Sirna conjugate and method for producing it

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and represents a conjugate used for siRNA intracellular delivery and containing siRNA, which is conjugated by a covalent bond with a hydrophilic compound on one side, e.g. PEG, and with a hydrophobic compound, e.g. cholesterol, on the other side. By self-assembly, the conjugates are able to form homogenous nanoparticles, micellas, wherein the hydrophobic compounds are packed inside the micella; siRNA - between the hydrophobic and hydrophilic compounds, and the hydrophilic compounds - outside. The present invention also discloses methods for producing the above conjugate, pharmaceutical compositions containing the above nanoparticles for the gene therapy of various diseases depending on specific siRNA delivered. What is also disclosed is a pharmaceutical composition for treating cancer, which contains the nanoparticles containing survivin-specific siRNA.

EFFECT: invention enables increasing the siRNA stability in a living body, providing thereby the effective delivery of therapeutic siRNA into cells and the shown activity of siRNA even in a low dose of a relatively low concentration.

25 cl, 19 dwg, 1 tbl, 6 ex

 

The technical field to which atsitsa invention

The present invention relates to a conjugate in which a polymer compound that will improve the delivery of miRNAs used in gene therapy of cancer and other infectious diseases, anywhereman with miRNAs through or split unsplittable connection, to a method for producing said conjugate and a method for delivery of miRNAs using the specified conjugate.

Art

The RNA interference mechanism means, which is a post-transcriptional suppression of genes induced double-stranded RNA (dcrk) specific sequence of nucleotides in the process of gene expression, and this mechanism was first discovered round worm and is generally found in plants, fruit flies Drosophila and vertebrates (Fire, etc., Nature, 1998, vol. 391, pp. 806-811; Novina &Sharp, Nature, 2004, vol. 430, pp. 161-164). As is well known, RNA interference occurs in such a way that dcrk 19-25 p. O. at the entrance to the cage forms a bond with RIK (RNA-induced complex suppression) and antisense (guide) chain is associated with mRNA, which is complementary to the sequence of nucleotides of the mRNA, thereby splitting machinewww mRNA endonuclease domains existing in RICP (Rana, T. M., Nat. Rev. Mol. Cell Biol., 2007, vol. 8, 23-36; Tomari, Y. &Zamore, P. D., Genes Dev., 2005, vol. 19, pp. 517-529).

When dcrk enters the cell, it forms a specific bond with mistaway sequence of mRNA to mRNA decompose, and therefore it is considered as a new tool capable of regulating gene expression. However, in the case of a person find it difficult to obtain the effect of RNA interference due to the impact of transport on interferon introduction dcrk in human cells. In 2001, Elbashir, Tuschl and others found that the introduction of small dcrk length of 21 NT. (nucleotide) in human cells has not led to the transport of interferon, but specifically destroyed the target mRNA (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T., Nature, 2001, vol. 411, pp. 494-498; Elbashir, S. M., Lendeckel, W., Tuschl, T., Genes & Dev., 2001, vol. 15, pp. 188-200; Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W., Tuschl, T., EMBO J., 2001, V. 20, p. 6877-6888). Subsequently dcrk length of 21 NT. adopted as a new tool for functional genomics and have termed "small interfering RNAS" (miRNAs).

Small interfering RNA is a substance that attracts great interest as a means of gene therapy since, as was shown her amazing effect of inhibiting the expression of specific genes in animal cells. In fact, due to its high activity and precise gene selectivity, expect that miRNAs will become drug alterna�actions are antisense to oligodeoxynucleotide (ODN), currently used as a drug, the result of twenty years of research (Dana J. Gary, etc., Journal of Controlled Release, 2007, vol. 121, pp. 64 to 73,). The use of miRNAs in the methods of treatment has great advantages, which include its lightweight design compared to other drugs, and high selectivity to the target and the property of inhibiting the expression of a particular gene. In addition, miRNAs are less toxic because interference RNA inhibits gene expression through a mechanism that naturally exists in the system. Bevasiranib (Bevasiranib), recently developed by OPKO Inc. as a means of treatment of the wet form of age-related macular degeneration, represents miRNAs that selectively affects the growth factor vascular endothelial (VEGF), including neovascularization, for inhibiting the expression of VEGF, and is currently undergoing a three-phase clinical trial (N. S. Dejneka, etc., Mol. Vis., 2008, vol. 28, No. 14, pp. 997-1005). In addition, recently developed drugs, including miRNAs and target different genes (Ryan P. Million, Nature Reviews Drug Discovery, 2008, vol. 7, p. 115-116).

Despite the varied results showing that the specific inhibition of gene expression induced in vivo by means of interferen�AI RNA, in vivo delivery of miRNAs requires the solution of numerous problems, including the destruction of enzymes of blood, interaction with blood components and non-specific delivery to cells (Shigeru Kawakami & Mitsuru Hashida, Drug Metab. Pharmacokinet., 2007, vol. 22, No. 3, pp. 142-151). Attempts to solve these problems, in particular, through the use of resistant to the nuclease of nucleoside analogues or improvement of delivery methods.

Examples of improved delivery methods include methods of gene delivery using viruses, including adenoviruses, retroviruses, etc., and methods of non-viral gene delivery carriers using liposomes, cationic lipids and cationic polymeric compounds. However, viral carriers associated with the issue of security, because the delivered genes can be introduced into the host chromosomes and create anomalies in the normal functioning of host genes and activation of oncogenes, and in addition, can cause autoimmune diseases due to subsequent expression of viral genes in small quantities or may not lead to effective protective immunity in the case when the modified viral infection caused by a viral media. While non-viral carriers are less efficient than viral carriers, but have the advantages of low-level�full-time effects and cheap production, taking into account the safety for the living organism and economic feasibility (S. Lehrman, Nature, 1999, vol. 401, No. 6753, pp. 517-518). In addition, non-viral methods of delivery require effective protection from enzymatic or non-enzymatic decomposition, in order to deliver RNA molecules, including miRNAs, and one of the ways is to use gene-expression plasmid DNA encoding the short form hairpin RNA (CSRC). The advantage of the system with the participation of DNA is the expression of miRNAs only in the presence of the expression vector. In addition, in a recent study of chemical modification of miRNAs has been proposed a method of increasing resistance to nucleases and reduce intracellular uptake (Shigeru Kawakami & Mitsuru Hashida, Drug Metab. Pharmacokinet., 2007, vol. 22, No. 3, pp. 142-151).

In one type of chemical modification of miRNAs postordering communication, which is a part of, the biodegradable nuclease, modified phosophorothioates bond or portion 2' of the pentose modified 2'-O-mark, 2'-deoxy-2'-fluoriding or closed nucleic acid (SNC) formed by the binding of the parts 2' and part 4', resulting in increased serum resistance (Braasch D. A., etc., Bioorg. Med. Chem. Lett., 2003, vol. 14, pp. 1139-1143; Chiu, Y. L. & T. M. Rana, RNA, 2003, vol. 9, pp. 1034-1048; M. Amarzguioui et, Nucl Acid Res., 2003, vol. 31, pp. 589-595). The chemical modification of a different type options�national group associated with the terminal region of the 3'-sense (direction) of the circuit, resulting in improved pharmacokinetic characteristics compared with control, and high efficiency is induced during use in a living organism by means of the balance between hydrophilicity and hydrophobicity miRNAs (Soutschek J. et, Nature, 2004, vol. 432, pp. 173-178).

However, the above methods still leave much to be desired in terms of protection of miRNAs from nucleases and increase the efficiency of the permeability of the cell membrane.

For this reason, the authors present invention found that the conjugate, in which a hydrophilic or hydrophobic polymer compound anywhereman with miRNAs using split or unsplittable communication, increases the resistance in a living organism miRNAs, and on this basis was established the present invention.

Description

The technical problem

The aim of the present invention is a conjugate in which a hydrophilic or hydrophobic polymer compound, which is a biocompatible polymeric compound, konjugierten by the end of the semantic chain or antisense chain miRNAs using split or unsplittable communication, to improve the efficiency of intracellular delivery of miRNAs.

Another objective of the present invention is a solid substrate comprising a polymeric compound, in particular, a polymer compound, resistant�resistance which proved, when introduced into the human body, for example, polyethylene glycol (PEG), and a method for efficient production of an oligonucleotide comprising RNA, DNA, chimeric RNA-DNA and its analogue, in which PEG is linked to its 3' end using the substrate.

Another objective of the present invention is a method of obtaining a conjugate of miRNAs and the method of delivery of miRNAs using conjugate miRNAs.

Technical solution

To achieve the objectives mentioned above, in the first paragraph present invention proposed a conjugate miRNAs and polymeric compounds having the following structure:

A-X-R-Y-B

(where A and B independently represent a hydrophilic polymer or a hydrophobic polymer compound; X and Y independently represent a simple covalent bond or a covalent bond via an intermediate linker; and R represents miRNAs).

In the second paragraph of the present invention proposed a conjugate miRNAs and polymeric compounds having the following structure:

A-X-R

(where A represents A hydrophobic polymeric compound; X represents a simple covalent bond or a covalent bond via an intermediate linker; and R represents miRNAs).

In the third paragraph of the present invention proposed a conjugate in which a single chain miRNAs (R) consists of 19 to 31 nucleotides.

In the fourth paragraph of the present invention offer�n conjugate, in which a hydrophobic polymer compound (A) has a molecular weight of from 250 to 1000.

In the fifth paragraph of the present invention proposed a conjugate in which a hydrophobic polymer compound (A) is a hydrocarbon, C16-C50or cholesterol.

In the sixth paragraph of the present invention proposed a conjugate in which a covalent bond (X, Y) is an unsplittable link or split the connection.

In the seventh paragraph, of the present invention proposed a conjugate in which the unsplittable link is an amide bond or a phosphate bond.

In the eighth paragraph of the present invention proposed a conjugate in which the degradable linkage is selected from disulfide bonds, split acid connection, ester bonds, anhydrite communication, bioassayset communication and biologically degradable by the enzyme connection.

In the ninth paragraph of the present invention proposed a conjugate in which a hydrophilic polymer compound (A or B) is a non-ionic polymeric compound having a molecular weight of from 1000 to 10000.

In the tenth paragraph of the present invention proposed a conjugate in which a hydrophilic polymer compound selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone and polyoxazole.

In the eleventh paragraph of the present invention proposed �knitted with polyethylene glycol solid substrate, having the following structure:

(where R represents alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl or heteroaryl; m is an integer from 2 to 18; n is an integer from 5 to 120; and X represents a hydrogen atom, 4-monomethacrylate, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl).

In the twelfth paragraph of the present invention associated with the proposed polyethylene glycol solid substrate, where the solid substrate is a glass with controlled pore size (UPC).

In the thirteenth paragraph of the present invention associated with the proposed polyethylene glycol solid substrate, in which the UPC has a diameter of from 40 to 180 μm and a pore size of from 500 to 3000 Å.

In the fourteenth paragraph of the present invention associated with the proposed polyethylene glycol solid substrate, which is associated with polyethylene glycol solid substrate is a compound of 3'-PEG(polyethylene glycol)-UPC, having the following structural formula IV:

[Structural formula IV]

In the fifteenth paragraph of the present invention a method of producing compound 3'-PEG-UPC, having the following structural formula IV, wherein the method involves:

1) reaction of CSP with 3-aminopropyltriethoxysilane, which produces compounds�s of long-chain alkylamine and glass with controlled pore size (DCAA-UPC);

2) the reaction of polyethylene glycol with 4,4'-dimethoxytrityl, which produces 2-[bis-(4-dimethoxytrityl)glycol];

3) reaction of the compound obtained in stage (2), with a compound having the following chemical formula 1, in which is formed a compound having the following structural formula I;

4) reaction of the obtained compound having the following structural formula I, with 4-nitrophenylphosphate in which is formed a compound having the following structural formula II;

5) the reaction of compounds having the following structural formula I and obtained in stage (3), with N-succinimidylester acid, in which is formed a compound having the following structural formula III; and

6) the reaction of a compound of DCAA-UPC, obtained in stage (1), compounds having the following structural formulas I, II and III, respectively, obtained at stages (3) to(5), respectively.

[Chemical formula 1]

[Structural formula I]

[Structural formula II]

[Structural formula III]

[Structural formula IV]

(where R represents alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl or heteroaryl; and n represents with�fight integer, of not less than 5 and not more than 120).

In the sixteenth paragraph of the present invention a method of producing the conjugate of miRNAs, wherein the method involves:

1) obtaining miRNAs against a gene target associated with the use of a polyethylene glycol solid substrate according to an eleventh item of the present invention; and

2) the connection terminal groups of miRNAs and polyethylene glycol covalent bonds.

In the seventeenth paragraph of the present invention provides nanoparticles consisting of conjugates of miRNAs according to the first or second paragraph, of the present invention.

In the eighteenth paragraph of the present invention a method of gene therapy, wherein the method involves:

1) preparation of the nanoparticles according to the seventeenth paragraph of the present invention; and

2) the introduction of nanoparticles into the body of the animal.

In the nineteenth paragraph of the present invention a method of gene therapy, in which the nanoparticles are administered by oral administration or intravenous injection.

In the twentieth paragraph present invention relates to pharmaceutical compositions containing pharmaceutically effective amount of the conjugates of miRNAs according to the first or second paragraphs of the present invention.

In the twenty-first paragraph present invention relates to the pharmaceutical�certification compositions containing a pharmaceutically effective amount of the nanoparticles according to the seventeenth paragraph of the present invention.

Hereinafter the present invention will be described in detail.

The present invention relates to a conjugate miRNAs and polymeric compounds having the following structure:

A-X-R-Y-B.

In the description of the application A and B independently represent a hydrophilic polymer or a hydrophobic polymer compound; X and Y independently represent a simple covalent bond or a covalent bond via an intermediate linker; and R represents miRNAs.

In addition, the present invention relates to conjugate miRNAs and polymeric compounds having the following structure:

A-X-R.

In the description of the application A is A hydrophobic polymer compound; X represents a simple covalent bond or a covalent bond via an intermediate linker; and R represents miRNAs.

In the conjugate of the present invention the oligonucleotide chain miRNAs may contain from 19 to 31 nucleotides. Any miRNAs derived from genes that is or can be used for gene therapy or research, can be used as miRNAs suitable for the present invention.

Hydrophobic polymer compound may be a hydrophobic polymer compound, Nieuwe� molecular weight of from 250 to 1000. Examples of the hydrophobic polymer compound may include a hydrocarbon, preferably a hydrocarbon, C16-C50, and cholesterol. In this document, a hydrophobic polymer compound is not limited to a hydrocarbon and cholesterol.

Hydrophobic polymeric compound function makes a hydrophobic interaction, which forms a micelle consisting of conjugates of miRNAs and hydrophobic polymer compound. Among hydrophobic polymer compounds, in particular, saturated hydrocarbon has the advantage that it can be easily conjugated to miRNAs in the process of production of miRNAs, and thus it is highly suitable for obtaining conjugates of the present invention.

In addition, covalent bond (i.e., X, Y) can represent one of unsplittable or split connection. In this case, there may exist an amide bond or a phosphate bond as unsplittable connection, and can exist disulfide bond, split by acid bond, ester bond, anhydride bond, virassamy communication and biologically degradable by the enzyme link as a split connection. However unsplittable or split the connection is not limited to the above examples.

Linker, whereby a bond covalently me�et hydrophilic polymer (or a hydrophobic polymer) and the end residue, derived from miRNAs, and is not limited in a certain way, provided that it can provide a degradable linkage within a particular environment as needed. Therefore, the linker can include any compound which may be associated with miRNAs and/or hydrophilic polymer (or a hydrophobic polymer), to activate them in the process of obtaining the conjugate.

Furthermore, the hydrophilic polymer compound may be a non-ionic polymeric compound having a molecular weight of from 1000 to 10000. For example, a hydrophilic polymer compound may include non-ionic hydrophilic polymer compound including polyethylene glycol, polyvinylpyrrolidone, polyoxazole and similar compounds, but is not limited thereto.

A functional group of a hydrophilic polymer compound can be substituted with another functional group as necessary. Among hydrophilic polymer compounds, in particular, PEG is a highly suitable to obtain the conjugates of the present invention, since it has different values of molecular weight, is able to limit the introduction of functional groups, has excellent biocompatibility, does not cause immune response and increases the solubility in water, which improves the efficiency of gene delivery in vivo.

In addition, �astasia the invention relates to linked with polyethylene glycol solid support, having the following structure:

In this case, the solid substrate includes, for example, UPC, polystyrene, silica gel, cellulose paper, etc., but not necessarily limited to these materials; R represents alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl or heteroaryl; m is an integer from 2 to 18; n is an integer from 5 to 120 (molar mass 282-5300); and X represents a 4-monomethacrylate, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl and removed after treatment of the acid with replacement of the hydrogen atom. In that case, if the solid substrate is a UPC, it may have a diameter of from 40 to 180 μm and a pore size of from 500 to 3000 Å.

In addition, the present invention relates to linked with polyethylene glycol solid support, which is associated with the connection 3'-PEG-UPC, having the following structural formula IV:

[Structural formula IV]

In addition, the present invention relates to a method for producing 3'-PEG-UPC with the following structural formula IV, having the following structural formula IV, wherein the method involves:

1) reaction of CSP with 3-aminopropyltriethoxysilane, which produces the connection of long-chain alkylamine and glass with controlled pore size (DCAA-UPC);

2) reaction polyeth�of langille with 4,4'-dimethoxytrityl, which produces 2-[bis-(4-dimethoxytrityl)glycol];

3) reaction of the compound obtained in stage (2), with a compound having the following chemical formula 1, in which is formed a compound having the following structural formula I;

4) reaction of the obtained compound having the following structural formula I, with 4-nitrophenylphosphate in which is formed a compound having the following structural formula II;

5) the reaction of compounds having the following structural formula I and obtained in stage (3), with N-succinimidylester acid, in which is formed a compound having the following structural formula III; and

6) the reaction of a compound of DCAA-UPC, obtained in stage (1), compounds having the following structural formulas I, II and III, respectively, obtained at stages (3) to(5), respectively.

[Formula 1]

[Structural formula I]

[Structural formula II]

[Structural formula III]

[Structural formula IV]

(where R represents alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl or heteroaryl; and n is an integer of not less than 5 and not more than 120).

Besides�, the present invention relates to a method of producing a conjugate comprising miRNAs and PEG using the associated with polyethylene glycol solid substrate. More specifically, a method of producing the conjugate of miRNAs, which provides:

1) obtaining miRNAs against a gene target associated with the use of a polyethylene glycol solid substrate according to the eleventh item of the present invention; and

2) the connection terminal groups of miRNAs and polyethylene glycol covalent bonds.

This way you can effectively get the oligonucleotides, including RNA, DNA, chimeric RNA-DNA and their equivalent.

According to a preferred embodiment of the present invention, miRNAs can be obtained by forming postordering relations that construct the basic structure of RNA, using β-cyanomethylphosphonate (Shina, etc., Nucleic Acids Research, 1984, vol. 12, pp. 4539-4557). For example, a number of procedures, including deblocking, coupling, oxidation and kupirovanie, running was performed on a solid support, to which was attached a nucleotide, using an RNA synthesizer to receive a reagent containing RNA desirable length. However, the present invention is not limited to the above.

In addition, the present invention relates to a nanoparticle comprising conjugates of miRNAs.

Conjugates of miRNAs and p�limanova compounds of the present invention can form the structure of nanoparticles through interaction among themselves, and conjugate miRNAs and polymeric compounds and nanoparticles, consisting of the thus obtained conjugates miRNAs and polymeric compounds, improve intracellular delivery of miRNAs and can be used to treat disease model. Obtaining conjugates, as well as the characteristics and effective intracellular delivery and the effect of nanoparticles consisting of conjugates, are described in detail in the examples below.

In addition, the present invention relates to a method of gene therapy using nanoparticles.

More specifically, the method of gene therapy involves preparation of nanoparticles, each consisting of conjugates of miRNAs and polymeric compounds, and the introduction of nanoparticles into the body of the animal.

In addition, the present invention relates to pharmaceutical compositions containing pharmaceutically effective amount of nanoparticles, each consisting of conjugates of miRNAs.

The composition of the present invention can be obtained, including one or more pharmaceutically acceptable carriers, in addition to the above active components, for reception. Pharmaceutically acceptable carrier must be compatible with the active components of the present invention. Pharmaceutically acceptable carrier can be used by mixing with physiological rustboro�, sterilized water, ringer's solution (Ringer) containing buffer saline solution, a dextrose solution, a maltodextrin solution, glycerol and ethanol, and one or more of these solutions, and also, as appropriate, you can enter other conventional additives, including antioxidants, buffers, prevents bacteria growth substances or similar substances. In addition, you can optionally type a diluents, dispersants, surfactants, binders and lubricants in order to obtain compositions for injection, including aqueous solutions, suspensions, emulsions or similar compounds. Furthermore, the composition of the present invention can preferably be depending on certain diseases or components using appropriate methods known in the art, or the methods described in pharmaceutical reference Remington's Pharmaceutical Science (Mack publishing company, Easton, Pennsylvania).

The pharmaceutical composition of the present invention can determine the specialists in the art based on the syndromes and severity of illness of patients. In addition, the pharmaceutical composition of the present invention can be in various forms including powders, tablets, capsules, liquids, injections, ointments, Siro�s, etc. and can be released in a single dose or multiple dose container, for example, in a closed ampoule, vial, etc.

The pharmaceutical composition of the present invention may be intended for oral or parenteral use. Route of administration of the pharmaceutical compositions of the present invention may include, but are not limited to, oral, intravenous, intramuscular, intramedullary, vnutriobolochechnoe, intracardiac, cutaneous, subcutaneous, intraperitoneal, enteral, sublingual, or topical application.

For clinical application of the pharmaceutical composition of the present invention can be in the respective forms, using known in the art methods. The dosage of compositions of the present invention has different limits depending on weight, age, sex, health condition, diet, time and route of administration, rate of excretion and the degree of the patient's disease, and it can easily identify experts in the field of technology.

Useful effects

Of nanoparticles, conjugates consisting of miRNAs and polymeric compounds of the present invention can increase the stability of miRNAs in vivo for the efficient delivery of therapeutic miRNAs in cells and can be very useful in basic research in the field of biotechnology�and and medical industries as a new type of delivery system miRNAs, and also as a carrier of miRNAs for the treatment of cancer and other infectious diseases, because it is able to be active miRNAs in a relatively low concentration dosage even without transfection reagents (transforming cells viral DNA).

Description of the drawings

Fig. 1 is the structural formula of the obtained compound 3'-PEG-RMS;

Fig. 2 represents the NMR data1H the compound obtained in example 1;

Fig. 3 represents the NMR data1H compound A which is a reagent 3'-PEG for connection to DCAA-UPC in example 1;

Fig. 4 represents the NMR data1H connection B, which is a reagent 3'-PEG for connection to DCAA-UPC in example 1;

Fig. 5 represents the NMR data1H connection C, which is a reagent 3'-PEG for connection to DCAA-UPC in example 1;

Fig. 6 is obtained by the method of mass spectrometry MALDIVE data molecular weight after the connection is obtained 3'-PEG-UPC and oligonucleotide (miRNAs) in example 1-3;

Fig. 7 is obtained by the method of mass spectrometry MALDIVE data molecular weight after the connection is obtained 3'-PEG-UPC and oligonucleotide (miRNAs) in example 1-4;

Fig. 8 is an electrophoretic photograph of unconjugated miRNAs in Kotor�th none of polymeric compounds not konjugierten, and conjugates miRNAs and polymeric compounds in which the hydrophilic or hydrophobic polymer compound konjugierten (miRNAs means unconjugated miRNAs and the corresponding conjugates are conjugates of miRNAs and polymeric compounds presented in table 1; in addition, or, or, or and or mean miRNAs consisting of 19, 23, 27 and 31 of the nucleotide, respectively, and they were used to obtain conjugates of miRNAs and polymeric compounds in the structure of the conjugate miRNAs 4);

Fig. 9 is an electrophoretic photograph reflecting the degree of decomposition of miRNAs depending on time in the presence of whey protein to assess the stability in the blood unconjugated miRNAs, which is not konjugierten none of polymeric compounds, and conjugates of miRNAs and polymeric compounds in which anywhereman hydrophilic polymer compound, namely PEG;

Fig. 10 is a schematic representation of a nanoparticle formed by the conjugates of miRNAs and polymeric compounds;

Fig. 11 represents the size distribution of nanoparticles consisting of unconjugated miRNAs that do not anywherevery polymeric compounds, according to the results obtained using the device for measuring ζ-potential;

Fig. 12 represents the size distribution of nanoparticles, each �W which consists of the conjugates of miRNAs and polymeric compounds 9, according to the results obtained using the device for measuring ζ-potential;

Fig. 13 represents the size distribution of nanoparticles, each consisting of conjugates of miRNAs and polymeric compounds 10, according to the results obtained using the device for measuring ζ-potential;

Fig. 14 represents the size distribution of nanoparticles, each consisting of conjugates of miRNAs and polymeric compounds 11, according to the results obtained using the device for measuring ζ-potential;

Fig. 15 represents the size distribution of nanoparticles, each consisting of conjugates of miRNAs and polymeric compounds 12, according to the results obtained using the device for measuring ζ-potential;

Fig. 16 represents the size distribution of nanoparticles, each consisting of conjugates of miRNAs and polymeric compounds 13, according to the results obtained using the device for measuring ζ-potential;

Fig. 17 is a graph comparing the degrees of expression of the mRNA of the gene of survivin after transfection with carrying out the transfection reagent to analyze the effects of RNA interference for unconjugated miRNAs and respective conjugates miRNAs and polymeric compounds in which anywhereman of hydrofil�first polymer compound, namely PEG;

Fig. 18 is a graph comparing the degrees of expression of the mRNA of the gene of survivin after transfection with carrying out the transfection reagent to analyze the effects of RNA interference for unconjugated miRNAs and corresponding with a long sequence of miRNAs, converted into conjugate miRNAs and polymer compound 4; and

Fig. 19 is a graph comparing the degrees of expression of the mRNA of the gene of survivin after transfection in the absence of carrying out the transfection reagent to analyze the effects of RNA interference for unconjugated miRNAs and conjugates miRNAs and polymeric compounds 1-5 and 9-14.

Best mode for carrying

Next will be described exemplary embodiments of the present invention. However, the following exemplary embodiments of the implementation describe the present invention merely by way of example, but not limiting it.

Example 1. Obtaining a solid substrate to obtain an oligonucleotide 3'-PEG

Example 1-1. Obtaining reagents 3'-PEG (compounds A, B and C) for connection to DCAA-UPC

In the following example, 3'-PEG-UPC received in this way, as shown by the following reaction scheme.

Example 1-1-1. Getting 2-[bis-(4-dimethoxytrityl)of polyethylene glycol]

�astarali 30 g (15 mmol) of polyethylene glycol 2000 (Alfa Aesar GmbH & Co. KG, Germany) as a starting material in 270 ml of pyridine (Sigma Aldrich, USA), was then added 3.55 ml (25.5 mmol) of triethylamine (Sigma Aldrich, USA) and 7.12 g (21 mmol) of 4,4'-dimethoxytrityl (GL Biochem, China) and then the obtained substance was reacted at room temperature for 20 hours. The reaction mixture once the reaction was concentrated and extracted with 450 ml of ethyl acetate and 450 ml of water followed by evaporation in vacuum with subsequent vacuum drying was obtained 2-[bis-(4-dimethoxytrityl)glycol] in an amount of 23 g (66%).

NMR data1H connection shown in Fig. 2,

NMR1H (δ, CDCl3): 1,93 (W, 1, OH), 3,20-of 3.80 (m, 186, PEG, DMT-OCH3), 6,80-6,83 (m, 4, DMT), of 7.19-7,47 (m, 9, DMT).

Example 1-1-2. The preparation of the compound of succinic acid and 2-[bis-(4-dimethoxytrityl)polyethyleneglycol] [compound A]

Was dissolved 3.9 g (1,672 mmol) of 2-[bis-(4-dimethoxytrityl)glycol], obtained in example 1-1-1, 20 ml of pyridine and then was cooled to 0°C. To a solution of reagent was added 351 mg (3,512 mmol) of succinic acid anhydride (Acros Organics, USA) and 42,5 mg (0,334 mmol) of DMAP (4-dimethylaminopyridine, Sigma Aldrich, USA) and stirred at 50°C for 3 days, and then the reaction was completed. The reaction mixture after completion of the reaction was stripped of solvent under vacuum and obtained compound of succinic acid and 2-[bis-(4-dimethoxytrityl)of polyethylene glycol] to [connect�tion A] in the form of a white solid in the amount of 3,65 g (90%).

NMR data1H connection shown in Fig. 3.

NMR1H (δ, CDCl3): 2,65 (m, 2, CH2CO), 3,20-3,88 (m, 186, PEG, DMT-OCH3), 4,25 (m, 2, CH2CO), 6,80-about 6,82 (m, 4, DMT), of 7.19-7,47 (m, 9, DMT).

Example 1-1-3. The preparation of the compound steam-nitrophenylarsonic acid and 2-[bis-(dimethoxytrityl)of polyethylene glycol] [compound B]

Was dissolved 1 g (0,411 mmol) of the compound obtained in example 1-1-2 in 20 ml of methylene chloride (DaeYeon Chemicals Co. Ltd., Korea) and cooled to 0°C. To a solution of reagent was added 143 μl (1.03 mmol) of triethylamine and 149 mg (0,740 mmol) 4-nitrophenylphosphate. Then the temperature was raised to room temperature and the resulting material was stirred for 4 hours, after which the reaction has ended. The reaction mixture after the reaction were washed once with 20 ml of saturated aqueous solution of NaHCO3and 20 ml of a 1 M solution of citric acid (Sigma Aldrich, USA), which was cooled to 0-4°C, and then dried over Na2SO4(Samchum Chemical Co., Korea). The resulting material was filtered using a filtering flask, Buchner funnel (Buchner) or aspirator, and then by evaporation under vacuum gave compound steam-nitrophenylarsonic acid and 2-[bis-(4-dimethoxytrityl)of polyethylene glycol] [compound B] in the form of a cream solid in an amount of 1.0 g (94%.

NMR data1H connection shown in Fig. 4.

YAM� 1H (δ, CDCl3): 2,80-2,90 (m, 2, CH2CO), 3,20-of 3.87 (m, 186, PEG, DMT-OCH3), 4,25 (m, 2, CH2CO), 6,80-about 6,82 (m, 4, DMT), of 7.19-7,47 (m, 9, DMT).

Example 1-1-4. The preparation of the compound 2,5-dioxopiperidin-1-Glafira succinic acid and 2-[bis-(4-dimethoxytrityl)(peg] [compound C]

Dissolved 500 mg (0,206 mmol) of the compound obtained in example 1-1-2 in 10 ml of methylene chloride was then added and 83,14 μl (1.03 mmol) of pyridine. To a solution of reagent was added 165 mg (0,781 mmol) N-succinimidylester acid (Sigma Aldrich, USA) and stirred at room temperature for 7 hours, after which the reaction has ended. The reaction mixture after completion of the reaction was stripped of solvent under vacuum and obtained compound 2,5-dioxopiperidin-1-refer succinic acid and 2-[bis-(4-dimethoxytrityl)of polyethylene glycol] [compound C] in the form of a white solid in the amount of 490 mg (94%).

NMR data1H connection shown in Fig. 5.

NMR1H (δ, CDCl3): 2,72-of 2.97 (m, 6, CH2CO, CH2CH2), 3,20-of 3.87 (m, 186, PEG, DMT-OCH3), 4,27-to 4.28 (m, 2, CH2CO), 6,80-6,83 (m, 4, DMT), 7,20-7,47 (m, 9, DMT).

Example 1-2. Connection DCAA-UPC and reagent 3'-PEG (compound A)

In the following example, received the UPC connection and reagent 3'-PEG, as shown by the following reaction scheme.

Example 1-2-1. The preparation of the compound of DCAA-RMS (2000 Å)

Evenly stirred 10 g of CSP (Silicycle Inc., Canada) with a particle diameter of 40-75 microns size nanopores 2000 Å and moistened 100 μl of toluene, and then added 2 ml of 3-aminopropyltriethoxysilane (TCI Org. Chem., Japan). Thereafter, the resulting substance with stirring to react at room temperature for 8 hours. After completion of the reaction the mixture was filtered and washed with methanol, water and methylene chloride in that order, and then by vacuum drying had 10 g compound DCAA-RMS (2000 Å).

Example 1-2-2. The preparation of the compound 3'-PEG-UPC (2000 Å) using compounds of succinic acid and 2-[bis-(4-dimethoxytrityl)of polyethylene glycol] [compound A]

Moistened 2 g of the compound of DCAA-RMS (2000 Å), obtained in example 1-2-1, 20 ml of methylene chloride. Moreover, the solution of DCA-RMS (2000 Å) was uniformly mixed with a solution containing 80 mg of compound A, 14 μl of tea (triethylamine, Sigma Aldrich, USA). Was dissolved 15 mg of BOP (hexaflurophosphate the benzotriazole-1-yloxytris(dimethylamino)phosphonium, TCI Org. Chem., Japan) and 5 mg HOBT (anhydrous 1-hydroxy-benzotriazole, TCI Org. Chem., Japan) in 2 ml of methylene chloride. The obtained substance was reacted by heating under reflux for 8 hours, and then the mixture once the reaction was filtered and washed with methanol, water and etilenglikolem in that order, followed by vacuum drying�.

Moistened 1 g of the substance 10 ml of pyridine and then added 1 ml of 1-methylimidazole (Sigma Aldrich, USA) and 1.6 ml of acetic anhydride (Sigma Aldrich, USA). The resulting material under uniform stirring, reacted at room temperature for 8 hours. Fully keperawanan UPC once the reaction was washed with methanol, water, methanol and methylene chloride in that order, and then by vacuum drying was obtained 1 g of compound 3'-PEG-UPC.

Example 1-3. Connection DCAA-RMS (2000 Å) and reagent 3'-PEG (compound B)

The preparation of the compound 3'-PEG-UPC (2000 Å) was carried out using compound B.

In particular, 1 g of compound DCAA-RMS (2000 Å), obtained in example 1-2-1, wetted sufficiently in 8 ml of pyridine. In addition, the solution containing 205 mg (2 EQ.) compounds B and 55 μl of triethylamine in 2 ml of pyridine, uniformly stirred with a solution of DCAA-UPC. The obtained substance was reacted at 50-60°C for 8 hours, and then the mixture was filtered after completion of the reaction. Filtered connection UPC was washed with methanol, water and methylene chloride in that order, followed by vacuum drying. After drying moistened 1G UPC connection in 10 ml of pyridine and then added 500 μl of 1-methylimidazole and 800 μl of acetic anhydride. The resulting material t�but stirred, and then it is reacted at room temperature for 8 hours. Mixture once the reaction was filtered, and then the connection UPC was washed with methanol, water and methylene chloride in that order, and after drying under vacuum gave 3'-PEG-UPC in the amount of 1.

Fig. 6 is obtained by the method MALDIVE mass spectrometry data on the distribution of molecular weight samples miRNAs obtained using 3'-PEG-UPC as a starting material, as shown in the following example 2.

The sequence for obtaining 3'-PEG-UPC:

semantic 5'-AAGGAGAUCAACAUUUUCA(dTdT)-PEG (6664,96 Yes + 2000 Da) (sequence No. 1)

antisense 5'-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG (6592,84 Yes + 2000 Da) (sequence No. 5)

You may find that measured by MALDIVE molecular weight increased by the molecular weight of PEG (2000 Da).

Example 1-4. Connection DCAA-RMS (2000 Å) and reagent 3'-PEG (compound C)

Obtaining 3'-PEG-UPC (2000 Å) was carried out using compound C.

In particular, 1 g of compound DCAA-RMS (2000 Å), obtained in example 1-2-1, wetted sufficiently in 8 ml of pyridine. In addition, a solution containing 200 mg of compound C and 55 μl of triethylamine in 2 ml of pyridine, uniformly stirred with a solution of DCAA-UPC. The obtained substance was reacted at 50-60°C for 8 h�owls, and then the mixture once the reaction was filtered. Filtered connection UPC was washed with methanol, water and methylene chloride in that order and dried in vacuum. After drying 1 g of the compound UPC moistened in 10 ml of pyridine, and then thereto was added 500 μl of 1-methylimidazole and 800 μl of acetic anhydride. The resulting material was uniformly mixed, and then reacted at room temperature for 8 hours. Fully keperawanan connection UPC once the reaction was washed with methanol, water and methylene chloride in that order, and after vacuum drying, the obtained 3'-PEG-UPC in the amount of 1.

Fig. 7 represents the data samples miRNAs obtained using 3'-PEG-UPC as a starting material, as shown in the following example 2.

The sequence for obtaining 3'-PEG-UPC:

semantic 5'-AAGGAGAUCAACAUUUUCA(dTdT)-PEG (6664,96 Yes + 2000 Da) (sequence No. 1)

antisense 5'-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG (6592,84 Yes + 2000 Da) (sequence No. 5)

You may find that measured by MALDIVE molecular weight increased by the molecular weight of PEG (2000 Da).

Example 2. Obtaining conjugates of miRNAs and polymeric compounds

The following examples of miRNAs survivin used to suppress survivin. Survivin is a protein, usually manifesting ex�ressio in most investigated to date neoplastic tumors or transformed cell lines, and, thus, expected that he would become an important target in anticancer treatment (Abbrosini G., etc., Nat. Med., 1997, vol. 3, No. 8, pp. 917-921). The sequence of miRNAs surviving of the present invention, when it contains 19 nucleotides, consists of a sense chain of sequence No. 1 and antisense chain having a sequence that is complementary to the sense chain, and, in addition, when it contains 23, 27 or 31 nucleotide has a base sequence corresponding to the sequence number 2, 3 or 4.

(Sequence No. 1) 5'-AAGGAGAUCAACAUUUUCA-3'

(Sequence No. 2) 5'-AGGAAAGGAGAUCAACAUUUUCA-3'

(Sequence No. 3) 5'-AGGAAAGGAGAUCAACAUUUUCAAAUU-3'

(Sequence No. 4) 5'-AAAGGAGAUCAACAUUUUCAAAUUAGAUGUU-3'

Getting miRNAs was performed by education postordering relations, creating the basic structure of RNA, using β-cyanomethylphosphonate (Shina, etc., Nucleic Acids Research, 1984, vol. 12, pp. 4539-4557). In particular, a number of procedures, including deblocking, coupling, oxidation and kupirovanie, re-performed on a solid support, to which was attached a nucleotide, using an RNA synthesizer (384 Synthesizer, BIONEER, Korea) to obtain a reagent containing RNA desirable length.

In addition, the conjugate miRNAs and polymeric compounds are obtained by attaching PEG to the end region 5' or saturated �of glendorado of hexadecane (C16) or octadecane (C18) to the end region 5' using dodecanol linker which is a hydrophobic polymer compound. In addition, the above-mentioned reaction was carried out using the obtained in example 1 3 PAG-UPC as the substrate to obtain the conjugate of miRNAs and polymeric compounds in which the PEG is attached to the end region 3'.

It was determined that the reactant should be proportionate to the sequence of nucleotides, RNA separation from the reactants using high performance liquid chromatography (HPLC) (liquid chromatograph LC-20A Prominence firm SHIMADZU, Japan) and measuring molecular weight by the method MALDIVE (mass spectrometer MALDI TOF-MS, SHIMADZU, Japan). After this semantic chain RNA and antisense strand of RNA is mixed in equal amounts and placed in hybridization buffer 1X (30 mm HEPES, 100 mm potassium acetate, 2 mm magnesium acetate, pH 7,0-7,5). The resulting material had reacted to the bath at a constant temperature of 90°C for 3 minutes, and then it is again reacted at 37°C to obtain a conjugate double-stranded miRNAs and polymeric compounds. The obtained conjugates miRNAs and polymeric compounds have a structures represented in table 1. Hybridization of the obtained conjugates miRNAs and polymeric compounds was confirmed using electrophoretic photographs (Fig. 8).

Table 1
Patterns and types of end modifications conjugates miRNAs and polymer compound
Names conjugatesNames of the structures of the conjugatesEnd of modifications
miRNAsUnconjugated miRNAsSemantic: none
Antisense: none
Conjugate miRNAs and polymeric compounds 15 PEG-sense miRNAsSemantic: 5 PEG
Antisense: none
Conjugate miRNAs and polymeric compounds 25 PEG-antisense miRNAsSemantic: none
Antisense: 5 PEG
Conjugate miRNAs and polymer compound 35'SS-antisense miRNAsSemantic: none
Antisense: 5'SS
Conjugate miRNAs and polymeric compounds 45 PEG+PEG miRNAsSemantic: 5 PEG
Antisense: 5 PEG
Conjugate miRNAs and polymeric compounds 55 PEG+SS miRNAsSemantic: 5 PEG
Antisense: 5'SS
Conjugate miRNAs and polymeric compounds 63 PEG-sense miRNAsSemantic: 3 PEG
Antisense: none
Conjugate miRNAs and polymeric compounds 73 PEG-antisense miRNAsSemantic: none
Antisense: 3 PEG
Conjugate miRNAs and polymeric compounds 83 PEG+PEG miRNAsSemantic: 3 PEG
Antisense: 3 PEG
Conjugate miRNAs and polymeric compounds 95'C18-sense miRNAsSemantic: 5'C18-C6-SS-C6
Antisense: none
Conjugate miRNAs and polymeric compounds 105'C18+PEG miRNAsSemantic: 5'C18-C6-SS-C6
Antisense: 5 PEG
Conjugate miRNAs and polymeric compounds 115'C16+PEG miRNAsSemantic: 5'C16-C6-SS-C6
Antisense: 5 PEG
Conjugate miRNAs and polymeric compounds 125'C18-antisense miRNAsSemantic: none
Antisense: 5'C18 - C6-SS-C6
Conjugate miRNAs and polymeric compounds 135 PEG+C18 miRNAsSemantic: 5 PEG
Antisense: 5'C18-C6-SS-C6
Conjugate miRNAs and polymeric compounds 145 PEG+C16 miRNAsSemantic: 5 PEG
Antisense: 5'C16-C6-SS-C6
* In the structures of the conjugates, ss means disulfidnuu, and C16 or C18 means hydrocarbon C16 or C18. Therefore, C18-C6-SS-C6 and C16-C6-SS-C6 mean hydrophobic polymer compound.

Example 3. Assessment of the stability of conjugates miRNAs and polymeric compounds in the conditions of a living organism

It was determined whether the conjugates miRNAs, and polymer compounds obtained and isolated in example 2, an increased stability compared to unconjugated miRNAs, which is not attached polymer compound. Unconjugated miRNAs without modifications and conjugates miRNAs and polymeric compounds 1 to 5 obtained in example 2, were incubated for 0, 1, 3, 6, 9, 12, 24, 36 or 48 hours in culture medium containing 10% fetal calf serum (ETS), which simulates the conditions of a living organism, and then evaluated the degree of decomposition of miRNAs using electrophoresis.

The results showed that the conjugates of miRNAs and polymeric compounds that contain an embedded PEG, showed the stability of miRNAs to 48 hours (Fig. 9). The stability of miRNAs appeared within 12 hours to 24 hours even in the 100% whey.

Example 4. Measuring the size of nanoparticles conjugates miRNAs and hydrophobic polymer compound

In each case, conjugates miRNAs and polymeric compounds 9-14 of nanoparticles, conjugates consisting of miRNAs and polymeric compounds, that is, with�agem, the micelle is formed by hydrophobic interaction between the hydrophobic polymer attached to the ends of miRNAs (Fig. 10). The particles size was determined using the instrument to measure the ζ-potential. Measuring the size of nanoparticles consisting of corresponding conjugates miRNAs and polymeric compounds 9-13, obtained in example 2 and miRNAs.

In particular, 2 nmol of miRNAs and conjugates miRNAs and polymer compound was dissolved in 1 ml of distilled water and then homogenized their nanoparticles (200 W, 40 kHz, 5 (C) using an ultrasonic homogenizer Wiseclean from the company DAIHAN, Korea). The size of homogenised nanoparticles was determined using an instrument for measuring ζ-potential of Nano-ZS from the company MALVERN (UK). Here the refractive index and absorption coefficient for the materials installed at the level of 1,454 and 0.001, respectively, and injected water temperature as the solvent at 25°C and its viscosity and refractive index. Simultaneous measurement consisted of 20 repeated measurements of the sizes, and this measurement was performed three times.

Fig. 11 represents the size distribution of nanoparticles containing unconjugated miRNAs, according to the results obtained using the device for measuring ζ-potential. It is shown that the sizes from 142 to 295 �m (maximum point: 164 nm) corresponded to 73.5% of the full amount of the nanoparticles, each of which consisted of miRNAs.

Fig. 12 represents the size distribution of nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 9, according to the results obtained using the device for measuring ζ-potential. It is shown that the sizes from 4,19 to 7,53 nm (the point of maximum: 6,50 nm) corresponded to 59.1% of the full amount of the nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 9.

Fig. 13 represents the size distribution of nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 10, according to the results obtained using the device for measuring ζ-potential. It is shown that the sizes from 5,61 to 10.1 nm (the point of maximum: 8,72 nm) corresponded to 58.9% of the full amount of the nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 10.

Fig. 14 represents the size distribution of nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 11, according to the results obtained using the device for measuring ζ-potential. It is shown that the sizes from 5,61 to 10.1 nm (the point of maximum: 8,72 nm) corresponded to 45.6% of the full amount of the nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 11.

Fig. 15 represents the distribution fit�RAM nanoparticles each of which consisted of a conjugate of miRNAs and polymeric compounds 12, according to the results obtained using the device for measuring ζ-potential. It is shown that the sizes from 4,85 to 5.61 nm corresponded to 23.6%, and the sizes from 21,0 to 32.7 nm corresponded to 23.5%, and the sizes from 68,1 to 78.8 nm corresponded to 23.1% of the full amount of the nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 12.

Fig. 16 represents the size distribution of nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 13, according to the results obtained using the device for measuring ζ-potential. It is shown that the size 4, 85 8,72 nm (the point of maximum: 5,61 nm) corresponded to 84,6% of the total number of nanoparticles, each of which consisted of a conjugate of miRNAs and polymeric compounds 13.

In cases conjugates miRNAs and polymeric compounds 9-13, except conjugate miRNAs and polymer compound 12, the sizes of the nanoparticles were mainly from 4 to 8 nm. In the case of conjugate miRNAs and polymeric compounds 12 specific dimensions of the nanoparticles differed, which considered the relevant aggregation of the nanoparticles over time in the measurement process, despite the fact that homogenization was performed using an ultrasonic homogenizer. As pok�shown in Fig. 12-16, certain sizes of nanoparticles, each of which consisted of conjugates miRNAs, was 100 nm or less that is an adequate size for endocytosis in cells by pinocytosis (Kenneth A. Dawson, etc., Nature Nanotechnology, 2009, vol. 4, p. 84-85).

Example 5. Inhibition of expression of target genes in tumor cell lines using conjugates of miRNAs and polymeric compounds with transfectional reagents

Cancer cell line human cervical that represent tumour cell lines were subjected, respectively, transfection conjugates miRNAs and polymeric compounds 1-8 obtained in example 2 and analyzed the gene expression levels of survivin subjected to transfection tumor cell lines.

Example 5-1. Culture of tumor cell lines

Tumor cells human cervical (HeLa), obtained from the American type culture collection (ACTP), were cultured in culture medium RPMI 1640 (GIBCO, Invitrogen, USA), which was added 10 vol.% fetal calf serum, 100 u/ml penicillin and 100 µg/ml streptomycin at 37°C in atmosphere containing 5 vol.% CO2.

Example 5-2. Inhibition of expression of the target genes using conjugates of miRNAs and polymer compound

Line HeLa tumor cells were subjected to transfection with the conjugates of miRNAs and polyester�rnogo compounds 1-8 sequence No. 1, obtained in example 2 and analyzed the gene expression levels of survivin subjected to transfection tumor cell lines.

Example 5-2-1. Transfection of tumor cell lines using conjugates of miRNAs and polymer compound

Cultivated 1,3•105of tumor cell lines, cultured in example 5-1, in the medium RPMI 1640 for 6-well plate at 37°C for 18 hours in an atmosphere containing 5 vol.% CO2, then deleted the environment and to each well was added 800 μl of medium Opti-MEM (GIBCO, USA).

At the same time were mixed with 2 µl of lipofectamine (Lipofectamine™ 2000 from Invitrogen company, USA) and 198 μl of medium Opti-MEM, which are then reacted among themselves at room temperature for 5 minutes, after which was added 0.8 or 4 µl of the respective conjugates miRNAs and polymeric compounds (25 pmol/μl), obtained in example 2 (the final treatment with 20 or 100 nm). Thereafter, the resulting substance is again reacted at room temperature for 20 minutes to prepare the solution.

After that, 200 μl of a solution for transfection was placed in each of the holes into which was placed a medium Opti-MEM, and tumor cells were cultured for 6 hours followed by removal of the medium Opti-MEM. The wells were placed in 2.5 µl of culture medium RPMI 1640, and then the tumor cells were cultured at 37°C in an atmosphere containing about 5�.% CO 2in the next 24 hours.

Example 5-2-2. Relative quantitative analysis of mRNA of the gene of survivin

The quantity of RNA was extracted from cell lines, subjected to transfection in example 5-2-1 to obtain cDNA, and then the relative amount of mRNA of the gene of survivin was determined using real-time PCR.

Example 5-2-2-1. Separation of RNA and obtaining cDNA from subjected to transfection cells

The quantity of RNA was extracted from cell lines, subjected to transfection in example 5-2-1, using the kit for RNA extraction (set for total RNA extraction AccuPrep Cell BIONEER company, Korea), and cDNA was obtained from extracted RNA using reverse transcriptase RNA (AccuPower CycleScript RT Premix/dT20from BIONEER company, Korea) as follows.

Specifically, 1 μg of extracted RNA was placed into each of the Eppendorf tubes (Eppendorf) with a volume of 0.25 ml containing reverse transcriptase AccuPower CycleScript RT Premix/dT20from BIONEER company (Korea), and distilled water-treated diethylpyrocarbonate (DEPC) was added to bring the full volume to 20 ál. Using PCR device with a unit thermal gradient MyGenie™ 96 Gradient Thermal Block from BIONEER company (Korea) two-stage hybridization priming RNA at 30°C for 1 minute and synthesis of cDNAs at 52°C for 4 minutes was repeated six times. Then the inactive�Oia of the enzyme was carried out at 95°C for 5 minutes, to complete the amplification reaction.

Example 5-2-2-2. Relative quantitative analysis of mRNA of the gene of survivin

The relative amount of mRNA of the gene of survivin was determined by real-time PCR using cDNA obtained in example 5-2-2-1, as a matrix as follows.

In particular, cDNA obtained in example 5-2-2-1, was diluted in the ratio of 1/5 with distilled water in each well of 96-hole tablet was then added to 3 ál of diluted cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál priming qPCR of survivin (10 pmol/ál) from BIONEER company (Korea), obtaining a liquid mixture to analyze the expression level of survivin. On the other hand, when using GMBS (hydroxymethylglycinate), HPRT ( 1), UBC (ubiquitin C) and YWHAZ (ζ-polypeptide protein activation of tyrosine-3-monooxygenases/tryptophan 5-monooxygenases), which represent the genes of the household" (hereinafter abbreviated referred to as "DH genes"), as a reference gene to normalize the expression level of the mRNA obtained in example 5-2-2-1 cDNA was diluted in the ratio 1/5 and then injected with 3 μl of diluted cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál W�weed CPCR each gene DX (10 pmol/μl BIONEER, Korea) to prepare a liquid mixture for analysis by real-time PCR of each gene hall sensor in each well of 96-hole tablet. The following reaction was carried out on 96-well plate containing liquid mixture, with using Exicycler™ 96 with heat block for quantitative real-time analysis from BIONEER company (Korea).

The activation of the enzyme and cDNA secondary structure is eliminated during the reaction at 95°C for 15 minutes. After that, four stages, including denaturing at 94°C for 30 seconds, hybridization at 58°C for 30 seconds, elongation at 72°C for 30 seconds and scan with the dye SYBR Green, re carried out 42 times and then carried out a final extension at 72°C for 3 minutes. Thereafter, the temperature was maintained at 55°C for 1 min and were analyzed by melting curve in the range of 55-95°C.

After PCR the values obtained threshold cycle Ct of survivin respectively corrected using the values of mRNA (normalized multiplier (NF), normalized by DH genes, and then to obtain the ΔCt of the difference between the Ct value of the control group treated with only one reagent for transfection, and the corrected Ct values. The rate of mRNA expression of survivin compared with each other using ΔCt values and the calculated ur�wnanie 2 (-ΔCt)•100 (Fig. 17). Fig. 17 simulation means of the control group, treated only reagent for transfection.

As a result, as shown in Fig. 17, the effect of interference RNA miRNAs varied depending on the types of end modifications conjugates miRNAs and polymeric compounds in which was anywhereman hydrophilic polymer compound, namely PEG. In particular, each of the conjugates 6-8 with a type of terminal modification, wherein the PEG is conjugated to the target region 3', showed the degree of inhibition of expression similar to those exhibits unconjugated miRNAs. Therefore, it is expected that conjugates 6-8 will create a small steric obstacle in the formation of complex with the RNA-induced complex of suppression (RICP) in the mechanism of RNA interference involving miRNAs. In addition, most conjugates miRNAs-PEG showed higher degree of inhibition of gene expression of the target mRNA under conditions of treatment with a low concentration (20 nm) than in processing conditions at high concentration (100 nm), and thus, it is expected that will be prevented binding of miRNAs with RIK thanks PEG under conditions of high concentrations of conjugate miRNAs-PEG.

Example 5-3. Inhibition of expression of the target genes using conjugates having a long sequence mi�NC and polymer compound

When cells were subjected to transfection with the conjugates of miRNAs and a hydrophilic polymer compound with a reagent for transfection, we analyzed the inhibition of gene expression of the target mRNA. Here miRNAs used in which end modification in the structure of the conjugate miRNAs and polymer compound 4 was induced for each sequence of bases miRNAs (sequence Nos. 1 to 4).

Example 5-3-1. Transfection of tumor cell lines using conjugates of miRNAs and polymer compound

Cultivated 1,3•105of tumor cell lines, cultured in example 5-1, in the medium RPMI 1640 for 6-well plate at 37°C for 24 hours in an atmosphere containing 5 vol.% CO2, then deleted the environment and to each well was added 800 μl of medium Opti-MEM.

At the same time were mixed with 2 µl of lipofectamine (Lipofectamine™ 2000) and 198 μl of medium Opti-MEM, which are then reacted among themselves at room temperature for 5 minutes, after which was added 0.8 or 4 µl of the respective conjugates miRNAs and polymeric compounds (25 pmol/μl), obtained in example 2 (the final treatment with 20 or 100 nm). Thereafter, the resulting substance is again reacted at room temperature for 20 minutes to prepare the solution.

After that, 200 μl of a solution for transfection was placed in each of the holes into which b�La placed Wednesday Opti-MEM, and tumor cells were cultured for 6 hours followed by removal of the medium Opti-MEM. The wells were placed in 2.5 µl of culture medium RPMI 1640, and then the tumor cells were cultured at 37°C in atmosphere containing 5 vol.% CO2in the next 24 hours.

An example of a 5-3-2. Relative quantitative analysis of mRNA of the gene of survivin

The quantity of RNA was extracted from cell lines, subjected to transfection in example 5-3-1 to obtain cDNA, and then the relative amount of mRNA of the gene of survivin was determined using real-time PCR.

An example of 5-3-2-1. Separation of RNA and obtaining cDNA from subjected to transfection cells

The quantity of RNA was extracted from cell lines, subjected to transfection in example 5-3-1, using the kit for RNA extraction (set for total RNA extraction AccuPrep Cell BIONEER company, Korea), and cDNA was obtained from extracted RNA using reverse transcriptase RNA (AccuPower CycleScript RT Premix/dT20from BIONEER company, Korea) as follows.

Specifically, 1 μg of extracted RNA was placed into each of the Eppendorf tubes (Eppendorf) with a volume of 0.25 ml containing reverse transcriptase AccuPower CycleScript RT Premix/dT20from BIONEER company (Korea), and distilled water-treated diethylpyrocarbonate (DEPC) was added to bring the full volume to 20 ál. With ispolzovaniya PCR with block of thermal gradient MyGenie™ 96 Gradient Thermal Block from BIONEER company (Korea) two-stage hybridization priming RNA at 30°C for 1 minute and synthesis of cDNAs at 52°C for 4 minutes was repeated six times. Then the inactivation of the enzyme was carried out at 95°C for 5 minutes to complete the amplification reaction.

Example 5-3-2-2. Relative quantitative analysis of mRNA of the gene of survivin

The relative amount of mRNA of the gene of survivin was determined by real-time PCR using cDNA obtained in example 5-2-2-1, as a matrix as follows.

In particular, cDNA obtained in example 5-3-2-1, was diluted in the ratio of 1/5 with distilled water in each well of 96-hole tablet, and then were added 3 μl of diluted cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál priming qPCR of survivin (10 pmol/ál) from BIONEER company (Korea), obtaining a liquid mixture to analyze the expression level of survivin. On the other hand, when using GMBS, HPRT, UBC and YWHAZ, which represent the genes of HHS as a reference gene to normalize the expression level of the mRNA obtained in example 5-3-2-1 cDNA was diluted in the ratio 1/5 and then injected with 3 μl of diluted cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál priming CPCR each gene DX (10 pmol/µl, BIONEER, Korea) to prepare a liquid mixture for analysis by real-time PCR of each gene of HHS in each lance-hole tablet. The following reaction was carried out on 96-well plate containing liquid mixture, with using Exicycler™ 96 with heat block for quantitative real-time analysis from BIONEER company (Korea).

The activation of the enzyme and cDNA secondary structure is eliminated during the reaction at 95°C for 15 minutes. After that, four stages, including denaturing at 94°C for 30 seconds, hybridization at 58°C for 30 seconds, elongation at 72°C for 30 seconds and scan with the dye SYBR Green, re carried out 42 times and then carried out a final extension at 72°C for 3 minutes. Thereafter, the temperature was maintained at 55°C for 1 min and were analyzed by melting curve in the range of 55-95°C.

After PCR the values obtained threshold cycle Ct of survivin respectively corrected using the values of mRNA (normalized multiplier (NF), normalized by DH genes, and then to obtain the ΔCt of the difference between the Ct value of the control group treated with only one reagent for transfection, and the corrected Ct values. The rate of mRNA expression of survivin compared with each other using ΔCt values and the estimated equation 2(-ΔCt)•100 (Fig. 18). Fig. 18 simulated mean of the control group, treated only reagent for transfection, and me, me, Mer, and or represent the sequence Nos. 1 to 4, respectively. 5'P+P represents the structure of the conjugate miRNAs and polymer compound 4. Cells were treated at concentrations of 20 nm and 100 nm, respectively, and the degree of inhibition of expression of target genes were compared with each other.

As a result, as shown in Fig. 18, long-chain non-conjugated miRNAs converted to form conjugates miRNAs and polymer compound 4, showed a smaller difference between the degree of inhibition of mRNA expression of target genes compared to unconjugated miRNAs. Therefore, it is possible to detect that transformed long-chain miRNAs reduces the phenomenon of steric hindrance due to the PEG, compared to the shorter-miRNAs.

In particular, in the case of long-chain miRNAs, miRNAs are cleaved in the structure of the 19+2 under the action of a cleaving enzyme (dasera) in the mechanism of action of RNA interference and miRNAs split joins the complex RIK, powering the mechanism of RNA interference. For this reason, long-chain miRNAs, in which the PEG is attached to both end regions, leads to the existence of a large number of miRNAs without the attachment of PEG and, thus, has a relatively high degree of interaction with the complex RIK compared with sequence number 1, which is considered support�ment the effect of inducing RNA interference.

Example 6. Inhibition of expression of the target genes in tumor cell lines using only the conjugates of miRNAs and polymeric compounds without reagents for transfection

Line HeLa tumor cells were subjected to transfection with the conjugates of miRNAs and polymeric compounds 1 to 14 obtained in example 2 and analyzed the gene expression of survivin subjected to transfection of tumor cell lines.

Example 6-1. Culture of tumor cell lines

Tumor cells human cervical (HeLa), obtained from the American type culture collection (ACTP), were cultured in culture medium RPMI 1640 (GIBCO/Invitrogen, USA), which was added 10 vol.% fetal calf serum, 100 u/ml penicillin and 100 µg/ml streptomycin at 37°C in atmosphere containing 5 vol.% CO2.

Example 6-2. Transfection of tumor cell lines using conjugates of miRNAs and polymer compound

Cultivated 1,3•105of tumor cell lines, cultured in example 6-1, in the medium RPMI 1640 for 6-well plate at 37°C for 24 hours in an atmosphere containing 5 vol.% CO2, then deleted the environment and to each well was added to 900 μl of medium Opti-MEM.

At the same time mixed 100 μl of medium Opti-MEM, 5 or 10 µl of the respective conjugates miRNAs and polymeric compounds 1-5 (1 pmol/µl), obtained in example 2 (finite obra�privileges denied at 500 nm or 100 μm), and the resulting material is again reacted at room temperature for 20 minutes to prepare the solution.

At the same time 100 μl of medium Opti-MEM, 5 or 10 µl of the respective conjugates miRNAs and polymeric compounds 9-14 (1 pmol/µl), obtained in example 2 (the ultimate treatment at 500 nm or 100 μm) and micelles consisting of conjugates of miRNAs and a hydrophobic polymer compound, homogenized under the action of ultrasound for the preparation of the solution.

After that, 100 µl of a solution for transfection was placed in each of the holes, which were medium Opti-MEM, and tumor cells were cultured for 24 hours followed by the addition of 1 ml of medium RPMI 1640 containing 20% FBS. Cells then were cultured at 37°C for 24 hours in an atmosphere containing 5 vol.% CO2were treated with the conjugates of miRNAs and polymeric compounds and then cultured 48 hours.

Example 6-3. Relative quantitative analysis of mRNA of the gene of survivin

The quantity of RNA was extracted from cell lines, subjected to transfection in example 6-2, to obtain cDNA, and then the relative amount of mRNA of the gene of survivin was determined using real-time PCR.

Example 6-3-1. Separation of RNA and obtaining cDNA from subjected to transfection cells

The quantity of RNA was extracted from cell lines, machined parts�bent transfection in example 6-2, using the kit for RNA extraction (set for total RNA extraction AccuPrep Cell BIONEER company, Korea), and cDNA was obtained from extracted RNA using reverse transcriptase RNA (AccuPower CycleScript RT Premix/dT20from BIONEER company, Korea) as follows.

Specifically, 1 μg of extracted RNA was placed into each of the Eppendorf tubes (Eppendorf) with a volume of 0.25 ml containing reverse transcriptase AccuPower CycleScript RT Premix/dT20from BIONEER company (Korea), and distilled water-treated diethylpyrocarbonate (DEPC) was added to bring the full volume to 20 ál. Using PCR device with a unit thermal gradient MyGenie™ 96 Gradient Thermal Block from BIONEER company (Korea) two-stage hybridization priming RNA at 30°C for 1 minute and synthesis of cDNAs at 52°C for 4 minutes was repeated six times. Then the inactivation of the enzyme was carried out at 95°C for 5 minutes to complete the amplification reaction.

Example 6-3-2. Relative quantitative analysis of mRNA of the gene of survivin

The relative amount of mRNA of the gene of survivin was determined by real-time PCR using cDNA obtained in example 6-3-1, as a matrix as follows.

In particular, cDNA obtained in example 6-3-1, was diluted in the ratio of 1/5 with distilled water in each well of 96-hole tablet was then added to 3 µl of RA�added cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál priming qPCR of survivin (10 pmol/ál) from BIONEER company (Korea), obtaining a liquid mixture to analyze the expression level of survivin. On the other hand, when using GMBS (hydroxymethylglycinate), HPRT ( 1), UBC (ubiquitin C) and YWHAZ (ζ-polypeptide protein activation of tyrosine-3-monooxygenases/tryptophan 5-monooxygenases), which represent the genes of the household" (hereinafter abbreviated referred to as "DH genes"), as a reference gene to normalize the expression level of the mRNA obtained in example 5-2-2-1 cDNA was diluted in the ratio 1/5 and then injected with 3 μl of diluted cDNA, 10 μl of a basic solution for PCR 2xGreenStar™ PCR from BIONEER company (Korea), 6 μl of distilled water and 1 ál priming CPCR each gene DX (10 pmol/µl, BIONEER, Korea) to prepare a liquid mixture for analysis by real-time PCR of each gene hall sensor in each well of 96-hole tablet. The following reaction was carried out on 96-well plate containing liquid mixture, with using Exicycler™ 96 with heat block for quantitative real-time analysis from BIONEER company (Korea).

The activation of the enzyme and cDNA secondary structure is eliminated during the reaction at 95°C in the�Linux 15 minutes. After that, four stages, including denaturing at 94°C for 30 seconds, hybridization at 58°C for 30 seconds, elongation at 72°C for 30 seconds and scan with the dye SYBR Green, re carried out 42 times and then carried out a final extension at 72°C for 3 minutes. Thereafter, the temperature was maintained at 55°C for 1 min and were analyzed by melting curve in the range of 55-95°C. After PCR values obtained threshold cycle Ct of survivin respectively corrected using the values of mRNA (normalized multiplier (NF), normalized by DH genes, and then to obtain the ΔCt of the difference between the Ct value of the control group treated with only one reagent for transfection, and the corrected Ct values. The rate of mRNA expression of survivin compared with each other using ΔCt values and the estimated equation 2(-ΔCt)•100 (Fig. 19).

As a result, as shown in Fig. 17, conjugates miRNAs-PEG 2-5 in a higher degree inhibit the mRNA expression level of survivin compared to the conjugate miRNAs and polymer compound 1, in contrast to the result in the case where the transfection was carried out by means of the reagent for transfection. Conjugates of miRNAs and polymeric compounds 1-5 showed in a higher degree the effect of RNA interference in low concentration� (500 nm), than high concentrations. In addition, conjugates of miRNAs and hydrophobic polymeric compounds 9-14 showed a lower degree of inhibition of the expression level of mRNA of the gene of survivin compared to conjugates miRNAs 1-5 in the case of treatment with the same concentration (500 nm). However, in the case of processing under conditions of high concentration (1 nm), in particular, limit the modification of the conjugate miRNAs and polymeric compounds 14 leads to a high degree the effect of inhibition of the expression level of mRNA of survivin.

The list of sequences

Cm. the list of sequences

1. Conjugate for intracellular delivery of miRNAs containing miRNAs polymer compound of the following structure:
A-X-R-Y-B,
(where one of A and B is a hydrophilic polymer compound, and the other is a hydrophobic compound; X and Y independently represent a simple covalent bond or a covalent bond via an intermediate linker; and R represents miRNAs),
where the hydrophobic compound is represented by hydrocarbon, C16-C50or cholesterol, having a molecular weight from 250 to 1000.

2. The conjugate according to claim 1, wherein the hydrophilic polymer compound and the hydrophobic compound anywherevery to the same chain miRNAs.

3. The conjugate according to claim 1, wherein the hydrophilic polymer compound and a hydrophobic soedinyayuschayasya to both circuits of miRNAs.

4. The conjugate according to claim 1, wherein the single chain miRNAs (R) consists of 19 to 31 nucleotides.

5. The conjugate according to claim 1, wherein the covalent bond (X, Y) is an unsplittable link or split the connection.

6. The conjugate according to claim 5, in which the unsplittable link is an amide bond or a phosphate bond.

7. The conjugate according to claim 5, in which the degradable linkage is selected from disulfide bonds, split acid connection, ester bonds, anhydrite communication, bioassayset communication and biologically degradable by the enzyme connection.

8. The conjugate according to claim 1, wherein the hydrophilic polymer compound is a nonionic polymer compound having a molecular weight of from 1000 to 10000.

9. The conjugate according to claim 8, in which the hydrophilic polymer compound selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone and polyoxazolines.

10. A method of obtaining a conjugate according to claim 1,
where the conjugate according to claim 1 in which the hydrophilic polymer compound is a polyethylene glycol (PEG),
which includes:
1) obtaining the conjugate single-stranded miRNAs-PEG using the associated with polyethylene glycol solid substrate has the following structure:

where R represents alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl or heteroaryl; m performance�exists an integer from 2 to 18; n is an integer from 5 to 120; and X represents a hydrogen atom, 4-monomethacrylate, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl;
2) connection of hydrophobic compounds with PEG-conjugated chain at the distal end via a covalent bond, where the hydrophobic compound is represented by hydrocarbon, C16-C50or cholesterol, having a molecular weight from 250 to 1000; and
3) annealing circuit, where PEG and hydrophobic compound konjugierten at both ends, with its complementary chain.

11. A method of obtaining a conjugate according to claim 1,
where the conjugate according to claim 1 in which the hydrophilic polymer compound is a polyethylene glycol (PEG), which provides:
1) the accession of the end groups of one chain of miRNAs to PEG or hydrophobic compound with covalent bonds, where the hydrophobic compound is represented by hydrocarbon, C16-C50or cholesterol, having a molecular weight from 250 to 1000;
2) connect another end of the group of other chain miRNAs to PEG (if the hydrophobic compound is joined on stage (1)) or to a hydrophobic compound (if the PEG is attached at the stage (1)) via a covalent bond;
3) annealing stage circuit (1) with another circuit stage (2).

12. Of nanoparticles for intracellular delivery of miRNAs consisting of conjugates containing miRNAs-polyester�RNA compound according to claim 1.

13. Pharmaceutical composition for gene therapy containing a pharmaceutically effective amount of the conjugates for intracellular delivery of miRNAs containing miRNAs polymer compound according to claim 1.

14. Pharmaceutical composition for gene therapy containing a pharmaceutically effective amount of the nanoparticles according to claim 12.

15. Conjugate survivin-specific miRNAs and the polymeric compound for the treatment of cancer having the following structure
A-X-R-Y-B,
(where one of A and B is a hydrophilic polymer compound, and the other is a hydrophobic compound; X and Y independently represent a simple covalent bond or a covalent bond via an intermediate linker; and R is survivin-specific miRNAs),
where the hydrophobic compound is represented by hydrocarbon, C16-C50or cholesterol, having a molecular weight from 250 to 1000.

16. The conjugate according to claim 15, in which one chain of survivin-specific miRNAs (R) consists of 19 to 31 nucleotides.

17. The conjugate according to claim 15, where survivin-specific miRNAs (R) represents any sequence selected from any of the sequences SEQ ID NO: 1-4.

18. The conjugate according to claim 16, where survivin-specific miRNAs has a chemical modification.

19. The conjugate according to claim 18, where the chemical modification comprises at least one modification�operators, selected from:
modified phosphorodithioic regard to phosphorothiate connection; modified-OH in 2' position of the pentose to a 2'-OCH3or 2'-dioxo-2'-pteridine; and
modified-OH in 2' position of the pentose to LNA type, obtained by linking 2'-position and 4'-position of the pentose.

20. The conjugate according to claim 15, in which the hydrophilic polymer compound is a nonionic polymer compound with a molecular weight of from 1,000 to 10,000; and a covalent bond is unsplittable link or split the connection.

21. The conjugate according to claim 20, in which the unsplittable link is an amide bond or a phosphate bond, and degradable linkage is selected from disulfide bonds, split acid connection, ester bonds, anhydrite communication, bioassayset communication and biologically degradable by the enzyme connection.

22. The conjugate according to claim 20, in which the hydrophilic polymer compound includes at least one compound selected from polyethylene glycol (PEG), polyvinylpyrrolidone and polyoxazolines.

23. Of nanoparticles for cancer treatment containing a conjugate according to claim 15.

24. Pharmaceutical composition for cancer treatment containing a conjugate according to claim 15 as a pharmaceutically effective component.

25. Pharmaceutical composition for cancer treatment containing nanoparticle according to claim 23 as a semiconductor�Eski effective component.



 

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