JP5355456B2 - Quantum dot composite-containing vesicle and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a quantum dot complex equipped with position identification by fluorescent imaging and high efficient drug release mechanism to living body tissues, and capable of being obtained without accompanying a complex synthesis process, and to provide vesicles containing the quantum dot complex. <P>SOLUTION: There is provided a quantum dot complex 1 which includes a quantum dot 10 having a core 12 composed of semiconductor nanoparticles and a shell 14 provided by bonding a polymer on the surface of the core 12 and having reactive groups on the surface, and an organism-relating molecules 20 bonded to the reactive groups of the surface of the quantum dot 10. Further, the quantum dot 1 is contained in the layer and/or on the surface of the vesicle composed of a phospholipid double layer. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a quantum dot composite, a quantum dot composite-containing vesicle, and a method for producing them.

  Quantum dots made of semiconductor nanocrystals are a material that attracts attention in various technical fields including electronic and optical devices and life sciences. Quantum dots have a very high molar extinction coefficient and fluorescence quantum yield, and the absorption and fluorescence spectra can be easily tuned depending on the particle size of the quantum dots. It shows a sharp spectral characteristic. Furthermore, quantum dots have characteristics not found in known fluorescent materials, such as excellent light fading resistance and light / chemical degradation resistance.

  Until now, in organic imaging, organic fluorescent dyes are generally used, and the photobleaching has been a factor that hinders long-term observation. Quantum dots can be used to overcome this problem. In addition, quantum dots emit fluorescence at different wavelengths depending on the particle size, so single quantum excitation can be achieved by introducing quantum dots of different sizes in living tissue. Multicolor observation of biomolecules can be performed simultaneously at the wavelength.

  As materials constituting the quantum dots, cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe), zinc sulfide (ZnS), and the like are used, and there are concerns about their harmfulness to biological systems. It has been. In addition, although quantum dots exhibit excellent fluorescence properties, they cannot be fused with biomolecules such as proteins, carbohydrates, and nucleic acids as they are.

  Therefore, biocompatible quantum dots have been proposed in which the surface of the quantum dots is coated with various hydrophilic organic molecules and polymers and detoxified (Non-Patent Documents 1 and 2). On the surface of such quantum dots, carboxyl groups, amino groups, cysteine groups, etc. are introduced, and by using these functional groups, proteins (enzymes, hormones, receptors, growth factors, antibodies), It can chemically bond to biomolecules such as nucleic acids (DNA and RNA), carbohydrates, oligopeptides, oligonucleotides and the like.

Phospholipid molecules, on the other hand, form lipid bilayers (phospholipid bilayers) by self-assembly and become the main component of the cell membrane, as well as small lipid vesicles (liposomes) used for mass transfer inside and outside the cell membrane. To do. A vesicle is a capsule-like structure in which a minute aqueous phase is encapsulated in a phospholipid bilayer, and various chemical substances such as anticancer agents can be encapsulated in the inner pores. Therefore, it is expected to be applied as an effective technical tool in the pharmaceutical, food and cosmetic industries, and as a component of the next generation nano / micro scale drug delivery system and biochip. In recent years, a production tool for a library substrate has been proposed in which a microarray in which vesicles are fixed at a high density on a substrate is prepared, and attoliter (1aL = 10 ⁻¹⁸ L) scale molecules are inspected at high throughput. (Non-Patent Document 3).

  In these biosystems, the vesicle plays a role of a vessel containing the drug or a carrier for transporting the drug. At that time, it is necessary to fluorescently label the vesicle in order to specify the position of the vesicle. Usually, as the means, a method of inserting a phospholipid molecule labeled with a fluorescent dye into a vesicle is used (Non-patent Documents 4 and 5).

Z. Zhelev et al., “Analytical Chemistry”, 78, 2006, 321-330. K. Susumu et al., "Journal of American Chemical Society," 129, 2007, 13987-13996. D. Stamou et al., “Angewandte Chemie International Edition”, 42, 2003, 5580-5583. J. et al. W. Nichols, “Biochemistry”, 24, 1985, 6390-6398. Y. Li et al., "Journal of American Chemical Society", 130, 2008, 12252-2253.

However, existing fluorescent dye compounds such as organic fluorescent dyes have a problem in photobleaching, and are not suitable for systems that observe the dynamic and static behavior of vesicles over a long period of time. Fluorescent dye-labeled phospholipid molecules have the disadvantage of causing photo / chemical degradation when irradiated with intense light, and vesicles may become unstable with the photo / chemical degradation and the structure may be destroyed. is there. In particular, in the drug delivery system, when the vesicle is collapsed, the encapsulated chemical substance is released at an undesired position, and therefore cannot be used for that purpose.
In addition, it was difficult to incorporate the quantum dots into the phospholipid bilayer of the vesicle even when trying to fluorescent label with quantum dots instead of the fluorescent dye compound.
Furthermore, when vesicles are used for drug delivery and biochip systems, a mechanism is required that allows the contained drug to act on biomolecules with high efficiency.

  The present invention has been made in view of the above circumstances, and includes a quantum dot composite having a position specified by fluorescence imaging and a high-efficiency drug release mechanism with respect to a living tissue and obtained without a complicated synthesis process. Quantum dot composite-containing vesicles and methods for producing them.

Quantum dot conjugates containing vesicle of the present invention, the lipid bilayer composed of phospholipids is formed in a capsule shape, quantum dot complex, are contained in one or both either the lipid bilayer of the layers in and on the surface A quantum dot composite-containing vesicle comprising a core part composed of semiconductor nanoparticles and a shell formed by binding polyethylene glycol having a carboxyl group as a reactive group to the surface of the core part. And a quantum dot having the reactive group on the surface of the shell, and a phospholipid bonded to the reactive group .
A chemical substance may be included in the lipid bilayer formed in the capsule shape, and the lipid bilayer preferably includes a polyether chain on a surface thereof, and the polyether chain has a biomolecule at a terminal. It is more preferable to provide a functional group that can be bonded to.

Method of manufacturing a quantum dot conjugates containing vesicle of the present invention includes a core portion formed of a semiconductor nanoparticle, and a shell portion which polyethylene glycol is engaged binding to the surface of the core part comprising a carboxyl group as a reactive group, and first obtain the quantum dot having the reactive groups on the surface of the shell portion, and a phospholipid, a pre Kihan応活groups and the phospholipids quantum dot conjugates bound by chemical bonds between the reactive group of After one step and the obtained quantum dot complex and another phospholipid are dispersed in a dispersion medium, the dispersion medium is removed and the other phospholipid containing the quantum dot complex is thinned. And a second step of encapsulating the other phospholipid in an aqueous solution.
In the second step, a chemical substance may be further added. In the second step, a phospholipid introduced with a polyether chain having a functional group capable of binding to a biomolecule at the end is further added. A dispersion is preferred.

  According to the quantum dot composite and quantum dot composite-containing vesicle of the present invention, the position can be specified by fluorescence imaging without involving a complicated synthesis process, and the efficiency of the drug release mechanism for living tissue can be improved.

It is sectional drawing of the quantum dot composite_body | complex concerning one Embodiment of this invention. (A) It is sectional drawing of the quantum dot composite containing vesicle concerning one Embodiment of this invention. (B) It is the elements on larger scale of (a). It is sectional drawing of the quantum dot composite containing vesicle concerning one Embodiment of this invention. It is sectional drawing of the quantum dot composite containing vesicle concerning one Embodiment of this invention. It is sectional drawing of the quantum dot composite containing vesicle concerning one Embodiment of this invention. It is a graph which shows the result of the absorption spectrum measurement in the quantum dot composite_body | complex of Example 1,2. It is a graph which shows the measurement result of the fluorescence spectrum in the quantum dot composite_body | complex of Example 1,2. (A) It is a graph which shows the measurement result of the dynamic light scattering in the quantum dot complex of Example 1. (B) It is a graph which shows the measurement result of the dynamic light scattering in the quantum dot 525 which is a reference product. (A) It is a photograph which shows the observation result with the atomic force microscope of the quantum dot composite_body | complex of Example 1. FIG. (B) It is a graph which shows the measurement result of the particle size of the particle | grains observed by (a). (C) It is a graph which shows the measurement result of the particle size of the particle | grains observed by (a). (A) It is a photograph which shows the observation result with the atomic force microscope of the quantum dot 525 which is a reference product. (B) It is a graph which shows the measurement result of the particle size of the particle | grains observed by (a). (C) It is a graph which shows the measurement result of the particle size of the particle | grains observed by (a). It is a photograph which shows the observation result of the confocal laser fluorescence microscope of the quantum dot composite containing vesicle of Example 5. (A) It is a photograph which shows the observation result in the atomic force microscope of the quantum dot composite containing vesicle of Example 5. FIG. (B) It is a graph which shows the measurement result of the film thickness of a vesicle. (C) It is a graph which shows the measurement result of the particle size of the dot structure observed by (a). (A) It is a photograph which shows the observation result in the atomic force microscope of the quantum dot composite containing vesicle of Example 5. FIG. (B) It is a graph which shows the measurement result of the particle size of the dot structure observed by (a).

(Quantum dot composite)
An example of the quantum dot composite of the present invention will be described below with reference to the drawings. As shown in FIG. 1, the quantum dot composite 1 includes a quantum dot 10 and a bio-related molecule 20 bonded to the surface of the quantum dot 10.

<Quantum dots>
The quantum dot 10 includes a core portion 12 made of semiconductor nanoparticles, a shell portion 14 in which a polymer is bonded to the surface of the core portion 12, and a reactive active group on the surface.
The semiconductor nanoparticles that are the core portion 12 are semiconductor nanometer-sized fine particles, and are light-emitting materials having a quantum confinement effect. Examples of the semiconductor nanoparticles include a combination of a metal element and a nonmetallic element or a transition metal element. For example, cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe), zinc sulfide ( IIB-VIB group such as ZnS) and IIIB-VB group such as indium phosphide (InP) and gallium phosphide (GaP).

The polymer constituting the shell portion 14 may be any polymer having a reactive group that binds to a reactive group of the biological molecule 20 described later. For example, biological polymers such as polyethylene glycol (PEG), polyacrylamide, and polymethacrylate Examples of the compatible polymer include a polymer having a carboxyl group or an amino group as a reactive group. Especially, when using phospholipid as the bio-related molecule 20, a polymer having a carboxyl group as a reactive group is preferable, and PEG having a carboxyl group as a reactive group is more preferable.
The semiconductor nanoparticles of the core 12 are likely to aggregate in a solution and are difficult to handle, and there are concerns about health damage to the human body and toxicity to the environment. The quantum dot 10 is rendered harmless by including the shell portion 14 made of a polymer having high biocompatibility, and can be used for general purposes in the medical and biotechnology fields.
In addition, the quantum dot 10 is a quantum dot composite in which a biologically active molecule 20 is directly chemically bonded to the surface of the quantum dot 10 by introducing a reactive group such as a carboxyl group on the surface of the shell portion 14. 1 can be produced.

 The emission wavelength of the quantum dots 10 varies significantly depending on the size of the semiconductor nanoparticles in the core portion 12. In general, since bio-imaging uses a wavelength in the visible range, the size of the core portion 12 is preferably about 1 to 20 nm showing light emission in the visible range. The thickness of the shell portion 14 is not particularly limited, but is preferably about 1 to 30 nm.

<Biologically related molecules>
The bio-related molecule 20 is a molecule existing in the living body and a derivative thereof. Examples of the biological molecule 20 include phospholipids, nucleic acids such as single-stranded or double-stranded DNA or RNA, polypeptides, oligopeptides, sugars, etc. Among them, phospholipids are preferable.

  In the phospholipid, the acyl chain length of the hydrophobic part, the presence or absence of a double bond in the acyl chain, and the site and number of the double bond are not particularly limited. However, the hydrophilic head of the phospholipid preferably has a structure including phosphaethanolamine (PE) that can chemically bond with an active reactive group such as a carboxyl group on the surface of the quantum dot 10. As such phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphaethanolamine (DOPE) represented by the following formula (I), 1,2- represented by the following formula (II) Examples include dipalmitoyl-sn-glycero-3-phosphaethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphaethanolamine (DSPE) represented by the following formula (III). .

<Method for producing quantum dot composite>
The method for producing a quantum dot composite of the present invention is a method in which a reactive group of a biologically relevant molecule is bonded to a reactive group of the surface of the quantum dot, that is, a shell portion. It is done. For example, when a phospholipid is used as a bio-related molecule and a quantum dot complex is obtained, the quantum dot and the phospholipid are combined with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) catalyst. Examples of the production method include dehydration condensation. On this occasion. The mass ratio represented by quantum dots / phospholipids is not particularly limited, and can be determined in consideration of the types of quantum dots or phospholipids.

(Quantum dot composite-containing vesicles)
The quantum dot complex-containing vesicle of the present invention comprises a vesicle in which a phospholipid bilayer is formed in a capsule shape and the quantum dot complex of the present invention. It is contained in both or any one of these.
The quantum dot composite-containing vesicle of the present invention will be described below with reference to the drawings. FIG. 2A is a cross-sectional view according to an embodiment of the quantum dot composite-containing vesicle of the present invention, and FIG.
As shown in FIG. 2A, the quantum dot composite-containing vesicle 100 includes a capsule-like vesicle 110 having an inner hole 120 and the quantum dot composite 1, and a liquid is held in the inner hole 120. ing. As shown in FIG. 2 (b), the vesicle 110 is composed of a phospholipid bilayer that is a bimolecular layer of phospholipid 112, and the quantum dot complex 1 is both in and / or on the surface of the phospholipid bilayer. It is contained in either.

<Vesicle>
The phospholipid 112 constituting the vesicle 110 is not particularly limited in terms of the acyl chain length of the hydrophobic part, the presence or absence of a double bond in the acyl chain, and the site and number of the double bond. Examples of the phospholipid constituting the vesicle 110 include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), Examples include phosphatidylglycerol (PG) and sphingolipids. These phospholipids may be used alone or in combination of two or more.

  The vesicles 110 are classified into small vesicles having an outer diameter of less than 50 nm and large vesicles having an outer diameter of 50 nm or more depending on the size. In particular, the vesicle 110 having an outer diameter of more than 10 μm is called a giant vesicle. The outer diameter (vesicle size) of the vesicle 110 can be controlled by a synthesis method or size separation and purification. The size of the vesicle 110 used in the present invention is not particularly limited, but is preferably a giant vesicle. In general, with a small vesicle size, the film thickness of the vesicle 110 is 4 to 5 nm, and if the vesicle size is 50 to 100 nm, the curvature of the spherical structure increases, so that defects are likely to occur on the film surface and the structure is not good. It is stable. For this reason, in the present invention, it is more preferable to use a large vesicle from the viewpoints of easy handling operation of the vesicle 110, stability of the vesicle structure, and controllability of chemical substance uptake into the inner hole 120. By making the vesicle 110 a giant vesicle, the chemical substance can be included in the inner hole 120 in a wide range of concentrations.

  The type of liquid in the inner hole 120 is not particularly limited, and examples thereof include a solvent used in the manufacturing process of the quantum dot composite-containing vesicle 100. Since the liquid is held in the inner hole 120, the form of the quantum dot composite-containing vesicle 100 is maintained.

<Chemical substances>
A chemical substance may be included in the inner hole 120.
FIG. 3 is a cross-sectional view of the quantum dot composite-containing vesicle 200, and the chemical substance 210 is contained in the inner hole 120 of the vesicle 110.
The chemical substance 210 is a substance in which a specific binding substance that causes some interaction such as formation and adsorption of a chemical bond and a target biological substance exists, that is, for example, a specific protein is specifically recognized, And specifically, various amino acids, ATP, ions, saccharides, proteins, peptides, low molecular organic compounds, and the like.

<Polyether chain>
The quantum dot composite-containing vesicle of the present invention can further comprise a polyether chain on the surface thereof. Since vesicles are flexible and have fluid-like characteristics, vesicles may be fused when left for a long time. In order to overcome this problem, it is preferable to introduce a polyether chain to the vesicle surface to prevent vesicle fusion. By providing the polyether chain, the vesicle structure can be stabilized for a long time by keeping the vesicle alone in the solution by the excluded volume effect (steric hindrance) of the polyether chain on the vesicle surface.
Examples of the polyether chain include a polyethylene glycol (PEG) chain and a polypropylene glycol chain. Among these, a polyethylene glycol chain is preferable.

The polyether chain preferably has a functional group chemically bonded to a biomolecule such as a protein at its end. By providing such a functional group, the surface of the quantum dot complex-containing vesicle is further functionalized and can specifically bind to a target substance. As a functional group that chemically bonds to a biomolecule, a functional molecule such as biotin and maleimide can be used.
As a polyether (functional polyether) having such a functional group, for example, 1,2-distearoyl-sn-glycero-3-phosphaethanolamine-N- [ Biotin-PEGylated phospholipid molecule such as biotinyl (polyethylene glycol) -2000] (ammonium salt) (DSPE-PEG2000-Biotin), 1,2-distearoyl-sn-glycero-3 represented by the following formula (V) -Maleimide-PEGylated phospholipid molecules such as phosphaethanolamine-N- [maleimide (polyethylene glycol) -2000] (ammonium salt) (DSPE-PEG2000-Maleimide).

  The chain length of the polyether chain is preferably one having a mass average molecular weight of 500 to 10,000 from the viewpoint of the yield in the production of the obtained quantum dot complex-containing vesicle and the stability of the vesicle structure.

  An example of a quantum dot complex-containing vesicle having a functional polyether chain is shown in FIG. As shown in FIG. 4, the quantum dot complex-containing vesicle 300 includes a biotin-PEGylated phospholipid molecule 310 on the surface of the vesicle 110. In this embodiment, a polyethylene glycol chain (PEG chain) 314 is bonded to the surface of the vesicle 110, and biotin 312 is provided at the end of the PEG chain 314, so that the outermost part of the vesicle 300 containing the quantum dot complex is provided. It is assumed that biotin 312 is located. The quantum dot complex-containing vesicle 300 includes the biotin-PEGylated phospholipid molecule 310 and can specifically and easily bind to streptavidin.

  Further, as shown in FIG. 5, the quantum dot complex-containing vesicle 400 including a polyether chain includes a quantum dot complex-containing vesicle 400 including a maleimide-PEGylated phospholipid molecule 410 on the surface of the vesicle 110. In the present embodiment, the PEG chain 314 is bonded to the surface of the vesicle 110, and the maleimide 412 is provided at the end of the PEG chain 314, so that the maleimide 412 is located on the outermost part of the quantum dot complex-containing vesicle 400. It is supposed to be. The quantum dot complex-containing vesicle 400 includes the maleimide-PEGylated phospholipid molecule 410, so that the maleimide 412 is a protein such as an antigen, an antibody, an enzyme, a membrane protein, a receptor protein, a fluorescent protein, a cytoskeleton, or a motor protein. It can be easily bound to the protein by reacting with the terminal amino group.

<Method for producing vesicles containing a quantum dot composite>
The method for producing a quantum dot complex-containing vesicle according to the present invention includes a first step of obtaining a quantum dot complex by binding a biological molecule to the surface of a quantum dot, and the obtained quantum dot complex and phospholipid. And a second step of encapsulating the phospholipid in a dispersion liquid.

  The first step is the same as the above-described “<Method for producing quantum dot composite>”.

  In the second step, the quantum dot complex obtained in the first step and the phospholipid (phospholipid for vesicle) constituting the vesicle are dispersed in a dispersion medium (dispersion operation), and then the dispersion medium is removed. -After drying, a vesicle phospholipid is formed in a capsule shape in water or a buffer solution (encapsulation operation) to form a vesicle.

<< Distributed operation >>
In the dispersion operation, the quantum dot composite and the phospholipid for vesicle are dispersed in a dispersion medium.
Examples of the dispersion medium include organic solvents such as chloroform.
Although the density | concentration of the quantum dot composite in a dispersion liquid is not specifically limited, For example, 0.001-1 g / L is preferable.
Moreover, the phospholipid concentration for vesicles in the dispersion is not particularly limited, but is preferably 0.1 to 10 g / L, for example.

  If necessary, a functional polyether such as polyether or biotin-PEGylated phospholipid molecule, maleimide-PEGylated phospholipid molecule can be added to the dispersion. By adding a polyether or a functional polyether to the dispersion, a polyether chain or a functional polyether chain can be introduced on the surface of the vesicle. The content of the polyether or functional polyether in the dispersion is preferably 0.1 to 1% by mass with respect to the phospholipid for vesicles.

≪Encapsulation operation≫
In the encapsulation operation, the organic solvent as a dispersion medium is removed and dried to form a phospholipid bilayer composed of phospholipids for vesicles, and this phospholipid bilayer is formed into a capsule shape in water or a buffer solution. Is produced. Examples of methods for producing vesicles include static hydration method and electro-swelling method (electric field forming method). Among them, large vesicles are easy to produce, and because of the reaction time and the simplicity of the reaction process, the electric field forming method. Is preferably adopted. The electric field forming method is, for example, P.I. Examples include known electrolysis methods described in Girard et al., “Biophysical Journal”, Vol. 87, 2004, pp. 419-429. In the electric field forming method, a dispersion liquid is applied on an electrode such as indium tin oxide (ITO), the dispersion medium is removed and dried to form a phospholipid film, and then a vesicle aqueous solution is added to the thin film to generate an alternating electric field. This is a technique for swelling giant vesicles in an aqueous solution by applying. The method for removing the dispersion medium is not particularly limited, and examples thereof include drying under reduced pressure. As the aqueous vesicle solution, water or a buffer solution such as a phosphate buffer solution or a borate buffer solution is used. In order to obtain vesicles of uniform size, it is preferable to form a uniform phospholipid thin film having a thickness of 10 nm to 100 μm on the ITO substrate. Moreover, the AC electric field to be applied is preferably under the conditions of a frequency of about 10 Hz and an amplitude of about 100 mV to 2 V. At lower amplitudes, the yield of vesicles is low, and at higher amplitudes, vesicles may break down or electrolyze water, and vesicles may not be manufactured.

This encapsulation operation is an operation for forming a vesicle composed of a phospholipid in a vesicular aqueous solution. A chemical substance can be added to the aqueous vesicle solution as necessary. By adding a chemical substance to the aqueous vesicle solution, a quantum dot complex-containing vesicle that encloses the chemical substance can be produced.
Through the above steps, a vesicle dispersion in which the quantum dot composite-containing vesicle is dispersed can be obtained.

According to the quantum dot composite of the present invention, the quantum dot has a shell portion made of a polymer, and has a bio-related molecule on the surface of the quantum dot. Fluorescence characteristics can be exhibited.
In addition, the quantum dot composite of the present invention can be obtained by a simple method of binding biologically related molecules to the surface of the quantum dots.

  Conventionally, biochips and the like for testing physiological activity have a problem that most of chemical substances to be added diffuse into the solution, and the action of the chemical substances on biomolecules becomes low efficiency. The quantum dot composite-containing vesicle of the present invention can be easily destroyed by adding an aqueous solution of 1 mmol / L to 100 mmol / L of sodium chloride, calcium chloride, magnesium chloride or the like. According to the quantum dot complex-containing vesicle of the present invention, the chemical substance to be contained is added to the biomolecule by adding an aqueous solution containing salts after the quantum dot complex-containing vesicle is directly bonded to the biomolecule. It can be released at a short distance, allowing chemical substances to act with high efficiency. In addition, since the vesicle is fluorescently labeled with quantum dots, it is possible to easily grasp the position information of the target biomolecule in the living tissue.

  The quantum dot composite-containing vesicle of the present invention can prevent fusion of quantum dot composite-containing vesicles by providing a polyether chain on the surface thereof. In addition, by making the polyether chain into a functional polyether chain, high selectivity can be imparted to the quantum dot composite-containing vesicle of the present invention depending on the target substance or candidate target substance.

 According to the method for producing a quantum dot complex-containing vesicle of the present invention, a quantum dot complex-containing vesicle having a quantum dot complex in and / or on the surface of the phospholipid bilayer of the vesicle is easily produced. it can. In addition, in the second step, the chemical substance can be included in the inner hole of the vesicle by adding the chemical substance to the dispersion. Furthermore, the quantum dot composite containing vesicle which functionalized the surface of the vesicle can be produced by adding a polyether or a functional polyether in the second step.

  The present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

Example 1
Carboxyl group-modified quantum dot 525 (quantum dot 525 (emission wavelength 525 nm), Qdot <R> ITK ^™ Carboxyl Quantum Dots, Invitrogen Corp., Carlsbad, CA, USA) containing 8 μmol of water, and 2.5 mg / mL of DOPE A chloroform dispersion, an aqueous solution containing 20 mg / mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and a boric acid buffer solution (10 mmol H ₃ BO ₃ , pH 7.4) were prepared, respectively. did. Into a glass bottle of the reaction vessel, 1.1 mL of chloroform was added, followed by 286 μL (960 nmol) of DOPE chloroform dispersion. While gently stirring with a stir bar, 700 μL of borate buffer solution, 100 μL (0.8 nmol) of quantum dot 525 aqueous solution, and 276 μL (28.8 μmol) of EDC aqueous solution were added, and methanol was added in order to make the reaction solution homogeneous. 5 mL was added dropwise. When this reaction solution was stirred at room temperature overnight, a yellow product deposited on the wall surface of the glass container was obtained. When the reaction solution was completely removed and chloroform was added, the product was uniformly dispersed in chloroform. By adding pure water to this chloroform dispersion, repeating centrifugation (10,000 rpm / min, 10 min, 10 ° C.) and removing the supernatant aqueous phase, only the chloroform phase in which the product is dissolved is extracted. Unreacted quantum dots 525 and excess EDC were removed. Thereafter, chloroform was completely removed to obtain a target quantum dot composite. This product could be redispersed in chloroform because the surface of the quantum dots was chemically modified with phospholipid molecules.

(Example 2)
A quantum dot composite was produced in the same manner as in Example 1 except that the phospholipid chemically modified on the surface of the carboxyl group-modified quantum dot 525 was changed to DPPE instead of DOPE.

<Absorption spectrum measurement>
The quantum dot composite obtained in Example 1 or 2 was dispersed in chloroform so as to be 50 μg / mL to prepare a chloroform dispersion of the quantum dot composite (complex-chloroform dispersion). The absorption spectrum (wavelength 300-700 nm) of this complex-chloroform dispersion was measured. For reference, the same measurement was performed on an aqueous dispersion of quantum dots 525 as a starting material.

In FIG. 6, the legend (a1) shows the measurement results of the quantum dots 525 measured as a reference, the legend (a2) shows the measurement results of the quantum dot composite obtained in Example 1, and the legend (a3) shows the measurement results of the quantum dot complex obtained in Example 2. It is. As shown in the legends (a2) and (a3), in each of the quantum dot complexes obtained in Examples 1 and 2, an absorption peak having a maximum at 508 nm is observed, and a short wavelength is observed in the vicinity of 300 nm to 480 nm. The absorption characteristics increased as the absorption intensity increased. The absorption characteristics of the starting quantum dot 525 showed a similar tendency. Thus, the shape of the absorption spectrum of the quantum dot composites of Examples 1 and 2 and the shape of the absorption spectrum of the quantum dot 525 coincide. That is, it was suggested that the optical properties of the quantum dots are retained even after chemical modification of phospholipid molecules on the surface of the quantum dots.
In addition, the light absorbency shown in the graph of FIG. 6 is the value which normalized the light absorbency measured about each solution with the peak top of 508 nm.

<Fluorescence spectrum measurement>
The quantum dot complex obtained in Example 1 or 2 was dispersed in chloroform so as to be 50 μg / mL to prepare a complex-chloroform dispersion. The fluorescence spectrum (excitation wavelength: 400 nm, measurement wavelength: 410-700 nm) of this complex-chloroform dispersion was measured. For reference, the same measurement was performed on an aqueous dispersion of quantum dots 525 as a starting material. The results are shown in FIG.

In FIG. 7, the legend (b1) shows the measurement results of the quantum dots 525 measured as a reference, the legend (b2) shows the measurement results of the quantum dot composite obtained in Example 1, and the legend (b3) shows the measurement results of the quantum dot composite. It is. As shown in the legends (b2) and (b3), in each of the quantum dot complexes obtained in Examples 1 and 2, a fluorescence peak having a maximum at 525 nm was observed. The fluorescence characteristics of the quantum dot 525, which is the starting material, showed similar fluorescence peaks. Thus, the shape of the fluorescence spectrum of the quantum dot composites of Examples 1 and 2 and the shape of the fluorescence spectrum of the quantum dot 525 coincide. That is, it was suggested that the optical properties of the quantum dots are retained even after chemical modification of phospholipid molecules on the surface of the quantum dots.
The fluorescence intensity shown in the graph of FIG. 7 is a value obtained by normalizing the fluorescence intensity measured for each solution with a peak top of 525 nm.

<Dynamic light scattering measurement>
The quantum dot complex obtained in Example 1 was dispersed in chloroform so as to be 50 μg / mL to prepare a complex-chloroform dispersion. Dynamic light scattering measurement of this complex-chloroform dispersion was performed, and the particle size distribution of the quantum dot complex was observed. For reference, the same measurement was performed on an aqueous dispersion of quantum dots 525 as a starting material. The results are shown in FIG.

FIG. 8A shows the measurement result of Example 1, and FIG. 8B shows the measurement result of the quantum dot 525.
As shown in FIG. 8A, in the quantum dot composite of Example 1, two peaks having an average particle diameter of 16.7 nm and 71.8 nm were observed. From this peak intensity ratio, it was suggested that in the quantum dot composite of Example 1, particles having a particle size of 71.8 nm were present in an amount 3.5 times that of particles having a particle size of 16.7 nm. On the other hand, as shown in FIG. 8B, two peaks having an average particle diameter of 16.3 nm and 143 nm were also observed in the aqueous dispersion of the quantum dots 525 as a starting material. From this peak intensity ratio, it was suggested that the quantum dot 525 contains 6.8 times the amount of particles having a particle size of 16.3 nm as compared to particles having a particle size of 143 nm. From these results, it was estimated that the particle size of the quantum dots 525 is about 16 to 17 nm. In general, nanoparticles tend to aggregate in a solution, but the reason why the abundance ratio of large particles in the quantum dot composite of Example 1 is large is that in the process of chemical modification of phospholipid molecules or the process of purification of products. There is a possibility that the quantum dot particles are aggregated.

<Atomic force microscope (AFM) measurement>
The quantum dot complex obtained in Example 1 was dispersed in chloroform so as to be 100 μg / mL to prepare a complex-chloroform dispersion. After 10 μL of this complex-chloroform dispersion was dropped on a cleaved mica substrate, AFM measurement was performed in air at room temperature for a sample dried in air. Further, as a reference, the same measurement was performed on a dried sample obtained by dropping an aqueous dispersion of quantum dots 525 as a starting material onto a mica substrate. The results are shown in FIGS.

FIG. 9A is a photograph by AFM of the quantum dot composite of Example 1, and FIG. 9B is a measurement result of the particle size of the particle A observed in FIG. 9A. 9 (c) is a measurement result of the particle size of the particle B observed in FIG. 9 (a). 10A is a photograph of the quantum dot 525 by AFM, FIG. 10B is a measurement result of the particle size of the particle C observed in FIG. 10A, and FIG. FIG. 11 is a measurement result of the particle diameter of the particle D observed in FIG.
As shown in FIG. 9A, in Example 1, a plurality of particles A and B were observed. As shown in FIGS. 9B and 9C, from the results of the particle size measurement of the particles A and B, the particles observed in FIG. 9A were 20 to 40 nm in height. However, since the distance in the XY plane direction reaches 100 nm or more in any case, these are not simple quantum dot complexes, but in the process of drying the measurement sample, the quantum dot complexes are two-dimensionally and three-dimensionally separated. Presumed to be aggregated.
As shown in FIG. 10A, a plurality of particles C and D were also observed for the quantum dot 525, which is the starting material. As shown in FIGS. 10B and 10C, from the results of the particle size measurement of the particles C and D, the particles observed in FIG. 10A have a height of 20 to 60 nm in the XY plane direction. The distance was over 100 nm. This suggested the possibility that the quantum dots 525 are aggregated in the aqueous dispersion. Further, in FIG. 10A, a string-like structure F derived from the polymer layer was observed on the particle surface. After the sample was dried, it was observed that the polymer chains were intertwined in a complicated manner by cross-linking the particles and forming a network structure with each other.

(Example 3)
Example except that in place of the quantum dot 525, a carboxyl group-modified quantum dot 625 (quantum dot 625 (emission wavelength 625 nm), Qdot <R> ITK ^™ Carboxyl Quantum Dots, Invitrogen Corp., Carlsbad, CA, USA) was used. In the same manner as in Example 1, a quantum dot composite was prepared.
When the absorption spectrum and the fluorescence spectrum of the obtained quantum dot composite were measured in the same manner as in Example 1, an absorption spectrum and a fluorescence spectrum equivalent to the aqueous dispersion of quantum dots 625 were shown.

Example 4
A quantum dot composite was prepared in the same manner as in Example 3 except that the phospholipid molecule chemically modified on the surface of the quantum dot 625 was changed to DPPE instead of DOPE.
When the absorption spectrum and the fluorescence spectrum of the obtained quantum dot composite were measured in the same manner as in Example 1, an absorption spectrum and a fluorescence spectrum equivalent to the aqueous dispersion of quantum dots 625 were shown.

(Example 5)
Complex-chloroform dispersion 200 μL containing 0.2 mg of egg yolk-derived phosphatidylcholine (L-α-PC) and 10.5 μg (5 mass%) of the quantum dot complex of the quantum dot 525 obtained in Example 1 Was prepared. Subsequently, L-α-PC and the complex-chloroform dispersion are uniformly applied on the ITO substrate (substrate obtained by thinning ITO with a thickness of 100 nm on SiO ₂ , size 40 × 40 nm, 50 to 100 Ω · cm). It was applied to. The substrate was dried under reduced pressure at room temperature for 2 hours to completely remove the chloroform dispersion, thereby forming a uniform phospholipid thin film on the ITO substrate. On this phospholipid thin film, a silicone rubber having a window portion (having a window portion in which a silicone rubber having an outer dimension of 30 × 30 mm and a thickness of 1 mm is cut through in a size of 20 × 20 mm) is disposed in close contact with the window portion. To the flask, 400 μL of 200 mmol aqueous sucrose solution was added dropwise. Further, an ITO substrate was placed on the top, and the solution in the silicone rubber window was sandwiched between the ITO substrates and sealed. Subsequently, a clip electrode is joined to the ITO substrate, and an alternating electric field (sine wave, 1 V, 10 Hz) is applied on a hot plate at 60 ° C. for 2 hours, whereby the quantum dot composite-containing vesicle is formed by an electric field forming method. A vesicle-sucrose dispersion dispersed in an aqueous sucrose solution was obtained. The inner hole of this vesicle contains a sucrose aqueous solution as a chemical substance.

<Confocal laser fluorescence microscope measurement>
300 μL of a 200 mmol glucose solution was dropped on the surface of a silicone-coated water-repellent glass slide to form droplets. 10 μL of the vesicle-sucrose dispersion obtained in Example 5 was added to the droplet. Using the difference in specific gravity between the sucrose solution inside the vesicle and the glucose solution outside the vesicle, the vesicle was allowed to stand for 5 minutes to settle near the slide glass surface. Then, this sample was observed using a confocal laser fluorescence microscope (excitation: 488 nm argon laser, observation: fluorescent filter for 505 to 525 nm region observation, 40 × objective lens). The result is shown in FIG.

  FIG. 11A is a photograph observed at a magnification of 40 times, and FIG. 11B is an enlarged view of a part of FIG. As shown in FIGS. 11A and 11B, many ring-shaped fluorescent images were observed from the confocal laser fluorescence micrographs. This ring shape is an image reflecting the cross section of the vesicle. The result of the fluorescence component being observed in a ring shape suggests that the quantum dot complex is introduced into the phospholipid bilayer of the vesicle structure or on the vesicle surface. A fluorescent component is not observed in the inner hole of the vesicle, and it is assumed that the quantum dot complex is hardly contained in the liquid in the vesicle.

<AFM measurement>
On the cleaved mica substrate, 3 μL of the vesicle-sucrose dispersion obtained in Example 5 and 50 μL of a 200 mmol glucose solution were dropped, and then allowed to stand for 15 minutes. After standing, the quantum dot complex-containing vesicles were allowed to settle on a mica substrate, 20 μL of 5 mmol calcium chloride aqueous solution was added to the droplets, and the mixture was allowed to stand for 15 minutes. By this treatment, the vesicle containing the quantum dot complex was broken and formed into a film on mica. With respect to this sample, AMF measurement was performed in a solution at room temperature. The result is shown in FIG.

FIG. 12A is a photograph by AFM of the vesicle containing the quantum dot composite of Example 5 in which the vesicles were collapsed, and FIG. 12B is the film thickness of the cross section at the phantom line G in FIG. FIG. 12C shows the measurement results, and FIG. 12C shows the measurement results of the particle size of the particles H observed in FIG. FIG. 13 (a) is a photograph by AFM of the vesicle containing the quantum dot composite of Example 5 in which the vesicle was disrupted, and FIG. 13 (b) is a measurement result of the particle size of the particle L in FIG. 13 (a). It is.
In FIGS. 12 (a) and 13 (a), symbol J indicates a film produced by collapsing the vesicle. As shown in FIG. 12A, a plurality of dot structures I and dot structures H were observed in the film J. In addition, as shown in FIG. 12 (b), the membrane J was derived from the phospholipid bilayer of the vesicle and had a cross-sectional thickness of 5 nm on the phantom line G. Furthermore, as shown in FIG. 12C, the dot structure H existing inside the film J was 9 nm in height derived from the quantum dot composite. Considering the possibility that the quantum dots are embedded in the film J, the particle size of the quantum dot composite is about 14 nm (a particle size that takes into account the sum of the height of 5 nm in the film and 9 nm outside the film). It is estimated that the height of the dot structure H substantially coincides with the particle size of the quantum dot composite of Example 1 observed by dynamic light scattering measurement.
In addition, as shown in FIG. 13A, a plurality of dot structures K and dot structures L were confirmed in the film J. As shown in FIG. 13B, the dot structure L existing inside the film J was 9 nm in height derived from the quantum dot composite. Considering the possibility that the quantum dots are embedded in the film J, the particle size of the quantum dot composite is estimated to be about 14 nm, and the height of the dot structure L is observed by dynamic light scattering measurement. It almost coincides with the particle size of the quantum dot composite of Example 1.
From the above results, it was confirmed that a quantum dot composite-containing vesicle in which the quantum dot composite was introduced into the vesicle was produced.

(Example 6)
A quantum dot composite-containing vesicle was produced in the same manner as in Example 5 except that the quantum dot composite introduced into the vesicle was changed to the quantum dot composite obtained in Example 2.
The obtained quantum dot composite-containing vesicle was subjected to confocal laser fluorescence microscope measurement and AFM measurement in the same manner as in Example 5. As a result, the quantum dot composite-containing vesicle was introduced into the vesicle. It was confirmed that it was made.

(Example 7)
A quantum dot composite-containing vesicle was produced in the same manner as in Example 5 except that the quantum dot composite introduced into the vesicle was changed to the quantum dot composite obtained in Example 3.
The obtained quantum dot composite-containing vesicle was subjected to confocal laser fluorescence microscope measurement and AFM measurement in the same manner as in Example 5. As a result, the quantum dot composite-containing vesicle was introduced into the vesicle. It was confirmed that it was made.

(Example 8)
A quantum dot composite-containing vesicle was produced in the same manner as in Example 5 except that the quantum dot composite introduced into the vesicle was changed to the quantum dot composite obtained in Example 4.
The obtained quantum dot composite-containing vesicle was subjected to confocal laser fluorescence microscope measurement and AFM measurement in the same manner as in Example 5. As a result, the quantum dot composite-containing vesicle was introduced into the vesicle. It was confirmed that it was made.

Example 9
Egg yolk-derived phosphatidylcholine (L-α-PC) 0.2 mg, and the quantum dot complex 10.5 μg (5% by mass) obtained in Example 1, and DSPE-PEG (2000) -Biotin 0.6 μg ( 200 μL of a chloroform dispersion containing 0.3% by mass) was prepared. Hereinafter, in the same manner as described in Example 5, the vesicle surface was modified with biotin-PEG molecules by the electric field forming method on the ITO substrate, and both or both of the inside and the surface of the phospholipid bilayer of the vesicle. The crab quantum dot complex was introduced, and a quantum dot complex-containing vesicle containing a sucrose solution in the inner vesicle hole was produced.
The obtained quantum dot composite-containing vesicle was subjected to confocal laser fluorescence microscope measurement and AFM measurement in the same manner as in Example 5. As a result, the quantum dot composite-containing vesicle was introduced into the vesicle. It was confirmed that it was made.

(Example 10)
A quantum dot complex-containing vesicle was prepared in the same manner as in Example 9 except that DSPE-PEG (2000) -Maleimide was used instead of DSPE-PEG (2000) -Biotin as a molecule for functionalizing the vesicle surface. .
The obtained quantum dot composite-containing vesicle was subjected to confocal laser fluorescence microscope measurement and AFM measurement in the same manner as in Example 5. As a result, the quantum dot composite-containing vesicle was introduced into the vesicle. It was confirmed that it was made.

DESCRIPTION OF SYMBOLS 1 Quantum dot composite_body | complex 10 Quantum dot 12 Core part 14 Shell part 20 Bio-related molecule | numerator 100, 200, 300, 400 Quantum dot composite containing vesicle 110 Vesicle 112 Phospholipid 120 Inner pore 210 Chemical substance 314 Polyethylene glycol chain 312 Biotin 412 Maleimide

Claims (7)

A phospholipid-containing lipid bilayer is formed in a capsule shape, and a quantum dot complex is contained in the lipid bilayer and / or on the surface thereof.
The quantum dot composite includes a core portion made of semiconductor nanoparticles, and a shell portion formed by bonding polyethylene glycol having a carboxyl group as a reactive group to the surface of the core portion, and the surface of the shell portion A quantum dot complex-containing vesicle comprising a quantum dot having a reactive group and a phospholipid bonded to the reactive group . The quantum dot complex-containing vesicle according to claim 1 , wherein a chemical substance is encapsulated in the lipid bilayer formed in the capsule shape. 3. The quantum dot complex-containing vesicle according to claim 1 or 2 , wherein the lipid bilayer comprises a polyether chain on a surface thereof. 4. The quantum dot complex-containing vesicle according to claim 3 , wherein the polyether chain has a functional group capable of binding to a biomolecule at a terminal. Quantum comprising a core portion made of semiconductor nanoparticles, and includes a shell portion of polyethylene glycol is engaged binding to the surface of the core part comprising a carboxyl group as reactive group and the reactive groups on the surface of the shell portion and dots, a first step of obtaining a phospholipid, a pre Kihan応活groups and the phospholipids quantum dot conjugates bound by chemical bonds between the reactive groups of,
A quantum dot conjugate obtained, and another phospholipid is dispersed in a dispersion medium, after another phospholipid of the containing quantum dot complex by removing the dispersion medium is thinned, the in aqueous solution And a second step of encapsulating another phospholipid of the method for producing a vesicle containing a quantum dot complex. The method for producing a vesicle containing a quantum dot complex according to claim 5 , wherein a chemical substance is further added in the second step. The quantum dot composite according to claim 5 or 6 , wherein the second step further comprises adding a phospholipid introduced with a polyether chain having a functional group capable of binding to a biomolecule at a terminal to obtain a dispersion. A method for producing a containing vesicle.