KR101451775B1 - Molecularly imprinted metal nanopaticles for serum amyloid P component protein - Google Patents

Abstract

The present invention relates to a molecule-imprinted metal nanoparticle that binds to serum amyloid P urea protein. More specifically, the present invention relates to a metal-imprinted metal nanoparticle that forms an azide-terminal self-assembled monolayer on a metal nanoparticle, (SAP) having a structure in which a layer is formed on the surface of the amorphous P urea protein (SAP).
According to the present invention, when a protein molecule imprinting layer is prepared on the surface of a nanoparticle using a click reaction, the insolubility of the azide material used in the reaction in the buffer solution has been a problem. Nanoparticles that have not undergone surface modification can form irreversible precipitates by aggregation reactions in organic solvents. In the case of a click raw material including an azide group, the solubility in water is low and it should be dissolved in an organic solvent such as ethanol. Thus, it is impossible to apply a click reaction to the surface of nanoparticles. In the present invention, the nanoparticles may be immobilized on a substrate, and the azide may be dissolved in an organic solvent to form a fixed layer. In this case, the nanoparticles may be reacted without aggregation reaction. Finally, Respectively.
The SAP-binding nanoparticles of the present invention had the property of inhibiting the binding of SAP to amyloid fibrils, and it was observed that the application of this SAP in vivo relieved the onset of dementia disease substantially. Molecular imprinting techniques for SAP and SAP-coupled nanoparticles have not been made in the prior art, and the particles prepared in the present invention can be used for SAP detection and concentration measurement, together with the ability to inhibit SAP binding of amyloid fibrils.

Description

Molecularly imprinted metal nanoparticles for serum amyloid P component protein binding to serum amyloid P element protein [

The present invention relates to a molecule-imprinted metal nanoparticle that binds to serum amyloid P urea protein. More specifically, the present invention relates to a metal-imprinted metal nanoparticle that forms an azide-terminal self-assembled monolayer on a metal nanoparticle, The present invention relates to a metal nanoparticle specifically binding to a serum amyloid P urea protein having a structure in which a layer is formed.

Various technologies have been developed in the fields of diagnosis and treatment of diseases using characteristics of nanoparticles such as drug delivery, bioimaging, and nano medicine. These nanoparticles are often prepared so as to have a specific binding force with various substances such as protein, DNA, RNA and cell membrane receptor present in vivo. For this purpose, immobilization of an antibody against a target substance is used as a main method. Antibodies used herein include murine, chimeric or humanized antibodies. Murine antibodies are entirely mouse-derived proteins and are generally removed by the immune response in a short period of time before reaching the target material. Therefore, chimeric antibodies in which human proteins and mouse proteins are combined (human: mouse = 70: 30) have been developed and used. Humanized antibodies are those in which more than 90% of humanized antibodies have human-derived protein sequences.

However, since the process of producing such a chimeric or humanized antibody to be active requires a complex process that consumes time and cost, an antibody manufacturing technique capable of providing a targeting function of a substance is very crucial for determining the efficacy of nanomedicine It plays a role.

Molecularly imprinted polymer (MIP) is known as a means of selectively sensing a desired substance. The molecule-imprinting polymer refers to a polymer having an inscribed space having the same shape as the template material by synthesizing a polymer using a monomer bonded to a suitable template as a starting material and removing the template material. The binding site of the polymer can be bound to only the morphologically identical substance, and the molecule having a different stereostructure from the template substance can be bound to the steric hindrance, so that only the desired substance can be selectively detected will be.

In the present invention, gold nanoparticles were prepared by immobilizing an organic molecule capable of substituting an antibody to nanoparticles by a molecular imprint method to impart specific binding force and selectivity at a level similar to that of the antibody.

Serum amyloid P component (SAP) was used as a target protein, which is one of five monoclonal-linked pentactoxin proteins and is known to be involved in various inflammatory diseases including Alzheimer's disease . It has a mechanism that interferes with fibril degradation by binding to amyloid fibril, which causes systemic amyloidosis such as dementia. (Bodin K et al., Nature , 468, 93-97, 2010) and the CPHPC specifically binding to SAP were found to be effective when amyloid- A decrease in the amyloid deposit when injected into the blood of patients with amyloidosis (Pepys, MB et al., Nature 417, 254259. 2002) has been reported.

Therefore, when the nanoparticles prepared according to the present invention bind preferentially to SAP binding to amyloid fibrils and decrease the serum concentration of SAP, they can be used for the treatment of amyloid diseases such as Alzheimer's disease. Until now, there has been no report on the production of SAP binding nanoparticles and the detection technique without antibody.

Accordingly, a main object of the present invention is to provide a metal-imprinted metal nanoparticle having high binding force to a serum amyloid P component (SAP) protein.

Another object of the present invention is to provide a chip for SAP detection comprising the SAP-binding metal nanoparticles.

Another object of the present invention is to provide a process for preparing the SAP-binding metal nanoparticles.

It is another object of the present invention to provide a pharmaceutical composition for preventing and treating amyloidosis diseases containing the SAP-binding metal nanoparticles as an active ingredient.

According to one aspect of the present invention, there is provided a metal nanoparticle comprising: a metal nanoparticle; A pinning layer formed on the metal nanoparticles and comprising R ₁ at the azide or acetylene terminal; And a molecular imprinting layer formed on the fixed layer and formed of R ₂ -X-proline (wherein R ₂ represents an acetylene or an azide end group and X represents a spacer group) The proline contained in the molecular imprinting layer is oriented to correspond to the proline binding site of each molecule of serum amyloid P component (SAP) protein, and the immobilization layer and the molecular imprinting layer are formed so as to correspond to R ₁ And R ₂ of the molecular imprinting layer. The metal nanoparticles specifically bind to the SAP protein.

In the present invention, the sensing substance for the SAP protein was prepared by 2D molecular imprint of a proline monomer having specific binding force to SAP in order to increase the binding force to SAP. For this purpose, proline monomer was immobilized on the surface of metal nanoparticles of 50 nm size by 'click' chemistry. (I) an azido-thiol having an azide functional group at one terminal and a thiol functional group at the other terminal, and (ii) an azido- Propargyl functional group and propargyl-proline having proline monomer at the other terminal were synthesized.

The target substance to be sensed in the present invention is a serum amyloid P component (hereinafter referred to as SAP) protein, which is a pentactaxin-based protein having five monomers bonded thereto. It is present in the blood, and has various inflammatory properties including Alzheimer's It is known as a protein involved in diseases. The SAP protein binds to and stabilizes amyloid fibrils and is known to inhibit the normal activity of each tissue. Injecting a substance that specifically binds to SAP into the body reduces the amount of SAP bound to the amyloid fiber by reducing the activity of SAP in the blood, thereby promoting the degradation of the amyloid fibrils. When a substance that binds to the same site as the active site to which the SAP binds to the amyloid fibrils is present in the blood, it inhibits the activity of the SAP, which can be used for the treatment of amyloidosis such as Alzheimer's disease. Each molecule of the SAP protein includes a binding site capable of specifically binding to five proline groups. Accordingly, in the SAP protein-binding metal nanoparticles according to the present invention, the ends of the molecular imprinting layer are oriented such that the proline groups correspond to the proline binding sites of each molecule of the SAP protein .

As used herein, the term 'molecular imprinting material', 'molecular imprinting layer' or 'molecular imprinting monolayer (MIM)' refers to the use of a monomer which is associated with a suitable template Means a monomer arrangement immobilized at the same position as the active site of the template material by removing the template material after forming the 2D monolayer layer. MIM can be used to separate template molecules and other molecules, since only template materials with structurally identical binding sites can be bound to the template material space and molecules with template structures and other stereostructures can not be interrupted. This is because Fischer's Lock-and-Key Concept, in which an antibody formed against an antigen selectively interacts with an antigen, or the Receptor Theory in which an enzyme in a living body is only active against a specific substrate It is. These molecular imprinting techniques have been studied extensively in the field of detecting and separating specific materials using molecularly imprinted polymers. In particular, the selective adsorption of molecular imprinted polymers is known to have a very good effect on the separation of optical isomers.

As used herein, the term " SAP protein " includes proteins capable of binding to the SAP molecule-imprinting metal nanoparticles of the present invention similar in structure to SAP proteins and SAP proteins.

In the present invention, when R ₁ of the fixed layer is an azide end group, R ₂ of the molecular imprinting layer is an acetylene end group, and when R ₁ of the fixed layer is an acetylene end group, R ₂ of the molecular imprinting layer is an azide end But is not limited thereto.

In the present invention, the fixing layer may be a polymer layer having an azide or acetylene terminal R1 on its surface, a self assembly membrane or a mica, but is not limited thereto. For example, the polymer layer, the self-assembled monolayer, or the mica is fixed on the substrate via a thiol group, the surface of which has an azide or acetylene group, Id or an acetylene group. Preferably, the immobilizing layer is a self assembly membrane comprising R ₁ at the azide or acetylene terminal on the surface.

The polymer layer can be used without particular limitation, provided that the polymer layer is fixed on the substrate through a functional group such as a thiol group and has an azide or acetylene group on its surface. For example, the polymer layer can be formed of a polymer selected from the group consisting of polyacrylate, polymethacrylate, ethylene glycol amine, ethylene glycol thiol, Nhydroxysuccinimide (NHS) -ethyleneglycol, maleimide-ethylene glycol, polyethylene glycol amine, polyethylene glycol thiol, - polyethylene glycol, maleimide-polyethylene glycol, polymers based on carbohydrate unit linkages, and combinations thereof.

A bond is formed between the azide group and the acetylene group to form a composite layer of the fixed layer (for example, a spot assembly layer) and the molecular imprinting layer. Therefore, when the azide terminal group is located in the fixed layer, the acetylene terminal group is located in the molecular imprinting layer, and when the acetylene terminal group is located in the fixed layer, the azide terminal group is located in the molecular imprinting layer.

In the present invention, the pinning layer comprises HS-YR ₁ , wherein Y may have 1 to 30 carbon atoms, or 1 to 25 carbon atoms, which may contain at least one of a double bond, a triple bond and an aromatic ring, To 20, or a hydrocarbon group or an oxyhydrocarbon group having 1 to 15 carbon atoms, but is not limited thereto. Preferably, Y is an alkylene group, an alkylene oxide group having 1 to 30 carbon atoms or a hybrid of an alkylene group and an alkylene oxide group, and R ₁ is an azide or acetylene terminal group. More preferably, HS-YR ₁ for the fixed bed is formed HS-C _{1 - 30} alkylene-azide, HS-C _{1 - 30} alkylene oxide-azide, HS-C _{1 - 20} alkylene -C _{1 - 10} alkylene oxide-azide, HS-C _{1 - 30} alkylene-acetylene, HS-C _{1 - 30} alkylene oxide-acetylene or HS-C _{1 - 20} alkylene -C _1-10 alkylene oxide - Acetylene. HS is thiol group or sulfhydryl group.

The metal which can be used as the material of the nanoparticles in the present invention is not particularly limited, and any metal used in the art can be used. For example, metals that can be used in the present invention include gold, silver, copper, platinum, copper, aluminum, palladium, metal alloys thereof (e.g., alloys of gold and copper), and metal oxides. Preferably, the metal is gold (Au).

In the present invention, X of R ₂ -X-proline contained in the molecular imprinting layer is a spacer having a length of 0.1-5.0 nm. The term 'spacer' means an appropriate length of an inorganic or organic molecular compound linking an azide group and proline, or an acetylene group and proline.

In the present invention, the layer imprinted molecule is R ₂ -X- proline (R ₂ comprises an acetylene or azide end groups, X is a spacer) is formed, including the proline contained in the molecule imprinting layer SAP protein are oriented (orientation) so as to correspond to the proline binding site of each molecule and, wherein the fixed layer and the inscription layer molecules click between the R ₂ included in the R ₂ -X- R ₁ proline of imprinting of a fixed bed and the molecular layer (click ) By chemical reaction.

In the present invention, the R ₂ -X-proline includes an acetylene or azide end group as R ₂ , an alkylene group, an alkylene oxide group or a hybrid of an alkylene group and an alkylene oxide group having 1 to 30 carbon atoms as X . Preferably, the R ₂ -X- proline azide -C _{1 - 30} alkylene-proline, azide -C _{1 - 30} alkylene oxide-proline, azide -C _{1 - 20} alkylene -C _{1 - 10} alkylene oxide-proline, acetylene -C _{1 - 30} alkylene-proline, acetylene -C _{1 - 30} alkylene oxide - a proline-proline or acetylenic -C _{1 - 20} alkylene -C _{1 - 10} alkylene oxide.

According to another aspect of the present invention, there is provided a chip for detecting serum amyloid P urea protein comprising the SAP-binding metal nanoparticles.

The SAP detection chip may be prepared by integrating the SAP-binding metal nanoparticles of the present invention on a substrate, or by performing the above-described preparation process of the SAP-binding metal nanoparticles of the present invention on a substrate, . ≪ / RTI > A method for producing SAP-binding metal nanoparticles on a substrate to produce a detection chip can be realized through steps a) to f) of the following production method.

According to another aspect of the present invention, the present invention provides a method for preparing serum amyloid P component (SAP) binding metal nanoparticles comprising the steps of:

a) forming a pinning layer (I) comprising a thiol or amine end on a substrate;

b) fixing metal nanoparticles on the substrate;

c) forming a pinning layer (II) on the nanoparticles comprising R ₁ at the azide or acetylene terminal;

d) R ₂ -X- proline (wherein, R ₂ is azide or acetylene end denotes the short-term, X is a spacer (spacer) represents a) molecular imprinted material and mixing the SAP protein the molecular imprinted material and SAP proteins comprising To form a mixed solution;

e) the molecular imprinting by immersing the material and the substrate provided that the fixing layer (II) to a mixed solution of SAP protein, clicking of the R ₂ contained in R ₁ and wherein the molecular imprinted material is R ₂ -X- proline of the fixed bed ( forming a composite layer of nanoparticles, a fixed bed, a molecular imprinting material and a SAP protein by a chemical reaction;

f) removing the SAP protein from the complex layer to form a molecular imprinting layer; And

g) separating the nanoparticles formed with the molecular imprinting layer from the substrate.

In the method of the present invention, the 'substrate' in step a) is a solid substrate for immobilizing metal nanoparticles during a click reaction and a molecular imprinting reaction, wherein the substrate is an organic polymer or Inorganic materials. Any substrate that can be used in the present invention can be used regardless of the surface characteristics.

The organic polymer may be selected from the group consisting of a polyamide homopolymer or copolymer (e.g., nylon), a heat resistant plastic fluorinated polymer (e.g., polyvinylidene fluoride PVDF), a polyvinyl halide (E.g., polyvinyl chloride PVC), polysulfone, cellulose materials such as paper, nitrocellulose or cellulose acetate, polyolefins, polyacrylamides such as poly (N-isopropylacrylamide), polyglycolic acid (PGA) (PDA), poly-4-hydroxybutyrate, carbon, carbon nanotubes, colloids, and natural polymers such as polylactic acid (PLA), polyglycine 910 (Vicryl), polygluconate Inorganic materials such as glass, quartz, silica, silver, gold, aluminum, copper, other silicon-containing materials (e.g., , A silicon oxide or nitride), metal oxides (e.g., aluminum oxide), metal alloys, Si / SiO2 wafer, germanium and gallium arsenide.

The inorganic substance which can be used as a solid substrate in the present invention is not particularly limited, and any substance used in the art can be used. The surface shape, size and chemical composition of the inorganic solid substrate used in the present invention are not particularly limited, and preferably the substrate is glass. The substrate may also include activating the substrate surface to react with a silane fixing layer (I) comprising a thiol or amine terminus to immobilize gold nanoparticles.

In the process of the invention, step c) and R ₂ of the molecule imprinting layer when the R ₁ of a fixed bed due to azide-terminal in step acetylene end groups, when R ₁ of the fixed bed is due acetylene terminal is R ₂ of the molecule imprinting layer aza Id end term, but is not limited thereto.

In the method of the present invention, the fixing layer in step c) may be, but not limited to, a polymer layer having an azide or acetylene terminal R ₁ on its surface, a self assembly membrane or a mica .

In the method of the present invention, after the complex layer is formed in step (e), the step of treating polyethylene glycol with an azide or acetylene terminal at the end of the fixed layer to which the molecular imprint material is not bonded may be further included. The polyethylene glycol treatment can block the azide or acetylene terminal which did not participate in the click reaction by inducing another click reaction. The non-immobilized portion of proline is blocked by treatment with polyethylene glycol at the azide or acetylene terminal. Preferably, the polyethylene glycol is mPEG350 or mPEG2000 at the azide or acetylene terminal, more preferably mPEG350 or mPEG2000 at the acetylene terminus, even more preferably mPEG350 or mPEG2000 at the propargyl end. The azide or acetylene terminal polyethylene glycol is prepared and polymerized on the fixed bed portion where the molecular imprinting material is not bonded as a click reaction.

The reason for the PEG treatment is as follows. In order to minimize nonspecific binding of proteins other than SAP in the production of SAP molecule-imprinted gold nanoparticles according to the present invention, a substance which inhibits non-specific binding to the azide terminal of the fixed layer (II) in which propargyl- Blocking process is required. mPEG polymers are well known as substances that inhibit non-specific binding of proteins such as albumin in vivo.

In the method of the present invention, pre-incubation of the molecular imprinting material and the SAP protein mixture solution before the step d) is carried out before the step e), so that the proline derived from the molecular imprinting material binds to the proline binding site to correspond to a binding site.

In the present invention, in the process of pre-incubation with the SAP protein, propargyline is arranged in a direction substantially perpendicular to the SAP cross section and polymerized by azide and click reaction in the state of binding to SAP, whereby proline is bound to five binding sites of SAP Can be oriented to fit well.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating amyloidosis diseases comprising the SAP-binding metal nanoparticles together with a pharmaceutically acceptable carrier.

The pharmaceutical composition may preferentially bind to SAP binding to amyloid fibrils in the body to decrease the concentration of SAP in the blood, thereby promoting the degradation of amyloid fibrils and thus being used for the treatment of amyloidosis diseases.

The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate into injectable solutions, powders, tablets, capsules, rings, granules or injection solutions such as aqueous solutions, suspensions and emulsions. Further, it can be suitably formulated according to the disease or component according to a method known in the art or using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In a preferred embodiment of the present invention, the SAP binding metal nanoparticles were used to inhibit SAP binding to amyloid fibrils in vitro. As a result, inhibition of binding of SAP-amyloid fibrils to a level similar to that of SAP antibodies (See FIG. 8). In addition, the SAP-binding metal nanoparticles of the present invention were administered to a model of amyloidosis-inducing animal such as dementia, and it was confirmed that they have the effect of alleviating dementia symptoms (see FIG. 9).

The pharmaceutical composition of the present invention may be administered orally or parenterally. Parenteral formulations preferably comprise a sterile aqueous solution or suspension comprising the SAP-binding metal nanoparticles of the invention, and various techniques for the preparation of pharmaceutical aqueous solutions or suspensions are known in the art. The solution may also contain pharmaceutically acceptable buffers, stabilizers, antioxidants and electrolytes such as sodium chloride. Parenteral formulations may be injected directly or mixed with large amounts of parenteral formulations.

Formulations for oral administration may vary widely and are known in the art. Such formulations generally comprise an aqueous solution or suspension comprising a therapeutically effective amount of an SAP-binding metal nanoparticle according to the present invention. The oral formulations may optionally include buffers, surfactants, adjuvants, thixotropic agents, and the like. Formulations for oral administration may also contain ingredients for increasing spices and other functionalities. The appropriate dosage of the nanoparticles contained in the pharmaceutical composition may be appropriately selected depending on the condition, age, age, sex, body weight, etc. of the patient. An effective amount can be administered over a period of days to days, for example, by adjusting the concentration and dose to include about 10 < ² > to about 10 < ¹⁸ > nanoparticles (NPs).

Hereinafter, the method for producing the SAP-binding metal nanoparticles of the present invention will be described in more detail by step.

a) Above the substrate The fixing layer (I)  Forming step

First, a pinning layer comprising a thiol or amine terminus is formed on a substrate. According to a preferred embodiment of the present invention, the immobilizing layer is a single membrane layer, a polymer layer, or a self assembly membrane containing a thiol or amine terminus on its surface.

b) Above the substrate Immobilized  step

The nanoparticles are treated to react with the ends of the fixed layer (I), and the metal nanoparticles are immobilized on the substrate by adsorption or chemical bonding using electrical bonding, ionic bonding, adsorption or van der Waals bonding force .

c) On nanoparticles The fixed bed (II)  Forming step

To form a complex layer with a molecular imprinting layer as a fixed layer containing an azide or acetylene terminal group on its surface. Therefore, when the azide terminal group is located in the fixed layer, the acetylene terminal group is located in the molecular imprinting layer, and when the acetylene terminal group is located in the fixed layer, the azide terminal group is located in the molecular imprinting layer.

d) Molecular imprint  Materials and SAP Lt; RTI ID = 0.0 >

Subsequently, the molecular imprinting material and the SAP protein are mixed to prepare a mixed solution of the molecular imprinting substance / SAP protein. The molecular imprinting material and the SAP protein are reacted in an appropriate ratio to induce the proline group to bind to each of the five SAP units. The ratio of the molecular imprinting material to the SAP protein is preferably 5: 1.

In addition, pre-incubating the molecular imprinting material and the SAP protein mixed solution after the step d) and orienting the proline derived from the molecular imprinting material so as to correspond to the proline binding site of each molecule of the SAP protein .

e) Fixed bed , Molecular imprint  Materials and SAP  Step of complex layer formation of protein

The substrate on which the fixed layer is formed is immersed in a mixed solution of molecular imprinting material / SAP protein in step d), and the click chemistry of R2 contained in R2 of the fixed layer and the molecular imprinting material R2-X- .

In step d), a complex of a molecule-imprinting material and a SAP protein is formed, and then the complex is added to a solution in which nanoparticles coated with a polymer having an azido or acetylene terminal are immersed to cause a click reaction between the azido and acetylene ends do. Through the click reaction, a proline group capable of binding SAP to the nanoparticle surface is arranged.

f) Molecular imprinting layer  Forming step

The SAP protein is removed from the complex layer to form a molecular imprinting layer. The method of removing the SAP protein from the complex layer can be performed by various known methods related to protein modification. For example, the SAP, which is the template protein, can be removed by immersing the substrate immobilized with the nanoparticles in a 2% SDS solution.

The ability of SAP binding to the SAP molecule imprinted nanoparticles produced can be measured using immune detection with the nanoparticles immobilized on the substrate. The SAP-binding nanoparticles of the present invention can provide nanoparticles that inhibit SAP-amyloid binding while exhibiting a higher binding amount than the surface that is not imprinted on the molecular-engraved surface.

g) Molecular imprint  Step of liberating nanoparticles from the substrate

The nanoparticles on the substrate can be advantageously treated by acid, base, heat, or ultrasonic waves depending on the binding method, and finally, SAP-bonded nanoparticles can be provided.

In the evaluation of bonding ability to SAP, high binding force was shown for the molecule-imprinted nanoparticles. Based on this binding force, it showed the property of inhibiting SAP binding to amyloid fibers. In conclusion, the nanoparticles immobilized with proline monomers in a SAP molecule-imprinted array inhibited the binding of SAP-amyloid fibers in vitro and delayed the dementia symptoms in vivo experiments.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a metal imprinting metal nanoparticle having binding ability to SAP.

(b) The SAP-binding metal nanoparticles of the present invention can be prepared by forming an azide-terminal self-assembled monolayer on the surface of nanoparticles and forming a molecule by azeotrope-self- To form an imprinting layer.

(c) The present invention relates to a method for immobilizing nanoparticles on a substrate to form a molecular imprinting layer, and finally separating the nanoparticles from the substrate, in order to solve problems caused by precipitation of nanoparticles in a click reaction.

(d) Propargyl-proline, which forms the molecular imprinting layer, forms a molecule imprinting layer on the azide-terminal self-assembled monolayer formed on the surface of the nanoparticles and is easily bonded by a click reaction to facilitate molecular imprinting .

(e) The SAP-binding metal nanoparticles of the present invention also have the property of inhibiting the SAP binding to the amyloid fiber and alleviating the production of amyloid fiber in vivo.

(f) In mouse in vivo experiments, it was observed to delay dementia when injected with SAP-conjugated nanoparticles.

FIG. 1 schematically shows a process for preparing SAP molecule-imprinting metal nanoparticles (SAM: self assembly membrane).
Fig. 2 schematically shows a schematic diagram of a click reaction in the molecular engraving process.
FIG. 3 shows the results of analyzing the amount of SAP binding to the surface of SAP molecule-imprinted gold nanoparticles.
FIG. 4 shows the result of analysis of the size distribution of molecular gold nanoparticles by dynamic light scattering (DLS): BG, bare gold; C, CM; N, NIM; M, MIM.
FIG. 5 is a TEM photograph of the gold nanoparticles separated by ultrasonic waves from a glass substrate; BG, bare gold; C, CM; N, NIM; M, MIM.
6 is a micrograph of the sequence of the amyloid peptide and the amyloid fibril. It is the result of comparison of SAP bonding amount by immunochemical method.
Figure 7 shows the result of confocal microscopy comparing the inhibition of SAP-amyloid fiber binding by molecularly imprinted gold nanoparticles.
FIG. 8 shows the result of analysis of the degree of inhibition of binding of SAP-amyloid fibers by molecular gold nanoparticles using a fluorescence analyzer.
FIG. 9 shows the results of observing inhibition of dementia by molecular imprint gold nanoparticles in dementia-induced rats.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Manufacturing example  One. Azayido Thiol  synthesis

[Reaction Scheme 1]

1.40 g (5.02 mmol) of 11-mercaptotung decanoic acid, 4.58 mmol (11-murcaptoundecanoic acid), trityl chloride and 0.95 g (6.87 mmol; potassium carbonate) of potassium carbonate were dissolved in acetonitrile 20 mL, and the mixture was stirred while heating to 100 ° C. After 7 hours of reaction, acetonitrile was removed by distillation under reduced pressure, dissolved in chloroform, and washed several times with 0.1 N hydrochloride aqueous solution. The chloroform was collected and dried with adding magnesium sulfate, and the dried chloroform was filtered under reduced pressure to remove magnesium sulfate. Thereafter, the chloroform was distilled off under reduced pressure, and the residue was loaded with a developing solution (hexane: ethyl acetate = 9: 1) to remove trityl chloride. The residue was purified by column chromatography using a developing solution (hexane: ethyl acetate = 1: 1) 1.76 g (yield, 83%) of Compound 1 (11- (tritythio) undecanoic acid) was obtained.

[Reaction Scheme 2]

5.00 g (33.3 mmol) of triethylene glycol and 2.70 mL (19.40 mmol) of triethylamine were added to 50 mL of chloroform, cooled to 0 ° C using an ice-bath, and then 1.00 mL 12.90 mmol; methane sulfonyl chloride) was added slowly. After the addition, the ice bath was removed and the mixture was stirred at room temperature. After the reaction for about 20 hours, the reaction solution was removed by distillation under reduced pressure, and impurities were removed as much as possible by using a developing solution (dichloromethane: methanol = 95: 5) to obtain 2.55 g of a product. 1.10 g (16.90 mmol; sodium azide) and 1.40 g (8.43 mmol; potassium iodide) of the product were added to 50 mL of ethanol and the mixture was stirred at 110 ° C. After about 14 hours of reaction, the reaction mixture was distilled off under reduced pressure, dissolved in water, and extracted several times with chloroform. The extracted chloroform was collected and dried with magnesium sulfate, and dried chloroform was filtered under reduced pressure to remove magnesium sulfate. Thereafter, the resultant was distilled under reduced pressure to remove chloroform to obtain 0.91 g (yield, 40%) of Compound 2 (2- (2- (2-azidoethoxy) ethoxy) ethanol).

Ethyl-3- (3-tert-butoxycarbonyl) ethoxy] ethanol and 1.76 g (3.81 mmol) of 1- (tritylthio) undecanoic acid, Dimethylaminopropyl) carbodiimide hydrochloride (1.10 g, 5.74 mmol) and N, N'-dimethylaminopyridine (0.70 g, 5.73 mmol) were added to 20 mL of chloroform and the mixture was stirred at room temperature. After about 17 hours of reaction, the reaction solution was diluted with chloroform and washed several times with 0.1 N aqueous hydrochloric acid solution and brine. The chloroform was collected and dried with magnesium sulfate, and the dried chloroform was filtered under reduced pressure to remove magnesium sulfate. The magnesium sulfate was removed by distillation under reduced pressure, and the residue was purified by column chromatography using a developing solution (hexane: ethyl acetate = 3: 1) (Yield: 79%) of 3 (2- (2- (2-azidoethoxy) ethoxy) ethyl 11- (tritylthio) undecanoate.

[Reaction Scheme 3]

1.80 g (2.92 mmol) of ethyl 11- (tritylthio) undecanoate, 0.56 mL (3.51 mmol) of triethylsilane and 2.00 mL of trifluoroacetic acid Was added to chloroform (8 mL), and the mixture was stirred at room temperature. After about 4 hours, the reaction solution was diluted with chloroform and washed several times with an aqueous sodium bicarbonate solution. The chloroform was collected and dried with magnesium sulfate, and the dried chloroform was filtered under reduced pressure to remove magnesium sulfate. The chloroform was distilled off under reduced pressure, and the residue was purified by column chromatography using a developing solution (hexane: ethyl acetate = 3: 1) (Yield: 43%) of 2- (2- (2-azidoethoxy) ethoxy) ethyl 11-thioundecanoate.

Manufacturing example  2. Propargill - Synthesis of proline monomer

2-1. One- Hex -5- Inoylpyrrolidine -2- Carboxylate  Synthesis method

[Reaction Scheme 4]

1.00 g (4.81 mmol) of D-proline t-butyl ester hydrochloride and 1.26 mL (7.23 mmol) of diisopropylethylamine were added to 10 mL of N, N'-dimethylformamide and stirred for about 10 minutes. 1.38 g (7.20 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.97 g (7.18 mmol) of 1-hydroxybenzotriazole were added, Lt; / RTI > After completion of the reaction, N, N'-dimethylformamide was removed by distillation under reduced pressure, dissolved in chloroform, and then washed with water and sodium bicarbonate aqueous solution. The washed aqueous solutions were back-extracted several times with chloroform, and all chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate and the chloroform was removed by distillation under reduced pressure. The residue was purified by column chromatography using a developing solvent (hexane: ethyl acetate = 2: 1) to obtain compound 5 (Tert-butyl 1-hex- ynoylpyrrolidine-2-carboxylate) (yield, 89%).

[Reaction Scheme 5]

Butyl-1-hex-5-inoylpyrrolidine-2-carboxylate (1.13 g, 4.26 mmol) and trifluoroacetic acid (1 mL) were added to 9 mL of dichloromethane and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was removed by distillation under reduced pressure, and the residue was purified by column chromatography using a developing solution (dichloromethane: methanol = 95: 5) to obtain 0.79 g of 1-hex-5-ynoylpyrrolidine- (Yield, 88%).

2-2. Rat -Butyl 1- Undec -10- Inoylpyrrolidine -2- Carboxylate  Synthesis method

[Reaction Scheme 6]

1.00 g (4.81 mmol) of D-proline t-butyl ester hydrochloride and 1.26 mL (7.23 mmol) of diisopropylethylamine were added to 10 mL of N, N'-dimethylformamide and stirred for about 10 minutes. 1.38 g (7.20 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.97 g (7.18 mmol) of 1-hydroxybenzotriazole And reacted for about 24 hours. After completion of the reaction, N, N'-dimethylformamide was removed by distillation under reduced pressure, dissolved in chloroform, and washed with water and sodium bicarbonate aqueous solution. The washed aqueous solutions were back-extracted several times with chloroform, and all the chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate, and the chloroform was removed by distillation under reduced pressure. The residue was purified by column chromatography using a developing solution (hexane: ethyl acetate = 2: 1) to obtain Compound 7 (Tert-butyl 1-undec- 10-ynoylpyrrolidine-2-carboxylate) (yield, 89%).

[Reaction Scheme 7]

Butyl-1-undec-10-inoylpyrrolidine-2-carboxylate (1.35 g, 4.02 mmol) and trifluoroacetic acid (1 mL) were dissolved in dichloromethane (9 mL), and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was removed by distillation under reduced pressure and purified by column chromatography using a developing solution (dichloromethane: methanol = 95: 5) to obtain 0.80 g of 1-undec-10-ynoylpyrrolidine- (Yield, 72%).

Manufacturing example  3. Propargill - mPEG Synthesis of

[Reaction Scheme 8]

1.00 g (2.86 mmol) of methoxypoly (ethylene glycol) Mn 350 and 0.27 g (5.72 mmol) of sodium hydride were added to 10 mL of N, N'-dimethylformamide and cooled to 0 ° C in an ice bath. 0.51 mL (11.2 mmol) of propargyl bromide was slowly added to the cooled reaction solution, and the reaction mixture was stirred at room temperature for 24 hours. After the completion of the reaction, the reaction solution was distilled off under reduced pressure, dissolved in water, and then extracted with chloroform several times. The extracted chloroform was collected and dried with magnesium sulfate, and the dried chloroform was filtered under reduced pressure to remove magnesium sulfate. The chloroform was removed by distillation under reduced pressure, and the residue was loaded with a developing solution (dichloromethane: methanol = 95: 5) Dichloromethane: methanol = 9: 1) afforded 0.89 g of compound 9 (yield, 80%).

1.00 g (1.33 mmol) of methoxypoly (ethylene glycol) Mn 750 and 0.13 g (5.42 mmol) of sodium hydride were added to 10 mL of N, N'-dimethylformamide and cooled to 0 ° C in an ice bath. 0.24 mL (2.69 mmol) of propargyl bromide was slowly added to the cooled reaction solution, the ice bath was removed, and the mixture was stirred at room temperature for 24 hours. After the completion of the reaction, the reaction solution was distilled off under reduced pressure, dissolved in water, and then extracted with chloroform several times. The extracted chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate, and chloroform was removed by distillation under reduced pressure. The residue was recrystallized in ether to obtain 0.50 g (yield, 48% .

1.00 g (0.50 mmol) of methoxypoly (ethylene glycol) Mn 2000 and 0.05 g (2.00 mmol) of sodium hydride were added to 10 mL of N, N'-dimethylformamide and cooled to 0 ° C in an ice bath. To the cooled reaction solution was slowly added 0.10 mL (1.12 mmol) of propargyl bromide, and the ice bath was removed, followed by stirring at room temperature for 24 hours. After the completion of the reaction, the reaction solution was distilled off under reduced pressure, dissolved in water, and then extracted with chloroform several times. The extracted chloroform was collected and dried with magnesium sulfate. The dried chloroform was filtered under reduced pressure to remove magnesium sulfate. The chloroform was removed by distillation under reduced pressure, and then recrystallized in ether (twice) to obtain 0.88 g of Compound 11 (yield: 86% .

Example  1. Click reaction SAP Molecular imprint  Manufacture of gold chips

The overall manufacturing process is the same as in FIG. The molecular formula of the substance used in the manufacturing process and the reaction process are shown in FIG.

1-1. Immobilization of gold nanoparticles on glass substrate

A glass plate treated with a pyran solution (3: 7 H ₂ O ₂ : Conc. H ₂ SO ₄ ) was immersed in a 1% APTMS (3-aminopropyl trimethoxysilane) solution and reacted at room temperature for 8 hours. The glass plate was rinsed well with toluene, immersed in distilled water containing 50 nm gold nanoparticles dispersed therein, and reacted at room temperature for 12 hours. The glass plate on which the gold nanoparticles were immobilized was washed well with ethanol and water, and immersed in a solution of 2 mM azido thiol (compound 4 produced in Preparation Example 1) to self-assemble the azodithiol layer to the gold nanoparticle surface .

1-2. Molecular imprint  Nanoparticle manufacturing

To prepare a molecularly imprinted monolayer (MIM), 3.32 nmol of propargyl proline (Compound 6 prepared in Preparation 2) was dissolved in 1 mL of binding buffer (0.1 M Tris / HCl , 150 mM NaCl, 5 mM CaCl ₂ , pH 8.0) at a molar ratio of 0.66 nmol SAP (propargyl-proline: SAP = 5: 1). The 1,3-dipolar cycloaddition reaction, which is one of the click chemistries, contained 1.66 nmol of copper sulfate (II) · pentahydrate, 3.32 nmol sodium ascorbate and 16 hours of preliminary Gold nanoparticles coated with a self-assembled monolayer of 11-azidodecane-1-thiol were immersed in a 20 mL PBS solution containing the cultured propargyl proline-SAP complex. The molar ratio of propargyl proline: copper sulfate: sodium ascorbate was 1: 0.5: 1.

To prepare a non-imprinted monolayer (NIM), all steps are the same as in a molecularly imprinted monolayer except that SAP is not added to the reaction mixture. After the reaction was completely finished, the surface was thoroughly rinsed with cold PBS and dried under a nitrogen gas.

Propargyl-mPEG was added to the substrate (gold-imprinted gold nanoparticles) for another click reaction to block the azide end that did not participate in the click response. The substrate was immersed in 20 mL PBS containing 20 μmol propargyl-mPEG, 10 μmol copper sulfate (II) · hexahydrate, and 20 μmol sodium ascorbate for 5 hours at room temperature.

To prepare a control monolayer (CM), gold nanoparticles coated by N ₃ -terminal self-assembled monolayers were modified directly by propargyl-mPEG (350 and 2000) by a click reaction.

In order to remove SAP from the molecule-imprinted monolayer, it was immersed in a 2% SDS solution at room temperature for 30 minutes. Finally, the substrate was thoroughly washed with deionized water, acetone, and ethanol, and dried under nitrogen gas.

1-3. DLS  And TEM Using Molecular imprint  Gold nanoparticle size analysis

As a result of dynamic light scattering (DLS) analysis, the particle size of CM, NIM and MIM was increased as compared to Bare gold (FIG. 3). The mean particle size of bare gold was 15 nm, whereas the particle size of CM, NIM and MIM was 54, 50 and 63 nm, respectively. In TEM (transmission electron microscope) analysis, a film appeared as an organic layer in the CM, NIM, and MIM, and the surface modification by the organic layer was observed (FIG. 4).

1-4. Molecular imprint  Separation of nanoparticles

The glass substrate on which gold nanoparticles were immobilized was placed in a beaker containing distilled water and sonicated for 30 minutes in an ultrasonic washing machine. The distilled water containing gold nanoparticles dispersed from the glass plate was centrifuged at 10,000 rpm for 5 minutes to remove the distilled water and re-dispersed in a buffer solution such as PBS.

Example  2. SAP Molecular imprint  For gold chip surface SAP  Binding ability assessment

To analyze the amount of SAP bound to CM, NIM and MIM, immunoassay was performed. The glass substrate immobilized with gold nanoparticles was immersed in 10 nM SAP solution to bind the SAP to the molecularly imprinted proline binding site, and the anti-rabbit IgG antibody (rabbit, IgG fraction) and HRP (horse raddish peroxidase) Were successively treated, developed with TMB solution, and absorbance was measured at 450 nm. NIM showed a background binding similar to that of CM, whereas MIM showed increased SAP binding in all of the mPEG350 and mPEG2000 blocked nanoparticles (FIG. 5).

Example  3. SAP Molecular imprint  Of gold nanoparticles SAP -amyloid Fibril  Binding inhibition effect

Experiments were conducted to determine whether the gold nanoparticles synthesized in the present invention inhibited binding between SAP-amyloid fibrils. The amyloid fibril bond of SAP is made in the presence of calcium ions and is known to be on the same plane as the binding site of the proline ligand. Amyloid fibrils were prepared by preparing amyloid beta peptide (42 amino acids) shown in Fig. 6 and reacting in 50 mM borate buffer (pH 9.0) for 7 days. The resulting amyloid fibrils were observed by reversed-phase microscopy, TEM, and fluorescence microscopy (Thioflavin T staining) (Fig. 6).

SAP (FITC-SAP) labeled with FITC was used to detect the degree of binding of SAP to amyloid fibrils. MIM-mPEG350 and MIM-mPEG2000 were added together with FITC-SAP and 0.5 mg of amyloid fibril, respectively, and the mixture was reacted at 37 DEG C for 30 minutes. As a control, a sample not containing gold nanoparticles and a sample to which gold nanoparticles coated with anti-SAP antibodies were added was used. Unreacted SAP was removed by passing through a 0.2 μm filter, washed twice with borate buffer, and the fraction remaining in a 0.2 μm filter was redissolved in borate buffer and observed with a confocal microscope (FIG. 7). As a result, it was observed that the amount of FITC-SAP binding was significantly reduced in samples containing molecularly-imprinted gold nanoparticles (MIM-mPEG350 and MIM-mPEG2000) compared to control samples not containing gold nanoparticles. This was observed in a similar pattern to that of amyloid fibrils with added gold nanoparticles coated with antibody.

To quantitatively analyze the binding amount of FITC-SAP, the FITC intensity included in the sample was measured using a fluorescence reader of each sample (FIG. 8). As a result, the intensity of the FITC fluorescence of the sample containing the gold nanoparticles with molecular imprint was reduced by about 30% compared with the negative control image similar to the image obtained by the confocal microscope. These results are similar to those obtained by adding gold nanoparticles coated with anti-SAP antibodies as observed in a confocal microscope.

As a result, molecularly imprinted gold nanoparticles have the property of inhibiting the binding of SAP-amyloid fibrils through a specific reaction with SAP, and this property is observed to be similar to that of SAP antibodies.

Example  4. Mouse in vivo Of dementia

Molecular imprinted gold nanoparticles (MIM-mPEG2000) were injected into the C57 mouse tail vein twice a week to induce dementia disease by injecting amyloid beta into the brain of C57 mice. The severity of dementia was assessed by measuring the escape latency and swimming distance in a Water maze experiment (Morris R, J Neurosci Methods 1984, 11: 47-60). As shown in FIG. 9, the mice injected with MIM showed a 25% decrease in the escape time and escape distance compared with the mice not injected, and the symptoms of dementia were alleviated.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (18)

Metal nanoparticles;
Formed on the metal nano-particles, HS-YR ₁ (where the Y is a hybrid containing 1 to 30 carbon atoms in the alkylene group, an alkylene oxide group or an alkylene group and an alkylene oxide group, wherein R ₁ is azide or An acetylene terminal group); And
A molecular imprinting layer formed on the fixed layer and formed of R ₂ -X-proline, wherein R ₂ represents acetylene or an azide end group, and X represents a spacer group,
The proline contained in the molecular imprinting layer is oriented to correspond to the proline binding site of each molecule of serum amyloid P component (SAP) protein,
Wherein the fixed layer and the molecular imprinting layer are bonded by a click chemistry between R ₁ of the fixed layer and R ₂ of the molecular imprinting layer.
The method of claim 1, wherein when the R ₁ of the fixed bed due to azide-terminal R ₂ of the molecule imprinting layer is acetylene end groups, when R ₁ of the fixed bed is due acetylene terminal is R ₂ of the molecule imprinting layer azide Wherein the SAP binding metal nanoparticle is an end group.
[2] The SAP binding metal nanoparticle according to claim 1, wherein the fixing layer is a polymer layer having an azide or acetylene terminal R ₁ on its surface, a self assembly membrane or a mica.
delete The method of claim 1, wherein the HS-YR ₁ is HS-C _1-30 alkylene-azide, HS-C _1-30 alkylene oxide-azide, HS-C _1-20 alkylene -C _1-10 Alkylene oxide-azide, HS-C _1-30 alkylene-acetylene, HS-C _1-30 alkylene oxide-acetylene or HS-C _1-20 alkylene-C _1-10 alkylene oxide- Features SAP-binding metal nanoparticles.
The SAP binding metal nanoparticle according to claim 1, wherein the metal nanoparticles are gold nanoparticles.
2. The SAP-binding metal nanoparticle according to claim 1, wherein X is a spacer having a length of 0.1-5.0 nm.
The method of claim 1, wherein the molecular imprinting layer comprises R ₂ -X-proline, R ₂ is acetylene or an azide end group, and X is an alkylene group, alkylene oxide group or alkyl Wherein the SAP binding metal nanoparticle is formed by incorporating a hybrid of an alkylene oxide group and an alkylene oxide group.
The method of claim 8, wherein R ₂ -X- proline azide -C _1-30 alkylene-proline, azide -C _1-30 alkylene oxide-proline, azide -C _{1 - 20} alkylene -C _{1 - 10} alkylene oxide-proline, acetylene -C _1-30 alkylene-proline, acetylene -C _{1 - 30} alkylene oxide-proline or acetylenic -C _{1 - 20} alkylene -C _1-10 alkylene oxide-proline ≪ RTI ID = 0.0 &
A chip for detecting serum amyloid P urea protein comprising SAP binding metal nanoparticles according to any one of claims 1 to 3 and 5 to 9.
A method for preparing serum amyloid P component (SAP) -binding metal nanoparticles comprising the steps of:
a) forming a pinning layer (I) comprising 3-aminopropyl trimethoxysilane (APTMS) on a substrate;
b) fixing metal nanoparticles on the substrate;
c) HS-YR ₁ on the nanoparticles, wherein Y is an alkylene, alkylene oxide or alkylene oxide group having 1 to 30 carbon atoms or an alkylene oxide group and R ₁ is an azide or acetylene Forming a pinned layer (II) formed thereon;
d) R ₂ -X- proline (wherein, R ₂ is azide or acetylene end denotes the short-term, X is a spacer (spacer) represents a) molecular imprinted material and mixing the SAP protein the molecular imprinted material and SAP proteins comprising To form a mixed solution;
e) the molecular imprinting by immersing the material and the substrate provided that the fixing layer (II) to a mixed solution of SAP protein, clicking of the R ₂ contained in R ₁ and wherein the molecular imprinted material is R ₂ -X- proline of the fixed bed ( forming a composite layer of nanoparticles, a fixed bed, a molecular imprinting material and a SAP protein by a chemical reaction;
f) removing the SAP protein from the complex layer to form a molecular imprinting layer; And
g) separating the nanoparticles formed with the molecular imprinting layer from the substrate.
12. The method of claim 11, wherein the step c) the R ₁ of a fixed bed azide R ₂ of the molecule imprinting layer if the terminal group is an acetylene end groups, when R ₁ of the fixed bed is due acetylene terminal is R ₂ of the molecule imprinting layer aza from Id end group.
[12] The method of claim 11, wherein the fixed layer in step c) is a polymer layer having an azide or acetylene terminal R ₁ on its surface, a self assembly membrane or a mica.
[12] The method of claim 11, further comprising, after forming the composite layer in step e), treating the polyethylene glycol having the azide or acetylene terminal at the fixed layer portion to which the molecular imprinting material is not bonded, Way.
12. The method according to claim 11, wherein the step of performing the step d) further comprises the step of pre-culturing the molecular imprinting material and the SAP protein mixed solution before the step e), so that the proline derived from the molecular imprinting material binds to the proline binding site to correspond to a binding site of the antibody.
12. The method according to claim 11, wherein X in step d) is a spacer having a length of 0.1-5 nm.
9. A pharmaceutical composition for the prevention and treatment of amyloidosis diseases comprising an SAP-binding metal nanoparticle according to any one of claims 1 to 3 and 5 to 9 together with a pharmaceutically acceptable carrier.
18. The pharmaceutical composition according to claim 17, which preferentially binds to SAP binding to amyloid fibrils in the body to promote the degradation of amyloid fibrils by reducing the concentration of SAP in the blood.

Images