KR100795100B1 - Polymeric nano particles for imaging of phosphorylation by protein kinase and uses thereof - Google Patents

Abstract

The present invention relates to polymeric nanoparticles that selectively fluoresce in phosphorylation by protein kinases, methods for their preparation, and their use. Since the polymer nanoparticles according to the present invention selectively fluoresce according to the phosphorylation of protein kinases, it is possible to diagnose various diseases caused by excessive expression of protein kinases, and to efficiently screen the efficacy of new drugs developed as protein kinase inhibitors. can do.

Polymer nanoparticles, protein kinases

Description

POLYMERIC NANO PARTICLES FOR IMAGING OF PHOSPHORYLATION BY PROTEIN KINASE AND USES THEREOF}

FIG. 1 is a schematic diagram of a method in which polymer nanoparticles that selectively fluoresce in phosphorylation by a protein kinase prepared in the present invention exhibit specific fluorescence. FIG.

2 is a particle diameter of the polymer nanoparticles prepared by combining PEI-FITC-Kemp and PAA-RITC prepared in Example 1 in various composition ratios, and shows an average of 300 nanometers.

3 is a particle diameter of the polymer nanoparticles prepared by combining PEI-FITC-Kemp-Cy5.5 and PAA prepared in Example 1 in various composition ratios, and shows an average of 300 nanometers.

FIG. 4 is a diagram illustrating the fluorescence intensity of FITC varying according to the amount of PAA-RITC in the image box when PEI-FITC-Kemp and PAA-RITC are combined at various composition ratios.

FIG. 5 shows the fluorescence intensity of Cy5.5 changing according to the amount of PAA when the PEI-FITC-Kemp-Cy5.5 and PAA are combined at various composition ratios through an image box.

6 is a diagram showing the fluorescence intensity of the FITC changes by adding PKA to the polymer nanoparticles containing PEI-FITC-Kemp and PAA-RITC, the lower number is the fluorescence intensity value confirmed through the fluorimeter.

7 is a graph showing the fluorescence intensity of Cy5.5 changed by adding PKA to the polymer nanoparticles containing PEI-FITC-Kemp-Cy5.5 and PAA, and the numbers at the bottom are the fluorescence intensity values confirmed through the fluorometer. to be.

The present invention provides polymer nanoparticles that selectively fluoresce in phosphorylation by protein kinase, a method for preparing the same, a method for imaging protein phosphorylation by protein kinase using the same, and effectively developed as a protein kinase inhibitor using the same. It is about how to select new drugs that will be.

Protein kinases play an important role in the control of information transfer within or between cells, which occurs by the protein kinases transferring phosphoryl groups from nucleoside triphosphates to protein receptors involved in the signaling pathway. In other words, numerous protein kinases exert cell stimulation and other stimuli through their pathways, allowing various cellular responses to occur within the cell. Many diseases are associated with abnormal cellular responses triggered by protein kinase mediated events. These diseases include autoimmune diseases, inflammatory diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease or hormone related diseases.

CAMP-dependent protein kinase (PKA), known to regulate many life functions, including energy metabolism, gene transcription, proliferation, differentiation, reproductive function, secretion, neuronal activity, memory, contraction and motility, binds to dimeric regulatory subunits Is a tetrameric proenzyme comprising two catalytic subunits. Binding of the cAMP separates the catalytic subunit from the regulatory subunit to form an active serine / threonine kinase. Three isomers of the catalytic subunits (C-α, C-β, C-γ) have been reported, especially the C-α subunits with increased expression in the first phase and metastatic melanoma. AKT (Rac protein kinase B (PKB)) and serine / threonine protein kinases are known to be overexpressed in many cancers and are mediators of normal cell function. Signs of modified AKT regulation appear in both injury and disease, with the most important role being in cancer. AKT has been found to be overexpressed in human ovarian cancer, pancreatic cancer and the like, suggesting that AKT may be associated with tumor targeting. Rho-associated coiled-coil forming kinase (ROCK) is a serine / threonine kinase that activates the small G-protein RhoA. Hypertension, erectile dysfunction, angiogenesis, nerve regeneration, metastasis, glaucoma, inflammation, arteriosclerosis, immunity It is involved in a number of diseases including inhibition, vascular restenosis, asthma and cardiac hypertrophy. 3-phosphoinositide-dependent protein kinase-1 (PDK-1) plays a key role in regulating the activity of many kinases belonging to the AGC subclass of protein kinases. PDK1 mediated signal transduction is activated as a response to insulin and growth factors and adhesion of cells to the extracellular matrix. Once activated, these enzymes mediate many different cellular events by phosphorylating key regulatory proteins that play important roles in controlling cell survival, growth, proliferation, and glucose regulation. In addition, a wide range of cancer cell genotypes may be used in cell physiology for tissue infiltration that leads to self-sufficiency in growth signal transduction, avoidance of apoptosis, desensitization to growth-inhibitory signal transduction, unlimited replication potential, inspired angiogenesis and metastasis It is due to the manifestation of six major variations. PDK1 is an important mediator of the PI3K signaling pathway, which regulates a number of cellular functions, including growth, proliferation, and survival. Thus, inhibition of this pathway may affect four or more of the six that have been shown to be necessary for cancer progression, whereby PDK1 inhibitors are expected to affect the growth of a very wide range of human cancers.

Accordingly, there is a need for developing inhibitors of these protein kinases along with many studies related to protein kinase activities such as PKA, AKT, ROCK and PDK1. In addition, the development of technology for the early diagnosis of protein kinase-related diseases by imaging these protein kinase activity, and the development of technology to easily evaluate the activity of inhibitors is very necessary.

This need has led to the early diagnosis of disease by imaging cellular responses by protein kinases. In addition, we attempted to identify effective protein kinase inhibitors as therapeutic agents for diseases derived from protein kinases, and in particular, attempts to identify protein kinase inhibitors that act selectively without causing unexpected side effects due to non-selectivity and new protein kinases. In order to develop inhibitors, efforts have been made to more easily identify inhibitor activity.

Recently, a technique for imaging phosphorylation of protein by protein kinase using concentration change of secondary messengers Ca2 ⁺ , cAMP, cGMP, and the like has been studied. Techniques for imaging phosphorylation of proteins occurring in cells using fluorescence resonance energy transfer have also been studied. However, The above method is not a real-time imaging method, and uses phosphors that are difficult to use in vivo research. In this regard, the development of a technique for imaging phosphorylation of proteins by protein kinases in vivo is urgently needed using phosphors that can be used in vivo.

The present invention provides novel polymeric nanoparticles capable of real-time imaging of the reaction of protein kinases in cells, which exhibit fluorescent signals through physical structural changes that appear selectively in protein phosphorylation by protein kinases, methods for their preparation, and their use.

In one embodiment, the present invention provides polymeric nanoparticles that can selectively fluoresce protein phosphorylation by protein kinases. The polymer nanoparticles of the present invention include a cationic polymer complex and an anionic polymer complex, and image the reaction of the protein kinase by intracellular protein kinase by selectively phosphorylating the protein kinase and causing a fluorescence signal by showing a fluorescence signal. can do.

Specifically, the present invention relates to a cationic polymer complex, an anionic polymer, and a fluorescence resonance energy in which a cationic polymer, a cationic polymer capable of selectively phosphorylating by a kinase capable of fluorescence resonance energy transfer, and a peptide derivative having a nucleotide sequence selectively phosphorylated by a protein kinase are chemically bound. Provided is a polymer nanoparticle comprising an anionic polymer composite bonded to a phosphor capable of transition.

In addition, the present invention provides a cationic polymer, a cationic polymer complex in which a peptide derivative having a nucleotide sequence selectively phosphorylated by a phosphor and a protein kinase having a quenching effect is chemically bonded to a polymer nanoparticle comprising an anionic polymer. to provide.

As the polymer according to the present invention, any polymer having biocompatibility may be used.

Polymers used in cationic polymer composites are preferably polyethylenimine, poly-L-lysine, chitosan and the like having a positron having high cell absorption efficiency. More preferably, it is polyethyleneimine, and polyethyleneimine contains a lot of positrons in the chain, so the cell absorption rate is very high, and the amine group has an advantage of inducing various chemical modifications.

The polymer used in the anionic polymer composite forming the cationic polymer complex and the ion aggregate is preferably polyaspartic acid, polyglutamic acid, heparin, or the like, or a chemical derivative thereof. More preferably, it is polyaspartic acid, because polyaspartic acid contains a lot of negative electrons in the chain and thus has a high cell uptake rate, and a carboxyl group can induce various chemical modifications.

The polymer can form nanoscale self-assembly through the balance of hydrophobicity and hydrophilicity and the action of cations and anions, and can be effectively absorbed into living cells. It is also bound to any functional group that, after being absorbed into the cell, reacts sensitively to phosphorylation of the protein by protein kinases and fluoresces.

The phosphor according to the present invention is a phosphor that generates a fluorescence resonance energy transfer or a phosphor that generates a quenching effect. These phosphors are in close proximity to each other in the polymer nanoparticles, resulting in fluorescence resonance energy transfer or quenching effect, thereby minimizing the fluorescence intensity of the phosphor. In particular, it is preferable to use a phosphor that emits red or near infrared fluorescence and has a high quantum yield. The phosphor of the present invention is preferably a phosphor of cyanine, rodamine, fluorescein and bodiphy. Phosphors exhibiting a fluorescence resonance energy transfer effect are preferably tetramethylrodamine and fluorescein, and fluorescein and cyanine are also preferable. For example, since the emission wavelength band of fluorescein overlaps with the excitation wavelength band of tetramethylrodamine, it is used as an excitation wavelength of tetramethylrodamine in which the emission wavelength of fluoresce is generated when providing an excitation wavelength for fluorescein. This is because tetramethylrodamine can also emit light at the same time. In addition, each phosphor, such as cyanine, fluorescein, rhodeamine, tetramethylrodamine, and Vodaf, is preferable as the phosphor exhibiting the quenching effect because the photoquenching effect occurs when they are close to each other. In particular, since the cyanine emits and absorbs near-infrared light, interference with or absorption of cells, blood, biological tissues, and the like can be minimized.

Peptide derivatives included in the cationic polymer complex according to the present invention is a peptide derivative having a nucleotide sequence selectively phosphorylated by protein kinase, Leu-Arg-Arg- the Asp-Ser-Leu-Gly ( kemptide, SEQ ID NO: 1), which takes place is selectively phosphorylated by protein kinase a. The present invention includes all peptide derivatives that include sequences that selectively phosphorylate by protein kinases of any length.

Protein kinases that can be imaged using polymeric nanoparticles according to the invention are all kinds of protein kinases that phosphorylate proteins. Such protein kinases include cAMP dependent protein kinase (PKA), AKT (PKA), low-linked coiled-coil forming kinase (ROCK), and 3-phosphoinositide-dependent protein kinase-1 (PDK1).

More specifically, the present invention provides a polymer nanoparticle comprising a cationic polymer composite having a structure of Formula 1 and an anionic polymer complex of Formula 2. Polymer nanoparticles having the above configuration is one example capable of imaging the phosphorylation reaction by protein kinases using fluorescence resonance energy transfer.

Wherein A is ethyleneimine, B is a phosphor such as fluorescein, rhodeamine, tetramethylrodamine, etc. capable of fluorescence resonance energy transfer to the amine group of ethyleneimine, and C is a base to which phosphorylation selectively occurs by protein kinase. A peptide derivative having a sequence, a is an integer between 25 and 40, b is an integer between 5 and 10, and c is an integer between 20 and 35.

In the above formula, A is aspartic acid, B is phosphor such as fluorescein, rodamine, tetramethylrodamine, etc. capable of fluorescence resonance energy transfer to the amine group of aspartic acid, a is an integer between 50 and 150, and b is from 5 to Is an integer between 10

The present invention also provides a polymer nanoparticle comprising a cationic polymer composite having a structure of Formula 3 and an anionic polymer of Formula 4. The polymer nanoparticle having the above configuration is one example capable of imaging the phosphorylation reaction by protein kinase using the quenching effect of the phosphor.

In the above formula, A is ethyleneimine, B is phosphor such as fluorescein, rhodeamine, tetramethylrodamine, etc. capable of fluorescence resonance energy transfer, and C is selectively phosphorylated by protein kinase. A peptide derivative having a nucleotide sequence, D is a cyanine phosphor exhibiting a quenching effect, a is an integer between 25 and 40, b is an integer between 1 and 5, c is an integer between 20 and 35, d is It is an integer between 5 and 10.

In the formula, it is aspartic acid. a is an integer between 50 and 150.

Phosphors capable of fluorescence resonance energy transfer of Formula 3 play a role in indicating the presence position when the polymer of the present invention is infiltrated in vivo.

When the protein kinase phosphorylates the peptide derivative contained in the polymer nanoparticles, anionicity is added to the cationic polymer complex to change the ionic environment so that the cationic polymer complex and the anionic polymer complex of the present invention dissociate with each other. When the polymer nanoparticles form ionic aggregates, the phosphors in the nanoparticles are in close proximity and show weak or no fluorescence due to the fluorescence resonance energy transfer or quenching effect of the phosphor. However, when the ion aggregate is dissociated by the protein kinase as described above, the phosphors in the nanoparticles are far apart and the fluorescence resonance energy transfer or quenching effect disappears. That is, the phosphors fluoresce specifically for phosphorylation of protein kinases, allowing selective imaging of the reaction of phosphorylation by protein kinases.

The particle size of the polymer nanoparticles according to the present invention has an average size of 50 to 500 nanometers, has excellent biocompatibility and control of the absorption efficiency in cells, and can also be passively accumulated in specific tissues or cells in vivo. It can be used for various purposes such as cell imaging, specific tissue imaging, drug delivery, and the like. In addition, since it can be applied both in vivo and in vitro, it can be used in various applications such as high-throughput screening method and early disease diagnosis necessary for drug development.

Accordingly, as another aspect, the present invention provides a method for imaging phosphorylation of a protein by protein kinase using the polymer nanoparticles according to the present invention. Such imaging methods will enable the diagnosis of various diseases manifested by overexpression of protein kinases.

In still another aspect, the present invention provides a method for selecting candidate drugs having protein kinase inhibitory activity by using the polymer nanoparticles according to the present invention. By adding a candidate drug having protein kinase inhibitory activity to the polymer nanoparticles according to the present invention, the degree of fluorescence may be confirmed, and thus the activity of the candidate drug may be confirmed, and thus may be usefully used in developing new drugs.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these embodiments.

Example  One. Cationic  Polymer composites and Anionic  Preparation of Polymer Composite

Peptide derivative Leu-Arg- having a nucleotide sequence selectively phosphorylated by protein kinase A as a peptide that binds polyethyleneimine having a molecular weight of 25000 as a cationic polymer, a polyaspartic acid having a molecular weight of 10000 as an anionic polymer to a polyethyleneimine Arg-Asp-Ser-Leu-Gly (kemptide, SEQ ID NO: 1) is a fluorescent material capable of fluorescence resonance energy transfer, fluorescein-isothiocyanate and rhodamine-isothiocyanate Cyanine 5.5 was used as a phosphor capable of exhibiting a quenching effect to prepare a cationic polymer composite and an anionic polymer composite.

Example  1-1. PEI - FITC Synthesis of -kemp

100 mg of polyethyleneimine (PEI) having a molecular weight of 25000 is completely dissolved in 5 mL of sodium bicarbonate (NaHCO ₃ ), and then 1 mg to 20 mg of fluorescein isothiocyanate (FITC) is 0.5 mL of dimethylsulfoxide (DMSO). After dissolving in), it was mixed with the polyethyleneimine solution and reacted for one day. After the reaction, the reactant was precipitated twice in acetone / diethyl ether mixed solution, and the scarlet precipitate was collected by centrifugation and dried to dryness. After dissolving the dried product in distilled water, the product polyethyleneimine-fluorescein derivative was separated from the PD10 column, lyophilized and stored in the refrigerator. The prepared polyethylenimine-fluorescein derivative was identified through fluorescence microscopy and named PEI-FITC. The absorption wavelength intensity of PEI-FITC at 494 nm, which is an intrinsic absorption wavelength of FITC, was quantitatively analyzed through UV-visible spectrum to confirm that five FITCs were chemically modified. 10 mg of synthesized PEI-FITC was completely dissolved in 20 ml of water, followed by solid phase synthesis, followed by Leu-Arg-Arg-Asp-Ser-Leu-Gly (kemptide) in the Fmoc strategy. Peptide synthesized with 10 mg, 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide; EDC) 5 mg, 420 mg 3 mg of N-hydrosuccinimide (NHS) was added thereto and reacted at room temperature for one day. After completion of the reaction the reaction was separated using a PD-10 column, named PEI-FITC-kemp. The amount of modification of Kemptide was confirmed by comparing the molecular weights of PEI-FITC and PEI-FITC-kemp using MALDI-TOF mass spectrometer, and 30 were chemically modified. The reaction is represented by the following Scheme 1.

Example  1-2. PEI - FITC -kemp- Cy5 .5 Preparation

After 10 mg of PEI-FITC-kemp prepared in Example 1-1 was completely dissolved in 2 ml dimethylsulfoxide (DMSO), 2 mg of near-infrared cyanine phosphor Cy5.5 was charged and reacted with nitrogen for one day at room temperature. After completion of the reaction the reaction was separated using PD-10 column and named PEI-FITC-kemp-Cy5.5. The absorption wavelength intensity of PEI-FITC-kemp-Cy5.5 at 675 nm, which is an intrinsic absorption wavelength of Cy 5.5, was quantitatively analyzed through UV-visible spectrum to confirm that five Cy 5.5 were chemically modified. The reaction is represented by the following Scheme 2.

Example  1-3. PAA - RITC Manufacture

After dissolving 50 mg of polyaspartic acid (PAA) having a molecular weight of 10000 in 25 ml DMSO, 5 mg of rodamine isothiocyanate (RITC) was added thereto, and charged with nitrogen to react at room temperature for one day. After completion of the reaction the reaction was separated using PD-10 column and named PAA-RITC. At 570 nm, the inherent absorption wavelength of RITC, the absorption wavelength intensity of PAA-RITC was quantitatively analyzed through UV-visible spectrum to confirm that five RITCs were chemically modified . The reaction is represented by the following Scheme 3.

Example  2. Preparation of Polymer Nanoparticles

PEI-FITC-kemp and PAA-RITC prepared in Example 1 were dissolved in PBS to prepare ion aggregate polymer nanoparticles. It was confirmed by the light scattering apparatus that the nanoparticles of 150 to 400 nanometers are made according to the composition ratio of the two polymers.

In addition, PEI-FITC-kemp-Cy5.5 and PAA prepared in Example 1 were dissolved in PBS to prepare an ion aggregate polymer nanoparticles. It was confirmed by the light scattering device that the nanoparticles of 200 to 500 nanometers are made according to the composition ratio of the polymer.

2 and 3 are particle diameters of the two ion aggregate polymer nanoparticles, and in both cases, it can be seen that they have an average particle of about 300 nanometers.

Example  3. Fluorescence Resonance Energy Transfer of Phosphor in Polymer Nanoparticles Quenching  Effect measurement

Example  3-1. Fluorescence Resonance Energy Transfer Measurement of Phosphor in Polymer Nanoparticles

In order to observe the fluorescence resonance energy transfer of the phosphors in the prepared polymer nanoparticles, the amount of PEI-FITC-kemp prepared in Example 1 was fixed and various amounts of PAA-RITC were added as shown in Table 1 to prepare the polymer nanoparticles. After the preparation, the fluorescence intensity of the FITC was measured using a FITC filter in a fluorometer. Table 1 shows that the light intensity is lowered according to the ratio of PEI-FITC-kemp and PAA-RITC. However, when the ratio of the two substances was increased to 1: 1 and the amount of PAA-RITC was increased, the fluorescence intensity of FITC increased again. This is due to the formation of ion aggregate polymer nanoparticles by the cationic polymer PEI and the anionic polymer PAA, in which FITC and RITC exhibit fluorescence resonance energy transfer. FIG. 4 induces the phenomenon in a well plate and visually confirms it through an image box. Similar to the results of Table 1, the change in fluorescence intensity of FITC was observed as the amount of PAA-RITC was increased.

One 2 3 4 5 6 7 8 9 PEI-FITC-kemp 50 50 50 50 50 50 50 50 50 PAA-RITC 0 5 10 20 30 40 50 75 100 Brightness (X1000) 47.3 41.2 36.3 28.4 18.7 7.6 4.3 9.1 15.3

Example  3-2. Phosphor in Polymer Nanoparticles Quenching  Effect measurement

In order to observe the quenching effect of the phosphors in the prepared polymer nanoparticles, the polymer nanoparticles were prepared by fixing the amount of PEI-FITC-kemp-Cy5.5 and adding various amounts of PAA. Fluorescence luminescence of Cy5.5 was measured. Table 2 shows the change in fluorescence of Cy5.5 with the ratio of PEI-FITC-kemp-Cy5.5 and PAA. As the amount of PAA increases, the fluorescence intensity of Cy5.5 decreases. The fluorescence intensity of Cy5.5 was observed to be strong. This is due to the formation of ionic aggregate polymer nanoparticles by the cationic polymer PEI and the anionic polymer PAA, and the fluorescence intensity of Cy5.5 was decreased by the quenching effect in the particles. 5 induces the phenomenon in the well plate and visually confirms through the image box. Similar to the results in Table 2, the change in fluorescence intensity of Cy5.5 was observed as the amount of PAA increased.

One 2 3 4 5 6 7 8 9 PEI-FITC-kemp-Cy5.5 50 50 50 50 50 50 50 50 50 PAA-RITC 0 5 10 20 30 40 50 75 100 Brightness (X1000) 46.9 39.2 29.0 15.3 20.7 30.9 32.9 43.2 45.1

Example  4. Protein Kinase  Analysis of Optical Properties of Polymer Nanoparticles by Phosphorylation of Proteins

In Example 3, each of the polymer nanoparticles exhibiting fluorescence resonance energy transfer and quenching effect was placed in a well plate, and protein kinase A was added to observe changes in the optical properties of the polymer nanoparticles.

Example  4-1. Protein Kinases of Polymeric Nanoparticles with Fluorescence Resonance Energy Transfer Optical Properties on  Optical characteristic analysis

PEI-FITC-kemp and PAA-RITC nanoparticles prepared in Example 2 and PKA were added under the conditions shown in Table 3 below and the optical properties were observed.

One 2 3 4 PEI-FITC-kemp 50 50 50 50 PAA-RITC 50 50 50 0 PKA 0 15 nM 30 nM 0

Figure 6 shows the change in the FITC fluorescence intensity of the polymer nanoparticles prepared in Example 3-1 according to the phosphorylation of kemptide by PKA, the lower number is the value of the fluorescence intensity measured using a fluorometer (fluorometer) Indicates. It was observed that the fluorescence intensity of the polymer nanoparticles in which PKA was added to the solution was greatly increased. This is because PKA phosphorylates kemptide, which adds anionicity to the cationic polymer composite PEI-FITC-kemp, which breaks down the ionic balance with PAA-RITC, thus dissociating each other without forming ionic aggregate polymer nanoparticles. The fluorescence resonance energy transfer between and RITC is gone. These results demonstrate that the polymer nanoparticles including PEI-FITC-kemp and PAA-RITC of the present invention can display specific images according to phosphorylation of proteins by protein kinases.

Example  4-2. Quenching  Optical Characterization by Protein Kinase of Polymer Nanoparticles with Effect Optical Properties

PEI-FITC-kemp-Cy 5.5 and PAA nanoparticles prepared in Example 2 and PKA were added under the conditions shown in Table 4 below, and the optical properties were observed.

One 2 3 4 PEI-FITC-kemp-Cy 5.5 50 50 50 50 PAA-RITC 50 50 50 0 PKA 0 15 nM 30 nM 0

Figure 7 shows the change in the Cy5.5 fluorescence intensity of the nanoparticles prepared in Example 3-2 according to the phosphorylation of kemptide by PKA, the lower number represents the value of the fluorescence intensity through a fluorometer (fluorometer) . It was observed that the fluorescence intensity of the nanoparticles to which PKA was added to the solution was greatly increased. This is because PKA phosphorylates kemptide, which adds anionicity to the cationic polymer complex PEI-FITC-kemp-Cy 5.5, which breaks down the ionic balance with PAA, thus dissociating each other without forming ionic aggregate polymer nanoparticles. The matting effect of Cy5.5 is gone. These results demonstrate that the polymer nanoparticles including PEI-FITC-kemp-Cy 5.5 and PAA of the present invention can display specific images according to phosphorylation of proteins by protein kinases.

As described above, the polymer nanoparticles including the cationic polymer complex and the anion polymer complex of the present invention have a characteristic of selectively fluorescence in phosphorylation reaction by protein kinase. These polymer nanoparticles are excellent in cell permeability and easy to change fluorescence even in cells, and thus can be injected into experimental animal models or human body particles, and by real-time imaging of protein phosphorylation by protein kinases in various tissues, cancer and dementia, etc. Can be used for early diagnosis of various intractable diseases. It can also be usefully used for the screening of new drugs such as protein kinase inhibitors.

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Claims (19)

Cationic polymers selected from the group consisting of polyethyleneimine, poly-L-lysine and chitosan; Phosphors capable of fluorescence resonance energy transfer selected from the group consisting of fluorescein, rhodeamine and tetramethylrodamine; And a cationic polymer complex to which a peptide derivative having a nucleotide sequence selectively phosphorylated by protein kinase is bound; Anionic polymers selected from the group consisting of polyaspartic acid, polyglutamic acid and heparin; And an anionic polymer composite in which a phosphor capable of fluorescence resonance energy transfer is selected from the group consisting of fluorescein, rodamine and tetramethylrodamine. delete delete The polymer nanoparticle of claim 1, wherein the nucleotide sequence selectively phosphorylated by the protein kinase is Leu-Arg-Arg-Asp-Ser-Leu-Gly. delete The polymer nanoparticle of claim 1, further comprising a cationic polymer composite having a structure of Formula 1 and anionic polymer composite having a structure of Formula 2. [Formula 1]

Wherein A is ethyleneimine, B is a phosphor selected from the group consisting of fluorescein, rhodeamine and tetramethylrodamine capable of fluorescence resonance energy transfer, and C is a nucleotide sequence which selectively phosphorylates by protein kinase A peptide derivative having a, a is an integer between 25 and 40, b is an integer between 5 and 10, c is an integer between 20 and 35, [Formula 2]

Wherein A is aspartic acid, B is a phosphor selected from the group consisting of fluorescein, rodamine and tetramethylrodamine capable of fluorescence resonance energy transfer, a is an integer from 50 to 150 and b is from 5 to 10 Is an integer between. The method according to claim 6, wherein A in Formula 1 is ethyleneimine, B in Formula 1 is fluorescein, C in Formula 1 is Leu-Arg-Arg-Asp-Ser-Leu-Gly, A is aspartic acid, B of the formula (2) is a rod amine polymer nanoparticles. The protein kinase of claim 1, wherein the protein kinase is cAMP dependent protein kinase (PKA), AKT (PKA), rho-associated coiled-coil forming kinase (ROCK), and 3-phosphinositide-dependent protein kinase-1 (PDK1). Polymer nanoparticles selected from the group consisting of). Cationic polymers selected from the group consisting of polyethyleneimine, poly-L-lysine and chitosan; Peptide derivatives having a nucleotide sequence where phosphorylation occurs selectively by protein kinases; And cyanine bonded cationic polymer complex; A polymer nanoparticle comprising an anionic polymer selected from the group consisting of polyaspartic acid, polyglutamic acid and heparin. delete The polymer nanoparticle of claim 9, wherein the nucleotide sequence selectively phosphorylated by the protein kinase is Leu-Arg-Arg-Asp-Ser-Leu-Gly. delete delete The polymer nanoparticle of claim 9, further comprising a cationic polymer composite having a structure of Formula 3 and an anionic polymer having a structure of Formula 4. [Formula 3]

Wherein A is ethyleneimine, B is a phosphor selected from the group consisting of fluorescein, rhodeamine and tetramethylrodamine capable of fluorescence resonance energy transfer, and C is a nucleotide sequence which selectively phosphorylates by protein kinase A peptide derivative having D, c is a cyanine phosphor, a is an integer between 25 and 40, b is an integer between 1 and 5, c is an integer between 20 and 35, and d is an integer between 5 and 10 ego; [Formula 4]

Wherein A is aspartic acid. a is an integer between 50 and 150. The method of claim 14, wherein A in Formula 3 is ethyleneimine, B in Formula 3 is fluorescein, C in Formula 3 is Leu-Arg-Arg-Asp-Ser-Leu-Gly, Is cyanine, wherein A in Formula 4 is asparagine. The protein kinase of claim 9, wherein the protein kinase is cAMP dependent protein kinase (PKA), AKT (PKA), rho-associated coiled-coil forming kinase (ROCK), and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Polymer nanoparticles selected from the group consisting of). The polymer nanoparticle according to any one of claims 1, 4, 6 to 9, 11, and 14 to 16 for imaging phosphorylation of proteins by protein kinases. 17. A method of imaging protein phosphorylation by protein kinases using the polymer nanoparticles of any one of claims 1, 4, 6-9, 11, 14-14. 17. A candidate drug having a protein kinase inhibitory activity is selected by using the polymer nanoparticles according to any one of claims 1, 4, 6, 9, 11, 14, and 16. Way.

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