JPWO2017213107A1 - Nanocapsule integration method and apparatus - Google Patents

Abstract

  The nanocapsule is integrated by irradiating an aqueous liquid (W) in which a plurality of nanocapsules (10) are dispersed with laser light (L1) outside the wavelength range of localized surface plasmon resonance of gold nanoparticles (1). A step (C) in which ELP2 is changed from hydrophilic to hydrophobic by increasing the temperature of ELP2 to agglomerate a part of the plurality of nanocapsules (10), and the nanocapsule aggregate (10A) formed by the aggregation is laser-induced The step (D) of capturing at the irradiation position of the light (L1) and the nanocapsule aggregate (10A) by converting the laser light (L1) into heat by the gold nanoparticles (10) contained in the nanocapsule aggregate (10A) ) Heating the surrounding liquid (E) and accumulating a plurality of nanocapsules (10) by convection generated by heating the liquid (G).

Description

  The present disclosure relates to a nanocapsule accumulation method and an accumulation device, and more particularly, to a nanocapsule accumulation method and an accumulation device including metal nanoparticles.

  In recent years, a drug delivery system (DDS: Drug Delivery System) for delivering a drug to a specific location has attracted attention. In a drug delivery system, a polymer containing a drug is used as a carrier. Such carriers are also referred to as “nanocapsules” because they generally have a size on the order of nanometers.

International publication WO2014 / 92937 International Publication WO2015 / 170758 JP 2012-232961 A

D. Fukushima, UH Sk, Y. Sakamoto, I. Nakase, C. Kojima, “Dual stimuli-sensitive dendrimers: Photothermogenic gold nanoparticle-loaded thermo-responsive elastin-mimetic dendrimers”, Colloids and Surfaces B: Biointerfaces, 132, 155 -160 (2015)

  There has been proposed a technique for accumulating minute objects having a size on the order of nanometers to the order of micrometers by irradiating light into a liquid in which metal nanoparticles are dispersed (for example, international publication by the present inventors). WO2014 / 92937 (patent document 1) and international publication WO2015 / 170758 (patent document 2)).

  When light is irradiated into the liquid in which the metal nanoparticles are dispersed, the metal-nanoparticles in the vicinity of the light irradiation position are captured by the light irradiation position due to the action of light-induced force. The captured metal nanoparticles heat the surrounding liquid by the effect of converting light into heat by localized surface plasmon resonance (hereinafter also referred to as “photothermal effect”). Due to the temperature gradient generated thereby, convection toward the light irradiation position is generated. A small object is carried to the light irradiation position by this convection and accumulated.

  In this way, micro objects can be integrated by using convection generated by the light heating effect of the metal nanoparticles. This photothermal effect of the metal nanoparticles can occur even when the metal nanoparticles are encapsulated in the nanocapsules. Therefore, it is conceivable to accumulate the nanocapsules by irradiating the nanocapsules encapsulating the metal nanoparticles with light.

  The intensity of the light-induced force acting on the metal nanoparticles depends on the size (volume) of the metal nanoparticles (details will be described later). The metal nanoparticles are less likely to be captured by light-induced force as the particle diameter is smaller. The particle diameter of the metal nanoparticles that can be captured by light irradiation is several tens of nanometers or more at a typical light intensity (for example, about several hundred mW).

  On the other hand, depending on the size of the nanocapsules, it may be desirable to use finer metal nanoparticles, ie, metal nanoparticles having a size on the order of “single nanometer” that indicates a range from 1 nm to 10 nm. However, it is difficult to capture single nanometer order metal nanoparticles. If the metal nanoparticles cannot be captured by light-induced force, convection does not occur and nanocapsules cannot be integrated. Therefore, a technique for using metal nanoparticles of the order of single nanometer is desired.

  In addition, the wavelength of localized surface plasmon resonance of metal nanoparticles in a liquid (for example, in water) is generally included in the wavelength region of visible light. Depending on the application field of nanocapsules (for example, the above-described drug delivery system), It may be desirable to be able to use non-resonant light (eg, infrared light) outside the surface plasmon resonance wavelength range. However, when using non-resonant light, capturing metal nanoparticles can be more difficult than when using resonant light.

  The present disclosure has been made in order to solve the above-described problems. The purpose of the present disclosure is to provide a nanocapsule including metal nanoparticles of the order of a single nanometer, outside the wavelength range of localized surface plasmon resonance of the metal nanoparticles. It is to provide a technology that can be integrated using non-resonant light having a wavelength.

  A nanocapsule accumulation method according to an aspect of the present disclosure accumulates a plurality of nanocapsules. Each of the plurality of nanocapsules is modified on the surface of a metal nanoparticle having a size on the order of a single nanometer indicating a range from 1 nanometer to 10 nanometer, a polymer shell enclosing the metal nanoparticle, and a surface of the polymer shell And organic molecules that change from hydrophilic to hydrophobic as the temperature rises. The above integration method irradiates non-resonant light outside the wavelength range of localized surface plasmon resonance of metal nanoparticles to an aqueous liquid in which a plurality of nanocapsules are dispersed, so that the organic molecules are made hydrophilic by raising the temperature of the organic molecules. The step of aggregating a part of a plurality of nanocapsules by changing to hydrophobicity, the step of capturing the nanocapsule aggregate formed by aggregation at the irradiation position of non-resonant light, and the metal nano contained in the nanocapsule aggregate The method includes converting non-resonant light into heat by particles to heat a liquid around the nanocapsule aggregate, and collecting a plurality of nanocapsules by convection generated by heating the liquid.

  A nanocapsule accumulation method according to another aspect of the present disclosure accumulates a plurality of nanocapsules. Each of the plurality of nanocapsules is modified on the surface of a metal nanoparticle having a size on the order of a single nanometer indicating a range from 1 nanometer to 10 nanometer, a polymer shell enclosing the metal nanoparticle, and a surface of the polymer shell And organic molecules that change from hydrophobic to hydrophilic as the temperature rises. The above integration method irradiates a liquid, which is an organic solvent in which a plurality of nanocapsules are dispersed, with non-resonant light outside the wavelength range of localized surface plasmon resonance of metal nanoparticles, thereby making the organic molecules hydrophobic due to the temperature rise of the organic molecules. The nanocapsule aggregate includes a step of aggregating a part of a plurality of nanocapsules by changing from a hydrophilic property to a hydrophilic property, a step of capturing the nanocapsule aggregate formed by the aggregation at a position irradiated with non-resonant light, and a nanocapsule aggregate Non-resonant light is converted into heat by the metal nanoparticles to heat a liquid around the nanocapsule aggregate, and a plurality of nanocapsules are accumulated by convection generated by heating the liquid.

  Preferably, in the collecting step, bubbles are generated by heating the liquid, and a plurality of nanocapsules are collected by generating convection toward the bubbles.

Preferably, the non-resonant light is laser light having a wavelength included in an infrared wavelength region.
Preferably, the organic molecule is a temperature responsive polymer that exhibits a polymer that changes from hydrophilic to hydrophobic with increasing temperature.

More preferably, the polymer shell is a dendrimer. The temperature-responsive polymer is a polypeptide including an amino acid sequence represented by (X ₁ PGX ₂ G) _n . X ₁ represents valine or isoleucine, X ₂ represents an amino acid excluding proline, P represents proline, G represents glycine, and n represents a natural number of 1 or more.

  A nanocapsule accumulation apparatus according to still another aspect of the present disclosure accumulates a plurality of nanocapsules. Each of the plurality of nanocapsules is modified on the surface of a metal nanoparticle having a size on the order of a single nanometer indicating a range from 1 nanometer to 10 nanometer, a polymer shell enclosing the metal nanoparticle, and a surface of the polymer shell And organic molecules that change from hydrophilic to hydrophobic as the temperature rises. The accumulation device includes a holding member configured to hold an aqueous liquid in which a plurality of nanocapsules are dispersed, and a light source that emits non-resonant light outside the wavelength range of localized surface plasmon resonance of metal nanoparticles. The light source was formed by agglomeration by irradiating non-resonant light to the liquid, causing the organic molecule to change from hydrophilic to hydrophobic by increasing the temperature of the organic molecule, and aggregating some of the nanocapsules. The nanocapsule aggregate is captured at the irradiation position of the non-resonant light, the non-resonant light is converted into heat by the metal nanoparticles contained in the nanocapsule aggregate, and the liquid around the nanocapsule aggregate is heated. A plurality of nanocapsules are accumulated by convection generated by heating.

  A nanocapsule accumulation apparatus according to still another aspect of the present disclosure accumulates a plurality of nanocapsules. Each of the plurality of nanocapsules is modified on the surface of a metal nanoparticle having a size on the order of a single nanometer indicating a range from 1 nanometer to 10 nanometer, a polymer shell enclosing the metal nanoparticle, and a surface of the polymer shell And organic molecules that change from hydrophobic to hydrophilic as the temperature rises. The accumulation device includes a holding member configured to hold a liquid, which is an organic solvent in which a plurality of nanocapsules are dispersed, and a light source that emits non-resonant light outside the wavelength range of localized surface plasmon resonance of metal nanoparticles. The light source was formed by agglomeration by irradiating the liquid with non-resonant light to change the organic molecule from hydrophobic to hydrophilic by increasing the temperature of the organic molecule and aggregating a part of the plurality of nanocapsules. The nanocapsule aggregate is captured at the irradiation position of the non-resonant light, the non-resonant light is converted into heat by the metal nanoparticles contained in the nanocapsule aggregate, and the liquid around the nanocapsule aggregate is heated. A plurality of nanocapsules are accumulated by convection generated by heating.

  Preferably, the light source generates bubbles attached to the holding member by converting non-resonant light into heat by the metal nanoparticles, and accumulates a plurality of nanocapsules by generating convection toward the bubbles.

Preferably, the non-resonant light is laser light having a wavelength included in an infrared wavelength region.
Preferably, the organic molecule is a temperature responsive polymer that exhibits a polymer that changes from hydrophilic to hydrophobic with increasing temperature.

More preferably, the polymer shell is a dendrimer. The temperature-responsive polymer is a polypeptide including an amino acid sequence represented by (X ₁ PGX ₂ G) _n . X ₁ represents valine or isoleucine, X ₂ represents an amino acid excluding proline, P represents proline, G represents glycine, and n represents a natural number of 1 or more.

  Preferably, the light source causes the cells to be killed by heat generation of the metal nanoparticles contained in the nanocapsule aggregates generated by irradiation with non-resonant light when cells exist in the vicinity of each of the plurality of nanocapsules. It is configured.

  According to the present disclosure, nanocapsules enclosing metal nanoparticles of the order of a single nanometer can be integrated using non-resonant light having a wavelength outside the wavelength range of localized surface plasmon resonance of metal nanoparticles.

It is a figure for demonstrating the mechanism by which a metal nanoparticle is trapped. It is a figure for demonstrating the nanocapsule which concerns on this Embodiment. It is a figure for demonstrating the nanocapsule which concerns on this Embodiment, and the nanocapsule which concerns on a comparative example. It is a figure which shows an example of the temperature dependence of the size of a nanocapsule. It is a figure which shows schematically the structure of the integration device of the nanocapsule which concerns on this Embodiment. It is a figure which shows the structure around a board | substrate in detail. It is a flowchart which shows the integration | stacking method of the nanocapsule which concerns on this Embodiment. It is a figure for demonstrating the principle of photothermal conversion. It is a figure for demonstrating each step of the integration mechanism of the nanocapsule in this Embodiment. It is a figure for demonstrating the mode of the laser spot vicinity at the time of convection generation | occurrence | production. It is a figure for demonstrating the integration conditions of the nanocapsule in the 1st-7th sample. It is a continuous image which shows the light irradiation result to the 1st sample. It is a continuous image which shows the light irradiation result to the 2nd sample. It is a continuous image which shows the light irradiation result to the 3rd sample. It is a continuous image which shows the light irradiation result to the 4th sample. It is a continuous image which shows the light irradiation result to the 5th sample. It is a continuous image which shows the light irradiation result to the 6th sample. It is a continuous image which shows the light irradiation result to the 7th sample. It is a figure for demonstrating an example of the result of having measured the extinction spectrum near the laser spot in the 6th sample using the spectroscope which is not illustrated. It is a figure which shows the structure of the integrated device used for the measurement for confirming the influence on a biological tissue. It is a figure for demonstrating the spectroscopy result before and behind light irradiation in the 6th sample. It is another figure for demonstrating the spectroscopy result before and behind light irradiation in the 6th sample. It is a figure for demonstrating the spectroscopy result before and behind light irradiation in the 8th sample. It is a figure for demonstrating the life-and-death determination result in a 6th sample.

  Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals and description thereof will not be repeated.

  In this disclosure and its embodiments, “single nanometer order” means a range from 1 nm to 10 nm. “Nanometer order” means a range from 1 nm to 1000 nm (= 1 μm). “Micrometer order” means a range from 1 μm to 1000 μm (= 1 mm).

  In the present disclosure and its embodiments, “nanocapsule” refers to a nanocapsule in which another substance (so-called guest molecule) having a shape and size is included in a void inside a substance (so-called host molecule). A structure having a size on the order of meters. A “nanocapsule aggregate” is a mass formed by aggregation of a plurality of nanocapsules. “Agglomeration” means a phenomenon in which substances dispersed and present in a medium take a dense aggregate state. “Accumulation” means a phenomenon in which the density of a substance in a predetermined area becomes higher than the density of a substance in another area when the substances are collected in the predetermined area.

  In the present disclosure and its embodiments, “resonant light” means light that causes a large light-induced polarization in a metal nanoparticle upon incidence on the metal nanoparticle. The light-induced polarization is, for example, electric polarization generated by exciting the electrons inside the metal nanoparticle with light. The “wavelength range of localized surface plasmon resonance of metal nanoparticles” is a wavelength range corresponding to the full width at half maximum of the peak of localized surface plasmon resonance of metal nanoparticles, for example. This wavelength range is often included in the visible light wavelength range of 400 nm to 700 nm. On the other hand, “non-resonant light” means light having a small light-induced polarization generated in metal nanoparticles upon incidence on the metal nanoparticles. “Outside the wavelength range of localized surface plasmon resonance of metal nanoparticles” is, for example, a wavelength range outside the full width at half maximum of the peak of localized surface plasmon resonance of metal nanoparticles. This wavelength range is typically included in the infrared wavelength range. The “infrared wavelength region” refers to a wavelength region of 700 nm to 10,000 μm (= 1 mm), preferably a wavelength region of 700 nm to 2,500 nm, more preferably a wavelength region of 700 nm to 1,400 nm. Point to. Note that “outside the wavelength range of localized surface plasmon resonance of metal nanoparticles” may be included in the ultraviolet wavelength range (for example, the wavelength range of 10 nm to 400 nm).

  In the present disclosure and the embodiment thereof, “biocompatibility” means that the substance performs its original function, does not cause adverse effects or strong stimulation on the living body over a long period of time, and does not cause abnormality in the living body. Means the nature of the substance.

[Embodiment]
In the present embodiment, gold nanoparticles are employed as one exemplary form of metal nanoparticles. However, the kind of metal nanoparticle is not specifically limited, For example, a silver nanoparticle or a copper nanoparticle may be sufficient.

<Capture of gold nanoparticles>
In the present embodiment, as will be described in detail below, a plurality of gold nanoparticles 1 dispersed in a medium (for example, a liquid) are captured by light-induced force.

  FIG. 1 is a diagram for explaining a mechanism by which gold nanoparticles 1 are captured. Referring to FIG. 1, when a medium in which a plurality of gold nanoparticles 1 are dispersed is irradiated with laser light L, the plurality of gold nanoparticles 1 are captured in the vicinity of the beam waist of the laser light L by light-induced force. “Photo-induced force” is used as a general term for dissipative force, gradient force, and inter-material photo-induced force. The “dissipative force” is a force generated by applying a momentum of light to the gold nanoparticle 1 in a dissipative process such as light scattering or light absorption. “Gradient force” is the movement of the gold nanoparticle 1 to the stable point (beam waist) of the electromagnetic potential when the gold nanoparticle 1 in which photo-induced polarization occurs is placed in a non-uniform electromagnetic field. It is power. “Inter-substance light-induced force” is the sum of a force caused by a longitudinal electric field and a force caused by a transverse electric field generated from induced polarization in a plurality of photo-excited gold nanoparticles 1. The photoinduced force F is represented by the following formula (1) (for details, see, for example, T. Iida and H. Ishihara, Phys. Rev. B77, 245319 (2008)).

  Equation (1) is a general expression of light-induced force including dissipative force, gradient force, and inter-substance light-induced force. V in Formula (1) represents the sum total of the volume of all the gold nanoparticles 1. For example, when only one gold nanoparticle 1 exists in the medium, the above formula (1) is expressed as the following formula (2).

  In the formula (2), v represents the volume of a certain gold nanoparticle 1. As can be seen from the equation (2), the light-induced force F increases as the volume v of the gold nanoparticle 1 increases.

<Nanocapsule>
For the nanocapsules according to the present embodiment, a dendrimer that is one type of a dendritic polymer having a three-dimensionally branched dendritic structure or a branched structure is used. In general, a dendrimer has a property of functioning as a host molecule so that a guest molecule can be included (that is, included) inside the dendrimer, and a surface of the dendrimer can be modified with various organic molecules.

  FIG. 2 is a diagram for explaining the nanocapsule 10 according to the present embodiment. As shown in FIG. 2A, the nanocapsule 10 includes a gold nanoparticle 1 as a guest molecule, a dendrimer 2 enclosing the gold nanoparticle 1 as a host molecule, and an organic molecule 3 modified on the surface of the dendrimer 2. Including.

  FIG. 2B schematically shows the internal structure of the dendrimer 2. The shape of the dendrimer 2 is a spherical shell. The diameter D2 of the dendrimer 2 is, for example, in the range from several nm to several tens of nm. Dendrimer 2 includes a core 2A, a branched portion 2B, and a terminal group 2C. The core 2A is formed by being derived from a compound having one or more functional groups.

  The branched portion 2B is formed by repeating a branched structure including an atom (carbon, nitrogen, silicon, phosphorus, etc.) having a valence of 3 or more. In FIG. 2B, a dendrimer having a branch structure repetition number (also referred to as “number of generations”) of 4 is shown. The 0th generation to the 4th generation are represented by G0 to G4, respectively. Gold nanoparticles 1 are encapsulated in the branched portion 2B.

  The particle diameter D1 of the gold nanoparticle 1 is smaller than the diameter D2 of the dendrimer 2 and is on the order of a single nanometer. The particle diameter D1 of the gold nanoparticle 1 is preferably 1 nm to 5 nm, and more preferably 1 nm to 3 nm. 2A shows an example in which only one gold nanoparticle 1 is included in the dendrimer 2, but two or more gold nanoparticles 1 may be included. Techniques for encapsulating metal nanoparticles in dendrimers are known, for example, disclosed in Y. Haba, C. Kojima, A. Harada, T. Ura, H. Horinaka, K. Kono, Langmuir 23 (2007) 5243. .

The terminal group 2 </ b> C is a partial structure (group) existing on the surface of the dendrimer 2. The end group 2C is modified with an organic molecule 3 containing a predetermined repeating structure. More specifically, in this embodiment, the polypeptide is modified to the end group 2C. This polypeptide includes a pentapeptide having an amino acid sequence represented by (X ₁ PGX ₂ G) _n . X ₁ is valine or isoleucine. X ₂ is an amino acid except proline. P is proline and G is glycine. The repetition number n is a natural number of 1 or more.

  The term “amino acids excluding proline” refers to alanine, glycine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine and tyrosine. (Refer to Japanese Patent Laid-Open No. 2012-232961 (Patent Document 3)).

The polypeptide may be modified to all terminal groups 2C of dendrimer 2 or may be modified to only some terminal groups 2C. The polypeptide may be naturally derived or obtained by a known synthesis method. Furthermore, when the repeating number n is 2 or more, X ₁ and X ₂ in the amino acid sequence may be the same or different between the repeating structures.

<Contrast of nanocapsules according to this embodiment and comparative example>
The nanocapsule 10 is characterized in that it undergoes thermal denaturation at a relatively low temperature to form an aggregate. This feature will be described in comparison with the nanocapsule 90 according to the comparative example.

  FIG. 3 is a diagram for explaining the nanocapsule 10 according to the present embodiment and the nanocapsule 90 according to the comparative example. With reference to FIG. 3, the nanocapsule 10 which concerns on this Embodiment is demonstrated first.

  The number of gold nanoparticles 1 included in each dendrimer 2 is, for example, 1. The particle diameter D1 of the gold nanoparticle 1 is, for example, 1.5 nm. The diameter D2 of the dendrimer 2 is, for example, 4.4 nm. The number of generations of dendrimer 2 is 4, and the molecular weight of dendrimer 2 is about 14,000.

As the organic molecule 3, an elastin-like peptide (ELP: Elastin-Like-Peptides) which is a polypeptide that mimics elastin contained in a living body (for example, ligament, skin, or artery) is used. More specifically, a polypeptide containing two pentapeptides represented by an amino acid sequence (VPGVG) (V is valine) is used. That is, in the above-mentioned amino acid sequence (X ₁ PGX ₂ G) _n , X ₁ and X ₂ are both valine and the repeat number n is 2. Hereinafter, this elastin-like peptide is also referred to as “ELP2”. The length (organic molecule length) L3 of ELP2 is about 3 nm.

  Next, the nanocapsule 90 according to the comparative example will be described. The number of gold nanoparticles 1 included in each dendrimer 2 is, for example, 1. The particle diameter D1 of the gold nanoparticle 1 is, for example, 2.0 nm. On the other hand, the dendrimer 2 is equivalent to the dendrimer 2 of the nanocapsule 10 according to the present embodiment. That is, the diameter D2 of the dendrimer 2 is, for example, 4.4 nm.

  As the organic molecule 3, polyethylene glycol (PEG) is used. Since PEG exhibits hydrophilicity, hydration of the nanocapsule 90 is enhanced by modifying the surface of the dendrimer 2 with PEG. Thereby, the dispersibility of the nanocapsule 90 in an aqueous liquid can be improved. In addition, in order to apply the nanocapsule to a drug delivery system, it is necessary for the nanocapsule to have biocompatibility. Therefore, the surface of dendrimer 2 is modified with PEG to impart biocompatibility to the nanocapsule. Can do. This can be seen from the fact that, for example, in the field of cell biology, PEG is used for DNA introduction into cells, cell fusion, and the like.

  FIG. 4 is a diagram illustrating an example of the temperature dependence of the size of the nanocapsules 10 and 90. In FIG. 4, the horizontal axis indicates the temperature (unit: ° C.) of an aqueous liquid (for example, pure water) in which the nanocapsules 10 or the nanocapsules 90 are dispersed. The vertical axis represents the size (average diameter) of nanocapsules 10 and 90 (unit: nm).

  The nanocapsule 10 undergoes thermal denaturation within a relatively low temperature range (for example, a range of 25 ° C. to 50 ° C.) in a neutral region (for example, a range of pH 6 to pH 8) to form an aggregate. More specifically, as the liquid temperature rises, the three-dimensional structure (conformation) of ELP2 changes from a hydrophilic random coil structure to a hydrophobic β-turn structure, so that the surface of the nanocapsule 10 becomes hydrophilic. Changes from hydrophobic to hydrophobic. When such heat denaturation occurs, the smaller the surface area of the hydrophobic portion in the aqueous liquid, the smaller the surface energy and the more stable the state. Therefore, the nanocapsules 10 aggregate to form nanocapsule aggregates. . In the example shown in FIG. 4, the size of the nanocapsule aggregate increases to about 500 nm by heating the liquid.

  Thus, by modifying the surface of the dendrimer 2 with ELP2, the nanocapsule 10 can be given temperature responsiveness. It is possible to impart biocompatibility to the nanocapsule 10 by modifying the surface of the dendrimer 2 with ELP2.

  On the other hand, since PEG does not cause thermal denaturation, nanocapsule aggregates are not formed in the nanocapsule 90 according to the comparative example even when the temperature of the liquid rises. Therefore, the size of the nanocapsule 90 is almost constant regardless of the temperature of the liquid.

  In this embodiment, an example in which a dendrimer is used as the “polymer shell” according to the present disclosure will be described, but other dendritic polymers may be used as the “polymer shell”. Examples of dendritic polymers other than dendrimers include polyethyleneimine, polyether, polyester, and polyamide. Alternatively, two or more of these dendritic polymers may be combined.

  In addition, if it is a structure formed of a polymer capable of encapsulating metal nanoparticles and capable of modifying the shell surface with organic molecules that change from hydrophilic to hydrophobic as the temperature rises, a dendritic polymer and It is also possible to use polymers having different structures instead of or in addition to dendritic polymers. Such polymers include liposomes (liposome membranes) or micelles. The liposome membrane is formed using, for example, DMPC (dimerystoyl phosphatidylcholine), DPPC (dipalmitoyl phophatidylcholine), DSPC (distearoyl phosphatidylcholine) or the like.

  Further, instead of the elastin-like polypeptide having the specific amino acid sequence described above, another substance that changes from hydrophilic to hydrophobic by heating may be used as the organic molecule 3. Such materials are also referred to as “temperature-responsive polymers”, and specific examples thereof include poly (N-isopropylacrylamide) (abbreviated as PNIPAAm), poly (N-alkylacrylamide), poly (N-vinyl). Alkylamide), polyvinyl alkyl ether, polyethylene glycol / polypropylene glycol block copolymers, various vinyl ether polymers having oxyethylene chains, and the like. Alternatively, another polypeptide or protein can be used as the temperature-responsive polymer (see, for example, Masamichi Nakamura, Drug Delivery System 23-6, 627-636 (2008)).

<Configuration of nanocapsule integration device>
FIG. 5 is a diagram schematically showing the configuration of the nanocapsule 10 accumulation apparatus according to the present embodiment. The integrated apparatus 100 includes a substrate 21, an XYZ axis stage 22, a sample supply unit 23, an adjustment mechanism 24, a laser light source 25, an optical component 26, an objective lens 27, an illumination light source 28, and a photographing device 29. And a control unit 30. Hereinafter, the x direction and the y direction represent the horizontal direction. The x direction and the y direction are orthogonal to each other. The z direction represents the vertical direction. The direction of gravity is downward in the z direction.

  The substrate 21 is disposed on the XYZ axis stage 22 and holds the sample S. The detailed configuration of the substrate 21 will be described with reference to FIG.

  The sample supply unit 23 supplies the sample S onto the substrate 21 in response to a command from the control unit 30. As the sample supply unit 23, for example, a dispenser can be used.

  The adjustment mechanism 24 adjusts the positions in the x, y, and z directions of the XYZ axis stage 22 on which the substrate 21 is arranged in accordance with a command from the control unit 30. Since the position of the objective lens 27 is fixed in the present embodiment, the relative positional relationship between the substrate 21 and the objective lens 27 is adjusted by adjusting the position of the XYZ axis stage 22. As the adjustment mechanism 24, for example, a drive mechanism such as a servo motor attached to the microscope and a focusing handle can be used, but the specific configuration of the adjustment mechanism 24 is not particularly limited. The adjusting mechanism 24 may adjust the position of the objective lens 27 with respect to the fixed substrate 21.

  The laser light source 25 emits, for example, near-infrared (for example, a wavelength of 1064 nm) laser light L1 in response to a command from the control unit 30. The laser light source 25 corresponds to a “light source” according to the present disclosure.

  The optical component 26 includes, for example, a mirror, a dichroic mirror, or a prism. The optical system of the integrated device 100 is adjusted so that the laser light L 1 from the laser light source 25 is guided to the objective lens 27 by the optical component 26.

  The objective lens 27 condenses the laser light L1 from the laser light source 25. The light condensed by the objective lens 27 is applied to the substrate 21. Here, “irradiate” includes the case where the laser light L1 passes through the sample S, and is not limited to the case where the beam waist of the light collected by the objective lens 27 is located in the sample S. The optical component 26 and the objective lens 27 can be incorporated into, for example, an inverted or upright microscope body.

  The illumination light source 28 emits white light L <b> 2 for illuminating the sample S in the substrate 21 in response to a command from the control unit 30. As one example, a halogen lamp can be used as the illumination light source 28. The objective lens 27 is also used for taking in the white light L <b> 2 irradiated on the substrate 21 from the illumination light source 28. White light L <b> 2 captured by the objective lens 27 is guided to the photographing device 29 by the optical component 26.

  The imaging device 29 captures the sample S in the substrate 21 irradiated with the white light L <b> 2 in response to a command from the control unit 30, and outputs the captured image to the control unit 30. As the photographing device 29, a video camera including a charge coupled device (CCD) image sensor or a complementary metal oxide semiconductor (CMOS) image sensor is used.

  The control unit 30 controls the sample supply unit 23, the adjustment mechanism 24, the laser light source 25, the illumination light source 28, and the imaging device 29. Further, the control unit 30 performs predetermined image processing on the image photographed by the photographing device 29. Although not shown, the control unit 30 is realized by a microcomputer including a CPU (Central Processing Unit), a memory, and an input / output buffer.

  If the optical system of the integrated device 100 can irradiate the substrate 21 with the laser light L1 from the laser light source 25 and can take the white light L2 from the substrate 21 into the photographing device 29. 5 is not limited to the configuration shown in FIG. 5, and may be configured to include an optical fiber or the like. In the integrated device 100, the sample supply unit 23, the illumination light source 28, and the imaging device 29 are not essential components.

  FIG. 6 is a diagram showing the configuration around the substrate 21 in more detail. The sample S held on the substrate 21 is an aqueous liquid (for example, ultrapure water) W in which the nanocapsules 10 are dispersed. The substrate 21 is made of a material that does not affect the localized surface plasmon resonance (described later) of the gold nanoparticles 1 by the laser light L1 and is transparent to the white light L2. Examples of such a material include quartz and silicon. For the substrate 21, for example, a cover glass can be used. In this case, the sample S dropped on the substrate 21 has a semi-elliptical spherical shape as shown in FIG.

  The objective lens 27 is disposed, for example, below the substrate 21 and condenses the laser light L1 from below. The condensed light is applied to the sample S above the objective lens 27. For example, the horizontal irradiation position of the laser beam L1 is preferably near the center of the circular (or elliptical) bottom surface of the sample S. Further, the vertical position of the focal point (beam waist) of the laser beam L1 is preferably in the vicinity of the interface between the sample S and the substrate 21. The state of the irradiation position (laser spot) of the laser light L1 is photographed from the top to the bottom by the photographing device 29 (see FIG. 5).

  Note that the configuration around the substrate 21 is not limited to the configuration shown in FIG. For example, the laser beam L1 may be irradiated from the top to the bottom. The substrate 21 corresponds to a “holding member” according to the present disclosure. The shape of the “holding member” is not particularly limited as long as the sample S can be held, and may have a three-dimensional shape. Specifically, a liquid level guide (not shown) that indicates the position where the sample S should be dropped and defines the shape of the dropped sample S may be provided on the substrate 21. Further, the “holding member” may be a container 41 (for example, a glass bottom dish) as illustrated in FIG.

<Nanocapsule integration flow>
In the integration apparatus 100 configured as described above, the nanocapsules 10 dispersed in the liquid W are integrated in the vicinity of the laser spot by controlling each device according to the flowchart shown below.

  FIG. 7 is a flowchart showing a method for integrating the nanocapsules 10 according to the present embodiment. Each step included in this flowchart is basically realized by software processing by the control unit 30, but part or all of the steps may be realized by hardware (electric circuit) produced in the control unit 30. Good. At the start of this flowchart, it is assumed that the sample S in which the nanocapsules 10 are dispersed is installed in the sample supply unit 23.

  Referring to FIGS. 1 and 7, in step S <b> 10, control unit 30 prepares substrate 21 and places it on XYZ axis stage 22. This process is realized by, for example, a feeding mechanism (not shown) for the substrate 21.

  In step S <b> 20, the control unit 30 controls the sample supply unit 23 so that the sample S is supplied (dropped) onto the substrate 21. The dropping amount of the sample S may be as small as several μL to several mL.

  In step S <b> 30, the control unit 30 controls the illumination light source 28 so as to emit white light L <b> 2 for irradiating the sample S, and controls the photographing device 29 so as to start photographing the sample S.

  In step S40, the control unit 30 controls the adjusting mechanism 24 so that the laser light L1 from the laser light source 25 is irradiated to an appropriate position (for example, the position described in FIG. 6) of the sample S. The position of the axis stage 22 is adjusted. As an example, in the horizontal direction, the contour of the droplet of the sample S is extracted from the image photographed by the photographing device 29 using the image processing technique of pattern recognition, so that an appropriate position (for example, the sample S of the sample S is obtained). The amount of adjustment up to the vicinity of the center of the circular contour can be calculated. On the other hand, in the vertical direction, since the current position of the substrate 21 is known by a servo motor (not shown) in the adjustment mechanism 24, the vertical direction is adjusted to a predetermined position (for example, the position of the interface between the sample S and the substrate 21). Is possible.

  In step S50, the control unit 30 controls the laser light source 25 to emit the laser light L1. The laser beam L1 is collected by the objective lens 27, and the sampled light is irradiated with the collected light. Thereby, convection is generated in the liquid W of the sample S, and the nanocapsules 10 dispersed in the liquid W are accumulated in the vicinity of the laser spot. The accumulation mechanism of the nanocapsules 10 and how they are accumulated will be described in detail with reference to FIGS. 8 to 19 and FIGS.

  In step S60, the control unit 30 controls the laser light source 25 to stop the irradiation of the laser light L1 onto the substrate 21. As a result, a series of processing ends.

  Note that the process of step S30 is a process for observing the sample S, and is not an essential process for accumulating the nanocapsules 10. Further, when the substrate 21 is fixed on the XYZ axis stage 22, the process of step S10 can be omitted. Accordingly, the nanocapsules 10 can be integrated even when a flowchart that does not include the processes of steps S10 and S30 is executed.

<Nanocapsule accumulation mechanism>
In the integrated device 100, when the sample S is irradiated with the laser light L1 from the laser light source 25, the gold nanoparticles 1 in the nanocapsule 10 generate heat by photothermal conversion.

  FIG. 8 is a diagram for explaining the principle of photothermal conversion. Referring to FIG. 8, the free electrons of gold nanoparticle 1 form localized surface plasmons and are vibrated by laser light L1. This causes polarization. This polarization energy is converted into lattice vibration energy by Coulomb interaction between the free electrons and the nucleus. As a result, the gold nanoparticles 1 generate heat. This effect is also referred to as a “photothermal effect”. It has been confirmed that even when the gold nanoparticles 1 are encapsulated in the dendrimer 2, heat is generated due to the photothermal effect.

  FIG. 9 is a diagram for explaining each stage of the accumulation mechanism of the nanocapsules 10 in the present embodiment. Before the start of laser beam L1 irradiation (hereinafter also abbreviated as “light irradiation”), the nanocapsules 10 are dispersed in the aqueous liquid W (see FIG. 9A). Thereafter, light irradiation is started (see FIG. 9B).

  As described with reference to FIG. 1, the greater the volume of the metal nanoparticles, the greater the light-induced force acting on the metal nanoparticles. In other words, metal nanoparticles are less likely to be captured by light-induced force as the particle diameter is smaller. The particle diameter of the metal nanoparticles that can be captured by light irradiation is several tens of nanometers or more with typical laser light intensity (for example, about several hundreds mW after passing through the objective lens).

  On the other hand, the particle diameter D1 of the gold nanoparticle 1 is on the order of a single nanometer (1.5 nm in the example shown in FIG. 3), and is generally an order of magnitude smaller than the particle diameter of metal nanoparticles that can be captured. In this case, the volume V of the gold nanoparticles 1 is generally three orders of magnitude smaller than the volume of the metal nanoparticles that can be captured. Therefore, when the gold nanoparticle 1 is dispersed alone in the liquid W, it is difficult to capture the gold nanoparticle 1 by light-induced force.

  Therefore, in the present embodiment, the property of the nanocapsules 10 in which the gold nanoparticles 1 are encapsulated is used. When light irradiation is started, the gold nanoparticles 1 in the nanocapsule 10 in the vicinity of the laser spot generate heat due to the light heating effect. At this stage, since the gold nanoparticles 1 present in the vicinity of the laser spot are few, the generated heat is considered to be a very small amount. However, as described with reference to FIG. 4, the thermal denaturation of the nanocapsules 10 occurs at a relatively low temperature. Therefore, even with a very small amount of heat, the nanocapsules 10 in the vicinity of the laser spot aggregate to form a nanocapsule aggregate 10A ( 10) is formed (see FIG. 9C).

  Since the nanocapsule aggregate 10 </ b> A includes a large number of gold nanoparticles 1, it can be said that the nanocapsule aggregate 10 </ b> A is an aggregate of gold nanoparticles 1. Therefore, the nanocapsule aggregate 10A can be captured by the light-induced force (see FIG. 9D).

  Since the gold nanoparticle 1 in the nanocapsule aggregate 10A captured by the laser spot continuously receives the laser beam L1, it emits more heat than the stage shown in FIG. 9C. Generated by heat generation effect. Thereby, the liquid around the nanocapsule aggregate 10A is heated (see FIG. 9E). As a result, the liquid W in the vicinity of the laser spot locally boils and microbubbles (bubbles of the order of micrometers) are generated in the laser spot (see FIG. 9F). Microbubbles MB can grow over time.

  The closer to the laser spot, the higher the temperature of the liquid W. That is, a temperature gradient is generated in the liquid W by light irradiation. Due to this temperature gradient, regular thermal convection (laminar flow) is constantly generated in the liquid W (see FIG. 9G). Hereinafter, this thermal convection is abbreviated as “convection”.

  FIG. 10 is a conceptual diagram for explaining the state near the laser spot when convection occurs. In FIG. 10, the substrate 21 and the liquid W are not shown in order to avoid complicated drawing.

  Referring to FIG. 10, the direction of convection is a direction that once heads toward microbubble MB (or laser spot) and then leaves microbubble MB. The reason why convection occurs in such a narrow region can be explained as follows. That is, the liquid existing above the microbubbles MB in the vertical direction becomes diluted by heating and rises by buoyancy. At the same time, a relatively low-temperature liquid existing in the horizontal direction of the microbubbles MB flows toward the heated region.

  Returning to FIG. 9, the nanocapsules 10 are transported toward the microbubbles MB in a convection, whereby the nanocapsules 10 are accumulated in the vicinity of the laser spot. More specifically, since a stagnation region R in which the convection flow velocity becomes zero is generated between the microbubble MB and the upper surface of the substrate 21, the nanocapsules 10 carried by the convection stay in the stagnation region R. Collected. After that, when the light irradiation is stopped, the convection is weakened (see FIG. 9H).

  Thus, according to the present embodiment, the aggregation action due to thermal denaturation (change in the three-dimensional structure of ELP2) of the nanocapsule 10 and the convection action due to the heat generation of the nanocapsule 10 (the light heating effect of the gold nanoparticles 1) By actively using the nanocapsules 10, the nanocapsules 10 can be integrated in the vicinity of the laser spot in a short time and at a high density. Note that “the nanocapsules 10 are integrated in the vicinity of the laser spot” may include the case where the nanocapsules 10 are integrated on the surface of the microbubble MB.

<Results of nanocapsule integration>
Subsequently, the result of integration of the nanocapsules 10 will be described in detail in comparison with a comparative example. In the present embodiment and the comparative example, the following eight samples were prepared.

  FIG. 11 is a diagram for explaining the nanocapsule accumulation conditions in the first to eighth samples. As shown in FIG. 11, first to fourth and eighth samples were prepared for the comparative example, and fifth to seventh samples were prepared for the present embodiment.

  The first sample and the second sample are common in that the gold nanoparticle 1 is not encapsulated in the nanocapsule, but the organic molecules 3 modified on the surface of the nanocapsule are different from each other. In the first sample, the nanocapsule surface is modified with PEG, and in the second sample, the nanocapsule surface is modified with ELP2.

  In both the third and fourth samples, the gold nanoparticle 1 is encapsulated in the nanocapsule, and the surface of the nanocapsule is modified with PEG. The concentration of nanocapsules differs by an order of magnitude between the third sample and the fourth sample.

  In the fifth to seventh samples, the point that the gold nanoparticle 1 is encapsulated in the nanocapsule 10 and the point that the surface of the nanocapsule 10 is modified by ELP2 are common, while the concentration of the nanocapsule 10 is Different by one digit.

  In the eighth sample, as in the second sample, the gold nanoparticles 1 are not encapsulated in the nanocapsules, and the surface of the nanocapsules is modified with ELP2. However, the concentration of nanocapsules differs by an order of magnitude between the second sample and the eighth sample.

  The intensity (laser output) of the laser beam L1 from the laser light source 25 was 1.0 W. The magnification of the objective lens 27 was 100 times. The laser output after passing through the objective lens 27 is lower than the laser output from the laser light source 25 and becomes about 20%. That is, the laser output to the sample S was about 200 mW.

  12 to 15 are continuous images respectively showing the results of light irradiation on the first to fourth samples. 12 to 15 and FIGS. 16 to 19 to be described later, the numerals in the drawings indicate the elapsed time (unit: second) from the light irradiation start time.

  Referring to FIG. 12 and FIG. 13, in the first and second samples not containing the gold nanoparticle 1, 110 seconds from the start of light irradiation regardless of the type of the organic molecule 3 modified on the surface of the nanocapsule. Even after elapse, no microbubble MB was generated.

  Next, referring to FIGS. 14 and 15, in the third and fourth samples, the gold nanoparticle 1 is encapsulated in the nanocapsule, but the surface of the nanocapsule is modified with PEG. In this case, the microbubble MB was not generated in the third sample having a relatively low concentration of nanocapsules. In the fourth sample with a higher concentration of nanocapsules, although generation of microbubbles MB was confirmed after about 60 seconds from the start of light irradiation, the size of microbubbles MB was significantly smaller than that of the sample described later.

  16 to 18 are continuous images showing the results of light irradiation on the fifth to seventh samples, respectively. In the fifth to seventh samples, the gold nanoparticle 1 is encapsulated in the nanocapsule 10 and the surface of the nanocapsule 10 is modified with ELP2. In the fifth sample having the lowest nanocapsule 10 concentration, no microbubble MB was generated.

  On the other hand, in the sixth sample whose concentration of nanocapsules 10 is one digit higher than that of the fifth sample, microbubbles MB were generated after several seconds from the start of light irradiation. Furthermore, it was confirmed that the nanocapsules 10 were annularly integrated around the contact region between the microbubble MB and the substrate 21 (the region where the microbubble MB adhered to the substrate 21). The concentration of nanocapsules 10 in the sixth sample is equal to the concentration of nanocapsules in the third sample (see FIG. 14). Comparing these samples, it can be seen that the nanocapsules are accumulated when the surface of the nanocapsules is modified with ELP2, while the nanocapsules are not accumulated when the surface of the nanocapsules is modified with PEG. .

  In the seventh sample having the highest concentration of nanocapsules 10, microbubbles MB were generated immediately after the start of light irradiation. The size of the microbubble MB was even larger than the size of the microbubble MB in the sixth sample. In addition, it was confirmed that convection was generated, and a large amount of nanocapsules 10 were accumulated in the vicinity of the microbubble MB. When the fourth and seventh samples having the same nanocapsule concentration are compared, there is a clear difference in the size of the microbubble MB depending on the type of the organic molecule 3 modified on the surface of the nanocapsule.

  From the above results, it can be seen that the gold nanoparticles 1 are essential for the generation of microbubbles MB and the subsequent accumulation of nanocapsules. In addition, when the concentration of nanocapsules is equal, the organic molecule 3 is more easily ELP2 than the organic molecule 3 is PEG, and microbubbles are more likely to be generated, and the subsequent nanocapsules are more likely to accumulate. I understand. Further, it can be seen that when the organic molecules 3 are of the same type, the higher the concentration of nanocapsules, the more likely the generation of microbubbles MB and the accumulation of nanocapsules occur.

  FIG. 19 is a diagram for explaining an example of the result of measuring the extinction spectrum in the vicinity of the laser spot in the sixth sample using a spectroscope (not shown). As shown in the continuous image of FIG. 19A, the microbubble MB was attached on the substrate 21 during the light irradiation, but immediately after the light irradiation was stopped after 180 seconds from the start of the light irradiation. In addition, the microbubbles MB floated in the liquid W and disappeared.

  The extinction spectrum was measured before the start of light irradiation and after 183 seconds, 210 seconds and 240 seconds from the start of light irradiation (that is, after 3 seconds, 30 seconds and 60 seconds from the stop of light irradiation). The measurement result of the extinction spectrum during the light irradiation is not shown because when the microbubble MB exists during the light irradiation, the intensity of the scattered light by the microbubble MB of the laser light L1 is too high, This is because measurement cannot be performed accurately, and measurement in a state where the microbubble MB does not exist is necessary. The measurement range of the extinction spectrum is the range shown in the image after 183 seconds from the start of light irradiation.

  In FIG. 19B, the horizontal axis represents wavelength (unit: nm), and the vertical axis represents intensity. The intensity of the extinction spectrum after 183 seconds from the start of light irradiation was about twice the intensity of the extinction spectrum before the start of light irradiation. According to Lambert-Beer's law, the higher the intensity of the extinction spectrum, the higher the degree of nanocapsule integration. Therefore, it can be said that the integration of the nanocapsules 10 was confirmed from the extinction spectrum in addition to the continuous image shown in FIG.

  On the other hand, the intensity of the extinction spectrum after 210 seconds or 240 seconds from the start of light irradiation (that is, after 30 seconds or 60 seconds from the stop of light irradiation) is the intensity of the extinction spectrum before the start of light irradiation. Almost equal. This is presumably because the temperature in the vicinity of the laser spot quickly decreased after the light irradiation stopped, and as a result, the nanocapsules 10 once accumulated were dispersed again in the liquid W. That is, since the change in the three-dimensional structure of ELP2 is reversible, it is considered that the surface of the nanocapsule 10 returns from hydrophobic to hydrophilic as the temperature decreases, and as a result, the nanocapsule 10 is dispersed again.

  If the laser light L1 has a wavelength included in the wavelength range of visible light, the laser light L1 can be an obstacle when photographing the vicinity of the laser spot by the photographing device 29 under the irradiation of the white light L2. On the other hand, in the present embodiment, laser light L1 having a wavelength of 1064 nm included in the near-infrared wavelength region is used. Thereby, it can be avoided that the laser beam L1 becomes an obstacle to photographing. Along with that, the following facts are shown.

  The wavelength range of localized surface plasmon resonance of the gold nanoparticle 1 is the wavelength range of visible light in water. Therefore, when using resonant light (visible light) in the wavelength range of localized surface plasmon resonance, the same laser is used compared to using non-resonant light (near infrared light) outside the wavelength range of localized surface plasmon resonance. A greater amount of heat can be generated at the output. Therefore, it is possible to more easily capture the nanocapsule aggregate 10 </ b> A, thereby causing convection due to the light heat generation effect and integrating the nanocapsules 10. From a reverse viewpoint, according to the present embodiment, even when near-infrared light, which is non-resonant light, is used (in other words, under more severe conditions) It can be seen that the capsule 10 can be accumulated.

<Influence on cells and living tissues>
Near-infrared light, for example, widely used in the field of biological imaging, is less susceptible to attenuation (scattering, etc.) due to biological tissue, and has high permeability within the biological tissue. According to the present embodiment, the nanocapsules 10 can be integrated using near-infrared light, suggesting the possibility of application to living tissue. Hereinafter, the applicability of the nanocapsule accumulation method according to the present disclosure to a living tissue (in particular, a cell that is a constituent element of the living tissue) will be described.

  FIG. 20 is a diagram illustrating a configuration of an accumulation device used for measurement for confirming the influence on cells. In this integrated apparatus, a container 41 is used instead of the substrate 21. 20A shows a side view of the container 41, and FIG. 20B shows a top view of the container 41.

  Referring to FIGS. 5, 20 (A), and 20 (B), container 41 is, for example, a glass bottom dish. The container 41 includes a cylindrical bottom portion 411, a wall portion 412 extending in the vertical direction (Z direction) from the bottom portion, and a measurement well 413 that is a cylindrical recess formed in the center of the bottom portion 411. The material of the measurement well 413 is glass. For example, a resin (for example, polypropylene) is used as the material of the bottom portion 411 and the wall portion 412.

  As the sample to be accumulated, any one of the sixth and eighth samples described in FIG. 11 was used. The sixth sample is a sample including nanocapsules 10 enclosing the gold nanoparticles 1. On the other hand, the eighth sample is a sample including nanocapsules that do not enclose the gold nanoparticles 1. In the measurement results shown below, the sixth and eighth samples further include cells 4. As the cell 4, a HeLa cell, which is a cell derived from human cervical cancer, was used. The environmental temperature is room temperature (specifically, 24.3 ° C.), and thermal denaturation (change from hydrophilicity to hydrophobicity) of the nanocapsules 10 has not occurred before light irradiation.

  Laser light L1 was irradiated in a state where 10 μL of the droplet of the sixth or eighth sample containing the healer cell (drop of the medium of the healer cell) was dropped on the measurement well 413. In this measurement, the objective lens 27 having a magnification of 40 times was used. The height of the beam waist of the laser beam L1 is set to only 5 scales from the upper surface of the measurement well 413 by controlling the drive mechanism of the adjustment mechanism 24 (specifically, a servo motor and a focusing handle attached to the microscope). It was set to be in the upper position. This one scale corresponds to 1 μm in the air. The laser output from the laser light source 25 was 1.0 W, and the laser output after passing through the objective lens 7 was 200 mW. The irradiation time of the laser beam L1 was set to 60 seconds. Although not shown in FIG. 5, FIG. 20 (A) and FIG. 20 (B), a spectroscope for measuring the absorption and scattering of the sample by the white light L2 from the illumination light source 28 is provided, An extinction spectrum (local extinction spectrum at a specific position) was measured.

  FIG. 21 is a diagram for explaining the spectral results before and after the light irradiation in the sixth sample. 21 to 23, the measurement region of the extinction spectrum is indicated by a white circle.

  FIG. 21A shows an image (optical microscopic image) and extinction spectrum (that is, an extinction spectrum before generation of microbubble MB) before light irradiation and an image immediately after the disappearance of microbubble MB generated by light irradiation. And the extinction spectrum. The intensity of the extinction spectrum is different between the occurrence of microbubbles MB and the disappearance of the microbubbles MB, although both are common in that no microbubbles MB exist. The intensity of the extinction spectrum after light irradiation is higher than the intensity of the extinction spectrum before light irradiation.

  FIG. 21B shows the extinction spectrum before light irradiation at the position of the cell 4 and the spectrum of the cell after light irradiation at the position of the cell 4. It can be seen that the intensity of the extinction spectrum at the position where the cells 4 are present is higher before light irradiation than after light irradiation.

  FIG. 22 is a diagram for explaining other spectral results before and after light irradiation in the sixth sample. In FIG. 22, the measurement result of the irradiation spectrum to cells 4a and 4b other than the cell 4 shown in FIG. 21 is shown. FIG. 22A shows an image of the cell 4a before light irradiation. FIG. 22B shows an image of the cell 4a after light irradiation. FIG. 22C shows an image of the cell 4b after light irradiation. FIG. 22D shows the extinction spectrum of the cells 4a and 4b. Also in the spectroscopic result shown in FIG. 22D, it is understood that the intensity of the extinction spectrum is higher after light irradiation than before light irradiation.

  FIG. 23 is a diagram for explaining the spectral results before and after the light irradiation in the eighth sample. FIGS. 23A and 22B show images of the eighth sample before and after light irradiation, respectively. In the eighth sample containing nanocapsules that did not encapsulate the gold nanoparticles 1, microbubbles MB were not generated. FIG. 23C shows an extinction spectrum. It can be seen that the extinction spectrum hardly changes before and after the light irradiation.

  Next, the determination result of the life or death of the cell 4 after light irradiation will be described. As fluorescent dyes for staining bacteria, Calcein-AM and PI (Propidium Iodide) are known. Calcein-AM itself emits little fluorescence, but when it is hydrolyzed in the cell, it emits strong yellow-green fluorescence when irradiated with light having an excitation wavelength. Therefore, the region observed in yellow-green in the fluorescence observation image indicates that the cell is alive. On the other hand, PI does not have membrane permeability. For this reason, only cells with damaged cell membranes (ie dead bacteria) are stained with PI. When PI is excited from the outside, red fluorescence is emitted.

  FIG. 24 is a diagram for explaining the life / death determination result in the sixth sample. Here, the measurement well 413 is virtually divided into four regions, and the laser beam L1 is irradiated in the order of region B, region C, and region D (see FIG. 20). The irradiation time of the laser beam L1 in each region B to D was set to 60 seconds. Region A is a region for a control experiment in which the laser beam L1 is not irradiated.

  FIG. 24A shows an image (optical microscope image), a phase contrast image, and a fluorescence observation image after light irradiation in the region C shown in FIG. 20 in order from the left. FIG. 24B shows an image, a phase difference image, and a fluorescence observation image after light irradiation in the region D. In the fluorescence observation image, the dead cells (cells emitting red fluorescence) are distinguished from the surviving cells.

  As shown in FIGS. 24A and 24B, it can be seen that the cells 4 near the laser spot are dead. As described above, the nanocapsules 10 including the gold nanoparticles 1 are accumulated at the irradiation position of the laser light L1 which is non-resonant light, and the cells 4 near the gold nanoparticles 1 are killed by the heat generation of the gold nanoparticles 1. be able to. That is, cancer cells at the targeted position can be selectively killed. This indicates that it can be used for a drug delivery system (DDS). The “near” of the gold nanoparticle 1 preferably means a region inside a few times (for example, 2 to 3 times) the diameter of the microbubble MB generated by irradiation with the laser light L1.

  In the present embodiment, the configuration in which the temperature-responsive polymer, which is an organic molecule that changes from hydrophilic to hydrophobic as the temperature rises, is modified on the nanocapsule surface has been described as an example. Thus, in a water-based liquid, a temperature-responsive polymer that dissolves at low temperatures due to hydrophilicity, but changes from hydrophilic to hydrophobic when heated to a certain temperature and becomes insoluble, the lower limit eutectic temperature ( It is also called LCST (Lower Critical Solution Temperature) type temperature responsive polymer.

  Instead of the LCST type temperature responsive polymer, the following temperature responsive polymer may be used. That is, using a temperature-responsive polymer that dissolves in a liquid (non-aqueous liquid) that is an organic solvent due to hydrophobicity at low temperatures, but changes from hydrophobic to hydrophilic when it rises to a certain temperature. Also good. Such a temperature-responsive polymer is also referred to as an upper critical solution temperature (UCST) type temperature-responsive polymer. The combination of the liquid that is an organic solvent and the UCST type temperature-responsive polymer is basically the same as the above-described embodiment although the direction of the hydrophilicity / hydrophobicity change is reversed. The explanation will not be repeated. In the organic solvent, the resonance wavelength of the localized surface plasmon resonance of the metal nanoparticles can be included in the wavelength range of visible light, so that the non-resonance wavelength range can be, for example, the infrared (or ultraviolet) wavelength range. .

  The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present disclosure is shown not by the above description but by the scope of claims, and is intended to include all modifications within the meaning and scope equivalent to the scope of claims.

  The present disclosure can be used, for example, as a method or apparatus for accumulating nanocapsules encapsulating a drug together with metal nanoparticles. As an example, in a new development, a small amount of drug can be accumulated at a light irradiation position to evaluate the influence on cells or living tissue, and can be used in the medical field. The present disclosure can also be applied to medical devices for performing so-called “thermotherapy” (hyperthermia). It is known that human cells die rapidly when the temperature rises above 42.5 ° C. Furthermore, “photothermal therapy” in which a metal nanomaterial is administered and thermotherapy is induced by light irradiation is also an example of thermotherapy. As in the present disclosure, photothermotherapy can be performed by selectively raising the temperature of cancer cells existing near the nanocapsule, for example, by killing the nanocapsule by light irradiation, thereby reducing the size of the cancer tissue. Or suppress the proliferation of cancer cells.

  1 gold nanoparticle, 2 dendrimer, 2A core, 2B branched part, 2C end group, 3 organic molecule, 10,90 nanocapsule, 10A nanocapsule aggregate, 21 substrate, 22 XYZ axis stage, 23 sample supply unit, 24 adjustment Mechanism, 25 Laser light source, 26 Optical component, 27 Objective lens, 28 Illumination light source, 29 Imaging device, 30 Control unit, 4, 4a, 4b Cell, 41 Container, 411 Bottom part, 412 Wall part, 413 Measurement well, 100 Integrated device , S sample, W liquid.

Claims (13)

A method for accumulating a plurality of nanocapsules, the method for accumulating nanocapsules,
Each of the plurality of nanocapsules is
Metal nanoparticles having a size on the order of a single nanometer exhibiting a range from 1 nanometer to 10 nanometers;
A polymer shell encapsulating the metal nanoparticles;
An organic molecule that is modified on the surface of the polymer shell and changes from hydrophilic to hydrophobic as the temperature rises;
The integration method includes:
By irradiating the aqueous liquid in which the plurality of nanocapsules are dispersed with non-resonant light outside the wavelength range of localized surface plasmon resonance of the metal nanoparticles, the organic molecules are changed from hydrophilic to hydrophobic by the temperature increase of the organic molecules. Aggregating a part of the plurality of nanocapsules by changing to a sex;
Capturing the nanocapsule aggregate formed by aggregation at the irradiation position of the non-resonant light; and
Heating the liquid around the nanocapsule aggregate by converting the non-resonant light into heat by the metal nanoparticles contained in the nanocapsule aggregate;
Integrating the plurality of nanocapsules by convection generated by heating the liquid. A method for accumulating a plurality of nanocapsules, the method for accumulating nanocapsules,
Each of the plurality of nanocapsules is
Metal nanoparticles having a size on the order of a single nanometer exhibiting a range from 1 nanometer to 10 nanometers;
A polymer shell encapsulating the metal nanoparticles;
An organic molecule that is modified on the surface of the polymer shell and changes from hydrophobic to hydrophilic as the temperature rises;
The integration method includes:
By irradiating the liquid, which is an organic solvent in which the plurality of nanocapsules are dispersed, with non-resonant light outside the wavelength range of localized surface plasmon resonance of the metal nanoparticles, the organic molecules become hydrophobic due to the temperature rise of the organic molecules. Agglomerating a part of the plurality of nanocapsules by changing from hydrophilic to hydrophilic,
Capturing the nanocapsule aggregate formed by aggregation at the irradiation position of the non-resonant light; and
Heating the liquid around the nanocapsule aggregate by converting the non-resonant light into heat by the metal nanoparticles contained in the nanocapsule aggregate;
Integrating the plurality of nanocapsules by convection generated by heating the liquid.   The nanocapsule accumulation according to claim 1 or 2, wherein in the accumulation step, bubbles are generated by heating the liquid, and the plurality of nanocapsules are accumulated by generating the convection toward the bubbles. Method.   The nanocapsule integration method according to claim 1, wherein the non-resonant light is laser light having a wavelength included in an infrared wavelength region.   5. The nanocapsule accumulation method according to claim 1, wherein the organic molecule is a temperature-responsive polymer indicating a polymer that changes from hydrophilic to hydrophobic as the temperature rises. The polymer shell is a dendrimer;
The temperature-responsive polymer is a polypeptide comprising an amino acid sequence represented by (X ₁ PGX ₂ G) _n ,
X ₁ represents a valine or isoleucine, X ₂ represents an amino acid except proline, P is indicated proline, G represents glycine, n represents shown a natural number of 1 or more, accumulation of nanocapsules according to claim 5 Method. A nanocapsule accumulating device for accumulating a plurality of nanocapsules,
Each of the plurality of nanocapsules is
Metal nanoparticles having a size on the order of a single nanometer exhibiting a range from 1 nanometer to 10 nanometers;
A polymer shell encapsulating the metal nanoparticles;
An organic molecule that is modified on the surface of the polymer shell and changes from hydrophilic to hydrophobic as the temperature rises;
The integrated device is:
A holding member configured to hold an aqueous liquid in which the plurality of nanocapsules are dispersed;
A light source that emits non-resonant light outside the wavelength range of localized surface plasmon resonance of the metal nanoparticles,
The light source is
By irradiating the liquid with the non-resonant light, the organic molecules are changed from hydrophilic to hydrophobic by increasing the temperature of the organic molecules, and a part of the plurality of nanocapsules is aggregated,
Capturing nanocapsule aggregates formed by aggregation at the irradiation position of the non-resonant light,
The non-resonant light is converted into heat by the metal nanoparticles contained in the nanocapsule aggregate to heat the liquid around the nanocapsule aggregate,
A nanocapsule accumulating device that accumulates the plurality of nanocapsules by convection generated by heating the liquid. A nanocapsule accumulating device for accumulating a plurality of nanocapsules,
Each of the plurality of nanocapsules is
Metal nanoparticles having a size on the order of a single nanometer exhibiting a range from 1 nanometer to 10 nanometers;
A polymer shell encapsulating the metal nanoparticles;
An organic molecule that is modified on the surface of the polymer shell and changes from hydrophobic to hydrophilic as the temperature rises;
The integrated device is:
A holding member configured to hold a liquid which is an organic solvent in which the plurality of nanocapsules are dispersed;
A light source that emits non-resonant light outside the wavelength range of localized surface plasmon resonance of the metal nanoparticles,
The light source is
By irradiating the liquid with the non-resonant light, the organic molecules are changed from hydrophobic to hydrophilic by increasing the temperature of the organic molecules, and a part of the plurality of nanocapsules is aggregated,
Capturing nanocapsule aggregates formed by aggregation at the irradiation position of the non-resonant light,
The non-resonant light is converted into heat by the metal nanoparticles contained in the nanocapsule aggregate to heat the liquid around the nanocapsule aggregate,
A nanocapsule accumulating device that accumulates the plurality of nanocapsules by convection generated by heating the liquid.   The light source generates bubbles attached to the holding member by converting the non-resonant light into heat by the metal nanoparticles, and accumulates the nanocapsules by generating convection toward the bubbles. The nanocapsule accumulation apparatus according to claim 7 or 8.   10. The nanocapsule integration device according to claim 7, wherein the non-resonant light is laser light having a wavelength included in an infrared wavelength region. 11.   11. The nanocapsule accumulation apparatus according to claim 7, wherein the organic molecule is a temperature-responsive polymer indicating a polymer that changes from hydrophilic to hydrophobic as the temperature rises. The polymer shell is a dendrimer;
The temperature-responsive polymer is a polypeptide comprising an amino acid sequence represented by (X ₁ PGX ₂ G) _n ,
The accumulation of nanocapsules according to claim 11, wherein X ₁ represents valine or isoleucine, X ₂ represents an amino acid excluding proline, P represents proline, G represents glycine, and n represents a natural number of 1 or more. apparatus.   The light source kills the cells by heat generation of the metal nanoparticles contained in the nanocapsule aggregates generated by irradiation with the non-resonant light when cells exist in the vicinity of each of the plurality of nanocapsules. 13. The nanocapsule accumulation apparatus according to claim 7, wherein the nanocapsule accumulation apparatus is configured to allow the nanocapsules to be accumulated.