microparticles

Abstract

To provide fluorocarbon-based core-shell microparticles that can be used as artificial oxygen carriers.SOLUTION: Core-shell microparticles comprising a shell including a polymer having a Young's modulus of 100 MPa or less and a core including a fluorocarbon.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to microparticles that can be used as artificial oxygen carriers. [Background technology]

[0002] Many oxygen carriers have been developed to artificially replace the oxygen-carrying capacity of red blood cells used for transfusion. Artificial oxygen carriers can be broadly classified into those that use hemoglobin and those that use PFCs (perfluorocarbons). Hemoglobin has four subunits in one molecule, and can transport oxygen by coordinating the heme iron in each subunit with the oxygen molecule and the imidazole group of the globin protein. Hemoglobin-based oxygen carriers have been developed, including those made from polymers cross-linked with glutaraldehyde and liposome-type oxygen carriers encapsulating hemoglobin. While these hemoglobin-based oxygen carriers have a high oxygen-carrying capacity, issues have been raised regarding their reduced oxygen-carrying capacity due to methemoglobinization (MetHb) and the risk of infection. PFCs can be used as oxygen carriers because they have a high dissolving capacity for gases such as oxygen and carbon dioxide and are chemically and biologically inert. Although PFC-based oxygen carriers are inferior to hemoglobin-based carriers in terms of oxygen transport capacity, they have advantages in terms of sterilization, storage stability, and / or raw material supply, and research and development efforts are being actively conducted toward their practical application.

[0003] In recent years, the use of artificial oxygen carriers as an alternative to red blood cells has been anticipated in the fields of regenerative medicine and tissue engineering. For example, recent advances in regenerative medicine and tissue engineering have led to attempts to regenerate large, three-dimensional tissues. In 3D tissue regeneration, oxygen supply cannot rely solely on diffusion. In particular, oxygen solubility is low in culture media, leading to insufficient oxygen diffusion into the growing tissue, which can lead to cell death due to hypoxia. Therefore, for tissue regeneration in tissues with a vascular network, such as the human body, medium perfusion is essential, both in the regeneration process and in the regeneration process. Of the three elements of tissue engineering—cells, scaffolds, and humoral factors—the development of scaffolds with a vascular network is anticipated, as is the development of artificial oxygen carriers that circulate through the channels of scaffolds.

[0004] Most of the oxygen carriers developed to date are approximately 200 nm in size, similar to DDS preparations, because such nanoscale oxygen carriers are safe from the perspective of blood circulation and do not pose the risk of aggregation or vascular embolism when administered into the body. On the other hand, natural platelets (approximately 2 μm) and red blood cells (approximately 8 μm) have sizes on the order of micrometers. Because such micro-sized particles can avoid cellular uptake by endocytosis or phagocytosis, attempts have been made to develop micro-sized oxygen carriers, and recent examples of the development of micro-sized oxygen carriers have been reported (e.g., Non-Patent Document 1: Xiong, Yu et al., Biomocromolecules (13) 3292-3300, 2012; Non-Patent Document 2: Xiong, Yu et al., ACS NANO (7) 7454-7461, 2013, etc.). Furthermore, it is preferable that the micro-sized oxygen carrier be deformable so that it can pass through microchannels such as capillaries. Deformable oxygen carriers have also been investigated. For example, Chen et al. created soft, disk-shaped gel particles with a diameter of 6 μm, imitating the 8 μm diameter disk shape of red blood cells and immobilizing hemoglobin gel (Non-Patent Document 3: Chen, K et al., Biomocromolecules (13) 2748-2759, 2012). The gel particles are said to be able to pass through capillaries due to the bending of their disk shape. However, all of these micro-sized oxygen carriers are based on hemoglobin, and hemoglobin is susceptible to inactivation due to methemoglobinization (MetHb) or infection. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Xiong, Yu et al., Biomocromolecules (13) 3292-3300, 2012 [Non-patent document 2] Xiong, Yu et al., ACS NANO (7) 7454-7461, 2013 [Non-patent document 3] Chen, K et al., Biomocromolecules (13)2748-2759, 2012 Summary of the Invention [Problem to be solved by the invention]

[0006] Under these circumstances, there is a need for novel fluorocarbon-based core-shell microparticles. [Means for solving the problem]

[0007] The present invention is, for example, as follows. [1] A core-shell microparticle comprising a shell containing a polymer having a Young's modulus of 100 MPa or less and a core containing a fluorocarbon. [1a] The particles according to [1], wherein the Young's modulus of the polymer is 0.5 to 100 MPa. [1b] The particles according to [1] or [1a], wherein the Young's modulus of the microparticles is 500 kPa or less. [2] A core-shell microparticle, comprising a shell containing a polymer and a core containing a fluorocarbon, wherein the microparticle has a Young's modulus of 500 kPa or less. [2a] The particles according to [2], wherein the Young's modulus of the polymer is 100 MPa or less. [2b] The particles according to [2] or [2a], wherein the Young's modulus of the polymer is 0.5 to 100 MPa. [3] The particles according to any one of [1] to [2], [1a] to [1b] and [2a] to [2b], wherein the size of the microparticles is in the range of 1 to 20 μm. [4] The particles according to [3], wherein the size of the microparticles is in the range of 1 to 10 μm. [5] The particles according to any one of [1] to [4], [1a] to [1b] and [2a] to [2b], wherein the molecular weight of the fluorocarbon is in the range of 100 to 2,000 g / mol. [5a] The particles according to any one of [1] to [5], [1a] to [1b] and [2a] to [2b], wherein the fluorocarbon is in a liquid or gel state at room temperature and normal pressure.

[0008] [6] The particles according to any one of [1] to [5], [1a] to [1b], [2a] to [2b] and [5a], wherein the content of the fluorocarbon in the microparticles is 30% by weight or more. [7] The particle according to any one of [1] to [6], [1a] to [1b], [2a] to [2b], and [5a], wherein the shell has a thickness in the range of 0.1 to 4 μm. [8] The particles according to any one of [1] to [7], [1a] to [1b], [2a] to [2b], and [5a], wherein the polymer is an elastomer. [9] The particles according to any one of [1] to [8], [1a] to [1b], [2a] to [2b], and [5a], wherein the polymer comprises at least one selected from the group consisting of a copolymer of polylactide and / or polyglycolide with polycaprolactone, and a copolymer of polylactide and / or polyglycolide with trimethylene carbonate.

[10] The particle according to any one of [1] to [8], [1a] to [1b], [2a] to [2b] and [5a], wherein the core contains a gas dissolved in the fluorocarbon. [10a] The particle according to [9], wherein the gas contains oxygen.

[0009]

[11] The particles according to any one of [1] to

[10] , [1a] to [1b], [2a] to [2b], [5a], and [10a], wherein the fluorocarbon has a boiling point of 35°C or higher (preferably 50°C or higher, more preferably 120°C or higher).

[12] The particle according to any one of [1] to

[11] , [1a] to [1b], [2a] to [2b], [5a] and [10a], wherein the core contains a fluorocarbon having an oxygen solubility of 30 vol / vol% or more.

[13] The particles according to any one of [1] to

[12] , [1a] to [1b], [2a] to [2b], [5a] and [10a], wherein the fluorocarbon comprises at least one selected from the group consisting of bis(fluoroalkyl)ethene, hydrofluorocarbon, hydrofluoroether, hydrochlorofluorocarbon, cyclic perfluorocarbon, perfluoroamine, perfluoro(2-butyltetrahydrofuran), brominated perfluorocarbon, iodinated perfluorocarbon, chlorinated perfluorocarbon, and perfluoroalkyl ether or polyether.

[14] The particle according to any one of [1] to

[13] , [1a] to [1b], [2a] to [2b], [5a] and [10a], wherein the particle is deformable.

[15] The particles according to any one of [1] to

[14] , [1a] to [1b], [2a] to [2b], [5a] and [10a], which have an anisotropic shape. [15a] The particle according to any one of [1] to

[15] , [1a] to [1b], [2a] to [2b], [5a] and [10a], wherein the particle has a shape having a depression.

[0010]

[16] The particles according to any one of [1] to

[15] , [1a] to [1b], [2a] to [2b], [5a], [5b], [10a] and [15a], wherein the CV value of the microparticles is 40% or less. [16a] The particles according to any one of [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a] and [15a], wherein the particles are biodegradable.

[17] A dispersion liquid containing particles according to any one of [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a], [15a] and [16a] in an aqueous medium.

[18] The dispersion liquid according to

[17] , which is used as an oxygen infusion solution, a perfusion solution for transplanted organs, a preservation solution for transplanted organs, an oxygen supply solution for regenerative medicine, or a culture medium for animal cells or microorganisms for producing biopharmaceuticals.

[19] A method for producing particles according to any one of [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a], [15a] and [16a], A dispersed phase obtained by dispersing a fluorocarbon and a polymer having a Young's modulus of 100 MPa or less in an organic solvent is membrane-emulsified into a continuous phase containing a surfactant through a porous membrane having a uniform pore size to obtain an emulsion; forming core-shell microparticles comprising a shell comprising the polymer and a core comprising the fluorocarbon; A method comprising:

[20] The method according to

[19] , wherein the core-shell microparticles are formed by removing the organic solvent phase contained in the emulsion.

[0011]

[21] Adding alcohol to a dispersion obtained by dispersing the particles according to any one of [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a], [15a] and [16a], or the particles obtained by the production method according to

[19] or

[20] in an aqueous medium; and resuspending the resulting particles in a solvent; A method for producing anisotropic microparticles, comprising: [21a] Adding alcohol to a dispersion obtained by dispersing the particles according to any one of [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a], [15a] and [16a], or the particles obtained by the production method according to

[19] or

[20] in an aqueous medium; and resuspending the resulting particles in a solvent; A method for producing microparticles having depressions, comprising:

[22] A core-shell microparticle comprising a shell comprising an elastomer and a core comprising a fluorocarbon. [22a] The particle according to

[22] , wherein the elastomer is a polymer according to any one of the embodiments described in [1] to

[16] , [1a] to [1b], [2a] to [2b], [5a], [10a] and [15a]. [Effects of the Invention]

[0012] The microparticles of the present invention have one or more of the following advantages. (1) Novel core-shell microparticles containing fluorocarbons are provided. The microparticles can function as carriers capable of transporting gases such as oxygen by occluding gases such as oxygen in the fluorocarbons present in the core. In one embodiment, the microparticles are deformable. In one embodiment, the microparticles have excellent flexibility and deformability, and can recover their shape after deformation. In a preferred embodiment, the microparticles can function as oxygen carriers with deformability similar to that of red blood cells. In a preferred embodiment, the microparticles have excellent channel-passing properties. In a particularly preferred embodiment, the microparticles can pass through passages of approximately 5 μm, similar to those of capillaries. (2) Microparticles that stably retain fluorocarbons inside and have superior stability compared to emulsion-type oxygen carriers can be obtained. In one embodiment, the microparticles have excellent flexibility and deformability, as well as excellent stability. (3) The particle size and particle size distribution of the microparticles are easily controlled. In one embodiment, the microparticles have a strictly controlled size and / or a uniform particle size distribution (monodispersion). In a preferred embodiment, microparticles having a size comparable to that of red blood cells can be obtained. In one embodiment, the shell thickness of the microparticles can be controlled within a desired range. (4) In one embodiment, it is possible to produce microparticles having anisotropy (e.g., having depressions). In one embodiment, microparticles having anisotropy (e.g., having depressions) can increase the specific surface area and improve gas permeability. In one embodiment, microparticles having anisotropy (e.g., having depressions) can improve channel passability. In one preferred embodiment, microparticles having anisotropy (e.g., having depressions) have a shape similar to that of red blood cells. In one embodiment, microparticles having anisotropy (e.g., having depressions) are particularly excellent in flexibility and deformability. In a preferred embodiment, microparticles having anisotropy (e.g., having depressions) have a low Young's modulus similar to that of red blood cells. (5) Novel fluorocarbon-based core-shell microparticles are provided. Fluorocarbon-based microparticles are less susceptible to inactivation than hemoglobin-based particles and are highly stable and sterilizable. [Brief explanation of the drawings]

[0013] [Figure 1] 1A and 1B are schematic cross-sectional views of a core-shell spherical microparticle according to one embodiment of the present invention, and a deformed microparticle according to one embodiment of the present invention. [Figure 2] FIG. 2A is a schematic cross-sectional view of a microparticle having a core-shell shaped depression according to one embodiment of the present invention, and FIG. 2B is a schematic top cross-sectional view of a microparticle having a core-shell shaped depression according to one embodiment of the present invention. [Figure 3] Fig. 3A is a schematic diagram of a production apparatus used in a method for producing microparticles according to one embodiment of the present invention, and Fig. 3B is a diagram illustrating the principle of membrane emulsification via a porous membrane. [Figure 4] FIG. 4 is a graph showing the measurement results of the particle size distribution of the PFOB / PLC-0.8s MPs obtained in Example 5. [Figure 5] FIG. 5 is a diagram showing the relationship between the volume average particle size of the microparticles obtained in Examples 5 to 8 and the pore size of the SPG membrane used in the production. [Figure 6]FIG. 6 shows an optical photograph of the mixture of PFOB / PLC-0.8s MPs and human red blood cells (hRBCs) obtained in Example 5. [Figure 7] Figure 7A shows an SEM photograph of the PFOB / PLC-0.8s MPs obtained in Example 5. Figure 7B shows a confocal laser scanning microscope fluorescence image of the PFOB / PLC-0.8S MPs obtained in Example 5. Figure 7C shows a confocal laser scanning microscope fluorescence image of the solid microparticle PLC MPs obtained in Comparative Example 1. [Figure 8] FIG. 8 shows the results of measuring the IR spectrum of the PFOB / PLC-0.8s MPs obtained in Example 5. [Figure 9] Figure 9A shows an SEM photograph of the concave-shaped PFOB / PLC-4d MPs of Example 11. Figure 9B shows an SEM photograph after deformation, and Figure 9C shows an SEM photograph before deformation. [Figure 10] Figure 10 shows the results of measuring the particle size distribution of PFOB / PLC-4d MPs (before shape change) and concave-shaped PFOB / PLC-4d MPs (after shape change) obtained in Example 11, as well as human red blood cells (hRBCs) as a control. [Figure 11] Fig. 11A is a diagram showing the results of compression experiments on the solid microparticles PLC MPs (No. 1) obtained in Comparative Example 1, the microparticles (Nos. 2 to 6) obtained in Examples 1 to 5, the microparticles PFOB / PLGA-0.8s MPs (No. 7) obtained in Comparative Example 2, and the microparticles PLGA MPs (No. 8) obtained in Comparative Example 3. Fig. 11B is a diagram showing the results of compression experiments on the microparticles with a dimpled shape obtained in Example 11 (cDFC in the diagram), the spherical microparticles (DFC in the diagram) obtained in Example 5, the microparticles (Rigid FC in the diagram) obtained in Comparative Example 2, and acrylic beads (acrylic beads in the diagram). [Figure 12] FIG. 12 is a diagram showing the measurement procedure and principle of the passing experiment. [Figure 13]13 is a diagram showing the Pper of microparticles, specifically, the microparticles obtained in Example 5 (PFOB / PLC-0.8s MPs; PFOB / PLC-4d MPs), the microparticles obtained in Comparative Example 2 (PFOB / PLGA-0.8s MPs; PFOB / PLGA-4d MPs), commercial MPs (product name: cross-linked acrylic medium-dispersion particles MZ-8HN, manufactured by Soken Chemical & Engineering Co., Ltd.; average particle size: 8 μm), the microparticles obtained in Example 11 (concave-shaped PFOB / PLC MPs; concave-shaped PFOB / PLC-4d MPs), and normal or crosslinked human red blood cells (natural or crosslinked hRBCs). [Figure 14] Figures 14A to 14C show the cake layers formed at the channel inlet in the flow-through experiment. Specifically, Figure 14A shows the cake layers formed when the microparticles obtained in Examples 5 to 8 were used, and from top to bottom, the results for Example 8 (PFOB / PLC-5d MPs), Example 5 (PFOB / PLC-4d MPs), Example 7 (PFOB / PLC-3d MPs), and Example 6 (PFOB / PLC-2d MPs) are shown. Figure 14B shows the cake layers formed when the microparticles obtained in Comparative Example 2 were used, and from top to bottom, the results for PFOB / PLGA-5d MPs, PFOB / PLGA-4d MPs, PFOB / PLGA-3d MPs, and PFOB / PLGA-2d MPs are shown. Figure 14C shows the cake layers formed when the microparticles obtained in Examples 9 to 12 were used. Specifically, the results for Example 9 (Concave-shaped PFOB / PLC-2d MPs) are shown in a, the results for Example 10 (Concave-shaped PFOB / PLC-3d MPs) are shown in b, the results for Example 11 (Concave-shaped PFOB / PLC-4d MPs) are shown in c, and the results for Example 12 (Concave-shaped PFOB / PLC-5d MPs) are shown in d. [Figure 15]Figure 15 shows the particle size distribution of microparticles before and after passing through a microchannel. In Figure 15, "b" shows the particle size distribution of the concave-shaped PFOB / PLC-3d MPs obtained in Example 10 before passing and after passing, and "c" shows the particle size distribution of the concave-shaped PFOB / PLC-4d MPs obtained in Example 11 before passing and after passing. [Figure 16] 16 shows the force-deformation curves of microparticles and crosslinked red blood cells obtained by AFM measurement. In the figure, cDFC represents the results for the microparticles with a dimple shape obtained in Example 11, DFC represents the results for the microparticles of Example 5 used as the raw material for producing the particles with a dimple shape in Example 11, and 0.005%RBC represents the results for red blood cells crosslinked with glutaraldehyde at a concentration of 0.005%. [Figure 17] 17 shows the Young's modulus of microparticles and crosslinked red blood cells calculated by AFM measurement. In the figure, cDFC represents the results for the microparticles having a dimple shape obtained in Example 11, DFC represents the results for the microparticles of Example 5 used as the raw material for producing the particles having a dimple shape in Example 11, and 0.005% RBC represents the results for red blood cells crosslinked with glutaraldehyde at a concentration of 0.005%. [Figure 18] 18A and 18B show the results of a storage stability test of microparticles. Specifically, the particle sizes (average diameter (μm)) of microparticles dispersed in three types of solvents (PBS (phosphate buffered saline), saline, and DMEM (Dulbecco's modified Eagle's medium)) when stored at 4°C for 4 weeks are plotted. In FIG. 18A, DFC represents the results using the spherical microparticles obtained in Example 5, and in FIG. 18A, Rigid FC represents the results using the microparticles obtained in Comparative Example 2. In FIG. 18B, cDFC represents the results using the microparticles with a dimpled shape obtained in Example 11. [Figure 19]FIG. 19a shows the oxygen storage capacity of the microparticles, and FIG. 19b shows the oxygen release capacity of the microparticles. [Figure 20] 20 shows the cell viability of HUVECs in the presence of microparticles. In the figure, DFC indicates the results using the spherical microparticles (PFOB / PLC-0.8s MPs; PFOB / PLC-4d MPs) obtained in Example 5, cDFC indicates the results using the concave-shaped microparticles (PFOB / PLC-4d MPs) obtained in Example 11, and Control indicates the results for the control group (HUVEC group only). [Figure 21] Figure 21 shows confocal laser scanning micrographs of HeLa cells under hypoxic conditions (2% O2). Figure 21A shows the results of HeLa cells cultured in the absence of microparticles, and Figure 21B shows the results of HeLa cells cultured in the presence of microparticles. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below with reference to embodiments and examples, but the present invention is not limited to the embodiments and examples shown below and can be modified as desired without departing from the spirit of the present invention. All documents and publications mentioned in this specification are incorporated herein by reference in their entirety, regardless of their purpose. Note that the dimensional proportions in the drawings are exaggerated for the sake of explanation and may differ from the actual proportions. As used herein, "alkyl" means a straight, cyclic, or branched chain saturated aliphatic hydrocarbon group having the specified number of carbon atoms.

[0015] 1. Microparticles One aspect of the present invention relates to core-shell microparticles comprising a shell containing a polymer and a core containing a fluorocarbon. In some embodiments, the polymer has a Young's modulus of 100 MPa or less. In some embodiments, the microparticles have a Young's modulus of 500 kPa or less. Such microparticles are highly flexible and deformable, and also have excellent stability.

[0016] As used herein, the term "microparticle" refers to a fine particle having a size on the micrometer scale (e.g., 1 to 1000 μm, or 1 to 500 μm, or 1 to 100 μm, or 1 to 50 μm). As used herein, the size of a microparticle refers to the volume average particle diameter measured by a laser diffraction scattering method.

[0017] The size of the microparticles is preferably 1 μm or more, more preferably 1.5 μm or more, even more preferably 2 μm or more, even more preferably 3 μm or more, and particularly preferably 5 μm or more. Within these ranges, cellular uptake by endocytosis or phagocytosis can be avoided or suppressed. On the other hand, in terms of flow through the channels of the scaffold material and regenerated capillaries, the size of the microparticles is preferably 30 μm or less, more preferably 20 μm or less, even more preferably 15 μm or less, even more preferably 12 μm or less, and particularly preferably 10 μm or less. For example, the size of the microparticles may be 1 to 30 μm, or 1 to 20 μm, or 1 to 15 μm, or 1 to 12 μm, or 1 to 10 μm. In another example, the size of the microparticles may be 2 to 30 μm, or 2 to 20 μm, or 2 to 15 μm, or 2 to 12 μm, or 2 to 10 μm. In another example, the size of the microparticles may be 3 to 30 μm, or 3 to 20 μm, or 3 to 15 μm, or 3 to 12 μm, or 3 to 10 μm. In another example, the size of the microparticles may be 5 to 30 μm, or 5 to 20 μm, or 5 to 15 μm, or 5 to 12 μm, or 5 to 10 μm. In a preferred embodiment, microparticles having a size similar to that of red blood cells (e.g., a particle diameter of 8 to 9 μm) can be obtained. In another embodiment, the size of the microparticles may range from 7.5 to 30 μm, or from 7.5 to 20 μm.

[0018] Furthermore, it is preferable that the microparticles have a controlled size and / or a uniform particle size distribution. In one embodiment, the microparticles have a monodisperse particle size distribution. Furthermore, the CV value of the microparticles is preferably 40% or less (more preferably 35% or less, and even more preferably 30% or less). The CV value of the microparticles is the coefficient of variation of the particle size distribution, calculated from the standard deviation of the particle size and the average particle size. A lower CV value indicates better uniformity of the particle size. Specifically, the coefficient of variation of the particle size distribution, CV, can be calculated using the following formula: CV value (%) = (standard deviation of particle size / average particle size) x 100 In this specification, the particle size distribution of microparticles can be measured by laser diffraction / scattering particle size distribution measurement.

[0019] The shape of the particles is not particularly limited, and may be spherical, non-spherical, ellipsoidal (spheroidal), rod-shaped, pyramidal, cubic, disc-shaped, or concave-shaped. In one embodiment, the microparticles are spherical or anisotropically shaped. In one embodiment, the microparticles are anisotropically shaped. In one embodiment, the microparticles are spherical or recessed. In one embodiment, the microparticles are recessed (concave). In one embodiment, the microparticles are disc-shaped and have a recess in at least one of the major surfaces of the disc. A preferred embodiment of the microparticles having cavities has a shape similar to that of red blood cells. Microparticles having cavities can increase the specific surface area, which can improve gas permeability to gases such as oxygen. In addition, the cavities can improve the particle's ability to pass through a microchannel. The shape of the microparticles and the presence of cavities in the microparticles can be observed using a microscope such as a scanning electron microscope (SEM).

[0020] The microparticles have a core-shell structure. Core-shell microparticles are different from conventional emulsion-type artificial oxygen carriers and non-encapsulated artificial oxygen carriers in which oxygen-storing components such as fluorocarbons are present on the particle surface. Core-shell microparticles stably retain fluorocarbons inside and are more stable than emulsion-type and non-encapsulated oxygen carriers.

[0021] 1A and 1B are schematic cross-sectional views of a core-shell spherical microparticle according to one embodiment of the present invention, and a deformed microparticle according to one embodiment of the present invention. 2A and 2B are schematic cross-sectional views showing a microparticle having a core-shell type depression according to one embodiment of the present invention, where Fig. 2A is a horizontal cross-sectional view of the microparticle and Fig. 2B is a top cross-sectional view of the microparticle. 1 and 2, the microparticle 1 is a microcapsule having a shell 11 and a core 12. The core 12 forms the interior of the microparticle 1 and is encapsulated or surrounded by the shell 11, which forms the exterior of the microparticle 1. The microparticle shown in Figure 1A has a spherical shape. As shown in Figures 2A and 2B, the microparticle can have an anisotropic shape. The microparticle 1 shown in Figure 2 has a roughly disk shape and has a depression 15 in the center.

[0022] The core 12 contains a fluorocarbon 14. The fluorocarbon 14 has excellent solubility for gases (e.g., oxygen). Therefore, the microparticle 1 can function as a carrier that can retain gas within the core and transport it to a target site or tissue. In one embodiment, the microparticle can function as a gas carrier that has deformable permeability similar to red blood cells.

[0023] Shell 11 includes polymer 13. Shell 11 including polymer 13 is highly elastic, deformable, and flexible, resulting in a deformable microparticle 1. As used herein, "deformable" refers to the ability to maintain a core-shell structure even when the particle is deformed. In one embodiment, the microparticle has a core component encapsulated inside or surrounded by the shell, without the shell collapsing even when the particle is deformed. For example, the microparticles can be compressed to a displacement of 25% or more (e.g., 30% or more, or 35% or more) of the particle size. For example, as shown in FIG. 1B, the spherical microparticles of FIG. 1A can be deformed to a smaller diameter d a and major axis d b The displacement of the microparticles during compression deformation can be measured by carrying out compression deformation under microscope observation and measuring the stress and displacement during compression deformation. Such deformable microparticles 1 have excellent channel permeability and can pass through microchannels. For example, the microparticles can deform to pass through microchannels smaller than the average particle diameter of the particles. In one embodiment, the microparticles 1 can pass through microchannels having a slit width of 75% or less (e.g., 70% or less, or 65% or less) of the average particle diameter of the particles. In one embodiment, the microparticles can pass through a passage having a slit width of approximately 5 μm (a size equivalent to pulmonary capillaries), similar to that of capillaries. Such microparticles can function as carriers of gas (e.g., oxygen) into microchannels that are difficult for conventional microscale particles to pass through, while avoiding cellular uptake by endocytosis or phagocytosis, which occurs with nanometer-scale particles. The polymer 11 also has excellent shape recovery properties. These properties enable the microparticle 1 to pass through a channel such as a capillary blood vessel and to recover its shape after passing through the channel. In one embodiment, the microparticles have excellent flexibility and deformability, and are capable of recovering their shape after deformation. Furthermore, the size of the microparticles that have recovered their shape after deformation changes little compared to the size of the microparticles before deformation. For example, the ratio (d1 / d2) of the size of the microparticles before deformation (particle diameter; d1) to the size of the microparticles after deformation and recovery (particle diameter; d2) is in the range of 100 / 80 to 100 / 120 (preferably 100 / 95 to 100 / 105).

[0024] The microparticles preferably have a Young's modulus of 500 kPa or less. A particle with a Young's modulus of 500 kPa or less exhibits excellent flexibility and deformability. The lower the Young's modulus, the better, but it is, for example, 1 kPa or more. In one embodiment, the Young's modulus of the microparticles may be 500 kPa or less, or 400 kPa or less, or 300 kPa or less, or 250 kPa or less, or 200 kPa or less, or 150 kPa or less, or 100 kPa or less, or 1 kPa or more, or 2 kPa or more, or 3 kPa or more. The Young's modulus of the microparticles may be, for example, in the range of 1 to 500 kPa, or 1 to 400 kPa, or 1 to 300 kPa, or 2 to 250 kPa, or 3 to 200 kPa, or 3 to 150 kPa, or 3 to 100 kPa. Within these ranges, the flexibility, deformability, and / or shape recovery of the shell are further improved, resulting in microparticles with excellent channel passability. Generally, the preferred range of Young's modulus of microparticles also depends on the Young's modulus of the microparticles. For example, the Young's modulus of microparticles having a particle size of 5 μm or less (e.g., 1 to 5 μm) can be in the range of 1 to 500 kPa (preferably 1 to 400 kPa, or 1 to 300 kPa, or 2 to 250 kPa, or 3 to 200 kPa, or 3 to 150 kPa, or 3 to 100 kPa). For example, the Young's modulus of microparticles having a particle size of 5 μm or more (e.g., 5 to 500 μm, or 5 to 100 μm, or 5 to 50 μm, or 5 to 30 μm, or 5 to 20 μm, or 5 to 15 μm, or 5 to 12 μm, or 5 to 10 μm) can be in the range of 1 to 300 kPa, or 2 to 250 kPa, or 3 to 200 kPa, or 3 to 150 kPa, or 3 to 100 kPa). The Young's modulus of the microparticles can be measured by a method using atomic force microscope (AFM) measurement, which will be described in the Examples below.

[0025] The shell thickness of the microparticles is not particularly limited, but from the viewpoint of particle stability, it is, for example, 0.05 μm or more, or 0.1 μm or more, or 0.5 μm or more. Furthermore, as long as stability is ensured, the shell thickness is preferably thinner, for example, 4 μm or less. The shell thickness of the microparticles is preferably, for example, 0.1 to 4 μm. The shell thickness of the microparticles can be, for example, 0.2 to 4 μm or 0.5 to 4 μm. The shell thickness can also be measured using a confocal laser scanning microscope or an electron microscope.

[0026] The polymer constituting the shell preferably has a Young's modulus of 100 MPa or less. A Young's modulus of 100 MPa or less provides excellent shell flexibility and deformability. It is generally known that the Young's modulus of rubber materials is 100 MPa or less. Using a polymer with such a Young's modulus can provide the shell with flexibility and deformability. In terms of shell flexibility and deformability, the lower the Young's modulus, the better, and it is, for example, 0.5 MPa or more. In one embodiment, the Young's modulus of the polymer is preferably 100 MPa or less, more preferably 50 MPa or less, even more preferably 30 MPa or less, even more preferably 20 MPa or less, preferably 0.5 MPa or more, and more preferably 1 MPa or more. The Young's modulus of the polymer is, for example, 0.5 to 100 MPa, more preferably 0.5 to 50 MPa, even more preferably 0.5 to 30 MPa, even more preferably 1 to 20 MPa, and particularly preferably 1 to 15 MPa. Within these ranges, the flexibility, deformability, and / or shape recovery of the shell are further improved, resulting in microparticles with excellent channel passability. The Young's modulus of a polymer can be measured by the following tensile test. (1) Preparation of test films A test piece for a tensile test is prepared by dissolving the polymer in a solvent such as chloroform, applying the solution to a glass substrate, and drying the solution to form a film of approximately 200 μm. (2) Tensile test Tensile tests are performed using a tensile testing machine (e.g., CR-3000EX-S, manufactured by San Scientific Co., Ltd.) at a displacement rate of 5 mm / min. After forming the test specimens, measurements are performed at room temperature (21±2°C) for 5 weeks, and then at room temperature and 50±5% relative humidity (RH) in accordance with ISO 527-3 / 1995 (ISO 527-3 / 2 / 5). The Young's modulus of the test specimens is obtained from the tensile test results.

[0027] The structure and type of the polymer are not particularly limited as long as it has a Young's modulus within the above range, and it may be any of a random, block, or alternating copolymer structure. In one embodiment, the polymer is an elastomer. For example, a copolymer containing a hard segment and a soft segment can be preferably used as the polymer (elastomer).

[0028] In some embodiments, the hard segment (H) is a polymer chain in a glassy or semi-crystalline state at room temperature (e.g., 10 to 35°C), and the soft segment (S) is a polymer chain in a rubbery state at room temperature. The term "glassy state" refers to a hard solid state in which the molecules are not crystallized (amorphous), have poor molecular fluidity, and do not flow even when an external force is applied. The term "semi-crystalline state" refers to a solid state in which crystallized portions are mixed with amorphous portions due to folded portions or entanglement between molecular chains. The term "rubbery state" refers to a soft, stretchable solid state in which the molecules are mobile and flow when pulled or pushed. The "glassy state," "semi-crystalline state," and "rubbery state" can be determined by differential scanning calorimetry or other methods. The temperature at the boundary between the rubbery state and the glassy or semi-crystalline state is the glass transition temperature (Tg). In one embodiment, the polymer is a copolymer containing hard segments and soft segments, and the ratio of the hard segments to the soft segments constituting the polymer (hard segments / soft segments; weight ratio) is in the range of 90 / 10 to 0 / 100 (more preferably in the range of 80 / 20 to 20 / 80, and even more preferably in the range of 75 / 25 to 45 / 55). Generally, the higher the ratio of the soft segments, the more the flexibility of the microparticles can be improved, while if the ratio of the soft segments is too high, the elastic deformability of the microparticles decreases, making them more susceptible to plastic deformation. By adjusting the ratio of the hard segments to the soft segments, microparticles that combine flexibility and shape recovery can be obtained. The structure of the copolymer containing hard and soft segments is not particularly limited, and examples thereof include a diblock copolymer represented by (S)-(H), a triblock copolymer represented by (H)-(S)-(H) or (S)-(H)-(S), and a multiblock copolymer composed of the hard segment (H) and the soft segment (S).

[0029] Examples of hard segments include polylactide (PLA), polyglycolide (PGA), and poly(lactide-co-glycolide) (PLGA). These hard segments can be used alone or in combination of two or more. Poly(lactide-co-glycolide) (PLGA) refers to a polymer composed of lactic acid units (-O-CH(CH3)-CO-) and glycolic acid units (-O-CH2-CO-) randomly linked together. PLA, PGA, and PLGA have excellent biodegradability. In this specification, "poly(lactide and / or glycolide)" refers to polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), or a mixture thereof. Examples of soft segments include polycaprolactone (PCL), poly(trimethylene carbonate) (PTMC), polydioxanone (PDO), poly(4-hydroxybutyrate) (PHB), and poly(butylene succinate) (PBS). These hard segments can be used alone or in combination of two or more. The polymers constituting the hard and soft segments may be modified or derivatized with a resin or elastomer containing silicon or fluorine. In addition to the above-mentioned hard and soft segments, the polymer may also contain a hydrophilic segment such as polyethylene glycol (PEG) or a segment with excellent biocompatibility such as 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0030] A preferred example of the polymer is at least one selected from the group consisting of a block copolymer of poly(lactide and / or glycolide) and polycaprolactone, and a block copolymer of poly(lactide and / or glycolide) and polytrimethylene carbonate, which are highly biodegradable and flexible.

[0031] (1) Block copolymer of poly(lactide and / or glycolide) and polycaprolactone Specific examples of block copolymers of poly(lactide and / or glycolide) and polycaprolactone include (i) block copolymers of PLA blocks and PCL blocks (PLC), (ii) block copolymers of PGA blocks and PCL blocks, and (iii) block copolymers of PLGA blocks and PCL blocks.

[0032] The ratio of the poly(lactide and / or glycolide) component to the polycaprolactone component in the block copolymer of poly(lactide and / or glycolide) and polycaprolactone is not particularly limited, but from the viewpoint of improving the mechanical strength (rigidity) and flexibility of the shell, the ratio (poly(lactide and / or glycolide) / polycaprolactone; molar ratio) is preferably in the range of 90 / 10 to 0 / 100 (more preferably in the range of 80 / 20 to 20 / 80, and even more preferably in the range of 75 / 25 to 45 / 55).

[0033] The block copolymer may have a diblock, triblock, or multiblock structure. For example, examples of the diblock copolymers (i) to (iii) above are shown in the following formulas (1) to (3). [ka] In the above formulas (1) to (3), R1 is selected from a hydrogen atom and an alkyl having 1 to 12 carbon atoms. R2 is selected from a hydroxyl group, a hydrogen atom and an alkyl having 1 to 12 carbon atoms. In one embodiment, R1 is a hydrogen atom and R2 is a hydroxyl group. In the above formulas (1) to (3), x and y each independently represent an integer of 1 to 300. The ratio of x to y (x / y) may be adjusted so that the elasticity (Young's modulus) falls within a desired range, and may be in the range of 99 / 1 to 1 / 99, for example. In the above formula (3), n is 0.1 to 0.9. In the above formula (3), the notation " / " indicates that the n and n-1 monomer units on either side of it can be arranged in any order.

[0034] (2) Copolymers of poly(lactide and / or glycolide) and polytrimethylene carbonate Specific examples of copolymers of poly(lactide and / or glycolide) and polytrimethylene carbonate include (iv) block copolymers of PLA blocks and PTMC blocks, (v) block copolymers of PGA blocks and PTMC blocks, and (vi) block copolymers of PLGA blocks and PTMC blocks.

[0035] The ratio of the poly(lactide and / or glycolide) component to the polytrimethylene carbonate component in the block copolymer of poly(lactide and / or glycolide) and polytrimethylene carbonate is not particularly limited, but the ratio (molar ratio) is, for example, in the range of 99 / 1 to 1 / 99.

[0036] The block copolymer may have a diblock, triblock, or multiblock structure. A block copolymer is preferred. For example, examples of the diblock copolymers (iv) to (vi) are shown in the following formulas (4) to (6). [ka] In the above formulas (4) to (6), R3 and R4 are selected from a hydrogen atom and an alkyl having 1 to 12 carbon atoms. In one embodiment, R3 and R4 are a hydrogen atom. In the above formulas (4) to (6), x and z each independently represent an integer of 1 to 300. The ratio of x to z (x / z) is, for example, in the range of 1 / 99 to 99 / 1. In the above formula (6), n is 0.1 to 0.9. In the above formula (6), the notation " / " indicates that the n and n-1 monomer units on either side of it can be arranged in any order.

[0037] The polymer may be synthesized by a conventionally known method, or may be a commercially available product. For example, commercially available products that can be used include Corbin's PURASORB PLC series (PURASORB PLC 7015, PURASORB PLC 7038, etc.), Taki Chemical Co., Ltd.'s lactone-based polymers (CG, CL), and Taki Chemical Co., Ltd.'s trimethylene carbonate-based polymers (T-100, TL-70, TL-50, TLG-442, etc.). The polymers can be used alone or in combination of two or more.

[0038] The weight average molecular weight (Mw) of the polymer is not particularly limited, but is preferably 3,000 to 300,000, and more preferably 4,000 to 240,000. In this specification, the weight average molecular weight (Mw) of the polymer is the weight average molecular weight measured by gel permeation chromatography (GPC) and converted into standard polystyrene.

[0039] By controlling the weight average molecular weight of the block copolymer, the length of each block chain, the block ratio, the lactide-glycolide ratio, etc., it is possible to adjust the Young's modulus of the polymer within the desired range.

[0040] The polymer may be biodegradable or non-biodegradable. In one embodiment, the microparticles are biodegradable or non-biodegradable. Preferably, the polymer is biodegradable. The use of a biodegradable polymer results in biodegradable microparticles. Such biodegradable microparticles can reduce or avoid health risks during physiological periods. In one embodiment, the polymer is a biodegradable elastomer.

[0041] The shell may be composed of only a polymer, or may contain other components in addition to the polymer. Examples of other components that may be contained in the shell include at least one selected from dyes used in imaging, drugs, and radical scavengers such as vitamin C and vitamin E. Radical scavengers are added to suppress side effects when there is a risk of toxicity due to oxygen exposure. The shell preferably contains 80 to 100% by weight of polymer relative to the total weight (100% by weight) of the shell. The proportion of other components is preferably 20% by weight or less relative to the total weight (100% by weight) of the shell.

[0042] The core is composed of a fluorocarbon. As used herein, "fluorocarbon" refers to an organic fluorine compound having a carbon-fluorine bond. Fluorocarbons can reversibly store and / or adsorb gases such as oxygen, carbon dioxide, nitrogen, and hydrogen. Due to the reversible nature of their gas storage ability, fluorocarbons can release gases under physiological conditions (especially in environments where gases such as oxygen are in short supply), and can release needed gases such as oxygen to ischemic tissues and / or organs. Furthermore, fluorocarbons are highly stable and sterilizable.

[0043] The type of gas to be occluded and / or adsorbed (dissolved) in the fluorocarbon is not particularly limited, and examples thereof include oxygen, carbon dioxide, nitrogen, etc. In a preferred embodiment, the gas is oxygen, and the microparticles function as an artificial oxygen carrier.

[0044] Fluorocarbons are preferably liquid or gel-like at room temperature and pressure, and more preferably liquid. In particular, fluorocarbons are preferably liquid under ambient or physiological conditions. Physiological conditions may include in vivo chemical conditions (temperature, pressure, pH). For example, fluorocarbons are preferably liquid under atmospheric pressure (e.g., 1 atm) and room temperature (e.g., 10 to 35°C). In one embodiment, the fluorocarbon preferably has a boiling point of 35°C or higher, more preferably 50°C or higher, and even more preferably 120°C or higher.

[0045] The type of fluorocarbon is not particularly limited, but fluorocarbons with excellent gas storage capacity (gas solubility) are preferred. For example, liquid fluorocarbons with an oxygen solubility of 30 vol / vol% or more (more preferably 40 vol / vol% or more, and even more preferably 60 vol / vol% or more) are preferred. Note that "oxygen solubility" refers to the solubility of oxygen molecules in fluorocarbons at 25°C.

[0046] From the viewpoint of gas solubility, the molecular weight of the fluorocarbon is preferably in the range of 100 to 2,000 g / mol, more preferably 150 to 1,000 g / mol, and even more preferably 200 to 700 g / mol.

[0047] The fluorocarbon is not particularly limited as long as it is capable of reversibly occluding and / or adsorbing gas, and fluorocarbons that have conventionally been known as oxygen carriers or candidate oxygen carriers can also be used in the present invention. Examples of such fluorocarbons include at least one selected from the group consisting of bis(fluoroalkyl)ethenes, hydrofluorocarbons, hydrofluoroethers, hydrochlorofluorocarbons, cyclic perfluorocarbons, perfluoroamines, perfluoro(2-butyltetrahydrofuran) (FC-75 or RM101), brominated perfluorocarbons, iodinated perfluorocarbons, chlorinated perfluorocarbons, and perfluoroalkyl ethers or polyethers. As used herein, the term "perfluoro" in "perfluorocarbon," "perfluoroamine," and "perfluoroalkyl" means that all of the hydrogen atoms of the hydrocarbon have been replaced with fluorine atoms.

[0048] Examples of bis(fluoroalkyl)ethene include C4F9CH=CH4CF9 (F-44E) and i-C3F9CH=CHCF 13 (F-i36E), and C6F 13 CH=CHC6F 13 (F-66E) etc. Examples of hydrofluorocarbons (HFCs) include 1,1,1,3,3-pentafluorobutane (HFC-365mfc). Examples of hydrofluoroethers (HFEs) include C4F9O-CH3, C4F9O-C2H5, and C6F 13 O-CH3, etc. These are manufactured by 3M under the name Novec TM (Novec TM ) series (e.g., Novec7100 (C4F9O-CH3), Novec7200 (C4F9O-C2H5), Novec7300 (C6F 13 O-CH3)) can be used. Examples of hydrochlorofluorocarbons (HCFCs) include CClF2CF2CHClF (HCFC-225cb). Examples of cyclic perfluorocarbons include perfluorodecalin (FDC), perfluoroadamantane (FA), perfluoromethyladamantane (FMA), F-1,3-dimethyladamantane (FDMA), perfluorodimethylbicyclo[3,3,1]nonane (FMN), and perfluorotrimethylbicyclo[3,3,1]nonane. Examples of perfluoroamines include perfluorotripropylamine (FTPA), perfluorotributylamine (FTBA), F-4-methyloctahydroquinolizine (FMOQ), Fn-methyl-decahydroisoquinoline (FMIQ), Fn-methyldecahydroquinoline (FHQ), Fn-cyclohexylpyrrolidine (FCHP), and Fn-(4-methylcyclohexyl)piperidine (FMCP). Brominated perfluorocarbons include perfluorooctyl bromide (PFOB), perfluorodecyl bromide (PFDB), perfluorohexyl bromide (PFHB), and 1-bromopentadecafluoroseptane (CF 15 Br), etc. Examples of perfluorocarbon iodides include perfluorooctyl iodide. An example of the chlorinated perfluorocarbon is 1,8-perfluorodichlorooctane (PFDCO). Examples of perfluoroalkyl ethers or polyethers include (CF3)2CFO(CF2CF2)2OCF(CF3)2, (CF3)2CFO(CF2CF2)3OCF(CF3), (CF3)CFO(CF2CF2)F), (CF3)2CFO(CF2CF2)2F, and the like. The fluorocarbons can be used alone or in combination of two or more. Mixed fluorocarbon-hydrocarbon compounds modified in various forms, such as esters, thioethers, and the like, are also included within the broad definition of "fluorocarbon" materials suitable for use in the present invention. Other "fluorocarbons" not listed here may also be used as long as they have the properties described herein. In addition, a gel-like fluorocarbon such as PFC gel with a gelling agent added can also be used.

[0049] Fluorocarbons are exemplified in the following documents, and the fluorocarbons described in these documents can also be preferably used in the present invention. (1) Jean G. Riess, Oxygen Carriers (“Blood Substitutes”)-Raison d'Etre, Chemistry, and Some Physiology, Chemical Reviews, 2001, Vol.101, No.9,p2797-2919. In particular, see the section "V. Fluorocarbon-based Oxygen Carriers" on pages 2865-2893. (2) Hachiro Ishigaki, "Development of Artificial Blood", Organic Synthetic Chemistry, Vol. 38, No. 6 (1980), p. 520-527 (3) Kunihiko Nakai et al., Perfluorocarbons (PFCs): Challenges with Second-Generation PFCs, ARTIFICAL BLOOD Vol.8, No.2, 2002, p43-54

[0050] From the viewpoint of gas storage capacity, the content (weight percentage) of fluorocarbon in the microparticles is preferably 30% by weight or more, more preferably 40% by weight or more, and even more preferably 60% by weight or more, relative to the weight (100% by weight) of the microparticles. Meanwhile, the content of fluorocarbon is, for example, 95% by weight or less, or 90% by weight or less, relative to the weight (100% by weight) of the microparticles. For example, the content of fluorocarbon is in the range of 30 to 95% by weight, or 30 to 90% by weight. The content (weight percentage) of fluorocarbon is the ratio of the weight of fluorocarbon to the total weight of the shell and the core (i.e., excluding gas dissolved in the fluorocarbon).

[0051] The weight ratio (core / shell) of the core component to the shell component constituting the microparticles is, for example, 30 / 70 to 95 / 5, preferably 30 / 70 to 90 / 10, more preferably 40 / 60 to 90 / 10, and even more preferably 60 / 40 to 90 / 10.

[0052] 2.Dispersion liquid Another aspect of the present invention relates to a dispersion containing microparticles in an aqueous medium. The microparticles can function as carriers for transporting gases such as oxygen, for example, oxygen carriers for regenerative medicine, oxygen carriers for tissue transplant preservation solutions, and DDS carriers for gas delivery. In one embodiment, the dispersion containing the microparticles is used as an oxygen infusion solution, a perfusion solution for transplant organs, a preservation solution for transplant organs, an oxygen supply solution for regenerative medicine, or a culture medium for animal cells or microorganisms for producing biopharmaceuticals.

[0053] One embodiment of the present invention relates to the use of the microparticles or dispersions for transfusion purposes, in particular as blood substitutes, as artificial oxygen carriers for the prophylactic and / or therapeutic treatment of subjects. For example, the microparticles or dispersions can be used for the prophylactic and / or therapeutic treatment of an oxygen-deficient condition in a subject (e.g., ischemia due to heart failure, cerebral infarction, respiratory failure, etc.). "An oxygen-deficient condition" refers to ischemic and hypoxic conditions. "Ischemic" or "ischemia" refers to a reduction or cessation of blood flow to a tissue or organ. Alternatively, the microparticles or dispersions can be used for prophylactic and / or therapeutic treatment in subjects during and / or after blood loss associated with surgery, accidents, injuries, etc. (e.g., hemorrhagic shock, bleeding during surgery). Alternatively, the microparticles or dispersions can be used for the prophylactic and / or therapeutic treatment of ischemic conditions or conditions following reperfusion, particularly tourniquet syndrome (reperfusion syndrome). One embodiment of the present invention relates to the use of the microparticles or dispersion as an artificial oxygen carrier to deliver oxygen to transplanted tissue for the purpose of tissue preservation. Alternatively, the microparticles or dispersions can be used as transplant organ perfusion and / or preservation solutions to protect organs during transplant surgery. One embodiment of the present invention relates to the use of microparticles or dispersions as artificial oxygen carriers for delivering oxygen to transplanted tissue for the purpose of tissue preservation in the field of regenerative medicine, for example, the microparticles or dispersions can be used to supply oxygen to regenerated tissue cells. Alternatively, the microparticles or dispersions can be used for the prophylactic and / or therapeutic treatment of gas bubble formation or gas clots in a subject's bloodstream (e.g., for supplementation of extracorporeal circuits such as cardiopulmonary bypass, liquid ventilation, detoxification, etc.). The subjects of treatment include various animals such as humans, mice, rats, rabbits, pigs, dogs, and cats, and there is no limitation on the subjects of treatment.

[0054] 3. Microparticle Production Method Another aspect of the invention relates to a method for producing microparticles. The microparticles are preferably produced, for example, by a membrane emulsification method using a porous membrane. The method of this embodiment using the membrane emulsification method has the advantages of (1) obtaining microparticles with a uniform particle size distribution (monodisperse, low CV value), and (2) enabling size control of the particle size (particle diameter) and shell thickness of the microparticles. Specifically, the method for producing microparticles includes the following steps: Step 1: A dispersed phase of a fluorocarbon and a polymer having a Young's modulus of 100 MPa or less (for example, 0.5 MPa to 100 MPa) dispersed in an organic solvent is membrane-emulsified into a continuous phase containing a surfactant through a porous membrane with uniform pore size to obtain an emulsion. Step 2: Forming core-shell microparticles comprising a shell containing the polymer and a core containing the fluorocarbon (Step 2)

[0055] Each step will be described below with reference to Figures 3A and 3B. Figure 3A is a schematic diagram of a manufacturing apparatus used in a method for manufacturing microparticles according to one embodiment of the present invention. Figure 3B is a diagram illustrating the principle of membrane emulsification via a porous membrane.

[0056] In step 1, a dispersion is prepared by dispersing a fluorocarbon-containing core component and a polymer-containing shell component in an organic solvent as the dispersed phase. The organic solvent is preferably a low-boiling solvent that dissolves both the fluorocarbon-containing core component and the polymer-containing shell component while being immiscible with water. Specifically, a low-boiling solvent that is immiscible with water, such as dichloromethane or chloroform, is preferred, with dichloromethane being more preferred. The shell thickness of the final microparticles can be controlled by adjusting the ratio of the fluorocarbon-containing core component to the polymer-containing shell component in the dispersion.

[0057] Next, as shown in Figure 3A, the dispersion liquid prepared as the dispersed phase is forced through a porous membrane into a continuous phase containing a surfactant by applying pressure, for example, using nitrogen gas, to form a membrane emulsification. Specifically, as shown in Figure 3B, the dispersed phase, which is an oil (O) layer, is forced through the porous membrane into the continuous phase, which is a water (W) phase, to form an O / W emulsion. The size of the formed emulsion is uniform (monodisperse), correlating with the uniform pore size of the porous membrane. Furthermore, since the size of the formed emulsion is proportional to the pore size of the porous membrane, adjusting the pore size of the porous membrane makes it possible to control the size of the formed emulsion and, therefore, the size (particle diameter) of the microparticles. It is preferable to maintain the volume ratio of the dispersed phase to the continuous phase (dispersed phase / continuous phase) at 1 / 2 to 1 / 1000 (more preferably 1 / 5 to 1 / 100, and particularly preferably approximately 1 / 10). The porous membrane is not particularly limited as long as it has uniform pore diameters, and various materials can be used, for example, those made of glass, resins such as polycarbonate, metals, etc. The pore diameter of the porous membrane can be determined depending on the desired size of the microparticles, but typically, a porous membrane having a pore diameter in the range of 0.05 to 20 μm (preferably 2 to 10 μm) can be used. Furthermore, the membrane emulsification is preferably carried out under stirring in order to separate droplets from the membrane surface.

[0058] The continuous phase may be, for example, an aqueous solution containing a surfactant. The surfactant is not particularly limited, and any known surfactant used for emulsion formation can be suitably used, such as polyvinyl alcohol and Pluronic surfactants, which are block copolymers of polyethylene oxide (PEO) and polypropylene oxide (PPO). Examples of aqueous solvents include water, PBS (phosphate buffered saline), culture medium, and physiological saline. The type and concentration of the surfactant used affect the size of the emulsion. Specifically, a low surfactant concentration tends to result in a larger emulsion. The concentration of the surfactant in the aqueous solution is preferably 0.01 to 10% by weight, more preferably 0.1 to 3% by weight, based on the total weight (100% by weight) of the aqueous solution (aqueous solvent and surfactant).

[0059] In step 2, core-shell microparticles are formed that include a shell that includes a polymer and a core that includes a fluorocarbon. In one embodiment, the microparticles are formed by removing the organic solvent phase from the emulsion obtained in step 1, for example by evaporation. During the evaporation process, phase separation occurs between a polymer-containing shell component (O phase), a fluorocarbon-containing shell component (F phase), and a continuous phase (W phase), resulting in the formation of core-shell particles having a polymer-containing shell and a fluorocarbon-containing core. After formation, the particles are washed, for example with pure water, to remove the surfactant. In one embodiment, the microparticles are formed, for example, by cross-linking the shell.

[0060] 4. Method for producing anisotropic microparticles A further aspect of the present invention relates to a method for producing anisotropic microparticles. Hereinafter, a method for producing microparticles having depressions according to one embodiment of the present invention will be described. The method comprises the following steps: Step 1: Add alcohol to a dispersion of microparticles in an aqueous medium. Step 2: Resuspending the resulting particles in a solvent A method for producing microparticles having depressions, comprising:

[0061] First, in step 1, a dispersion liquid (first dispersion liquid) in which microparticles are dispersed in an aqueous medium is prepared, and alcohol is added to this to obtain a dispersion liquid (second dispersion liquid) in which microparticles are dispersed in a mixture of alcohol and an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include water, a mixed solution of water and alcohol (for example, a lower alcohol), PBS (phosphate buffered saline), culture medium, and physiological saline. The type of alcohol to be added is not particularly limited, but lower alcohols (for example, C1 to C6 alcohols, C1 to C3 alcohols) are preferred. Among them, isopropanol, ethanol, methanol, etc. are desirable in terms of compatibility with water. The concentration of microparticles in the dispersion is not particularly limited, but it is preferable that the microparticles be contained in an amount of, for example, 0.1 to 20% by weight relative to the total weight of the dispersion (aqueous medium and microparticles). The greater the amount of alcohol added, the higher the yield of microparticles having depressions tends to be. On the other hand, if the amount of alcohol added is too large, the particles tend to aggregate. From this perspective, the amount of alcohol added is preferably 1 to 60 parts by volume, more preferably 10 to 50 parts by volume, relative to the amount of water in the aqueous medium (100 parts by volume). After the addition of the alcohol, the second dispersion is preferably allowed to stand under stirring (preferably at a slow speed so that the particles do not settle) for preferably 6 to 48 hours (more preferably 8 to 36 hours, and even more preferably 10 to 24 hours). The temperature at which the dispersion is allowed to stand is not particularly limited, but is usually room temperature (for example, 10 to 35°C).

[0062] In step 2, the particles obtained in step 1 are centrifuged at 1000 rpm for 3 minutes to recover them and then resuspended in a solvent, thereby obtaining particles with depressions. The solvent is not particularly limited, but from the viewpoint of safety, examples thereof include aqueous media such as water, PBS (phosphate buffered saline), and physiological saline, ethanol, and dimethyl sulfoxide (DMSO), with aqueous media such as water and PBS (phosphate buffered saline) being preferred, and water being particularly preferred. There are no particular limitations on the amount of solvent used to suspend the particles, and for example, an amount of aqueous medium is used that will result in a particle concentration in the suspension of 0.1 to 50% by weight. The weight ratio of particles to solvent (particles / solvent) is preferably 1 / 10000 to 3 / 2, and more preferably 1 / 1000 to 1 / 1, from the viewpoint of shape control. [Example]

[0063] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples. In this specification, "room temperature" generally refers to about 10° C. to about 35° C. "%" refers to percent by weight unless otherwise specified. As used herein, the term "about" can mean ±10%.

[0064] I. Microparticle Production 1. Spherical microparticles The following materials were used to prepare the microparticles: (i) PFOB: Perfluorooctyl bromide, product name: 325-51352, manufactured by Wako [ka] (ii) DCM: dichloromethane, product name: 135-02441, manufactured by Wako (iii) PVA: Polyvinyl alcohol, product name: 363170-500G, manufactured by Sigma-Aldrich (iv) Pure water (v) SPG membrane Hydrophilic, Material: Glass, Product Name: SPG Pipe, Made by SPG Techno Four types of pore sizes were used: 2 μm, 3 μm, 4 μM, and 5 μM. (vi) PLC: Poly(lactide-co-caprolactone), weight average molecular weight: approximately 130 kDa, Young's modulus: approximately 12 MPa, product name: PURASORB PLC 7015, manufactured by Taki Chemical Industry Co., Ltd. [ka] x / y=70 / 30 (molar ratio) (vii) PLGA: Poly(lactic acid-co-glycolic acid), weight average molecular weight: 78 to 90 kDa, Young's modulus: approximately 1 GPa, product name: PLGA75-75, manufactured by Mitsui Chemicals [ka] x / y=75 / 25 (molar ratio) (viii) Nile Red, product name: 140-08813, manufactured by Wako (ix) Microchannel system (x) Syringe, 20 mL

[0065] Example 1: Core / shell microparticles PFOB / PLC-2.0s MPs 120 mg of PLC and 0.12 g of PFOB were dissolved in 2 mL of DCM. Nile Red was added for fluorescence measurement. The resulting solution was then extruded through an SPG membrane (pore size: 4 μm) into 20 mL of 2% PVA aqueous solution stirred at 150 rpm to obtain an O / W emulsion. The DCM was then evaporated at room temperature while stirring at 200 rpm, and the emulsion was then centrifuged at 1000 rpm for 3 minutes. The resulting particles were washed three times with pure water to obtain PFOB / PLC-2.0s MPs, which consist of a core made of PFOB and a shell made of PL.

[0066] Example 2: Core / shell microparticles PFOB / PLC-1.6s MPs PFOB / PLC-1.6s MPs were obtained in the same manner as in Example 1, except that 80 mg of PLC and 0.12 g of PFOB were used.

[0067] Example 3: Core / shell microparticles PFOB / PLC-1.2s MPs PFOB / PLC-1.2s MPs were obtained in the same manner as in Example 1, except that 50 mg of PLC and 0.12 g of PFOB were used.

[0068] Example 4: Core / shell microparticles PFOB / PLC-1.0s MPs PFOB / PLC-1.0s MPs were obtained in the same manner as in Example 1, except that 40 mg of PLC and 0.12 g of PFOB were used.

[0069] Example 5: Core / shell microparticles PFOB / PLC-0.8s MPs PFOB / PLC-0.8s MPs were obtained in the same manner as in Example 1, except that 30 mg of PLC and 0.12 g of PFOB were used. Note that "PFOB / PLC-0.8s MPs" is synonymous with "PFOB / PLC-4d MPs."

[0070] Example 6: Core / shell microparticles PFOB / PLC-2d MPs PFOB / PLC-2d MPs were obtained in the same manner as in Example 5, except that an SPG membrane with a pore size of 2 μm was used.

[0071] Example 7: Core / shell microparticles PFOB / PLC-3d MPs PFOB / PLC-3d MPs were obtained in the same manner as in Example 5, except that an SPG membrane with a pore size of 3 μm was used.

[0072] Example 8: Core / shell microparticles PFOB / PLC-5d MPs PFOB / PLC-5d MPs were obtained in the same manner as in Example 5, except that an SPG membrane with a pore size of 5 μm was used.

[0073] [Comparative Example 1: Microparticles PLC MPs] Except for using 50 mg of PLC and not using PFOB, PLC MPs were obtained in the same manner as in Example 1. PLC MPs are solid particles composed of PLC.

[0074] [Comparative Example 2: Core / shell type microparticles PFOB / PLGA-0.8s MPs] PFOB / PLGA-0.8s MPs (PFOB / PLGA-4d MPs), particles consisting of a core made of PFOB and a shell made of PLGA, were obtained in the same manner as in Example 5 (SPG membrane: pore size 4 μm), except that 30 mg of PLGA and 0.12 g of PFOB were used instead of PLC. PFOB / PLGA-2d MPs were obtained in the same manner as above, except that an SPG membrane with a pore size of 2 μm was used. PFOB / PLGA-3d MPs were obtained in the same manner as above, except that an SPG membrane with a pore size of 3 μm was used. PFOB / PLGA-5d MPs were also obtained in the same manner as above, except that an SPG membrane with a pore size of 5 μm was used.

[0075] [Comparative Example 3: Microparticles PLGA MPs] Except for using 50 mg of PLGA instead of PLC and not using PFOB, PLGA MPs were obtained in the same manner as in Example 1. PLGA MPs are solid particles composed of PLGA.

[0076] 2. Microparticles with a dimpled shape The following materials were used to prepare the microparticles with a dimpled shape: (i) PFOB / PLC MPs Aqueous dispersions of spherical core / shell microparticles of different sizes, PFOB / PLC-4d MPs, PFOB / PLC-2d MPs, PFOB / PLC-3d MPs, and PFOB / PLC-5d MPs, were used, which were prepared by the methods of Examples 5 to 8. (ii) Isopropanol, 166-04831, Wako (iii) Microchannel system (iv) Syringe, 20 mL

[0077] [Example 9: Concave-shaped core / shell microparticles (PFOB / PLC-2d MPs)] PFOB / PLC-2d MPs were produced in the same manner as in Example 6. 5 mL of isopropanol was slowly added to 5 mL of a 5% by volume aqueous dispersion of PFOB / PLC-2d MPs while stirring slowly. The mixture was left overnight (approximately 12 hours) at room temperature while stirring at a rate that did not cause the microparticles to settle. The resulting particles were resuspended in pure water at a concentration of 5 wt % to obtain microparticles with a dimpled shape.

[0078] [Example 10: Concave-shaped core / shell microparticles (PFOB / PLC-3d MPs)] PFOB / PLC-3d MPs were produced by the same method as in Example 7. Microparticles with a dimpled shape were obtained in the same manner as in Example 9, except that PFOB / PLC-3d MPs were used instead of PFOB / PLC-2d MPs.

[0079] [Example 11: Concave-shaped core / shell microparticles (PFOB / PLC-4d MPs)] PFOB / PLC-4d MPs were produced by the same method as in Example 5. Microparticles with a dimpled shape were obtained in the same manner as in Example 9, except that PFOB / PLC-4d MPs were used instead of PFOB / PLC-2d MPs.

[0080] Example 12: Concave-shaped core / shell microparticles (PFOB / PLC-5d MPs) PFOB / PLC-5d MPs were produced by the same method as in Example 8. Microparticles with a dimpled shape were obtained in the same manner as in Example 9, except that PFOB / PLC-5d MPs were used instead of PFOB / PLC-2d MPs.

[0081] II. Evaluation of Microparticles 1. Evaluation of particle size, particle size distribution, and particle structure (1) Measurement of particle size and particle size distribution The particles obtained in the examples were measured using a laser diffraction / scattering particle size distribution analyzer (Partica LA-950V2, Horiba) to measure the particle size (volume average particle size) and CV value. The CV value was calculated from the standard deviation of the particle size and the average particle size according to the following formula. CV value (%) = (standard deviation of particle size / average particle size) x 100

[0082] The results are shown in Table 1 and FIGS. Table 1 summarizes the particle sizes of the particles obtained in the above examples, along with the composition of the particles. [Table 1]

[0083] Figure 4 shows the results of measuring the particle size distribution of PFOB / PLC-0.8s MPs obtained in Example 5. In Figure 4, the particle size distribution of human red blood cells (human RBCs) is shown as a control. FIG. 4 confirms that the uniform pore size distribution of the SPG membrane results in a uniform size distribution of the microparticles. FIG. 5 is a diagram showing the relationship between the volume average particle size of the microparticles obtained in Examples 5 to 8 and the pore size of the SPG membrane used in the production. Figure 5 confirms that the volume-average particle size of the microparticles increases in proportion to the pore size of the SPG membrane, suggesting that precise control of the microparticles is possible by adjusting the pore size of the SPG membrane.

[0084] (2) Optical photography The PFOB / PLC-0.8s MPs obtained in Example 5 and human red blood cells (hRBCs) were dispersed in physiological saline and mixed. Optical photographs were taken using an inverted phase-contrast microscope (IX73, Olympus). Figure 6 shows an optical photograph of the mixed system of PFOB / PLC-0.8s MPs and human red blood cells (hRBCs).

[0085] (3) SEM observation of spherical particles and fluorescence observation using a confocal scanning microscope The morphology of each microparticle obtained in Examples 1 to 8 was observed using a scanning electron microscope (SEM) (Hitachi TM3030Plus Miniscope). As a representative example, an SEM photograph of the PFOB / PLC-0.8s MPs of Example 5 is shown (FIG. 7A). Furthermore, a confocal laser scanning microscope (device name: confocal laser scanning microscope [LSM510 META NLO, Carl Zeiss]) was used to irradiate the samples with laser light at a wavelength of 488 nm and perform fluorescence observation. Figure 7B is a confocal laser scanning microscope fluorescence image of PFOB / PLC-0.8s MPs. Figure 7C is a confocal laser scanning microscope fluorescence image of the solid microparticles (PLC MPs) obtained in Comparative Example 1. From these images, the shell thickness of each microparticle was measured and is shown in Table 1. Ring-shaped fluorescence was observed in Figure 7B, whereas circular fluorescence was observed in Figure 7C, confirming that the PFOB / PLC-0.8s MPs obtained in Example 5 formed a core-shell structure.

[0086] (4) FT-IR measurement (Measurement procedure) IR spectra were measured using a Fourier transform infrared spectrophotometer (FT / IR-4200ST, JASCO). The microparticles were mixed with KBr to form pellets, which were then used for the measurements. Liquid PFOB was measured using a liquid measurement cell with a NaCl window. Figure 8 shows the IR spectrum of the PFOB / PLC-0.8s MPs obtained in Example 5. From Figure 8, the PFOB peaks (1243, 1216 cm) -1 [-CF2], 1153 cm -1 [-CF3]) was confirmed, suggesting that PFOB is present in the core of the microparticles.

[0087] (5) SEM observation of particles with dimples The morphology of the microparticles obtained in Examples 9 to 12 was observed by SEM. As a representative example, an SEM photograph of concave-shaped PFOB / PLC-4d MPs of Example 11 is shown (FIG. 9A). FIG. 9B shows an SEM photograph after deformation, and FIG. 9C shows an SEM photograph before deformation. 9A to 9C, a shape similar to that of a red blood cell can be seen.

[0088] (6) Measurement of particle size distribution of particles with a concave shape The particle size distribution of the particles obtained in Examples 9 to 12 was measured using a laser diffraction / scattering particle size distribution measuring device, and the particle size (volume average particle size) was measured. Figure 10 shows the measurement results of the particle size distribution of the concave-shaped PFOB / PLC-4d MPs obtained in Example 11. In Figure 10, "After shape change" shows the measurement results of the particles obtained in Example 11, "Before shape change" shows the measurement results of the microparticles used as the raw material for producing the concave-shaped particles, and "hRBCs" shows the measurement results of human red blood cells used as a control. 10, it can be seen that the size of the deformed particles does not change significantly from that before deformation. It can also be seen that microparticles with a shape similar to that of red blood cells (a shape with depressions) were produced.

[0089] II. Evaluation of deformability (1) Compression experiment The deformability of microparticles was measured using a microcompression tester (MCT series, Shimadzu). One particle was selected from the particles on the stage under microscope observation (50x objective lens), and the particle was compressed from above with a probe to evaluate the stress and displacement during compression.

[0090] 11A is a diagram showing the results of compression experiments on the solid microparticles PLC MPs (No. 1) obtained in Comparative Example 1, the microparticles (Nos. 2 to 6) obtained in Examples 1 to 5, the microparticles PFOB / PLGA-0.8s MPs (No. 7) obtained in Comparative Example 2, and the microparticles PLGA MPs (No. 8) obtained in Comparative Example 3. Fig. 11A confirms that the microparticles composed of a PLC shell can achieve large displacement at low compressive stress and have excellent deformability.

[0091] 11B shows the results of compression experiments on concave-shaped microparticles (PFOB / PLC-4d MPs) obtained in Example 11 (cDFC in the figure), spherical microparticles (PFOB / PLC-0.8s MPs; PFOB / PLC-4d MPs) obtained in Example 5 (DFC in the figure), microparticles (PFOB / PLGA-0.8s MPs) obtained in Comparative Example 2 (Rigid FC in the figure), acrylic beads (acrylic beads in the figure), and red blood cells cross-linked with 0.05% or 0.5% glutaraldehyde (0.05% RBC and 0.5% RBC, respectively). Due to the difference in shape, the concave-shaped microparticles (cDFC) reached their deformation limit at a shorter displacement than the spherical microparticles (DFC). At the initial stage of compression, a cross point was observed between the curves of the dimpled microparticles (cDFC) and spherical microparticles (DFC), suggesting that the dimpled microparticles (cDFC) have higher deformability than the spherical microparticles (DFC) at the initial stage of the compression experiment (deformation up to about 0.4 μm).

[0092] (2) Passing experiment P per indicates the percentage of particles that can pass through the channel (Percent of permeated MPs or hRBCs), and indicates the percentage of particles that can pass through the channel. It is an index of particle passing ability, and P per The larger the value, the harder the material is and the better the passability.

number

[0093] (Measurement procedure) A passing test was carried out according to the method described in Takahiro Kawakatsu et al., Regular-sized cell creation in microchannel emulsification by visual microprocessing method, Journal of the American Oil Chemists' Society, March 1997, Volume 74, Issue 3, pp. 317-321. Figure 12 shows the measurement procedure and principle of the passing test. As shown in Figure 12, 10 mL (10 mL) of particle dispersion was poured into a microchannel with a slit width of 4.5 μm. 5 The particle dispersion liquid was collected after passing through the microchannel, and the number of particles was measured by microscopic observation. per was measured.

[0094] The results are shown in Figures 13 to 15. Figure 13 shows the P per Specifically, the P values ​​of the microparticles (PFOB / PLC-0.8s MPs) obtained in Example 5, the microparticles (PFOB / PLGA-0.8s MPs) obtained in Comparative Example 2, and the microparticles (concave-shaped PFOB / PLC MPs) obtained in Example 11 are shown. per As a control, human red blood cells (Natural hRBCs) and those crosslinked with glutaraldehyde at different concentrations (crosslinked hRBCs), as well as commercial MPs (product name: crosslinked acrylic medium-dispersion particles MZ-8HN, manufactured by Soken Chemical & Engineering Co., Ltd.; average particle size: 8 μm) were used. per From Figure 13, P perIt was confirmed that this depends on the particle type and size. Compared with commercial MPs, which are commercially available solid particles, and the PFOB / PLGA-0.8s MPs obtained in Comparative Example 2, the PFOB / PLC-0.8s MPs obtained in Example 5 were found to have high microchannel permeability. In addition, the shape-altered concave-shaped PFOB / PLC MPs obtained in Example 11 showed even higher permeability than spherical MPs, confirming that their permeability is closer to that of normal human red blood cells (Natural hRBCs).

[0095] Figure 14 shows the cake layer formed at the channel inlet in a microparticle passage experiment. Specifically, Figure 14A shows the cake layer formed when the microparticles obtained in Examples 5 to 8 were used. From top to bottom, the results are those of Example 8 (PFOB / PLC-5d MPs), Example 5 (PFOB / PLC-4d MPs), Example 7 (PFOB / PLC-3d MPs), and Example 6 (PFOB / PLC-2d MPs). Figure 14B shows the cake layer formed when the microparticles obtained in Comparative Example 2 were used. From top to bottom, the results are those of PFOB / PLGA-5d MPs, PFOB / PLGA-4d MPs, PFOB / PLGA-3d MPs, and PFOB / PLGA-2d MPs. Figure 14C shows the cake layer formed when the microparticles obtained in Examples 9 to 12 were used. Specifically, the results of Example 9 (Concave-shaped PFOB / PLC-2d MPs) are shown in a, the results of Example 10 (Concave-shaped PFOB / PLC-3d MPs) are shown in b, the results of Example 11 (Concave-shaped PFOB / PLC-4d MPs) are shown in c, and the results of Example 12 (Concave-shaped PFOB / PLC-5d MPs) are shown in d. Figure 14 confirms that particles with low passage ability form a cake layer at the channel entrance, and that cake layer formation is suppressed as the passage ability increases.

[0096] 15 shows the particle size distribution of microparticles before and after passing through a microchannel. Specifically, "b" shows the particle size distribution of the concave-shaped PFOB / PLC-3d MPs obtained in Example 10 before passing and after passing, and "c" shows the particle size distribution of the concave-shaped PFOB / PLC-4d MPs obtained in Example 11 before passing and after passing. The particle size distribution of the microparticles was measured using a laser diffraction / scattering particle size distribution analyzer. Figure 15 confirms that the size of the particles that passed through the microchannel did not change significantly from before passing through, suggesting that the microparticles returned to their original shape after passing through the microchannel.

[0097] (3) Young's modulus measurement of microparticles using AFM Atomic force microscopy (AFM) measurements were performed in contact mode using a scanning probe microscope (SPM-9700HT, Shimadzu Corporation) with a Pyrex nitride cantilever (spring constant 2.0 N / m). The tip half angle was 45°, and the probe curvature radius was 10 nm. Highly oriented pyrolytic graphite was used as the substrate and fixed to the bottom of a 15 mm inner diameter Petri dish. The particles were dispersed in pure water, and 500 μL of the resulting dispersion was dropped onto the substrate to fix the particles to the substrate. After leaving it to stand for 5 minutes, an additional 500 μL of pure water was dropped, and measurements were performed. The obtained deformation curve is shown in Figure 16.

[0098] The Young's modulus was also calculated using the Hertz contact model (Kuznetsova, TG, Starodubtseva, MN, Yegorenkov, NI, Chizhik, SA, Zhdanov, RI. Atomic force microscopy probing of cell elasticity. Micron 2007, 38, 824-833; Yeow, N., Tabor, RF, Garnier, G. Atomic force microscopy: From red blood cells to immunohematology. Advances in Colloid and Interface Science 2017, 249, 149-162.) as shown below.

number

[0099] In Figures 16 and 17, cDFC represents the results for the concave-shaped microparticles obtained in Example 11 (concave-shaped PFOB / PLC-4d MPs), DFC represents the results for the microparticles (PFOB / PLC-4d MPs) of Example 5 used as the raw material for producing the concave-shaped particles of Example 11, and 0.005%RBC represents the results for red blood cells cross-linked with glutaraldehyde at a concentration of 0.005%.

[0100] As can be seen in Figure 16, the dimpled microparticles (cDFC) exhibited superior deformability due to their dimpled shape and thin shell. The dimpled microparticles (cDFC) exhibited greater deformation than the spherical microparticles (DFC) at the same load, similar to that of slightly cross-linked red blood cells (0.005% RBC). The dimpled microparticles (cDFC) were more deformable than the spherical microparticles (DFC) over the measured range, suggesting that they share similar deformability to slightly cross-linked red blood cells. This result is also consistent with the cross-point shown in the compression curves in Figure 11B, confirming that the dimpled microparticles (cDFC) exhibited higher deformability than the spherical microparticles (DFC) in the initial stage (deformation up to approximately 0.4 μm). Considering the high microchannel passing properties of the microparticles with a dimpled shape (cDFC) shown in Figure 13, it is suggested that deformability at the submicron scale is an important factor for the channel passing properties of microparticles with a dimpled shape. As shown in Figure 17, the calculated Young's modulus of the dimpled microparticles (cDFC) (92.8 ± 16.9 kPa) was lower than that of the spherical microparticles (DFC) (286.9 ± 46.4 kPa) and similar to that of slightly cross-linked human red blood cells (0.005% RBC) (94.3 ± 28.8 kPa). As a critical indicator of the elasticity of cells or artificial particles, Young's modulus plays an important role in aiding disease diagnosis and can characterize the deformability of particles in this study. Table 2 below summarizes literature values ​​for Young's modulus for healthy, aging, and pathological red blood cells. [Table 2] The Young's modulus of the dimpled microparticles (cDFC) was approximately three times higher than that of healthy red blood cells and was comparable to that of red blood cells with hereditary spherocytosis and G6PD deficiency. Furthermore, as shown in Figure 13, the dimpled microparticles (cDFC) of the present invention exhibited good channel passage properties comparable to those of red blood cells. Also, as shown in Figure 13, the spherical microparticles (DFC) passed through the channel more smoothly than spherical particles with a PLGA shell (Rigid FC in Comparative Example 2). These results suggest that the higher elasticity of the PLGA shell contributed to the smooth deformation and passage properties. It has been reported that red blood cells with a Young's modulus of around 26 kPa can easily pass through capillaries. Microparticles with thinner shells are more deformable when compressed, and further improvement in deformability is expected by thinning the shell of microparticles with dimpled shapes. Alternatively, using a softer shell material (the polymer that makes up the shell) is expected to improve deformability. The flexibility of a polymer depends on the proportion of soft segments, so increasing the soft segment content is expected to result in more flexible microparticles. By adjusting the Young's modulus and thickness of the shell material, it is possible to further improve deformability and flexibility, resulting in microparticles that more closely resemble the Young's modulus of healthy red blood cells. As shown in Table 2, it is suggested that microparticles having a Young's modulus of up to 300 kPa have good flexibility and deformability. Also, considering that microparticles having a higher Young's modulus can be used as the particle size decreases, in the present invention, microparticles having a Young's modulus of up to 500 kPa can have good flexibility and deformability.

[0101] (4) Storage stability test To evaluate storage stability, the microparticles were dispersed in three solvents commonly used in biomedical applications: phosphate-buffered saline (PBS), saline, and Dulbecco's modified Eagle's medium (DMEM). The particles were stored at 4°C for several weeks, and changes in particle size were monitored. A laser diffraction / scattering particle size distribution analyzer was used to measure particle size (volume-average particle diameter). Figure 18A shows the results (DFC in the figure) for the spherical microparticles (PFOB / PLC-0.8s MPs; PFOB / PLC-4d MPs) obtained in Example 5 and the results (Rigid FC in the figure) for the microparticles (PFOB / PLGA-0.8s MPs) obtained in Comparative Example 2, and Figure 18B shows the results (cDFC in the figure) for the concave-shaped microparticles (PFOB / PLC-4d MPs) obtained in Example 11. 18A and 18B confirm that the microparticles are highly stable in all media without significant size fluctuations during storage. Because poly(lactide-co-caprolactone) (PLC), used to prepare the microparticles, is a biodegradable polymer, it is important to evaluate the biodegradability of the microparticles. Considering that the shelf life of RBCs provided for transfusion is 21 days, the microparticles of the present invention are expected to be usable for potential applications in terms of storage stability.

[0102] (5) Evaluation of oxygen storage and release capacity (Production of microparticles for evaluation) Spherical microparticles loaded with Ru(ddp), Ru(ddp)-loaded DFC, were obtained in the same manner as in Example 5, except that 0.1 mg / mL Ru(ddp) (manufactured by Santa Cruz Biotech) was used instead of Nilelet. A Ru(ddp)-loaded cDFC was obtained in the same manner as in Example 11, except that 0.1 mg / mL Ru(ddp) (manufactured by Santa Cruz Biotech) was used instead of Nilete.

[0103] (Oxygen storage and release capacity measurement) The oxygen storage and release capacity was measured by measuring the time change in dissolved oxygen (DO) concentration. Specifically, to measure the oxygen storage capacity, 20 mL of a dispersion (concentration: 45% by volume) of Ru(ddp)-loaded particles (Ru(ddp)-loaded DFC and Ru(ddp)-loaded cDFC) in phosphate-buffered saline (PBS) was deoxygenated with nitrogen and then immersed in the atmosphere (50 cm 3 / min) (oxygen storage process). For the measurement of oxygen release capacity, the same amount of sample was oxygenated and then sparged with nitrogen (50 cm 3 The particles were deoxygenated at a rate of 1 / min (oxygen release process). For the measurements of oxygen storage capacity and oxygen release capacity, 0.2 mL of sample was taken at various time points, and the fluorescence intensity from the Ru(ddp) loaded on the particles was measured using a spectrofluorometer (FP-8200, JASCO). The obtained fluorescence intensity was converted to DO concentration using a calibration curve prepared using a DO meter (Visiferm DO ARC 120, Hamilton). The DO concentration during oxygen storage and release was also monitored by inserting the DO meter into the sample. Experiments were performed on three individuals under each condition, and the data are expressed as the mean ± standard deviation.

[0104] The results of dissolved oxygen concentration measurements using a DO meter are shown in Figure 19a (oxygen absorption process) and Figure 19b (oxygen release process). In contrast to PBS (phosphate buffered saline), the microparticles of the present invention (Ru(ddp)-loaded DFC and Ru(ddp)-loaded cDFC) exhibited high oxygen storage and release capacities, indicating their oxygen supply function. Furthermore, comparison of Ru(ddp)-loaded DFC and Ru(ddp)-loaded cDFC showed that the oxygen absorption and release capacities remained almost unchanged even after the shape change. These results suggest that the microparticles of the present invention can function as promising oxygen carriers.

[0105] (6) Biocompatibility (cell viability) The biocompatibility of the microparticles was evaluated by culturing them with HUVECs (human umbilical vein endothelial cells). Specifically, the cell viability of the microparticles was evaluated as follows: HUVECs (human umbilical vein endothelial cells) were cultured at a density of 10 5 Cells were cultured in 24-well flat-bottom plates (IWAKI, Asahi Glass Co., Ltd.) at 100 cells / well and maintained at 37°C in 20% O2 and 5% CO2 for 1 day. The culture medium was then replaced with fresh medium, and a 45% (volume) dispersion of microparticles in PBS (UV radiation for sterilization before use) was added to the cells using cell inserts. The cells were maintained at 37°C in 20% O2 and 5% CO2 for 1 day. Cell viability was measured using the WST-8 assay (Cell Counting Kit-8, Dojindo Laboratories). Absorbance at 450 nm was measured using a plate reader (2030 ARVO V3; PerkinElmer). The absorbance value for each well was normalized to that of the control group (HUVECs only). The microparticles used were spherical microparticles (PFOB / PLC-4d MPs) obtained in Example 5 (DFC in the figure) and concave-shaped microparticles (concave-shaped PFOB / PLC-4d MPs) obtained in Example 11 (cDFC in the figure).

[0106] The results are shown in Figure 20. As shown in Figure 20, the cell viability in the presence of microparticles was not reduced compared to the control group (HUVECs only), and was in fact slightly higher. This confirmed that the microparticles of the present invention are biocompatible. Furthermore, a comparison of spherical microparticles (DFC) and microparticles with a dimpled shape (cDFC) suggests that the biocompatibility of the microparticles does not change significantly depending on the shape of the microparticles.

[0107] (7) Oxygen supply for cell culture Oxygen supply for cell culture was evaluated using HeLa cells as follows: 750 μL of HeLa cells were cultured at 2 × 10 6Cells were seeded onto a 24-well glass-bottom plate at a density of 100 cells / well and maintained at 37°C in 20% O2 and 5% CO2 for 1 day. Next, 500 μL of a 45% by volume dispersion of microparticles in PBS was added to the cells after changing the culture medium, and the cells were maintained at 37°C in 2% O2 and 5% CO2 for 2 days. Fluorescence images were taken using a confocal laser scanning microscope (LSM880 AiryScan, Carl Zeiss). As a control, HeLa cells were cultured in the same manner except that no microparticles were added. The microparticles used were the concave-shaped microparticles (concave-shaped PFOB / PLC-4d MPs) obtained in Example 11 (cDFC in the figure).

[0108] The results are shown in Figure 21. As shown in Figure 21, the fluorescence of the cells shows a response to oxygen supply. HeLa cells cultured in the absence of microparticles exhibited strong fluorescence, suggesting insufficient oxygen supply (Figure 21A). In contrast, HeLa cells cultured with the addition of microparticles exhibited faint fluorescence, suggesting that oxygen supply from the microparticles alleviated hypoxia (Figure 21B). Because EGFP expression in HeLa cells can reflect oxygen levels at the subcellular level, these results suggest that microparticles can function as promising oxygen carriers. [Industrial Applicability]

[0109] The microparticles of the present invention can be used as artificial oxygen carriers. The size of the microparticles of the present invention can be controlled according to the application, and they have a narrow particle size distribution, so they can be used in a variety of medical fields. The microparticles of the present invention combine stability and channel permeability, and are therefore highly practical and useful. [Explanation of symbols]

[0110] 1. Microparticles 11 Shell 12 cores 13 Polymers 14 Fluorocarbon 15 hollow d a Short diameter d b Long diameter

Claims

1. A core-shell microparticle comprising a shell containing a polymer having a Young's modulus of 100 MPa or less and a core containing a fluorocarbon.

2. A core-shell microparticle, comprising a shell comprising a polymer, a core comprising a fluorocarbon, and a core comprising a fluorocarbon, wherein the microparticle has a Young's modulus of 500 kPa or less.

3. 3. The particles of claim 1 or 2, wherein the size of the microparticles ranges from 1 to 20 μm.

4. The particles of claim 3, wherein the size of the microparticles ranges from 1 to 10 μm.

5. 5. The particle according to claim 1, wherein the molecular weight of the fluorocarbon is in the range of 100 to 2,000 g / mol.

6. 6. The particles according to claim 1, wherein the content of the fluorocarbon in the microparticles is 30% by weight or more.

7. 7. The particle according to claim 1, wherein the shell has a thickness in the range of 0.1 to 4 μm.

8. The particle according to any one of claims 1 to 7, wherein the polymer is an elastomer.

9. The particles according to any one of claims 1 to 8, wherein the polymer comprises at least one selected from a copolymer of polylactide and / or polyglycolide with polycaprolactone, and a copolymer of polylactide and / or polyglycolide with trimethylene carbonate.

10. A particle according to any one of claims 1 to 9, wherein the core comprises a gas dissolved in the fluorocarbon.

11. The particles according to any one of claims 1 to 10, wherein the fluorocarbon has a boiling point of 35°C or higher.

12. 12. The particle according to claim 1, wherein the core comprises a fluorocarbon having an oxygen solubility of 30 vol / vol % or more.

13. The fluorocarbon comprises at least one selected from the group consisting of bis(fluoroalkyl)ethene, hydrofluorocarbon, hydrofluoroether, hydrochlorofluorocarbon, cyclic perfluorocarbon, perfluoroamine, perfluoro(2-butyltetrahydrofuran), brominated perfluorocarbon, iodinated perfluorocarbon, chlorinated perfluorocarbon, and perfluoroalkyl ether or polyether. The particles according to any one of claims 1 to 12.

14. The particle of any one of claims 1 to 13, wherein the particle is deformable.

15. The particle according to any one of claims 1 to 14, wherein the particle has an anisotropic shape.

16. The particles according to any one of claims 1 to 15, wherein the CV value of the microparticles is 40% or less.

17. A dispersion comprising the particles according to any one of claims 1 to 16 in an aqueous medium.

18. The dispersion according to claim 17, which is used as an oxygen infusion solution, a perfusion solution for transplanted organs, a preservation solution for transplanted organs, an oxygen supply solution for regenerative medicine, or a culture medium for animal cells or microorganisms for producing biopharmaceuticals.

19. A method for producing particles according to any one of claims 1 to 16, comprising: A dispersed phase obtained by dispersing a fluorocarbon and a polymer having a Young's modulus of 100 MPa or less in an organic solvent is membrane-emulsified into a continuous phase containing a surfactant through a porous membrane having a uniform pore size to obtain an emulsion; forming core-shell microparticles comprising a shell comprising the polymer and a core comprising the fluorocarbon; A method comprising:

20. 20. The method of claim 19, wherein the formation of the core-shell microparticles is carried out by removing an organic solvent phase contained in the emulsion.

21. Adding an alcohol to a dispersion in which the particles according to any one of claims 1 to 16 or the particles obtained by the production method according to claim 19 or 20 are dispersed in an aqueous medium; and resuspending the resulting particles in a solvent; A method for producing anisotropic microparticles, comprising:

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