JP2008189784A - Stimulus-responsive protein nanoparticles - Google Patents

Abstract

A stimulus-responsive cross-linked protein micelle comprising a cross-linked protein structure is provided.
The protein constituting the structure preferably has an aspartic acid or glutamic acid residue, and a lysine residue; and the cross-linking is preferably a -COOH group of the aspartic acid or glutamic acid residue And a lysine residue or an N-terminal -NH ₂ group. A particularly preferred example of the protein used is β-casein. The cross-linked protein micelle of the present invention has an inherent lower critical solution temperature (LCST) that dissolves on the low temperature side and insolubilizes on the high temperature side at a specific temperature. The stimuli-responsive crosslinked protein micelle of the present invention is expected to be applied in biological separation, biosensing, immobilized enzyme, and drug delivery system.
[Selection figure] None

Description

  The present invention relates to a stimulus-responsive crosslinked protein micelle comprising a crosslinked protein structure. The cross-linked protein micelle of the present invention has an inherent lower critical solution temperature (LCST) that dissolves on the low temperature side and insolubilizes on the high temperature side at a specific temperature. The stimuli-responsive crosslinked protein micelle of the present invention is expected to be applied in biological separation, biosensing, immobilized enzyme, and drug delivery system.

  In recent years, research in a new biotechnology (nanobiotechnology) field that utilizes the properties of materials on a nanoscale has become active. In this field, by designing the composition and arrangement of macromolecules and peptides, and adding the self-organization ability peculiar to the arrangement or composition, a nanoscale structure consisting of these self-organizations is bottom-up. Research to create is actively conducted.

  Above all, nanostructures that exhibit some kind of response to external stimuli such as temperature, electricity, and photochemical stimuli are attracting attention as intelligent materials, and are applied to encapsulation, drug delivery, nano or micro size reactors, etc. Is expected. For example, poly-N-isopropylacrylamide (PNIPAAm) is known as a biocompatible material that responds to external stimuli such as temperature and pH (Patent Document 1). The solubility of PNIPAAm changes discontinuously and reversibly with temperature change of the aqueous solution (temperature-responsive polymer). In addition, a temperature-responsive protein called Elastin-Like Protein (ELP) (Non-patent Document 1) has attracted attention, and research utilizing the temperature responsiveness has been underway. For example, various applications have been studied by incorporating peptide sequences exhibiting ELP temperature responsiveness into heterologous proteins by genetic recombination using genetic recombination techniques.

In addition, polymer gel fine particles (nanogels) obtained by subjecting nanosize polymer fine particles to three-dimensional network crosslinking are attracting attention in the fields of life science and bio-nanotechnology. Nanogels are more resistant to degradation than macromolecules, can incorporate substances into the gel matrix, and have features such as the size being suitable as the size of the drug carrier, Application to drug delivery systems is expected. For example, nanoparticles in which micelles formed by a block copolymer having a carboxyl group or an amino group are crosslinked with diamine and EDC or glutaraldehyde have been reported (Non-patent Document 2). The particles are said to be responsive to swelling. In addition, it has been reported that block copolymers are made into micelles with Ca ²⁺ ions to form micelles, and then crosslinked with diamine and EDC to produce nanoparticles (Non-patent Document 3). Since the swelling behavior of these particles changes in response to pH, the possibility of use in DDS is considered. Furthermore, a material in which a gold nanoparticle is used as a core and the surface thereof is coated with a polyacrylic acid derivative polymer is surface-crosslinked with diamine using EDC (Non-patent Document 4).

Meanwhile, casein is the major phosphoprotein milk _{protein, α s1 -, α s2 -} , β-, and a four-casein components called κ- casein. Each of these casein components alone does not have a specific higher order structure, but due to its high amphiphilicity and high surface activity, it is known that it forms a unique molecular assembly in water depending on its concentration and environment. (Non-Patent Document a). That is, casein has a highly flexible micelle-like form. Casein is also an inexpensive protein that is abundant in nature. The present inventors tried to prepare a nanoscale protein structure by covalently crosslinking the micelle structure formed by casein by a chemical method. According to this method, since a synthetic polymer or a synthetic peptide is not required, nanoparticles can be prepared extremely easily, in a short time, and at a low cost compared to existing nanomaterials. Applications are also expected. The present inventors have reported that nanoparticles can be prepared by enzymatic crosslinking of casein micelles with transglutaminase (Patent Document 2).
Japanese Patent Laid-Open No. 9-16950 (Patent No. 3807765) Japanese Unexamined Patent Publication No. 2006-115751 Arthur S. Tatham and Peter R. Shewry., Trends in Biochemical Sciences, 25, 567-571, 2000 Biomacromolecules, 6, 2213-2220, 2005) J. Am. Chem. Soc., 127, 8236-8237, 2005) Angew. Chem. Int. Ed., 44, 409-412, 2005) David S. Horne., Current Opinion in Colloid and Interface Science, 7, 456-461, 2002

None of the nanoparticles described above exhibit thermal responsiveness.
The present inventors have now obtained a nanoscale protein structure by chemically crosslinking the micelle structure formed by β-casein using a water-soluble carbodiimide (WSC) solution. As a result of functional evaluation, it was found that the cross-linked β-casein micelle exhibits responsiveness to external factors such as temperature and pH, and the present invention has been completed.

[Crosslinked protein micelle]
The present invention provides the following:
1) A stimuli-responsive crosslinked protein micelle comprising a crosslinked protein structure.
2) The protein constituting the structure has an aspartic acid residue or glutamic acid residue, and a lysine residue or an N-terminal -NH ₂ group; and the cross-linking is an aspartic acid residue or a glutamic acid residue- The cross-linked protein micelle according to 1) above, which is formed by a condensation reaction between a COOH group and a lysine residue or an N-terminal -NH ₂ group.
3) The crosslinked protein micelle according to 2) above, wherein the protein is β-casein.
4) A modified crosslinked protein micelle, wherein the surface of the crosslinked protein micelle according to any one of 1) to 3) above is modified.

The crosslinked protein micelle of the present invention (in the present specification, sometimes referred to as “nanoparticle” of the present invention) is composed of an amphipathic protein capable of forming micelles in a suitable solvent as a material. Proteins that are cross-linked. The protein that can be used as a material is not particularly limited as long as it can form micelles and can crosslink proteins that constitute micelles by an appropriate method. From the viewpoint that the obtained crosslinked protein micelle can have sufficient stimulus responsiveness described later, a protein having a free —COOH group and —NH ₂ group, for example, an aspartic acid residue or It is preferable to use a protein having a glutamic acid residue and a lysine residue or an N-terminal —NH ₂ group (α-amino group). When such a protein is used, the cross-linking can be formed by a condensation reaction between a —COOH group and a —NH ₂ group.

An example of a protein that can be used in the present invention is casein. β-casein is a particularly preferred example because the micelles that are formed have the highest monodispersity compared to other casein components. β-casein consists of 4 aspartic acid residues (-COOH group), 19 glutamic acid residues (-COOH group), 11 lysine residues (-NH ₂ group) and N-terminal in one molecule. It has an α-amino group, and β-casein micelles can be crosslinked by using WSC (water-soluble carbodiimide, chemical name: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) solution. It is.

  The cross-linked β-casein micelle obtained by the present invention is almost spherical. The particle diameter of the crosslinked β-casein micelle can be 5 to 100 nm (for example, 10 to 30 nm), and can be almost the same as the particle diameter (15 to 20 nm) of normal β-casein micelle.

  For the measurement of the particle size of the crosslinked protein micelle of the present invention, a technique well known to those skilled in the art can be used. For example, observation using a dynamic light scattering photometer (DLS) or atomic force microscope (AMF) Can be measured. In the present specification, when the particle size of micelles is indicated, an average value obtained from the scattering intensity by DLS is indicated unless otherwise specified.

  In the crosslinked β-casein micelle of the present invention, usually 30 to 98% of the amino groups are crosslinked. The degree of crosslinking can be prepared depending on the concentration of the crosslinking agent used in the crosslinking reaction described below. For example, in the case of β-casein micelles crosslinked with 25 mM WSC, 20-40% amino groups can be crosslinked, and in the case of β-casein micelles crosslinked with 250 mM WSC, 50-70% amino groups can be crosslinked.

  The surface of the crosslinked protein micelle of the present invention can be modified. Modifications include labeling (eg, enzyme labeling, FITC labeling, biotin labeling, gold colloid labeling).

[Method for producing crosslinked protein micelle]
The present invention also provides the following:
5) A method for producing a cross-linked protein micelle, comprising a step of cross-linking proteins constituting the micelle using a cross-linking agent.
6) The protein has an aspartic acid residue or glutamic acid residue, and a lysine residue or an N-terminal -NH ₂ group; and the cross-linking is an aspartic acid residue or a glutamic acid residue -COOH group and a lysine residue. or those formed by the condensation reaction of the -NH ₂ group at the end N, the manufacturing method according to claim 5.
7) The production method according to 6) above, wherein the protein is casein; and the crosslinking agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

  Crosslinking between proteins constituting the micelles can be performed so as to form a covalent bond by various methods. A known method can be used for crosslinking, but it is necessary to confirm whether or not the crosslinked micelle has a desired property, i.e., stimulus responsiveness, as described later. The present inventors have confirmed that when a certain enzymatic method is used, the resulting cross-linked protein micelles did not show thermal responsiveness. In the present invention, it is preferable to perform crosslinking so as to form a covalent bond chemically using a crosslinking agent.

In cross-linking, a solvent in which the protein to be used can form micelles and a protein concentration capable of forming micelles are selected. A well-known thing can be used as a crosslinking agent.
When a protein having an aspartic acid residue or glutamic acid residue and a lysine residue or N-terminal -NH ₂ group is used as a protein, the cross-linking is performed with the -COOH group of the aspartic acid residue or glutamic acid residue. It can be formed by a condensation reaction with a lysine residue or N-terminal -NH ₂ group, and as a crosslinking agent, water-soluble carbodiimide (chemical name: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) Can be used. Such a crosslinking reaction usually proceeds within 1 to 48 hours at a temperature of 0 to 60 ° C. in an aqueous solvent having a pH of 5.0 to 9.0. Although glutaraldehyde can be used as a crosslinking agent, it is necessary to confirm the properties of the resulting crosslinked micelle.

  According to the study by the present inventors, when water-soluble carbodiimide is used, the reaction proceeds stably at 4 ° C. If it is 15 ° C. or higher, it is difficult to control the cross-linking between micelles, the aggregates increase, and the yield may be inferior. In general, casein micelle formation seems to be promoted at high temperatures, and it has been reported that at 4 ° C, the equilibrium between monomer and micelle is almost biased to the monomer side (J. Colloid. Int. Sci., 258 , 33-39, 2003). It is surprising that spherical nanoparticles can be formed by reaction at 4 ° C. In addition, there is an optimum range for the buffer solution concentration during the crosslinking reaction, and if it is too high, the crosslinking reaction itself may be inhibited, resulting in a low crosslinking rate and resulting crosslinking. Micelle may not show thermal responsiveness. On the other hand, if it is too low, the crosslinking rate will be high, but the reason is not clear, but the thermal responsiveness of the resulting crosslinked micelles will deteriorate (the phase transition temperature described later will be greatly shifted to the high temperature side) There is. Considering the results, nanoparticles may be formed, but nanoparticles with different structures may have been prepared. Furthermore, in the cross-linking reaction, the influence of the concentration of the cross-linking agent can be taken into consideration, and nanoparticles can be stably prepared by setting the stock solution of water-soluble carbodiimide to about 1M.

Confirmation of the progress of the crosslinking reaction can be performed by confirming the molecular weight of the reaction product by, for example, SDS-denaturing polyacrylamide gel electrophoresis (SDS-PAGE).
For specific conditions for the crosslinking reaction when β-casein is used as the protein, the examples of the present invention can be referred to.
The prepared cross-linked protein micelle can be purified by passing through a membrane filter having a pore size of about 0.2 μm or by a known technique such as gel filtration.

[Functions and uses of cross-linked protein micelles]
The cross-linked protein micelles of the present invention are responsive to stimuli (eg, temperature, pH, salt concentration). For example, β-casein micelles that are not crosslinked are not aggregated even when the aqueous solution is heated to 70 ° C, but those that have undergone crosslinking treatment exhibit thermal responsiveness, and micelles aggregate at high temperatures. It becomes insoluble and becomes a cloudy solution.

Also, assuming that the temperature at which the turbidity takes an intermediate value is the transition temperature (T _t ), the cross-linked β-casein micelle greatly shifts to a higher temperature when the pH of the solution increases, and the pH is shifted from 7 to 8 ΔT _t = 40 ° C can be reached just by changing to (see Figure 1-4). This is considered to be because the surface charge is greatly shifted negatively as the pH is increased, and aggregation is suppressed by suppressing contact between particles due to electrostatic repulsion between cross-linked β-casein micelles. .

Further examination of the salt concentration of the cross-linked β-casein micelle solution shows that the transition temperature gradually decreases with the addition of NaCl (see Figure 1-5).
Thus, the cross-linked protein micelle of the present invention represented by the cross-linked β-casein micelle can control the transition temperature by changing the solution pH, and is also capable of coexisting ion species and buffer species (the present inventors). The phosphate buffer and the Tris buffer show slightly different behaviors), or the transition temperature can also be controlled by the concentration.

Thus, the present invention also provides:
8) A method for separation, detection, drug delivery, stabilization, purification, or reaction control using the crosslinked protein micelle according to any one of claims 1 to 3.
9) The method for controlling the stimulus responsiveness of a crosslinked protein micelle according to any one of claims 1 to 3, comprising a step of modifying the surface of the crosslinked protein micelle.

  The stimulus-responsive cross-linked protein micelle of the present invention is expected to be applied to biological separation, target molecule detection, biosensing, immobilized enzyme, and drug delivery system (DDS).

  For example, when the stimulus-responsive crosslinked protein micelle of the present invention is used as a separation material, it is possible to present a ligand on the surface of a nanoparticle, bind a target substance, and precipitate by aggregation by heating to be separated. is there. There are various techniques for selectively recovering a target having a specific affinity for a certain ligand (which is not limited by molecular weight and needs only to be displayed on the surface of a nanoparticle) from a biological sample. This is an extremely important technique in analysis. In fact, application examples using ELP are aimed at immunoassay (eg, Anal. Chem., 77, 2318-2322, 2005) through affinity separation in combination with antibody-binding proteins and proteome analysis ( For example, J. Proteome Res., 4, 2355-2359, 2005). The same use can be expected when using the nanoparticles of the present invention. Moreover, in using the nanoparticles of the present invention, application examples of magnetic nanoparticles and temperature-responsive magnetic particles can be referred to.

In the present invention, when casein is used as a material, it should be noted that the obtained nanoparticles are inherently biodegradable.
When the drug or the like is retained inside the nanoparticles of the present invention, a desired substance can be retained in the micelle before crosslinking, and then crosslinked, and the micelle can be crosslinked after crosslinking. Since hydrophobicity increases (ANS fluorescence intensity increases), it may be possible to partition into micelles by adding a hydrophobic drug to the system after crosslinking. Considering that the inside of the micelle is hydrophobic, it seems that the substance to be retained inside is preferably hydrophobic to some extent. By using the nanoparticles of the present invention, it is possible to solubilize a hydrophobic substance transparently and stably even at a supersaturated concentration.

  On the other hand, since the casein micelle itself does not have a highly hydrophobic core, the substance held inside the micelle is not limited to a hydrophobic substance. Regarding the substance to be held inside, there seems to be no molecular weight limitation. For example, the nanoparticles of the present invention may be used as a carrier for relatively small physiologically active substances such as peptides and siRNA.

  If the nanoparticles of the present invention are used in DDS, it may be preferable to use a human-derived amphiphilic protein having no antigenicity (for example, breast milk-derived casein) as the protein. In addition, the crosslinked protein micelle of the present invention is a stable micelle having a relatively uniform particle size in water and is responsive to stimuli. Therefore, in particular, it transports drugs that require long-term administration and drugs that are easily degraded. As a body, it can be suitably used as an agent for injection, oral administration, transdermal or transmucosal administration. The cross-linked protein micelle of the present invention is used for controlling the accumulation of drugs in the affected area in response to an external stimulus (for example, temperature), for example, local administration of the drug in the affected area where the temperature has increased due to an inflammatory reaction or the like. Can be used for

  In repeated experiments of aggregation and solubilization of cross-linked β-casein micelles, reversibility was observed up to several times, but it has been found that the aggregate is less likely to be resolubilized thereafter. This is because aggregation-solubilization uses hydrophobic interaction as a driving force, and during repeated heating-cooling cycles, some of the cross-linked β-casein micelles gradually denatured irreversibly, and cross-linked β- It is assumed that a stable aggregate structure is formed between the casein micelles.

1. Preparation of cross-linked β-casein micelles:
β-casein (5 mg) was dissolved in 950 μL of 50 mM phosphate buffer (pH 7.0) to prepare a β-casein aqueous solution. Thereafter, 50 μL of a predetermined concentration of water-soluble carbodiimide (WSC) aqueous solution prepared in advance was added to the β-casein aqueous solution while stirring at room temperature (20 ° C.). After the addition, the mixture was stirred at room temperature for 12 hours to prepare crosslinked β-casein micelles. Finally, the prepared crosslinked β-casein micelle solution was purified by gel filtration through a PD10 column and used.

  Crosslinking of the prepared sample was confirmed by SDS-denatured polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in Figure 1-1. The band of β-casein alone (lane B) is located at approximately 25 kDa, and β-casein + WSC (lane C, lane D) has a higher molecular weight than lane B. Was shown to be crosslinked by WSC.

  Next, the number of amino groups in the crosslinked β-casein was quantified. Since the amino group in β-casein is involved in the crosslinking reaction, the crosslinking rate can be calculated by quantifying the amino group. Here, amino group quantification was performed based on TNP conversion of amino groups with trinitrobenzenesulfonic acid. As a result, in the case of β-casein micelles crosslinked with 25 mM WSC, 20-40% amino groups are crosslinked, and in the case of β-casein micelles crosslinked with 250 mM WSC, 50-70% amino groups are crosslinked. I found out.

2. Structural evaluation of cross-linked β-casein micelles:
It was found that β-casein micelles can be cross-linked by WSC. Next, in order to obtain information on the structure of the crosslinked β-casein micelle, DLS measurement and AFM observation were performed.

  Figure 1-2 shows changes in particle size distribution (scattering intensity distribution) due to cross-linking by DLS measurement. From the figure, the particle size distribution of the cross-linked β-casein micelle was slightly smaller than that of the β-casein micelle not subjected to the cross-linking treatment. However, the particle size distribution of β-casein micelles (particle size distribution: 5 to 200 nm) and cross-linked β-casein micelles (average particle size: about 20 nm) differ greatly, and more monodisperse nanoparticles can be quantitatively analyzed. I was able to get it.

  Next, the results of AFM observation are shown in Figure 1-3. As a result of AFM observation, cross-linked β-casein micelles have a spherical shape, and it is considered that cross-linking between protein molecules constituting the micelles mainly occurred.

  From the above results, it was shown that by using β-casein micelles with WSC, β-casein constituting the micelles can be intermolecularly crosslinked to prepare spherical nanostructures. It was.

3. External factor responsiveness of cross-linked β-casein micelles:
Figure 1-4 shows changes in turbidity due to heating. First, the casein aqueous solution not subjected to the crosslinking operation was heated, but no aggregation was observed when the temperature was raised to 70 ° C. A similar study was performed on a sample crosslinked with 25 mM WSC, but no thermal aggregation was observed. On the other hand, those subjected to crosslinking treatment with 250 mM WSC showed remarkable thermal response. Therefore, first, the temperature responsiveness to the pH change of the solution was examined. Specifically, the effect of pH using the prepared cross-linked β-casein micelles with solvent substitution with pH 6 (50 mM phosphate buffer), pH 7-9 (50 mM Tris-HCl buffer) I investigated. The effect of salt concentration was examined on cross-linked β-casein micelles (Tris-HCl buffer pH 7). The heating rate was 0.5 ° C./min.

The relationship between temperature and turbidity at different pH was investigated. The temperature at which the intermediate value of turbidity was obtained was defined as the transition temperature (T _t ). It was found that when the pH of the cross-linked β-casein micelle solution was increased, the transition temperature was greatly shifted to the higher temperature side, and ΔT _t = 40 ° C was reached only by changing the pH from 7 to 8. In addition, the relationship between the transition temperature and the surface charge of the cross-linked β-casein micelle was examined, and it was found that the surface charge shifted greatly negatively with increasing pH. Therefore, the transition temperature increased with the increase in pH because the electrostatic repulsion between the cross-linked β-casein micelles prevented the contact between the particles, thereby suppressing the aggregation, resulting in an increase in the transition temperature. it is conceivable that.

Next, the relationship between the salt (NaCl) concentration and the transition temperature was examined. From Fig. 1-5, the transition temperature gradually decreased with the addition of NaCl. The decrease in transition temperature accompanying the addition of salt is thought to be because thermal aggregation was promoted by the destruction of the hydration structure on the nanoparticle surface.
From the above, it was confirmed that the transition temperature can be controlled by changing the surface charge of the nanoparticles as the pH changes, and that the transition temperature changes depending on the coexisting ion species.

4). Control of aggregation of cross-linked β-casein micelles by temperature:
Repeated experiments of aggregation-solubilization of cross-linked β-casein micelles were performed. Specifically, the procedure of stirring the cross-linked β-casein micelle solution at 37 ° C. for 10 minutes, measuring the absorbance at 600 nm, stirring at 4 ° C. for 1 hour, and measuring the absorbance at 600 nm again was performed. Repeated times. As a result, sufficient reversibility was observed up to the third time, but the aggregates were difficult to re-solubilize after the third time. This is because aggregation-solubilization is driven by hydrophobic interaction, and during repeated heating-cooling cycles, some of the cross-linked β-casein micelles are gradually irreversibly denatured and cross-linked β -It is assumed that a stable aggregate structure is formed between the casein micelles.

A photograph of the state of aggregation by heating is shown in FIG. 1-6.
[Conclusion]
We succeeded in preparing proteinaceous nanostructures by cross-linking between casein molecules composed of β-casein micelles using a water-soluble crosslinking agent (WSC). It was revealed that the obtained crosslinked β-casein micelle has temperature responsiveness and that the transition temperature can be controlled by a slight pH change.

[Experimental operation]
1. Preparation of cross-linked β-casein micelles:
β-casein (40 mg) was dissolved in 6 mL of 50 mM phosphate buffer (pH 7.0) to prepare a β-casein aqueous solution. Thereafter, 2 mL of a 1M aqueous solution of water-soluble carbodiimide (WSC) prepared in advance was added to the β-casein aqueous solution while stirring at 4 ° C. After the addition, the mixture was stirred at 4 ° C. for 24 hours to prepare crosslinked β-casein micelles. Finally, the prepared crosslinked β-casein micelle solution was passed through a membrane filter (pore size 0.2 μm), and the filtrate was purified by gel filtration through a PD10 column and used.

2. Evaluation of cross-linked β-casein micelle solution:
2.1 Measurement of protein concentration The protein concentration of the prepared cross-linked β-casein micelle solution was measured by BCA method and absorbance in the ultraviolet region.

2.2 Evaluation of thermal responsiveness The prepared cross-linked β-casein micelle solution was taken out into a 400 μl microtube and heated with a dryer to determine the presence or absence of thermal responsiveness.

2.3 Measurement of turbidity change The turbidity change of the prepared crosslinked β-casein micelle solution was measured using a spectrophotometer. The measurement wavelength was 600 nm where no protein was absorbed, and the temperature was varied between 15-75 ° C at a rate of 1 ° C or 2 ° C / min.

2.4 Measurement of cross-linking rate Take 1 ml of each prepared cross-linked β-casein micelle solution, add 1 ml of 2% sodium dodecyl sulfate (SDS) aqueous solution, and then add 1 ml of 4% sodium hydrogen carbonate solution (pH 8.0). did. Further, 1 ml of a 0.1% trinitrobenzenesulfonic acid (TNBS) aqueous solution was added, light-shielded with aluminum foil, and reacted for 2 hours with stirring in a 40 ° C. constant temperature bath. 1 ml of 1N hydrochloric acid was added to the solution after the reaction to stop the reaction, and the absorbance at 340 nm was measured. At the same time, a calibration curve was prepared with a β-casein micelle solution that had not been crosslinked, and the crosslinking rate was calculated from the number of amino groups involved in the reaction.

2.5 Measurement of hydrophobic strength Add 8-anilino-1-naphthalenesulfonic acid (ANS) to 1 ml of cross-linked β-casein micelle solution to a final concentration of 10 μM, and excite it at a wavelength of 350 nm. The hydrophobic intensity of casein micelles was evaluated by measuring the fluorescence intensity at nm.

2.6 Measurement of particle size and zeta potential The particle size distribution and zeta potential of the prepared cross-linked β-casein micelle solution were measured by dynamic light scattering and electrophoresis using Zetasizer-nano ZS90.

3. Confirmation of nanoparticle structure of cross-linked β-casein micelles:
The 1 M MgCl ₂ solution was diluted with 10 mM Tris-HCl (pH 7.5) to prepare a 2 mM MgCl ₂ solution. A β-casein micelle solution crosslinked with this solution was diluted to 1 μg / ml. 10 ml of this diluted β-casein micelle solution was dropped on a Mica substrate and gently washed with distilled water. After washing with distilled water, the water droplets around the Mica substrate were removed with Air, and the Mica substrate was vacuum dried for about 2.5 hours. Two and a half hours later, the prepared Mica substrate was observed using an atomic force microscope (AFM).

4). Effect of protein concentration on thermal response of cross-linked β-casein micelles:
The prepared crosslinked β-casein micelle solution was diluted to prepare 0.5, 1.0, 1.5, and 2.0 mg / ml crosslinked β-casein micelle solutions. The turbidity change of the prepared cross-linked β-casein micelle solution was measured using a spectrophotometer. The measurement wavelength was 600 nm without protein absorption, and the temperature was varied between 15-75 ° C at a rate of 2 ° C / min.

5. Functionalization and evaluation of cross-linked β-casein micelle particle surface:
5.1 FITC labeling The prepared cross-linked β-casein micelle solution was replaced with 100 mM sodium carbonate buffer (pH 8.5) using a PD 10 column, and then 1.0 mg / ml FITC dissolved in dimethyl sulfoxide. 200 μl of solution was added. After stirring at 4 ° C. for 24 hours, impurities were removed and the buffer was replaced by gel filtration using a PD 10 column with 50 mM phosphate buffer (pH 7.0). The prepared FITC-labeled casein micelle solution was evaluated by the method shown in 2 above, and a precipitate was obtained by applying centrifugation after heating.

5.2 Biotin labeling After replacing the prepared cross-linked β-casein micelle solution with a 100 mM sodium carbonate buffer (pH 8.5) using a PD 10 column, 1.0 mg / ml Biotin dissolved in dimethyl sulfoxide 200 μl of -OSu (BI) and Biotin- (AC ₅ ) ₂ -Osu (BII) solution were added. After stirring at 4 ° C. for 24 hours, impurities were removed and the buffer was replaced by gel filtration using a PD 10 column with 50 mM phosphate buffer (pH 7.0). The prepared biotin-labeled casein micelle solution was evaluated by the method shown in 2 above, and the difference in the length of the linker site of the biotinylated reagent in the thermal response was examined.

6). Biotin-labeled cross-linked β-casein micelles evaluated for avidin targeting:
Prepared biotin-labeled cross-linked β-casein micelle solution diluted to 0.5 mg / ml, 1.0 mg / ml avidin solution dissolved in 50 mM phosphate buffer (pH 7.0) was added in 10 μl aliquots to 150 μl, and 20 The change in turbidity at 0 ° C. was measured. In addition, for each solution, the thermal responsiveness at the avidin addition amount (150 μl in the 25 mM WSC cross-linked sample, 30 μl addition in the 250 mM WSC cross-linked sample) and the avidin addition amount (250 mM WSC cross-linked) that caused white turbidity did not occur. Each sample was examined for thermal response at 150 μl). In the experiment of avidin trap, FITC-labeled avidin (Sigma) was added to a cross-linked β-casein micelle solution or biotin-labeled cross-linked β-casein micelle solution, and after heat aggregation, centrifuged, and the supernatant The fluorescence intensity of was measured.

[Results and discussion]
1. Atomic force microscopy of cross-linked β-casein micelle solution:
The results are shown in Fig. 2-1. From the figure, the average particle diameter radii on the substrate surface in the AFM image were 20.0 nm and 20.7 nm for the crosslinked samples with 25 mM and 250 mM WSC, respectively. From these results, it was confirmed by calculation that particle sizes of the crosslinked β-casein micelles were 20.0 nm and 21.5 nm, respectively, and it was confirmed that nano-sized particles could be prepared. The previously measured dynamic light scattering (DLS) particle size results in Z-Average values of 24.6 nm and 20.9 nm, respectively, which are somewhat consistent with the current AFM and DLS particle size measurements. It was confirmed that there is sex.

2. Effect of protein concentration on thermal response of cross-linked β-casein micelles:
The results are shown in Fig. 2-2. From the figure, it became clear that the higher the protein concentration, the sharper the thermal response. At 0.5 mg / ml, the phase transition temperature was 34 ° C, while at 2.0 mg / ml, a faster response was seen at 25 ° C. A relatively similar response was seen at 1.5 mg / ml and above, and it was revealed that a certain level of protein concentration was required for a sharp response. From this result, it is considered that there is a difference in the change in the association state of micelles depending on the protein concentration and the strength of interaction between micelles.

3. FITC labeling on the surface of crosslinked β-casein micelle particles and its effects:
As a result, when the sample that became cloudy by heating was centrifuged, an orange precipitate was obtained as shown in Fig. 2-3, and it was confirmed that the particle surface was modified.

4). Biotin labeling on the surface of crosslinked β-casein micelle particles and its effects:
As biotinylation reagents, the following two types of reagents having different linker chain lengths were used.

  Changes in the thermoresponsive behavior due to biotin labeling are shown in Figure 2-4. It was clarified that the thermal responsiveness was slightly decreased by biotin labeling. All of the 250 mM WSC cross-linked samples showed thermal response, but BII with a longer linker site showed a phase transition behavior at a lower temperature than BI with a shorter linker site. Figure 2-5 shows that the hydrophobic strength is weakened by labeling. It is thought that the phase transition temperature has shifted to a high temperature due to this change in properties.

5. Biotin-labeled cross-linked β-casein micelles evaluated for avidin targeting:
Fig. 2-6 shows the results of measurement of turbidity changes associated with the addition of avidin to biotin-labeled samples. As shown in the figure, in the 250 mM WSC cross-linked sample, white turbidity started to occur when about 40 μl of avidin was added. On the other hand, in the 25 mM WSC cross-linked sample, although light turbidity could be visually confirmed, strong turbidity due to aggregation was not observed. This suggests that biotin-avidin specific binding occurs in both samples due to the addition of avidin, and in the 250 mM WSC cross-linked sample, slight aggregation occurs in the sample even without heating. Has been confirmed to occur.

  Next, the results of the thermoresponsive behavior of biotin-labeled samples containing avidin are shown in Fig. 2-7. The turbidity gradually increased by heating even in the sample that had already been clouded by the addition of avidin. The behavior was equivalent to that in the absence of avidin, and it was confirmed that there was no significant change in the thermoresponsive behavior inherent to the crosslinked micelles.

  Based on the above results, it was confirmed whether or not avidin could actually be trapped using FITC-labeled avidin (Sigma). From the measurement of fluorescence intensity remaining in the soluble fraction after heat aggregation and centrifugation, it was confirmed that BII-labeled crosslinked casein micelles could trap 85% of 35 nM FITC-labeled avidin. BI-labeled cross-linked casein micelles had a slightly lower trap rate (Figure 2-8).

  From the above, it was confirmed that the crosslinked casein micelle solution was about 20 nm nanoparticles. In addition, the nanoparticle surface can be modified, and the modification revealed various changes in properties such as surface charge and thermal response behavior. Furthermore, it became clear that avidin targeting is possible by biotin modification.

Fig. 1-1 is a photograph showing the results of SDS-PAGE. Fig. 1-2 is a graph showing changes in particle size distribution due to crosslinking. Fig. 1-3 is a graph showing the details of the particle size distribution by DLS. Fig. 1-4 is a graph showing the results of turbidity measurement. FIG. 1-5 is a graph showing the influence of [Na ⁺ ] on the phase transition behavior. Fig. 1-6 is a photograph showing the state of aggregation by heating. A: (Left) Buffer solution; (Right) Casein nanoparticle aqueous solution before heating. B (left) buffer; (right) casein nanoparticle aqueous solution after heating. Protein concentration: 1 mg / mL in 50 mM Tris-HCl buffer, pH 7 FIG. 2-1 shows the results of AFM observation of cross-linked β-casein micelles at 25 mM (top) and 250 mM (bottom) WSC. Fig. 2-2 is a graph showing the relationship between protein concentration and turbidity change. Β-casein micelles were cross-linked with 50 mM WS. Temperature rise: 2.0 ℃ Fig. 2-3 is a photograph of a system containing FITC-labeled nanoparticles heated and the resulting aggregates precipitated by centrifugation. FIG. 2-4 is a graph showing changes in thermoresponsive behavior due to biotin labeling. Fig. 2-5 is a graph showing changes in hydrophobicity due to biotin labeling. FIG. 2-6 is a graph showing the results of measuring turbidity change of a biotin-labeled nanoparticle accompanying avidin addition. FIG. 2-7 is a graph showing the thermoresponsive behavior of biotin-labeled nanoparticles in the presence of avidin. FIG. 2-8 is a graph showing the results of an experiment in which it was confirmed whether BII-labeled crosslinked casein micelles can trap avidin using FITC-labeled avidin.

Claims (9)

  A stimulus-responsive cross-linked protein micelle comprising a cross-linked protein structure. The protein constituting the structure has an aspartic acid residue or glutamic acid residue, and a lysine residue or an N-terminal -NH ₂ group; and the cross-linking is an aspartic acid residue or a glutamic acid residue -COOH group _2. The cross-linked protein micelle according to claim 1, which is formed by a condensation reaction of lysine residues or an N-terminal -NH ₂ group.   The cross-linked protein micelle according to claim 2, wherein the protein is casein.   A modified cross-linked protein micelle, wherein the surface of the cross-linked protein micelle according to any one of claims 1 to 3 is modified.   A method for producing a cross-linked protein micelle comprising a step of cross-linking proteins constituting a micelle using a cross-linking agent. The protein has an aspartic acid residue or glutamic acid residue, and a lysine residue or N-terminal -NH ₂ group; and the cross-linking is an aspartic acid residue or glutamic acid residue -COOH group and a lysine residue or N it is those formed by the condensation reaction of youngest -NH ₂ group, the process according to claim 5. The production method according to claim 6, wherein the protein is casein; and the crosslinking agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.   A method for separating or detecting a target molecule, or delivering, stabilizing, purifying, or controlling a reaction using the cross-linked protein micelle according to any one of claims 1 to 3.   The method for controlling stimulation responsiveness of a crosslinked protein micelle according to any one of claims 1 to 3, comprising a step of modifying the surface of the crosslinked protein micelle.