KR20120113694A - A molecular imaging system using chemical conjugation of biocompatibility polymer mediator - Google Patents

Abstract

PURPOSE: A system for molecular imaging of subject cells is provided to connect the subject cells with a contrast medium and to enhance cell bioavailability. CONSTITUTION: A composite for molecular imaging comprises a biocompatibility polymer mediator with different fractional groups at both ends, wherein the one end is conjugated with surface modified-iron oxide system nanoparticles, and the other end is chemically conjugated with the surface of the subject cells. The biocompatibility polymer mediator is polyethylene glycol(PEG) with a hetero functional group. The subject cells are islet cells. The molecular imaging is MRI(magnetic resonance imaging).

Description

Molecular Imaging System Using Chemical Conjugation of Biocompatibility Polymer Mediator

The present invention relates to an effective system for molecular imaging of a target cell, wherein the molecule is characterized by chemically conjugating the target cell and a contrast agent using a biocompatible mediator polymer material having different functional groups at both ends. Imaging methods and complexes for molecular imaging of such chemically bonded structures.

Molecular imaging (Molecular Imaging) is a rapidly developing field. It is a technique for evaluating various molecular-level changes in cells, such as gene expression, biochemical phenomena, and biological changes. Molecular imaging is made possible by combining molecular cell biology and advanced imaging technology, and is a new field created by integrating medicine, genetics, molecular biology, cytology, chemistry, pharmacy, physics, computer science, medical engineering, radiology, and nuclear medicine.

While conventional medical imaging methods produce video signals using nonspecific physical and chemical properties and clinically estimate the meaning of these signals, molecular imaging uses video signals from the molecular and gene levels. Because of this, there is a characteristic of a more specific image.

Molecular imaging techniques are the basic technology necessary for the first time in the medical field to evaluate gene therapy and each function of the human body, and furthermore, it is a core technology that will revolutionize biotechnology by providing information on life phenomena. The development of biochemistry and molecular biology and imaging techniques over the past 20-30 years is expected to become an important field that opens the horizon of future biomedical research.

On the other hand, diabetes is an important disease with chronic hyperglycemia, and accordingly, complications such as stroke, heart failure, blindness, renal failure, neuropathy and lower limb cutting are social and economically important diseases.

There is no cure for diabetes until now, and insulin fortification can reduce the complications of diabetes due to hyperglycemia, but it is not practical to prevent it completely. Recently, the most physiological method is the pancreatic or pancreatic islet transplantation. The pancreatic islet transplantation is simpler than the pancreas transplantation and the manipulation of immunity and proliferation in vitro is difficult. It is possible to repeat the procedure, and pancreatic islet transplantation is a trend that is recommended.

However, there is a problem of recognizing when about 10% of the transplanted pancreatic islets remain after islet cell transplantation. At that time, the islets are late to be transplanted, and thus molecular imaging is needed to confirm the survival of the transplanted islets.

However, currently, there is no effective way to determine the location and survival of islet cells after islet cell transplantation.

Identifying the location and survival of islet cells is an important issue for islet cell transplantation. Even if 10% of the islet cells are transplanted, this function is not a problem, but 10% of the remaining islet cells are rapidly deteriorated. Location is important.

In this case, until now, the method of entering the pancreatic islets into the pancreatic islet cells, that is, the iron oxide contrast agent has been invaded into the cells to identify the location of the pancreatic islets. However, there are some problems with the existing method.

First, the iron oxide contrast agent stays in the cells for a long time, affecting the problem of toxicity and thus the survival rate. This causes the cells to know the presence of pancreatic islets, resulting in problems on the other side for the effect.

Second, I think this iron oxide contrast agent will not be worried because it does not affect the living body at nano size, but since this iron oxide contrast agent has a strong tendency to stick together, the method of invading the cell and iron oxide contrast agent into the cell on the media for 24 hours without rule is especially It is not known what form of iron oxide contrast agent is in the cell, and it also causes the iron oxide contrast agent to become entangled with each other, causing the size to be larger than the nanosize, which affects cell viability.

Third, 100% labeling of cell and iron oxide contrast agents incubated under media conditions for 24 hours. This fact has been reported in previous studies, and it is confirmed that the distribution of transplanted pancreatic islets and the distribution of pancreatic islets as a result of MRI are different and this is a problem.

Therefore, the present inventors made an effort to develop a method of connecting iron oxide contrast agent to the cell surface rather than invading into the cell, which is a chemical method of connecting a cell and iron oxide contrast agent using a biocompatible mediated polymer material. As a result, using PEG with different functional groups, it is confirmed that the cells can be chemically connected to each other by allowing the functional group NHS to react with the islet cells and the other reactor maleimide with the polysaccharide of the contrast agent. The present invention has been completed.

The present invention relates to a new method of molecular imaging technology to solve the problems of contrast agents used in conventional molecular imaging,

The main object of the present invention is to provide a composite for molecular imaging having a structure in which the iron oxide nanoparticle contrast agent and the target cell chemically connected by using a biocompatible mediated polymer.

Another object of the present invention is to provide a method for producing the composite for molecular imaging, characterized in that for modifying the surface of the target cell using a biocompatible mediated polymer.

Another object of the present invention to provide a molecular imaging method of the target cell using the complex for molecular imaging.

In order to solve the above problems, one end of the biocompatible mediated polymer having different functional groups at both ends is combined with iron oxide-based nanoparticles (contrast agent) surface-modified with a modifying polymer or polysaccharide; The other end provides a molecular imaging complex, characterized in that it has a configuration that is chemically bonded to the surface of the target cell.

A preferred molecular imaging technique for use in the present invention is magnetic resonance imaging (MRI).

Biocompatible media polymers constituting the complex for molecular imaging is selected from the group consisting of polyethylene glycol (PEG), alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO And preferably polyethylene glycol (PEG).

In particular, the polyethylene glycol (PEG) has different functional groups at both ends (hetero functional groups), and may have any one of the following modified forms:

In one embodiment of the present invention, one end of the polyethylene glycol (PEG) is bound to iron oxide nanoparticles having a maleimide group and surface-modified with a polysaccharide; The other end has an NHS group and is characterized by having a structure chemically bonded to the surface of the target cell.

On the other hand, for the surface modification of the iron oxide-based nanoparticles (SPIO, contrast agent) can be used for modifying polymers or polysaccharides, preferably polysaccharides.

The modifying polymer may be at least one polymer selected from the group consisting of PEG, alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO.

In addition, the polysaccharide is alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate. ), Heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives and carrageenan It may be selected from the group consisting of, preferably heparin (heparin).

In addition, the target cell is not particularly limited as long as the cell can be a molecular imaging target. For example, it may be islet cells, stem cells, inverted differentiation induced stem cells (IPS), adipocytes, chondrocytes, liver cells, cardiomyocytes, and the like. In particular, the present invention can be usefully used when applying the molecular imaging method to confirm the pathological state after transplantation of specific cells.

In one embodiment of the present invention, pancreatic islet cells, which are a kind of complex cells, were used as target cells.

Therefore, in the present invention, a maleimide group of polyethylene glycol (PEG) having maleimide groups and NHS groups at both ends is chemically bonded to iron oxide nanoparticles surface-modified with heparin; The NHS group provides a complex for islet cell molecular imaging, characterized in that it has a configuration that is chemically bonded to the amine group on the surface of the islet cells.

In addition, the present invention for use in molecular imaging of the target cell,

Modifying the surface of the iron oxide nanoparticles with a modifying polymer or polysaccharide,

Chemically binding a target cell and a biocompatible median polymer, and

It provides a method for producing a composite for molecular imaging comprising the step of chemically bonding the surface-modified iron oxide nanoparticles and the biocompatible mediated polymer coupled to the target cell.

The components used in each step are as described above.

In addition, the present invention also provides a method for molecular imaging of a target cell, characterized in that it is used by chemically connecting a contrast agent of the iron oxide-based nanoparticles and the target cell through a biocompatible mediated polymer having different functional groups at both ends. do.

A description of each configuration used in the method is also as described above.

The present invention can solve the problems of conventional contrast agents applied to cells. By using biocompatible mediated polymer material on the cell, the material is connected to the target cell and the contrast agent at the same time as the cell surface modification, thereby solving the toxicity of the existing cell-invasive contrast agent to the cell to increase the survival rate of the cell, 100% labeling is possible.

Therefore, the present invention can be usefully used for diagnosing a disease or checking a pathology after transplantation of a specific cell through very safe and efficient molecular imaging.

1 is a diagram showing a schematic structure of a composite for molecular imaging of the present invention.
2 is a transmission electron micrograph of an iron oxide contrast agent modified surface with polysaccharides.
Figure 3 is a uranil acetate staining to confirm the presence of the polysaccharide coated on the surface modified iron oxide contrast agent.
Figure 4 is an MRImage results of the iron oxide contrast agent modified surface with polysaccharides.
Figure 5 is a plyan blue staining and electron micrographs to confirm the presence of iron oxide contrast agent present on the surface of the islet cells.
Figure 6 is a photograph of the complex of the present invention after the FITC fluorescent Dye attached to the contrast medium confirmed by confocal microscopy.
7 is MRImage results for the complex of the present invention.
8 is a graph showing the results of confirming the M2 T2 attenuation signal.
9 is an MRImage result of pancreatic islets according to the iron oxide contrast agent concentration.
10 is a result of comparing the islet cell survival rate according to the concentration-specific treatment of the contrast agent.
11 is an MR T2 image immediately after and one month after implantation of the complex of the present invention into a mouse.

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

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, diagnosis involves identifying the state after transplantation of specific cells in the body.

"Biocompatibility" means a substance that interacts with the human body without undesirable subsequent effects.

"Molecular imaging" is a technique that analyzes and evaluates various molecular-level changes in a cell, such as gene expression, biochemical changes, and biological changes, and is made possible by combining molecular biology and advanced imaging technology. It is an integrated technology of molecular biology, cytology, chemistry, pharmacy, physics, computer science, medical engineering, radiology and nuclear medicine. By using molecular imaging, it is possible to evaluate both temporal and spatial information for imaging the fundamental phenomena and biomechanism of cells, increasing understanding of the disease, as well as many intractable diseases such as tumors, neurological diseases and immune diseases. Early diagnosis is possible. In addition, it can be used for the development of new drugs that act on specific molecules, genes and signaling systems, and contributes to gene therapy since non-invasive imaging and easy quantification of the expression of externally injected genes in target organs or tissues .

"Target cell" is a target cell to be molecularly imaged using a contrast agent to confirm the presence of any pathological state, in one embodiment of the present invention molecular imaging for the purpose of confirming the survival after transplantation of pancreatic islet cells Use the law.

"Contrast" is a substance used to make certain organs or tissues easy to observe in contrast with the surroundings, and is generally divided into negative contrast agent and positive contrast agent. Negative contrast agent absorbs more X-rays, radiation, etc. than the surrounding tissue to display an image, and positive contrast agent is the opposite.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates the detection of a reagent conjugated to, fused, conjugated or fused to a reagent, eg, a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use of unscrupulous infringers.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have devised for the first time a method of connecting iron oxide contrast agent to the surface of a target cell, rather than the conventional method of invading into cells, using a biocompatible mediated polymer material to connect the target cell and the iron oxide contrast agent. .

The present invention is a technique related to molecular imaging.

Molecular imaging is a technique for evaluating the changes of various molecular levels occurring in a cell. Molecular imaging technology has been studied in three approaches: optical imaging and nuclear imaging. And radiological imaging techniques such as CT and ultrasound (US), including magnetic resonance imaging (MRI).

Optical imaging techniques are the most widely used reporter genes using fluorescent proteins and bioluminescent imaging using an enzyme called luciferase.

In the study using nuclear medical imaging techniques, Tjuvajev of the Gambhir of UCLA and Tjuvajev of the Memorial Sloan-Kettering CancerCenter developed the reporter gene imaging method, which transferred the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene into specific cells. A method of imaging gene expression using a radiolabeled substrate and PET was presented. The image made using TK is a PET image, and unlike the optical imaging method, it has a high permeability, making it easy to acquire or quantify tomographic images.

In addition, there are radiographic imaging techniques using MR, CT, and ultrasound, and these conventional diagnostic radiographs and methods have high resolution and have an advantage of being able to image deeper.

In the present invention, a radiological molecular imaging technique is preferably used. In particular, it is most preferable to use magnetic resonance imaging (MRI).

Magnetic resonance imaging (MRI) is a molecular imaging technology suitable for imaging cells. MRI measures signals from protons. Diagnostic images can be taken at any angle, at any angle, in any cross section, coronal plane, and sagittal plane. As a method, there is no risk of radiation exposure of existing Xray-related imaging equipment.

Therefore, the present invention is, in one aspect,

One end of the biocompatible mediator having different functional groups at both ends is combined with iron oxide-based nanoparticles surface-modified with polysaccharides, and the other end is chemically bonded to the surface of the target cell. It relates to a composite for molecular imaging.

Iron oxide  Nanoparticles (Contrast Agents)

Contrast agent is used to increase the clarity and accuracy of the MRI image, which is a molecular imaging method that can be preferably used in the present invention. In particular, colloidal nanoparticle solutions based on superparamagnetic iron oxide (SPIO) series using nanotechnology among MRI contrast agents are excellent in contrast and have great potential for research as MRI contrast agents. In addition, in line with current research trends, a highly sensitive diagnostic method can be additionally combined with SPIO contrast agents to provide a more accurate and clear diagnosis, or to develop a form of contrast medium that can simultaneously diagnose and treat the treatment effect. This seems to be lively.

The material used as the contrast agent in the present invention is iron oxide nanoparticles, preferably superparamagnetic iron oxide nanoparticles (SPIO). The term "iron oxide nanoparticles" is used herein interchangeably with terms such as "iron oxide nanoparticles", "iron oxide contrast agent" or "contrast agent."

Iron oxide nanoparticles have a magnetizing property and thus have a very high biomedical value. Iron oxide has been studied as MRI contrast agent because it shows high magnetic resonance efficiency even at nano molar concentration.

In particular, superparamagnetic iron oxide nanoparticles have been in the spotlight recently in biomedical fields such as magnetic resonance imaging (MRI) diagnostics, drug delivery and treatment. Because of its superparamagnetic properties, SPIO can exhibit higher contrast in MRI than conventional paramagnetic Gd-based contrast agents. These superparamagnetic iron oxide particles have a longer blood half-life than gadolinium (Gd) and manganese (Mn), and the magnetic resonance efficiency is about 10 to 100 times higher than that of the paramagnetic composite, so that a smaller amount can be used.

The superparamagnetic iron oxide nanoparticles can be prepared using any of known co-precipitation method, sol-gel method, pyrolysis method or emulsion method without limitation, but preferably pyrolysis method can be used. The thermal decomposition method is easier to control the size of the superparamagnetic iron oxide nanoparticles than the coprecipitation method, it is possible to produce nanoparticles excellent in dispersibility, crystallinity and paramagnetic properties. In addition, the growth of the superparamagnetic iron oxide nanoparticles may be manufactured to a desired size by applying a seed mediated growth method again.

However, the superparamagnetic iron oxide nanoparticles may cause toxicity in vivo such as chemical bonding, viscosity and immunity due to aggregation of contrast agent, and inhibition of enzymatic action such as lysozyme, and thus cannot be used as such. Therefore, the use of superparamagnetic iron oxide nanoparticles as an MRI contrast medium requires high imaging images, low toxicity, high efficiency, long half-life in blood, dispersion in tissue specificity, good solubility and stability in physiological fluids. It should be coated with a biocompatible polymer.

On the other hand, for the surface modification of the iron oxide-based nanoparticles (SPIO, contrast agent) can be used for modifying polymers or polysaccharides, in the present invention, it is preferable to use iron oxide nanoparticles surface-modified with polysaccharides.

The modifying polymer may be at least one polymer selected from the group consisting of PEG, alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO.

The polysaccharide may be alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, Group consisting of heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan Can be selected from. Most preferably, iron oxide nanoparticles surface-modified with heparin are used.

Heparin consists of sulfated linear polysaccharides, mainly composed of uronic acid. Heparin is a mixture of various structures and has various properties such as antithrombogenicity, inflammation inhibition, angiogenic cancer growth inhibition and immunosuppression. Heparin used in the present invention preferably has a molecular weight of about 20,000 Da, but is not limited thereto.

On the other hand, the iron oxide-based nanoparticles, preferably superparamagnetic iron oxide-based nanoparticles (SPIO) on the surface, a ligand capable of chemically binding to one end of the biocompatible median polymer acting as a mediator in the present invention and connects them It comprises a linker that serves to make.

In one embodiment, the iron oxide-based nanoparticles surface-modified with heparin convert the functional group of heparin into a sulfide group to chemically bind to one end of a biocompatible mediator, for example, a maleimide group, which acts as a mediator in the present invention.

That is, in the present invention, the contrast agent, the iron oxide-based nanoparticles have a structure that is chemically bonded to a biocompatible polymer that acts as a mediator, unlike the conventional case in which the surface-modified polysaccharide and directly invade into the target cell.

At this time, the size of the (superparamagnetic) iron oxide nanoparticles is not particularly limited, but is preferably 4 to 200 nm. If the (superparamagnetic) iron oxide-based nanoparticle size exceeds 200 nm, there is a problem that is easily recognized by macrophages in vivo, and if it is less than 4 nm there is a problem that its production is not easy.

In addition, in the present invention, a fluorescent probe may be further bonded to the surface of the iron oxide nanoparticles.

The fluorescent dyes used as the fluorescent probes are FITC, RITC, IRDye, Cy2 (trade name), Cy3 (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), Cy-chrome, pico Eritrin, PerCP (Peridinine Chlorophyll-a Protein), PerCP-Cy5.5, Alexa Fluor (R) 350, Alexa Floor (R) 430, Alexa Floor (R) 488, Alexa Floor (R) 532, Alexa Floor (trade name) 546, Alexa Floor (trade name) 568, Alexa Floor (trade name) 594, Alexa Floor (trade name) 633, Alexa floor (trade name) 647, Alexa floor (trade name) 660, Alexa floor (trade name) 680 Can

Target cell

In the present invention, the target cell is a target for molecular imaging, and there is no particular limitation. Multiple cell bodies can be used as target cells, and single cell bodies can be used.

That is, any cell may be a target, and preferably, islet cells, stem cells, reverse differentiation induced stem cells (IPS), adipocytes, chondrocytes, liver, which require molecular imaging to confirm survival after transplantation. Cells, or cardiomyocytes and the like. In one embodiment of the present invention, pancreatic islet cells were used as target cells.

In the structure of the molecular imaging complex of the present invention, the functional group on the surface of the target cell and the other end of the biocompatible medial polymer are chemically bound.

In one embodiment of the present invention, the amine group present in the collagen tissue on the surface of the islet cells has a configuration in which the NHS group of the biocompatible mediator polymer is chemically bonded.

Particularly, in the present invention, the biocompatibility-mediated polymer material is applied to each of the target cells so that 100% adheres to the material, and then 100% labeling is performed because the surface polysaccharide functional group of the iron oxide-based nanoparticles (contrast agent) is combined. Do. That is, in the present invention, the surface of the target cell has the effect of surface modification with biocompatible mediated polymers.

Biocompatible Mediated Polymers

In the present invention, any biocompatible polymer that can have two functional groups capable of chemically and firmly mediating a target cell and a contrast agent (iron oxide nanoparticles) may be used as the biocompatible media polymer.

As a preferred example, polyethylene glycol (PEG), alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, PEO or the like can be used. Among these, polyethylene glycol (PEG) is especially preferable.

The biocompatible mediated polymer is a material that plays a role of mediating a target cell and iron oxide nanoparticles (contrast agent), and has different functional groups at both ends.

In this case, different functional groups having both ends of the biocompatible mediated polymer may be appropriately modified according to the type of the material to which the target cell and the contrast agent are surface-modified.

Hereinafter, the case of polyethylene glycol (PEG) is described as an example.

PEG protects the nanostructures from lymphocyte attack and protein adsorption, thereby increasing the stability and retention time of the nanostructures. Therefore, PEG mainly consists of amine groups, carboxyl groups, hydroxyl groups, thiol groups, maleimide, etc. It can be used as a multi-branched PEG having one or more functional groups selected from the group, and PEGs having various numbers of branches such as two, three, and six are commercialized, and various ends thereof are commercialized. Functional groups can be applied.

That is, all of the following PEG may be used to bind the cell surface by chemical conjugation with a contrast agent.

(i) Monofunctioncal activated PEG

Carboxylated mPEGs: NHS Active Ester

Other monofunctional activated PEGs

(ii) multi-arm activated PEG

? 2 Functional PEG Series

? 4 Functional PEG Series

? 8 Functional PEG Series

(iii) branched active PEG

(iv) heterofunctional PEG

In one embodiment of the present invention, PEG having maleimide group and NHS group at both ends was used.

That is, to apply the conjugation method that connects the pancreatic islet cells with the contrast agent, PEG, a biocompatible mediator polymer, was selected to have two different sites with different functional groups, that is, a PEG having a Heterobifuctional group. In order to prevent the PEG from sticking to one side or entanglement with each other, PEG of the other bidentate functional group was selected.

The NHS group is chemically bound to the amine group of the target cell, and the maleimide group is chemically bonded to the SH group of the iron oxide nanoparticle (contrast agent) surface polysaccharide.

In this way, the composite for molecular imaging of the present invention takes the structure shown in FIG.

As such, the composite for molecular imaging of the present invention comprises a composition in which the target cell and the iron oxide-based contrast agent are chemically connected by using a biocompatible mediated polymer material, which is a structure in which the surface of the iron oxide-based contrast agent is modified to invade into a conventional cell. Compared with the above, it is characterized by taking the configuration of modifying the surface of the target cell.

That is, in the present invention, by modifying the surface of the target cell using a biocompatible mediator such as PEG, it is used to increase the survival rate of the target cell by blocking it from external immune responses.

In another aspect, the present invention relates to a method for preparing the composite for molecular imaging.

In one embodiment, the present invention, for use in molecular imaging of the target cell,

Modifying the surface of the iron oxide nanoparticles with a modifying polymer or polysaccharide,

Chemically binding a target cell and a biocompatible median polymer, and

It provides a method for producing a composite for molecular imaging comprising the step of chemically bonding the surface-modified iron oxide nanoparticles and the biocompatible media polymer coupled to the target cell.

Detailed description of each component is as mentioned above.

Techniques for surface-modifying iron oxide nanoparticles with modified polymers or polysaccharides, or techniques for modifying functional groups at both ends of a biocompatible mediated polymer so as to bind surface-modified contrast agents or functional groups on the surface of a target cell, are known to those skilled in the art. Use them appropriately.

For example, a bond may be formed to the contrast agent and the target cell through functional groups on the biocompatible mediated polymer or crosslinking agent, respectively.

The crosslinking agent may be activated using an activating material capable of introducing a functional group (cross-linker) capable of binding to a functional group bonded to the biocompatible median polymer at the end of the crosslinking agent, and vice versa. In this case, the biocompatible media may be crosslinked through a crosslinking linker introduced into the crosslinking agent or a functional group bonded thereto, or crosslinked through a crosslinking linker introduced into the crosslinking agent. The functional group bound to the biocompatible median polymer may be any functional group capable of chemically binding to the target cell and the surface-modified contrast agent, and may be, for example, an amine group, a carboxyl group, a hydroxyl group, a thiol group, a maleimide group, or the like. .

The average size of the composite for molecular imaging prepared by the above method is 50nm to 10㎛, preferably 50nm to 200nm. In one embodiment it was 60 nm.

The complex for molecular imaging according to the present invention shows excellent contrast effect in MRI images, and can be used as a sensor for early diagnosis of real-time tissue images and non-invasive diseases.

In another aspect, the present invention provides a method for molecular imaging of a target cell, comprising using a biocompatible mediator having different functional groups at both ends, and chemically connecting the contrast agent of the iron oxide-based nanoparticles to the target cell. It is about.

The molecular imaging method includes the use of a composition comprising a complex for a molecular imaging of the present invention described above or a pharmaceutically acceptable carrier with it.

Carriers include carriers and vehicles commonly used in the medical arts, and specifically include ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg, various phosphates, glycine, sorbic acid). , Potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, poly Vinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like. The composite composition of the present invention may also further include lubricants, wetting agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

In one embodiment, the complex composition according to the invention can be prepared in an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the complex composition of the present invention may be in the form of sterile injectable preparations of aqueous or oily suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose any non-irritating non-volatile oil, including synthetic mono or diglycerides can be used.

More specifically, the molecular imaging method comprises administering the complex of the present invention to a living body or a sample; And obtaining an image by sensing a signal emitted by the complex from the living body or the sample.

"Sample" means a tissue or cell isolated from a subject to be diagnosed. In addition, the step of injecting the complex composition into a living body or a sample may be administered via a route commonly used in the medical field, parenteral administration is preferred, for example, intravenous, intraperitoneal, intramuscular, subcutaneous or topical route. It can be administered through.

In this method, the signal emitted by the complex can be detected by various equipment using a magnetic field, and in particular, a magnetic resonance imaging device (MRI) is preferable.

The magnetic resonance imaging apparatus puts a living body in a strong magnetic field and irradiates radio waves of a specific frequency so that atomic nuclei such as hydrogen in biological tissues absorb energy to make energy high, and then stops propagating the nuclear energy such as hydrogen. The energy is converted into a signal, processed by a computer, and imaged. Since the magnetic or radio waves are not disturbed by the bone, a sharp three-dimensional tomographic image can be obtained at the end, transverse, or any angle with respect to the hard bone around or the brain or the bone marrow. In particular, the magnetic resonance imaging apparatus is preferably a T2 spin-spin relaxation magnetic resonance imaging apparatus.

As described above, the present invention reacts the target cell and the contrast agent with the target cell at one functional group thereof using a biocompatible mediated polymer having different functional groups such as PEG, and the other reactor is a surface polysaccharide of the contrast agent (iron oxide nanoparticles). It relates to a chemical conjugation technique that connects a target cell and a contrast agent to each other by making it react with each other.

The present invention solves the following existing problems by featuring such a configuration.

First, by conjugating the iron oxide contrast agent with a biocompatible mediator polymer on the surface of the target cell, the contrast agent directly invades the target cell and affects the organelles, thereby solving the problem of viability.

Second, the problems caused by the non-constant existence and morphology of the conventional iron oxide contrast agent were solved by taking a conjugation method on the surface of the target cell. This is because the conjugation method of the surface of the target cell does not cause a problem in the size and size of the contrast iron oxide.

third. It solved the problem of not being 100% labeling (cover), which was considered as a disadvantage. That is, 100% labeling was made possible by changing the polysaccharide functional group of the iron oxide contrast agent so that the target cell was treated with a biocompatible mediated polymer material and adhered to the material 100%.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One: Pancreatic islets  Cell separation

The pancreas removed from the experimental animals (SD-rat rats) was reacted in a water bath for 15 minutes, after which the M199 media (sigma) was added to 50 ml and placed in a centrifuge set at 1400 RPM / 2 minutes. After centrifugation, the supernatant was slowly discarded into a beaker, repeated twice and vortexed 25ml M199 Media.

The vortexing was over and entered filtration. The funnel was placed in a 50 ml tube, the kit was placed in the funnel, the vortexed islet cells were poured, the 25 ml buffer was added to the empty islet cell tube, shaked again, placed in a 50 ml tube and placed in a centrifuge set at 1400 RPM / 2 minutes.

The supernatant was thrown back into the beaker and the 50 ml tube was turned upside down. After 30 seconds, the inlet was cleaned with a tissue and 5 ml of picol solution (sigma) was added. In addition, 10 m of picol solution was added and 10 ml of M199 media was slowly poured on it.

It was placed in a centrifuge set at RPM: 2400/17 min / 4 deg / Break: 0, and the pancreatic islets were slowly extracted between layers with a 10 ml disposer pipette. 50 ml M199 media was added to the tube, which was then turned into a centrifuge set at 1800 RPM / 2 minutes and removed with a disposer pipette.

Pour 25ml M199 media again and gently loosen it by hand, pour about 10ml of M199 media every 4 minutes (put it in a beaker), and repeat the process of adding 10ml again. The 10ml was discarded at the 7th and divided into 3 parts. 9ml (①), 5ml (②) and 1ml (③) were put into 60mm dish (mostly islet), and they were adjusted to 10ml each.

Using a pipette and a microscope, the pancreatic islets were picked out one by one and filled with 5 ml RPMIMedia (Sigma) in a clean bench. Allow to settle for 3-6 minutes, then wash and place in 150mm dish. RPMI + FBS + P / S Media was added to the dish in 20 ml and incubated.

Example  2: Preparation of Iron Oxide Contrast Agent Using Various Polysaccharides

Insert 30 ml of 3rd DW into a 3 neck, Angled Flask 100ml, and then dressing for 30 minutes while flowing nitrogen. Iron (III) chloride Hexahydrate Eisen (III) chloride Hexahydrate (sigma) 0.5g FeCl ₃ , 6H ₂ O (sigma) Pipetteade was added with 7.5 ml of iron (II) chloride Tetrahydrate Eisen (II) chloride Tetrahydrate (sigma) 0.184g FeCl ₂ , 4H ₂ O (sigma), and ammonium hydroxide solution ammoniumhydroxide-Losung (Fluka). The mixture was stirred for 30 minutes under flowing nitrogen gas.

The prepared polysaccharide (see formulas below) was put in 250 mg of 25 ml of 3rd DW. At this time, the preparation method is slightly different depending on the characteristics of the polysaccharides, the magnetite was stirred for 30 minutes with distilled water, the final washing 30ml DW and put the prepared polysaccharides in the process of coating in the beaker. Magnetic bar was added and stirred for 2 hours, and then washed. The wash was done and sonicated for 50 minutes. At this time, the last washing was put into 20ml DW was sonicated.

[Formula 1] Dextran

[Formula 2] Alginate

[Formula 3] glycol chitosan

[Formula 4] methyl cellulose

Polysaccharide: high molecular weight heparin

[Formula 6] low molecular weight heparin

Polysaccharide modified by functional group of high molecular weight heparin

The modified heparin (see schematic) in polysaccharide of iron oxide contrast agent was used. Because heparin functional group is an amine group, heparin functional group is the same as that of membrane of pancreatic islet, and heparin functional group is used as 2-iminothiolane and sulfide group (-SH). ). Maleimide was a functional group that effectively binds sulfides, and was used to select the other functional group of PEG as maleimide.

After the completion of the sonication, it was placed on a magnetic magnet for 12 hours. Thereafter, only the supernatant was placed in a centrifuge tube and centrifuged three times for 10 minutes at RPM10.000. The tube was centrifuged and filtered by inserting a 25 mm filter into a syringe, and 1 ml of iron oxide contrast agent (vial) and 2 ml of D.W were placed in a stirrer heated to 80 ° C., heated for 60 minutes, and then sized with a zetasizer.

If the range was correct, it was concentrated to about 10ml in 100K ultrafiltration (Amicon stirred cell model 8200). After concentrating, 1 mlDW and droplet concentrate were added to determine the size. In this way, the iron oxide contrast agent was completed.

Table 1 shows the results of measuring the size of iron oxide contrast agent using various polysaccharides. The zeta potential of the polysaccharides is also measured and shown.

As can be seen from the above table, all of the polysaccharides used were in the size of nm and harmless size of the human body.

Experimental Example  1: Polysaccharide of Iron Oxide Contrast Agent Surface modification  Confirm

(1) Transmission electron microscope Transmission Electron Microscope Confirmation by)

The prepared iron oxide contrast agent was observed by transmission electron microscope.

The results are shown in Fig. The transmission electron microscope is a microscope suitable for observing the iron oxide contrast agent under a microscope that can detail the size and shape of the particles. As shown in FIG. 2, the uncoated iron oxide contrast agent is nanosized in size but the particles are gathered together, which is an important reason for showing the problem of the iron oxide contrast agent. On the other hand, the iron oxide contrast agent coated with polysaccharide dextran and high molecular weight heparin is uniform. You can see that it keeps its shape.

Through this, the size of the nanoparticles of the iron oxide contrast agent and the shape of the nanoparticles were confirmed.

(2) Checking the presence of polysaccharides on the surface Uranil  Acetate dyeing)

Uranyl acetate stained samples were confirmed by transmission electron microscopy after staining the iron oxide contrast agent. Uranyl acetate dye sample is a dye sample that shows the characteristic of blackening on a transmission electron microscope by dyeing a polysaccharide.

The results are shown in Fig.

In the case of the iron oxide contrast agent coated with dextran, it was confirmed that dextran, a polysaccharide, was present in the line of gum. In the case of high molecular weight heparin, the boundary between the black line and the undyed white part was clear, and it was confirmed that the high molecular weight heparin was present in the iron oxide contrast agent.

In order to confirm more precisely, the contrasted dyes and contrasted dyes were compared.

As shown in Figure 3, it was confirmed that the result of staining with low molecular weight heparin and the result without staining is clearly different.

(3) of iron oxide contrast agent MRI

4 shows the results of confirming the iron oxide contrast agent coated with the polysaccharide by MRI.

As a result, the prepared iron oxide contrast agent detected T2 attenuation and T2 attenuation was black. When the contrast agent was not treated, T2 attenuation effect was not shown, which resulted in white color. In contrast, as the concentration of the contrast agent was increased, the T2 attenuation effect was increased and appeared black as MRI.

Example  3: PEG Using Pancreatic islets  Chemical Bonding of Cells with Iron Oxide Contrast

In order to apply the conjugation method that connects the islet cells and the contrast agent, PEG, which is a biocompatible mediator, was selected to have two different sites with different functional groups, that is, a PEG having a Heterobifuctional group. If it is not different sites, PEG may stick to only one side or become entangled with each other, so we chose a PEG with a different double site functional group for the purpose of this study.

The islet cell membrane is surrounded by the collagen layer, and the collagen layer contains a lot of amine groups. Therefore, one functional group of the other bidentate functional groups of PEG was selected as NHS by using the functional group NHS that effectively binds the amine group.

First, the pancreatic islet cells and PEG were reacted for 1 hour under the conditions of PH8.0 HBSS buffer.Then, after the reaction, the iron oxide contrast agent made by modifying the functional group of heparin in high molecular weight was put into 100 mm dish and then added to PH 6.0. HBSS buffer was added and reacted for 1 hour to conjugate.

(One) Pancreatic islets  Cells and PEG  Combination

Cultured pancreatic islets were run for 2 min at RPM1400 in a centrifuge and buffered HBSS PH8.0 (Gibco) was poured into centrifuge tubes and placed in a 100 mm culture dish.

25 mg of PEG (NOF) was placed in an E-tube, and 2 ml of HBSS PH8.0 buffer was added to the syringe, and 1 ml was added to the E-Tube. Once again sucked, put a syringe filter in an 8ml dish containing pancreatic islets. It was left in the incubator for one hour.

After an hour, RPMI1640 (Gibco) was added a little to stop the reaction, and then placed in a 50ml tube. Centrifuge for 2 minutes at RPM1400.

(2) contrast agent PEG  Combination

5 ml of PH 6.0 HBSS was added to the dish and PEGylated islet cells were added to the dish. And 450 ml of self-made contrast agent (600 mg / ml) was added and placed in an incubator for one hour. After an hour, RPMI1640 (Gibco) was added a little, the reaction was stopped, and then placed in a 50ml tube. Centrifuge for 2 minutes at RPM1400.

Experimental Example  2: check the structure of the complex

(1) Observation by staining and electron microscope

Islets were observed by staining with Prussian blue staining sample. The Prussian blue dye sample is a dye stained blue color in the presence of iron, and is an experiment to check whether iron oxide contrast agent is present on the surface of the islet cells.

The results are shown above in FIG. The untreated pancreatic islet cells were stained with a Prussian blue staining sample, but no staining was confirmed.

In addition, when the pancreatic islet cells were incubated for 24 hours as in the conventional method, the iron oxide contrast agent was physically induced into the cells. However, when chemically conjugated the islets and iron oxide contrast agent with the biocompatible mediator of the present invention, it was confirmed that the iron oxide contrast agent present on the surface of the islet cells was stained and the edges were colored blue.

And the result observed with the electron microscope is shown in the lower part of FIG.

In other words, despite the same treatment time, the result of incubation for 1 hour was confirmed to enter into the cell through the mechanism of endocytosis, and the conjugation complex of the present invention was placed on the cell surface even if incubated for the same time. It was confirmed.

(2) Confocal  Observation with a microscope

The polysaccharide used for surface modification of iron oxide contrast agent was attached to the FITC fluorescent dye, and the contrast agent conjugated using PEG to the islets was confirmed by confocal microscopy.

As a result, as shown in Figure 6, the FITC fluorescent sample is green light, it was confirmed that the fluorescent sample is distributed around the cell in a round. Through this, it was confirmed that the configuration of the present invention was chemically conjugated to the islet cells and the iron oxide contrast agent with a biocompatible mediated polymer.

Example  3: Molecular Imaging MRI Observation by

Next, the pancreatic islet cells were treated with iron oxide contrast agent, and confirmed by MRI.

As a result, as shown in Figure 7, it was confirmed that there is no T2 attenuation effect in the case of the islet cells not treated with the iron oxide contrast agent, the islet cells physically uptake iron oxide contrast agent as in the conventional method has a T2 attenuation effect It was confirmed. In the complex of the present invention, the T2 attenuation was higher than that of the uptake islets.

Through this, it can be seen that the function of the iron oxide contrast agent is much deteriorated when the conventional physical method up-taken into the cell.

In addition, the results of confirming the degree of T2 attenuation signal on the MRI by treating the iron oxide contrast agent in the islet cells in FIG.

Lower T2 attenuation values indicate that MRI is more sensitive to MRI. That is, the lower the T2 value, the better the contrast agent and the image. The higher the R2 value, the better the result.

As can be seen in Figure 8, in the case of pancreatic islets that were not treated with the iron oxide contrast agent, the attenuation was higher than that of the uptake pancreatic islet cells, and the MRI showed no clouding effect as indicated by the T2 attenuation effect. . However, in the complex of the present invention, T2 attenuation was about four times more effective than islet cells uptake of conventional iron oxide contrast medium.

Example  4: according to the iron oxide contrast agent concentration Pancreatic islet cells MRI

As shown in FIG. 9, the pancreatic islet cells were treated at different concentrations of the iron oxide contrast agent. And, the conventional contrast agent invaded into the islet cells and the case of the complex of the present invention was compared.

As a result, it was confirmed that the MR image appears even at a small concentration of the complex of the present invention. In other words, by treating the iron oxide contrast agent in a small concentration, it is possible to reduce the effect on the cells as well as molecular imaging, and is more effective in the survival rate of the islets.

Hereinafter, the survival rate of the pancreatic islet cells was directly compared.

Example  5: Pancreatic islet cells  Survival Rate Comparison

Iron oxide contrast agent was treated according to the concentration, as described in Figure 10 and compared the survival rate after 1 day, 5 days, 10 days.

As a result, as shown in Figure 10, the complex of the present invention did not show a difference in survival rate with the untreated islet cells even after 1 day, 5 days, it was confirmed that the survival rate is rather high when treated with a low concentration of iron oxide contrast agent . However, it was confirmed that the survival rate of the conventional uptake pancreatic islets is poor.

That is, the complex configuration of the present invention can be seen that the biocompatible mediators play a role in protecting the islet cells.

Example  6: For molecular imaging  Complex To the mouse  transplantation

In the pancreatic islet, 300 complexes of the present invention conjugated with an iron oxide contrast agent were implanted into the left kidney of the nude mouse.

As a result, as can be seen in Figure 11, it was clearly confirmed that the islet cells were transplanted.

After one month, the results of molecular imaging with MRI were also shown in FIG. 11. The presence and condition of the transplanted pancreatic islets could be clearly confirmed.

As such, the complex of the present invention can be very usefully used for molecular imaging to confirm the cell state after transplantation.

Claims (22)

Biocompatible mediated polymers with different functional groups at both ends
One end is combined with iron oxide nanoparticles surface-modified with a modifying polymer or polysaccharide,
The other end has a configuration that is chemically bonded to the surface of the target cell complex for molecular imaging.
The method of claim 1, wherein the biocompatible media is selected from the group consisting of polyethylene glycol (PEG), alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO A composite for molecular imaging, characterized in that at least one polymer.
The composite of claim 2, wherein the biocompatible mediated polymer is polyethylene glycol (PEG).
The method of claim 3, wherein one end of the polyethylene glycol (PEG) has a maleimide group and is bonded to the iron oxide-based nanoparticles modified with a polymer or polysaccharide for modification; The other end has an NHS group, and the molecular imaging complex characterized in that it is chemically bonded to the surface of the target cell.
The complex of claim 3, wherein the polyethylene glycol (PEG) has a structure having any one of the following hetero functional groups:

The method of claim 1, wherein the modifying polymer is one or more polymers selected from the group consisting of PEG, alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO A composite for molecular imaging, characterized in that.
The method of claim 1, wherein the polysaccharide is alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin, heparin sulfpy. Heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives and carrageenan (carrageenan) is a composite for molecular imaging, characterized in that selected from the group consisting of.
The complex for molecular imaging according to claim 7, wherein the polysaccharide is heparin.
The method of claim 1, wherein the target cell is any one selected from the group consisting of islet cells, stem cells, reverse differentiation induced stem cells (IPS), branched cells, chondrocytes, and cardiomyocytes. Complex.
The complex of claim 9, wherein the target cell is a pancreatic islet cell.
The complex of claim 1, wherein the molecular image is magnetic resonance imaging (MRI).
Of polyethylene glycol (PEG) which has maleimide group and NHS group in both ends, respectively
Maleimide groups are chemically bound to iron oxide-based nanoparticles surface-modified with heparin,
The NHS group is a pancreatic islet cell molecular imaging complex, characterized in that it has a configuration that is chemically bonded to the amine group on the surface of the islets.
To use for molecular imaging of target cells,
Modifying the surface of the iron oxide nanoparticles with a modifying polymer or polysaccharide,
Chemically binding a target cell and a biocompatible median polymer, and
Chemically bonding the surface-modified iron oxide nanoparticles and the biocompatible media polymer coupled to the target cells, the method of claim 1, wherein the molecular imaging complex.
The method of claim 13, wherein the biocompatible media is selected from the group consisting of polyethylene glycol (PEG), alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO Method of producing a composite for molecular imaging of claim 1, characterized in that at least one polymer.
The method of claim 13, wherein the polysaccharide is alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin, heparin sulfpy. Heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water-soluble cellulose derivatives and carrageenan The method for preparing a complex for molecular imaging according to claim 1, which is selected from the group consisting of (carrageenan).
The method of claim 13, wherein the modifying polymer is at least one polymer selected from the group consisting of PEG, alginate, hydrogel, fibrinogen gel, collagen, gelatin, chitosan, alginate, hyaluronic acid, PLGA, PEI, and PEO. Method for producing a composite for molecular imaging of claim 1, characterized in that.
The method of claim 1, wherein the target cell is any one selected from the group consisting of islet cells, stem cells, reverse differentiation induced stem cells (IPS), branched cells, chondrocytes, and cardiomyocytes. Method for preparing a composite for molecular imaging.
A method of molecular imaging of a target cell, comprising using a biocompatible mediated polymer having different functional groups at both ends and chemically connecting the contrast agent of the iron oxide-based nanoparticles to the target cell.
The method of claim 18, wherein the molecular image is magnetic resonance imaging (MRI).
19. The method of claim 18, wherein the contrast agent of the iron oxide nanoparticles is surface modified with polysaccharides.
19. The method of claim 18, wherein the biocompatible mediated polymer is polyethylene glycol.
19. The method of claim 18, wherein the target cell is a pancreatic islet cell.

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