JP2008285624A - METHOD FOR PRODUCING NANOPARTICLE COMPRISING HYDROPHOBED POLY (gamma-GLUTAMIC ACID) - Google Patents
METHOD FOR PRODUCING NANOPARTICLE COMPRISING HYDROPHOBED POLY (gamma-GLUTAMIC ACID) Download PDFInfo
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本発明は、疎水化ポリ（γ−グルタミン酸）からなるナノ粒子の製造方法に関する。 The present invention relates to a method for producing nanoparticles made of hydrophobic poly (γ-glutamic acid).
ポリ（γ−グルタミン酸）はバチルス（Bacillus）属の微生物の発酵により非常に効率よく（例えば、培養液１ｌ当たり５０ｇ）生産され、α−カルボキシル基に由来するカルボキシル基を側鎖に有する直鎖の水溶性の生分解性ポリマーである。このような特性を生かし、ポリ（γ−グルタミン酸）（以下、γ−ＰＧＡと略す。）は、制ガン剤に化学結合を介して担持させるか、化学的な架橋によるゲル化を介してゲルマトリックス中に薬物を内包させるために薬物担体としての用途が提案されている（例えば、特許文献１と２）。 Poly (γ-glutamic acid) is produced very efficiently by fermentation of microorganisms of the genus Bacillus (for example, 50 g per liter of culture solution), and is a linear chain having a carboxyl group derived from an α-carboxyl group in the side chain. It is a water-soluble biodegradable polymer. Taking advantage of these characteristics, poly (γ-glutamic acid) (hereinafter abbreviated as γ-PGA) is supported in an anticancer agent via a chemical bond or gelled by chemical crosslinking in a gel matrix. In order to encapsulate a drug, use as a drug carrier has been proposed (for example, Patent Documents 1 and 2).
これに対し、本発明者らは、γ−ＰＧＡのさらなる機能化を目的として、γ−ＰＧＡの化学修飾の検討を行っているが、γ−ＰＧＡの側鎖カルボキシル基の一部を介して疎水性基を導入した疎水化γ−ＰＧＡが水性媒体中で平均粒径が２００ｎｍ程度のナノ粒子を生成することを見出し、新たな薬物担体として用いることを提案している（例えば、非特許文献１）。疎水化γ−ＰＧＡのナノ粒子は、水性媒体中で、表面にγ−ＰＧＡが局在し、内部には疎水性基が存在する構造をとっている。 In contrast, the present inventors have been studying chemical modification of γ-PGA for the purpose of further functionalization of γ-PGA. However, hydrophobicity is partially introduced through a part of the side chain carboxyl group of γ-PGA. It has been found that hydrophobized γ-PGA introduced with a functional group produces nanoparticles having an average particle size of about 200 nm in an aqueous medium, and has been proposed to be used as a new drug carrier (for example, Non-Patent Document 1). ). Hydrophobized γ-PGA nanoparticles have a structure in which γ-PGA is localized on the surface and a hydrophobic group is present inside in an aqueous medium.
その疎水化γ−ＰＧＡのナノ粒子を製造するためには、一旦、疎水化γ−ＰＧＡを良溶媒に溶解させた後、その溶解した溶液と貧溶媒とを混合することにより、溶解した疎水化γ−ＰＧＡを不溶化させてナノ粒子を生成させる方法を用いている。その良溶媒には、ジメチルスルホキシド、ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチル−２−ピロリドン等が知られている。特にジメチルスルホキシドは優れた溶解能を有しており、疎水化γ−ＰＧＡのナノ粒子の製造に一般に用いられている。
しかしながら、ジメチルスルホキシドは、疎水化γ−ＰＧＡを溶解させる点では優れた溶媒であるが、毒性を有している。そして、ジメチルスルホキシドは、ナノ粒子回収時に残留溶剤としてナノ粒子中に残留し除去するのは容易ではない。そのため、無害であることを要求される医薬品の製造に用いることはできない。 However, dimethyl sulfoxide is an excellent solvent in terms of dissolving hydrophobized γ-PGA, but has toxicity. And dimethyl sulfoxide remains in a nanoparticle as a residual solvent at the time of nanoparticle collection | recovery, and it is not easy to remove. Therefore, it cannot be used for the manufacture of pharmaceutical products that are required to be harmless.
そこで、本発明は、ジメチルスルホキシド等の有害な溶剤を用いることなく医薬品の製造に使用可能な疎水化γ−ＰＧＡのナノ粒子を提供することが可能な、疎水化γ−ＰＧＡのナノ粒子の製造方法を提供することを目的とした。 Therefore, the present invention provides a hydrophobized γ-PGA nanoparticle capable of providing a hydrophobized γ-PGA nanoparticle that can be used in the manufacture of a pharmaceutical without using a harmful solvent such as dimethyl sulfoxide. Aimed to provide a method.
上記課題を解決するため、本発明者らは鋭意検討した結果、良溶媒として、弱アルカリ性と炭素数１から３のアルコールとの混合溶媒を用いることにより、疎水化γ−ＰＧＡのナノ粒子を製造可能なことを見出して本発明を完成させたものである。
すなわち、本発明に係る疎水化γ−ＰＧＡのナノ粒子の製造方法は、疎水化γ−ＰＧＡを弱アルカリ性水溶液と炭素数１から３のアルコールとの混合溶媒からなる良溶媒に溶解させた溶液と、貧溶媒とを混合して、ナノ粒子を生成させることを特徴とする。
In order to solve the above problems, the present inventors have intensively studied, and as a result, using a mixed solvent of weak alkalinity and alcohol having 1 to 3 carbon atoms as a good solvent produces hydrophobized γ-PGA nanoparticles. The present invention has been completed by finding out what is possible.
That is, the method for producing hydrophobized γ-PGA nanoparticles according to the present invention comprises a solution obtained by dissolving hydrophobized γ-PGA in a good solvent composed of a mixed solvent of a weak alkaline aqueous solution and an alcohol having 1 to 3 carbon atoms. And mixing with a poor solvent to produce nanoparticles.
本発明では、弱アルカリ性水溶液に、溶質として、クエン酸ナトリウムと炭酸水素ナトリウムからなる群から選択された少なくとも１種を含むことができる。 In the present invention, the weak alkaline aqueous solution may contain at least one selected from the group consisting of sodium citrate and sodium hydrogen carbonate as a solute.
また、炭素数１から３のアルコールには、メタノール、エタノール、１−プロパノール、２−プロパノールからなる群から選択された少なくとも１種を用いることができる。 Moreover, at least 1 sort (s) selected from the group which consists of methanol, ethanol, 1-propanol, and 2-propanol can be used for C1-C3 alcohol.
また、貧溶媒には、弱酸性水溶液を用いることができる。 Further, a weakly acidic aqueous solution can be used as the poor solvent.
本発明によれば、ジメチルスルホキシド等の有害な溶剤を用いることなく、疎水化γ−ＰＧＡのナノ粒子を製造することができる。ジメチルスルホキシドに代えて用いる弱アルカリ性水溶液と炭素数１から３のアルコールとの混合溶媒は、それぞれ生体適合性と生物分解性を有しているので、医薬品製造に使用可能な疎水化γ−ＰＧＡのナノ粒子を製造することが可能となる。 According to the present invention, hydrophobic γ-PGA nanoparticles can be produced without using a harmful solvent such as dimethyl sulfoxide. Since a mixed solvent of a weak alkaline aqueous solution and an alcohol having 1 to 3 carbon atoms used in place of dimethyl sulfoxide has biocompatibility and biodegradability, respectively, hydrophobized γ-PGA that can be used for pharmaceutical production is used. Nanoparticles can be produced.
本発明に係る疎水化γ−ＰＧＡのナノ粒子の製造方法は、疎水化γ−ＰＧＡを弱アルカリ性水溶液と炭素数１から３のアルコールとの混合溶媒からなる良溶媒に溶解させた溶液と、貧溶媒とを混合して、ナノ粒子を生成させることを特徴とするものである。 The method for producing nanoparticles of hydrophobized γ-PGA according to the present invention comprises a solution obtained by dissolving hydrophobized γ-PGA in a good solvent composed of a mixed solvent of a weak alkaline aqueous solution and an alcohol having 1 to 3 carbon atoms, A nanoparticle is produced by mixing with a solvent.
本発明に用いる疎水化γ−ＰＧＡは、γ−ＰＧＡの側鎖カルボキシル基の一部を介して疎水性基を導入したものである。原料のγ−ＰＧＡには、微生物による培養法、すなわち、バチルス・ズブチルスの発酵により得られる高分子量のもの、あるいは必要に応じ、その発酵由来のものを機械的あるいは化学的に加水分解して分子量を低下させたものを用いることができる。本発明に用いるγ−ＰＧＡの分子量は特に限定されるものはないが、１０万Ｋｄａ〜５００万Ｋｄａの分子量のものを好適に用いることができる。 The hydrophobized γ-PGA used in the present invention has a hydrophobic group introduced through a part of the side chain carboxyl group of γ-PGA. The raw material γ-PGA has a high molecular weight obtained by microbial culture, ie, fermentation of Bacillus subtilis, or, if necessary, mechanically or chemically hydrolyzes the one derived from the fermentation to obtain a molecular weight. Those having a reduced value can be used. Although the molecular weight of (gamma) -PGA used for this invention is not specifically limited, The thing of the molecular weight of 100,000 Kda-5 million Kda can be used suitably.
また、疎水性基には、−(Ｘ)−ＣＨＲ２Ｒ３の一般式で表され、（Ｘ）は−Ｏ−または−ＮＨ−を表し、Ｒ２は水素またはＣ１−Ｃ２１アルキルを表し、かつ、Ｒ３は水素またはＣ１−Ｃ６アルキルオキシカルボニルを表す基や、カルボキシル基をエステル化した疎水性アミノ酸残基（以下、エステル化疎水性アミノ酸残基という。）を用いることができるが、エステル化疎水性アミノ酸残基を用いることが好ましい。生体分解性や生体適合性に優れるからである。ここで、疎水性アミノ酸残基には、バリン残基、ロイシン残基、イソロイシン残基、メチオニン残基、フェニルアラニン残基を挙げることができる。より好ましくは、フェニルアラニン残基である。疎水性基は、カルボジイミド等の縮合剤を用いて、γ−ＰＧＡに導入することができる。エステル化疎水性アミノ酸残基のエステルには、炭素数１から６のアルキル基を含むエステルを用いることができるが、好ましくは、メチルエステル又はエチルエステルである。疎水性基の導入率は、全体の側鎖カルボキシル基に対する疎水性基が導入された側鎖カルボキシル基の比率（モル％）で定義され、ＮＭＲ測定に基づき算出することができる。本発明に用いる疎水化γ−ＰＧＡでは、原料のγ−ＰＧＡの分子量と導入する疎水性基の種類に応じて広い範囲で制御することが可能である。例えば、分子量３０万Ｋｄａのγ−ＰＧＡを用い、エステル化フェニルアラニン残基を導入した場合、１０〜８０モル％である。 Further, the hydrophobic group, - (X) represented by the general formula -CHR 2 R 3, (X) represents -O- or -NH-, R 2 represents hydrogen or C1-C21 alkyl, R 3 may be a group representing hydrogen or C1-C6 alkyloxycarbonyl or a hydrophobic amino acid residue obtained by esterifying a carboxyl group (hereinafter referred to as an esterified hydrophobic amino acid residue). It is preferable to use a hydrophobized amino acid residue. It is because it is excellent in biodegradability and biocompatibility. Here, examples of the hydrophobic amino acid residue include a valine residue, a leucine residue, an isoleucine residue, a methionine residue, and a phenylalanine residue. More preferably, it is a phenylalanine residue. The hydrophobic group can be introduced into γ-PGA using a condensing agent such as carbodiimide. As the ester of the esterified hydrophobic amino acid residue, an ester containing an alkyl group having 1 to 6 carbon atoms can be used, and methyl ester or ethyl ester is preferable. The introduction rate of the hydrophobic group is defined by the ratio (mol%) of the side chain carboxyl group into which the hydrophobic group is introduced relative to the entire side chain carboxyl group, and can be calculated based on NMR measurement. The hydrophobized γ-PGA used in the present invention can be controlled in a wide range depending on the molecular weight of the raw γ-PGA and the type of the hydrophobic group to be introduced. For example, when γ-PGA having a molecular weight of 300,000 Kda is used and an esterified phenylalanine residue is introduced, it is 10 to 80 mol%.
疎水化γ−ＰＧＡのナノ粒子を製造するには、一旦、疎水化γ−ＰＧＡを良溶媒に溶解させ、その溶液と貧溶媒とを混合することにより、疎水化γ−ＰＧＡのナノ粒子を生成させる。本発明によれば、平均粒径１μｍ以下、より好ましくは０．５μｍ、さらに好ましくは０．３μｍ以下のナノ粒子を製造することができる。 In order to produce hydrophobized γ-PGA nanoparticles, hydrophobized γ-PGA nanoparticles are generated by dissolving hydrophobized γ-PGA in a good solvent and mixing the solution with a poor solvent. Let According to the present invention, nanoparticles having an average particle diameter of 1 μm or less, more preferably 0.5 μm, and even more preferably 0.3 μm or less can be produced.
本発明では、弱アルカリ性水溶液と炭素数１から３のアルコールとの混合溶媒を良溶媒に用いることができる。弱アルカリ性水溶液のみ、あるいは炭素数１から３のアルコールのみを用いても疎水化γ−ＰＧＡを溶解することはできないが、それらの混合溶媒を用いることにより、疎水化γ−ＰＧＡを溶解することができる。弱アルカリ性水溶液のｐＨは７．５〜１０、より好ましくは７．５〜９である。また、ｐＨ７．５〜１０が得られる溶質であれば特に限定されない。例えば、クエン酸ナトリウムと炭酸水素ナトリウムからなる群から選択された少なくとも１種を含むことができるが、医薬品添加物として認められているクエン酸ナトリウムが好ましい。また、炭素数１から３のアルコールには、メタノール、エタノール、１−プロパノール、２−プロパノールからなる群から選択された少なくとも１種を用いることができるが、医薬品添加物としての使用が認められているエタノール又は２−プロパノールが好ましい。また、弱アルカリ性水溶液と炭素数１から３のアルコールとの混合比率は、アルコールの種類により異なるが、体積比率で、弱アルカリ性水溶液／メタノールでは、１／１〜１／５、弱アルカリ性水溶液／エタノールでは、１／１〜１／２、弱アルカリ性水溶液／２−プロパノールでは、２／１〜１／２、が好ましい。 In the present invention, a mixed solvent of a weak alkaline aqueous solution and an alcohol having 1 to 3 carbon atoms can be used as a good solvent. Hydrophobized γ-PGA cannot be dissolved by using only weakly alkaline aqueous solution or only alcohol having 1 to 3 carbon atoms. However, by using a mixed solvent thereof, hydrophobized γ-PGA can be dissolved. it can. The pH of the weak alkaline aqueous solution is 7.5 to 10, more preferably 7.5 to 9. Moreover, if it is a solute from which pH 7.5-10 is obtained, it will not specifically limit. For example, at least one selected from the group consisting of sodium citrate and sodium bicarbonate can be included, but sodium citrate recognized as a pharmaceutical additive is preferred. In addition, as the alcohol having 1 to 3 carbon atoms, at least one selected from the group consisting of methanol, ethanol, 1-propanol, and 2-propanol can be used, but its use as a pharmaceutical additive is recognized. Preferred is ethanol or 2-propanol. The mixing ratio of the weak alkaline aqueous solution and the alcohol having 1 to 3 carbon atoms varies depending on the type of alcohol, but is a volume ratio of 1/1 to 1/5 for the weak alkaline aqueous solution / methanol, and the weak alkaline aqueous solution / ethanol. Then, 1/1 to 1/2, and 2/1 to 1/2 is preferable for the weak alkaline aqueous solution / 2-propanol.
なお、クエン酸ナトリウムと、炭素数１から３のアルコールが医薬品製造に使用可能な安全性を有することは、例えば、日本医薬品添加剤協会編，「医薬品添加物事典」，薬事日報社，２００５年や、厚生労働省発行通知文「医薬審第39号平成12年2月8日」に記載されている。 It should be noted that sodium citrate and alcohols having 1 to 3 carbon atoms are safe for use in pharmaceutical production, for example, edited by Japan Pharmaceutical Additives Association, “Pharmaceutical Additives Encyclopedia”, Yakuji Nipposha, 2005. Or, it is described in a notice issued by the Ministry of Health, Labor and Welfare, “Pharmaceutical Trial No. 39, February 8, 2000”.
また、本発明に用いる貧溶媒は、疎水化γ−ＰＧＡを溶解させた良溶媒と混合することにより、疎水化γ−ＰＧＡの溶解性を低下させるものであれば特に限定されない。例えば、貧溶媒には、弱酸性水溶液やクエン酸緩衝液等を用いることができるが、弱酸性水溶液を用いることが好ましい。疎水化γ−ＰＧＡのカルボキシル基の解離を抑制して、疎水化γ−ＰＧＡの溶解性を低下させることができるからである。弱酸性水溶液のｐＨは３．０〜５．０、より好ましくは３．５〜４．０である。ｐＨ３．０より小さいと凝集が起こり易くなり、またｐＨ５．０より大きいと粒子が生成しにくくなるからである。この弱酸性水溶液は、さらに塩化物イオンを含む塩、例えば、塩化ナトリウムや塩化カリウムを含むことが好ましい。疎水化γ−ＰＧＡのカルボキシル基の電荷反撥を抑制し、疎水性側鎖の核形成を促進できるからである。より好ましくは、塩化ナトリウムである。塩化ナトリウムの濃度は、５０〜２００ｍＭ、より好ましくは７５〜１００ｍＭである。塩濃度が５０ｍＭより小さいと粒子が生成せず、また塩濃度が２００ｍＭより大きいと粒子が凝集し易くなるからである。また、塩化カルシウムのように疎水化γ−ＰＧＡのカルボキシル基をキレートして沈殿を生成するような２価以上の金属イオンを含む塩化物は適さない。 Moreover, the poor solvent used for this invention will not be specifically limited if it mixes with the good solvent which melt | dissolved hydrophobized (gamma) -PGA, and reduces the solubility of hydrophobized (gamma) -PGA. For example, a weakly acidic aqueous solution or a citrate buffer solution can be used as the poor solvent, but a weakly acidic aqueous solution is preferably used. This is because dissociation of the carboxyl group of the hydrophobized γ-PGA can be suppressed and the solubility of the hydrophobized γ-PGA can be reduced. The pH of the weakly acidic aqueous solution is 3.0 to 5.0, more preferably 3.5 to 4.0. This is because aggregation is likely to occur when the pH is less than 3.0, and particles are less likely to be generated when the pH is greater than 5.0. This weakly acidic aqueous solution preferably further contains a salt containing chloride ions, such as sodium chloride or potassium chloride. This is because the charge repulsion of the carboxyl group of the hydrophobized γ-PGA can be suppressed and the nucleation of the hydrophobic side chain can be promoted. More preferred is sodium chloride. The concentration of sodium chloride is 50 to 200 mM, more preferably 75 to 100 mM. This is because if the salt concentration is lower than 50 mM, particles are not generated, and if the salt concentration is higher than 200 mM, the particles are likely to aggregate. Further, a chloride containing a divalent or higher metal ion that chelates the carboxyl group of the hydrophobized γ-PGA and produces a precipitate such as calcium chloride is not suitable.
本発明に用いる良溶媒と貧溶媒との好ましい組合せは、良溶媒にクエン酸ナトリウム水溶液とエタノール又は２−プロパノールとの混合溶媒を用い、貧溶媒に塩化ナトリウムの弱酸性水溶液を用いる組合せである。さらに好ましくは、貧溶媒にｐＨ３．７の１００ｍＭ塩化ナトリウム水溶液を用いる。 A preferable combination of the good solvent and the poor solvent used in the present invention is a combination in which a mixed solvent of a sodium citrate aqueous solution and ethanol or 2-propanol is used as the good solvent, and a weakly acidic aqueous solution of sodium chloride is used as the poor solvent. More preferably, a 100 mM sodium chloride aqueous solution having a pH of 3.7 is used as the poor solvent.
また、疎水化γ−ＰＧＡを溶解させた良溶媒と貧溶媒の混合方法は、良溶媒へ貧溶媒を滴下する方法、あるいは貧溶媒へ良溶媒を滴下する方法等、ナノ粒子が生成可能であれば特に限定されない。貧溶媒へ良溶媒を滴下する方法が好ましい。より短時間でナノ粒子を生成させることができるからである。また、良溶媒と貧溶媒の割合は、混合後の最終的な比率が３／１〜１／３となることが好ましい。 In addition, the mixing method of the good solvent and the poor solvent in which the hydrophobized γ-PGA is dissolved is capable of producing nanoparticles such as a method of dropping the poor solvent into the good solvent or a method of dropping the good solvent into the poor solvent. If it does not specifically limit. A method of dropping a good solvent into a poor solvent is preferred. This is because nanoparticles can be generated in a shorter time. The ratio of the good solvent and the poor solvent is preferably 3/1 to 1/3 after mixing.
また、疎水化γ−ＰＧＡを溶解させた良溶媒と貧溶媒とを混合する際の温度は、２０〜３７℃が好ましい。２０℃より低いと凝集し易くなり、３７℃より高いと疎水性相互作用により疎水化γ−ＰＧＡが凝集し易くなるからである。 Moreover, as for the temperature at the time of mixing the good solvent which dissolved hydrophobized (gamma) -PGA, and a poor solvent, 20-37 degreeC is preferable. This is because when the temperature is lower than 20 ° C., aggregation easily occurs, and when the temperature is higher than 37 ° C., hydrophobic γ-PGA easily aggregates due to hydrophobic interaction.
また、良溶媒に溶解させる疎水化γ−ＰＧＡの濃度は溶解する限り特に制限はない。ただ、凝集を抑制し単分散のナノ粒子を得るため、５〜５０ｍｇ／ｍｌ、より好ましくは、１５〜２５ｍｇ／ｍｌである。５ｍｇ／ｍｌより小さいと、ナノ粒子が生成するのに長時間を要し、５０ｍｇ／ｍｌより大きいと良溶媒に疎水化γ−ＰＧＡが溶けにくくなるからである。 The concentration of the hydrophobized γ-PGA dissolved in the good solvent is not particularly limited as long as it dissolves. However, in order to suppress aggregation and obtain monodisperse nanoparticles, it is 5 to 50 mg / ml, more preferably 15 to 25 mg / ml. This is because if it is smaller than 5 mg / ml, it takes a long time to produce nanoparticles, and if it is larger than 50 mg / ml, the hydrophobized γ-PGA hardly dissolves in a good solvent.
また、疎水化γ−ＰＧＡを溶解させた良溶媒と貧溶媒とを混合することにより生成させたナノ粒子は乳白色の分散液の状態で存在する。この分散液を遠心分離により上澄みと沈殿とに分離し、上澄みを除去する。得られた沈殿を超純水中に再分散し、遠心分離を行い、沈殿を回収する洗浄操作を１回以上行う。回収した沈殿を凍結乾燥等の乾燥操作により乾燥してナノ粒子を得ることができる。 Moreover, the nanoparticle produced | generated by mixing the good solvent and poor solvent which dissolved hydrophobized (gamma) -PGA exists in the state of a milky white dispersion liquid. This dispersion is separated into a supernatant and a precipitate by centrifugation, and the supernatant is removed. The obtained precipitate is redispersed in ultrapure water, centrifuged, and a washing operation for collecting the precipitate is performed once or more. The collected precipitate can be dried by a drying operation such as freeze drying to obtain nanoparticles.
以下、実施例を用いて本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。
合成例１．
図１の反応式に示すように、γ−ＰＧＡにＬ−フェニルアラニンエチルエステル（ＰＡＥ）を縮合反応により導入した。すなわち、１．２１４ｍｇのγ−ＰＧＡ（分子量３８万）を５０ｍＭ炭酸水素ナトリウム水溶液２００ｍｌに溶解させ、１．８０２ｍｇのＷＳＣ（水溶性カルボジイミド）を加え攪拌した。２．１６０ｍｇのＰＡＥを加え１時間氷冷下で攪拌した。その後室温で２４時間攪拌した。反応後、排除分子量（ＭＷＣＯ）５０，０００の透析膜を用い反応液を純水で３日間透析した。その後、反応液を凍結乾燥した。凍結乾燥した生成物にエタノール２００ｍｌを加え６時間攪拌した。その後、その溶液を遠心分離し、上澄みのエタノールを除去し、沈殿物を減圧乾燥して疎水化γ−ＰＧＡ（Ｉ）を得た。ＮＭＲ測定に基づき算出したＰＡＥの導入率は５２％であった。
EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to a following example.
Synthesis Example 1
As shown in the reaction formula of FIG. 1, L-phenylalanine ethyl ester (PAE) was introduced into γ-PGA by a condensation reaction. That is, 1.214 mg of γ-PGA (molecular weight 380,000) was dissolved in 200 ml of 50 mM aqueous sodium hydrogen carbonate solution, and 1.802 mg of WSC (water-soluble carbodiimide) was added and stirred. 2. 160 mg of PAE was added and stirred for 1 hour under ice cooling. Thereafter, the mixture was stirred at room temperature for 24 hours. After the reaction, the reaction solution was dialyzed against pure water for 3 days using a dialysis membrane having an excluded molecular weight (MWCO) of 50,000. Thereafter, the reaction solution was freeze-dried. 200 ml of ethanol was added to the lyophilized product and stirred for 6 hours. Thereafter, the solution was centrifuged, the supernatant ethanol was removed, and the precipitate was dried under reduced pressure to obtain hydrophobized γ-PGA (I). The introduction rate of PAE calculated based on NMR measurement was 52%.
実施例１．
疎水化γ−ＰＧＡ（Ｉ）を０．１Ｍクエン酸ナトリウム水溶液（ｐＨ８）１００μｌに分散させた。これにメタノール、エタノール、２−プロパノール、そして比較例として１−ブタノールを所定量添加して、疎水化γ−ＰＧＡ（Ｉ）が溶解するかどうかを調べた。結果を表１に示す。ここで、溶解性の判定基準は、以下の通りである。
○：溶解する。△：若干不透明。×：溶解せず沈殿が生成。
Example 1.
Hydrophobized γ-PGA (I) was dispersed in 100 μl of 0.1 M aqueous sodium citrate (pH 8). A predetermined amount of methanol, ethanol, 2-propanol, and 1-butanol as a comparative example were added thereto to examine whether the hydrophobized γ-PGA (I) was dissolved. The results are shown in Table 1. Here, the criteria for determining solubility are as follows.
○: Dissolved. Δ: Slightly opaque. X: A precipitate was formed without dissolving.
クエン酸ナトリウム水溶液のみ、そしてアルコールのみでは溶解しなかった。しかし、クエン酸ナトリウム水溶液／メタノール比が１／１〜１／５、クエン酸ナトリウム水溶液／エタノール比が１／１〜１／２、クエン酸ナトリウム水溶液／２−プロパノール比が１／０．５〜１／２の範囲で疎水化γ−ＰＧＡ（Ｉ）は溶解した。一方、１−ブタノールでは疎水化γ−ＰＧＡ（Ｉ）を溶解させることはできなかった。 It did not dissolve only with aqueous sodium citrate solution and with alcohol alone. However, the sodium citrate aqueous solution / methanol ratio is 1/1 to 1/5, the sodium citrate aqueous solution / ethanol ratio is 1/1 to 1/2, and the sodium citrate aqueous solution / 2-propanol ratio is 1 / 0.5 to Hydrophobized γ-PGA (I) dissolved in the range of 1/2. On the other hand, hydrophobized γ-PGA (I) could not be dissolved with 1-butanol.
実施例２．
疎水化γ−ＰＧＡ（Ｉ）を０．１Ｍクエン酸ナトリウム水溶液（ｐＨ８）５０ｍｌに分散させた。これにエタノール５０ｍｌを加え溶解させた。最終濃度は、０．１Ｍクエン酸ナトリウム／エタノール＝１／１（体積比）の混合溶媒を用いて、１０ｍｇ／ｍｌである。
Example 2
Hydrophobized γ-PGA (I) was dispersed in 50 ml of 0.1 M aqueous sodium citrate (pH 8). To this, 50 ml of ethanol was added and dissolved. The final concentration is 10 mg / ml using a mixed solvent of 0.1 M sodium citrate / ethanol = 1/1 (volume ratio).
疎水化γ−ＰＧＡ（Ｉ）溶液を、同体積の弱酸性塩溶液（ｐＨ３．７、ＮａＣｌ１００ｍＭ）に滴下し、乳白色の分散液を得た。 The hydrophobized γ-PGA (I) solution was added dropwise to the same volume of weakly acidic salt solution (pH 3.7, NaCl 100 mM) to obtain a milky white dispersion.
得られた分散液を遠沈管に移し遠心分離し（１５，０００ｒｐｍで１５分）、上澄みを捨て沈殿物を得た。得られた沈殿に超純水を加え、ピペッティング操作で再分散させた。この再分散液をさらに遠心分離し、上澄みを捨て沈殿物を得た（沈殿１）。得られた沈殿に超純水を加え、ピペッティング操作で再分散させた。この再分散液をさらに遠心分離し、上澄みを捨て沈殿物を得た（沈殿２）。沈殿２に超純水を加え十分に分散させた状態にし、これを凍結乾燥した。 The obtained dispersion was transferred to a centrifuge tube and centrifuged (15 minutes at 15,000 rpm), and the supernatant was discarded to obtain a precipitate. Ultrapure water was added to the resulting precipitate and redispersed by pipetting. This re-dispersed liquid was further centrifuged, and the supernatant was discarded to obtain a precipitate (precipitation 1). Ultrapure water was added to the resulting precipitate and redispersed by pipetting. This re-dispersed liquid was further centrifuged, and the supernatant was discarded to obtain a precipitate (precipitation 2). Ultrapure water was added to the precipitate 2 to make it sufficiently dispersed, and this was freeze-dried.
ナノ粒子の平均粒径は動的光散乱装置を用いて測定した。また、凍結乾燥したナノ粒子の形態を走査型電子顕微鏡を用いて観察した。 The average particle size of the nanoparticles was measured using a dynamic light scattering device. In addition, the morphology of the freeze-dried nanoparticles was observed using a scanning electron microscope.
比較例１．
疎水化γ−ＰＧＡ（Ｉ）を１０ｍｇ／ｍｌとなるようにＤＭＳＯに溶解した。そのＤＭＳＯ溶液１００μｌを３００ｍＭ ＮａＣｌ溶液１００μｌに滴下し、白濁した分散液を得た。その分散液を遠心分離し（１４，５００ｒｐｍで５分）、上澄みを捨て、沈殿を２００μｌの超純水に再分散させた（洗浄１）。再度遠心分離を行った後、上澄みを捨て、沈殿を２００μｌのＮａＣｌ溶液に再分散させた（洗浄２）。再度遠心分離を行った後、沈殿を１００μｌのＰＢＳに再分散させた。
Comparative Example 1
Hydrophobized γ-PGA (I) was dissolved in DMSO to a concentration of 10 mg / ml. 100 μl of the DMSO solution was added dropwise to 100 μl of a 300 mM NaCl solution to obtain a cloudy dispersion. The dispersion was centrifuged (5 minutes at 14,500 rpm), the supernatant was discarded, and the precipitate was redispersed in 200 μl of ultrapure water (Wash 1). After centrifugation again, the supernatant was discarded and the precipitate was redispersed in 200 μl NaCl solution (Wash 2). After centrifugation again, the precipitate was redispersed in 100 μl PBS.
（結果）
得られた沈殿についてＮＭＲ測定を行い、疎水化γ−ＰＧＡ（Ｉ）中の残存ＤＭＳＯ量を測定したところ、２４ｎｍｏｌ／ｍｇであった。
(result)
It was 24 nmol / mg when NMR measurement was performed about the obtained precipitation and the amount of residual DMSO in hydrophobized (gamma) -PGA (I) was measured.
比較例２．
疎水化γ−ＰＧＡ（Ｉ）を１０ｍｇ／ｍｌとなるようにＤＭＳＯに溶解した後、当量の水を添加した。その後、透析膜を用い精製した。この溶液にｐＨ４．７〜４．９の１０ｍＭのＮａＣｌ溶液を添加し、白濁させた。その分散液を遠心分離して沈殿を生成させ、その沈殿を乾燥した。
Comparative Example 2
Hydrophobized γ-PGA (I) was dissolved in DMSO to 10 mg / ml, and then an equivalent amount of water was added. Then, it refine | purified using the dialysis membrane. A 10 mM NaCl solution having a pH of 4.7 to 4.9 was added to the solution to make it cloudy. The dispersion was centrifuged to form a precipitate, and the precipitate was dried.
（結果）
得られた沈殿についてＮＭＲ測定を行い、疎水化γ−ＰＧＡ（Ｉ）中の残存ＤＭＳＯ量を測定したところ、６ｎｍｏｌ／ｍｇであった。
(result)
It was 6 nmol / mg when NMR measurement was performed about the obtained precipitation and the amount of residual DMSO in hydrophobized (gamma) -PGA (I) was measured.
図１と図２は、それぞれ、実施例２と比較例１で回収したナノ粒子の粒径分布を示す図である。本発明の方法によっても、ＤＭＳＯを用いた場合と同様に、平均粒径が２００ｎｍ程度のナノ粒子を得ることができた。また、図３と図４は、それぞれ、実施例２と比較例１で回収したナノ粒子のＳＥＭ写真である。本発明の方法によっても、ＤＭＳＯを用いた場合と同様の粒径の揃ったナノ粒子が得られた。 1 and 2 are diagrams showing the particle size distributions of the nanoparticles collected in Example 2 and Comparative Example 1, respectively. Also according to the method of the present invention, nanoparticles having an average particle diameter of about 200 nm could be obtained as in the case of using DMSO. 3 and 4 are SEM photographs of the nanoparticles collected in Example 2 and Comparative Example 1, respectively. Also by the method of the present invention, nanoparticles having the same particle diameter as in the case of using DMSO were obtained.
以上説明したように、本発明によれば、良溶媒に弱アルカリ性水溶液と炭素数１から３のアルコールとの混合溶媒を用いることにより、ＤＭＳＯを用いることなくＤＭＳＯを用いた場合と同様の平均粒径と粒径分布を有する疎水化γ−ＰＧＡのナノ粒子を製造することができる。本発明によれば、ＤＭＳＯを用いることなく、医薬品製造に使用可能な疎水化γ−ＰＧＡのナノ粒子を製造することができる。 As described above, according to the present invention, by using a mixed solvent of a weak alkaline aqueous solution and an alcohol having 1 to 3 carbon atoms as a good solvent, the same average particle size as in the case of using DMSO without using DMSO is obtained. Nanoparticles of hydrophobized γ-PGA having a diameter and a particle size distribution can be produced. According to the present invention, it is possible to produce hydrophobic γ-PGA nanoparticles that can be used in pharmaceutical production without using DMSO.
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| CN112717138A (en) * | 2021-01-15 | 2021-04-30 | 齐齐哈尔医学院 | Gamma-polyglutamic acid nano-carrier and preparation method and application thereof |
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| CN112717138A (en) * | 2021-01-15 | 2021-04-30 | 齐齐哈尔医学院 | Gamma-polyglutamic acid nano-carrier and preparation method and application thereof |
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