JP4963221B2 - Cyanoacrylate polymer particles and method for producing the same - Google Patents

Description

  The present invention relates to a cyanoacrylate polymer particle capable of conjugating a desired substance such as a medicine therein and a method for producing the same.

  It is known that particles composed of cyanoacrylate-based polymers can be conjugated with a desired substance such as a medicine inside, and it is also known to use this as a drug delivery system (DDS) by utilizing this property. (Non-Patent Document 1). The cyanoacrylate polymer particles are produced by anionic polymerization of a cyanoacrylate monomer. As a polymerization initiator and stabilizer for this anionic polymerization, a surfactant such as dextran, a polyoxyethylene sorbitan fatty acid ester (trade name Tween), a cyclodextrin, and the like are used (Non-patent Document 1). .

Douglas et al., Journal of Colloid and Interface Science, Vol.103, No. 1, January 1985

  However, in the known method for producing cyanoacrylate polymer particles, the particle diameter of the produced particles varies. When the particles are used as DDS, it is preferable to use particles having a size that can be easily taken up by tissues and cells. In addition, it is easier to handle if the particle size is uniform, and when used as a DDS, it is easier to control the amount of drug that is actually taken up by tissues or cells. However, the known methods do not always satisfy the uniformity of the size of the produced particles.

  Accordingly, an object of the present invention is to provide a method for producing cyanoacrylate polymer particles, and cyanoacrylate polymer particles produced thereby, which can produce particles having a high particle size uniformity.

As a result of earnest research, the inventor of the present application has used a monosaccharide or disaccharide having a hydroxyl group at a concentration of 2 w / v% to 10 w / v% as an initiator / stabilizer for anionic polymerization of a cyanoacrylate monomer. The inventors have found that the variation in the particle diameter of the acrylate polymer particles can be reduced, and completed the present invention.

That is, the present invention is a method for producing particles comprising a cyanoacrylate polymer by anionic polymerization of a cyanoacrylate monomer, wherein the anionic polymerization is performed from a cyanoacrylate monomer, a monosaccharide having a hydroxyl group, and a disaccharide. comprising rows that have in the presence of at least one sugar selected from the group, the concentration of the at least one sugar in the polymerization reaction mixture at the start of the reaction is a 2w / v% ~10w / v% , cyanoacrylate A method for producing a polymer particle is provided. Further, the present invention provides the above-mentioned desired substance in the above-described method of the present invention, wherein the anionic polymerization is performed in the presence of the cyanoacrylate monomer, the sugar, and the desired substance to be conjugated in the produced particles. A method for producing cyanoacrylate-based polymer particles conjugated with is provided. Furthermore, this invention provides the cyanoacrylate type polymer particle manufactured by the method of the said invention. Furthermore, the present invention provides cyanoacrylate polymer particles conjugated with a desired substance produced by the method of the present invention.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel method for producing cyanoacrylate polymer particles capable of producing particles with high particle size uniformity and cyanoacrylate polymer particles with high particle size uniformity produced thereby are provided. It was done. Since the cyanoacrylate polymer particles produced by the method of the present invention have high uniformity in particle size, they are highly effective when used as DDS and are convenient to handle. Further, the produced particles contain a small amount of monosaccharide and / or disaccharide used as a polymerization initiator / stabilizer, but since these sugars are derived from living organisms, they are highly safe for the human body.

  The cyanoacrylate monomer used in the method of the present invention is preferably an alkyl cyanoacrylate monomer (the alkyl group preferably has 1 to 8 carbon atoms), and is particularly used as an adhesive for sutures in the surgical field. N-butyl-2-cyanoacrylate (nBCA) represented by the following formula is preferred.

  In the method of the present invention, a cyanoacrylate monomer is polymerized by anionic polymerization, and at least one selected from the group consisting of monosaccharides and disaccharides having a hydroxyl group for the initiation of polymerization and stabilization of polymerization. Use sugar. The sugar used may be any sugar as long as it is a monosaccharide or disaccharide having a hydroxyl group. Preferred examples include glucose, mannose, ribose, fructose, maltose, trehalose, lactose and sucrose. These sugars may be either cyclic or chain-like, and when they are cyclic, they may be any one of pyranose type, furanose type and the like. In addition, there are various isomers of sugar, and any of them may be used. Usually, monosaccharides exist in a pyranose type or furanose type form, and disaccharides are those in which they are α-bonded or β-bonded, and sugars in such a normal form can be used as they are. Monosaccharides and disaccharides can be used alone or in combination of two or more.

  As a solvent for the polymerization reaction, water is usually used. Since anionic polymerization is initiated by hydroxide ions, the pH of the reaction solution affects the polymerization rate. When the pH of the reaction solution is high, the hydroxyl ion concentration is high, so that the polymerization is fast, and when the pH is low, the polymerization is slow. Usually, a moderate polymerization rate can be obtained under acidic conditions with a pH of about 2 to 4. Although it does not specifically limit as an acid added in order to make a reaction liquid acidic, Hydrochloric acid which does not have a bad influence on reaction but volatilizes after reaction can be used preferably.

The concentration of the cyanoacrylate monomer in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.5 wt% to 2 wt%, preferably about 0.8 wt% to 1.2 wt%. In addition, the concentration of sugar in the polymerization reaction solution at the start of the reaction (the total concentration when multiple types are used) is 2 w / v% to 10 w / v%, preferably about 4 w / v% to 6 w / v% . In addition, the reaction temperature is not particularly limited, but it is convenient and preferable to carry out the reaction at room temperature. The reaction time is not particularly limited, but is usually about 1 hour to 6 hours, preferably about 2 hours to 4 hours. The polymerization reaction is preferably performed with stirring. Since the particles are usually used as neutral particles, it is preferable to neutralize the reaction solution by adding a base such as an aqueous sodium hydroxide solution to the reaction solution after completion of the reaction.

  By the above polymerization reaction, the cyanoacrylate monomer is anionically polymerized to produce particles composed of the cyanoacrylate polymer. The size (diameter) of the particles obtained by the above method is not particularly limited, but is usually nano size (less than 1 μm), preferably about 40 nm to 500 nm. The particle size can be adjusted by adjusting the concentration of the cyanoacrylate monomer in the reaction solution and the reaction time. Also, depending on the type of sugar to be used, if other conditions are the same, the particle size is usually smaller when a monosaccharide is used than when a disaccharide is used (see Examples below). The generated particles can be collected by a conventional method such as centrifugal ultrafiltration.

  The cyanoacrylate-based polymer particles produced by the above method are porous and can be conjugated with a desired substance inside, so that they are known to be used as DDS or the like. After the particles are formed by the above method, the desired substance can be conjugated to the inside of the particles by immersing the particles in an aqueous solution of the desired substance. By performing the polymerization, a desired substance can be conjugated to the produced particles. The latter method is preferred because it is simpler and the conjugation rate is higher.

  The desired substance to be conjugated within the particle is not particularly limited, but is an agent desired to be directly administered to the tissue, for example, antibacterial agents such as ampicillin, vancomycin, levofloxanecin, various anticancer agents, iRNA, antisense RNA, etc. And various nucleic acids, various antibody drugs, various vaccines, physiologically active proteins, chemical mediators, and the like. In addition, it is not limited to pharmaceuticals, various reagents or diagnostic agents for detecting or measuring substances in vivo, various cosmetic ingredients such as hormones and hyaluronic acid used in the cosmetics field, deodorants, fragrances, paints Examples include metals, pigments, and organometallic dyes used in the field.

  The concentration of the desired substance to be coexisted in the polymerization reaction solution can be appropriately set according to the properties of the desired substance, the dose required at the time of use, and the like. For example, in the case of an antibacterial agent, it is usually about 0.1 to 0.5% by weight, and in the case of a nucleic acid, it is usually 0.001% to 0.01% by weight, but is not limited to these ranges.

  Since the size of the particles of the present invention is usually nano-sized (less than 1 μm) as described above, when applied to a tissue or cell, it easily passes through the membrane and enters the inside of the tissue or cell. And the substance conjugated inside the particles is gradually released. Therefore, the particles of the present invention encapsulating a drug exhibit an excellent effect when used for DDS.

  The amount of the particles conjugated with the desired substance is appropriately set according to the nature of the conjugated desired substance. When the desired substance is a medicine, the application amount of the medicine prescribed for the medicine is conjugated. Is the amount to be.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Examples 1-4 Manufacture of particles using monosaccharides As sugars, D (+)-glucose (Example 1), D (+)-mannose (Example 2), L (+)-ribose are monosaccharides. (Example 3) or D (-)-fructose (Example 4) was used.

  1.00 g of each sugar was dissolved in 20 ml of 0.01N HCl solution (pH 2.10) at the time of use. Under stirring, 0.20 ml of nBCA was added, and the mixture was polymerized by stirring at room temperature for 3 hours. 0.1N NaOH was added dropwise to the reaction solution, and the mixture was neutralized and stirred for 10 minutes. Since it did not pass through the 5μm size Milex filter (trade name), it was diluted 5 times with distilled water, filtered, and centrifuged at 2000 rpm for 10 minutes using a commercially available centrifugal ultrafiltration device (trade name Centriprep). Filtered. To the sedimentation, 5 ml of distilled water was added, and centrifugal filtration was performed again at 2000 rpm for 10 minutes using a centrifugal ultrafiltration apparatus (trade name Centriprep). Further, 5 ml of distilled water was added each time, and centrifugal filtration was performed three times to obtain washed particles.

  The particle diameter of the obtained particles was measured with laser scattered light. The results are shown in Table 1 below. Further, when the particles were observed with an electronic scanning microscope, it was confirmed that spherical particles having a highly uniform particle size were generated.

Examples 5 to 8 Production of particles using disaccharides As sugars, maltose (Example 5), D (+)-trehalose (Example 6), lactose (Example 7) or sucrose (Examples) which are disaccharides Particles were produced by the same operation as in Examples 1 to 4 except that 8) was used.

  The particle diameter of the obtained particles was measured with laser scattered light. The results are shown in Table 1 below. Further, when the particles were observed with an electronic scanning microscope, it was confirmed that spherical particles having a highly uniform particle size were generated.

Example 9 Production of Ampicillin Conjugated Particles
To 50 ml of 0.001N hydrochloric acid solution, 2.5 g of glucose was added and dissolved. Ampicillin (ABPC) 100 mg was added to the obtained glucose hydrochloride solution and completely dissolved. While stirring, 500 μl of nBCA was added to the solution, and the polymerization reaction was continued at room temperature for 2 hours. 0.1N NaOH was added dropwise to the reaction solution, and the mixture was neutralized and stirred for 10 minutes. After filtration through a 5 μm filter, the filtrate was centrifuged at 2000 rpm for 10 minutes using a Centriprep (trade name) filter.

Centriprep (trade name) filter passing liquid is a, and liquid not passing is b. In order to determine the ABPC conjugation rate, the absorbance (254 nm) of the obtained solution a was measured, and the amount of ABPC that was not conjugated was determined by the absorbance method. Therefore,
ABPC conjugate amount = ABPC dosage-ABPC amount not conjugated
ABPC conjugation rate = ABPC conjugation amount ÷ ABPC dosage was calculated.

  The liquid b was further centrifuged at 2000 rpm for 10 minutes each time by adding distilled water and centrifuged, and the same operation was repeated 4 times to obtain ABPC-conjugated particles.

  The average particle size of the obtained ABPC-conjugated particles was 207 nm. Further, when the particles were observed with an electronic scanning microscope, it was confirmed that spherical particles having a highly uniform particle size were generated. Moreover, when the conjugation rate was calculated | required as above-mentioned, it was 32.3%.

Example 10 Production of DNA-conjugated particles
1 g of glucose was dissolved in 20 ml of 0.001N hydrochloric acid solution. A physiological saline solution containing 1 mg of E. coli-derived DNA was added to the resulting glucose hydrochloride solution with stirring. To the solution, 200 μl of nBCA was added with stirring, and the polymerization reaction was continued for 2 hours. 0.1N NaOH was added dropwise to the reaction solution, and the mixture was neutralized and stirred for 10 minutes. After filtration through a 5 μm filter, the filtrate was centrifuged at 2000 rpm for 10 minutes using a Centriprep (trade name) filter.

Centriprep (trade name) filter passing liquid is a, and liquid not passing is b.
In order to determine the DNA conjugation rate, the absorbance (260 nm) of the obtained solution a was measured, and the amount of unconjugated DNA was determined by the absorbance method. Therefore,
DNA conjugation amount = DNA addition amount-DNA amount not conjugated
DNA conjugation rate = DNA conjugation amount / DNA addition amount.

  For solution b, centrifugation was further performed at 2000 rpm for 10 minutes, and after adding 5% glucose solution and centrifugation, the same operation was repeated 4 times to obtain DNA-conjugated particles.

  The conjugation rate determined by the above method was 87.6%.

Comparative Examples 1 to 3 Production of Particles Using Dextran As the sugar, dextran having a molecular weight of 10 kDa (Comparative Example 1), 70 kDa (Comparative Example 2) or 150 kDa (Comparative Example 3) was used.

  Particles were produced in the same manner as in Examples 1 to 8, except that 1 g of dextran was added to 100 mL of 0.01N hydrochloric acid, and centrifugal filtration with Centriprep (trade name) was performed for 15 minutes. The measurement results of the particle size are shown in Table 3 below.

Comparative Example 4 Production of ampicillin-conjugated particles using dextran
Ampicillin-conjugated particles were produced in the same manner as in Example 9, except that 500 mg of dextran having a molecular weight of 70 kDa was used instead of 2.5 g of glucose. The conjugation rate was 28.7%.

Claims (8)

A method for producing particles comprising a cyanoacrylate polymer by anionic polymerization of a cyanoacrylate monomer, wherein the anionic polymerization is at least selected from the group consisting of a cyanoacrylate monomer, a monosaccharide having a hydroxyl group, and a disaccharide. rows that have in the presence of one sugar, the concentration of the at least one sugar in the polymerization reaction mixture at the start of the reaction is a 2w / v% ~10w / v% , the production method of the cyanoacrylate polymer particles .   The method according to claim 1, wherein the sugar is at least one selected from the group consisting of glucose, mannose, ribose, fructose, maltose, trehalose, lactose and sucrose.   The method according to claim 1 or 2, wherein the cyanoacrylate monomer is n-butyl cyanoacrylate.   The method according to any one of claims 1 to 3, wherein the average particle diameter of the produced particles is 40 nm to 500 nm.   The method according to any one of claims 1 to 4, wherein the anionic polymerization is performed in the presence of the cyanoacrylate monomer, the sugar, and a desired substance to be conjugated in the produced particles. A method for producing cyanoacrylate polymer particles conjugated with a desired substance.   6. The method according to claim 5, wherein the desired substance is a medicine or a nucleic acid.   The cyanoacrylate type polymer particle manufactured by the method of any one of Claims 1 thru | or 4.   A cyanoacrylate polymer particle conjugated with a desired substance produced by the method according to claim 5 or 6.