KR20100035062A - A disease targeted peptide-conjugated amphiphilic chitosan based nanoparticle imaging agent for diagnosis of disease - Google Patents

Abstract

PURPOSE: A nanoparticle contrast media in which fluorescence and disease target peptide are conjugated to an amphiphilic chitosan derivative is provided to improve targeting efficiency and increase efficiency of preventing and treating diseases. CONSTITUTION: An amphiphilic chitosan nanoparticle contrast media has fluorescence and disease target peptide which are conjugated to bile acid derivative or fatty acid derivative. The disease target peptide is IL-4 receptor target peptide or derivative thereof; a target peptide of apoptotic cell comprising an amino acid of sequence number 2 or derivative thereof; a target peptide of bladder cancer comprising an amino acid of sequence number 3 or derivative thereof; a peptide having a peptide RGD sequence targeting aptamer, folic acid, and integrin; or transferring or derivative thereof. The peptide having a peptide RGD sequence targeting integrin is cyclo (Arg-Gly-Asp-D-Phe-Val), cyclo(Arg-Gly-Asp-D-Phe-Lys), cyclo(Arg-Gly-Asp-D-Phe-Cys), cyclo(Arg-Gly-Asp-D-Phe-Glu), cyclo(Arg-Gly-Asp-D-Tyr-Lys), cyclo(Arg-Gly-Asp-D-Tyr-Glu), Arg-Gly-Asp-Ser or Gly-Arg-Gly-Asp-Ser. A contrast medium contains radioactive isotope, quantum dot or MRI contrast media.

Description

Amphiphilic chitosan nanoparticle contrast agent bound to target peptide for diagnosis of disease {A DISEASE TARGETED PEPTIDE-CONJUGATED AMPHIPHILIC CHITOSAN BASED NANOPARTICLE IMAGING AGENT FOR DIAGNOSIS OF DISEASE}

The present invention binds a disease target peptide having high specificity, selectivity, and affinity to specific disease tissues and cells to bile acid-chitosan nanoparticles having high selectivity for neovascularization induced by inflammatory diseases, and at the same time to bind near-infrared phosphors to inflammatory diseases. The present invention relates to a disease-targeting nanoparticle contrast agent that can be usefully used for the early diagnosis, prevention and treatment of cancer.

To date, various nanoparticles such as gold nanoparticles, quantum dot nanoparticles, and iron oxide nanoparticles, which are polymer nanoparticles, micelles, liposomes, and inorganic nanoparticles, have been developed. Using various nanoparticles currently being developed, nanoparticles containing a drug carrier for treating a disease and a contrast agent for diagnosing a disease are used for diagnosis and prevention of a disease.

Typically, nanoparticles are selectively accumulated in diseased tissue by enhanced permeability and retention (EPR) effects because of the high permeability of neovascularization formed at the disease site. Using these properties, nanoparticles containing radioisotopes, phosphors, anticancer agents and various disease therapeutic agents have been developed as cancer diagnosis and therapeutic agents. However, these nanoparticles have a low ability to selectively deliver contrast and therapeutic agents to the disease site because they passively deliver contrast and therapeutic agents.

Meanwhile, biomarkers having targets for diseased tissues, for example, various antibodies, aptamers, folate derivatives, disease-targeted peptides, and the like, have been searched for and discovered. Using nanoparticles having such disease-targeted biomarkers bound thereto, When combined with a variety of disease imaging equipment, namely near infrared fluorescence imaging equipment, PET, CT, MRI, etc., it is possible to develop improved disease diagnosis, prevention, and treatment than conventional contrast agents and disease treatments. Accordingly, the present inventors have high specificity, selectivity, and affinity for fatty acids or bile acid derivative-chitosan nanoparticles selectively accumulating in diseased tissues by high permeability of neovascularization in diseased tissues. By combining peptides and chemically binding Cy5.5, which is capable of near-infrared vision at the same time, a novel disease-targeting nanoparticle contrast agent according to the present invention has been developed for the early diagnosis, prevention, and treatment of diseases.

It is an object of the present invention to provide a nanoparticle contrast agent in which a phosphor and a disease target peptide are bound to an amphiphilic chitosan derivative to which a bile acid derivative or fatty acid derivative is bound.

It is also an object of the present invention to provide a drug carrier in which a hydrophobic drug is encapsulated in a nanoparticle contrast agent in which a phosphor and a disease target peptide are bound to an amphiphilic chitosan derivative to which a bile acid derivative or a fatty acid derivative is bound.

In order to achieve the above object, the present invention is a nanoparticle contrast agent in which a fluorescent substance and a target peptide is coupled to an amphiphilic chitosan derivative in which a bile acid derivative or a fatty acid derivative is bound, wherein the disease target peptide is an atherosclerotic plaque or an IL-4 receptor target. Peptides, target peptides of apoptosis cells, bladder cancer cell target peptides, aptamers, folic acid, peptide derivatives comprising the sequence of Arg-Gly-Asp, an integrin target peptide expressed in vascular endothelial cells of blood vessels in inflammatory disease tissues, transferrin, It provides a nanoparticle contrast agent selected from the group consisting of glucose and derivatives thereof.

In one embodiment of the present invention, the disease-targeted peptide is an atherosclerotic plaque or IL-4 receptor target peptide consisting of SEQ ID NO: 1, a dead cell target peptide consisting of SEQ ID NO: 2 and bladder cancer cell target peptide consisting of SEQ ID NO: 3 It may be a peptide having an amino acid sequence selected from the group consisting of.

In one embodiment of the present invention, the nanoparticle may further comprise a radioisotope, a quantum dot or an MRI contrast agent.

In another aspect, the present invention is a squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, Drug delivery agents for cancer treatment selected from the group consisting of colorectal cancer, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, and gallbladder cancer, or osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis, pancreatic cell antibodies Provided are drug delivery agents for treating inflammatory diseases selected from the group consisting of insulin-dependent childhood diabetes, chronic thyroiditis, ulcerative colitis, multiple sclerosis, autoimmune encephalomyelitis or fibromyalgia syndrome and atherosclerosis.

In one embodiment of the invention, the hydrophobic drug is daunomycin (daunomycin), docetaxel (Docetaxel), cisplatin (cis-platin), camptothecin (camptothecin), paclitaxel (paclitaxel), tamoxifen (tamamoxifen), Anaste Anasterozole, Gleevec, 5-Fluorouracil (5-FU), Fluxuridine, Leuprolide, Flotamide, Zoledronate Doxorubicin, Vincristine, Gemcitabine, Streptozocin, Carboplatin, Topotecan, Belotecan, Irinotecan, Vinote Levin (Vinorelbine), hydroxyurea, Valrubicin, retinoic acid series, Methotrexate, Mechloretamine, Chlorambucil, Bu Busulfan, Doxifluridine, Vinblastin, Itomycin, Prednisone, Testosterone, Mitoxantron, Aspirin and Salicylates, Ibuprofen, Naproxen, Naproxen, Phenopro Fenoprofen, indomethacin, phenyltazone, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib , Valdecoxib, nimesulide, cortisone, corticosteroid, and the like.

As described above, the nanoparticle contrast agent to which the disease target peptide is bound according to the present invention is significantly improved in targeting the disease than conventional nanoparticles, and screens and diseases of cells and tissues of specific diseases in vivo as well as cells. Can significantly increase the effectiveness of diagnosis, prevention and treatment. In addition, it is possible to combine or encapsulate various isotopes, inorganic nanoparticles, or disease therapeutic agents to the nanoparticles to which the target peptide is bound, and thus, it is possible to apply them to the development of multifunctional nanoparticles that can simultaneously diagnose and treat diseases. .

Hereinafter, the present invention will be described in detail.

The present invention provides a nanoparticle contrast agent in which a phosphor and a disease target peptide are bound to an amphipathic chitosan derivative to which a bile acid derivative or a fatty acid derivative is bound.

The amphiphilic chitosan polymer nanoparticles can form nano-sized self-assemblies through a balance of hydrophobicity and hydrophilicity, and can be selectively accumulated in diseased tissue. In addition, the chemical modification of the near-infrared phosphor which is capable of near-infrared light in the polymer nanoparticles should be easy, and the near-infrared light should show strong near-infrared fluorescence selective to diseased tissue.

In addition to the chitosan polymers, all polymers having biocompatibility may be used, particularly dextran and chitosan, which are biopolymers having high tissue accumulation efficiency through neovascularization generated in diseased tissues and used as drug carriers for anticancer drugs. ), Glycol chitosan, poly-L-lysine, poly-aspartic acid, and the like, and synthetic polymers include poly (N-2- (hydroxypropyl). ) (Methacrylamide) (poly (N-2- (hydroxypropyl) methacrylamide), poly (divinyl ehter-co-maleic anhydride), poly (styrene-co) -Maleic anhydride (poly), poly (ethylene glycol), etc. The polymer nanoparticles are biocompatible / biodegradable in vivo. It should be excellent in stability and excellent in vivo stability. The high degree of biodistribution in the blood is required to accumulate continuously in cancer tissues for a sufficient time, in particular, the glycol chitosan contains a lot of positrons in the polymer chains, resulting in a high efficiency of cancer tissue accumulation when producing nanoparticles. Very high, the amine groups of the polymer can induce chemical modification of hydrophobic materials and near-infrared phosphors.

In the present invention, a "disease specific target peptide" refers to phage clones into which peptides are inserted by repeatedly searching for phages that specifically bind to specific disease cells and tissues using a phage display technique. By peptide, it refers to a peptide to be discovered by measuring specificity and selectivity.

As disease-targeted peptides that are bound to the polymer nanoparticles, arteriosclerosis plaques or IL-4 receptor target peptides (SEQ ID NO: 1: CRKRLDRNC) and derivatives thereof, which are detected and discovered using phage display method, are killed (apoptotic). ) A target peptide (SEQ ID NO: 2: CQRPPR) and a derivative thereof, a bladder cancer cell target peptide (SEQ ID NO: 3: CSNRDARRC), and a peptide derivative including a peptide sequence thereof may be bound to the cell.

Here, coronary artery disease such as atherosclerosis plaque and atherosclerosis occurs when fat components such as cholesterol accumulate in the body, and when these fat components are deposited in blood vessels, blood vessel walls become thick and blood clots (blood clots) are caught. It narrows and interferes with smooth blood circulation, and the accumulation of these fats is called atherosclerotic lesions, also called plaques.

In addition, it is a substance that can be used for cancer targets known through various literatures (Jae Hyung Park et al, Progress in polymer science, 33, 113-137, 2008; Peptides International. Inc.) and the surface of vascular endothelial cells in inflammatory diseases. As RGD peptides and derivatives thereof capable of targeting integrins expressed in cyclic RGDfV (cyclo (Arg-Gly-Asp-D-Phe-Val)), cyclic RGDfK (cyclo (Arg-Gly-Asp) -D-Phe-Lys)), cyclic RGDfC (cyclo (Arg-Gly-Asp-D-Phe-Cys)), cyclic RGDfE (cyclo (Arg-Gly-Asp-D-Phe-Glu)), cyclic RGDyK ( cyclo (Arg-Gly-Asp-D-Tyr-Lys)), cyclic RGDyE (cyclo (Arg-Gly-Asp-D-Tyr-Glu)), RGDS (Arg-Gly-Asp-Ser), GRGDS (Gly- Arg-Gly-Asp-Ser). Details of the RGD peptides are shown in Table 1 below.

TABLE 1

(Source: Peptides International, Inc.)

In addition, aptamers having a highly targeted RNA or DNA structure may be coupled to prostate cancer, and folate receptors overexpressed in various cancer cells and cancer tissues, such as the rectum, lung, prostate, uterus, and brain. receptors and folic acid with high affinity and selectivity can be combined.

In addition, transferrin is a protein synthesized in the liver and secreted into the plasma, and binds to iron in the body to form iron-transferin chelate and is an important factor for supplying iron to cells. Transferrin receptors are overexpressed on the surface of malignant tumor cells and are recognized by iron-transferin chelate and are taken up intracellularly by endocytosis and applied to cancer therapy. Transferrin proteins are also cancer-targeting and can bind to nanoparticles. In addition, various biomarkers used for specific diseases can be combined.

Phosphors such as cyanine, allophycocyanine, fluorescein, tetramethylrodamine, bodiphy, alexa, and the like are chemically introduced into the amphiphilic polymer for in vivo optical viewing. The phosphor binds to a hydrophobic material in the amphiphilic polymer. Among the phosphors, cyanine emits and absorbs near-infrared light, and thus, interference with cells, blood, biological tissues, and the like is most preferable, and a good example is Cy5.5. Polymeric nanoparticles modified with near-infrared phosphors can be used to image autoimmune disease tissues in vivo using near-infrared irradiation, and various complex molecular images can be obtained when radioisotopes, quantum dots, and MRI contrast agents are simultaneously introduced. .

The polymer nanoparticles in which the near-infrared phosphors prepared in the present invention are bonded have a stable nanoparticle structure in water and preferably have high disease tissue accumulation efficiency. Moreover, it is preferable that near-infrared perspective shows high near-infrared fluorescence intensity which has disease tissue targeting.

In particular, as an example of the present invention, the polymer nanoparticles composed of hydrophilic glycol chitosan chains and hydrophobic bile acid derivatives can be used because various disease therapeutic agents can be encapsulated in the nanoparticles. Glycol chitosan contains a lot of positrons in the polymer chain, so when the nanoparticles are prepared, the accumulation efficiency is remarkably excellent in inflammatory disease tissues, and the amine group of the glycol chitosan is most effective for inducing chemical modification of hydrophobic substances and near-infrared phosphors. Suitable.

Specifically, in the present invention, amphiphilic chitosan derivatives are prepared by first introducing a hydrophobic substance into the hydrophilic glycol chitosan, which is a chitosan polymer. The hydrophobic material bound to the hydrophilic polymer is bile acid and deoxycholic acid (deoxycholic acid), lithocholic acid (lithocholic acid), taurodeoxycholic acid (taurodeoxycholic acid), taurocholic acid (taurocholic acid), glycokenodidi Bile acid derivatives, such as glycochenodeoxyhoclic acid; Fatty acid derivatives such as stearic acid, cholesterol (cholesterol), palmitic acid and olelic acid may be used, and bile acids are preferred.

Amphiphilic chitosan refers to a chitosan derivative having both hydrophilicity and hydrophobic properties by introducing a hydrophobic substance into hydrophilic chitosan as described above. The amphiphilic chitosan easily forms nanoparticles in an aqueous solution, and high accumulation efficiency appears around diseased tissues according to the characteristics of the polymer nanoparticles. In some cases, for early diagnosis, Cy5.5, a near-infrared phosphor, is chemically bound to the hydrophobic chitosan complex, thereby enabling the location and quantitative analysis of nanoparticles in vivo during near-infrared vision. In addition, amphipathic chitosan nanoparticles can be used as drug carriers, and since hydrophobic anticancer drugs can be easily encapsulated physically and chemically, they can be used as a new drug delivery system for cancer treatment as well as for early diagnosis of cancer. In addition, by combining a peptide with high specificity, selectivity and affinity to the disease, thereby improving the target efficiency to the disease, the disease diagnosis, prevention, and treatment efficacy can be excellently enhanced.

A preferred example for preparing a bile acid-chitosan-Cy5.5 complex in which the target peptide forming the polymer nanoparticles as described above is bound may be represented by the following Chemical Formula 1.

Wherein A and B are composed of N-aceylglucosamine and N-glucosamine derivatives, which are repeat structures of glycol chitosan polymers, and C is hydrophobic bile acids and fatty acids of N-glucosamine derivative amines for introducing hydrophobic groups into hydrophilic glycol chitosan. A hydrophobic substance is chemically bound to a derivative, D is a N-glucosamine derivative amine is a disease-specific, selective or friendly target peptide is bound, E is N-glucosamine derivative amine N-glucosamine for in vivo imaging Near-infrared phosphors, radioisotopes, quantum dots and the like are chemically bound to amines of derivatives. In addition, a and b have a composition of 10-50% of each monomer according to the molecular weight of glycol chitosan, and c has a hydrophobic ratio of 10-40% to prepare nanoparticles of glycol chitosan. Has a composition. Composition d of D comprising the target peptide has a composition of 5-20%, and composition e of E for imaging has a composition of 0.1-2%. However, Formula 1 is represented by a rough structural formula to explain the structure of the nanoparticle contrast agent according to the present invention, as shown in Formula 1, each element of A, B, C, D, E is connected in series with each other As shown in FIG. 1, as a three-dimensional structure, bile acids, phosphors, and target peptides are coupled to glycol chitosan.

Specifically, as shown in Figure 1, the glycol chitosan polymer derivative is composed of N-aceylglucosamine and N-glucosamine derivatives of the repeating structure of the polymer, in order to introduce a hydrophobic group to the hydrophilic glycol chitosan, Hydrophobic substances such as bile acids and fatty acids are chemically bound to colchitosan, and disease-specific, selective or friendly target peptides are coupled to the glycol chitosan, and near-infrared phosphors for in vivo imaging of nanoparticles. , Radioisotopes, quantum dots, etc. are chemically bound derivatives.

Depending on the molecular weight of glycol chitosan, the number of moles of the composition of each monomer of N-aceylglucosamine and N-glucosamine has a value of 100 to 10,000, and the number of moles of hydrophobic material is used to prepare nanoparticles of glycol chitosan. Has a value of 20-100, C is 10-30.

The bile acid-chitosan-Cy5.5 complex is made of nanoparticles of self-assembled self-aggregated form and has a size of 100 to 800 nm. The prepared polymer nanoparticles have excellent biocompatibility and excellent accumulation efficiency in cancer tissues, and can be imaged in vivo by near-infrared, PET / SPECT, and CCD cameras. Phosphors that can bind in place of Cy5.5 in the nanoparticles include Cy3, TAMRA, Texas Red, ROX, Cy5, Cy5.5, Rhodamine, Alexa Fluor  568, Alexa Fluor  594, Alexa Fluor  610, Alexa Fluor  647, Alexa Fluor  660, Alexa Fluor  680, Alexa Fluor  700, BODIPY  493/503, BODIPY  530/550, BODIPY  558/568, BODIPY  581/591, Cy7, Cy7.5 and the like.

In addition, the bile acid-chitosan-Cy5.5 complex is a nanoparticle having an amphipathic surface composed of a hydrophobic portion inside a hydrophilic polymer and a hydrophobic portion in a nanoparticle, and anti-rheumatic agents, anti-inflammatory agents, etc. Possible, in particular, daunomycin, docetaxel, cis-platin, camptothecin, paclitaxel, tamoxifen, tamoxifen, anasterozole, gleevec Gleevec, 5-Fluorouracil (5-FU), Floxuridine, Leuprolide, Flotamide, Zoledronate, Doxorubicin, Vincristine (Vincristine), gemcitabine, streptozocin, carboplatin, topotecan, belotecan, irinotecan, vinorelbine, and hirohileurea (hydroxyurea), Valrubicin, Retinoic acid series, Mesotrexate, Mechlorethamine, Chlorambucil, Busulfan, Doxifluridine, Vinblastin , Mitomycin, prednisone, testosterone, testosterone, mitoxantron, aspirin and salicylates, ibuprofen, naproxen, napexen Fenoprofen, indomethacin, phenyltazone, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib Drugs such as celecoxib, valdecoxib, nimesulide, cortisone and corticosteroids can be encapsulated, osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis, and pancreas. three Childhood insulin dependent diabetes, thyroid chronic salt by antibodies, ulcerative colitis, multiple sclerosis (multiple sclerosis), the self-use is possible for a variety of disease treatment such as immune encephalomyelitis or fibromyalgia (Fibromyalgia syndrome) and arteriosclerosis or various cancers.

In addition, the polymer nanoparticles bonded to the near-infrared fluorescent substance prepared in the present invention have a nanoparticle structure that is stable in the water system, and thus exhibits hydrophobicity and inorganic nanoparticles having a size of 1 to 20 nm or less, for example, gold nanoparticles. , Iron oxide nanoparticles, quantum dots can be encapsulated, and can be used as optical imaging contrast agents, CT contrast agents, and MRI contrast agents.

In the nanoparticle contrast agent, the bile acid-chitosan-Cy5.5 complex prepared by using a hydrophilic polymer as chitosan, a hydrophobic material as bile acid, and a near-infrared phosphor as Cy5.5 is prepared as nanoparticles in self-assembled or self-aggregated form. Its size ranges from tens to hundreds of nanometers. The composite of the polymer nanoparticles is excellent in biocompatibility and excellent in the efficiency of accumulation in autoimmune diseases and cancer tissues, and can be imaged in vivo by near-infrared, PET / SPECT and CCD cameras.

In addition, the bile acid-chitosan-Cy5.5 complex has a high selectivity to the inflammatory tissues due to the EPR effect of the inflammatory tissues compared to the general low molecular weight contrast agent, and the accumulation efficiency of the inflammatory disease tissues is remarkably excellent, and the retention period in vivo is low. Significant increases over molecular weight contrast agents allow long-term imaging of various inflammatory and cancerous tissues.

The nanoparticles conjugated to the disease target peptide developed in the present invention can be applied to diagnosis and prophylactic treatment of various inflammatory diseases, and examples of various inflammatory diseases include osteoarthritis, rheumatoid arthritis and other autoimmune diseases, and squamous epithelium. Cell cancer, cervical cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer It is possible to apply to the diagnosis, prevention and treatment of cancer, or intractable diseases including dementia, stroke and the like. The polymer nanoparticles may form nano-sized self-assemblies through the balance of hydrophobicity and hydrophilicity, and may be selectively accumulated in inflammatory disease tissues. In addition, the polymer nanoparticles of the present invention are easy for chemical modification of near-infrared phosphors capable of near-infrared light, and may exhibit selective strong near-infrared fluorescence of inflammatory disease tissues by near-infrared light.

The polymer nanoparticles are excellent in biocompatibility / biodegradability in vivo, and have excellent stability in vivo, and thus have high biodistribution in blood and accumulate in inflammatory disease tissues for a sufficient time.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. The following examples and experimental examples are only examples of the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Nanoparticle Contrast Agents According to the Present Invention

1-1. Preparation of Bile Acid-Glycol Chitosan-Cy5.5 Complex

500 mg of glycol chitosan was dissolved in 60 ml of water and 90 ml of methanol was added. Then, 260 mg of bile acid (5-β-cholanic acid) was dissolved in 100 ml of methanol and slowly added dropwise to the glycol chitosan solution. 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC) and 420 mg of N-hydrosuccinimide (NHS) ) Was dissolved in 50 ml of methanol, added to the reaction solution, and stirred at room temperature for 24 hours. Thereafter, the reaction solution was dialyzed for 2 days to remove unreacted bile acid, and then freeze-dried to prepare a bile acid-glycol chitosan complex.

100 mg of the bile acid-glycol chitosan complex prepared above was dissolved in 20 ml of water and 20 ml of DMSO was added, followed by addition of 2 mg of near-infrared phosphor Cy-5.5-NHS (monoreactive hydroxysuccimimide ester of Cy5.5) for 6 hours. After reacting for 2 days, the solution was dialyzed for 2 days to remove unreacted Cy5.5 and freeze-dried to prepare a bile acid-glycol chitosan-Cy5.5 complex, the preparation process of which is shown in Scheme 1 below. same.

1-2. Synthesis of Bile Acid-Glycol Chitosan-Cy5.5 Derivatives Bound to Disease Target Peptides

80 mg of a complex consisting of bile acid, chitosan and Cy5.5 was dispersed in 32 ml of distilled water having a pH of 8.0, and then 7 mg of N-succinimidyl 4- (maleimidomethyl) cyclohexane carboxylate (SMCC) was dissolved in 1 ml of dimethyl sulfoxide. Add gin solution. Thereafter, the mixed solution is adjusted to pH 6.7, and then reacted for 2 hours by adding a solution of target peptide in 1 ml of distilled water, followed by dialysis and lyophilization. The number of moles of the target peptide bound to 1 mole of the bile acid-chitosan derivative is shown in Table 2 below.

TABLE 2

Sample Particle Size (nm) Number of moles of target peptide bound to 1 mole of bile acid-chitosan derivative CRKRLDRNC-HGC-Cy5.5 298 5.5 CQRPPR-HGC-Cy5.5 305 4.5 CSNRDARRC-HGC-Cy5.5 310 4.2

Experimental Example 1. Analysis of cell affinity of vascular endothelial cells of CRKRLDRNC-HGC-Cy5.5 nanoparticles to which atherosclerotic plaque target peptide was bound

Atherosclerotic plaque target peptides have excellent binding ability to IL-4 receptors, and atherosclerotic plaques secrete cytokine TNF-alpha, activating vascular endothelial cells by TNF-alpha and overexpressing IL-4R at the cell surface do.

To determine the binding capacity of CRKRLDRNC-HGC-Cy5.5 in cells expressing IL-4R overexpression, 10 ⁴ Bovine aortic endothelial cells (BAEC) were placed in the laminar flow chamber and the cells were about 80% filled in the chamber. Incubated until. Thereafter, TNF-alpha was treated with a concentration of 10 ng / ml for 1 hour to activate BAEC, and then 100 μg / ml of CRKRLDRNC-HGC-Cy5.5 or HGC-Cy5.5 nanoparticles were contained. The culture solution was flowed into the flow chamber for about 2 hours using a pump. After 2 hours, the cells were washed with the culture solution, and the cells were fixed with the fixed solution, and then analyzed for binding of the nanoparticles through a fluorescence microscope.

As shown in FIG. 2, HGC-Cy5.5 nanoparticles were not treated with TNF-alpha or did not bind to treated cells. On the other hand, CRKRLDRNC-HGC-Cy5.5 particles did not bind to inactivated BAEC cells that were not treated with TNF-alpha, but were well bound to activated BAEC treated with TNF-alpha. This means that CRKRLDRNC-HGC-Cy5.5 nanoparticles selectively bind to activated BAEC overexpressing IL-4R by cytokine TNF-alpha, and thus may be applicable to disease diagnostic screening in cells. .

Experimental Example 2. Atherosclerotic plaque imaging in atherosclerotic rats

Atherosclerotic plaque images of CRKRLDRNC-HGC-Cy5.5 nanoparticles were observed in atherosclerotic rat (LDLr-/-) and normal atherosclerotic arteries. Atherosclerotic rats and normal rats were treated with tail vein with 5 mg / kg of CRKRLDRNC-HGC-Cy5.5 nanoparticles. After 6 hours, the atherosclerotic arteries of atherosclerotic rats and normal rats were revealed and eXplore Optix was used. Imaging.

As a result, as shown in Fig. 3a, in the atherosclerotic artery of the atherosclerotic rat (3a-1), the fluorescence was strongly observed in the atherosclerotic artery of the normal rat (3a-2). 3b was confirmed by Oil red-O staining that plaques were formed in atherosclerotic atherosclerosis as compared to normal rats. FIG. 3c shows CRKRLDRNC-HGC on plaques formed in atherosclerotic arteries of atherosclerotic rats. -Cy5.5 nanoparticles were found to be significantly higher binding force.

In view of the above results, CRKRLDRNC-HGC-Cy5.5 nanoparticles will be available as atherosclerosis diagnostic contrast agents.

Experimental Example 3 Targeting Evaluation in Cancers Overexpressing IL-4R

To demonstrate that atherosclerosis target nanoparticle contrast agent, CRKRLDRNC-HGC-Cy7.5, has high target efficiency in other inflammatory diseases, an animal model was established using H226, a cancer cell overexpressing IL-4R. H226 cells (3 × 10 ⁶ cells) were implanted subcutaneously in the flank of the mouse, and CRKRLDRNC-HGC-Cy7.5 was injected at 5 mg / kg through the tail vein when the cancer was about 5-7 mm in size. It was. HGC-Cy7.5 was administered as a control.

As shown in FIG. 4, cancer targeting was increased in the group administered with CRKRLDRNC-HGC-Cy7.5 than the nanoparticles to which the peptide was not bound. This, when combined with the target peptide can significantly increase the disease target efficiency than conventional nanoparticles, thereby improving the efficacy of the diagnosis, prevention, and treatment of various inflammatory diseases.

Figure 1 shows a glycol chitosan-bile acid polymer composite nanoparticles coupled to a disease target peptide prepared in the present invention, and also bonded to a phosphor and a method for producing the same.

FIG. 2 shows TNF in which glycol chitosan-bile acid-Cy5.5 nanoparticles (2b) bound to atherosclerotic plaque target peptides and glycol chitosan-bile acid nanoparticles (2a) in which peptides are not bound are cytokines. Cell images showing the binding force to coronary vascular endothelial cells according to the presence or absence of -alpha treatment.

FIG. 3 shows glycol chitosan-bile acid-Cy5.5 nanoparticles combined with atherosclerotic plaque target peptides in arteriosclerosis-induced rats (LDLr ^{− / −} ) (3a-1) and normal mice (3a-2). Target and near-infrared images of plaque in atherosclerosis are shown. 3b is an oil red-O staining of atherosclerosis-induced and normal atherosclerosis, and 3c is atherosclerosis. Tissue sections of the atherosclerotic artery extracted from induced and normal rats were made and confirmed by the presence of nanoparticles using a fluorescence microscope.

4 shows glycol chitosan-bile acid-Cy7.5 nanoparticles to which atherosclerotic plaque target peptides are bound in cancer tissues expressing a large amount of interleukin 4-receptor (IL-4R) and nanoparticles to which peptides are not bound. NIR images comparing cancer targets are shown.

<110> KIST <120> A DISEASE TARGETED PEPTIDE-CONJUGATED AMPHIPHILIC CHITOSAN BASED <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> atheroscleroti plaque-homing peptide <400> 1 Cys Arg Lys Arg Leu Asp Arg Asn Cys   1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> apoptotic cell target peptide <400> 2 Cys Gln Arg Pro Pro Arg   1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> bladder cancer target peptide <400> 3 Cys Ser Asn Arg Asp Ala Arg Arg Cys   1 5  

Claims (5)

As nanoparticle contrast agent in which phosphor and disease target peptide are bound to amphiphilic chitosan derivatives bound to bile acid derivatives or fatty acid derivatives, The disease-target peptide is an atherosclerotic plaque or an IL-4 receptor target peptide or derivative thereof consisting of the amino acid sequence of SEQ ID NO: 1, a target peptide or a derivative thereof of the dead cell consisting of the amino acid sequence of SEQ ID NO: 2, an amino acid sequence of SEQ ID NO: 3 Bladder cancer cell target peptide or derivatives thereof, peptides targeting aptamers, folic acid, integrin, peptides having a RGD sequence cyclo (Arg-Gly-Asp-D-Phe-Val), cyclo (Arg-Gly-Asp-D -Phe-Lys), cyclo (Arg-Gly-Asp-D-Phe-Cys), cyclo (Arg-Gly-Asp-D-Phe-Glu), cyclo (Arg-Gly-Asp-D-Tyr-Lys) , nanoparticles selected from the group consisting of cyclo (Arg-Gly-Asp-D-Tyr-Glu), Arg-Gly-Asp-Ser or Gly-Arg-Gly-Asp-Ser, and transferrin or derivatives thereof Contrast agent. The nanoparticle contrast agent of claim 1, further comprising a radioisotope, quantum dot, or MRI contrast medium in the nanoparticle. Osteoarthritis, rheumatoid arthritis, progressive systemic sclerosis, insulin dependent childhood diabetes mellitus, chronic thyroiditis, ulcerative colitis, multiple Drug delivery drug for the treatment of inflammatory diseases selected from the group consisting of multiple sclerosis, autoimmune encephalomyelitis or fibromyalgia syndrome and atherosclerosis. Squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, and colon cancer with hydrophobic drugs embedded in the nanoparticle contrast agent according to claim 1 Drug delivery agent for cancer treatment selected from the group consisting of, rectal cancer, stomach cancer, kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, and gallbladder cancer. According to claim 3 or 4, wherein the hydrophobic drug, Prednisone (Testredone), Testosterone (Testosterone), Mitoxantron (Mitoxantron), Aspirin (salicylates), Ibuprofen (ibuprofen), B Naproxen, fenoprofen, indomethacin, phenyltazone, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone (prednisolone) prednisolone, celecoxib, valdecoxib, valdecoxib, nimesulide, cortisone, corticosteroid, daunomycin, docetaxel, docetaxel, cisplatin (cis-platin), camptothecin, paclitaxel, tamoxifen, anastozole, anasterozole, Gleevec, 5-fluorouracil (5-FU), phloxuridine Floxuridine, Leuprolide, Flotamide amide, Zoledronate, Doxorubicin, Vincristine, Gemcitabine, Streptozocin, Carboplatin, Topotecan, Velotecan (Belotecan), Irinotecan, Vinorelbine, Hyrogyurea, hydroxyurea, Valrubicin, Retinoic acid series, Methotrexate, Meclorethamine , Chlorambucil, Chulrambucil, Busulfan, Doxifluridine, Doxifluridine, Vinblastin, or mitomycin.

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