JP2008056878A - Thermo-responsive polylysine - Google Patents
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Description
温度の外部刺激に応答してその物性を大きく変化させる刺激応答性高分子(ポリリシン)に関する。 The present invention relates to a stimulus-responsive polymer (polylysine) that greatly changes its physical properties in response to an external stimulus of temperature.
温度、pH、光、電場などの外部刺激に応答してその物性を大きく変化させる刺激応答性高分子は、クロマトグラフィー担体やインテリジェント型のドラッグデリバリーシステム(DDS)担体などの生医学材料として数多く研究されている。 Many stimuli-responsive polymers that greatly change their physical properties in response to external stimuli such as temperature, pH, light, and electric field have been studied as biomedical materials such as chromatography carriers and intelligent drug delivery system (DDS) carriers. Has been.
近年では高機能ドラッグキャリアーとして生体内での使用を想定し、生分解性と生体適合性を有する刺激応答性高分子に関する研究が国内外で行われている。しかし、これまでに報告されている方法は、1)合成過程が多段階であり複雑である、2)分解生成物の安全性に疑問が残るなどの問題点を有している。 In recent years, research on stimuli-responsive polymers having biodegradability and biocompatibility has been conducted in Japan and overseas, assuming use in vivo as a high-performance drug carrier. However, the methods reported so far have the following problems: 1) the synthesis process is multistage and complicated, and 2) the safety of decomposition products remains questionable.
例えば、納豆の粘りの主成分であるポリ(γ−グルタミン酸)(γ−PGA)側鎖のカルボキシル基にアルキル基を適量導入してγ−PGA分子鎖全体の親・疎水バランスを制御することより、温度刺激に応答可能なプロピル化γ−PGAの合成がなされている(非特許文献1)。しかしながらγ−PGAは生理条件下(生体内の環境と同じpH(7.4)、塩濃度(150mM)の環境)で感熱応答性を発現しないなどの問題点がある。 For example, by introducing an appropriate amount of an alkyl group into the carboxyl group of the poly (γ-glutamic acid) (γ-PGA) side chain, which is the main ingredient of natto's stickiness, to control the parent-hydrophobic balance of the entire γ-PGA molecular chain In addition, a propylated γ-PGA that can respond to temperature stimulation has been synthesized (Non-patent Document 1). However, γ-PGA has problems such as that it does not exhibit heat-responsiveness under physiological conditions (the same pH (7.4) and salt concentration (150 mM) environment as in vivo).
また、ポリ(αL−リシン)をコレステロールで疎水化した例も報告されている(非特許文献2)。しかしこの例は、コレステロールをコアとして水中でナノサイズの粒子を作る技術であり、刺激応答性を有する高分子を提供するものではない。
本発明は上記事情に鑑みなされたものであり、生理条件下で感熱応答性を発現する生分解性と生体適合性を有する新規な刺激応答性高分子を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel stimulus-responsive polymer having biodegradability and biocompatibility that develops thermosensitive response under physiological conditions.
本発明者らが鋭意研究した結果、上記目的は、ポリリシンを両親媒化することにより達成できることを見出し、本発明をなすに至った。 As a result of intensive studies by the present inventors, it has been found that the above object can be achieved by amphiphilization of polylysine, and the present invention has been made.
すなわち、本発明は1,2−エポキシアルカンを導入したポリリシン誘導体に関する。 That is, the present invention relates to a polylysine derivative into which 1,2-epoxyalkane is introduced.
本発明で使用するポリリシンの構成モノマーであるリシンは、L体、D体またはそれらの混合物いずれの構成単位でもよい。すなわちポリリシンとしてはポリ(ε−L−リシン)、ポリ(ε−D−リシン)、ポリ(ε−D、L−リシン)、ポリ(α−L−リシン)、ポリ(α−D−リシン)、ポリ(α−D、L−リシン)いずれであってもよい。L体が自然に存在する形態であり、従って、L−リシンで構成されるポリリシンが通常使用される。中でも、ポリ(ε−L−リシン)(ε−PL)はstreptomyces albulus 346により産生される微生物由来、即ち天然由来のポリアミノ酸であり、L−リシンのホモポリマーであり、側鎖に修飾可能な1級アミノ基を有している。このε−PLは食品添加物として使用されているなど生体への安全性が高い。 The lysine, which is a constituent monomer of polylysine used in the present invention, may be any constituent unit of L-form, D-form or a mixture thereof. That is, as polylysine, poly (ε-L-lysine), poly (ε-D-lysine), poly (ε-D, L-lysine), poly (α-L-lysine), poly (α-D-lysine) Poly (α-D, L-lysine) may be used. L-form is a naturally occurring form, and therefore polylysine composed of L-lysine is usually used. Among them, poly (ε-L-lysine) (ε-PL) is a microorganism-derived polyamino acid produced by streptomyces albulus 346, that is, a naturally occurring polyamino acid, is a homopolymer of L-lysine, and can be modified to the side chain. Has a primary amino group. This ε-PL is highly safe to living bodies, such as being used as a food additive.
生理条件下で感熱応答性を発現する生分解性と生体適合性を有する新規な刺激応答性高分子を提供することを目的としていることから、ポリリシンは、水に溶けるものを使用するようにする。生理条件下、生体適合性という条件に限定されず、感熱応答性という特性を目的とするのであれば、水に溶けるものだけに限定される必要はない。 The purpose of the present invention is to provide a novel stimulus-responsive polymer having biodegradability and biocompatibility that develops thermosensitive response under physiological conditions. Therefore, polylysine should be soluble in water. . The physiological condition is not limited to the condition of biocompatibility, and it is not necessary to be limited to only those that are soluble in water as long as the purpose is a heat-responsive property.
ポリリシンの分子量は、リシンの重合度を調整することにより調整可能であり、必要に応じて適宜選択すればよい。 The molecular weight of polylysine can be adjusted by adjusting the polymerization degree of lysine, and may be appropriately selected as necessary.
通常は数平均分子量(Mn)が数千〜200000、好ましくは数千〜100000のものを使用するようにすればよい。分子量が低いものを使用すると、感熱応答性を発現するための駆動力である分子間相互作用が弱く、応答性が発現しにくい可能性が予想される。分子量が高すぎるものを使用すると、合成過程において高分子量体のポリリシン同士がコンプレックスを形成し、効率的に疎水基を導入できない可能性がある。 Usually, the number average molecular weight (Mn) may be several thousand to 200,000, preferably several thousand to 100,000. If a material having a low molecular weight is used, the intermolecular interaction, which is the driving force for expressing the heat-sensitive response, is weak, and it is expected that the response may be difficult to express. If the molecular weight is too high, high molecular weight polylysine may form a complex in the synthesis process, and the hydrophobic group may not be efficiently introduced.
ポリリシンの商品としては、微生物由来のポリ(ε−L−リシン)(数平均分子量4700:商品名ポリリジン塩酸塩:チッソ社製)等を入手可能である。 As a product of polylysine, microorganism-derived poly (ε-L-lysine) (number average molecular weight 4700: trade name polylysine hydrochloride: manufactured by Chisso Corporation) and the like are available.
ポリリシンに導入される1,2−エポキシアルカンは、アルカンの炭素数が4以上、好ましくは4又は5の1,2−エポキシブタン又は1,2−エポキシペンタン、より好ましくは1,2−エポキシブタンである。アルカンの炭素数が3以下では、感熱応答性を発現させることができない。また、炭素数が多すぎる1,2−エポキシアルカンは、水に溶けないので、それをポリリシンに導入する際には、合成的工夫が必要となる。 The 1,2-epoxyalkane introduced into polylysine is 1,2-epoxybutane or 1,2-epoxypentane, more preferably 1,2-epoxybutane, wherein the alkane has 4 or more carbon atoms, preferably 4 or 5. It is. When the carbon number of the alkane is 3 or less, the thermal sensitivity cannot be expressed. In addition, 1,2-epoxyalkane having too many carbon atoms does not dissolve in water, and therefore, a synthetic contrivance is required when introducing it into polylysine.
ポリリシンへの1,2−エポキシアルカンへの導入は、ポリリシンを適当な溶媒に溶解させ、所定量の1,2−エポキシアルカンを添加溶解させて、ポリリシンの第1アミンのエポキシ基への求核開環反応させることにより行うことができる。結果的にポリリシンの第1アミンにヒドロキシアルキル基が導入される。 The introduction of 1,2-epoxyalkane into polylysine is accomplished by dissolving polylysine in a suitable solvent, adding and dissolving a predetermined amount of 1,2-epoxyalkane, and then nucleophilicity of the polylysine to the epoxy group of the primary amine. This can be done by ring-opening reaction. As a result, a hydroxyalkyl group is introduced into the primary amine of polylysine.
例えば、ポリリシンとしてポリ(ε−L−リシン)、1,2−エポキシアルカンとして1,2−エポキシブタンを使用する場合は、水を溶媒とする水溶液中で一段階で合成可能である。1,2−エポキシアルカンとして1,2−エポキシペンタンを使用する場合は、1,2−エポキシペンタンが水に溶けないので、ポリ(ε−L−リシン)に一旦1,2−エポキシブタン導入して、1,2−エポキシペンタンが溶解可能な有機溶媒に溶解可能な程度まで疎水性を付与し、有機溶媒中で1,2−エポキシペンタンを反応導入させるようにすればよい。 For example, when poly (ε-L-lysine) is used as polylysine and 1,2-epoxybutane is used as 1,2-epoxyalkane, it can be synthesized in an aqueous solution using water as a solvent. When 1,2-epoxypentane is used as the 1,2-epoxyalkane, 1,2-epoxypentane does not dissolve in water, so once 1,2-epoxybutane is introduced into poly (ε-L-lysine). Thus, hydrophobicity is imparted to such an extent that it can be dissolved in an organic solvent in which 1,2-epoxypentane can be dissolved, and 1,2-epoxypentane may be introduced by reaction in the organic solvent.
ポリリシンへの1,2−エポキシアルカンへの導入により、ポリリシンに疎水性を付与することができる。 By introducing 1,2-epoxyalkane into polylysine, hydrophobicity can be imparted to polylysine.
例えば、ポリ(ε−L−リシン)は、1.2−エポキシブタンの導入率(ポリリシンの第1アミンへの1,2−エポキシアルカンの導入の割合)が、14%を超えると、DMSO,エタノール等の有機溶媒溶解性が改善される。導入率は、合成の際の1.2−エポキシブタン等の1,2−エポキシアルカンの仕込量を変えることにより調整できる。 For example, when poly (ε-L-lysine) has an introduction rate of 1.2-epoxybutane (ratio of 1,2-epoxyalkane introduction into the primary amine of polylysine) of more than 14%, DMSO, The solubility of organic solvents such as ethanol is improved. The introduction rate can be adjusted by changing the amount of 1,2-epoxyalkane such as 1.2-epoxybutane in the synthesis.
さらに、ポリリシンの親疎水バランスを制御することにより、感熱応答性を付与することができる。本発明において「感熱応答性」とは、温度刺激により可逆的に引き起こされる相分離現象を意味している。相分離温度は疎水基の導入率59%以上)、ポリマー濃度(0.125〜2.0wt%)、pH(7以上、好ましくは7.4以上)を変化させることにより制御可能である。相分離温度は導入率の上昇に伴い低温側にシフトする。例えばポリ(ε−L−リシン)に1,2−エポキシブタンを59%程度以上導入すると、NaCl(1.0M)、pH12の緩衝液(塩濃度150mM)中で相分離温度が観測される。なお、本実施例で使用したpH12の緩衝液(塩濃度150mM)の組成は、リン酸水素二ナトリウム・水酸化ナトリウム水溶液を意味しており、塩濃度が、150mMであることは生理条件に近い。
Furthermore, thermoresponsiveness can be imparted by controlling the hydrophilicity / hydrophobicity balance of polylysine. In the present invention, “thermosensitive” means a phase separation phenomenon reversibly caused by temperature stimulation. The phase separation temperature can be controlled by changing the hydrophobic group introduction rate of 59% or more, the polymer concentration (0.125 to 2.0 wt%), and the pH (7 or more, preferably 7.4 or more). The phase separation temperature shifts to a lower temperature side as the introduction rate increases. For example, when about 59% or more of 1,2-epoxybutane is introduced into poly (ε-L-lysine), a phase separation temperature is observed in a buffer solution (salt concentration 150 mM) of NaCl (1.0 M) and
感熱応答性は、主鎖の構造によりそのメカニズムが異なる。例えばポリ(ε−L−リシン)に1,2−エポキシブタンを導入したポリリシン誘導体(ε−PL−B)は、コアセルベートにより感熱応答性を発現し、ポリ(α−L−リシン)に1.2−エポキシブタンを導入したポリリシン誘導体は、コイル・グロビュール転移により感熱応答性を発現する。 The mechanism of thermal sensitivity varies depending on the structure of the main chain. For example, a polylysine derivative (ε-PL-B) in which 1,2-epoxybutane is introduced into poly (ε-L-lysine) exhibits a thermosensitive response due to coacervate, and 1 is added to poly (α-L-lysine). A polylysine derivative into which 2-epoxybutane is introduced exhibits heat-responsiveness due to the coil-globule transition.
ε−PL−Bは、相分離状態で形成するコアセルベート滴内に疎水性相互作用と静電相互作用を駆動力としてアニオン性化合物、例えばアニオン性色素、アニオン性のタンパク質、DNA、RNA等を分離、濃縮することが可能である。 ε-PL-B separates anionic compounds such as anionic dyes, anionic proteins, DNA, RNA, etc., using hydrophobic and electrostatic interactions as driving forces in coacervate droplets formed in a phase-separated state It is possible to concentrate.
1,2−エポキシアルカンを導入した新規なポリリシン誘導体を提供した。 A novel polylysine derivative incorporating a 1,2-epoxyalkane was provided.
(実施例1)
ポリ[Nα−(2−ヒドロキシブチル)リシン](ε−PL−B)の合成
ポリ(ε−L−リシン)(ε−PL)(商品名:ポリリジン塩酸塩:チッソ株式会社製)820mg(5.0unit mmol)を25mlの超純水に溶解後、所定量(下記表1に記載)のブチレンオキサイドを加えて40℃で24時間反応させた。
Synthesis of poly [ Nα- (2-hydroxybutyl) lysine] (ε-PL-B) Poly (ε-L-lysine) (ε-PL) (trade name: polylysine hydrochloride: manufactured by Chisso Corporation) 820 mg ( 5.0 unit mmol) was dissolved in 25 ml of ultrapure water, a predetermined amount (described in Table 1 below) of butylene oxide was added, and the mixture was reacted at 40 ° C. for 24 hours.
反応終了後、分子量分画500の透析膜を使用して蒸留水で透析を行い、凍結乾燥によりポリマーを精製した。生成物の構造確認は1H−NMR、FT−IRを使用して行った。 After completion of the reaction, dialysis was performed with distilled water using a dialysis membrane having a molecular weight fraction of 500, and the polymer was purified by lyophilization. The structure of the product was confirmed using 1 H-NMR and FT-IR.
図1にε−PL−Bの1H−NMRチャートを示した。すべてのピークはε−PLとε−PL−Bに由来する。 FIG. 1 shows a 1 H-NMR chart of ε-PL-B. All peaks are from ε-PL and ε-PL-B.
2−ヒドロキシブチル基の導入率はε−PL−B主鎖のメチンに起因するピークと側鎖のメチル基に起因するピークの積算値の比から算出した。2−ヒドロキシブチル基の導入率は14から93%であった。 The introduction rate of 2-hydroxybutyl group was calculated from the ratio of the integrated value of the peak attributed to methine in the ε-PL-B main chain and the peak attributed to the methyl group in the side chain. The introduction rate of 2-hydroxybutyl group was 14 to 93%.
溶解性試験
ポリマー濃度0.2重量%の濃度で、ε−PLとε−PL−Bの種々の溶媒に対する溶解性を試験した。結果を表1に示した。
Solubility test The solubility of ε-PL and ε-PL-B in various solvents was tested at a polymer concentration of 0.2 wt%. The results are shown in Table 1.
ε−PLは超純水に対しては溶解性を示したが、有機溶媒に対しては不溶であった。一方、導入率14〜93%のε−PL−Bは、超純水に対して溶解性を示し、エタノール、ジメチルホルムアミド(DMF)、ジメチルスルホキシド(DMSO)への溶解性が改善された。これらの結果からε−PL側鎖に2−ヒドロキシブチル基を導入することでε−PLの分子間相互作用を解消し、有機溶媒に対する溶解性を向上させることが可能となったことがわかる。 ε-PL showed solubility in ultrapure water, but was insoluble in organic solvents. On the other hand, ε-PL-B having an introduction rate of 14 to 93% showed solubility in ultrapure water and improved solubility in ethanol, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). From these results, it can be seen that by introducing a 2-hydroxybutyl group into the ε-PL side chain, it was possible to eliminate the intermolecular interaction of ε-PL and improve the solubility in organic solvents.
ε−PL−Bの感熱応答性評価
ε−PL−Bの感熱応答性は、UV−visスペクトルを用いて温度変化に対する溶液の光透過率測定を行うことにより評価した。
Evaluation of heat-sensitive response of ε-PL-B The heat-sensitive response of ε-PL-B was evaluated by measuring the light transmittance of the solution with respect to temperature change using a UV-vis spectrum.
ε−PL−Bを超純水、NaCl水溶液(1.0M)、pH7.4,10,12の緩衝液(塩濃度150mM)に溶解(ポリマー濃度は超純水およびNaCl水溶液では1wt%とし、緩衝液中では0.25wt%とした)させ、スリット幅1mmの石英セルに入れて500nmの波長の吸光度を測定した。加熱・冷却速度は1℃/minとし、相分離温度は透過率90%の時の温度とした。 ε-PL-B is dissolved in ultrapure water, NaCl aqueous solution (1.0 M), pH 7.4, 10, 12 buffer solution (salt concentration 150 mM) (polymer concentration is 1 wt% in ultrapure water and NaCl aqueous solution, The absorbance at a wavelength of 500 nm was measured in a quartz cell having a slit width of 1 mm. The heating / cooling rate was 1 ° C./min, and the phase separation temperature was the temperature at a transmittance of 90%.
2−ヒドロキシブチル基導入率14,37,59,73,80,93%のε−PL−B水溶液の透過率測定から得られた相分離温度を表2に示した。
NaCl水溶液(1.0M)(ポリマー濃度1wt%)のε−PL−B水溶液の光過率変化と93%ε−PL−B水溶液(pH7.4,10,12)(ポリマー濃度0.25wt%)の光透過率変化(相分離温度)をそれぞれ図2(a)−(c)と図3(a)−(c)に示した。図2(a)、図3(a)は、加熱過程での透過率変化、図2(b)、図3(b)は、冷却過程での透過率変化、図2(c)は、相分離温度の疎水基の導入率依存性を示す。図3(c)は、相分離温度の水素イオン濃度依存性を示している。
Table 2 shows the phase separation temperatures obtained from the measurement of the transmittance of an ε-PL-B aqueous solution having a 2-hydroxybutyl group introduction rate of 14, 37, 59, 73, 80, and 93%.
Change in light rate of ε-PL-B aqueous solution of NaCl aqueous solution (1.0 M) (
超純水に溶解したε−PL−B(14〜93%)のすべてのサンプルは、超純水中で感熱応答性を示さなかった。NaCl水溶液(1.0M)、pH7.4,10,12の緩衝液(塩濃度150mM)中では2−ヒドロキシブチル基の導入率59%以上のε−PL−Bは感熱応答性を発現した。また、図2(c)によると相分離温度は導入率の上昇に伴い低温側にシフトしていることがわかる。 All samples of ε-PL-B (14 to 93%) dissolved in ultrapure water did not show thermal sensitivity in ultrapure water. In a NaCl aqueous solution (1.0 M) and a pH 7.4, 10, and 12 buffer solution (salt concentration of 150 mM), ε-PL-B having a 2-hydroxybutyl group introduction rate of 59% or more exhibited heat-responsiveness. Moreover, according to FIG.2 (c), it turns out that the phase-separation temperature has shifted to the low temperature side with the raise of the introduction rate.
NaCl水溶液(1.0M)中では導入率59,73,80,93%のε−PL−B水溶液において温度変化に対する透過率変化が観察された。また、pH7.4,10,12の緩衝液中でも導入率59,73,80,93%のε−PL−B水溶液は可逆的な透過率変化が観察された。図3(c)によると、pHを変化させることにより相分離温度をコントロールできることが分かる。データは示していないが相分離温度はポリマー濃度、pH、イオン強度、導入率により制御することが可能であった。 In an aqueous NaCl solution (1.0 M), a change in transmittance with respect to a change in temperature was observed in an ε-PL-B aqueous solution having an introduction rate of 59, 73, 80, and 93%. In addition, reversible changes in transmittance were observed in the ε-PL-B aqueous solutions with introduction rates of 59, 73, 80, and 93% even in pH 7.4, 10, and 12 buffer solutions. FIG. 3C shows that the phase separation temperature can be controlled by changing the pH. Although no data is shown, the phase separation temperature could be controlled by the polymer concentration, pH, ionic strength, and introduction rate.
また、相分離温度以上において示差走査型熱重量測定により相分離温度付近での水の明確な吸熱ピークが観察されず、顕微鏡写真から、濃厚相による液滴が観察されたことからコアセルベートにより感熱応答性が発現していることが示唆された。 In addition, no clear endothermic peak of water near the phase separation temperature was observed by differential scanning thermogravimetry above the phase separation temperature, and droplets due to the concentrated phase were observed from the micrographs. It was suggested that sex was expressed.
以上の結果から2−ヒドロキシブチル基を導入してε−PL分子鎖全体の親・疎水バランスを適度に調整し、無機塩の添加により水の極性を上げる、またはpHを上昇させることにより側鎖のアミノ基の静電反発を低下させることにより、2−ヒドロキシブチルの疎水性相互作用を強めれば、ε−PLに感熱応答性を付与できることが明らかとなった。 From the above results, 2-hydroxybutyl group is introduced to adjust the hydrophilic / hydrophobic balance of the entire ε-PL molecular chain appropriately, and by adding inorganic salt, the polarity of water is increased or the side chain is increased by increasing pH. It has been clarified that by reducing the electrostatic repulsion of the amino group of ε-PL, heat-responsiveness can be imparted to ε-PL by increasing the hydrophobic interaction of 2-hydroxybutyl.
ε−PL−B水溶液のイオン性色素との複合化挙動
コアセルベートによる感熱応答性を発現したε−PL−Bは側鎖にアミノ基が残存している。そこでイオン性色素であるトリパンブルー(TB:アニオン性)とメチレンブルー(MB:カチオン性)を使用してε−PL−B水溶液の相分離温度前後での複合化挙動について観察した。
Complexing Behavior of ε-PL-B Aqueous Solution with Ionic Dye ε-PL-B that expresses thermal sensitivity by coacervate has an amino group remaining in the side chain. Therefore, the complexing behavior of the ε-PL-B aqueous solution before and after the phase separation temperature was observed using trypan blue (TB: anionic) and methylene blue (MB: cationic) which are ionic dyes.
導入率93%のε−PL−B(ε−PL−B93)水溶液(pH7.4,LCST(相分離温度)45℃)中にTBとMBをそれぞれ10μMとなるように添加して相分離温度前後でのイオンコンプレックス形成について観察した。 TB and MB were each added to an aqueous solution of 93% ε-PL-B (ε-PL-B93) (pH 7.4, LCST (phase separation temperature) 45 ° C.) to a concentration of 10 μM, and the phase separation temperature. The formation of ion complexes before and after was observed.
相分離温度以下である20℃ではε−PL−B93水溶液ではTBとMBはともに均一に溶解していることが確認された。これに対して相分離温度以上である50℃ではアニオン性色素であるTBがコアセルベート滴に濃縮されて沈殿する様子が確認された。これは相分離温度以上では疎水性相互作用を駆動力としてε−PL−B93が会合してコアセルベートによる濃厚相を形成し、静電相互作用によりアニオン性色素であるTBのみを液滴内に濃縮したために起こったと考えられる。 It was confirmed that both TB and MB were uniformly dissolved in the ε-PL-B93 aqueous solution at 20 ° C., which is lower than the phase separation temperature. On the other hand, at 50 ° C., which is higher than the phase separation temperature, it was confirmed that TB, which is an anionic dye, was concentrated and precipitated in coacervate droplets. Above this phase separation temperature, ε-PL-B93 associates with a hydrophobic interaction as a driving force to form a concentrated phase by coacervate, and only TB, which is an anionic dye, is concentrated in the droplet by electrostatic interaction. It is thought that it happened because of it.
TBの吸収波長である570nmの吸光度を測定したところ50℃のε−PL−B93水溶液の上澄み液からはTBの吸収は観察されなかった。 When the absorbance at 570 nm, which is the absorption wavelength of TB, was measured, no TB absorption was observed from the supernatant of the 50 ° C. aqueous ε-PL-B93 solution.
以上の結果よりε−PL−B93は感熱応答により形成するコアセルベート滴内に静電相互作用を介してアニオン性化合物、例えばアニオン性のタンパク質、DNAを捕捉できることも予想され、温度刺激を利用した分離材料としての利用が期待される。 From the above results, it is expected that ε-PL-B93 can capture anionic compounds such as anionic proteins and DNAs via electrostatic interaction in coacervate droplets formed by thermal response, and separation using temperature stimulation. Use as a material is expected.
ε−PL−B水溶液のCDスペクトル測定
ε−PL−B(導入率93%)のCDスペクトルを、pH7.4,10,12、ポリマー濃度0.005wt%、バッファー濃度150mMの条件下で測定した。結果を図4に示した。
CD spectrum measurement of ε-PL-B aqueous solution The CD spectrum of ε-PL-B (introduction rate: 93%) was measured under the conditions of pH 7.4, 10, 12, polymer concentration 0.005 wt%, buffer concentration 150 mM. . The results are shown in FIG.
pH7.4,12の条件下でε−PLとε−PL−Bはβ−シート構造をとることが明らかとなった。さらに加熱により、ε−PL−Bのβ構造は安定化することが分かる。 It was revealed that ε-PL and ε-PL-B have a β-sheet structure under the conditions of pH 7.4 and 12. Furthermore, it turns out that the beta structure of (epsilon) -PL-B is stabilized by heating.
ε−PL−B水溶液のDLS測定
ε−PL−B(導入率93%)、pH7.4、相分離温度44℃、ポリマー濃度0.5wt%、バッファー濃度150mMの溶液を使用しε−PL−B(導入率93%)のDLSを、20、40、60℃条件下で測定した。結果を図5に示した。
DLS measurement of ε-PL-B aqueous solution ε-PL-B (
ε−PL−B水溶液は相分離温度以上で2−ヒドロキシブチル基の疎水性相互作用を駆動力として粒径400nm程度の会合体を形成した。 The ε-PL-B aqueous solution formed an aggregate having a particle size of about 400 nm at a temperature equal to or higher than the phase separation temperature and using the hydrophobic interaction of 2-hydroxybutyl groups as a driving force.
ポリ[Nε−(2−ヒドロキシブチル)リシン](α−PL−B)の合成
ポリ[Nα−(2−ヒドロキシブチル)リシン](ε−PL−B)と同様の方法でポリ[Nε−(2−ヒドロキシブチル)リシン](α−PL−B)を合成した。表3に合成結果と水に対する溶解性を示した。
Synthesis of poly [N ε- (2-hydroxybutyl) lysine] (α-PL-B) Poly [N α- (2-hydroxybutyl) lysine] (ε-PL-B) [epsilon] -(2-hydroxybutyl) lysine] ([alpha] -PL-B) was synthesized. Table 3 shows the synthesis results and water solubility.
2−ヒドロキシブチル基の導入率は50,72,83,95%であった。導入率が50%,72%のα−PL−Bは超純水に溶解したが、導入率83%のα−PL−Bは超純水中で分散し、95%のα−PL−Bは、超純水に対して不溶であった。 The introduction rate of 2-hydroxybutyl group was 50, 72, 83, 95%. Α-PL-B having an introduction rate of 50% and 72% was dissolved in ultrapure water, but α-PL-B having an introduction rate of 83% was dispersed in ultrapure water, and 95% α-PL-B was dissolved. Was insoluble in ultrapure water.
また導入率50,72%のα−PL−BはpH7.4の緩衝液に対する溶解性を示したが、導入率83%のα−PL−BはpH7.4の緩衝液中で沈殿した。α−PL−Bは導入率の増加に伴い、水に対する溶解性が低下した。合成したα−PL−Bはε−PL−Bと比較して、同程度の導入率の場合では水に対する溶解性が低下することが明らかとなった。これはα−PL−Bは側鎖の疎水基の鎖長が長いためε−PL−Bと比較して強い疎水性相互作用が働くことに起因すると考えられる。 In addition, α-PL-B having an introduction rate of 50, 72% showed solubility in a buffer solution having a pH of 7.4, whereas α-PL-B having an introduction rate of 83% was precipitated in the buffer solution having a pH of 7.4. As α-PL-B was introduced, the solubility in water decreased. It has been clarified that the synthesized α-PL-B has a lower solubility in water in the case of the same introduction rate as compared with ε-PL-B. This is considered to be due to the fact that α-PL-B has a strong hydrophobic interaction as compared with ε-PL-B because the side chain has a long chain length.
pH7.4の緩衝液に対する溶解性を示した導入率50,72%のα−PL−Bの感熱応答性について検討し、水に分散した導入率83%の動的光散乱(DLS)測定を行った。 The thermal responsiveness of α-PL-B having an introduction rate of 50,72% showing solubility in a buffer solution of pH 7.4 was examined, and dynamic light scattering (DLS) measurement with an introduction rate of 83% dispersed in water was performed. went.
図6に導入率50%(ポリマー濃度:1wt%),導入率72%(ポリマー濃度:1wt%,0.2wt%)のα−PL−B水溶液(pH7.4)の温度変化に対する光透過率測定の結果を示した。 FIG. 6 shows the light transmittance with respect to temperature change of an α-PL-B aqueous solution (pH 7.4) having an introduction rate of 50% (polymer concentration: 1 wt%) and an introduction rate of 72% (polymer concentrations: 1 wt%, 0.2 wt%). The measurement results are shown.
導入率72%のサンプルのみ官能応答性を示すことが確認されたが、透過率変化の乱れが観察された。相分離温度以上でのα−PL−B水溶液を観察するとポリマーの析出および沈殿が観察された。 Only the sample with an introduction rate of 72% was confirmed to exhibit a sensory response, but a disturbance in transmittance change was observed. When the α-PL-B aqueous solution above the phase separation temperature was observed, polymer precipitation and precipitation were observed.
DSC測定
ε−PL−B(導入率93%)、α−PL−B(導入率72%)について、pH7.4、ポリマー濃度0.5wt%、バッファー濃度150mM、加熱・冷却速度2℃/分の条件下で、DSC測定を行った。結果を図7に示す。
DSC measurement ε-PL-B (
DSC測定の結果より、ε−PL−Bは、液−液相分離であるコアセルベートにより感熱応答性を発現し、α−PL−Bは、液−固相分離であるコイル・グロビュール移転により感熱応答性を発現することが確認された。また、図7よりα−PL−B水溶液(pH7.4)の相分離温度は約65℃であることがわかる。 From the results of DSC measurement, ε-PL-B expresses thermal sensitivity by coacervate which is liquid-liquid phase separation, and α-PL-B has thermal response by coil-globule transfer which is liquid-solid phase separation. It was confirmed to express sex. Further, FIG. 7 shows that the phase separation temperature of the α-PL-B aqueous solution (pH 7.4) is about 65 ° C.
ε−PL−Bの相分離状態では液一液相分離であったのでポリマーの沈殿は確認されなかったが、α−PL−Bは、側鎖の疎水基の鎖長が長いため、ε−PL−Bと比較して強い疎水性相互作用が働き、α−PL−B72水溶液は温度刺激に応答して疎水性相互作用を駆動力としたポリマーの会合が起こり析出したと考えられる。 In the phase separation state of ε-PL-B, since the liquid-liquid phase separation was performed, polymer precipitation was not confirmed. However, since α-PL-B has a long chain length of the hydrophobic group, ε- Compared with PL-B, a strong hydrophobic interaction acts, and in the α-PL-B72 aqueous solution, it is considered that a polymer association was caused by the hydrophobic interaction as a driving force in response to temperature stimulation and precipitated.
図8に、水分散性を示したα−PL−B83のDLS測定の結果を示した。20℃と50℃で測定したα−PL−B83の平均粒径はそれぞれ177nmと183nmであった。α−PL−B83は2−ヒドロキシブチル基の疎水性相互作用を駆動力として水中で比較的単分散な会合体を形成することが確認された。また、分散状態でのα−PL−B83のゼータ電位測定を行ったところ表面電位は35mVであり、表面にアミノ基が集積された会合体を形成していることが明らかとなった。 In FIG. 8, the result of the DLS measurement of (alpha) -PL-B83 which showed water dispersibility was shown. The average particle diameters of α-PL-B83 measured at 20 ° C. and 50 ° C. were 177 nm and 183 nm, respectively. It was confirmed that α-PL-B83 forms a relatively monodisperse aggregate in water using the hydrophobic interaction of 2-hydroxybutyl groups as a driving force. Further, when the zeta potential of α-PL-B83 in a dispersed state was measured, the surface potential was 35 mV, and it was revealed that an aggregate in which amino groups were accumulated on the surface was formed.
本実施例においては、ポリリシンとして、ポリ(ε−L−リシン)およびポリ(α−L−リシン)を使用した実施例を示しているが、ポリ(ε−D−リシン)、ポリ(ε−D、L−リシン)(リシンモノマー単位がD体とL体の混合物)、ポリ(α−D−リシン)、ポリ(α−D、L−リシン)も同様に使用できると考えている。 In this example, poly (ε-L-lysine) and poly (α-L-lysine) are used as polylysine. However, poly (ε-D-lysine), poly (ε- D, L-lysine) (a mixture of D and L lysine monomer units), poly (α-D-lysine), and poly (α-D, L-lysine) are also considered to be usable.
本発明のポリリシン誘導体は、新規な生分解性分離材料、アニオン性ドラッグのキャリアへの応用が可能であると考えられる。 The polylysine derivative of the present invention is considered to be applicable to a novel biodegradable separation material, an anionic drug carrier.
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