the present invention exhibits high binding ability to therapeutic nucleic acids such as DNA by copolymerizing two or more spermines (SPEs) using a linker compound comprising two or more hydroxyl groups, and a nuclease. By effectively protecting DNA from physicochemical properties and exhibiting suitable physicochemical properties for use as nucleic acid carriers, as well as improving the stability of the nucleic acid carriers in the blood by preventing the hydroxyl groups of the linker from accessing and resulting aggregation of other blood proteins, It exhibits very low cytotoxicity in vitro and in vivo, and has a high transduction efficiency, which can be usefully used as a nucleic acid carrier for gene therapy.

Figure 1 schematically shows a method for producing a spermine copolymer (GPT-SPE) according to an embodiment of the present invention.

2 is a result of analyzing the composition of the spermine copolymer (GPT-SPE) prepared according to an embodiment of the present invention by ¹ H-NMR, at δ = 1.6-1.7 ppm [-CH ₂ -CH _2- ] Peaks for spermine protons, peaks for protons of the methylene group of GPT of [-CH-] at δ = 3.9-4.0 ppm confirmed that the spermine copolymer was effectively formed.

Figure 3a is agarose gel electrophoresis analysis of the mobility of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios The result is.

Figure 3b is a nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios can effectively protect DNA from nucleases It is a result that indicates that there is.

Figure 3c is an energy filtering electron microscope (Energy Filtering) of the form of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios Transmision Electron Microscopy (EF-TEM).

Figure 3d is a result of analyzing the particle size distribution of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios.

Figure 3e is a surface charge (n = 3, error bars of nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios Standard deviation).

Figure 4a is investigated the influx of cells of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention at various weight ratios using FITC labeling The result is.

Figure 4b is the transduction efficiency of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting the spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention in various weight ratios investigated the A549 cell line Result (mean ± SD, n = 3). In the figure, RLU stands for 'relative light unit' and indicates the degree of light emission.

Figure 4c is an experimental result to determine the effect of serum on gene transduction efficiency. A549 cells were cultured with the copolymer / pGL3 complex in the presence / absence of 10% serum (mean ± SD, n = 3).

Figure 4d shows the buffering capacity (buffering capacity) of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting spermine copolymer (GPT-SPE) and DNA of various embodiments of the present invention at various weight ratios. (bafilomycin) is the result of investigation through the treatment of A1.

Figure 5 shows the cytotoxicity of the spermine copolymer (GPT-SPE) of one embodiment of the present invention at various concentrations (a: A549 cell line; b: HepG2 cell line; c: MCF7 cell line; d: HeLa cell line) The results of the survey.

Figure 6 after aerosol administration of the mouse pulmonary nucleic acid delivery complex (GPT-SPE / GFP) prepared by reacting the spermine copolymer (GPT-SPE) and green fluorescent protein (GFP) of one embodiment of the present invention As a result of analyzing the expression of GFP in vivo (magnification: 200 ×, scale bar shows 50 μm), a indicates transduction efficiency and b indicates lung histopathological results.

7 shows tissues of various organ tissues including lungs of mice aerosol-administered with a nucleic acid delivery complex (GPT-SPE / GFP) prepared by reacting spermine copolymer (GPT-SPE) with GFP according to one embodiment of the present invention. It is the result of pathological examination (magnification: 200 *, scale bar shows 50 micrometers).

Figure 8 is aerosol administration of the lung tumor-bearing k-ras mouse nucleic acid delivery complex (GPT-SPE / shAkt1) prepared by reacting the spermine copolymer (GPT-SPE) and Akt1 shRNA of one embodiment of the present invention After confirming the therapeutic effect (magnification: 200 ×, scale bar indicates 50 μm), FIG. 8A shows lung lesions after aerosol administration (red circles indicate tumor tissue); Figure 8b is the result of measuring the total number of tumors after aerosol administration (n = 4, * is p <0.05 compared to GPT-SPE / Scr, ** is p <0.01 compared to Akt1 shRNA alone, p <0.01 compared to the control), And a result of measuring the number of tumors having a diameter of 1 mm or more after aerosol administration (n = 4, * is p <0.05 compared to GPT-SPE / Scr, *** is p <0.001 compared to Akt1 shRNA alone, and ** is a control group) Relative to p <0.01); FIG. 8C shows histopathologically as a result of red lesions in the lung (magnification: 200 ×, scale bar showing 50 μm); Figure 8d is the result of Western blot analysis of the Akt1 protein expression level in the lung after aerosol administration by densitometer (n = 4, ** p <0.01, *** p <0.001).

In order to achieve the above object, in one aspect, the present invention is a primary amine or a secondary amine of spermine at at least one, preferably at least two acrylate end of the linker represented by the following formula (1) To provide a combined spermine copolymer.

[Formula 1]

The linker of the present invention comprises at least two hydroxyl groups and at least two acrylate groups. The linker of the present invention exhibits hydrophilicity by the hydroxyl group, and can improve the stability of the nucleic acid carrier in the blood by preventing the access of the blood protein in the blood and consequent aggregation. In addition, the linker of the present invention, the primary amine or secondary amine of spermine to the acrylate group (acting as a Michael acceptor), preferably NH ₂ (acting as a Michael donor) and Michael can add reaction. As a result of the Michael addition reaction with the linker, spermine can be polymerized through a simple process. The linker of the present invention may be represented by Formula 1, but is within the scope of the present invention, as long as it contains at least two hydroxyl groups and at least two acrylate groups. In the general formula (1), it is possible to control the carbon number between the acrylate group and the hydroxyl group, preferably, in order to further improve the nucleic acid compressive ability may be three acrylate groups and hydroxyl groups, more preferably the following formula It may be glycerol propoxylate triacrylate represented by 2 (glycerol propoxylate triacrylate, GPT, C ₂₁ H ₃₂ O ₉ , 428 Da).

[Formula 2]

In one embodiment of the present invention, glycerol propoxylate triacrylate can react with spermine to polymerize spermine at three acrylate ends by Michael addition reaction.

As used herein, the term "spermine" is a biogenic tetra-amine represented by the following formula (3) and having two primary and secondary amino groups.

[Formula 3]

Spermine is naturally present in all eukaryotic cells, especially in tissues rich in protein and nucleic acid synthesis, involved in cell metabolism, stabilizing DNA or RNA structure, stabilizing ribosomes and rRNA binding, histone acetyl It exhibits physiological effects such as promoting stimulation and promoting phosphorylation of non-histone proteins. As a nucleic acid carrier, spermine has a low molecular weight compared to polyethyleneimine (25kDa), which is used as a nucleic acid carrier, and thus has low cytotoxicity and excellent safety. Since monomeric spermine is short in length, it is necessary to polymerize spermine in order to compress the nucleic acid effectively and efficiently enter the cell.

The spermine copolymer of the present invention has excellent nucleic acid compressibility by connecting a plurality of spermines, and since the binding between the linker and spermine is hydrolyzed after being introduced into the cell, the nucleic acid releasing ability is also excellent. Is hydrolyzed into monomers to have an excellent safety effect. In addition, by the hydroxyl group of the linker compound, it is possible to prevent the access of the blood protein in the blood and consequent aggregation, thereby improving the stability of the nucleic acid carrier in the blood.

In one embodiment of the present invention, the spermine copolymer is added by addition reaction to the acrylate end of the linker, wherein the primary or secondary amine that is not linked to the linker in spermine is another It may be combined with, to be a spermine copolymer to form a dendrimer (dendrimer) through a linker represented by the formula (1). The dendrimer may be, for example, the following structure as a repeatedly branched structure.

In one embodiment of the present invention, the spermine copolymer may be represented by the following formula (4).

[Formula 4]

In one embodiment of the present invention, the spermine copolymer has a form in which three molecules of spermine are bound based on one molecule of glycerol propoxylate triacrylate. In one embodiment of the invention, the spermine copolymer preferably has a molecular weight ranging from 3 to 15 kDa for effective nucleic acid delivery. In addition, in one embodiment of the present invention, it is preferred for the spermine copolymer to have a zeta potential in the range of 5 to 30 mV for effective nucleic acid delivery. When exhibiting the physicochemical properties of the above range, the spermine copolymer of the present invention can be effectively introduced into the endosome of the cell as a nucleic acid carrier.

The spermine copolymer of the present invention may further be combined with polyethylene glycol (PEG), galactosylated polyethylene glycol (gal-PEG), folate, mannose, or a combination thereof. The bond may be a physical or chemical bond, and the chemical bond may be a covalent bond, an ionic bond, an intermolecular bond, or the like, but is not particularly limited.

Modification by PEG binding can be expected to effect the spermine copolymer of the present invention to block the interaction with the plasma components to improve blood stability to extend the half-life of the nucleic acid carrier. As used herein, the term "polyethylene glycol (PEG)" is a straight chain polymer having a terminal hydroxyl group, the formula HO-CH ₂ CH _2- (CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH (wherein , n is about 8 to about 4000). Suitable PEGs for use in the present invention may be bi-functional PEGs having the same or different functional groups at both ends, preferably having an amine group (NH ₂ ) at the N-terminus and at the C-terminus. Hetero-functional PEG having a carboxyl group (COOH). The molecular weight of PEG useful in the present invention may preferably be 2 to 10 kDa, more preferably 5 kDa.

Galactosylated polyethyleneglycol is bound to impart hepatocyte targeting ability and can be usefully used for gene therapy as a hepatocyte target nucleic acid carrier. Because it contains galactose residues that are involved in receptor-mediated interactions with asialo glycoproteins (ASGPR), which are present only in hepatocytes at high density, hepatocyte-specific incorporation into cells allows for efficient cell delivery. Galactosylated polyethyleneglycol refers to a conjugate synthesized by an amide reaction between an activated carboxyl group of a galactose-containing glycoprotein and an amine group of a bifunctional PEG, which can be represented by the following formula (5). The galactose-containing glycoprotein is any glycoprotein having any galactose residue as a terminal sugar group, and any galactose-containing glycoprotein can be used without limitation as long as it can impart galactose residue to the copolymer obtained by modification to PEG.

[Formula 5]

The terminal carboxyl group and the amine group of the bifunctional PEG are functional groups, and the amine group may form an amide bond with the carboxyl group of the galactose-containing glycoprotein, and the carboxyl group may form an amide bond with the amine of the spermine copolymer.

Incorporation of mannose into the spermine polymer can specifically bind cells with mannose receptors, thereby enhancing gene transfer efficiency and inducing receptor mediated endocytosis. Known cells with mannose receptors are macrophage cells and dendritic cells.

Folic acid is a type of vitamin, also called vitamin B9 or vitamin M. Folic acid forms specific binding with folic acid receptors. "Folic acid receptor" is a tumor-associated glycosylphosphatidylinositol anchor protein that allows bound folic acid or folate bound material to be ingested through receptor-mediated endocytosis.

In one embodiment, the present invention comprises the steps of mixing the linker compound represented by the formula (1) dissolved in a solvent with spermine dissolved in a solvent to perform a Michael addition reaction; And separating the spermine copolymer formed from the reactant.

As shown in Figure 1, the spermine copolymer according to an embodiment of the present invention,

1) dissolving each of spermine and glycerol propoxylate triacrylate in a solvent;

2) adding glycerol propoxylate triacrylate solution to spermine solution under stirring to perform Michael addition reaction; And

3) can be prepared through the step of obtaining a spermine copolymer by separating the copolymer formed from the reactant.

Specifically, step 1) is a step of preparing a reaction solution by dissolving each of spermine and a linker compound in a solvent for the Michael addition reaction between spermine as the Michael donor and the acrylate group of the linker as the Michael acceptor. The reaction solvent usable in the present invention may be suitably selected and employed as long as it can react with spermine and a linker compound (e.g., glycerol propoxylate triacrylate) or dissolve them without decomposing them. . Examples of such a solvent include anhydrous methyl alcohol, ethyl alcohol and the like.

Step 2) is a step of performing the Michael addition reaction by adding the linker compound reaction solution while stirring the spermine reaction solution prepared in step 1). Michael addition reactions suitable for the present invention are carried out for 1 to 3 hours at 3 to 5 ° C, then for 12 to 24 hours at 20 to 25 ° C, preferably for 2 hours at 4 ° C and for 12 hours at 25 ° C. The reaction molar ratio of spermine and the linker compound (eg glycerol propoxylate triacrylate) in the Michael addition reaction suitable for the present invention is 4: 3 to 8: 3, preferably 5: 3. In a preferred embodiment of the present invention, 1.5 M glycerol propoxylate triacrylate reaction solution is slowly added to 2.5 M spermine reaction solution under stirring at 4 ° C. for 2 hours, followed by overnight reaction at room temperature to add Michael. Do this.

Step 3) is a step of separating the copolymer formed by the Michael addition reaction of step 2) to obtain a spermine copolymer. In a preferred embodiment of the present invention, the obtained reactant is vacuum-dried for 24 hours at 4 ° C. After dissolving in distilled water at 4 ℃ for 24 hours to perform dialysis (MWCO = 3500) for distilled water to separate the spermine copolymer. The spermine copolymer thus separated can be freeze-dried and stored at -25 ° C, where freeze-drying can be carried out using commonly used freeze-drying methods or freeze-dryers, and as a result of by-products Nanoparticles of the spermine copolymer having this removed viscosity can be obtained.

The spermine copolymer of the present invention prepared in the preferred embodiment of the present invention has a molecular weight of 3,000 to 6,000 Da, methylene of glycerol propoxylate triacrylate at δ = 3.9-4.0 ppm by ¹ H-NMR analysis The peak for the proton of the group was detected at δ = 1.6-1.7 ppm and the peak for the proton of the ethylene group of spermine was detected (see Figure 2), resulting in Michael addition reaction between spermine and glycerol propoxylate triacrylate. It was confirmed that the spermine copolymer according to the present invention was effectively formed. As a result of gel permeation chromatography analysis, the weight-average molecular weight (Mw) of the spermine copolymer of the present invention was 5170, the number-average molecular weight (Mn) was 3870, and the polydispersity index, PDI = Mw / Mn) was found to be 1.34.

According to the production method of the present invention, since the spermine copolymer of the present invention can be produced through a simple process, the spermine copolymer of the present invention can be mass-produced. Since the ester bond between spermine is hydrolyzed and the polymer nucleic acid carrier is hydrolyzed into the monomer, the nucleic acid releasing ability and safety are excellent.

In one embodiment, the present invention provides a nucleic acid delivery complex wherein a therapeutic nucleic acid is bound to the spermine copolymer. The kind of therapeutic nucleic acid that can be bound to the spermine copolymer of the present invention is not particularly limited, and any nucleic acid that can be delivered to a desired target and exerts a desired therapeutic effect according to the purpose of the present invention is included in the scope of the present invention. do. For example, nucleic acids that can be delivered in a complex form with the spermine copolymer of the present invention include siRNAs (small interfering RNAs), shRNAs (small hairpins), and the like. RNA), antisense oligonucleotides, DNA, single stranded RNA, double stranded RNA, DNA-RNA hybrids and ribozymes.

For effective formation of the nucleic acid delivery complex according to the invention the therapeutic nucleic acid and spermine copolymer are 1: 1 to 1:50, preferably 1: 1 to 1:20, more preferably 1: 5 to 1: It is preferable to react at a weight ratio of 10 so that the charge of the nucleic acid carrier is an appropriate charge. If the positive charge of the nucleic acid carrier is excessively high, the nucleic acid compressive capacity is increased, but the cell surface is damaged and the cytotoxicity is increased.

In order to investigate the condensation capability of the spermine copolymer according to the present invention to the therapeutic nucleic acid, the spermine copolymer and DNA were reacted at various weight ratios. The nucleic acid delivery complex (GTP-SPE / DNA) of the spermine copolymer and DNA is most effectively formed (see FIG. 3A), and the DNA in the nucleic acid delivery complex is effectively protected from attack by nucleases (see FIG. 3B). It was confirmed that the formed nucleic acid delivery complex had a spherical compressed structure (see FIG. 3C). In addition, the nucleic acid delivery complex according to the present invention exhibits a relatively uniform particle size distribution (PDI: 1.938e-001) of 120-200 nm on average (see FIG. 3D), which has a particle size suitable for use as a nucleic acid carrier. Rather, the surface charge exhibits a positive zeta potential (see FIG. 3E), confirming that it can effectively bind to the anion cell surface.

In addition, in order to confirm the influx of the spermine copolymer according to the present invention, the spermine copolymer was labeled with FITC and then reacted with DNA to express the FITC-labeled nucleic acid delivery complex (GTP-SPE / DNA-FITC). ) Was prepared. After transducing the prepared FITC-labeled nucleic acid delivery complex to the cells, the cells were observed by confocal microscopy to confirm that the FITC-labeled nucleic acid delivery complexes were detected in the cells (see FIG. 4A).

In addition, in order to investigate the transduction efficiency of the spermine copolymer according to the present invention by reacting the spermine copolymer with DNA in various weight ratios to form a nucleic acid delivery complex (GTP-SPE / DNA) after transduction thereof The efficiency was investigated through luciferase activity analysis. As a result, the transduction efficiency of the nucleic acid delivery complex increases as the weight ratio of the DNA and spermine copolymer increases from 1: 5 to 1:10, and similar high transduction efficiency at a weight ratio of 1:10 or more. In particular, it was confirmed that the nucleic acid delivery complex of the present invention exhibits a transduction efficiency of 1800 times higher than the DNA treatment group alone (see FIG. 4B). This means that the spermine copolymer of the present invention has excellent binding ability to DNA to effectively form nanoscale nucleic acid delivery complexes, thereby exhibiting high transduction efficiency. In addition, when the transduction efficiency of GPT-SPE / pGL3 according to an embodiment of the present invention in the presence of serum compared with the transduction efficiency of PEI 25K / pGL3 and lipofectamine / pGL3, in the presence of 10% serum, The transduction efficiency of GPT-SPE / pGL3 was slightly decreased (12.8-folds) according to one embodiment of the present invention, whereas the transduction efficiency of PEI 25K / pGL3 and lipofectamine / pGL3 was significantly reduced (192.7-folds, respectively). And 3468.4-folds) (see FIG. 4C).

In order to determine the transduction mechanism of the spermine copolymer according to the present invention, the buffering capacity of the copolymer was investigated using a bacillomycin A1, an endosome proton pump inhibitor. As a result, the transduction efficiency of the spermine copolymer according to the present invention was drastically decreased after the treatment with bacillomycin A1 (see FIG. 4D). This indicates that the spermine copolymer of the present invention can be effectively released from the endosome into the cytoplasm by the proton sponge effect.

In addition, as a result of investigating the cytotoxicity of the spermine copolymer according to the present invention in various cell lines, the cytotoxicity was lower than that of polyethyleneimine (PEI, 25kDa) used as a comparative group, and high concentration (100 ㎍ / Ml) also showed high cell viability (see FIGS. 5A-5D).

Thus, the spermine copolymer of the present invention not only forms a spherical uniform particle size nucleic acid delivery complex that exhibits high binding capacity to DNA, effectively protects DNA from nucleases, and is suitable for use as a nucleic acid carrier. In addition, it shows very low cytotoxicity and high transduction efficiency in vitro and in vivo and can be usefully used as a nucleic acid carrier for gene therapy.

Accordingly, the present invention, as one embodiment, provides a pharmaceutical composition for gene therapy containing a nucleic acid delivery complex in which the therapeutic nucleic acid is coupled to the spermine copolymer as an active ingredient. The pharmaceutical composition of the present invention may be administered together with a pharmaceutically acceptable carrier, and in the case of oral administration, in addition to the active ingredient, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments , Fragrances and the like may be further included. In the case of injectables, the pharmaceutical compositions of the invention may be used in combination with buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like. In the case of topical administration, the composition of the present invention may use a base, excipients, lubricants, preservatives and the like.

The formulation of the composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, they may be prepared in unit dosage ampules or in multiple dosage forms. It may be formulated into other solutions, suspensions, tablets, pills, capsules, sustained release preparations and the like.

Examples of suitable carriers, excipients, and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, preservatives and the like may be further included.

The pharmaceutical composition of the present invention can be administered orally or parenterally. Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal Sublingual, inhalation or topical administration is possible. For such clinical administration, the pharmaceutical compositions of the present invention can be formulated into suitable formulations using known techniques. For example, in oral administration, it can be administered by mixing with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active ingredient may be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

The effective dosage of the pharmaceutical composition of the present invention varies in the range depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, etc. of the patient, It can be easily determined by an expert.

The pharmaceutical composition of the present invention may have different routes of administration depending on the target disease type of the therapeutic nucleic acid bound to the nucleic acid carrier, and may be used for treating various diseases by combining targeting factors according to the target cell.

In one embodiment, the present invention provides a formulation for inhalation administration (aerosol formulation) of the pharmaceutical composition formulated to deliver a nucleic acid delivery complex conjugated with a spermine copolymer to a target nucleic acid via aerosol delivery. .

Drug delivery through inhalation is one of the non-invasive methods of delivering drugs directly through the airways and through the mucous membranes of the lungs to lung cells, particularly nucleic acid delivery through aerosol delivery for a wide range of treatment of lung diseases. This can be used to advantage. This is because the anatomical structure and location of the lung allows for an immediate and non-invasive approach and can be subject to topical application of the nucleic acid delivery system without affecting other organs. Therefore, when the nucleic acid delivery complex in which the spermine copolymer of the present invention is a nucleic acid associated with lung disease, in particular, lung cancer as a therapeutic nucleic acid is delivered to the lung in an aerosol manner, a prophylactic or therapeutic effect on the disease can be expected. For formulations for inhalation administration, it is preferred that the nucleic acid delivery complex has an average particle size of 120 to 200 nm, and for this purpose, the nucleic acid may be prepared by mixing with the nucleic acid carrier in a weight ratio of at least 1: 5.

In a preferred embodiment of the present invention, in order to confirm the in vivo delivery efficiency of the spermine copolymer according to the present invention by aerosol, the spermine copolymer and the green fluorescent protein (GFP) nucleic acid 10 Nucleic acid delivery complexes (GTP-SPE / GFP) were prepared by mixing at a weight ratio of: 1 and normal mice were exposed to aerosols containing them. As a result, a significantly increased GFP signal was detected in the nucleic acid delivery complex (GTP-SPE / GFP) treatment group of the present invention compared to the untreated control group and GFP alone treatment group, and it was confirmed that no cytotoxicity was observed in all treatment groups. (See FIGS. 6A, 6B and 7).

In one embodiment, the invention includes delivering, via small particle aerosol, an aqueous dispersant of a genetic macromolecule such as a therapeutic nucleic acid complexed with a spermine copolymer via an individual's airway in need of gene therapy. In addition, the present invention provides a method for targeting gene therapy therapy through the airways.

Methods of delivery of the pharmaceutical compositions according to the present invention may include oral and nasal inhalation methods, methods of facial masks or oral tubes, and methods of oxygen hoods for children that have been used to administer oxygen. Generally, small particle aerosols are created by a nebulization process, which is carried out by a jet nebulizer. In addition, the therapeutic nucleic acid preferably comprises operably linked elements necessary for expression, and representative therapeutic nucleic acids include nucleic acid encoding tumor suppressor such as gallbladder fibrosis transmembrane conductance regulator nucleic acid, p53 or retinoblastoma. Or variants of these nucleic acids that have been genetically engineered, cytokine nucleic acids such as interleukin-2 or interleukin-12, and nucleic acids that will be used as DNA vaccines to stimulate a direct immune response to tumors or other infectious agents. do. Another exemplary therapeutic nucleic acid is the Akt1 nucleic acid agonist, a serine / threonine kinase.

In a preferred embodiment of the present invention, nucleic acid delivery complexes in which a spermine copolymer is coupled to an agent that inhibits the expression of Akt1 nucleic acid as a therapeutic nucleic acid are used for the treatment of lung cancer by aerosol delivery. Akt is an important regulator of cell survival and proliferation and plays a key role in carcinogenesis through stimulation of cell proliferation and inhibition of apoptosis. Many tumors have been found to have amplified expression of nucleic acids encoding Akt isoforms Akt1, 2 and 3. Thus, inhibition of Akt activity or expression may be a reasonable strategy for the treatment of cancer by blocking the survival and proliferation of cancer cells and inducing apoptosis of cancer cells. Agents that inhibit the expression or activity of Akt1 suitable for the present invention may include small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense oligonucleotides, antagonists, ribozymes, inhibitors, peptides, small molecules and the like. In a preferred embodiment of the present invention, oligonucleotides (Meng et al., Cell Signal 2006, 18 (12): 2262e71) encoding the 19-mer hairpin sequence represented by SEQ ID NO: 1 as shRNA constructs targeting Akt1 It was used as an agent that inhibits the expression or activity of Akt1.

It is particularly contemplated that the pharmaceutical compositions of the present invention may be prepared for aerosol delivery of an aqueous dispersant to an individual or animal airway. One of ordinary skill in the art would be able to readily determine the appropriate dosage of the aerosol formulation without undue experimentation. When used in vivo for a targeted gene therapy regimen, the pharmaceutical compositions of the present invention are administered to a patient or animal in an amount that eliminates or alleviates the disease by efficiently expressing a therapeutically effective amount, ie, a therapeutic nucleic acid. Dosage and dosage regimen will depend on the type of disease, the characteristics of the particular spermine copolymer / DNA complex, for example its therapeutic index, the patient, the patient's history and other factors. The dosage of the pharmaceutical composition according to the invention will typically be from about 0.01 to about 0.1 g / kg body weight. The compositions and formulations may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives.

In another aspect, the present invention provides a dosage form for intravenous injection of the pharmaceutical composition formulated to deliver a nucleic acid delivery complex bound to a spermine copolymer to a target site via intravenous injection. Since the nucleic acid delivery complex of the present invention has high stability in blood, it can be efficiently used for systemic delivery, and PEG can be further bound to spermine copolymer to further improve stability in blood.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Spermine Copolymer

The spermine copolymers according to one embodiment of the present invention are subjected to Michael addition reactions with a slight modification of the method reported in Arote RB, et al., Bioconjug. Chem. 2009, 20 (12): 2231-41. According to the synthesis (Fig. 1).

Briefly, 2.5 M spermine (MW: 202.34 Da, Sigma-Aldrich) and 1.5 M glycerol propoxylate triacrylate (GPT) (MW: 428 Da, Sigma-Aldrich) were each anhydrous ethyl alcohol at 4 ° C. After dissolving in, the prepared GPT solution was slowly added to the prepared spermine solution with stirring at 4 ° C. for 2 hours and reacted overnight at room temperature. The copolymer obtained therefrom was vacuum-dried for 24 hours and dissolved in distilled water at 4 ° C. The obtained solution was dialyzed five times with distilled water at 4 ° C. for 24 hours using a dialysis membrane (Spectra / Por ^® , MWCO = 3500) to remove unreacted material, and separated spermine copolymer (GPT-SPE). Was freeze-dried at -20 ° C.

Analyzed the composition of the spermine copolymer (GPT-SPE) dried ¹ H- nuclear magnetic resonance ^{(1 H-nuclear magnetic resonance,} 1 H-NMR) (Avance 600, Bruker, Germany) - freezing. ^{For 1} H-NMR analysis, spermine copolymer (GPT-SPE) was dissolved in distilled water at a concentration of 10 mg / ml. As a result, as shown in Fig. 2A, the peak for the proton of the methylene group of glycerol propoxylate triacrylate at δ = 3.9-4.0 ppm, the proton of the ethylene group of spermine at δ = 1.6-1.7 ppm The peak was detected to confirm that the copolymer of spermine and glycerol propoxylate triacrylate was effectively formed.

In addition, the molecular weight of the copolymer was measured at 690 nm laser wavelength using a gel-permeation chromatography column (GPC-MALS, Dawn Eos, Wyatt, USA), and the results are shown in Table 1 below. .

Table 1

	Mw	Mn	PDI
GPT-SPE	5170	3870	1.34

As shown in Table 1, the weight-average molecular weight (Mw) of the spermine copolymer of the present invention is 5170, the number-average molecular weight (Mn) is 3870, and the polydispersity index (PDI = Mw / Mn). ) Was found to be 1.34.

Example 2: Characterization of the spermine copolymer / DNA complex

<2-1> DNA condensation ability of spermine copolymer

One of the most important features of nucleic acid carriers is their ability to interact with and condense plasmid DNA. The DNA condensation ability of the spermine copolymer prepared in Example 1 was confirmed by agarose gel electrophoresis. First, pGL3 plasmid (5.3 kb, Promega) is mixed in a solution of spermine copolymer (GPT-SPE) at various weight ratios of 0.01, 0.5, 1, 5, and 10, and then vortexed lightly. Incubate at room temperature for 30 minutes. After incubation, each reaction solution was subjected to electrophoresis on a 1% agarose gel and observed for DNA mobility under ultraviolet light. In this case, pGL3 plasmid alone, which was not reacted with the spermine copolymer (GPT-SPE) of the present invention, was used as a control.

As a result, as shown in Figure 3a, when the spermine copolymer of the present invention and the plasmid DNA reacted at a weight ratio of 1: 1 or less, retardation due to DNA condensation is not caused or is minimal, When the weight ratio of is greater than 5: 1, the mobility of DNA was completely delayed, indicating that the spermine copolymer of the present invention effectively condensed the plasmid DNA to form a nucleic acid transfer complex (GPT-SPE / DNA). The control unreacted plasmid DNA moved through the gel without any delay. From the above results, it can be seen that it is preferable to mix the spermid copolymer of the present invention and the plasmid DNA in a weight ratio of 5: 1 or more for effective formation of the nucleic acid delivery complex.

<2-2> DNA protection effect of spermine copolymer / DNA complex

For effective nucleic acid expression, the DNA in the nucleic acid delivery complex must be protected from enzymatic attacks such as nucleases. In order to confirm the DNA protective effect, 4 μl of a nucleic acid delivery complex (GPT-SPE / DNA) solution prepared by reacting the spermine copolymer with DNA in a weight ratio of 5: 1 or To 4 μl of unreacted plasmid DNA solution, 1 μl of DNase I (2 units) or 1 μl of DNase / Mg ²⁺ digestion buffer containing PBS (phosphate-buffered saline) (50 mM TrisHCl, pH 7.6 and 10 mM MgCl ₂ ) was added. Incubate with stirring at 100 rpm for 30 minutes at 37 ° C. To stop the enzymatic reaction, treat all reaction solutions with 4 μl of EDTA (250 mM) for 10 minutes, then mix 1% sodium dodecyl sulfate (pH 7.2) to adjust the final volume to 15 μl. Incubate at 25 ° C. for 2 hours. After incubation, the reaction solution was subjected to electrophoresis at 50 V for 1 hour on a 1% agarose gel using Tris-acetate-EDTA buffer.

As a result, as shown in FIG. 3B, the control unreacted plasmid DNA was completely degraded by DNase I, whereas the DNA in the nucleic acid delivery complex (GPT-SPE / DNA) of the present invention was effectively protected from degradation of DNase I. Able to know. These results indicate that the nucleic acid delivery complex of the present invention can be effectively delivered into cells while protecting DNA from the attack of nucleases.

<2-3> Morphological Analysis of Spermine Copolymer / DNA Complex

In Example <2-1>, the form of the nucleic acid delivery complex (GPT-SPE / DNA) prepared by reacting a spermine copolymer with a DNA at a weight ratio of 5: 1 was EF-TEM (LIBRA 120, Carl Zeiss, Germany).

As a result, as shown in Figure 3b, the nucleic acid delivery complex (GPT-SPE / DNA) according to an embodiment of the present invention that the spermine copolymer and the plasmid DNA effectively condensed to have a spherical compressed structure Confirmed.

The particle size of the nucleic acid delivery complex is essential for the influx of the complex into the cell as an important factor for the complex to access and pass through the target site. Since most nucleic acid delivery complexes are introduced into cells by endocytosis or pinocytosis, the size of the complex is required to be below a certain level for optimal intracellular entry. The particle size and distribution of the nucleic acid delivery complex at various weight ratios were measured using a dynamic light scattering spectrophotometer (ELS8000, Otsuka Electronics, Osaka, Japan). At this time, the scattering angle (scattering angle) was set to 90 ° and 20 °.

As a result, as shown in FIG. 3D, the nucleic acid delivery complex (GPT-SPE / DNA) of the present invention at a weight ratio of 5: 1 has a relatively uniform particle size distribution (PDI: 1.938e-001) having an average of 120 to 200 nm. It was confirmed that the particles have a particle size suitable for use as a nucleic acid carrier.

<2-4> Surface charge measurement of spermine copolymer / DNA complex

In addition, the positive surface charge of the nucleic acid delivery complex is essential for binding to the anion cell surface, which facilitates the influx of the complex into the cell. The zeta potential of the nucleic acid delivery complex (GPT-SPE / DNA) formed by reacting the spermine copolymer of the present invention with plasmid DNA at various weight ratios is determined by a dynamic light scattering spectrophotometer (ELS8000, Otsuka Electronics, Osaka, Japan).

As a result, as shown in Figure 3e, when the spermine copolymer of the present invention and the plasmid DNA reacted at a weight ratio of 0.1: 1, the complex between them was not completely formed, showing a negative zeta potential, As the weight ratio increased above 5: 1, the complex showed a positive zeta potential (zeta potential: +9.14 Mv; mobility: 6.922e-005 cm 2 / Vs), the value of which rapidly increased. This positive charge means that the negatively charged DNA is completely enclosed within the nucleic acid delivery complex, and the positive surface charge not only facilitates the incorporation of the nucleic acid delivery complex into the cell, but also between the particles. It causes the electrostatic repulsive force has the advantage of reducing the aggregation phenomenon between the particles.

Example 3: Transduction efficiency of spermine copolymer

<3-1> In Vitro Inflow Analysis

In order to confirm the in vitro cellular uptake of spermine copolymer (GPT-SPE) of one embodiment of the present invention, the plasmid DNA was labeled with FITC and then reacted with spermine copolymer to FITC -Marked nucleic acid delivery complexes (GTP-SPE / DNA-FITC) were prepared. After transducing the prepared FITC-labeled nucleic acid delivery complex to the cells and observing the cells under confocal microscopy, as shown in FIG. 4A, the FITC-labeled nucleic acid delivery complex was detected in the cell, It was confirmed that the fermin copolymer effectively introduced DNA into the cells.

<3-2> Transduction Efficiency Analysis

In order to investigate the transduction efficiency of the spermine copolymer (GPT-SPE) of one embodiment of the present invention, an in vitro luciferase activity assay was performed.

First, RPMI 1640 (Gibco BRL) medium containing 10% FBS (HyClone), 100 μg / ml streptomycin and 100 U / ml penicillin was added to a 24-well plate, and A549 cells were loaded at 1.5 × 10 ⁵ / well. Inoculation at a density was incubated for 18 hours in a 37 ℃, 5% CO ₂ incubator. In addition, after mixing the pGL3 expression vector (5.3 kb, Promega) containing luciferase nucleic acid induced expression by the SV40 promoter and enhancer in the spermine copolymer solution of 1, 5 and 10 lightly Nucleic acid delivery complex (GPT-SPE / GL3) was prepared by vortexing and incubating at room temperature for 30 minutes. The medium of the well plate was replaced with a medium containing 1 μg of serum-free medium or prepared nucleic acid delivery complex (GPT-SPE / GL3) and incubated in 37 ° C., 5% CO ₂ incubator for 4 hours. At this time, PEI (polyethylene imine) 25K (25 kDa, Sigma-Aldrich) and lipofectamine (lipofectamine) treated with the same concentration was used as a cell culture solution as a comparison group. Thereafter, the medium of the well plate was replaced with fresh 10% FBS containing DMEM medium and again incubated in 37 ° C., 5% CO ₂ incubator for 48 hours. Subsequently, after removing the medium, 100 μl of cell lysis buffer was added to each well and left at −20 ° C. for 24 hours, after which the medium was transferred to an e-tube and contained at 10,000 rpm. Centrifugation was performed for 15 minutes to obtain a supernatant. Luciferase activity of the obtained supernatant was measured using a luciferase assay kit (Promega).

As a result, as shown in Figure 4b, the weight ratio of DNA and spermine copolymer is 1: 5 to 1:10, the transduction efficiency of the nucleic acid delivery complex increases as the weight ratio is increased, at a weight ratio of 1:10 or more It showed a similar level of high transduction efficiency, in particular, the nucleic acid delivery complex of the present invention was confirmed to exhibit a transduction efficiency of 1800 times higher than the DNA alone treatment group. This means that the spermine copolymer of the present invention has excellent binding ability to DNA to effectively form nanoscale nucleic acid delivery complexes, thereby exhibiting high transduction efficiency.

In addition, when the transduction efficiency of GPT-SPE / pGL3 according to an embodiment of the present invention in the presence of serum compared with the transduction efficiency of PEI 25K / pGL3 and lipofectamine / pGL3, in the presence of 10% serum, The transduction efficiency of GPT-SPE / pGL3 was slightly decreased (12.8-folds) according to one embodiment of the present invention, whereas the transduction efficiency of PEI 25K / pGL3 and lipofectamine / pGL3 was significantly reduced (192.7-folds, respectively). And 3468.4-folds) (FIG. 4C).

<3-3> Buffer Capacity Analysis

 This high transduction efficiency of the spermine copolymer according to the invention is due to the buffering capacity of the copolymer. In order to determine the transduction mechanism of the spermine copolymer (GPT-SPE) according to an embodiment of the present invention, the buffering capacity (buffering capacity) of the copolymer was investigated. Bafilomycin A1 (bafilomycin A1, specific inhibitor of vacuolar type H + ATPase, 200 nM) diluted in DMSO was added to A549 cells cultured in a well plate as described above for 10 minutes, followed by transduction in the same manner. The efficiency was measured. As a result, as shown in Figure 4d, the transduction efficiency of the spermine copolymer (GPT-SPE) according to an embodiment of the present invention was drastically decreased after the treatment with bacillomycin A1, which is the The Min copolymer (GPT-SPE) has a high buffering capacity, and thus can be rapidly released from the endosome and introduced into the cytoplasm by the proton sponge effect.

Example 4: Cytotoxicity of Spermine Copolymers

To examine the cytotoxicity of one embodiment of spermine copolymer (GPT-SPE) of the present invention, by performing a cell proliferation assay ^{(Cell Titer 96 ® Aqueous One Solution} Cell Proliferation Assay) while varying the concentration of the copolymer Cell viability was investigated.

First, A549 (human lung adenocarcinoma cell (ATCC) cells and MCF7 (human breast cancer cell (ATCC) cells) cells, RPMI 1640 (Gibco BRL, Paris, France) medium, HepG2 (human hepatoblastoma cells, Korea Cell Line Bank) cells and HeLa Human cervical carcinoma cells (ATCC) cells were inoculated in Dulbecco's modified Eagle medium (Gibco BRL) medium and cultured in a 37 ° C., 5% CO ₂ incubator. At this time, 10% fetal calf serum (FBS, HyClone, Logan, UT, USA), 100 μg / ml streptomycin and 100 U / ml penicillin were added to all the medium. Cultured A549, HepG2, MCF7 and HeLa cells were seeded in 96-well plates at a density of 1 × 10 ⁴ / well and incubated in 37 ° C., 5% CO ₂ incubator for 18 hours. The medium of the well plate was replaced with serum-free medium containing the spermine copolymers of the present invention at concentrations of 5, 10, 20, 50 and 100 μg / ml, respectively, and then again at 37 ° C., 5% CO for 24 hours. Incubated in ₂ incubators. At this time, untreated cell culture was used as a control, and cell cultures treated with spermine (202.34 Da, Sigma-Aldrich) and PEI 25K (25 kDa, Sigma-Aldrich) at the same concentration were used as a comparison group. Thereafter, the mixture was replaced with growth medium (growth medium) that contains the culture medium of the cells 20 ㎕ potency assay reagent ^{(Cell Titer 96 ® Aqueous One Solution} Reagent) of the well plate. After incubation for 3 hours, the metabolic activity of the cells was examined by measuring the absorbance of the well plate at 590 nm using an ELISA plate reader (GLR 1000, Genelabs Diagnostics, Singapore).

As a result, as shown in Figure 5, compared to the control group treated with the spermine copolymer (GPT-SPE) of one embodiment of the present invention all showed a high cell viability even at a high concentration of 100 ㎍ / ㎖ ( 62.48% in A549 cells; 76.33% in HepG2 cells; 85.05% in MCF7 cells; and 79.00% in HeLa cells); cell viability of cells treated with PEI 25K decreased rapidly with increasing concentration (100 μg / ml 12.32% in A549 cells; 18.92% in HepG2 cells; 29.58% in MCF7 cells; and 19.71% in HeLa cells). From the above results, it was confirmed that the spermine copolymer of the present invention has very low cytotoxicity and excellent biocompatibility.

Example 5 In Vivo Aerosol Delivery of Spermine Copolymers

In order to confirm the in vivo delivery efficiency of the spermine copolymer according to the present invention by the aerosol was performed.

First, a plasmid pcDNA3.1 / CT-GFP (6.1 kb, Invitrogen) expressing a spermine copolymer and a green fluorescent protein (GFP) according to the method described in Example 2 was weight ratio of 10: 1. The nucleic acid delivery complex (GPT-SPE / GFP) was prepared by mixing.

Six-week-old male BALB / c mice (Breeding and Research Center) were stored in laboratory animal facilities maintaining a 12 hour night / day cycle at a temperature of 23 ± 2 ° C. and a relative humidity of 50 ± 20%. All experimental procedures in this study were approved by the Animal Care and Use Committee at Seoul National University (SNU-101211-2). To confirm the efficiency of nucleic acid delivery through the aerosol, a chamber (NOEC; Dusturbo, Seoul, Korea) was used to expose only the nasal area of the mouse, and 12 mice were randomly divided into three groups (four in each group) as follows. Divided by: untreated control; GFP only treatment group; And spermine copolymer and GFP complex (GPT-SPE / GFP) treatment group. The mice in each group were exposed to aerosols based on the method used in the literature to deliver 1 mg of DNA (HL Jiang, et al. Biomaterials 30: 5844-5852, 2009).

Two days after aerosol exposure, mice in each group were sacrificed and lungs removed. The extracted lungs were soaked in 4% paraformaldehyde and fixed for 12 hours, and then immersed in 30% sucrose solution and stored at 4 ° C. for 48 hours. The immobilized lung was embedded with an OCT compound (Tissue-Tek OCT, Sakura) at a temperature of -20 ° C. or lower, and cut into a microtome to prepare 10 μm thick lung tissue sections. For histopathological analysis, the prepared lung tissue sections were stained with hematoxylin and eosin (H & E) and then observed GFP signals with a confocal microscope (Zeiss LSM510, Carl Zeiss). In addition, for toxicity testing, lung, heart, liver, spleen, brain and kidney tissues extracted from each group of mice were fixed with 10% neutral formalin and paraffin sections of 3 μm were prepared, followed by H & E staining. It was observed by confocal microscopy (Zeiss LSM510, Carl Zeiss).

As a result, as shown in FIG. 6A, the GFP signal was not detected or very weak in the untreated control group and the GPT-treated group (without spermine copolymer), whereas the spermine copolymer and GFP of the present invention In the complex (GPT-SPE / GFP) treatment group, a strong GFP signal is detected, indicating that the aerosol delivery efficiency is very good.

As a result of histopathological examination of the lung, as shown in b of FIG. 6, it was confirmed that no pathological abnormality occurred in the lung upon aerosol delivery using the spermine copolymer according to the present invention. In addition, as shown in Figure 7, no pathological abnormalities were found in the major organs, such as the heart, liver, spleen, brain, kidney, including the lung, by aerosol delivery using the spermine copolymer according to the present invention It was confirmed that no toxicity was induced.

Example 6: Gene Therapy of Lung Cancer with Spermine Copolymer

In order to confirm the gene therapy effect of lung cancer using the spermine copolymer according to the present invention, the following experiment was performed.

Oligonucleotides encoding the 19-mer hairpin sequence of shRNA constructs targeting Akt1 were synthesized according to the method disclosed in Meng et al., Cell Signal 2006, 18 (12): 2262e71 and scrambled as a control thereof. (scramble) shRNA was prepared. A nucleic acid delivery complex (GPT-SPE / shAkt1 or GPT-SPE / Scr) was prepared by mixing 4 mg of spermine copolymer and 0.4 mg of Akt1 shRNA or scrambled shRNA in distilled water according to the method described in Example 2.

Six-week-old female k-ras mice with lung cancer (Breeding and Research Center) were stored in laboratory animal facilities maintaining a 12 hour night / day cycle at a temperature of 23 ± 2 ° C. and a relative humidity of 50 ± 20%. All experimental procedures in this study were approved by the Animal Care and Use Committee at Seoul National University (SNU-101211-2). To confirm the efficiency of nucleic acid delivery through the aerosol, a chamber (NOEC; Dusturbo, Seoul, Korea) was used to expose only the nose area of the mouse, and 16 mice were randomly divided into four groups (four in each group) as follows. Divided by: untreated control; Akt1 shRNA treatment group; Spermine copolymer and Akt1 shRNA complex (GPT-SPE / shAkt1) treatment group; And spermine copolymer and scrambled shRNA complex (GPT-SPE / Scr) treated group. The mice in each group were exposed to aerosols based on the method used in the literature to deliver 1 mg of DNA (HL Jiang, et al. Biomaterials 30: 5844-5852, 2009).

Mice were exposed to aerosol twice weekly for four weeks, after which mice in each group were sacrificed. At necropsy, tumor lesions on the lung surface were counted under a microscope according to the method described by Singh RP et al., J. Natl. Cancer Inst. 2006, 98 (12): 846-55. At the same time, the lungs were perfused with PBS, the lobes of the left lung were recovered, and fixed with 10% neutral buffered formalin to perform histopathological examination.

As a result, as shown in Figures 8a, 8b and 8c, the spermine aerial of the present invention compared to the untreated control group, Akt1 shRNA treated group and spermine copolymer and scrambled shRNA complex (GPT-SPE / Scr) treated group It was confirmed that the number of tumors, in particular, the number of tumors having a size of 1 mm or more was significantly reduced in the combination and Akt1 shRNA complex (GPT-SPE / shAkt1) treatment groups. In addition, as shown in Figure 8d, the spermine copolymer and Akt1 shRNA complex of the present invention compared to the untreated control group, Akt1 shRNA treatment group and spermine copolymer and scrambled shRNA complex (GPT-SPE / Scr) treatment group. It was confirmed that the expression of Akt1 protein in the lung was significantly reduced in the (GPT-SPE / shAkt1) treated group. In addition, as can be seen in Table 2 below, little toxicity was detected in the blood. This indicates that the spermine copolymer (GPT-SPE) of one embodiment of the present invention can be effectively used as an Akt1 shRNA delivery vehicle in vivo and can be usefully used for the treatment of lung cancer.

TABLE 2

CBC: complete blood count, WBC: white blood cell, RBC: red blood cell, HGB: hemoglobin, HCT: hematocrit, MCV: mean cell volume, MCH: mean Corpuscular Hemoglobin , MCHC: mean corpuscular hemoglobin concentration, CHCM: mean cell hemoglobin concentration, RDW: red cell distribution width, PLT: platelet, MPV: mean platelet volume (Mean platelet volume), PDW: platelet distribution width, PCT: plateletcrit, MPC: mean platelet component, MPM: mean platelet mass, Large Pit: large platelet (large platelets)

Claims (19)

1. A spermine copolymer in which a primary amine or a secondary amine of spermine is bonded by an addition reaction to at least one acrylate terminal of a linker represented by Formula 1 below.

   [Formula 1]

   
2. The spermine copolymer of claim 1, wherein the linker is a compound represented by the following Chemical Formula 2.

   [Formula 2]

   
3. The spermine copolymer according to claim 1, wherein a primary amine or a secondary amine in the spermine that is not linked to a linker is bonded to an acrylate end of the linker represented by another formula (1).
4. The spermine copolymer according to claim 3, wherein spermine forms a dendrimer through a linker represented by the formula (1).
5. The spermine copolymer of claim 1, wherein the copolymer has a molecular weight ranging from 3 to 15 kDa.
6. The spermine of claim 1, wherein spermine is further bound to polyethylene glycol (PEG), galactosylated polyethylene glycol (gal-PEG), folate, mannose, or a combination thereof. Copolymer.
7. A nucleic acid delivery complex wherein a therapeutic nucleic acid is bound to the spermine copolymer of any one of claims 1 to 6.
8. 8. The nucleic acid delivery complex according to claim 7, wherein said therapeutic nucleic acid and spermine copolymer are combined in a weight ratio of 1: 5 to 1:50.
9. 8. The nucleic acid delivery complex according to claim 7, wherein said nucleic acid delivery complex has an average particle size of 120 to 200 nm.
10. 8. The nucleic acid delivery complex of claim 7, wherein said nucleic acid delivery complex exhibits a zeta potential in the range of 5 to 30 mV.
11. 8. The method of claim 7, wherein the therapeutic nucleic acid is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), an antisense oligonucleotide, a DNA, a single stranded RNA, a double stranded RNA, a DNA-RNA hybrid and a ribozyme. A nucleic acid delivery complex that is in a form selected from the group consisting of.
12. 8. The nucleic acid delivery complex of claim 7, wherein the therapeutic nucleic acid targets the inhibition of expression of Akt1.
13. The nucleic acid delivery complex according to claim 12, wherein the therapeutic nucleic acid is a shRNA having a nucleotide sequence set forth in SEQ ID NO: 1 that inhibits expression of Akt1.
14. Claim 7 gene therapy pharmaceutical composition comprising the nucleic acid delivery complex as an active ingredient.
15. The pharmaceutical composition of claim 14, wherein the nucleic acid delivery complex is formulated for inhalation or intravenous administration.
16. Performing a Michael addition reaction by mixing the linker compound represented by Formula 1 dissolved in a solvent with spermine dissolved in a solvent; And

    A process for preparing the spermine copolymer of any one of claims 1 to 6 comprising the step of separating the spermine copolymer formed from the reactant.

    [Formula 1]

    
17. The method of claim 16, wherein the solvent is anhydrous ethyl alcohol or anhydrous methyl alcohol.
18. The method of claim 16, wherein the Michael addition reaction is carried out at 3 to 5 ° C. for 1 to 3 hours and then at 20 to 25 ° C. for 12 to 24 hours.
19. The method of claim 16, wherein the reaction molar ratio of spermine and the linker compound is from 4: 3 to 8: 3.

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