Method for producing fine particles, and fine particles

Abstract

This method for producing fine particles includes: a step for forming a polyion complex by ion-bonding an ionic active material and an ionic polymer; and a step for forming fine particles which contain the polyion complex and at least one polymer (P) that is selected from among a polylactic acid and a lactic acid-glycolic acid copolymer. The step for forming fine particles includes: a step for mixing the polyion complex and the polymer (P) in an organic solvent; a step for forming an o / w emulsion by adding the organic solvent after mixing, the organic solvent containing the polyion complex and the polymer (P), to an aqueous solution in which a dispersant for preventing bonding between emulsions is dissolved; and a step for removing the organic solvent from the aqueous solution that contains the o / w emulsion.

Description

Method for producing microparticles, and microparticles

[0001] This application claims priority to Japanese Patent Application No. 2023-146876, filed on September 11, 2023, the contents of which are incorporated herein by reference.

[0002] Conventionally, a technique has been known in which active substances such as vaccine antigens and pharmaceutical ingredients are encapsulated in microparticles containing at least one of polylactic acid (hereinafter referred to as "PLA") and lactic acid-glycolic acid copolymer (hereinafter referred to as "PLGA").

[0003] For example, Patent Document 1 discloses a method for encapsulating a water-soluble (ionic) protein or peptide (active substance) in fine particles made of a polymer such as PLGA.

[0004] In the method described in Patent Document 1, an aqueous solution containing an ionic active substance is first mixed with an organic solvent containing PLGA to form a w / o (water-in-oil) emulsion, which is then mixed with water containing a dispersant to form a w / o / w (water-in-oil-in-water) emulsion (w / o / w double emulsion method).

[0005] Finally, the organic solvent is evaporated to obtain microparticles with a polymer such as PLGA on the outside and an active substance inside.

[0006] Special Publication No. 10-511957

[0007] However, the method described in Patent Document 1 uses a w / o / w double emulsion method that includes a step of forming a w / o emulsion and a step of forming a w / o / w emulsion, which poses the problem of time-consuming encapsulation of the active substance in PLGA.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing microparticles that can easily encapsulate ionic active substances in particles containing PLA and / or PLGA, and microparticles that can be produced by the method.

[0009] One aspect of the present invention includes the following aspects.

[0010] [1] A method for producing microparticles, comprising: a step of forming a polyion complex by ionically bonding an ionic active substance with an ionic polymer; and a step of forming microparticles containing the polyion complex and at least one polymer (P) selected from polylactic acid and a lactic acid-glycolic acid copolymer, wherein the step of forming the microparticles comprises a step of mixing the polyion complex and the polymer (P) in an organic solvent; a step of adding the organic solvent containing the mixed polyion complex and the polymer (P) to an aqueous solution in which a dispersant that prevents emulsions from bonding together is dissolved to form an o / w emulsion; and a step of removing the organic solvent from the aqueous solution containing the o / w emulsion.

[0011] [2] The method for producing microparticles according to [1], wherein the active substance is a protein having a molecular weight of 1,000 or more.

[0012] [3] The method for producing microparticles according to [1], wherein the active substance is a nucleic acid having a molecular weight of 1,000 or more.

[0013] [4] The method for producing microparticles according to [1] or [2], wherein the active substance is cationic and the ionic polymer is an anionic polymer.

[0014] [5] The method for producing microparticles according to [4], wherein the anionic polymer comprises at least one selected from the group consisting of polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyvinyl sulfate, polyvinyl phosphonic acid, and copolymers containing at least one of these, as well as polyglutamic acid, polyaspartic acid, alginic acid, hyaluronic acid, heparin, chondroitin sulfate, and dextran sulfate.

[0015] [6] The method for producing microparticles according to any one of [1] to [3], wherein the active substance is anionic and the ionic polymer is a cationic polymer.

[0016] [7] The method for producing microparticles according to [6], wherein the cationic polymer comprises at least one selected from the group consisting of polyvinylamine, polyallylamine, polyvinylpyridine, and a copolymer containing at least one of these, and polylysine, polyarginine, polyhistidine, polytryptophan, polyethyleneimine, and chitosan.

[0017] [8] Microparticles comprising at least one polymer (P) selected from polylactic acid and lactic acid-glycolic acid copolymer, and a polyion complex, wherein the polyion complex comprises an ionic active substance and an ionic polymer ionically bonded to the active substance.

[0018] According to the present invention, it is possible to provide a method for producing microparticles that can easily encapsulate ionic active substances in particles containing PLA and / or PLGA, and microparticles that can be produced by the method.

[0019] 1A and 1B are bright-field and fluorescent images showing microparticles produced under the conditions of System 13 (no polyethyleneimine used) shown in Table 2. 1C are bright-field and fluorescent images showing microparticles produced under the conditions of System 15 (polyethyleneimine used as a cationic polymer) shown in Table 2. 1D are graphs showing the loading rate of an active substance in microparticles produced under the conditions of System 13 or System 15. 1E are fluorescent images showing microparticles containing PLA and an active substance.

[0020] Preferred embodiments of the present invention will now be described in detail.

[0021] <Method for Producing Microparticles> The method for producing microparticles according to this embodiment includes a step of forming a polyion complex by ionic bonding between an ionic active substance and an ionic polymer (polyion complex formation step), and a step of forming microparticles containing the formed polyion complex and at least one polymer (P) selected from PLA (polylactic acid) and PLGA (lactic acid-co-glycolic acid copolymer).

[0022] The process of forming microparticles containing a polyion complex and the polymer (P) includes a step of mixing the polyion complex formed in the polyion complex formation step with the polymer (P) in an organic solvent (mixing step), a step of adding the organic solvent containing the mixed polyion complex and the polymer (P) to an aqueous solution in which a dispersant that prevents emulsions from bonding together has been dissolved to form an o / w emulsion (o / w emulsion formation step), and a step of removing the organic solvent from the aqueous solution containing the o / w emulsion (organic solvent removal step).

[0023] (Polyion Complex Formation Step) In the polyion complex formation step, an ionic active substance and an ionic polymer are ionic bonded to form a polyion complex, thereby canceling out the charges of the charged active substance and making it closer to hydrophobicity.

[0024] This makes it easier for the active substance to be dissolved in an organic solvent in the subsequent mixing step, and allows it to be easily encapsulated in PLA and / or PLGA (the polymer (P)).

[0025] As used herein, the term "active substance" encompasses chemical substances that induce physiological effects in living organisms, as well as substances that produce chemical effects or catalyze chemical reactions in non-living worlds.

[0026] The type of active substance is not particularly limited, but examples include substances introduced into the body, plant protection agents, cosmetic active agents, aroma chemicals, chemicals in the construction field, and the like.

[0027] Examples of substances introduced into the body include, but are not limited to, pharmaceutical ingredients such as anticancer drugs and anti-inflammatory drugs, antigens used in vaccines, and cosmetic ingredients. The active substance may be cationic or anionic as a whole. Examples of chemicals in the construction field include catalysts used in polymerization or crosslinking.

[0028] The active substance is a polymer, and may be, for example, a protein, a nucleic acid, or another substance having a charge. The molecular weight of the active substance may be 1,000 or more, 5,000 or more, or 10,000 or more. The upper limit of the molecular weight of the active substance is not particularly limited, but may be, for example, 100,000. Forming a polyion complex using an ionic polymer can suppress the degradation of the active substance by degradative enzymes such as nucleases and proteases compared to forming an ion pair using an ionic low molecular weight compound. In addition to the sustained release provided by PLA and / or PLGA, the polyion complex can suppress the initial burst and provide long-term sustained release. Additionally, while low molecular weight ions are prone to cytotoxicity, using an ionic polymer can improve safety.

[0029] Even such high molecular weight active substances can be easily encapsulated in PLA and / or PLGA by forming a polyion complex.

[0030] Furthermore, when a protein is used as an active substance, the formation of a polyion complex is expected to have the effect of suppressing denaturation caused by organic solvents.

[0031] When the active substance is cationic, the ionic polymer is an anionic polymer, and when the active substance is anionic, the ionic polymer is a cationic polymer.

[0032] When the active substance is an ampholyte such as a protein, both an anionic polymer and a cationic polymer may be used as the ionic polymer.

[0033] When both an anionic polymer and a cationic polymer are used, the anionic polymer and the cationic polymer can be ionically bonded to the active substance, respectively, to prevent the anionic polymer and the cationic polymer from bonding to each other.

[0034] When the microparticles are applied to a living body, the anionic polymer may be a substance with low toxicity to the living body. Examples of anionic polymers with relatively low toxicity to the living body include, but are not limited to, polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyvinyl sulfate, polyvinyl phosphonic acid, and copolymers containing at least one of these, as well as at least one selected from the group consisting of polyglutamic acid, polyaspartic acid, alginic acid, hyaluronic acid, heparin, chondroitin sulfate, and dextran sulfate.

[0035] The copolymer may contain one or more blocks of polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyvinyl sulfate, or polyvinyl phosphonic acid (for example, a block of polyacrylic acid and a block of polymethacrylic acid), and may also contain one or more blocks of other anionic homopolymers.

[0036] The anionic polymer may have both cationic groups and anionic groups, but must be anionic as a whole.

[0037] When the microparticles are applied to a living body, the cationic polymer may be a substance with low toxicity to the living body. Examples of cationic polymers with relatively low toxicity to the living body include, but are not limited to, polyvinylamine, polyallylamine, polyvinylpyridine, copolymers containing at least one of these, and at least one selected from the group consisting of polylysine, polyarginine, polyhistidine, polytryptophan, polyethyleneimine, and chitosan.

[0038] The copolymer contains one or more blocks of polyvinylamine, polyallylamine, or polyvinylpyridine (for example, a block of polyvinylamine and a block of polyallylamine), and may also contain one or more blocks of other cationic homopolymers.

[0039] Polycaprolactone having a cationic group bonded thereto can also be used as the cationic polymer. Examples of polycaprolactone having a cationic group bonded thereto include polycaprolactone having a structure represented by the following formula (3):

[0040]

[0041] The compound having the structure represented by the above formula (3) can be obtained by reacting a copolymer of caprolactone and halogenated caprolactone with N,N-dimethylethylamine, as shown in the following chemical reaction formula (4).

[0042] (In chemical reaction formula (4), X represents a halogen atom.)

[0043] The cationic polymer may have both cationic groups and anionic groups, but must be cationic as a whole.

[0044] The molecular weight of the ionic polymer is not particularly limited, but may be, for example, 1,000 or more, 5,000 or more, or 10,000 or more. The molecular weight of the ionic polymer may also be 100,000 or less.

[0045] The active substance and the ionic polymer are preferably mixed in an organic solvent to form a polyion complex. The organic solvent is not particularly limited as long as it is capable of dissolving the active substance and the ionic polymer. The organic solvent is preferably one that can solubilize the polyion complex and has a low boiling point or other ease of removal. Examples of such organic solvents include hexafluoroisopropanol (hereinafter referred to as "HFIP"), N-methyl-2-pyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide, with HFIP being particularly preferred. By subjecting the polyion complex to the mixing step in a state where it is dissolved in HFIP, the polyion complex can be made more soluble in the organic solvent. The solvent for forming the polyion complex is not limited to HFIP. Alternatively, the solvent for forming the polyion complex may be a mixture of an organic solvent and water. As will be described in detail in the examples below, adding water to the solvent can sometimes improve the solubility of the active substance.

[0046] The amount of ionic polymer is preferably set so that the total amount of the active substance used can form a polyion complex. For example, as will be described in detail later in the Examples, when a polyion complex is formed from an active substance labeled with a labeling substance such as FITC (fluorescein isothiocyanate) and an ionic polymer, and the polyion complex is encapsulated in particles containing PLA and / or PLGA, the particles emit fluorescence evenly and appear nearly perfectly spherical when observed under a fluorescence microscope.

[0047] When applying the microparticles to a living body, the ionic polymer may be bound to a molecule (so-called pilot molecule) such as an antibody to guide the active substance to a desired location (target site). Furthermore, the ionic polymer may be bound to a compound that enhances hydrophobicity, such as a fatty acid. By binding a compound that enhances hydrophobicity to the ionic polymer, the hydrophobicity of the polyion complex can be adjusted.

[0048] (Mixing Step) In the mixing step, the polyion complex formed in the polyion complex forming step is mixed with PLA and / or PLGA in an organic solvent.

[0049] In the mixing step of this embodiment, an organic solvent such as HFIP containing a polyion complex is mixed with an organic solvent in which PLA and / or PLGA has been dissolved. However, PLA and / or PLGA in the form of powder or the like may also be mixed with the organic solvent containing the polyion complex.

[0050] The type of organic solvent for dissolving PLA and / or PLGA is not particularly limited, but a volatile organic solvent is preferred. Examples of such organic solvents include ethyl acetate, dichloromethane, and chloroform. Among these organic solvents, ethyl acetate is particularly preferred because of its low toxicity to living organisms.

[0051] PLA is a polymer of lactic acid as represented by the following formula (1), in which l is an integer of 10 or more and 10,000 or less, and an asterisk (*) represents an adjacent repeating unit or any other group.

[0052] PLGA is a copolymer of lactic acid and glycolic acid, and contains m units derived from lactic acid and n units derived from glycolic acid, as shown in the following formula (2): In formula (2), an asterisk (*) represents an adjacent repeating unit or any other group.

[0053]

[0054] In formula (2), m and n are integers of 1 or more and 10,000 or less, and the number of lactic acid molecules polymerized is equal to the number m, and the number of glycolic acid molecules polymerized is equal to the number n.

[0055] The molar ratio (m:n) of the constitutional units derived from lactic acid to the constitutional units derived from glycolic acid in PLGA may be 1:10 to 10:1, 1:8 to 8:1, 1:5 to 5:1, 1:4 to 4:1, 1:2 to 2:1, 1.5:1 to 1:1.5, 1.2:1 to 1:1.2, 1.1:1 to 1:1.1, or 1:1. PLGA may be a random copolymer or a block copolymer.

[0056] PLA and PLGA may be used alone or in any mixture. The concentration of PLA and / or PLGA in the organic solvent is not particularly limited, but may be, for example, 0.01 g to 0.5 g per mL of organic solvent. When both PLA and PLGA are used, this concentration refers to the combined concentration of PLA and PLGA. The higher the concentration of PLA and / or PLGA, the larger the particle size of the microparticles. The weight-average molecular weight of PLA and the weight-average molecular weight of PLGA may each be 1,000 to 200,000. Unless otherwise specified, the weight-average molecular weight in this specification refers to the weight-average molecular weight calculated in terms of standard polystyrene as determined by gel permeation chromatography (GPC).

[0057] (O / W Emulsion Formation Step) In the o / w emulsion formation step, the organic solvent containing the polyion complex mixed with the organic solvent in the mixing step is added to an aqueous solution in which a dispersant that prevents the emulsions from bonding together is dissolved, thereby forming an o / w emulsion.

[0058] In order to prevent the emulsions from bonding together, it is important that the dispersant has surface activity, and examples of substances having such properties include polyvinyl alcohol (PVA) and polyethylene glycol. The concentration of the dispersant is not particularly limited, but when polyvinyl alcohol is used as the dispersant, for example, it may be an aqueous polyvinyl alcohol solution with a concentration of 1 mg / mL to 50 mg / mL.

[0059] Although the timing at which the polyion complex is encapsulated in PLA and / or PLGA is not limited to a particular theory, it is believed that when an organic solvent containing the polyion complex and PLA and / or PLGA is added to an aqueous solution, the polyion complex aggregates with the PLA and / or PLGA and is encapsulated in the PLA and / or PLGA.

[0060] The aqueous solution to which the organic solvent containing the polyion complex is added may contain components other than the dispersant.

[0061] In the organic solvent removal step, the organic solvent is removed from the aqueous solution containing the o / w emulsion formed in the o / w emulsion formation step, thereby removing the organic solvent that may also be contained in the fine particles from the aqueous solution.

[0062] The method for removing the organic solvent from the aqueous solution includes, but is not limited to, a submerged drying method.

[0063] In the submerged drying method, an aqueous solution containing an o / w emulsion is continuously stirred to prevent the emulsions in the aqueous solution from bonding together and becoming enlarged, while the organic solvent is removed.

[0064] The temperature during stirring is not particularly limited, but stirring is preferably carried out at room temperature (for example, 10 to 30°C) or a temperature slightly higher than room temperature (for example, 30 to 50°C).

[0065] The stirring speed is not particularly limited, but may be, for example, 10 rpm or more and 1000 rpm or less.

[0066] The stirring time may be any time sufficient to remove the organic solvent, and is not particularly limited, but may be, for example, 5 hours to 24 hours. Note that, since the above-mentioned HFIP is completely miscible with water, it is hardly contained in the particles containing PLA and / or PLGA, and even if it is contained, it is removed in the organic solvent removal step.

[0067] As described above, in this embodiment, the oil-in-water emulsion method is used to form particles containing PLA and / or PLGA with an active substance encapsulated therein, which reduces the labor required for the microparticle manufacturing process compared to when particles containing PLA and / or PLGA are formed using the w / o / w double emulsion method, thereby enabling the active substance to be easily encapsulated in PLA and / or PLGA.

[0068] <Fine Particles> The fine particles of this embodiment are produced by the above-described production method.

[0069] That is, the microparticles contain at least one polymer (P) selected from PLA and PLGA and a polyion complex, and the polyion complex contains an ionic active substance and an ionic polymer ionically bonded to the active substance. The microparticles may contain only one of PLA and PLGA, or may contain PLA and PLGA in any ratio. The polyion complex may be located inside the microparticles, or a portion of the polyion complex may be exposed on the surface of the microparticles.

[0070] The microparticles of this embodiment can be used to introduce active substances, such as antigens for vaccines, various pharmaceutical ingredients such as anti-cancer agents, and cosmetic ingredients, into the body.

[0071] The active substance, ionic polymer, PLA and PLGA in the microparticles of this embodiment are the same as the ionic polymer, PLA and PLGA described in detail in the previous embodiment relating to the manufacturing method.

[0072] The particle size of the fine particles is not particularly limited, and may be 1 μm or more and 100 μm or less, 50 nm or more and 1000 nm or less, or 50 nm or more and 100 μm or less.

[0073] For particles containing PLGA (PLGA particles), multiple types of PLGA microparticles may be used, each having a different ratio of lactic acid-derived structural units to glycolic acid-derived structural units (the ratio of m to n in formula (2)). For example, a first type of PLGA particles having a ratio of m:n=2:3 may be used, and a second type of PLGA particles having a ratio of m:n=4:1 may be used.

[0074] In this case, the second type of microparticles, which have a larger molecular weight, take longer to decompose in the body, so after the PLGA of the first type of microparticles is decomposed and the active substance inside is released, the PLGA of the second type of microparticles is decomposed and the active substance inside is released.

[0075] In this way, by using multiple types of microparticles with different ratios of lactic acid-derived structural units and glycolic acid-derived structural units in the PLGA covering the active substance, the active substance functions in the body for a long period of time, with time intervals between each type of microparticle, thereby reducing the number of times the active substance is administered to the body.

[0076] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments, and various modifications are possible within the scope of the invention described in the claims.

[0077] For example, in the embodiment of the method for producing microparticles described above, PLA and / or PLGA particles encapsulating an active substance are formed by performing an organic solvent removal step, but the method may further include a step of separating the PLA and / or PLGA particles (microparticles) in the resulting aqueous solution from the aqueous solution. Methods for separating the PLA and / or PLGA particles include, but are not limited to, a method of separation by suction filtration, as shown in the examples detailed below.

[0078] Hereinafter, the present invention will be described with reference to examples relating to PLGA particles or PLA particles, but the present invention is not limited to the following examples.

[0079] <Study on Production Conditions for PLGA Particles> The production conditions for each system are shown in Table 1 below.

[0080]

[0081] Table 1 shows PLGA (Musashino Chemical Laboratory, weight average molecular weight: 11.2 × 10) added to ethyl acetate as an example of an organic solvent in each system. ４ , PDI: 4.00), the amount of ethyl acetate, and polyvinyl alcohol (manufactured by Mitsubishi Chemical Corporation, saponification degree: 86.5-89.0 mol%, viscosity: 3.4 mm ２ / s) and the processing time when homogenizing using a homogenizer after the o / w emulsion formation step are shown.

[0082] First, PLGA was dissolved in chloroform and added to hexane to precipitate and purify the PLGA, which was then dissolved in ethyl acetate, an example of an organic solvent.

[0083] Next, polyvinyl alcohol was added to 100 mL of pure water and dissolved.

[0084] The polyvinyl alcohol aqueous solution thus obtained was stirred at a speed of 500 rpm using a stirrer, and the above ethyl acetate solution in which PLGA had been dissolved was added dropwise, followed by emulsification (homogenization treatment) using a homogenizer at 8100 rpm.

[0085] Thereafter, the aqueous polyvinyl alcohol solution containing PLGA was stirred overnight at a speed of 500 rpm using a stirrer, and ethyl acetate was removed from the PLGA by submerged drying.

[0086] After removing the ethyl acetate, the mixture was filtered by suction, and the resulting white powder (PLGA particles) was washed with the same amount of pure water as the aqueous polyvinyl alcohol solution and dried under reduced pressure.

[0087] After drying, the PLGA particles were observed under a stereomicroscope. As a result, most of the observed PLGA aggregates were in a particulate form, and the conditions of System 7 shown in Table 1 were found to be the most suitable conditions for controlling the particle shape of PLGA particles. It was also revealed that the particle size of PLGA particles can be controlled by changing the amount of PLGA added to ethyl acetate (changing the concentration).

[0088] <Experiment for producing PLGA particles containing an active substance> The production conditions (amounts of reagents) for each system are shown in Table 2 below. Systems 12 and 14 are comparative examples in which no active substance was added. System 13 is a comparative example in which no ionic polymer was added and the polyion complex formation step was not performed. System 15 is an example in which each step, including the polyion complex formation step, was performed.

[0089] First, polyethyleneimine (PEI, weight-average molecular weight: 1800) as an example of a cationic polymer and FITC-labeled bovine serum albumin (FITC-BSA, albumin-fluorescein isothiocyanate conjugate) as an example of an anionic active substance were dissolved in HFIP (polyion complex formation step). The liquid thus obtained is referred to as Liquid A.

[0090] Next, PLGA dissolved in chloroform was added to hexane to precipitate and purify the obtained PLGA (Musashino Chemical Laboratory, weight average molecular weight: 11.2 × 10 ４ 0.1 g of the compound (PDI: 4.00) was dissolved in 5 mL of ethyl acetate as an example of an organic solvent, and mixed with the solution A to prepare a solution B (mixing step).

[0091] Then, 100 mL of polyvinyl alcohol (manufactured by Mitsubishi Chemical Corporation, saponification degree: 86.5-89.0 mol%, viscosity: 3.4 mm ２ The solution B was added dropwise to an aqueous solution (10 mg / mL) of PEG-100 / s (o / w emulsion formation step).

[0092] Next, the aqueous polyvinyl alcohol solution to which the solution B had been added dropwise was stirred overnight with a stirrer to remove ethyl acetate (organic solvent removal step).

[0093] Thereafter, the mixture was centrifuged at 2000 rpm for 10 minutes, and the resulting precipitate was washed.

[0094] Specifically, the precipitate was first dispersed in 100 mL of PBS (phosphate buffer solution), stirred for 1 hour, and then collected by suction filtration. This procedure was repeated twice, after which the precipitate was dispersed in pure water, stirred for 10 minutes, and then collected by suction filtration.

[0095] The residue was then dried under reduced pressure, and the resulting powder was observed under a fluorescence microscope. The absorbance of the supernatant after centrifugation and the two washings was measured to calculate the loading rate of FITC-labeled albumin.

[0096] In Example 15, PLGA particles (microparticles) encapsulating a polyion complex were produced as described above, but in the comparative examples of Examples 12 to 14, polyethyleneimine and / or FITC-labeled bovine serum albumin were not added to HFIP. Therefore, in Example 13, an attempt was made to encapsulate FITC-labeled albumin, an example of an active substance, into PLGA without forming a polyion complex.

[0097] FIG. 1 is a bright-field image and a fluorescent image showing microparticles produced under the conditions of System 13 (no polyethyleneimine used), and FIG. 2 is a bright-field image and a fluorescent image showing microparticles produced under the conditions of System 15 (polyethyleneimine used as a cationic polymer).

[0098] Furthermore, FIG. 3 is a graph showing the loading rate of the active substance when the microparticles were prepared under the conditions of System 13 or System 15.

[0099] In the fluorescence images of Figures 1 and 2, the areas that glow green due to the presence of FITC are shown in bright colors. As shown in Figure 1, when the active substance was encapsulated in PLGA without forming a polyion complex, it was confirmed that the microparticles emitted fluorescence non-uniformly (partially).

[0100] In contrast, as shown in FIG. 2, when an active substance was encapsulated in PLGA in a state where a polyion complex was formed, it was confirmed that the entire microparticle emitted fluorescence uniformly.

[0101] These results demonstrate that the active substance can be encapsulated uniformly within the microparticles by encapsulating it in PLGA after forming a polyion complex. Naturally, no fluorescence was observed under the conditions of systems 12 and 14, in which FITC-labeled bovine serum albumin was not added.

[0102] In addition, the loading rate of System 13, in which the active substance was encapsulated in PLGA without forming a polyion complex, was 43.5%, while the loading rate of System 15, in which the active substance was encapsulated in PLGA after forming a polyion complex, was 78.3% (see Figure 3).

[0103] From the above results, it became clear that by encapsulating an active substance in PLGA in a state where a polyion complex has been formed, a larger amount of active substance can be encapsulated in PLGA.

[0104] <Experiment for manufacturing PLA particles encapsulating active substances> In this experiment, PLA particles encapsulating active substances were manufactured using the same procedure as in the above ``Experiment for manufacturing PLGA particles encapsulating active substances'', except that PLA was used instead of PLGA and polylysine was used instead of polyethyleneimine.

[0105] In this experiment, microparticles encapsulating FITC-labeled bovine serum albumin were produced using 0.1 g of PLA, 1.0 mg of polyvinyl alcohol, 100 mL of water, 1 mg of polylysine, 5 mg of FITC-labeled bovine serum albumin, and 0.5 mL of HFIP, with reference to the conditions of the above systems 7 and 15. As a comparative example, a system was also prepared in which no polylysine was added and the polyion complex formation step was not performed.

[0106] After the microparticles were produced, they were purified and the resulting powder was observed under a fluorescent microscope.

[0107] Figure 4 is a fluorescence image showing microparticles containing PLA and an active agent, where the areas that glow green due to the presence of FITC are indicated by a bright color.

[0108] As shown in FIG. 4, in the powder (fine particles) containing polylysine, brighter green luminous areas due to the presence of FITC were observed compared to the powder not containing polylysine.

[0109] From the above results, it became clear that even when PLA was used instead of PLGA, a larger amount of active substance could be encapsulated in PLA by forming a polyion complex.

[0110] <Experiment for producing PLGA particles using cationic polymers other than PEI> In this experiment, PLGA particles were produced in the same manner as in the above-mentioned "Experiment for producing PLGA particles encapsulating an active substance," except for the following points.

[0111] As the cationic polymer, polylysine (PLL, weight-average molecular weight: 1800) or polycaprolactone bound to a cationic group (a compound having a structure represented by formula (3), cPCL, weight-average molecular weight: 3000) was used. The cationic polymer was mixed with FITC-BSA as an anionic active substance to form a polyion complex, and PLGA particles were produced. The experimental conditions for each system in this experiment are shown in Table 3 below.

[0112] *In Table 3, "PLGA Mw" is the weight average molecular weight of the PLGA particles.

[0113] To produce the particles, 0.1 g of PLGA, a total of 0.6 mL of a mixture of 0.5 mL of HFIP and 0.1 mL of pure water as a solvent for forming a polyion complex, 5.0 mL of ethyl acetate as a solvent for dissolving PLGA, and 100 mL of an aqueous solution containing 1 wt% polyvinyl alcohol as a dispersant were used.

[0114] After the production of the particles, centrifugation and washing were carried out twice. The supernatant from the centrifugation and the two washings were collected and subjected to absorbance measurement. The amount of FITC-BSA not encapsulated in the particles was quantified, and the FITC-BSA content of the PLGA particles was calculated by subtracting this amount from the initially added weight of 5 mg of FITC-BSA, as shown in the following formula (i): Content = Amount of BSA initially added - (Amount of BSA in the supernatant + Amount of BSA in the first washing solution + Amount of BSA in the second washing solution)...(i)

[0115] Furthermore, as shown in the following formula (ii), the ratio of the FITC-BSA content calculated by formula (i) to the amount of FITC-BSA initially added was calculated as the loading rate: Loading rate [%] = content / amount of FITC-BSA initially added × 100 (ii)

[0116] As shown in Table 1 above, when a polyion complex was formed with an active substance using a cationic polymer (PLL or cPCL) (Systems 17-19 and 21-23), the loading rate was significantly improved compared to when no cationic polymer was added and no polyion complex was formed. This result suggests that, regardless of the type of cationic polymer, forming a polyion complex allows a larger amount of active substance to be encapsulated in PLGA.

[0117] According to the present invention, it is possible to provide a method for producing microparticles that can easily encapsulate an ionic active substance in PLA and / or PLGA, and microparticles that can be produced by the method.

Claims

1. A method for producing microparticles, comprising the steps of: forming a polyion complex by ionically bonding an ionic active substance with an ionic polymer; and forming microparticles containing the polyion complex and at least one polymer (P) selected from polylactic acid and a lactic acid-glycolic acid copolymer, wherein the step of forming the microparticles comprises the steps of: mixing the polyion complex and the polymer (P) in an organic solvent; adding the organic solvent containing the mixed polyion complex and the polymer (P) to an aqueous solution in which a dispersant that prevents emulsions from bonding together is dissolved to form an o / w emulsion; and removing the organic solvent from the aqueous solution containing the o / w emulsion.

2. The method for producing microparticles according to claim 1, wherein the active substance is a protein having a molecular weight of 1,000 or more.

3. The method for producing microparticles according to claim 1, wherein the active substance is a nucleic acid having a molecular weight of 1,000 or more.

4. The method of claim 1, wherein the active substance is cationic and the ionic polymer is an anionic polymer.

5. The method for producing microparticles described in claim 4, wherein the anionic polymer comprises at least one selected from the group consisting of polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyvinyl sulfate, polyvinyl phosphonic acid, and copolymers containing at least one of these, as well as at least one selected from the group consisting of polyglutamic acid, polyaspartic acid, alginic acid, hyaluronic acid, heparin, chondroitin sulfate, and dextran sulfate.

6. The method of producing microparticles according to claim 1, wherein the active substance is anionic and the ionic polymer is a cationic polymer.

7. The method for producing microparticles described in claim 6, wherein the cationic polymer comprises at least one selected from the group consisting of polyvinylamine, polyallylamine, polyvinylpyridine, and a copolymer containing at least one of these, as well as polylysine, polyarginine, polyhistidine, polytryptophan, polyethyleneimine, and chitosan.

8. Microparticles comprising at least one polymer (P) selected from polylactic acid and lactic acid-glycolic acid copolymers, and a polyion complex, the polyion complex comprising an ionic active substance and an ionic polymer ionically bonded to the active substance.

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