WO2014051318A2 - Short interference rna gene delivery system for systemic circulation - Google Patents

Abstract

The present invention relates to a gene delivery system which improves siRNA delivery and the in vivo systemic circulation efficiency thereof More particularly, the present invention is a siRNA gene delivery system for systemic circulation based on polyethylene glycol (PEG) and an arginine 9 (R9) peptide.

Description

Short Interfering RNA Gene Carriers for Systemic Circulation

The present invention relates to a gene carrier with improved efficiency of siRNA delivery and its systemic circulation in the body. More specifically, the present invention is based on polyethylene glycol (PEG) and R9 (arginine) peptides, and systemic circulation. Invention for siRNA gene delivery system.

Although various gene therapy techniques have been developed as a substitute for conventional protein therapy, there are still important challenges to be solved. One of the major challenges of gene therapy is to achieve efficient influx through plasma membranes (animal cells) and nuclear membranes with minimal cytotoxicity.

Gene therapy systems can be broadly classified into viral vector-mediated systems and non-viral vector-mediated systems. Viral vectors made using retroviruses or adenoviruses have the advantage of having high transfection efficiency into cells, but have problems with immunogenicity in vivo and Inherent problems such as genetic recombination. To overcome the stability problem of these viral vectors, various polymeric gene delivery systems have been developed as an alternative to traditional viral vector-based gene delivery methods. However, polymeric vectors have the problem of having intracellular trafficking barriers such as endosomal escape and nuclear localization.

Gene delivery systems based on synthetic peptides can overcome the above problems associated with polymeric gene delivery systems by condensing DNA by causing leakage in the endosomal membrane at low pH and also promoting endosomal escape.

For this reason, various synthetic peptides have been developed to promote gene transfer in different cell lines in vitro . However, there are also problems such as toxicity and serum instability in in vivo applications. In particular, studies on vectors using short cationic peptides have been conducted in this regard, but in this case, there is a problem that the nucleic acid is unstable in the extracellular space, and that the stability of the complex with the nucleic acid and the level of gene expression are insufficient. There is a problem.

Among them, in the case of RNAi nucleic acid delivery, since the stability of the complex with the nucleic acid was low, the delivery to the synthetic peptide was difficult. Therefore, it was made by using lipids or liposomes, and it was difficult to effectively systemic systemic circulation.

Therefore, the present inventors, while studying a gene delivery system using a peptide, in particular, by PEGylating the vector of the R9 structure in which Cys is bound to one or both ends, it is possible to significantly improve the siRNA delivery efficiency. And the present invention was completed.

The main object of the present invention is to provide a siRNA carrier containing polyethylene glycol (PEG) and R9 (arginine) peptide and a method for delivering it into the body by systemic administration.

Another object of the present invention is polyethylene glycol (PEG) and R9 (arginine) peptide; And to provide a complex containing the desired siRNA.

Still another object of the present invention is to provide various uses of the complex containing siRNA carriers and siRNA for systemic circulation.

In order to solve the above problems,

Polyethyleneglycol (PEG) and R9 (arginine) peptide base structure (PEG-R9), provides a variety of uses for siRNA delivery system for systemic circulation. In particular, the present invention relates to an effective method of delivering siRNA genes for treating a desired disease into the body by systemic administration.

In one specific aspect, the present invention provides a siRNA delivery system for systemic circulation (PEG-R9) containing polyethylene glycol (PEG) and R9 (arginine) peptide.

In another specific aspect, the present invention provides a systemic circulating complex (PEG-R9-siRNA) containing polyethylene glycol (PEG), an R9 (arginine) peptide and a siRNA gene for treating a desired disease; And it provides a pharmaceutical composition for systemic circulation containing the same.

In particular, the R9 (arginine) peptide is characterized in that Cys is bonded to one or both ends, and preferably has a Cys- (D-R9) -Cys or Gly- (D-R9) -Cys structure. . Most preferably it forms a Cys- (D-R9) -Cys structure.

At this time, PEG has a structure in which PEG is bonded to one or both ends of the peptide by the amine group (-NH ₂ ) of Cys.

The polyethylene glycol (PEG) preferably has a molecular weight of 500 Daltons.

In addition, the systemic circulation complex of the present invention has an advantage of having a small size as a siRNA carrier by having a nano size of 200 nm or less in diameter, and having an excellent charge ratio (+/-) of 6: 1 to 15: 1. Has transfection efficiency. In this case, when the charge ratio (+ /-) of 12: 1, the transfection efficiency is the best.

The complex can exhibit an excellent therapeutic effect by increasing the body's circulation time of siRNA for the treatment of the desired disease.

In one embodiment of the present invention, siVEGF was used as the cancer treatment gene siRNA.

As such, PEG-R9-siRNA of the present invention can be used as an siRNA delivery system in which systemic circulation in the body is effective, having high transfection efficiency, low cytotoxicity, and high target gene expression efficiency.

PEG-R9 of the present invention may be the basis for siRNA delivery systems that significantly increase the delivery efficiency in the body, particularly by systemic administration of siRNA. Effective systemic administration can be achieved by PEGylating the ends of the R9 construct having Cys bound to one or both ends of the present invention, among various protein delivery domain types, thereby significantly improving the systemic delivery efficiency of siRNA. It is very useful as siRNA gene delivery system.

1 is a schematic diagram of a nucleic acid carrier using a conjugate of polyethylene glycol (PEG) and oligo arginine (R9) of the present invention.

Figure 2 is a result of confirming the conjugate formation and stability of PEG-R9.

3 shows the results of size and zeta potential measurements after PEG-R9-siVEGF complexes (polyplexes) formation.

4 shows the structure of C- (D-R9) -C in PEG-R9, which is a result of comparing the efficiency of transfection with D-R9 of another structure.

5 shows the results of luciferase analysis using PEG500 and PEG1000 to find the optimal PEG molecular weight.

6 is a luciferase assay (a) for intracellular gene transfer efficiency and an MTT assay (b) for cytotoxicity.

Fig. 7 shows the results of size and zeta potential analysis in the delivery vehicle using cys-R9-cys, PEG-R9 and PEG-cys-R9-cys.

Fig. 8 shows the results of size and zeta potential analysis in the delivery vehicle using PEG-cys-R9-cys, PEG-TAT, and PEG-cys-TAT-cys.

9 is a graph comparing the growth rate of cancer cells according to PEG-R9-siVEGF complex (polyplexes) administration.

10 is a photograph observing the inhibition of cancer cell growth 10 days after administration in nude mice transplanted with cancer.

Definitions of terms used in the present invention are as follows.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

The term "polynucleotide" refers to nucleotide polymers of all lengths, including ribonucleotides as well as deoxyribonucleotides.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. One or both strands of a double stranded nucleic acid molecule and any fragment or portion of an intact nucleic acid molecule. Representative nucleic acids intended for delivery in the present invention is siRNA.

The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is associated. The term "expression vector" includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector. Preferred vectors are those capable of self replication and expression of the associated nucleic acid.

"Transfection" refers to a method of expressing genotypes in a cell by directly introducing nucleic acids (DNA, RNA, etc.) into a culture animal cell. Putting it in is a common way. When the introduced gene was stabilized in cells, it was often interrupted by chromosomes. A cell into which a nucleic acid is introduced is called a transducer. Since the transduction efficiency is very low, several methods have been developed to increase the efficiency. Among them, calcium phosphate coprecipitation and DEAE-textlan treatment. Electroporation, redistribution (fusion method with cells that make artificial membranes and DNA complexes called liposomes).

By "zeta potential" is meant an electrodynamic potential difference resulting from the difference in positive charge density in the diffusion bilayer of immobilized moisture that is easily attached to the charged particle surface and movable moisture that is easily separated from the particle. Sometimes referred to as the electrical potential difference or zeta potential between the cell surface and the surrounding culture.

"Charge rate" refers to the proportion of each charge used in a complex that functions as a gene carrier, in which negatively charged DNA binds to a positively charged carrier or carrier through electrostatic attraction. After the complex is formed, the transfer efficiency is good when the overall charge is positive, because the cell membrane is negatively charged.

"Amino acid" and "amino acid residue" refer to natural amino acids, unnatural amino acids, and modified amino acids. Unless stated otherwise, all references to amino acids include references to both D and L stereoisomers (where the structure allows such stereoisomeric forms), either generically or by name. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) and Valine (Val) is included. In the present invention, it is preferable to use the D isomer of arginine (Arg).

The term “gene expression” generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in a cell. In that sense, gene expression includes not only transcriptional and translational processes, but also posttranscriptional and posttranslational processes that can affect the biological activity of a gene or gene product. The processes include RNA synthesis, processing and transport, as well as post-translational modifications of polypeptide synthesis, transport and polypeptides.

Luciferase is an enzyme that promotes the oxidation of luciferin and converts chemical energy into light energy to emit light. It is used to measure expression continuously and in real time in vivo. It functions as a reporter gene that allows you to verify the effect. It can be obtained directly from insects such as firefly or glow-worm or by expression from microorganisms comprising recombinant DNA fragments encoding such enzymes.

A "carrier" or "carrier" refers to a polymer material that is responsible for transporting a carrier when an active substance in an organism is present in combination with another substance or when a substance is transferred through a cell membrane. Examples of carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins such as serum albumin, gelatin or immunoglobulins, polyvinylpyrrolidone Hydrophilic polymers, such as glycine glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, sodium Salt-forming counterions, and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®.

"Treatment" is an approach for obtaining beneficial or desired results, including clinical results. "Treatment" or "mitigation" of a disease, disorder or condition is a progression of progression with less and / or less clinical signs and / or undesirable clinical signs of the condition, disorder or disease state as compared to not treating the disorder. Means slowing down or getting longer. For the purposes of the present invention, beneficial or desirable clinical outcomes, whether detectable or non-detectable, may alleviate or ameliorate one or more symptoms, reduce the extent of disease, or stabilize (ie, not worsen) a disease. , Delaying or slowing disease progression, ameliorating or alleviating disease state, and soothing (partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if untreated. In addition, treatment does not need to occur by administration of a single dose and often occurs upon administration of a series of doses. Thus, a therapeutically effective amount, an amount sufficient to alleviate, or an amount sufficient to treat a disease, disorder or condition may be administered in one or more administrations.

The term "disorder" is any condition that would benefit from treatment with molecules identified using the transgenic animal model of the present invention. This includes chronic and acute diseases or conditions, including pathological conditions that make mammals susceptible to mysterious diseases. Examples of diseases to be dealt with herein are, but are not limited to, cancer and the like.

A "therapeutically effective amount" means an active compound in a composition that will elicit a biological or medical response in a tissue, system, subject or human being sought by a researcher, veterinarian, physician or other clinician, which includes alleviating the symptoms of the disorder to be treated. Means the amount of.

"Geneology" refers to treating a genetic disease by correcting a mutated gene or treating a disease by controlling protein expression using a gene or RNAi. In other words, it is a method of treating a disease by transplanting a normal gene to a patient's cell and changing the phenotype of the cell.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to intracellular transfection of nucleic acid, and more particularly, to various uses, including a carrier and a method for enhancing the systemic delivery efficiency of siRNA by systemic administration.

The present invention in one aspect, non-viral siRNA gene delivery or vector for systemic circulation, including polyethylene glycol (PEG) and poly (oligo-arginine), in particular R9 (arginine) peptide; And a method of using the same.

A non-viral gene transfer vector is a generic term for a carrier that carries a gene into a cell without using a virus. The non-viral gene transfer vector uses a negative charge property of the nucleic acid constituting the gene. Vectors in the form of coating nucleic acids using interactions are representative examples.

The poly (oligo-arginine) of the present invention includes poly (oligo-L-arginine) and poly (oligo-D-arginine), and among them, poly (oligo-D-arginine) is most preferred. High molecular weight poly (oligo-D-arginine) effectively promotes condensation of DNA to form stable complexes and internalization of DNA into cells, and after internalization the complexes escape from the endosome to the cytoplasmic space by reduction of disulfide bonds. do.

The reducing poly (oligo-D-arginine) of the present invention is preferably composed of a cationic oligomer including terminal cysteine crosslinked with disulfide, but is not limited thereto. Cysteine is the only amino acid that contains sulfhydryl groups that form disulfide cross-linking with other adjacent cysteine molecules, and the protein delivery domain (PTD) moiety other than the disulfide-crosslinked terminal cysteine is any cationic peptide. Can be

More preferably, the reducing poly (oligo-D-arginine) of the present invention is characterized in that Cys is bonded to one or both ends. Most preferably, Cys is bonded to both ends. For example, it may consist of Cys- (D-R9) -Cys or Gly- (D-R9) -Cys repeat units. Such reducing poly (oligo-D-arginine) can be prepared by DMSO oxidation of the terminal cysteine-thiol groups of Cys- (D-R9) -Cys or Gly- (D-R9) -Cys repeat units, and reduction It can be fragmented into Cys- (DR) 9-Cys as a reagent.

That is, the reducing poly (oligo-arginine) of the present invention has a structure composed of nine arginine (R9) in which Cys is bonded at one or both ends thereof, preferably Cys- (D-R9) -Cys or Gly- (D-R9) -Cys structure, most preferably Cys- (D-R9) -Cys. Cys is located at both ends such that the effective condensation of the oligo peptoplex (peptoplex) and thus the neutral charge properties of the complex can be retained.

The gene delivery system of the present invention is characterized by binding polyethylene glycol (PEG) to an R9 (arginine) structure in which Cys is bound to one or both ends for systemic circulation.

About the carrier of this invention which has such a structure, it may be described in the specification of this invention by the abbreviation of "PEG-R9." That is, in the present invention, PEG-R9 refers to a structure in which polyethylene glycol (PEG) is bonded to one or more ends of R9 (arginine) in which Cys is bonded to one or both ends. However, in the experiment for determining the binding effect of Cys in the comparative example, Cys was separately indicated.

PEGylation (PEGylation) of protein is used to improve the systemic circulation efficiency of the transfer gene, the conjugation reaction occurs mainly through the four functional groups of the protein (carboxyl PEGylation, amine, respectively) It is called Amine PEGylation, N-terminal PEGylation, or Thiol PEGylation.

In the present invention, it is based on the R9 peptide structure, for example, Cys- (D-R9) -Cys or Gly- (D-R9) -Cys peptide structure, in which Cys is bonded at one or both ends, Amine PEGylation was used.

On the other hand, the PEG-R9 vector of the present invention is efficient for in vivo delivery of nucleic acid. Among them, it is very preferable for efficient in vivo delivery of siRNA.

RNA interference (RNAi) is a natural mechanism that involves specifically down-regulating expression of a gene of interest by a double helix, short interfering RNA (siRNA), which is an example of the type of RNAi reagent that mediates such RNAi. (Short interfering RNA) or miRNA (microRNA) or small hairpin RNA (shRNA), and the present invention is particularly preferred for in vivo delivery of siRNA (short interfering RNA).

Liposomes, which have been used for siRNA delivery in the body, are lost in the circulatory system by phagocytosis of macrophages in the liver or spleen due to the adsorption of blood proteins, as in the case of other particulate drug carriers for systemic circulation. Or, there was a problem that the drug is released from liposomes during the blood circulation. In particular, phagocytosis of macrophages occurs due to the adsorption of opsonic proteins on the surface of liposomes. As a technique to solve this problem, phospholipid-PEG derivatives incorporating PEG (poly [ethylene glycol]) at the end of phospholipid Liposomes that can inhibit the adsorption of opsonine proteins have been developed by using as a constituent of liposomes or by coating the surface of the prepared liposomes with PEG or polysaccharides.

However, when such a phospholipid-PEG derivative is used as a constituent of liposomes, liposomes have a low self-stability and have low transfection efficiency and toxicity. Therefore, it is difficult to apply in vivo for in vivo administration. In addition, since most of them go to the liver after administration, there was also a problem of low distribution of tumors compared to liver.

In contrast, the construct of PEG-R9 of the present invention, most preferably PEG-Cys-R9-Cys construct, is very effective in the systemic circulation of siRNA without such problems.

In another aspect, the present invention, polyethylene glycol (PEG); R9 (arginine) peptides; And siRNA complexes for systemic circulation, consisting of siRNA; And it relates to a composition for treating a target disease comprising the same, to a configuration in which the target gene siRNA is coupled to the gene carrier. In this case, it is preferable that Cys is bonded to one or both ends of the R9 peptide.

As the gene siRNA delivered by the complex of the present invention, any desired siRNA for the desired treatment can be inserted, and they may exist or be synthesized in nature, and may exist in various sizes from oligonucleotides to chromosomes in size. have. These genes come from humans, animals, plants, bacteria, viruses and the like. These can be obtained using methods known in the art.

The complex may also include therapeutic gene expression regulators of a desired disease, such as cancer, such as transcriptional promoters, enhancers, silencers, operators, terminators, attenuators and other expression regulators.

In one embodiment of the present invention, polyethylene glycol (PEG); R9 (arginine) peptides; And it can provide a composition for treating a target disease comprising a siRNA delivery complex consisting of a siRNA for treating the desired disease.

In some embodiments, siRNAs are expressed from transcriptional units inserted into nucleic acid vectors (commonly referred to as recombinant vectors or expression vectors). Vectors can be used to deliver nucleic acid molecules encoding siRNA into cells to target specific genes.

In addition, many suitable methods are known in the art. In general, transfection of cells expressing a target gene can be carried out using a variety of methods for transfection, such as, for example, electroporation, the use of cationic lipids or cationic polymers as helpers for transfection. Can be. Thereafter, the cells are cultured under suitable conditions to allow expression of the target gene. The expression of the target gene is then measured using a suitable technique such as, for example, measuring the amount of RT-PCR or reporter gene. Basically, any type of cell can be used for transfection, but in a preferred embodiment the cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell, most preferably a human cell.

In one embodiment of the present invention, siVEGF for anticancer was used as siRNA.

VEGF (vascular endothelial growth factor) is a factor that is overexpressed in most cancer (tumor) cells such as thyroid cancer, breast cancer, prostate cancer, colon cancer and cervical cancer, and is closely related to the occurrence, recurrence and metastasis of cancer. Therefore, the PEG-R9 transporter of the present invention, which can efficiently deliver siVEGF to cancer cells, may be usefully used for anticancer purposes.

Cancers that overexpress VEGF, such as lung cancer, gynecologic malignancies, melanoma, breast cancer, pancreatic cancer, ovarian cancer, uterine cancer, colorectal cancer, prostate cancer, kidney cancer, head cancer, pancreatic cancer, liver cancer (hepatocellular cancer) ), Cervical cancer, neck cancer, kidney cancer (renal cell cancer), sarcoma, myeloma, lymphoma and the like can be usefully used.

A therapeutically effective amount of the complex or composition according to the invention can be provided by known routes of administration. Embodiments according to the invention are applied to enable the transfection of PEG-R9-siVEGF into cancer cells.

The composition of the present invention may include or use any of the above means for transfecting genetic material into target cells, but not limited thereto. The siRNA may further include a cationic amphiphilic material to be released into cancer cells.

In addition, the nucleic acid molecules and promoters of the present invention may be formulated into pharmaceutical compositions prepared according to conventional pharmaceutical synthesis techniques.

The composition may comprise the active agent or a pharmaceutically acceptable salt of the active agent. The composition may be administered simultaneously or sequentially. These compositions may comprise, in addition to one active substance, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances well known in the art. These substances should be nontoxic and should not interfere with the efficacy of the active ingredient.

The carrier can take a variety of forms depending on whether it is the form of preparation desired for administration, eg, topical, intravenous, oral, meninges, neural epithelium, or parenteral. It can generally be prepared for oral or parenteral administration by mixing with fillers, dilators, binders, wetting agents, disintegrating agents, diluents such as surfactants, excipients.

Dosages and schedules effective for the administration of the complex and compositions comprising the complex can be determined empirically and can be appropriately determined by one skilled in the art. Single or multiple doses may be used.

In the present invention, PEG-R9 based transporters and complexes for the systemic circulation of siRNA have the following advantages.

(1) "PEG-R9" of the present invention is well conjugated with siRNA and excellent in its protective effect.

The transfection efficiency of PEG-R9 of the present invention is a charge ratio (+/-) of 6: 1 to 15: 1, moreover, because condensation of PEG-R9 and gene siRNA occurs well above a charge ratio of 6: 1. Preferably it has a charge ratio (+ /-) of 9: 1 to 15: 1. In particular, in one embodiment the carrier of the present invention exhibited the highest gene transfection efficiency at a charge ratio of 12: 1.

In the present invention, the charge ratio is a phosphate (phosphate) of the components of the gene (nucleic acid) is a negative charge and arginine of the components of the carrier is a positive charge, the negative charge of the gene as a reference to 1 and then reacted By increasing the amount of the carrier 6 to 15 times means the rate of reaction with the gene. In other words, having a charge ratio of 6: 1 to 15: 1 means that the amount of the carrier is 6 to 15 times larger than the target gene.

This is because the cell membrane is negatively charged, so that when the gene transfer complex is positively charged as a whole, the transfer efficiency is excellent. When the complex is negatively charged, it does not easily pass through the cell membrane, whereas when the complex is positively charged, it can easily pass through the cell membrane by a charge-to-charge reaction. In addition, the amount of charge affects cell membrane permeability, and the higher the amount of charge, the greater the ability to pass through the cell membrane.

On the other hand, PEG-R9 of the present invention has a much higher gene expression efficiency compared to the cationic carrier PEI that is commonly used. That is, PEG-R9 showed more effective siRNA condensation ability, and protected from DNA degradation in serum at an appropriate charge ratio.

(2) "PEG-R9" of the present invention combines with siRNA to form a nano sized complex and has a positive zeta potential value.

PEG-R9, when bound with siRNA, concentrates DNA more effectively by electrostatic interaction. In the case of including the Cys structure at both ends of the R9 of the present invention, the positive charge is displayed in the neutral condition, so that the complex can be effectively concentrated to the nano-size (about 200 nm or less), and the zeta of the charge ratio of about 5 or more (positive) It can have potential.

As described above, when the complex is negatively charged, the negative electrode cannot easily pass through the cell membrane having the same negative charge, while the positively charged complex can easily pass through the cell membrane by a charge-to-charge reaction, thereby providing a positive amount of the present invention. The property of zeta potential suggests excellent gene transfer efficiency.

In particular, through the embodiments of the present invention the particle size of the PEG-bonded complex is smaller than when PEG is not bound; And when the cysteine (cyctein, Cys) is bonded to both ends of R9 was confirmed that the size of the particles are smaller and uniform. In other words, the most preferred form of the present invention is that cysteine is bonded to both ends of the R9 oligopeptide, and furthermore, PEG is bonded thereto.

(3) "PEG-R9" of the present invention is excellent in the siRNA delivery efficiency and expression rate in the cell, there is no cytotoxicity.

When Cys and PEG were coupled to R9 of the present invention than other gene carriers, TAT, siRNA delivery efficiency was higher (FIG. 8), and intracellular expression rate was also confirmed using luciferase DNA (FIG. 6). ).

In addition, compared with the conventional polymeric cationic carrier PEI and the like, PEG-R9 of the present invention is low in cytotoxicity so that it can be used in vivo. In one embodiment of the invention PEG-R9 showed at least 95% cell viability compared to toxic PEI (FIG. 6).

(4) "PEG-R9" of the present invention shows an excellent anticancer effect by increasing the circulating time of siRNA.

In the present invention, the PEG-R9 and the siRNA conjugate were observed by intravenous injection into nude mice, and it was confirmed that cancer cell growth was significantly inhibited.

In short, the PEG-R9 and PEG-R9-siRNA of the present invention can be used as an siRNA delivery system in which systemic circulation in the body is effective, having high transfection efficiency, low cytotoxicity, and high target gene expression efficiency.

EXAMPLE

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

Example 1: PEG-R9-siRNA Complex Formation

A schematic diagram of a nucleic acid carrier using a conjugate of polyethylene glycol and oligo arginine is shown in FIG. 1.

In the Cys- (D-R9) -Cys structure, N-Hydroxysuccinimide (NHS) ester PEG (molecular weight 500, Thermo Fisher Scientific Inc. Rockford, IL USA) is bonded to the amine group (-NH2) of Cys. Peptide binding occurs when the peptide reacts with NHS ester PEG at a reaction ratio of 1: 1 at slightly basic conditions of pH 7-8.

siVEGF (5-AUGUGAAUGCAGACCAAAGAA dTdT-3) was purchased from Bioneer and the peptide (PEG-Cys- (D-R9) -Cys) was synthetically ordered from Anigen.

50 pmol siVEGF, deionized water and PEG-R9 (charge ratio: 6, 9) were incubated at room temperature for 20 minutes to prepare oligo peptoplexes (oligo-peptoplexe). After incubation, electrophoresis was performed at 2% agarose gel at 100V for 20 minutes. In addition, after incubating siVEGF and PEG-R9, the mouse serum was added at a volume ratio of 90% to perform a stability test for up to 24 hours. Heparin was added to isolate PEG-R9 and siVEGF to determine whether the band of siVEGF was maintained.

As a result, as shown in Figure 2, PEG-R9 form also showed that the conjugate with the nucleic acid is well formed, it was confirmed that the protective effect of the nucleic acid to the serum is excellent.

Example 2 PEG-R9-siRNA Complex Size and Zeta Potential

Zeta potential and mean diameter of PEG-R9 / siVEGF oligo-peptoplex (complex) were measured using dynamic light scattering (DLS).

5 μg siVEGF, deionized water and PEG-R9 (charge ratio: 6, 9, 12, 15) were incubated at room temperature for 30 minutes to prepare oligo peptoplexes. The average diameter and surface zeta potential of the oligo peptoplexes were determined using a DLS with Zetasizer-Nano ZS (Malvern Instruments, UK).

As a result, as shown in FIG. 3, the zeta potential showed a positive value at the charge ratio 6 or more, and the average diameter was less than 200 nm at the charge ratio 6 or more. The average diameter was the smallest at charge ratio 12 (100 nm).

Example 3: Evaluation of Intracellular Delivery (Transfection) Efficiency of PEG-R9

R9 used for PEG-R9 is bound to PEG in the structure of C- (D-R9) -C. In order to check whether the structure of C- (D-R9) -C is optimal, the following groups were compared. A complex of the carrier of G- (D-R9) -G, G- (D-R9) -C, C- (D-R9) -G, C- (D-R9) -C and luciferase DNA Formation was evaluated for transfection efficiency in cells. The composite used in the present example was formed by forming a charge ratio of 1: 5.

The result is shown in FIG.

Among the C- (D-R9) -G and C- (D-R9) -C structures, transfection efficiency was high, and C- (D-R9) -C was selected to evaluate the efficiency.

Example 4 Evaluation of Intracellular Delivery (Transfection) Efficiency of PEG-R9

Intracellular permeability of PEG-R9 / FITC-siVEGF was assessed using flow cytometry (FACS). FITC-siVEGF was purchased from Bioneer.

First, Squamous Cell Carcinoma (SCC7) cells were purchased from ATCC to induce differentiation. SCC7 cells were cultured in 37 ° C., 5% CO ₂ atmosphere in complete medium supplemented with RPMI 1640, 10% FBS, 1% penicillin and streptomycin. Cells were passaged three times a week.

The obtained SCC7 cells were seeded in 12 well plates at 1 × 10 ⁵ . After 24 hours of seeding, complexes were formed and transfected with 100 pmol of FITC-siVEGF and PEG-R9 (charge ratio: 8, 15, 23). PEI complex was used as control at charge ratio 8. After 4 hours of incubation, the cells were obtained by washing with PBS, trypsinized, transferred to 1.5 ml microtubes, and then centrifuged at 1,300 rpm for 3 minutes. After washing with FACS (Fluorescence Activated Cell Sorter) buffer, it was measured by FACS.

As a result, as shown in the following table, PEG-R9 / FITC-siVEGF was confirmed that the intracellular delivery efficiency as high as PEI / FITC-siVEGF positive control.

Table 1

Transfection Efficiency
4H	Control	Naked FITC-siVGF	PEI / FITC-siVEGF	PEG-9R / FITC-siVEGF (8)	PEG-9R / FITC-siVEGF (15)	PEG-9R / FITC-siVEGF (23)
%	0.17	10.3	96.37	71.51	86.87	90.32

Example 5: Expression efficiency and toxicity evaluation of PEG-R9

Luciferase gene expression was measured with a luciferase assay kit, and cell viability assays were performed by MTT assay.

Luciferase assay kit was purchased from Promega (USA) and DC protein assay kit and bovine serum albumin standard were purchased from Bio-Rad Laboratories (USA). SCC7 cells were seeded 2 × 10 ⁴ in 24 well plates. After 24 hours of seeding, cells were transfected with 4 μg of plasmid luciferase and R9 and PEG-R9 complexes with a charge ratio of 12.

PEI complex was used as control at charge ratio 8. After 48 hours of incubation, cells were washed with PBS and treated with 150 μl of 1 × cell lysis buffer reagent for 20 minutes. Cell lysates were scraped off and transferred to 1.5 ml microtubes and centrifuged for 3 minutes at 13,000 rpm. Luminescence of cell lysates was measured by 96-well plate photometer (Berthold Detection Systems, Germany) by 20 seconds integration and expressed as relative luminescence units (RLU) per mg of cell protein. .

Proteins were determined with a DC protein assay kit using bovine serum albumin standard. The above method was used to compare PEG500 and PEG1000, and the optimum PEG was selected.

The results are shown in FIG. As a result of evaluating the efficiency of transduction, PEG 500 showed better gene transfer efficacy compared to PEG 1000, and PEG 500 was used in the experiment of the present invention.

Meanwhile, cell viability was measured by MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide] assay.

SCC7 cells were cultured in 24 well plates and treated with R9, PEG-R9 and PEI complexes at defined charge rates. After 48 hours of transfection, 50 μl MTT reagent and 500 μl medium were added to each well and incubated at 37 ° C. for 3 hours. The culture medium was removed and 500 μl dimethyl sulfoxide (DMSO) was added to each well and incubated for 20 minutes at room temperature. Absorbance was measured at 570 nm.

The results are shown in FIG.

As a result of the luciferase assay, PEG-R9 expresses the gene well in the cell, and the cytotoxicity is very low compared to other gene carriers (ex. PEI). .

Comparative Example 1 Identification of Optimal Transporter for siRNA Delivery

In addition, after conjugation of siVEGF and various types of transporters, their size and zeta potential analysis were used to find an optimal transporter for siRNA delivery.

each case of cys-R9-cys, PEG-R9, PEG-cys-R9-cys; And PEG-cys-R9-cys, PEG-TAT, PEG-cys-TAT-cys, were compared using the delivery group and.

The results are shown in FIGS. 7 and 8.

First, the particle size of the PEG-coupled carrier was smaller than when PEG was not bound, and when cysteine was not present at the terminal of R9, the particle size was rather increased (FIG. 7). Through this, it was found that cysteine is bonded to both ends of R9, and furthermore, the binding rate of siRNA is improved when PEG is bound.

In addition, as a result of comparing and evaluating gene binding ability with R9 using TAT, it was found that when PEG is bound to R9 rather than TAT, it is an optimal carrier for siRNA delivery (FIG. 8).

As such, it was found that when cysteine and PEG are conjugated to the terminal of R9 of the present invention, the binding with siRNA is most efficient, and the size of nanoparticles is uniform.

Example 6: Anticancer Efficacy of PEG-R9-siVEGF

The cancer model was induced by injecting 2 × 10 ⁶ SCC7 cells into the hind limbs of 7 week old nude mice. Treatment started when the tumor volume reached 100 mm ³ or more. PEG-R9, R9 and PEI complexes at 10 μg of siVEGF and defined charge ratios were administered as tail vein three times a week for 2 weeks. The number of nude mice in each group was five.

As a result, as shown in Figure 9 and 10, when cancer was implanted in the nude mouse and administered the siVEGF complex with the tail vein, it was confirmed that the growth rate of tumors in the PEG-R9 / siVEGF administration group is slow compared to other groups This proved the anticancer effect.

Claims (15)

1. A siRNA delivery system for systemic circulation, comprising polyethylene glycol (PEG) and R9 (arginine) peptide.
2. The siRNA delivery system for systemic circulation according to claim 1, wherein the R9 (arginine) peptide has Cys attached to one or both ends thereof.
3. The siRNA delivery system for systemic circulation according to claim 2, wherein the R9 (arginine) peptide has a Cys- (D-R9) -Cys or Gly- (D-R9) -Cys structure.
4. Polyethylene glycol (PEG); R9 (arginine) peptides; And siRNA delivery complex for systemic circulation consisting of siRNA.
5. The complex of claim 4, wherein the R9 (arginine) peptide has Cys attached to one or both ends.
6. The complex of claim 5, wherein the R9 (arginine) peptide has a Cys- (D-R9) -Cys or Gly- (D-R9) -Cys structure.
7. The complex of claim 6, wherein the R9 (arginine) peptide has a Cys- (D-R9) -Cys structure.
8. The composite according to claim 4, wherein the polyethylene glycol (PEG) is PEG 500 having a molecular weight of 500 daltons.
9. The composite according to claim 4, wherein the composite has a diameter of 200 nm or less.
10. The complex of claim 4, wherein the complex has a charge ratio (+/−) of 6: 1 to 15: 1.
11. The complex of claim 10, wherein the complex has a charge ratio (+/−) of 12: 1.
12. Polyethylene glycol (PEG); R9 (arginine) peptides; And consisting of siVEGF, anti-cancer siRNA delivery complex for systemic circulation.
13. The complex of claim 12, wherein the R9 (arginine) peptide has a Cys- (D-R9) -Cys structure.
14. A composition for treating VEGF overexpressing cancer, comprising the complex of claim 12 as an active ingredient.
15. 15. The method of claim 14, wherein the VEGF overexpression cancer is lung cancer, gynecological malignancy, melanoma, breast cancer, pancreatic cancer, ovarian cancer, uterine cancer, colorectal cancer, prostate cancer, kidney kidney, head cancer, pancreatic cancer, liver cancer Cancer), cervical cancer, neck cancer, kidney cancer (renal cell cancer), sarcoma, myeloma and lymphoma.

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