WO2008029739A1 - Thermo-responsive polylysine - Google Patents
Thermo-responsive polylysine Download PDFInfo
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- WO2008029739A1 WO2008029739A1 PCT/JP2007/067041 JP2007067041W WO2008029739A1 WO 2008029739 A1 WO2008029739 A1 WO 2008029739A1 JP 2007067041 W JP2007067041 W JP 2007067041W WO 2008029739 A1 WO2008029739 A1 WO 2008029739A1
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- polylysine
- lysine
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- phase separation
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- C08L101/00—Compositions of unspecified macromolecular compounds
- C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable
Definitions
- the present invention relates to a stimulus-responsive polymer (polylysine) that greatly changes its physical properties in response to an external stimulus of temperature.
- Stimulus-responsive polymers that greatly change their physical properties in response to external stimuli such as temperature, pH, light, and electric field are biomedical products such as chromatography carriers and intelligent drug delivery system (DDS) carriers. It has been studied a lot as a material!
- ⁇ PGA poly ( ⁇ -glutamic acid)
- ⁇ PGA poly ( ⁇ -glutamic acid)
- ⁇ PGA poly ( ⁇ -glutamic acid)
- the main component of natto's stickiness controls the parent-hydrophobic balance of the entire ⁇ PGA molecular chain by introducing an appropriate amount of alkyl groups into the lupoxyl group.
- propylated ⁇ PGA that can respond to temperature stimulation has been synthesized (Non-patent Document 1).
- ⁇ PGA has problems such as not exhibiting thermosensitive response under physiological conditions ( ⁇ ⁇ ⁇ (7.4), environment with salt concentration (150 mM)).
- Non-Patent Document 2 an example in which poly (a L-lysine) is hydrophobized with cholesterol has also been reported.
- This example is a technology that makes nano-sized particles in water with cholesterol as a core, and does not provide a polymer having stimuli-responsiveness.
- Non-Patent Literature l Shimokuri, ⁇ ⁇ ; aneko, ⁇ ⁇ ; Akashi, ⁇ . J. Polym. Sci. Part A: Polym. Chem. 2004, 42, 4492-4501.
- Non-Patent Document 2 Akiyoshi,. Et al., Macromolecules 2000, 33, 6752-6756
- 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. To do.
- the present invention relates to a polylysine derivative into which 1, 2 epoxyalkane is introduced.
- the lysine that is a constituent monomer of the polylysine used in the present invention may be a constituent unit of any of L-form, D-form, or a mixture thereof. That is, as polylysine, any of poly ( ⁇ L lysine), poly ( ⁇ D lysine), poly ( ⁇ DL lysine), poly ( ⁇ L lysine), poly ( ⁇ -D-lysine), poly-DL-lysine) There may be.
- L-form is a naturally occurring form, so polylysine composed of L-lysine is usually used.
- poly (L-lysine) ( ⁇ -PL) is a polyamino acid derived from microorganisms produced by streptomyces albulus 346, that is, a naturally occurring polyamino acid, a homopolymer of L-lysine, and a primary amino acid that can be modified in the side chain. Has a group.
- This ⁇ -PL is used as a food additive and is highly safe for living bodies.
- polylysine is used that is soluble in water.
- the physiological condition is not limited to the biocompatibility condition, and it is not necessary to be limited to only those that are soluble in water if the purpose is to have a heat-responsive property.
- the molecular weight of polylysine can be adjusted by adjusting the degree of polymerization of lysine, and can be selected as needed.
- Mn number average molecular weight
- 200,000 preferably several thousand to 100,000
- Mn number average molecular weight
- the intermolecular interaction which is the driving force for developing the thermosensitive response, is weak and it is difficult to express the response.
- the molecular weight is too high, high molecular weight polylysine is used in the synthesis process. There is a possibility that they form a complex and the hydrophobic group cannot be efficiently introduced.
- microorganism-derived poly ( ⁇ -L-lysine) (number average molecular weight 4700: trade name polylysine hydrochloride: manufactured by Chisso Corporation) and the like are available.
- the 1,2-epoxyalkane introduced into the polylysine is 1,4-epoxybutane or 1,2-epoxypentane of 4 or 5 or more, preferably 1,2 or more, more preferably 1, 2—Epoxybutane. If the alkane has 3 or less carbon atoms, it is impossible to develop thermal response. In addition, 1,2-epoxyalkanes with too many carbon atoms are not soluble in water, and therefore a synthetic contrivance is required when introducing them into polylysine.
- the introduction of 1,2-epoxyalkane into polylysine involves dissolving polylysine in a suitable solvent, adding and dissolving a predetermined amount of 1,2-epoxyalkane, and then adding it to the epoxy group of the first amine of polylysine.
- the nucleophilic ring-opening reaction can be carried out. As a result, a hydroxyalkyl group is introduced into the primary amine of polylysine.
- 1,2-epoxypentane when poly (E-L-lysine) is used as polylysine and 1,2 epoxybutane is used as 1,2 epoxyalkane, it can be synthesized in one step in an aqueous solution using water as a solvent.
- 1,2-epoxypentane is used as 1,2-epoxyalkane, 1,2-epoxypentane is insoluble in water, so once 1,2-epoxybutane is introduced into poly ( ⁇ -L lysine) It is only necessary to impart hydrophobicity to such an extent that 1,2-epoxypentane can be dissolved in an organic solvent in which 1,2-epoxypentane can be dissolved, and to introduce 1,2-epoxypentane in the organic solvent.
- poly ( ⁇ -L-lysine) has an introduction rate of 1.2-epoxybutane (a ratio of 1,2-epoxyalkane introduction into the first amine of polylysine) exceeding 14%
- the solubility of organic solvents such as DMSO and ethanol is improved.
- the introduction rate can be adjusted by changing the amount of 1,2-epoxyalkane such as 1.2-epoxybutane at the time of synthesis.
- thermoresponsiveness can be imparted by controlling the hydrophilicity / hydrophobicity balance of polylysine.
- “thermosensitive” means a phase separation phenomenon reversibly caused by temperature stimulation. Phase separation temperature is more than 59% hydrophobic group introduction rate), polymer It can be controlled by changing one concentration (0 ⁇ 125 to 2 ⁇ 0 wt%) and 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 1,2-epoxybutane is introduced into poly ( ⁇ -L-lysine) at about 59% or more, the phase separation temperature is observed in NaCl (1.0 M) and pH 12 buffer (salt concentration 150 mM). . Note that the composition of the PH12 buffer solution (salt concentration 150 mM) used in this example means a sodium hydrogen phosphate / sodium hydroxide aqueous solution, and the salt concentration of 150 mM is close to physiological conditions. .
- the mechanism of heat sensitivity varies depending on the structure of the main chain.
- a polylysine derivative ( ⁇ -PL- ⁇ ) in which 1,2-epoxybutane is introduced into poly ( ⁇ L-lysine) exhibits heat-responsiveness due to coacervate
- ⁇ PL— ⁇ is an anionic compound such as an anionic dye, an anionic protein, DNA, using a hydrophobic interaction and an electrostatic interaction as driving forces in a coacervate droplet formed in a phase-separated state.
- RNA, etc. can be separated and concentrated.
- a novel polylysine derivative incorporating a 1,2-epoxyalkane was provided.
- FIG. 1 1 H-NMR chart of ⁇ -PL-B.
- FIG. 2a is a graph showing the temperature dependence (heating process) of the transmittance of an ⁇ PL--aqueous solution.
- FIG. 2b A graph showing the temperature dependence (cooling process) of the transmittance of ⁇ PL- aqueous solution.
- FIG. 2c is a graph showing the dependence of the phase separation temperature on the introduction rate of the ⁇ PL-sodium aqueous solution.
- FIG. 3a is a graph showing the temperature dependence (heating process) of the transmittance of an aqueous solution of ⁇ PL- ⁇ 93.
- FIG. 3b A graph showing the temperature dependence (cooling process) of the transmittance of ⁇ PL- ⁇ 93 aqueous solution.
- FIG. 3c is a graph showing the dependence of the phase separation temperature of ⁇ PL- ⁇ 93 aqueous solution on the hydrogen ion concentration.
- FIG.4 A diagram showing the CD spectrum of ⁇ -PL- ⁇ 93.
- FIG. 6 is a graph showing light transmittance with respect to temperature change of a PL-B aqueous solution.
- FIG. 7 is a diagram showing DSC measurement results of ⁇ PL——93% ⁇ PL— ⁇ 72%.
- Poly L-lysine ( ⁇ -PL) (trade name: Polylysine hydrochloride: manufactured by Chisso Corporation) After dissolving 820 mg (5. Ounit mmol) in 25 ml of ultrapure water, a predetermined amount (described in Table 1 below) Butylene oxide was added and reacted at 40 ° C for 24 hours.
- FIG. 1 shows a 1 H-NMR chart of ⁇ PL-B. All peaks are ⁇ —PL and ⁇
- the introduction rate of 2-hydroxybutyl group was calculated from the ratio of the integrated value of the peak attributed to the methine of the ⁇ -PL-B main chain and the peak attributed to the methyl group of the side chain.
- the introduction rate of 2-hydroxybutyl group was 14 to 93%.
- ⁇ PL has a solubility in ultrapure water. It was insoluble in organic solvents.
- ⁇ -PL-B with 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 ⁇ PL side chain, it was possible to eliminate the intermolecular interaction of ⁇ PL and improve the solubility in organic solvents.
- the thermal response of ⁇ PL- PL was evaluated by measuring the light transmittance of the solution with respect to temperature change using UV-vis spectrum.
- ⁇ PL- ⁇ is ultrapure water, NaCl aqueous solution (1.0 M), pH 7.4, 10, 12 buffer solution (salt concentration
- the polymer concentration was 1wt% for ultrapure water and NaCl aqueous solution and 0.25wt% for buffer solution
- the absorbance at 500nm wavelength was measured in a quartz cell with slit width lmm.
- the heating / cooling rate was l ° C / min
- the phase separation temperature was the temperature at a transmittance of 90%.
- ⁇ PL— ⁇ ⁇ which has developed a thermosensitive response due to coacervate, has an amino group remaining in the side chain. Therefore, we observed the complexation behavior of ⁇ PL-— aqueous solution around the phase separation temperature using trypan blue (TB: anionic) and methylene blue (MB: cationic), which are ionic dyes.
- TB anionic
- MB methylene blue
- TB and MB were added to a solution of 93% in an ⁇ PL-% ( ⁇ PL- ⁇ 93) aqueous solution ( ⁇ 7 ⁇ 4, LCST (phase separation temperature) 45 ° C) at 93% introduction rate, respectively.
- ⁇ PL-% ⁇ PL- ⁇ 93
- LCST phase separation temperature 45 ° C
- ⁇ PL- ⁇ 93 is also expected to capture anionic compounds, such as anionic proteins and DNA, via electrostatic interactions in coacervate droplets formed by the thermal response. It is expected to be used as a separation material using temperature stimulation.
- CD spectrum of ⁇ —PL—— (introduction rate: 93%) was measured under the conditions of ⁇ 7, 4, 10, 12, polymer concentration of 0.005 wt% and buffer concentration of 150 mM. The results are shown in FIG.
- the ⁇ PL-sodium aqueous solution formed an aggregate having a particle size of about 400 nm at a temperature higher than the phase separation temperature and driven by the hydrophobic interaction of 2-hydroxybutyl groups.
- the introduction rate of 2 hydroxybutyl groups was 50, 72, 83, 95%.
- a PL-B with an introduction rate of 50% and 72% is a force S dissolved in ultrapure water
- a PL-B with an introduction rate of 83% is dispersed in ultrapure water
- a PL-B of 95% is It was insoluble in ultrapure water.
- a PL-B with an introduction rate of 50, 72% showed solubility in the buffer solution of ⁇ 7.4.
- the solubility of water for a-PL-B decreased with increasing penetration. It was clear that the synthesized a-PL-B had a lower solubility in water when the introduction rate was similar to that of ⁇ -PL B. This is considered to be due to the fact that a-PL-B has a longer hydrophobic chain side chain and thus has a stronger hydrophobic interaction than ⁇ -PL-B.
- the introduction rate of 50%, 72% ⁇ PL-B, which showed solubility in pH 7.4 buffer solution was investigated! Light scattering (DLS) measurement was performed.
- DLS Light scattering
- Figure 6 shows the change in temperature of PL-B aqueous solution ( ⁇ 7 ⁇ 4) with an introduction rate of 50% (polymer concentration: lwt%) and an introduction rate of 72% (polymer concentration: lwt%, 0.21%). The results of light transmittance measurement are shown.
- ⁇ PL— ⁇ exhibits thermal responsiveness due to coacervate, which is liquid-liquid phase separation
- a PL-B is coiled globule, which is liquid-solid phase separation. It was confirmed that heat responsiveness was expressed by the transfer.
- Fig. 7 shows that the phase separation temperature of ⁇ PL-— aqueous solution ( ⁇ 7.4) is about 65 ° C.
- FIG. 8 shows the result of DLS measurement of ⁇ -PL-—83 that showed water dispersibility.
- the average particle diameters of ⁇ -PL-B83 measured at 20 ° C and 50 ° C were 177 nm and 183 nm, respectively.
- ⁇ PL- ⁇ 83 was confirmed to form relatively monodisperse aggregates in water using the hydrophobic interaction of 2-hydroxybutyl groups as the driving force.
- the surface potential was 35 mV, and it was revealed that an aggregate in which amino groups were accumulated was formed on the surface.
- poly-L lysine and poly-L lysine are used as polylysine.
- the polylysine derivative of the present invention is considered to be applicable to a carrier of a novel biodegradable separation material, an anionic drug.
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Abstract
Disclosed is a novel stimuli-responsive polymer which has biodegradability and biocompatibility and which can develop thermo-responsibility under physiological conditions. Specifically disclosed are: a novel polylysine (particularly, poly(ε-L-lysine)) derivative having a 1,2-epoxyalkane (particularly, 1,2-epoxybutane) introduced therein; an aqueous composition comprising the derivative and having thermo-responsibility; and a method for condensing/separating an anionic compound by using the polylysine derivative.
Description
明 細 書 Specification
感熱応答性ポリリシン Thermosensitive polylysine
技術分野 Technical field
[0001] 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。 [0001] The present invention relates to a stimulus-responsive polymer (polylysine) that greatly changes its physical properties in response to an external stimulus of temperature.
背景技術 Background art
[0002] 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!/、る。 [0002] Stimulus-responsive polymers that greatly change their physical properties in response to external stimuli such as temperature, pH, light, and electric field are biomedical products such as chromatography carriers and intelligent drug delivery system (DDS) carriers. It has been studied a lot as a material!
[0003] 近年では高機能ドラッグキャリア一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。 [0003] In recent years, research on stimuli-responsive polymers having biodegradability and biocompatibility has been conducted in Japan and overseas, assuming use in vivo as one of high-performance drug carriers. However, the methods reported so far have the following problems: 1) The synthesis process is multi-step and complicated, and 2) the safety of the degradation products remains unclear.
[0004] 例えば、納豆の粘りの主成分であるポリ ( γ グルタミン酸)( Ί PGA)側鎖の力 ルポ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)。し力、しながら γ PGAは生理条件下(生体内の環境と同じ ρ Η (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 [0004] For example, poly (γ-glutamic acid) ( Ί PGA), the main component of natto's stickiness, controls the parent-hydrophobic balance of the entire γ PGA molecular chain by introducing an appropriate amount of alkyl groups into the lupoxyl group. As a result, propylated γ PGA that can respond to temperature stimulation has been synthesized (Non-patent Document 1). However, γ PGA has problems such as not exhibiting thermosensitive response under physiological conditions (ρ 同 じ (7.4), environment with salt concentration (150 mM)).
[0005] また、ポリ( a L—リシン)をコレステロールで疎水化した例も報告されて!/、る(非特許 文献 2)。し力、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レ、。 [0005] In addition, an example in which poly (a L-lysine) is hydrophobized with cholesterol has also been reported (Non-Patent Document 2). This example is a technology that makes nano-sized particles in water with cholesterol as a core, and does not provide a polymer having stimuli-responsiveness.
非特許文献 l : Shimokuri, Τ·; aneko, Τ·; Akashi, Μ. J. Polym. Sci. Part A: Polym. Chem. 2004, 42, 4492-4501. Non-Patent Literature l: Shimokuri, Τ ·; aneko, Τ ·; Akashi, Μ. J. Polym. Sci. Part A: Polym. Chem. 2004, 42, 4492-4501.
非特許文献 2 : Akiyoshi, . et al., Macromolecules 2000, 33, 6752-6756 Non-Patent Document 2: Akiyoshi,. Et al., Macromolecules 2000, 33, 6752-6756
発明の開示 Disclosure of the invention
発明が解決しょうとする課題
[0006] 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。 Problems to be solved by the invention [0006] 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. To do.
[0007] 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, leading to the present invention.
課題を解決するための手段 Means for solving the problem
[0008] 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 [0008] That is, the present invention relates to a polylysine derivative into which 1, 2 epoxyalkane is introduced.
[0009] 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 —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 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レ、。 [0009] The lysine that is a constituent monomer of the polylysine used in the present invention may be a constituent unit of any of L-form, D-form, or a mixture thereof. That is, as polylysine, any of poly (ε L lysine), poly (ε D lysine), poly (ε DL lysine), poly (α L lysine), poly (α-D-lysine), poly-DL-lysine) There may be. L-form is a naturally occurring form, so polylysine composed of L-lysine is usually used. Among them, poly (L-lysine) (ε-PL) is a polyamino acid derived from microorganisms produced by streptomyces albulus 346, that is, a naturally occurring polyamino acid, a homopolymer of L-lysine, and a primary amino acid that can be modified in the side chain. Has a group. This ε-PL is used as a food additive and is highly safe for living bodies.
[0010] 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 [0010] Since it aims to provide a novel stimuli-responsive polymer having biodegradability and biocompatibility that develops thermosensitive response under physiological conditions, polylysine is used that is soluble in water. Like that. The physiological condition is not limited to the biocompatibility condition, and it is not necessary to be limited to only those that are soluble in water if the purpose is to have a heat-responsive property.
[0011] 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ょレ、。 [0011] The molecular weight of polylysine can be adjusted by adjusting the degree of polymerization of lysine, and can be selected as needed.
[0012] 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ぐ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。 [0012] Usually, those having a number average molecular weight (Mn) of several thousand to 200,000, preferably several thousand to 100,000 are used. If a low molecular weight is used, it is expected that the intermolecular interaction, which is the driving force for developing the thermosensitive response, is weak and it is difficult to express the response. If the molecular weight is too high, high molecular weight polylysine is used in the synthesis process. There is a possibility that they form a complex and the hydrophobic group cannot be efficiently introduced.
[0013] 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.
[0014] ポリリシンに導入される 1 , 2—エポキシアルカンは、アルカンの炭素数力 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 せること力 S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。 [0014] The 1,2-epoxyalkane introduced into the polylysine is 1,4-epoxybutane or 1,2-epoxypentane of 4 or 5 or more, preferably 1,2 or more, more preferably 1, 2—Epoxybutane. If the alkane has 3 or less carbon atoms, it is impossible to develop thermal response. In addition, 1,2-epoxyalkanes with too many carbon atoms are not soluble in water, and therefore a synthetic contrivance is required when introducing them into polylysine.
[0015] 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ァミンの 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ァミンにヒドロキシアルキル基が導入される。 [0015] The introduction of 1,2-epoxyalkane into polylysine involves dissolving polylysine in a suitable solvent, adding and dissolving a predetermined amount of 1,2-epoxyalkane, and then adding it to the epoxy group of the first amine of polylysine. The nucleophilic ring-opening reaction can be carried out. As a result, a hydroxyalkyl group is introduced into the primary amine of polylysine.
[0016] 例えば、ポリリシンとしてポリ( E —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—ェ 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 (E-L-lysine) is used as polylysine and 1,2 epoxybutane is used as 1,2 epoxyalkane, it can be synthesized in one step in an aqueous solution using water as a solvent. When 1,2-epoxypentane is used as 1,2-epoxyalkane, 1,2-epoxypentane is insoluble in water, so once 1,2-epoxybutane is introduced into poly (ε-L lysine) It is only necessary to impart hydrophobicity to such an extent that 1,2-epoxypentane can be dissolved in an organic solvent in which 1,2-epoxypentane can be dissolved, and to introduce 1,2-epoxypentane in the organic solvent.
[0017] 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 すること力 Sでさる。 [0017] By introducing 1,2-epoxyalkane into polylysine, it is possible to impart hydrophobicity to polylysine with force S.
[0018] 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 ァミンへの 1 , 2 エポキシアルカンの導入の割合)が、 14%を超えると、 DMSO,ェ 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。 [0018] For example, when poly (ε-L-lysine) has an introduction rate of 1.2-epoxybutane (a ratio of 1,2-epoxyalkane introduction into the first amine of polylysine) exceeding 14%, The solubility of organic solvents such as DMSO and ethanol is improved. The introduction rate can be adjusted by changing the amount of 1,2-epoxyalkane such as 1.2-epoxybutane at the time of synthesis.
[0019] 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
一濃度(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、リン酸水素ニナト 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。 [0019] 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. Phase separation temperature is more than 59% hydrophobic group introduction rate), polymer It can be controlled by changing one concentration (0 · 125 to 2 · 0 wt%) and 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 1,2-epoxybutane is introduced into poly (ε-L-lysine) at about 59% or more, the phase separation temperature is observed in NaCl (1.0 M) and pH 12 buffer (salt concentration 150 mM). . Note that the composition of the PH12 buffer solution (salt concentration 150 mM) used in this example means a sodium hydrogen phosphate / sodium hydroxide aqueous solution, and the salt concentration of 150 mM is close to physiological conditions. .
[0020] 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ーリ 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— Β)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[0020] The mechanism of heat sensitivity varies depending on the structure of the main chain. For example, a polylysine derivative (ε-PL-Β) in which 1,2-epoxybutane is introduced into poly (εL-lysine) exhibits heat-responsiveness due to coacervate, and 1 (poly- α -L-lysine) 2-—Polylysine derivatives containing epoxybutane exhibit thermal responsiveness due to coil-globule transition
〇 Yes
[0021] ε PL— Βは、相分離状態で形成するコアセルべ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ァニオン性化合物、例えばァニオン性色素、ァニオン性 のタンパク質、 DNA、 RNA等を分離、濃縮することが可能である。 [0021] ε PL—Β is an anionic compound such as an anionic dye, an anionic protein, DNA, using a hydrophobic interaction and an electrostatic interaction as driving forces in a coacervate droplet formed in a phase-separated state. RNA, etc. can be separated and concentrated.
発明の効果 The invention's effect
[0022] 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。 [0022] A novel polylysine derivative incorporating a 1,2-epoxyalkane was provided.
図面の簡単な説明 Brief Description of Drawings
[0023] [図 1] ε —PL— Bの1 H— NMRチャート。 [0023] [Fig. 1] 1 H-NMR chart of ε-PL-B.
[図 2a] ε PL— Β水溶液の透過率の温度依存性 (加熱過程)を示す図。 FIG. 2a is a graph showing the temperature dependence (heating process) of the transmittance of an ε PL--aqueous solution.
[図 2b] ε PL— Β水溶液の透過率の温度依存性 (冷却過程)を示す図。 [Fig. 2b] A graph showing the temperature dependence (cooling process) of the transmittance of ε PL- aqueous solution.
[図 2c] ε PL— Β水溶液の相分離温度の導入率依存性を示す図。 FIG. 2c is a graph showing the dependence of the phase separation temperature on the introduction rate of the ε PL-sodium aqueous solution.
[図 3a] ε PL— Β93水溶液の透過率の温度依存性 (加熱過程)を示す図。 FIG. 3a is a graph showing the temperature dependence (heating process) of the transmittance of an aqueous solution of ε PL-Β93.
[図 3b] ε PL— Β93水溶液の透過率の温度依存性 (冷却過程)を示す図。 [Fig. 3b] A graph showing the temperature dependence (cooling process) of the transmittance of ε PL- Β93 aqueous solution.
[図 3c] ε PL— Β93水溶液の相分離温度の水素イオン濃度依存性を示す図。 FIG. 3c is a graph showing the dependence of the phase separation temperature of ε PL- Β93 aqueous solution on the hydrogen ion concentration.
[図 4] ε —PL— Β93の CDスぺクトノレを示す図。 [Fig.4] A diagram showing the CD spectrum of ε -PL- Β93.
[図 5] εー?しー893の01^測定の結果を示す図。 [Figure 5] ε-? The figure which shows the result of 01 ^ measurement of shi-893.
[図 6] a PL— B水溶液の温度変化に対する光透過率を示す図。
[図 7] ε PL— Β93% α PL— Β72%の DSC測定結果を示す図。 FIG. 6 is a graph showing light transmittance with respect to temperature change of a PL-B aqueous solution. FIG. 7 is a diagram showing DSC measurement results of ε PL——93% α PL—Β72%.
[図 8] aー?しー883の01^測定の結果を示す図。 [Figure 8] a? The figure which shows the result of 01 ^ measurement of shi-883.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
(実施例 1 ) (Example 1)
ポリ [Ν α—(2—ヒドロキシブチル)リシン] ( ε —PL— B)の合成 Synthesis of poly [Ν α — (2-hydroxybutyl) lysine] (ε —PL— B)
ポリ 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) 820 mg (5. Ounit 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°Cで 24時間反応させた。 Poly L-lysine) (ε-PL) (trade name: Polylysine hydrochloride: manufactured by Chisso Corporation) After dissolving 820 mg (5. Ounit mmol) in 25 ml of ultrapure water, a predetermined amount (described in Table 1 below) Butylene oxide was added and reacted at 40 ° C for 24 hours.
[化 1] NH- CH2- CH2 CH2- CH2- ?H- + H 」CH CH2 CH3 [Chemical 1] NH- CH 2 -CH 2 CH 2 -CH 2- ? H- + H "CH CH 2 CH 3
NH2 NH 2
超純水 Ultrapure water
[0025] 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1 H— NMR FT— IRを使用して 行った。 [0025] After completion of the reaction, a dialysis membrane having a molecular weight fraction of 500 was used to dialyze with distilled water, and the polymer was purified by freeze drying. The structure of the product was confirmed using 1 H-NMR FT-IR.
[0026] 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 FIG. 1 shows a 1 H-NMR chart of ε PL-B. All peaks are ε —PL and ε
PL— Βに由来する。 PL—Derived from firewood.
[0027] 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。 [0027] The introduction rate of 2-hydroxybutyl group was calculated from the ratio of the integrated value of the peak attributed to the methine of the ε-PL-B main chain and the peak attributed to the methyl group of the side chain. The introduction rate of 2-hydroxybutyl group was 14 to 93%.
[0028] 溶解性試験 [0028] Solubility test
ポリマー濃度 0· 2重量%の濃度で、 ε PLと ε PL— Β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。 Ε PL and ε PL— for various solvents at a polymer concentration of 0.2% by weight Solubility was tested. The results are shown in Table 1.
[0029] [表 1] 合成 溶解性 [0029] [Table 1] Synthesis Solubility
1,2-ェボキ 導入率 e) 収 超 ェタノ ァセト DMF DMSO 1Ή¥ ク ρ サンプノレ シブタン (%) 率 純 ール ン ホノレム 1,2-Eboki Introduction rate e) Revenue Ethanoacetate DMF DMSO 1Ή ク ρ Sampnore Sibutane (%) Rate Pure Rune Honolem
(mmol' (%) 水 (mmol '(%) water
ε-PL 0 一 - - - - - i 2.5 14 82 - - + - 一 ε-PL 0 one-----i 2.5 14 82--+-one
2 5.0 37 76 + - - - + 2 5.0 37 76 +---+
3 10—0 59 80 + + - - 3 10—0 59 80 + +--
4 15—0 73 81 - + + ^ 4 15—0 73 81-+ + ^
5 25.0 80 84 + + - + + - - 5 25.0 80 84 + +-+ +--
6 30—0 85 89 + 十 + - -6 30—0 85 89 + tens +--
7 50.0 93 88 + + - + 7 50.0 93 88 + +-+
a)6-PL (5.0ュュット mmol) と 1,2-エポキシブタンを水中 40 °Cで 24時間反応させた。 b) 溶解性試験の時のボリマー濃度は 0.2 wt%とした。 +: 溶解- 不溶- c) C は 2-ヒ ドロキ シブチル基の導入率を示しており ^-NMR により算出した。 a) 6-PL (5.0 μt mmol) and 1,2-epoxybutane were reacted in water at 40 ° C for 24 hours. b) The polymer concentration at the solubility test was 0.2 wt%. +: Dissolved-insoluble- c) C represents the introduction rate of 2-hydroxybutyl group and was calculated by ^ -NMR.
[0030] ε PLは超純水に対しては溶解性を示した力 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。 [0030] ε PL has a solubility in ultrapure water. It was insoluble in organic solvents. On the other hand, ε-PL-B with 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.
[0031] ε PL— Βの感熱応答性評価 [0031] ε PL— Evaluation of thermal response of rice cake
ε PL— Β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。 The thermal response of ε PL- PL was evaluated by measuring the light transmittance of the solution with respect to temperature change using UV-vis spectrum.
[0032] ε PL— Βを超純水、 NaCl水溶液(1. 0M)、 pH7. 4, 10, 12の緩衝液(塩濃度 [0032] ε PL-Β is ultrapure water, NaCl aqueous solution (1.0 M), pH 7.4, 10, 12 buffer solution (salt concentration
150mM)に溶解(ポリマー濃度は超純水および NaCl水溶液では lwt%とし、緩衝 液中では 0. 25wt%とした)させ、スリット幅 lmm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 l°C/minとし、相分離温度は透過率 90 %の時の温度とした。 150mM) (the polymer concentration was 1wt% for ultrapure water and NaCl aqueous solution and 0.25wt% for buffer solution), and the absorbance at 500nm wavelength was measured in a quartz cell with slit width lmm. . The heating / cooling rate was l ° C / min, and the phase separation temperature was the temperature at a transmittance of 90%.
[0033] 2 ヒドロキシフ、、チノレ基導人率 14, 37, 59, 73, 80, 930/0の ε—PL— B水溶 ί夜の 透過率測定から得られた相分離温度を表 2に示した。 [0033] 2-hydroxyphenyl ,, Chinore Motoshirube human factor 14, 37, 59, 73, 80, 93 0/0 of epsilon-PL-phase separation temperature obtained from the measurement of transmittance B water ί night Table 2 It was shown to.
NaCl水溶液(1 · 0M) (ポリマー濃度 1 1%)の ε PL Β水溶液の光過率変化と 93% ε PL— Β水溶液(ρΗ7· 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、相分離温度の水素ィ オン濃度依存性を示してレ、る。 Change in light excess of ε PL Β aqueous solution of NaCl aqueous solution (1 · 0M) (polymer concentration 1 1%) and 93% ε PL—Β aqueous solution (ρΗ7 · 4, 10, 12) (polymer concentration 0.25 wt%) Light transmittance The changes (phase separation temperature) are shown in Fig. 2 (a)-(c) and Fig. 3 (a)-(c), respectively. Figures 2 (a) and 3 (a) show the change in transmittance during the heating process, Figures 2 (b) and 3 (b) show the change in transmittance during the cooling process, and Figure 2 (c) shows the phase. The dependence of the separation temperature on the introduction rate of hydrophobic groups is shown. Figure 3 (c) shows the dependence of the phase separation temperature on the hydrogen ion concentration.
[表 2] 導入率 NaCl aq (1.0 M) b) 緩衝液ひ 50 m ) c) [Table 2] Introduction rate NaCl aq (1.0 M) b ) Buffer solution 50 m) c )
{%) a) pH 7.4一 pFi 10 pH 12 加熱 冷却 加熱 冷却 加熱 冷却 加熱 冷却 (%) a) pH 7.4 1 pFi 10 pH 12 Heating Cooling Heating Cooling Heating Cooling Heating Cooling
14 - - 14 - -
37 - -37--
59 88 d) 7 Φ - - - - 84.1 81.859 88 d) 7 Φ----84.1 81.8
73 83 64 76.5 73 70.4 66.3 52.3 48.173 83 64 76.5 73 70.4 66.3 52.3 48.1
80 67 54 61 58.1 54.9 51.8 39.7 37.480 67 54 61 58.1 54.9 51.8 39.7 37.4
93 34 32 46.2 44 39.5 36.6 30.1 28.4 a) 2-ヒドロキシブチル基の ¾入率は NMRにより算出した。 b) ポリマー濃度は 1 wt% とした。 c) ボリマー濃度は 0.25 wtu/0とした。 d) 相分離温度は透過率 %の時の温度とした。 93 34 32 46.2 44 39.5 36.6 30.1 28.4 a) The rate of incorporation of 2-hydroxybutyl groups was calculated by NMR. b) The polymer concentration was 1 wt%. c) The polymer concentration was 0.25 wt u / 0 . d) The phase separation temperature was the temperature at the transmittance%.
[0035] 超純水に溶解した ε PL— Β ( 14〜93%)のすベてのサンプルは、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力、る。 [0035] All the samples of ε PL-Β (14 to 93%) dissolved in ultrapure water did not show thermal sensitivity in ultrapure water. In a NaCl aqueous solution (1.0 M) and pH 7.4, 10, and 12 buffer solutions (salt concentration 150 mM), ε-PL-B with a 2-hydroxybutyl group introduction rate of 59% or more exhibited heat-responsiveness. In addition, according to Fig. 2 (c), the phase separation temperature shifts to the low temperature side as the introduction rate increases.
[0036] NaCl水溶液(1 · 0M)中では導入率 59, 73, 80, 93%の ε PL— Β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— Β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 ること力 S分力、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。 [0036] In NaCl aqueous solution (1 · 0M), changes in transmittance with respect to temperature change were observed in ε PL-sodium aqueous solutions with introduction rates of 59, 73, 80, and 93%. In addition, the reversible transmittance change was observed in the ε PL-sodium aqueous solution with the introduction rate of 59, 73, 80, 93% even in the pH 7.4, 10, 12 buffer solution. According to Fig. 3 (c), it is possible to control the phase separation temperature by changing the pH. Although the data is not shown, the phase separation temperature could be controlled by polymer concentration, pH, ionic strength, and introduction rate.
[0037] 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べ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。
[0038] 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 ノ 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ァミノ基の静電反発を低下させることにより、 2—ヒドロキシプチ 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。 [0037] In addition, a clear endothermic peak of water near the phase separation temperature was not observed by differential scanning thermogravimetry at or above the phase separation temperature, and droplets due to the concentrated phase were observed from the micrographs. It was suggested that heat response was expressed by the bait. [0038] From the above results, by introducing a 2-hydroxybutyl group to adjust the parent-hydrophobic tolerance of the entire ε PL molecular chain appropriately, and increasing the polarity of water or increasing the pH by adding inorganic salts It has been clarified that by reducing the electrostatic repulsion of the side chain amino groups, the thermal response of εPL can be imparted by increasing the hydrophobic interaction of 2-hydroxypropyl.
[0039] ε PL— Β水溶液のイオン性色素との複合化挙動 [0039] ε PL— Complexation behavior of Β aqueous solution with ionic dye
コアセルべートによる感熱応答性を発現した ε PL— Βは側鎖にァミノ基が残存し てレ、る。そこでイオン性色素であるトリパンブルー(TB:ァニオン性)とメチレンブルー (MB :カチオン性)を使用して ε PL— Β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。 Ε PL— し た, which has developed a thermosensitive response due to coacervate, has an amino group remaining in the side chain. Therefore, we observed the complexation behavior of ε PL-— aqueous solution around the phase separation temperature using trypan blue (TB: anionic) and methylene blue (MB: cationic), which are ionic dyes.
[0040] 導入率 93%の ε PL— Β ( ε PL— Β93)水溶液(ρΗ7· 4, LCST (相分離温 度) 45°C)中に TBと MBをそれぞれ 10 M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っレ、て観察した。 [0040] TB and MB were added to a solution of 93% in an ε PL-% (εPL-Β93) aqueous solution (ρΗ7 · 4, LCST (phase separation temperature) 45 ° C) at 93% introduction rate, respectively. The formation of an ion complex around the phase separation temperature was observed.
[0041] 相分離温度以下である 20°Cでは ε PL— Β93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°Cではァ 二オン性色素である TBがコアセルべ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— Β93が会合 してコアセルべートによる濃厚相を形成し、静電相互作用によりァニオン性色素であ 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。 [0041] It was confirmed that TB and MB were uniformly dissolved in the εPL-—93 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 into coacervate droplets and precipitated. Above the phase separation temperature, ε PL- Β93 associates with a hydrophobic interaction as a driving force to form a dense phase by coacervate, and only TB, which is an anionic pigment, drops by electrostatic interaction. It is thought that it happened because it concentrated inside.
[0042] TBの吸収波長である 570nmの吸光度を測定したところ 50°Cの ε PL— Β93水 溶液の上澄み液からは TBの吸収は観察されな力、つた。 [0042] When the absorbance at 570 nm, which is the absorption wavelength of TB, was measured, the absorption of TB was observed from the supernatant of the ε PL- PL93 aqueous solution at 50 ° C.
[0043] 以上の結果より ε PL— Β93は感熱応答により形成するコアセルべート滴内に静 電相互作用を介してァニオン性化合物、例えばァ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。 [0043] Based on the above results, ε PL- Β93 is also expected to capture anionic compounds, such as anionic proteins and DNA, via electrostatic interactions in coacervate droplets formed by the thermal response. It is expected to be used as a separation material using temperature stimulation.
[0044] ε PL— Β水溶液の CDスペクトル測定 [0044] ε PL— Measurement of CD spectrum of aqueous solution
ε — PL— Β (導入率 93%)の CDスぺクトノレを、 ρΗ7·4, 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 of ε—PL—— (introduction rate: 93%) was measured under the conditions of ρΗ7, 4, 10, 12, polymer concentration of 0.005 wt% and buffer concentration of 150 mM. The results are shown in FIG.
[0045] ρΗ7·4, 12の条件下で ε —PLと ε —PL— Βは /3 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— Βの /3構造は安定化することが分かる。 [0045] It is clear that ε —PL and ε —PL— を have a / 3 sheet structure under the conditions of ρΗ7 · 4, 12. It became. Furthermore, it can be seen that the / 3 structure of ε PL— Β is stabilized by heating.
[0046] 8 PL— B水溶液の DLS測定 [0046] DLS measurement of 8 PL- B aqueous solution
ε PL— Β (導入率 93%)、 ρΗ7· 4、相分離温度 44°C、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、 ε PL— Β (introduction rate 93%), ρΗ7 · 4, phase separation temperature 44 ° C, polymer concentration 0.5 wt%, buffer concentration 150 mM DLS with ε —PL— B (introduction rate 93%) The 20,
40、 60°C条件下で測定した。結果を図 5に示した。 Measurements were made at 40 and 60 ° C. The results are shown in FIG.
[0047] ε PL— Β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。 [0047] The ε PL-sodium aqueous solution formed an aggregate having a particle size of about 400 nm at a temperature higher than the phase separation temperature and driven by the hydrophobic interaction of 2-hydroxybutyl groups.
[0048] ポリ [N E—(2 ヒドロキシブチル)リシン] ( a—PL— B)の合成 [0048] Synthesis of poly [N E — (2 hydroxybutyl) lysine] (a—PL— B)
ポリ [Να— (2—ヒドロキシブチル)リシン] ( ε —PL— B)と同様の方法でポリ [Ν Ε -Poly [Ν α - (2- hydroxybutyl) lysine] (ε -PL- B) similar to the manner in poly [New E -
(2 ヒドロキシブチル)リシン] ( α— PL— Β)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。 (2 Hydroxybutyl) lysine] (α-PL-Β) was synthesized. Table 3 shows the synthesis results and solubility in water.
[0049] [表 3] [0049] [Table 3]
1,2-ュ -ボキ 収率 超純水 pH 7.4 pH 12 サンプ 1 シブタン 1,2-Bo-Yield Ultrapure water pH 7.4 pH 12 Sump 1 Sibutane
mmol % % mmol%%
1 2.9 50 75 + + 十- 1 2.9 50 75 + + Ten-
2 4.3 72 78 + + -2 4.3 72 78 + +-
3 7.2 83 86 +— - -3 7.2 83 86 + —--
4 14.4 95 83 - - - -PL(1.44ュュット nunc °Cで 24時間反応させた。4 14.4 95 83----PL (reacted for 24 hours at 1.44 nuts nunc ° C.
+:溶解 +-:分散, -:不溶 +: Dissolved +-: Dispersed,-: Insoluble
[0050] 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% , 7 2%の a PL— Bは超純水に溶解した力 S、導入率 83%の a PL— Bは超純水中 で分散し、 95%の a PL— Bは、超純水に対して不溶であった。 [0050] The introduction rate of 2 hydroxybutyl groups was 50, 72, 83, 95%. A PL-B with an introduction rate of 50% and 72% is a force S dissolved in ultrapure water, a PL-B with an introduction rate of 83% is dispersed in ultrapure water, and a PL-B of 95% is It was insoluble in ultrapure water.
[0051] また導入率 50, 72%の a PL— Bは ρΗ7· 4の緩衝液に対する溶解性を示した 1S 導入率 83%の a—PL— Bは ρΗ7· 4の緩衝液中で沈殿した。 a—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 a—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 らカ、となった。これは a—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。
[0052] 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!/、て検討し、水に分散した導入率 83 %の動的光散乱 (DLS )測定を行つ た。 [0051] Moreover, a PL-B with an introduction rate of 50, 72% showed solubility in the buffer solution of ρΗ7.4. 1a a-PL-B with an introduction rate of 83% precipitated in the buffer solution of ρΗ7.4 . The solubility of water for a-PL-B decreased with increasing penetration. It was clear that the synthesized a-PL-B had a lower solubility in water when the introduction rate was similar to that of ε-PL B. This is considered to be due to the fact that a-PL-B has a longer hydrophobic chain side chain and thus has a stronger hydrophobic interaction than ε-PL-B. [0052] The introduction rate of 50%, 72% α PL-B, which showed solubility in pH 7.4 buffer solution, was investigated! Light scattering (DLS) measurement was performed.
[0053] 図 6に導入率 50 % (ポリマー濃度: lwt% ) ,導入率 72 % (ポリマー濃度: lwt% , 0 . 2 1% )の《 PL— B水溶液(ρΗ7 · 4)の温度変化に対する光透過率測定の結 果を示した。 [0053] Figure 6 shows the change in temperature of PL-B aqueous solution (ρΗ7 · 4) with an introduction rate of 50% (polymer concentration: lwt%) and an introduction rate of 72% (polymer concentration: lwt%, 0.21%). The results of light transmittance measurement are shown.
[0054] 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力 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— Β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。 [0054] Disturbance in the force transmittance change, in which it was confirmed that only a sample with an introduction rate of 72% exhibited sensory response, was observed. When the α PL-sodium solution above the phase separation temperature was observed, polymer precipitation and precipitation were observed.
[0055] DS C測定 [0055] DS C measurement
ε —PL— Β (導入率 93%)、 a—PL— B (導入率 72 % )について、 ρΗ7 · 4、ポリマ 一濃度 0. 5wt%、バッファー濃度 1 50mM、加熱 ·冷却速度 2°C/分の条件下で、 DSC測定を行った。結果を図 7に示す。 ε -PL- Β (introduction rate 93%), a-PL- B (introduction rate 72%), ρΗ7 · 4, polymer concentration 0.5 wt%, buffer concentration 1 50 mM, heating and cooling rate 2 ° C / DSC measurement was performed under the condition of minutes. The results are shown in FIG.
[0056] DS C測定の結果より、 ε PL— Βは、液 液相分離であるコアセルべートにより感 熱応答性を発現し、 a 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— Β水溶液 (ρΗ7 . 4)の相分離温度は約 65°Cであることがわかる。 [0056] From the results of DSC measurement, ε PL—Β exhibits thermal responsiveness due to coacervate, which is liquid-liquid phase separation, and a PL-B, is coiled globule, which is liquid-solid phase separation. It was confirmed that heat responsiveness was expressed by the transfer. In addition, Fig. 7 shows that the phase separation temperature of α PL-— aqueous solution (ρΗ7.4) is about 65 ° C.
[0057] ε PL— Bの相分離状態では液一液相分離であつたのでポリマーの沈殿は確認 されなかったが、 a—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、 a PL— B 72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。 [0057] In the phase separation state of ε PL-B, liquid-liquid separation was used, so no polymer precipitation was confirmed. However, a-PL-B has a long side chain hydrophobic group, Stronger hydrophobic interaction than ε-PL-B works, and a PL-B 72 aqueous solution is considered to be precipitated by polymer association in response to temperature stimulation and using hydrophobic interaction as a driving force. .
[0058] 図 8に、水分散性を示した α— PL— Β83の DLS測定の結果を示した。 20°Cと 50 °Cで測定した α— PL— B83の平均粒径はそれぞれ 1 77nmと 183nmであった。 α PL— Β83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《 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、表面にァミノ基が集積さ れた会合体を形成していることが明らかとなった。 FIG. 8 shows the result of DLS measurement of α-PL-—83 that showed water dispersibility. The average particle diameters of α-PL-B83 measured at 20 ° C and 50 ° C were 177 nm and 183 nm, respectively. α PL- Β83 was confirmed to form relatively monodisperse aggregates in water using the hydrophobic interaction of 2-hydroxybutyl groups as the driving force. When the zeta potential of << 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 was formed on the surface.
[0059] 本実施例においては、ポリリシンとして、ポリ —L リシン)およびポリ —L リ
シン)を使用した実施例を示しているが、ポリ 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( a—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。 [0059] In this example, poly-L lysine) and poly-L lysine are used as polylysine. Examples of using poly (D-lysine), poly (εD, L-lysine) (mixture of lysine monomer units D and L), poly (a-D-lysine), We believe that poly (αD, L-lysine) can be used as well.
産業上の利用可能性 Industrial applicability
本発明のポリリシン誘導体は、新規な生分解性分離材料、ァ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 carrier of a novel biodegradable separation material, an anionic drug.
Claims
[1] 1 , 2—エポキシアルカンを導入したポリリシン誘導体。 [1] 1,2—Polylysine derivative introduced with epoxyalkane.
[2] ポリリシンがポリ( ε L リシン)またはポリ(α L リシン)である、請求項 1に記 載のポリリシン誘導体。 [2] The polylysine derivative according to claim 1, wherein the polylysine is poly (εL-lysine) or poly (αL-lysine).
[3] ポリリシンがポリ( ε—L リシン)である、請求項 1に記載のポリリシン誘導体。 [3] The polylysine derivative according to claim 1, wherein the polylysine is poly (ε-L-lysine).
[4] 1 , 2—エポキシアルカン力 1 , 2—エポキシブタンである、請求項 1または 2に記載 のポリリシン誘導体。 [4] The polylysine derivative according to claim 1, which is 1,2-epoxyalkane force 1,2-epoxybutane.
[5] 1 , 2 エポキシアルカン力 ポリリシン誘導体の有する第 1ァミンに対して 14%以上 導入された、請求項;!〜 4いずれかに記載のポリリシン誘導体。 [5] 1, 2 Epoxyalkane force The polylysine derivative according to any one of claims 1 to 4, wherein 14% or more is introduced with respect to the first amine of the polylysine derivative.
[6] 1 , 2 エポキシアルカン力 ポリリシン誘導体の有する第 1ァミンに対して 14%以上 導入された、請求項;!〜 4いずれかに記載のポリリシン誘導体。 [6] 1, 2 Epoxyalkane force The polylysine derivative according to any one of claims 1 to 4, wherein 14% or more is introduced with respect to the first amine of the polylysine derivative.
[7] 請求項;!〜 6いずれかに記載のポリリシン誘導体を含有し、 ρΗが 7以上に維持され た感熱応答性を有する水溶液組成物。 [7] An aqueous solution composition comprising the polylysine derivative according to any one of claims 6 to 6 and having a heat-responsive property in which ρΗ is maintained at 7 or more.
[8] 請求項 1〜6いずれかに記載のポリリシン誘導体を使用することを特徴とする、ァニ オン性化合物の濃縮、分離方法。
[8] A method for concentrating and separating an anionic compound, characterized by using the polylysine derivative according to any one of [1] to [6].
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JPH0790080A (en) * | 1993-09-22 | 1995-04-04 | Yamanouchi Pharmaceut Co Ltd | D-galactopyranosyl-gluconic acid derivative of poly-epsilon-substituted-l-lysine |
JPH11152330A (en) * | 1997-11-19 | 1999-06-08 | Chisso Corp | Polylysine, production of polylysine, polylysine composition, and production of medicine which removes endotoxin |
JPH11255892A (en) * | 1998-03-12 | 1999-09-21 | Asahi Denka Kogyo Kk | Easily soluble acylated polylysine and preparation thereof |
JP2003171464A (en) * | 2001-12-06 | 2003-06-20 | Chisso Corp | Polylysine and method for producing the same |
JP2003335857A (en) * | 2002-05-22 | 2003-11-28 | Chisso Corp | URETHANIZED epsilon-POLYLYSINE, CHEMICALLY MODIFIED URETHANIZED epsilon-POLYLYSINE, CHEMICALLY MODIFIED epsilon- POLYLYSINE AND PROCESS FOR PRODUCING THEM |
JP2004035791A (en) * | 2002-07-05 | 2004-02-05 | Univ Kyoto | Temperature responsive polymer and temperature responsive gel |
JP2007230871A (en) * | 2006-02-27 | 2007-09-13 | Osaka Univ | Stimuli-responsive material |
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ATE229992T1 (en) * | 1999-05-19 | 2003-01-15 | Basf Ag | POLYMERS CONTAINING ALKOXYLATED, CONDENSED BASIC AMINO ACIDS AND METHOD FOR THE PRODUCTION THEREOF |
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JPH0790080A (en) * | 1993-09-22 | 1995-04-04 | Yamanouchi Pharmaceut Co Ltd | D-galactopyranosyl-gluconic acid derivative of poly-epsilon-substituted-l-lysine |
JPH11152330A (en) * | 1997-11-19 | 1999-06-08 | Chisso Corp | Polylysine, production of polylysine, polylysine composition, and production of medicine which removes endotoxin |
JPH11255892A (en) * | 1998-03-12 | 1999-09-21 | Asahi Denka Kogyo Kk | Easily soluble acylated polylysine and preparation thereof |
JP2003171464A (en) * | 2001-12-06 | 2003-06-20 | Chisso Corp | Polylysine and method for producing the same |
JP2003335857A (en) * | 2002-05-22 | 2003-11-28 | Chisso Corp | URETHANIZED epsilon-POLYLYSINE, CHEMICALLY MODIFIED URETHANIZED epsilon-POLYLYSINE, CHEMICALLY MODIFIED epsilon- POLYLYSINE AND PROCESS FOR PRODUCING THEM |
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JP2007230871A (en) * | 2006-02-27 | 2007-09-13 | Osaka Univ | Stimuli-responsive material |
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