JP2013227444A - Composite particle and cell preparation using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide composite particles which can sufficiently hold cells thereon for a period of time until an effect of the transplanted cells is developed, and to provide a cell preparation including the composite particles with the cells supported thereon.SOLUTION: The purpose of the invention can be achieved by composite particles each of which includes inorganic particles adsorbed on a surface of a particle composed of an L-lactide-ε-caprolactone copolymer, wherein the constitutional molar ratio of L-lactide to an ε-caprolactone in the copolymer is approximately 75/25. Furthermore, a cell preparation including cell-supporting composite particles, which includes cell-supporting composite particles produced by supporting cells on the composite particles is provided.

Description

  The present invention relates to a composite particle capable of sufficiently retaining cells until the effect of cell transplantation is obtained, and a cell preparation containing cell-supported composite particles in which cells are supported on the composite particles.

  Pluripotent stem cells or vascular endothelial progenitor cells present in the bone marrow have been shown to be involved in physiological and pathological angiogenesis in each tissue. By using the angiogenic ability of these cells, stem cells isolated from bone marrow and peripheral blood are directly transplanted to ischemic lower limbs, ischemic myocardium, etc. Yes. Although such treatments have been studied to increase the effectiveness by a cell engineering technique or a transplantation method combined with cytokines, a sufficient effect has not been obtained yet.

  In recent years, in the mechanism of angiogenesis therapy, only a few transplanted cells are taken up into the structure of the neovascularization, but it is clear that it is important for angiogenesis that the transplanted cells secrete multiple cytokines at the transplant site. became. However, it was confirmed that 70 to 80% of transplanted cells migrated into the systemic circulation within 48 hours, and few remained at the transplanted site. These findings are reported in Non-Patent Documents 1-7. Therefore, in order to realize a sufficient therapeutic effect, there has been a demand for a technique for allowing transplanted cells to remain at a site requiring revascularization (that is, a transplant site) until a transplant effect is obtained.

  In regenerative medicine, a scaffold for cell transplantation (cell carrier) for supporting cultured cells to be administered is indispensable when cells are used to regenerate tissues or organs that have deteriorated or malfunctioned. Known scaffolds for cell transplantation include gelatin and collagen. Gelatin and collagen are very effective materials, but have problems such as antigenicity because they are derived from animals. In addition, since a cell scaffold using gelatin or collagen is easily decomposed in the body, it is difficult to hold cells at a transplant site for a long period of time.

  In contrast, inorganic substances such as hydroxyapatite are non-animal, so there is no concern about antigenicity and the biocompatibility is excellent. Moreover, since it enables cell adhesion and proliferation, it is suitable as a scaffold for cell transplantation. However, inorganic materials have the drawback of low strength and toughness, and composite particles in which inorganic materials and bioabsorbable polymers are combined have been developed to compensate for these disadvantages. For example, Patent Document 1 discloses non-spherical fine particles in which nanoparticles formed of calcium salt are immobilized on the surface of a support formed of polyester or a polymer containing polar end groups. In addition, Patent Document 2 discloses that inorganic particles are not formed on the surface of thermoplastic resin particles without using an organic solvent that may be affected by a living body and additives such as molecular level surfactants and dispersion stabilizers. Discloses a method for producing spherical composite fine particles with adsorbed particles.

  Thus, also in the field of composite particles composed of inorganic substances and bioabsorbable polymers, various properties for imparting suitable physical properties and high safety as cell scaffolds to composite particles supporting cells. Technology has been developed. From such a background, it is possible to hold the transplanted cells for a sufficient period until the transplantation effect is obtained in vivo, and it can be absorbed quickly after the cell transplantation effect is obtained. There has been a demand for a cell scaffold having even more excellent physical properties as a cell scaffold.

JP 2010-235686 A JP 2011-184615 A

Weel V, et al. Ann Vasc Surg 2008; 22: 582-597 Phelps EA and Garcia AJ. Regen Med 2009; 4: 65-80 Andrew S, et al. Semin Thorac Cardiovas Surg 2008; 20: 49-101 Sieveking DP and Ng MKC Vascular Medicine 2009; 14: 153-166 Mheid IAI et al. Angiology 2009; 59: 705-716 Aranguren XL et al. J Mol Med 2009; 87: 3-16 Shinya Fukumoto, Hidenori Koyama, Shinji Tanaka, Takaaki Maeno, Takuhito Shoji et al .: Efficacy of autologous bone marrow mononuclear cell transplantation for severe limb ischemia—including dialysis patients—. Journal of Japanese Society for Dialysis Therapy 2004, 37: 1493-1501.

  The main object of the present invention is to provide composite particles capable of retaining cells at the transplant site until a cell transplant effect is obtained. Furthermore, an object of the present invention is to provide a cell preparation containing cell-supporting composite particles obtained by supporting cells on the composite particles.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that particles comprising an L-lactide-ε-caprolactone copolymer having a constituent molar ratio of L-lactide to ε-caprolactone of approximately 75:25. When cells are transplanted by carrying them on composite particles having inorganic particles such as hydroxyapatite adsorbed on the surface, the cells are sufficiently retained for a period of time until the effect of cell transplantation is obtained at the transplantation site. It has been found that the effect is remarkably enhanced. The present invention has been completed as a result of further research based on such knowledge. That is, this invention provides the composite particle and cell preparation of the following aspect.
Item 1. Composite particles in which inorganic particles are adsorbed on the surface of particles made of an L-lactide-ε-caprolactone copolymer, and the constituent molar ratio of L-lactide and ε-caprolactone in the copolymer is approximately 75:25.
Item 2. Item 2. The composite particle according to Item 1, wherein the inorganic particle is hydroxyapatite.
Item 3. Item 3. The composite particle according to Item 1 or 2, which is used as a scaffold for transplanted cells.
Item 4. Item 4. A cell preparation comprising cell-supporting composite particles obtained by supporting cells on the composite particles according to any one of Items 1 to 3.
Item 5. Item 5. The cell preparation according to Item 4, wherein the cell is a bone marrow mononuclear cell.
Item 6. Item 6. The cell preparation according to Item 5, which is used for promoting angiogenesis.

  The composite particle of the present invention is formed by adsorbing inorganic particles on the surface of a particle composed of an L-lactide-ε-caprolactone copolymer having a constitutional molar ratio of L-lactide and ε-caprolactone of about 75:25. Features. By using such an L-lactide-ε-caprolactone copolymer, it is possible to suppress dropping of inorganic particles after cell transplantation, and a sufficient period until the effect of cell transplantation is obtained. It is possible to hold cells over a period of time (for example, for one week to several months). Therefore, by carrying the cells on the composite particles of the present invention, the cells can be stably held in the living body (that is, excellent in cell carrying power), and the tissue regeneration efficiency at the transplant destination is dramatically improved. Can be increased. Further, since the composite particles of the present invention are rapidly decomposed and absorbed after the effect of cell transplantation is obtained, it is not necessary to remove them from the living body even after the effect of cell transplantation is obtained.

  Furthermore, since the composite particles of the present invention are prepared without using animal materials, there are no problems such as antigenicity. Moreover, when cells are carried on the composite particles of the present invention, high cell adhesion is realized simply by mixing the composite particles and the transplanted cells, so that the cell preparation of the present invention can be easily prepared in a short period of time. it can. In particular, the cell preparation in which bone marrow mononuclear cells are supported on the composite particles of the present invention is useful for promoting angiogenesis and can be suitably used for the treatment of lower limb ischemia.

FIG. 1 is a graph showing evaluation results of cell carrying power in a tissue per unit surface area of particles based on a composite particle material and an average particle diameter. In the figure, P <0.05 indicates a significant difference by ANOVA Scheffe test. FIG. 2 is a graph showing the relationship between the GFP concentration (that is, the amount of cells carried by the composite particles) and the blood flow improvement degree (blood flow ratio). In the figure, the GFP concentration indicates the GFP concentration in the unit muscle tissue and is represented by an Arbitrary Unit. FIG. 3 is a graph showing the blood flow improvement degree (blood flow ratio) due to the difference in the material of the composite particles. In the figure, P <0.05 indicates a significant difference by ANOVA Scheffe test.

1. Composite Particle The composite particle of the present invention is a composite particle obtained by adsorbing inorganic particles on the particle surface made of an L-lactide-ε-caprolactone copolymer, and the composition of L-lactide and ε-caprolactone in the copolymer The molar ratio is approximately 75:25. Hereinafter, the composite particles of the present invention will be described in detail.

  In the present invention, as a polymer constituting the core particle in the composite particle, an L-lactide-ε-caprolactone copolymer (hereinafter referred to as “PLCL (75) having an L-lactide: ε-caprolactone (molar ratio) of about 75:25”. / 25) ”may be abbreviated as“). By using PLCL (75/25) as a core particle for adsorbing inorganic particles, it is possible to make the surface adsorbing action excellent on inorganic particles. In addition, PLCL (75/25) can retain cells for a period of time until the effect of cell transplantation is expressed when the cells are transplanted while carrying cells, and is further decomposed and absorbed in vivo. Physical properties suitable as a cell scaffold can be realized.

  PLCL (75/25) used in the present invention is obtained by ring-opening polymerization of L-lactide (a cyclic dimer of L-lactic acid) and ε-caprolactone as monomers at a predetermined molar ratio. The weight average molecular weight of the L-lactide-ε-caprolactone copolymer used in the present invention is not particularly limited, but is, for example, 20,000 to 1,000,000, preferably 300,000 to 900,000, more preferably 500,000 to 800,000 can be mentioned. Here, the weight average molecular weight is a value calculated by GPC analysis.

  In the composite particles of the present invention, the content ratio of PLCL (75/25) is not particularly limited, but is, for example, 50% by mass or more, preferably 70% by mass or more, more preferably 80%, based on the total weight of the composite particles. The mass% or more, More preferably, 80-99.9 mass% is mentioned.

  In the composite particle of the present invention, each particle constituted by PLCL (75/25) may have either a hollow structure or a non-hollow structure, and preferably a non-hollow structure.

  The composite particles of the present invention have a structure in which inorganic particles are adsorbed on the surface of particles made of PLCL (75/25).

  The type of the inorganic compound constituting the inorganic particles used in the present invention is not particularly limited, but for example, calcium phosphate, silica, titania, alumina, zirconia, iron oxide, tin oxide, zinc oxide, gold, silver, palladium, Platinum, carbon black, calcium carbonate, clay (for example, montmorillonite), etc. are mentioned. These inorganic compounds may be used individually by 1 type, and may be used in combination of 2 or more type. Among these inorganic compounds, calcium phosphate is preferable.

Specific examples of calcium phosphate include hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ), calcium metaphosphate (Ca (PO ₃ ) ₂ ), Examples thereof include fluorapatite (Ca ₁₀ (PO ₄ ) ₆ F ₂ ) and chloroapatite (Ca ₁₀ (PO ₄ ) ₆ Cl ₂ ). These calcium phosphates may be used individually by 1 type, and may be used in combination of 2 or more type. Among these calcium phosphates, hydroxyapatite is preferably used from the viewpoint of excellent adsorption to PLCL (75/25), biocompatibility, retention / proliferation of transplanted cells, and the like.

  These inorganic compounds are preferably sintered products that have been subjected to a sintering treatment. By using a sintered product of the above inorganic compound, it becomes possible to provide heat resistance and chemical stability.

  The average particle size of the inorganic particles used in the present invention is appropriately set according to the type of inorganic compound constituting the inorganic particles, the use of the composite particles of the present invention, etc., but is adsorbed to PLCL (75/25). From the viewpoint of improving the property, 10 to 1000 nm, preferably 10 to 800 nm, and more preferably 15 to 500 nm can be mentioned. The said average particle diameter is a value calculated | required by measuring the particle diameter of 50 or more inorganic particles using a scanning electron microscope, and calculating an average value. Inorganic particles having such an average particle diameter can be obtained by a production method such as a wet method, a hydrothermal method, a thermal decomposition method, a sol-gel method, or an alkoxide method.

  In the composite particles of the present invention, the content ratio of the inorganic particles is not particularly limited. For example, 0.001 to 50% by mass, preferably 0.01 to 30% by mass, and more preferably, per total weight of the composite particles. Is 0.1 to 20% by mass.

  About the average particle diameter of the composite particle of this invention, although suitably set according to the use etc. of the said composite particle, 1-1,000 micrometers, Preferably it is 5-500 micrometers, More preferably, 25-150 micrometers is mentioned. An average particle diameter is a value calculated | required by measuring the particle diameter of 50 or more composite particles of this invention using an optical microscope, and calculating an average value. Moreover, as a particle size distribution in the composite particle | grains of this invention, the ratio of the particle | grains of 75 micrometers or more and less than 300 micrometers with respect to all the particles is 5-100%, Preferably 10-100% is mentioned. These particle size distributions are measured using a scanning electron microscope.

  In the composite particles of the present invention, the average particle diameter of the core particles made of PLCL (75/25) and the thickness of the surface layer made of inorganic particles are not particularly limited, and the average particle diameter of the composite particles, PLCL (75/25). 25), the content of inorganic particles, and the like.

2. Production Method The production method of the composite particles of the present invention is not particularly limited, and inorganic particles may be adsorbed on particles made of PLCL (75/25) according to a conventionally known method. A preferred example of the method for producing composite particles of the present invention includes a method including the following first step and second step.

First Step In the first step, a mixed solution containing a hydrophobic solution in which the PLCL (75/25) is dissolved in a hydrophobic solvent, inorganic particles, and a hydrophilic solution is stirred using a homogenizer. A pickering emulsion is formed.

  In the first step, the hydrophobic solvent used in the hydrophobic solution is not particularly limited as long as it can dissolve PLCL (75/25). For example, monochloromethane, dichloromethane, trichloromethane, tetrachloromethane, Examples thereof include trifluoroacetic acid, halogenated hydrocarbons such as 1,1,1,3,3,3-hexafluoro-2-propanol, and toluene. These hydrophobic solvents may be used individually by 1 type, and may be used in combination of 2 or more type. Among these, halogenated hydrocarbons are preferable, and dichloromethane is more preferable.

  The viscosity of the hydrophobic solution used in the first step can affect the average particle size of the resulting composite particles. In order to adjust the average particle diameter of the composite particles to a desired range by the stirring speed described later, the blending ratio of PLCL (75/25) mixed with the hydrophobic solvent is adjusted to a predetermined range to adjust the hydrophobic solution. It is desirable to set the viscosity to about 700 mPa · S or less. Here, the viscosity is measured at room temperature (about 20) ° C. using dichloromethane as a solvent with a rotational vibration viscometer (specifically, Viscomate VM-10A-L: manufactured by CBC Materials Co., Ltd.). It is a value obtained by measuring with. Further, at the time of measurement, it is desirable to measure immediately after the probe of the viscometer is immersed in the hydrophobic solution in order to avoid the fluctuation of the viscosity due to the volatilization of the solvent. The content ratio of PLCL (75/25) for adjusting the hydrophobic solution to such a viscosity is, for example, 0.01 to 30% by mass, preferably 2 to 20% by mass, and more preferably 4 to 10% by mass. Is mentioned.

  The hydrophilic solution used in the first step is not particularly limited as long as it is not compatible with the hydrophobic solution, and examples thereof include water, methanol, ethanol and the like. These hydrophilic solutions may be used individually by 1 type, and may be used in combination of 2 or more type. Among these, water is preferable.

  In the first step, first, the hydrophobic solution, the inorganic particles, and the hydrophilic solution are mixed to prepare a mixed solution. In the mixed solution, the content of the hydrophobic solution, inorganic particles, and hydrophilic solution is not particularly limited. For example, the hydrophobic solution is 1 to 20% by mass, preferably 5 to 15% by mass, Preferably 8 to 12% by mass; the inorganic particles are 0.05 to 2% by mass, preferably 0.1 to 1% by mass, more preferably 0.2 to 0.5% by mass; and the hydrophilic solution is 78% by mass. What is necessary is just to set so that -98.95 mass%, Preferably it is 84-94.9 mass%, More preferably, 87.5-91.8 mass% is satisfied.

  Further, in the preparation of the mixed solution, the order of addition of the hydrophobic solution, the inorganic particles, and the hydrophilic solution is not particularly limited, but from the viewpoint of more uniformly adsorbing the inorganic particles on the surface of the composite particles, After mixing the particles and the hydrophilic solution, it is desirable to add the hydrophobic solution thereto. Moreover, when mixing the said inorganic particle and a hydrophilic solution, it is desirable to use for ultrasonic processing etc. as needed to suppress aggregation of an inorganic particle.

  In the first step, a pickering emulsion is formed by stirring the mixed solution using a homogenizer. The stirring speed by the homogenizer is preferably 4.5 to 8 m / s, more preferably 4.5 to 7 m / s. Moreover, as stirring time by a homogenizer, 0.1 to 30 minutes, for example, Preferably it is 0.5 to 25, More preferably, 1 to 20 minutes is mentioned. By adjusting the stirring speed within the above range, the average particle diameter of the obtained composite particles can be adjusted. For example, when the average particle diameter of the composite particles is 50 μm, the stirring speed may be 3 to 10 m / s, preferably 3.5 to 8 m / s, more preferably 4.5 to 7 m / s, and the stirring time. Examples include 10 to 30 minutes, preferably 15 to 25 minutes. For example, when the average particle diameter of the composite particles is 100 μm, the stirring speed is 2 to 9 m / s, preferably 2.5 to 7 m / s, more preferably 3.5 to 6 m / s. Examples of the time include 5 to 20 minutes, preferably 5 to 15 minutes. Stirring may be performed in multiple stages, preferably in two stages, depending on the particle size of the composite particles obtained. For example, when the particles obtained after the first stirring are larger than the target average particle size, the particles may be adjusted so as to have a desired average particle size by stirring at a higher speed.

  By performing the stirring treatment as described above, a Pickering emulsion is formed in which the composite particles in which the PLCL (75/25) particle surfaces are coated with inorganic fine particles are dispersed in a hydrophilic solution.

Second Step In the second step, the hydrophobic particles are removed from the pickering emulsion formed in the first step, and the composite particles are recovered.

  The method for removing the hydrophobic solvent from the pickering emulsion is not particularly limited, depending on the type of the hydrophobic solvent to be used, a method of evaporating at normal temperature and normal pressure, a method of distilling the dispersant by vacuum distillation, What is necessary is just to set suitably from the methods etc. which heat-dry.

  If a halogen-based hydrocarbon is used as the hydrophobic solvent, a suitable example of the method for removing the dispersion medium in the second step is halogen at a rotation speed of 1 to 50 rpm at room temperature using a pot mill rotary table. The method of volatilizing a system hydrocarbon is mentioned.

  In this second step, the hydrophobic solvent forming the core layer of the composite particle is removed, and then the hydrophilic solution is separated by centrifugation, distillation, etc., to obtain the target composite particle. . In the second step, the hydrophilic solution may be removed simultaneously with the removal of the hydrophobic solvent.

  The composite particles thus recovered may be subjected to further cleaning treatment such as ultrasonic treatment and washing treatment with a washing liquid such as ethanol or water. Furthermore, the composite particle adjusted to the desired particle diameter can be obtained by fractionating by sieving etc. as needed. Although sieving can be performed by a conventionally known method and is not limited, it can be performed by using a vibrating sieve equipped with a sieve screen having a mesh for dividing a desired particle diameter.

  For example, in the case of obtaining composite particles having an average particle size of 50 μm, a sieve mesh having a mesh size of 10 to 90 μm, preferably 20 to 90 μm, more preferably 25 to 75 μm, according to JIS Z8801-1 (specifically, THE. It can be fractionated by using IIDA TESTING SIEVE (exemplified by IIDA MANUFACTURING CO. LTD.). More specifically, for example, particles that pass through a 75 μm sieve screen and do not pass through a 25 μm sieve screen can be obtained as composite particles having an average particle diameter of 50 μm. Further, when obtaining composite particles having an average particle diameter of 100 μm, fractionation can be performed by using a sieve screen having an opening of 20 to 180 μm, preferably 53 to 180 μm, more preferably 75 to 150 μm. More specifically, for example, particles that pass through a sieve screen having a 150 μm mesh and do not pass through a sieve mesh having a 75 μm mesh can be obtained as composite particles having an average particle diameter of 100 μm.

3. Cell preparation The composite particles of the present invention adhere to cultured cells or cells or tissues in the living body and are used as a scaffold for cell proliferation, and therefore are suitably used as medical materials. Specific examples of the medical material include a cell culture carrier, a bone supplement, a soft tissue supplement, and a cell carrier. In particular, the composite particles of the present invention are suitable as a scaffold for cell transplantation (a carrier for supporting transplanted cells) when cells are transplanted by regenerative medicine or the like. That is, the present invention further provides a cell preparation containing cell-supporting composite particles obtained by supporting cells on the composite particles.

  The type of cells to be carried on the composite particles of the present invention is not particularly limited. For example, ES cells, iPS cells, various mesenchymal stem cells, bone marrow mononuclear cells, peripheral blood mononuclear cells, vascular endothelial progenitor cells, platelets Vascular endothelial cells, smooth muscle cells, mesothelial cells, skeletal myoblasts, osteoblasts, preosteoblasts, fibroblasts, adipocytes, nerve cells and the like. ES cells, iPS cells, and mesenchymal stem cells may be differentiated. Among these cells, mononuclear cells are preferable, and bone marrow mononuclear cells are more preferable. By supporting bone marrow mononuclear cells on the composite particles of the present invention and using them as a cell preparation, angiogenesis can be remarkably amplified and angiogenesis therapy by more effective cell transplantation becomes possible. . That is, a cell preparation in which bone marrow mononuclear cells are supported on the composite particles of the present invention can be suitably used for promoting angiogenesis. Conventionally, lower limb ischemia in which ulcers and necrosis occur due to arteriosclerosis of the lower limb blood vessels has been a risk of lower limb amputation. By transplanting around, it promotes angiogenesis in the affected area and enables effective treatment of lower limb ischemia.

  The number of cells to be carried on the composite particles of the present invention is appropriately set according to the type and use of the cells to be carried. For example, the number of cells to be carried is 10 to 100,000 per composite particle, Preferably 100-10,000 pieces, More preferably, 500-5,000 pieces are mentioned.

  The method of supporting the cells on the composite particles of the present invention is not particularly limited, and examples thereof include a method of mixing the composite particles and the cells to be supported in an appropriate liquid. The liquid used for mixing such composite particles and cells can be appropriately prepared according to the type of cells to be supported, but is not limited to a medium such as DMEM, physiological saline, phosphate buffer solution, plasma (heparinized). Plasma, sodium citrate plasma and the like). Since the cell preparation of the present invention is mainly used for transplantation into adults, among these liquids, preferably a medium, physiological saline, phosphate buffer solution, plasma and the like are mentioned, more preferably plasma. Is mentioned.

  In addition, as another method of supporting the cells on the composite particles of the present invention, for example, a method of culturing cells to be supported in the presence of the composite particles and adhering them through the interaction between the composite particles and the cells. Can be mentioned.

In addition, since the composite particles of the present invention are excellent in cell adhesion, cells can be sufficiently retained in vivo without incubation before transplantation. However, incubation may be performed as necessary from the time the cells are carried on the composite particle to the time of transplantation. When incubation is performed, the incubation time and conditions are not particularly limited as long as the cells are supported on the composite particles, and preferably include about 1 to 3 hours at 37 ° C. under 5% CO ₂ conditions.

  The cell-supporting composite particles in which cells are supported on the composite particles of the present invention may be mixed with a pharmaceutically acceptable carrier and used as a cell preparation, if necessary. Examples of such carriers include physiological saline, phosphate buffer solution, plasma and the like. About the administration method etc. of the said cell formulation, it sets suitably according to the kind of cell to be used, the kind of disease etc. which are applied.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited to the following embodiment. For use in test examples described later, composite particles of Example 1 and Comparative Examples 1 to 3 below were prepared.

Example 1: HAp-PLCL (75/25) composite particles Preparation of Low Crystalline Hydroxyapatite Particles Low crystalline hydroxyapatite particles having a spherical shape were prepared by the wet method shown below. In addition, Ca (NO ₃ ) ₂ .4H ₂ O and (NH ₄ ) ₂ HPO ₄ are manufactured by Nacalai Tesque Co., Ltd., and 25% ammonia water is manufactured by Wako Pure Chemical Industries, Ltd. Milli-Q water was used as water.

First, a Ca (NO ₃ ) ₂ aqueous solution (42 mN, 80 mL) whose pH was adjusted to 12 with 25% aqueous ammonia was poured into a 1 L flask connected with a condenser and a semicircular stirring blade, and kept at room temperature. (NH ₄ ) ₂ HPO ₄ aqueous solution (100 mN, 200 mL) adjusted to pH 12 with aqueous ammonia was added to this flask at room temperature and allowed to react for 10 hours. Next, the obtained reaction product was separated and washed by centrifugation to obtain low crystalline hydroxyapatite particles.

2. Preparation of highly crystalline hydroxyapatite particles Highly crystalline hydroxyapatite nanoparticles were produced by firing the low crystalline hydroxyapatite particles obtained above by the following method.

First, as an anti-fusing agent, 0.5 g of polyacrylic acid (manufactured by ALDRICH, weight average molecular weight 15,000 g / mol), pH 7.0 aqueous solution (hereinafter referred to as Aqueous Solution A) 100 ml, By dispersing crystalline hydroxyapatite nanoparticles, polyacrylic acid was adsorbed on the surface of the particles. Next, 500 ml of a saturated aqueous solution of calcium hydroxide [Ca (OH) ₂ ] was added to the dispersion prepared above to precipitate calcium polyacrylate on the particle surface. The resulting precipitate was collected and dried at 80 ° C. under reduced pressure to obtain mixed particles.

  The mixed particles were put in a crucible and sintered at a sintering temperature of 800 ° C. for 1 hour. At this time, calcium polyacrylate was thermally decomposed to calcium oxide [CaO].

  Next, the anti-fusing agent is obtained by suspending the obtained sintered body in 500 ml of the aqueous solution A prepared above, separating and washing by centrifugation, further suspending in distilled water, and similarly performing separation and washing by centrifugation. Then, ammonium nitrate was removed, and highly crystalline hydroxyapatite nanoparticles were recovered.

  As a result of observing the obtained highly crystalline hydroxyapatite nanoparticles with a scanning electron microscope and measuring the average particle size, the average particle size was 48 nm. The scanning electron microscope was observed at a magnification of 90,000 using a model name JSM-6301F manufactured by JEOL Ltd.

3. Preparation of Composite Particles Composite particles having hydroxyapatite particles adsorbed on the surface of particles made of L-lactide-ε-caprolactone copolymer (PLCL) were prepared according to the following method.
In the L-lactide-ε-caprolactone copolymer, lactide and ε-caprolactone (molar ratio 75:25) are placed in a glass reaction tube, and 300 ppm of tin octylate (tin metal conversion: 87 ppm) is added thereto, under a nitrogen atmosphere. To obtain a copolymer having a weight average molecular weight of 780,000. The obtained copolymer was pulverized by a pulverizer to obtain L-lactide-ε-caprolactone copolymer particles (average particle diameter: 3.0 mm) serving as core particles. Hereinafter, the particles of the L-lactide-ε-caprolactone copolymer may be abbreviated as “PLCL (75/25)”.

(Preparation of composite particles having an average particle diameter of 50 μm)
1 g of the highly crystalline hydroxyapatite particles obtained above was dispersed in 500 mL of pure water (Milli-Q water) and subjected to ultrasonic treatment for 1 minute. To this was added 280 g of a dichloromethane solution in which 15 g of PLCL (75/25) was dissolved in 265 g (200 mL) of dichloromethane, and the stirring speed was 6 with a homogenizer (Hiscotron NS-56 manufactured by Microtech Nichion; generator shaft NS-20). A pickering emulsion was formed by stirring for 20 minutes at 28 m / s. Next, while the obtained pickering emulsion was rotated for 72 hours at room temperature (about 20 ° C.) at a speed of 4.5 rpm on a pot mill rotary elevated platform (ANZ-51S; manufactured by Nichito Kagaku Co., Ltd.), dichloromethane was added. Removal was performed to obtain a dispersion of composite particles.

  Next, the obtained dispersion of composite particles was subjected to an ethanol washing treatment. First, the dispersion liquid of each composite particle was centrifuged (300 rpm, 2 minutes), and then the supernatant was removed to collect a precipitate (composite particle). The recovered precipitate was added to ethanol and subjected to ultrasonic treatment for 30 seconds, and then the precipitate (composite particles) was recovered by centrifugation (300 rpm, 2 minutes). The obtained precipitate was repeatedly washed with ethanol four times and then dried to obtain composite particles. Composite particles in which hydroxyapatite particles are adsorbed on the surface of L-lactide-ε-caprolactone copolymer particles (PLCL (75/25)) may be abbreviated as “HAp-PLCL (75/25)”.

  Thereafter, composite particles having an average particle diameter of 50 μm were fractionated by sieving using THE IIDA TESTING SIEVE (IIDA MANUFACTURING CO. LTD. (JIS Z8801-1)). The composite particles having an average particle size of 50 μm were obtained by collecting particles obtained by passing through a sieve having an opening of 75 μm and not passing through a sieve having an opening of 25 μm. The yield of composite particles having an average particle size of 50 μm thus obtained was 48% with respect to the charged amount (total amount of high crystalline hydroxyapatite and PLCL used: 16 g).

(Preparation of composite particles having an average particle size of 100 μm)
1 g of the highly crystalline hydroxyapatite particles obtained above was dispersed in 520 mL of pure water (Milli-Q water) and subjected to ultrasonic treatment for 1 minute. To this was added 254 g of a dichloromethane solution in which 15 g of PLCL (75/25) was dissolved in 239 g (180 mL) of dichloromethane, and the stirring speed was 4 with a homogenizer (Hiscotron NS-56 manufactured by Microtech Nichion; generator shaft NS-20). The mixture was stirred for 10 minutes at 19 m / s, and further stirred with a homogenizer (Hiscotron NS-56 manufactured by Microtech Nichion; generator shaft NS-20) at a stirring speed of 6.28 m / s for 10 minutes. Formed. Next, while the obtained pickering emulsion was rotated for 72 hours at room temperature (about 20 ° C.) at a speed of 4.5 rpm on a pot mill rotary elevated platform (ANZ-51S; manufactured by Nichito Kagaku Co., Ltd.), dichloromethane was added. Removal was performed to obtain a dispersion of composite particles.

  Subsequently, the obtained dispersion liquid of composite particles was subjected to the same ethanol washing treatment and sieving as the preparation of composite particles having an average particle diameter of 50 μm. Sifting of composite particles having an average particle diameter of 100 μm was performed by collecting particles that passed through a sieve having an opening of 150 μm but did not pass through a sieve having an opening of 75 μm. The yield of composite particles having an average particle diameter of 100 μm thus obtained was 73% with respect to the charged amount (total amount of high crystalline hydroxyapatite and PLCL used: 16 g).

Comparative Example 1: Composite particles of hydroxyapatite and poly-L-lactide (PLLA) 1 g of the highly crystalline hydroxyapatite particles obtained above was dispersed in 500 mL of pure water (Milli-Q Water) and sonicated for 1 minute. Went. To this, 14 g of a dichloromethane solution in which 1 g of poly-L-lactide (PLLA: weight average molecular weight = 100,000) was dissolved in 13 g of dichloromethane (about 8.8 mL) was added, and a homogenizer (Dispergerated T10 basic (manufactured by IKA) was added. The pickering emulsion was formed by stirring for 3 minutes at a peripheral speed of 6.54 m / s. Next, the mixture was stirred overnight using a magnetic stirrer, and dichloromethane was removed from the resulting pickering emulsion to obtain a dispersion of composite particles.

  The obtained dispersion is subjected to the ethanol washing treatment described in Example 1, and then sieved according to the same method as in Example 1 (preparation of composite particles having an average particle diameter of 100 μm) to obtain an average particle diameter. 100 μm composite particles were obtained. Hereinafter, the composite particles of hydroxyapatite and poly-L-lactide may be abbreviated as “HAp-PLLA”.

Comparative Example 2: Composite particles of hydroxyapatite and L-lactide-ε-caprolactone copolymer (50/50) 1.5 g of the highly crystalline hydroxyapatite particles obtained above were added to 800 mL of pure water (Milli-Q Water). And was sonicated for 1 minute. 8 g of L-lactide-ε-caprolactone copolymer (50/50) (L-lactide: caprolactone molar ratio 50:50, weight average molecular weight 280,000) was dissolved in 92 g of dichloromethane (about 62 mL). Pickering emulsion was formed by adding 100 g of dichloromethane solution and stirring with a homogenizer (Hiscotron NS-56 (Generator Shaft NS-20) manufactured by Microtech Nichion) at a stirring speed of 2.93 m / s for 3 minutes. . Next, the bottle was fixed on a shaker and shaken overnight, whereby dichloromethane was removed from the obtained pickering emulsion to obtain a dispersion of composite particles.

  The obtained dispersion was subjected to the ethanol washing treatment described in Example 1, and then fractionated with an average particle size of 50 μm or 100 μm by sieving according to the same method as in Example 1, Composite particles having a particle size were obtained. Hereinafter, the composite particles of hydroxyapatite and L-lactide-ε-caprolactone copolymer (50/50) may be abbreviated as “HAp-PLCL (50/50)”.

Comparative Example 3: Composite particles of hydroxyapatite and L-lactide / glycolide copolymer (90/10) 3.5 g of the highly crystalline hydroxyapatite particles obtained above were dispersed in 700 mL of pure water (Milli-Q Water). And sonicated for 1 minute. To this, 24 g of L-lactide / glycolide copolymer (90/10) (L-lactide: glycolide molar ratio 90:10, weight average molecular weight = 150,000) was dissolved in 176 g of dichloromethane (about 119 mL). A pickering emulsion was formed by adding 200 g of the solution and stirring with a homogenizer (Hiscotron NS-56 (Generator Shaft NS-20) manufactured by Microtech Nichion) at a stirring speed of 4.19 m / s for 3 minutes. Next, dichloromethane was removed from the obtained pickering emulsion while rotating at a room temperature (about 20 ° C.) for 72 hours at a speed of 37 rpm on a pot mill rotary elevated platform (ANZ-51S; manufactured by Nissho Science Co., Ltd.) A dispersion of composite particles was obtained.

  The obtained dispersion was subjected to the ethanol washing treatment described in Example 1, and then fractionated with an average particle size of 50 μm or 100 μm by sieving according to the same method as in Example 1, Composite particles having a particle size were obtained. The yield of the composite particles having an average particle diameter of 50 μm thus obtained was 18.2% with respect to the charged amount (total amount of high crystalline hydroxyapatite and PLGA used: 27.5 g). The yield of 100 μm composite particles was 53.5% with respect to the charged amount. Hereinafter, the composite particles of hydroxyapatite and L-lactide / glycolide copolymer (90/10) may be abbreviated as “HAp-PLGA (90/10)”.

[Test Example 1]
Under anesthesia (pentobarbital 50 μg / body weight (g), intraperitoneal administration), the left femoral artery and its branch of C57BL6-Cr-Slc (male, 8 weeks old: obtained from Japan SLC Co., Ltd.) were ligated and excised. A mouse model of lower limb ischemia was prepared.

GFP-producing bone marrow mononuclear cells (BMC) derived from green mice (EGFP-transgenic mice) were used to evaluate the cell carrying capacity of the composite particles. GFP producing bone marrow mononuclear cells were prepared as follows. That is, after killing the EGFP transgenic mouse by cervical dislocation, bilateral femur and tibia were excised and bone marrow fluid was extracted. The obtained bone marrow fluid was treated with Ficoll solution (Ficoll-Paque Plus: (GE Healthcare AB (Sweden)), and only bone marrow mononuclear cells were separated and collected. It was suspended in serum (−)) and adjusted to 1 × 10 ⁶ cells / 100 μl (incubation time was 0 hour).

  The cell suspension (100 μl) obtained above was mixed with each composite particle (each 1.5 mg) of Example 1 and Comparative Examples 1 to 3, thereby supporting GFP-producing bone marrow mononuclear cells. Composite particles were obtained. In addition, as a control, the cell suspension (100 μl) obtained above was prepared from particles (average particle size: 100 μm) of poly-L-lactide (PLLA) (PLA-Particles; manufactured by Micromod Partechtechnologies) only (1. 5 mg) to obtain PLLA particles carrying GFP-producing bone marrow mononuclear cells.

  Composite particles carrying GFP-producing bone marrow mononuclear cells were injected intramuscularly (using a 24G needle) at four locations in the mouse ischemic limb. On day 7 after transplantation, the mice were sacrificed by cervical dislocation, and the thigh muscles were collected to prepare a muscle lysate. The preparation of thigh muscle lysate is described in Circulation. 2004; 109: 2454-2461.

  The GFP concentration in the lysate was measured using a GFP ELISA kit (manufactured by Cell Biolabs (San Diego, Calif.)). In addition, the total protein concentration in the lysate was measured by Bicinchonic (BCA) Protein Assay method. A value obtained by dividing the GFP concentration in each lysate by the total protein concentration was calculated as the GFP concentration in the unit muscle tissue. Furthermore, based on the measurement results, the GFP concentration (Arbitrary Unit) per unit surface area of the composite particles was calculated, and this was compared as the bone marrow mononuclear cell carrying ability of the composite particles in muscle tissue. The results are shown in FIG.

  As is clear from FIG. 1, when Comparative Example 2 (HAp-PLCL (50/50); average particle size 100 μm) was used, Comparative Example 1 (composite particles in which hydroxyapatite was coated on PLLA particles: average particles) The cell carrying power was about the same as the diameter of 100 μm. On the other hand, when the composite particles of Example 1 (HAp-PLCL (75/25)) were used, the cell carrying power in the tissue was dramatically improved. In addition, the composite particles of Example 1 showed a significantly high cell carrying capacity in the tissue even when compared with the HAp-PLGA (90/10) of Comparative Example 3.

  That is, composite particles obtained by adsorbing hydroxyapatite to an L-lactide-ε-caprolactone copolymer having a molar ratio of L-lactide and ε-caprolactone of 75:25 may have an extremely high cell carrying ability. Indicated.

[Test Example 2]
By mixing a cell suspension (100 μl) of GFP-producing bone marrow mononuclear cells prepared by the same method as in Test Example 1 with each composite particle (1.5 mg each) of Example 1 and Comparative Example 2 above, Composite particles carrying GFP-producing bone marrow mononuclear cells were obtained.

  Composite particles carrying GFP-producing bone marrow mononuclear cells were injected intramuscularly into lower limb ischemic mice. Injection (transplantation) The blood flow in the lower limb on the 14th day was measured by the blood flow Doppler method (Omega Flow FLO-N1: manufactured by OMEGA WAVE), and the blood flow in the ischemic limb (left lower limb) ) Divided by the blood flow volume was taken as the blood flow improvement degree (blood flow ratio) (FIG. 2). Moreover, when comparing the blood flow improvement degree of each group, the blood flow improvement degree (blood flow ratio) of each group is set with 1 as the blood flow improvement degree of the lower limb of the mouse treated (injected with 100 μl of DMEM serum (−)). ) As an Arbitrary Unit (FIG. 3). The lower limb ischemia mouse used in this test is the same as that used in Test Example 1. The GFP concentration in each unit muscle tissue was measured by the same method as in Test Example 1 after measuring the blood flow of the lower limbs. The obtained results are shown in FIGS.

  FIG. 2 is a graph showing the relationship between the GFP concentration in the unit muscle tissue and the blood flow ratio when GFP-producing bone marrow mononuclear cells are carried on HAp-PLCL (75/25) and injected into an ischemic limb. FIG. 2 shows that the higher the GFP concentration, the higher the blood flow improvement degree. That is, it was shown that the higher the cell carrying power, the higher the blood flow improvement degree.

  FIG. 3 shows that the improvement in blood flow varies depending on the material used for the composite particles (PLCL (75/25) or PLCL (50/50)). That is, when the composite particles of Example 1 having a high cell-carrying power are used, the blood flow is remarkable as compared with the case of using the composite particles of the vehicle treatment and Comparative Example 2 (HAp-PLCL (50/50)). The improvement effect was shown. Accordingly, it was shown that the cell preparation in which bone marrow mononuclear cells are supported on the composite particles of Example 1 can provide a high blood flow improvement effect in the ischemic leg.

[Test Example 3]
Using the composite particles of Example 1 and Comparative Example 3 described above, the hydroxyapatite desorption rate was evaluated. Specifically, the composite particles of Example 1 or Comparative Example 3 were immersed in a phosphate buffer solution (PBS) at 37 ° C., and the surface morphology change from immediately after immersion (0 weeks) to 24 weeks was observed. . Observation was performed using a 3D real surface view microscope (manufactured by Keyence Corporation) at a magnification of 10,000 times. The printed material of 3D real surface view photograph is divided into a hydroxyapatite coated part and an uncoated part, and the hydroxyapatite coated particles cover the PLCL particle surface by calculating the weight percentage of the hydroxyapatite coated part relative to the total weight of the printed material. The percentage was determined. The results are shown in Table 1 below.

  As shown in Table 1, when PLCL (75/25) was used as particles to coat hydroxyapatite, it maintained a hydroxyapatite coverage of 48% even after being immersed in PBS at 37 ° C. for 24 weeks. . In contrast, when PLGA (90/10) particles were coated with hydroxyapatite, the coverage decreased significantly to 48% after 4 weeks of immersion and 7% after 24 weeks.

  Since hydroxyapatite is useful for cell adhesion, the slow desorption rate of hydroxyapatite in vivo keeps the cells stable on the composite particles until the effect of transplanted cells is obtained. It is thought that it is effective to do. That is, from this test result, it was shown that PLCL (75/25) is suitable as a particle to coat hydroxyapatite.

[Summary]
By supporting cells on composite particles in which inorganic particles are adsorbed on particles made of L-lactide-ε-caprolactone copolymer (L-lactide: ε-caprolactone constituent molar ratio: 75/25), In the body, the cells can remain on the composite particles for a period sufficient for tissue regeneration, and the efficiency of tissue regeneration can be increased. In addition, as shown in the above results, bone marrow mononuclear cells were supported on composite particles in which hydroxyapatite was adsorbed on PLCL (75/25) particles, and transplanted to ischemic limb model mice. Blood flow improvement effect was obtained.

Claims (6)

Composite particles in which inorganic particles are adsorbed on the surface of particles made of an L-lactide-ε-caprolactone copolymer, and the constituent molar ratio of L-lactide and ε-caprolactone in the copolymer is approximately 75:25. The composite particle according to claim 1, wherein the inorganic particle is hydroxyapatite. The composite particle according to claim 1 or 2, which is used as a scaffold for transplanted cells. A cell preparation comprising cell-supporting composite particles obtained by supporting cells on the composite particles according to claim 1. The cell preparation according to claim 4, wherein the cell is a bone marrow mononuclear cell. The cell preparation according to claim 5, which is used for promoting angiogenesis.

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