WO2025057883A1 - Method for producing fine particles, and fine particles - Google Patents

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

The present invention relates to a method for producing fine particles, and to fine particles.
This application claims priority based on Japanese Patent Application No. 2023-146876, filed on September 11, 2023, the contents of which are incorporated herein by reference.

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

For example, Patent Document 1 discloses a method for encapsulating water-soluble (ionic) proteins or peptides (active substances) in microparticles made of polymers such as PLGA.

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

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

Special Publication No. 10-511957

However, the method described in Patent Document 1 uses the w/o/w double emulsion method, which includes a step of forming a w/o emulsion and a step of forming a w/o/w emulsion, and therefore has the problem that it is time-consuming to encapsulate the active substance in PLGA.

The present invention has been made in consideration of these 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.

Aspects of the present invention include the following:

[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 lactic acid-glycolic acid copolymer, the step of forming the microparticles comprising 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 described in [1], wherein the active substance is a protein having a molecular weight of 1,000 or more.

[3] The method for producing microparticles described in [1], wherein the active substance is a nucleic acid having a molecular weight of 1000 or more.

[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.

[5] The method for producing microparticles described in [4], wherein the anionic polymer includes 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.

[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.

[7] The method for producing microparticles described in [6], wherein the cationic polymer includes 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.

The present invention provides a method for producing microparticles that can easily encapsulate an ionic active substance in particles containing PLA and/or PLGA, and microparticles that can be produced by the method.

1 shows bright field and fluorescent images of microparticles produced under the conditions of System 13 (no polyethyleneimine) shown in Table 2. 1 shows bright field and fluorescent images of microparticles produced under the conditions of System 15 shown in Table 2 (polyethyleneimine was used as the cationic polymer). 1 is a graph showing the loading rate of an active substance when microparticles are produced under the conditions of System 13 or System 15. 1 is a fluorescent image showing microparticles comprising PLA and an active agent.

The following describes in detail a preferred embodiment of the present invention.

<Method of producing fine particles>
The method for producing microparticles according to this embodiment includes a step of forming a polyion complex by ionically bonding an ionic active substance with 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-glycolic acid copolymer).

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 the emulsions from bonding is 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).

(Polyion Complex Formation Step)
In the polyion complex formation step, an ionic active substance and an ionic polymer are ionically bonded to form a polyion complex, which offsets the charge of the active substance having a charge and makes it closer to hydrophobicity.

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

In this specification, the term "active substance" includes chemical substances that induce physiological effects in living organisms, and substances that produce chemical effects or catalyze chemical reactions in non-living organisms.

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 industry, etc.

Examples of substances to be introduced into the body include, but are not limited to, medicinal ingredients such as anticancer drugs and anti-inflammatory drugs, antigens used in vaccines, cosmetic ingredients, etc. The active substance may be cationic as a whole or anionic as a whole.
Chemicals in the construction sector include, for example, catalysts used in polymerization or crosslinking.

The active substance is a polymer, and may be, for example, a protein, a nucleic acid, or other substance having an electric 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.
By forming a polyion complex using an ionic polymer, the decomposition of an active substance by decomposition enzymes such as nucleases and proteases can be suppressed compared to the case where an ion pair is formed using an ionic low molecular weight compound, and in addition to the sustained release property of PLA and/or PLGA, the polyion complex can suppress the initial burst and exhibit long-term sustained release property. In addition, although low molecular weight ions are prone to cause cytotoxicity, the use of an ionic polymer can improve safety.

Even if the active substance is a polymer with a large molecular weight, it can be easily encapsulated in PLA and/or PLGA by forming a polyion complex.

In addition, 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.

If the active substance is cationic, an anionic polymer is used as the ionic polymer. If the active substance is anionic, a cationic polymer is used as the ionic polymer.

In addition, 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.

When using both an anionic polymer and a cationic polymer, 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.

When the microparticles are applied to a living organism, the anionic polymer may be a substance that is less toxic to the living organism. Examples of anionic polymers that are relatively less toxic to the living organism 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.

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

The anionic polymer may have both cationic and anionic groups, but must be anionic overall.

When the microparticles are applied to a living organism, the cationic polymer may be a substance that is less toxic to the living organism. Examples of cationic polymers that are relatively less toxic to the living organism include, but are not limited to, polyvinylamine, polyallylamine, polyvinylpyridine, and copolymers containing at least one of these, as well as at least one selected from the group consisting of polylysine, polyarginine, polyhistidine, polytryptophan, polyethyleneimine, and chitosan.

The copolymer may contain one or more blocks of polyvinylamine, polyallylamine, or polyvinylpyridine (e.g., a block of polyvinylamine and a block of polyallylamine), and may also contain one or more blocks of other cationic homopolymers.

Polycaprolactone having a cationic group bonded thereto can also be used as the cationic polymer. An example of polycaprolactone having a cationic group bonded thereto is polycaprolactone having a structure represented by the following formula (3).

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).

（化学反応式（４）中、Ｘはハロゲン原子を表す。）

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

The cationic polymer may have both cationic and anionic groups, but must be cationic overall.

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.

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 an organic solvent 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 and 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, and HFIP can be particularly preferably used. 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.
The solvent for forming the polyion complex may be a mixture of an organic solvent and water. As will be described later in detail in the Examples, the inclusion of water in the solvent may improve the solubility of the active substance.

It is preferable that the amount of ionic polymer is set so that the total amount of active substance used is an amount that allows the formation of a polyion complex. For example, as 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 a particle containing PLA and/or PLGA and observed under a fluorescent microscope, the amount is set so that the microparticles emit fluorescence evenly overall and are close to perfect spheres.

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 the desired location (target site). In addition, a compound that increases hydrophobicity, such as a fatty acid, may be bound to the ionic polymer. By binding a compound that increases hydrophobicity to the ionic polymer, the hydrophobicity of the polyion complex can be adjusted.

(Mixing process)
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.

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

The type of organic solvent that dissolves PLA and/or PLGA is not particularly limited, but an organic solvent that is easily volatile is preferred. Examples of such organic solvents that can be used include ethyl acetate, dichloromethane, and chloroform. Among these organic solvents, ethyl acetate is particularly suitable for use because of its low toxicity to living organisms.

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 repeat unit or any other group.

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.

In formula (2), m and n are integers between 1 and 10,000, and the number of lactic acid molecules polymerized is m, and the number of glycolic acid molecules polymerized is n.

The molar ratio (m:n) of the constituent units derived from lactic acid to the constituent 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 or block copolymer.

PLA and PLGA may be used alone or in any combination in any proportion.
The concentration of PLA and/or PLGA in the organic solvent is not particularly limited, but for example, 0.01 g to 0.5 g of PLA and/or PLGA may be used per 1 mL of organic solvent. Note that when both PLA and PLGA are used, the concentration is 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 1000 or more and 200,000 or less. In this specification, unless otherwise specified, the weight average molecular weight is a weight average molecular weight calculated in terms of standard polystyrene obtained by measurement using gel permeation chromatography (GPC).

(O/W emulsion formation step)
In the o/w emulsion formation step, the organic solvent containing the polyion complex mixed in 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, to form an o/w emulsion.

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

The timing at which the polyion complex is encapsulated in PLA and/or PLGA is not limited to a particular theory, but 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.

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

(Organic solvent removal process)
In the organic solvent removing step, the organic solvent in the aqueous solution containing the o/w emulsion formed in the o/w emulsion forming step is removed, thereby making it possible to remove the organic solvent that may also be contained in the fine particles from the aqueous solution.

Methods for removing the organic solvent from the aqueous solution include, but are not limited to, the submerged drying method.

In the submerged drying method, the aqueous solution containing the 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.

The temperature during stirring is not particularly limited, but it is preferable to stir at room temperature (e.g., 10 to 30°C) or at a temperature slightly higher than room temperature (e.g., 30 to 50°C).

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

The stirring time is not particularly limited as long as it is long enough to remove the organic solvent, but it can be, for example, from 5 hours to 24 hours.
In addition, 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.

As described above, in this embodiment, the o/w emulsion method is used to form particles containing PLA and/or PLGA with an active substance encapsulated therein, so the labor involved in the microparticle manufacturing process can be reduced compared to when the w/o/w double emulsion method is used to form particles containing PLA and/or PLGA. Therefore, the active substance can be easily encapsulated in PLA and/or PLGA.

<Microparticles>
The microparticles of this embodiment are produced by the above-mentioned production method.

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 may be partially exposed on the surface of the microparticles.

The microparticles of this embodiment can be used to introduce active substances, such as antigens for vaccines, various medicinal ingredients such as anticancer drugs, and cosmetic ingredients, into the body.

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 above embodiment relating to the manufacturing method.

The particle size of the microparticles 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.

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

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 in the first type of microparticles is decomposed and the active substance inside is released, the PLGA in the second type of microparticles is decomposed and the active substance inside is released.

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 that covers the active substance, the active substance functions in the body for a long period of time with time intervals between each type of microparticle. This makes it possible to reduce the number of times the active substance needs to be administered to the body.

The above describes in detail preferred embodiments of the present invention, but 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.

For example, in the embodiment of the method for producing the microparticles, the organic solvent removal step is performed to form PLA and/or PLGA particles encapsulating an active substance, 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.

The present invention will be explained below using examples relating to PLGA particles or PLA particles, but the present invention is not limited to the following examples.

<Study of manufacturing conditions for PLGA particles>
The preparation conditions for each system are shown in Table 1 below.

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

First, PLGA was dissolved in chloroform and added to hexane to precipitate and purify it, and the resulting PLGA was then dissolved in ethyl acetate, an example of an organic solvent.

Then, polyvinyl alcohol was added to 100 mL of pure water and dissolved.

The polyvinyl alcohol aqueous solution thus obtained was stirred at a speed of 500 rpm using a stirrer, while the above ethyl acetate solution in which PLGA had been dissolved was added dropwise, and the mixture was emulsified (homogenized) using a homogenizer at 8100 rpm.

Then, the aqueous polyvinyl alcohol solution containing PLGA was stirred overnight at 500 rpm using a stirrer, and the ethyl acetate was removed from the PLGA by submerged drying.

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

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 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).

<Production experiment of PLGA particles containing active substances>
The manufacturing conditions (amounts of reagents) for each system are shown in Table 2 below. Systems 12 and 14 are comparative examples where no active substance was added. System 13 is a comparative example where no ionic polymer was added and the polyion complex formation step was not performed. System 15 is an example where each step including the polyion complex formation step was performed.

　まず、カチオン性ポリマーの一例としてのポリエチレンイミン（ＰＥＩ、重量平均分子量：１８００）と、アニオン性の活性物質の一例としての、ＦＩＴＣで標識した牛血清アルブミン（ＦＩＴＣ－ＢＳＡ、アルブミン－フルオレセインイソチオシアン酸コンジュゲート）とをＨＦＩＰに溶解させた（ポリイオンコンプレックス形成工程）。こうして得られた液をＡ液という。

First, polyethyleneimine (PEI, weight average molecular weight: 1800) as an example of a cationic polymer and bovine serum albumin labeled with FITC (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 called Liquid A.

Next, 0.1 g of PLGA (Musashino Chemical Laboratory, weight average molecular weight: 11.2 × 10 ⁴ , PDI: 4.00) obtained by precipitation purification by adding PLGA dissolved in chloroform to hexane was dissolved in 5 mL of ethyl acetate as an example of an organic solvent, and mixed with liquid A to prepare liquid B (mixing process).

Thereafter, the above liquid B was added dropwise to 100 mL of a stirred aqueous solution (10 mg/mL) of polyvinyl alcohol (manufactured by Mitsubishi Chemical Corporation, degree of saponification: 86.5-89.0 mol %, viscosity: 3.4 mm ² /s) (o/w emulsion formation step).

Then, the polyvinyl alcohol aqueous solution to which liquid B had been added was stirred overnight with a stirrer to remove the ethyl acetate (organic solvent removal process).

Then, the mixture was centrifuged at 2000 rpm for 10 minutes and the resulting precipitate was washed.

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

The filter cake was then dried under reduced pressure, and the resulting powder was observed under a fluorescent microscope. The loading rate of FITC-labeled albumin was calculated by measuring the absorbance of the supernatant from the centrifugation and the two washings.

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

Figure 1 shows bright-field and fluorescent images of microparticles produced under the conditions of System 13 (no polyethyleneimine used), and Figure 2 shows bright-field and fluorescent images of microparticles produced under the conditions of System 15 (polyethyleneimine used as a cationic polymer).

Furthermore, Figure 3 is a graph showing the loading rate of the active substance when the microparticles were produced under the conditions of System 13 or System 15.

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

In contrast, as shown in Figure 2, when an active substance was encapsulated in PLGA after forming a polyion complex, it was confirmed that the entire microparticle emitted fluorescence uniformly.

These results demonstrate that by encapsulating an active substance in PLGA while a polyion complex is formed, the active substance can be encapsulated uniformly inside the microparticles. Naturally, no fluorescence was observed under the conditions of systems 12 and 14, in which FITC-labeled bovine serum albumin was not added.

In addition, in the case of System 13 in which the active substance was encapsulated in PLGA without forming a polyion complex, the loading rate was 43.5%.
In contrast, in the case of System 15 in which the active substance was encapsulated in PLGA in a state in which a polyion complex was formed, the loading rate was 78.3% (see FIG. 3).

These results demonstrate that by encapsulating an active substance in PLGA after forming a polyion complex, a larger amount of active substance can be encapsulated in PLGA.

<Production experiment of PLA particles containing active substances>
In this experiment, PLA particles encapsulating an active substance were produced using the same procedure as in the above ``Experiment for producing PLGA particles encapsulating an active substance'', using PLA instead of PLGA and polylysine instead of polyethyleneimine.

In addition, in this experiment, referring to the conditions of the above systems 7 and 15, microparticles containing 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. As a comparative example, a system was also prepared in which no polylysine was added and the polyion complex formation process was not performed.

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

Figure 4 is a fluorescence image showing microparticles containing PLA and an active agent. In the image shown in Figure 4, areas that glow green due to the presence of FITC are shown in light color.

As shown in Figure 4, in the powder (microparticles) using polylysine, areas that glowed a brighter green due to the presence of FITC were observed compared to the powder not using polylysine.

These results demonstrate that even when PLA is used instead of PLGA, the formation of a polyion complex allows more active substances to be encapsulated in PLA.

<Experiment for manufacturing 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 points described below.

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.

※表３において「ＰＬＧＡ　Ｍｗ」はＰＬＧＡ粒子の重量平均分子量である。

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

To manufacture 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.

After the production of the particles, centrifugation and washing were performed twice. The supernatant from the centrifugation and the two washing solutions 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 weight of 5 mg of FITC-BSA initially added, 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 + Amount of BSA in the second washing) ... (i)

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 added initially × 100 (ii)

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, by forming a polyion complex, more active substance can be encapsulated in PLGA.

The present invention provides a method for producing microparticles that can easily encapsulate ionic active substances in PLA and/or PLGA, and microparticles that can be produced by the method.

Claims (8)

A step of forming a polyion complex by ionically bonding an ionic active substance with an ionic polymer;
forming microparticles containing the polyion complex and at least one polymer (P) selected from polylactic acid and lactic acid-glycolic acid copolymer;
The step of forming the fine particles includes 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 for preventing the emulsions from bonding is dissolved to form an oil-in-water emulsion;
and removing the organic solvent in the aqueous solution containing the o/w emulsion. The method for producing microparticles according to claim 1, wherein the active substance is a protein having a molecular weight of 1000 or more. The method for producing microparticles according to claim 1, wherein the active substance is a nucleic acid having a molecular weight of 1000 or more. The method for producing microparticles according to claim 1, wherein the active substance is cationic and the ionic polymer is an anionic polymer. The method for producing microparticles according to 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 polyglutamic acid, polyaspartic acid, alginic acid, hyaluronic acid, heparin, chondroitin sulfate, and dextran sulfate. The method for producing microparticles according to claim 1, wherein the active substance is anionic and the ionic polymer is a cationic polymer. The method for producing microparticles according to 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. The composition comprises at least one polymer (P) selected from polylactic acid and a lactic acid-glycolic acid copolymer, and a polyion complex;
The polyion complex is a microparticle comprising an ionic active substance and an ionic polymer ionically bonded to the active substance.

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