JP6914487B2 - Composite porous material of nanoparticles for cancer hyperthermia and bioabsorbable polymer and its manufacturing method - Google Patents
Composite porous material of nanoparticles for cancer hyperthermia and bioabsorbable polymer and its manufacturing method Download PDFInfo
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Description
本発明は、外科手術でがん組織を除去した部位に埋め込まれ、あるいはがん組織を直接覆い、近赤外光を体外から照射されて発熱することにより、多孔質材料内あるいは周囲のがん細胞を死滅に至らしめる多孔質材料及びその製造方法に関する。 The present invention is implanted in a site from which cancer tissue has been removed by surgery, or directly covers the cancer tissue and is irradiated with near-infrared light from outside the body to generate heat, thereby causing cancer in or around the porous material. The present invention relates to a porous material that causes cell death and a method for producing the same.
がんはすべての病気の中で第一位の死因であり、現在でも３人に１人ががんで死亡している。高齢化社会の進行に伴い、この割合は今後増加すると予測されている。現在、がんの治療法として、手術療法、化学療法、放射線療法が三大療法といわれているが、がんの根本的治療に対する社会のニーズはきわめて高く、様々な治療方法と治療薬が開発されている。 Cancer is the leading cause of death among all illnesses, and one in three people still die of cancer. This percentage is projected to increase in the future as the aging society progresses. Currently, surgical therapy, chemotherapy, and radiation therapy are said to be the three major therapies for cancer, but the social needs for the fundamental treatment of cancer are extremely high, and various treatment methods and drugs have been developed. Has been done.
がん三大療法のうち、がん組織を手術で切除する治療法は最も直接的である。手術療法では、がん組織を切除し、その周辺組織やリンパ節に転移があれば、それらも一緒に切除する。早期のがんやある程度の進行がんであっても、切除可能であれば手術療法が積極的に行われている。手術療法には、塊状のがん組織を一気に切除できるというメリットがある。 Of the three major cancer therapies, the most direct treatment is surgical removal of the cancerous tissue. In surgery, the cancerous tissue is removed, and if there are metastases in the surrounding tissue or lymph nodes, they are also removed. Even if the cancer is early stage or advanced to some extent, surgical treatment is actively performed if it can be resected. Surgical therapy has the advantage of being able to remove massive cancerous tissue at once.
ただし、手術療法では、手術メスを入れることによって生じた創部の治癒や全身機能の回復にある程度時間がかかる。さらに、切除後の正常部位どうしをそのまま継ぎ合わせるため、切除した部位の大きさによっては臓器の機能が低下してしまう懸念もある。こうしたデメリットを低減するために、最近では、切除する範囲をできるだけ最小限にとどめる手術や、内視鏡による腹腔鏡下・胸腔鏡下手術のように体への負担を少なくする手術も行われるようになった。 However, in surgical therapy, it takes some time to heal the wound and restore systemic function caused by inserting a scalpel. Furthermore, since the normal parts after excision are joined as they are, there is a concern that the function of the organ may deteriorate depending on the size of the excised part. In order to reduce these disadvantages, recently, surgery to minimize the excision area and surgery to reduce the burden on the body such as endoscopic laparoscopic and thoracoscopic surgery are being performed. Became.
しかし現実には手術の後にかなりの頻度でがんが再発する。これは、肉眼では見えないがん細胞や微小ながん組織が手術後も体内に残存してしまうためである。ただし、手術で切除する範囲を小さくすればするほどがん細胞を取り残してしまう可能性が高くなり、再発のリスクが上昇する。 However, in reality, the cancer recurs quite often after surgery. This is because cancer cells and minute cancer tissues that are invisible to the naked eye remain in the body even after surgery. However, the smaller the surgical excision area, the more likely it is that cancer cells will be left behind, increasing the risk of recurrence.
手術療法では、取り残したがん細胞や微小ながん組織をどのように治療するかが問題となる。通常、手術後には抗がん剤による治療、すなわち化学療法や放射線療法を併用することがある。このように、複数の治療法を組み合わせて、総合的に治療を進める集学的治療が行われている。 In surgical therapy, how to treat leftover cancer cells and minute cancer tissues becomes a problem. Usually, after surgery, treatment with anticancer drugs, that is, chemotherapy or radiation therapy, may be used in combination. In this way, multidisciplinary treatment is performed in which a plurality of treatment methods are combined to promote comprehensive treatment.
前記の三大療法以外には、がん細胞が正常細胞に比べて熱に弱いという性質を利用し、がん細胞を死滅させる温熱療法が開発されている。高周波誘電加温法は、生体を電極の間に置き、全身を４２℃程度に加温する方法である。ただし、血流の冷却作用のため、がん組織内部の温度は上がらず、がん組織を死滅させるには十分ではない。そこで、近赤外光を照射したときに発熱する金ナノ粒子をがん組織に取り込ませるため、近年、ナノサイズの金微粒子（非特許文献１、２）が検討されている。 In addition to the above three major therapies, hyperthermia has been developed that kills cancer cells by utilizing the property that cancer cells are more sensitive to heat than normal cells. The high-frequency dielectric heating method is a method in which a living body is placed between electrodes and the whole body is heated to about 42 ° C. However, due to the cooling effect of blood flow, the temperature inside the cancer tissue does not rise, which is not enough to kill the cancer tissue. Therefore, in recent years, nano-sized gold particles (Non-Patent Documents 1 and 2) have been studied in order to incorporate gold nanoparticles that generate heat when irradiated with near-infrared light into cancer tissues.
これらのナノ粒子を血管に注射し、血流を通じて金ナノ粒子をがん組織に集積させる方法があるが、大きながん組織を死滅させるのに十分な金ナノ粒子の集積量を確保するのは難しい。 There is a method of injecting these nanoparticles into blood vessels and accumulating gold nanoparticles in cancer tissue through the bloodstream, but it is necessary to secure a sufficient amount of gold nanoparticles to kill large cancer tissues. difficult.
以上のような従来技術の現状から、がん組織を切除する範囲を大きくするほど、がん細胞を取り残してしまう可能性は低くなる一方で、切除した部位や切除部位の大きさによっては臓器の機能が大きく低下してしまう場合がある。逆に、切除する範囲を小さくするほど、がん細胞を取り残してしまう可能性が高くなり、再発のリスクが上昇する。また術後に、がん再発を予防する目的でがん治療薬を注射投与すると、薬剤は血流を通じて全身を循環するため、がん組織への集積効率の低さや副作用の惹起という問題がある。このように従来技術では、手術による組織・臓器機能が低下する場合があるうえに、放射線療法や化学療法による副作用の問題があった。 From the current state of the prior art as described above, the larger the area where the cancer tissue is excised, the less likely it is that the cancer cells will be left behind. The function may be greatly reduced. Conversely, the smaller the area to be resected, the more likely it is that the cancer cells will be left behind, increasing the risk of recurrence. In addition, when a cancer therapeutic drug is injected after surgery for the purpose of preventing cancer recurrence, the drug circulates throughout the body through the bloodstream, which causes problems such as low accumulation efficiency in cancer tissues and the induction of side effects. .. As described above, in the prior art, there is a problem that the tissue / organ function due to surgery may be deteriorated and side effects due to radiation therapy or chemotherapy.
最近、四酸化三鉄ナノ粒子と複合化した多孔質材料を体内に埋め込むことにより、体外からの近赤外光の照射や磁場の印加によって、がん細胞を死滅させることが報告されている（例えば、非特許文献３）。しかし、心臓ペースメーカー、体内神経刺激装置、骨成長刺激装置、体内自動除細動器、人工内耳など電気的、磁気的もしくは機械的に動作する埋入物が体内にある場合は、磁性酸化鉄ナノ粒子の使用は禁忌となる。このような埋入物のある患者のがん治療には磁性ナノ粒子にかわる手段が求められる。 Recently, it has been reported that by embedding a porous material composited with triiron tetroxide nanoparticles in the body, cancer cells are killed by irradiation with near-infrared light from outside the body or application of a magnetic field ( For example, Non-Patent Document 3). However, if there are electrical, magnetic or mechanical implants in the body such as cardiac pacemakers, internal nerve stimulators, bone growth stimulators, internal automatic defibrillators, cochlear implants, magnetic iron oxide nano The use of particles is contraindicated. Cancer treatment for patients with such implants requires alternatives to magnetic nanoparticles.
本発明は、このような実情に鑑み、外科手術でがん組織を切除した後に移植、あるいはがん組織に直接被覆するために使用できる金ナノ粒子／生体吸収性高分子複合多孔質材料およびその製造方法を提供することを目的とする。 In view of such circumstances, the present invention is a gold nanoparticle / bioabsorbable polymer composite porous material that can be used for transplantation or direct coating on cancer tissue after surgical excision of cancer tissue. It is an object of the present invention to provide a manufacturing method.
本発明による複合多孔質材料は、生体吸収性高分子と、近赤外線照射で発熱するナノ粒子とを含み、これにより上記課題を解決する。
前記ナノ粒子は金ナノ粒子であってもよい。
前記ナノ粒子の粒径が１ｎｍから１０００ｎｍの範囲であってもよい。
前記ナノ粒子は、球状、ロッド状、スター状、うに状、紡錘状、三角柱、直方体および立方体からなる形状を有してもよい。
前記ナノ粒子は、緻密体、多孔質体またはケージ体であってもよい。
前記生体吸収性高分子は、ゼラチン、コラーゲン、フィブリン、ヒアルロン酸、コンドロイチン硫酸、フィブロネクチン、ビトロネクチン、ラミニン、細胞成長因子、細胞分化制御因子、ポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体、ポリ（ε−カプロラクトン）、ポリ（グリセロールセバシン酸）及びこれらの共重合体からなる群から選択されてもよい。
孔径が０．１〜１０００μｍの範囲である空孔を有してもよい。
前記生体吸収性高分子は、架橋されていてもよい。
前記生体吸収性高分子の溶液に対する前記ナノ粒子の含有量は、０．１ｍＭ以上１００ｍＭ以下の範囲であってもよい。
前記生体吸収性高分子の溶液に対する前記ナノ粒子の含有量は、１．０ｍＭ以上５０ｍＭ以下の範囲が好ましい。
外科的な手術でがん組織を除去した部位に埋め込まれ、またはがん組織を直接覆い、外部から近赤外光が照射されることによって発熱し、内部或いは周囲のがん細胞を死滅してもよい。あるいは、多孔質体が吸収され、遊離した金ナノ粒子ががん細胞に取込まれ、外部から近赤外光が照射されることによって発熱し、金ナノ粒子を取り込んだがん細胞を死滅してもよい。
本発明による上述の複合多孔質材料の製造方法は、ナノ粒子と生体吸収性高分子とを混合し、多孔質化する工程を包含し、これにより上記課題を解決する。
前記多孔質化する工程は、前記ナノ粒子と前記生体吸収性高分子との混合溶液を凍結乾燥してもよい。
空孔形成剤を使用し、空孔を形成してもよい。
前記空孔形成剤は氷であってもよい。
空孔形成剤を使用せずに空孔を形成させてもよい。
前記多孔質化する工程に続いて、前記ナノ粒子と生体吸収性高分子との複合多孔質材料を架橋する工程をさらに包含してもよい。
The composite porous material according to the present invention contains a bioabsorbable polymer and nanoparticles that generate heat when irradiated with near infrared rays, thereby solving the above problems.
The nanoparticles may be gold nanoparticles.
The particle size of the nanoparticles may be in the range of 1 nm to 1000 nm.
The nanoparticles may have a shape consisting of a spherical shape, a rod shape, a star shape, a sea urchin shape, a spindle shape, a triangular prism, a rectangular parallelepiped and a cube.
The nanoparticles may be dense, porous or caged.
The bioabsorbable polymer is gelatin, collagen, fibrin, hyaluronic acid, chondroitin sulfate, fibronectin, vitronectin, laminin, cell growth factor, cell differentiation regulator, polylactic acid, polyglycolic acid, and a copolymer of lactic acid and glycolic acid. , Poly (ε-caprolactone), poly (glycerol sebacic acid) and copolymers thereof.
It may have pores having a pore diameter in the range of 0.1 to 1000 μm.
The bioabsorbable polymer may be crosslinked.
The content of the nanoparticles in the solution of the bioabsorbable polymer may be in the range of 0.1 mM or more and 100 mM or less.
The content of the nanoparticles in the solution of the bioabsorbable polymer is preferably in the range of 1.0 mM or more and 50 mM or less.
It is implanted in the site where the cancer tissue has been removed by surgery, or it covers the cancer tissue directly, and when it is irradiated with near-infrared light from the outside, it generates heat and kills the cancer cells inside or around it. May be good. Alternatively, the porous body is absorbed, the free gold nanoparticles are taken into the cancer cells, and heat is generated by irradiation with near-infrared light from the outside, and the cancer cells that have taken in the gold nanoparticles are killed. May be good.
The above-mentioned method for producing a composite porous material according to the present invention includes a step of mixing nanoparticles and a bioabsorbable polymer to make them porous, thereby solving the above-mentioned problems.
In the step of making the porous material, a mixed solution of the nanoparticles and the bioabsorbable polymer may be freeze-dried.
Pore-forming agents may be used to form pores.
The pore-forming agent may be ice.
Pore formation may be formed without using a pore forming agent.
Following the step of making the porous material, a step of cross-linking the composite porous material of the nanoparticles and the bioabsorbable polymer may be further included.
本発明により、がん組織の切除部位に複合多孔質材料を移植し、あるいは複合多孔質材料でがん組織を覆うことにより、明確に局限され、しかも自由な形状の領域だけを発熱させることができる。また、場所毎の発熱量についても、場所毎に複合多孔質材料の試料量（厚さ等）やそこに使用する材料中のナノ粒子の含有量を変えるなどの処置により、調節可能である。従って、複雑な形状の領域に存在していたり、あるいは熱による損傷が深刻な障害をもたらす部位の近傍に存在するがん組織であっても、効率的に死滅させることができる。また、多孔質構造により、がん細胞を複合多孔質材料内に侵入せしめることができ、がん細胞を効率的に死滅させることも可能となる。しかも、ナノ粒子は多孔質材料に担持されているため、ナノ粒子がすばやく拡散して光熱効率が低下してしまうことを防ぎ、繰り返しがん組織の切除部位やがん組織を局所的に加熱することが可能である。よって、近赤外光を照射することで、がん組織とがん細胞を繰り返し加熱することにより死滅させることが可能である。また、複合多孔質材料が体内で分解吸収されるようにすることもできる。更に、それにともなって放出された金ナノ粒子はがん細胞に取り込まれ、外科手術で取り残されたがん細胞を死滅させることができる。これにより、手術などにより更に散らばりやすくなったがん細胞を、体内に拡散する前に多孔質材料中に取り込んで、ここで加熱によって死滅させることができるようになる。 According to the present invention, by transplanting a composite porous material to the excision site of the cancer tissue or covering the cancer tissue with the composite porous material, it is possible to generate heat only in a clearly localized and freely shaped region. can. Further, the calorific value for each place can also be adjusted by taking measures such as changing the sample amount (thickness, etc.) of the composite porous material and the content of nanoparticles in the material used therefor for each place. Therefore, even cancerous tissue that is present in a region of complex shape or in the vicinity of a site where thermal damage causes serious damage can be efficiently killed. In addition, the porous structure allows cancer cells to invade the composite porous material, and can kill cancer cells efficiently. Moreover, since the nanoparticles are supported on a porous material, they prevent the nanoparticles from diffusing quickly and reducing the photothermal efficiency, and repeatedly heat the excision site of the cancer tissue and the cancer tissue locally. It is possible. Therefore, it is possible to kill cancer tissues and cancer cells by repeatedly heating them by irradiating them with near-infrared light. It is also possible to allow the composite porous material to be decomposed and absorbed in the body. Furthermore, the gold nanoparticles released accordingly can be taken up by the cancer cells and kill the cancer cells left behind in the surgery. As a result, cancer cells that have become more easily dispersed by surgery or the like can be taken into a porous material before being diffused into the body, and can be killed by heating here.
以下、本発明をさらに詳述する。本発明における複合多孔質材料の基材には、生体吸収性天然高分子、生体吸収合成高分子のいずれも用いることができる。生体吸収性天然高分子は生体に由来し、生体吸収性合成高分子は人工物を原料に合成した生体吸収性をもつもので、生体吸収性と生体適合性を示すものであれば何れも使用できる。 Hereinafter, the present invention will be described in more detail. As the base material of the composite porous material in the present invention, either a bioabsorbable natural polymer or a bioabsorbable synthetic polymer can be used. Bioabsorbable natural polymers are derived from living organisms, and bioabsorbable synthetic polymers have bioabsorbability synthesized from artificial materials, and any bioabsorbable polymer that exhibits bioabsorbability and biocompatibility is used. can.
本発明において好ましく使用される生体吸収性高分子はゼラチン、コラーゲン、フィブリン、ヒアルロン酸、コンドロイチン硫酸、フィブロネクチン、ビトロネクチン、ラミニン、細胞成長因子、細胞分化制御因子、ポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体、ポリ（ε−カプロラクトン）、ポリ（グリセロールセバシン酸）及びこれらの共重合体である。これらの生体吸収性合成高分子を１種類、あるいは２種類以上を混合してから用いることができる。本発明において好ましく使用される生体吸収性高分子はゼラチン、コラーゲン、あるいはゼラチンとコラーゲンを主成分とする混合物である。コラーゲンにはＩ、ＩＩ、ＩＩＩ、ＩＶ、Ｖ、ＶＩ、ＶＩＩ、ＶＩＩＩ、ＩＸ、Ｘ型などのものがあるが、本発明においてはこれらの何れも使用でき、これらの誘導体を使用してもよい。 Bioabsorbable polymers preferably used in the present invention are gelatin, collagen, fibrin, hyaluronic acid, chondroitin sulfate, fibronectin, vitronectin, laminin, cell growth factor, cell differentiation regulator, polylactic acid, polyglycolic acid, lactic acid and glycol. Acid copolymers, poly (ε-caprolactone), poly (glycerol sebacic acid) and copolymers thereof. These bioabsorbable synthetic polymers can be used after one type or a mixture of two or more types. The bioabsorbable polymer preferably used in the present invention is gelatin, collagen, or a mixture containing gelatin and collagen as main components. Collagen includes I, II, III, IV, V, VI, VII, VIII, IX, X type, etc., but any of these can be used in the present invention, and derivatives thereof may be used. ..
金ナノ粒子と生体吸収性合成高分子との複合多孔質材料の空孔の孔径は０．１〜１０００μｍ、好ましくは１〜８００μｍ程度とするのがよい。また、複合多孔質材料の大きさは、複合多孔質材料の用途によって適宜定めればよいが、通常一辺は０．０１〜２０ｃｍで、好ましくは０．０２〜１０ｃｍである。その気孔率は、通常５〜９９．９％、好ましくは２０〜９９．９％である。 The pore size of the pores of the composite porous material of the gold nanoparticles and the bioabsorbable synthetic polymer is preferably 0.1 to 1000 μm, preferably about 1 to 800 μm. The size of the composite porous material may be appropriately determined depending on the intended use of the composite porous material, but is usually 0.01 to 20 cm on a side, preferably 0.02 to 10 cm. Its porosity is usually 5-99.9%, preferably 20-99.9%.
上記の金ナノ粒子として、従来公知のものの何れも使用してよい。これらの金ナノ粒子は公知の方法で合成してもよい。市販の金ナノ粒子を用いてもよいし、例えば、既報（Ｊ． Ｌｉ Ｊ． ｅ ｔ ａｌ．， Ｎａｎｏｓｃａｌｅ， ８， ７９９２−８００７（２０１６））に従って合成してもよい。上述の方法で合成すれば、異なるサイズ（例えば、約３５．０ｎｍ、６５．０ｎｍ、１１５．０ｎｍ）と形状（例えば、ロッドとスター）とを有する金ナノ粒子を得ることができる。 Any of the conventionally known gold nanoparticles may be used as the above-mentioned gold nanoparticles. These gold nanoparticles may be synthesized by a known method. Commercially available gold nanoparticles may be used, or may be synthesized according to, for example, previously reported (J. Li J. et al., Nanoscale, 8, 7992-8007 (2016)). When synthesized by the method described above, gold nanoparticles having different sizes (for example, about 35.0 nm, 65.0 nm, 115.0 nm) and shapes (for example, rod and star) can be obtained.
上記の金ナノ粒子は、表面修飾していないものでも、表面修飾したものでもいずれも利用できる。金ナノ粒子の表面修飾は、多孔質体の原料である生体吸収性物質の溶液と混合するときに混合溶液における金ナノ粒子の分散性を向上させるために行われるものである。表面修飾に用いられる分子として、クエン酸、ポリビニルアルコール、ポリエチレングリコール、ポリアクリル酸、ポリリジン、ポリグルタミン酸、ポリエチレンイミン、アルブミン、ゼラチン、コラーゲン、アルブミン、葉酸などが挙げられる。 The gold nanoparticles described above can be either unsurface-modified or surface-modified. The surface modification of the gold nanoparticles is performed in order to improve the dispersibility of the gold nanoparticles in the mixed solution when mixed with the solution of the bioabsorbable substance which is the raw material of the porous body. Examples of the molecule used for surface modification include citric acid, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, polylysine, polyglutamic acid, polyethyleneimine, albumin, gelatin, collagen, albumin, folic acid and the like.
金ナノ粒子の粒径は１ｎｍから１０００ｎｍのものを利用できるが、発熱効率や分散性を考えると望ましいのは２ｎｍから５００ｎｍである。金ナノ粒子は粒径の均一なものでも良いし、不均一なものでもよい。 Gold nanoparticles having a particle size of 1 nm to 1000 nm can be used, but are preferably 2 nm to 500 nm in consideration of heat generation efficiency and dispersibility. The gold nanoparticles may have a uniform particle size or may have a non-uniform particle size.
金ナノ粒子の形状は、球状、ロッド状、スター状、うに状、紡錘状、三角柱、直方体、立方体などいずれも利用することができる。これらを１種類あるいは２種類以上の混合物として用いることができる。ナノ粒子は、緻密体、多孔質体あるいはケージ体であってもよい。 As the shape of the gold nanoparticles, any of spherical, rod-shaped, star-shaped, sea urchin-shaped, spindle-shaped, triangular prism, rectangular parallelepiped, cube and the like can be used. These can be used as one kind or a mixture of two or more kinds. The nanoparticles may be dense, porous or caged.
本発明の複合多孔質材料において、生体吸収性高分子に対する金ナノ粒子の含有量は、好ましくは、多孔質材料調製時の生体吸収性高分子の溶液に対してナノ粒子の含有量は０．１ｍＭ以上１００ｍＭ以下の範囲である。金ナノ粒子の含有量が０．１ｍＭ未満である場合、近赤外光の照射による発熱が十分でなく、がん細胞の死滅の効果が小さくなり得る。ナノ粒子の含有量が１００ｍＭを超えると、すべてのナノ粒子を多孔質材料に保持するのは困難で、所定の空孔を有する複合多孔質材料を得られない場合があり得る。生体吸収性高分子に対する金ナノ粒子の含有量は、さらに好ましくは、１．０ｍＭ以上５０ｍＭ以下の範囲である。金ナノ粒子の含有量が１．０ｍＭ以上であれば、がん細胞の死滅の効果が高くなる。生体吸収性高分子に対する金ナノ粒子の含有量は、さらに好ましくは、３．０ｍＭ以上４０．０ｍＭ以下の範囲である。この範囲であれば、がん細胞を確実に死滅できる。例えば、後述する実施例に示されるように、金ナノ粒子の含有量が３．０ｍＭ以上６．０ｍＭ以下の範囲であれば、少ない金ナノ粒子によっても確実にがん細胞を死滅できることが分かった。なお、複合多孔質材料中の金ナノ粒子の含有量を高精度に求めることは困難であるため、簡易的に、複合多孔質材料の作製に用いた金ナノ粒子の濃度と同義とみなすことができる。 In the composite porous material of the present invention, the content of gold nanoparticles with respect to the bioabsorbable polymer is preferably 0. The content of nanoparticles with respect to the solution of the bioabsorbable polymer at the time of preparing the porous material. The range is 1 mM or more and 100 mM or less. When the content of gold nanoparticles is less than 0.1 mM, the heat generated by irradiation with near-infrared light is not sufficient, and the effect of killing cancer cells may be reduced. When the content of nanoparticles exceeds 100 mM, it is difficult to retain all the nanoparticles in the porous material, and it may not be possible to obtain a composite porous material having predetermined pores. The content of the gold nanoparticles in the bioabsorbable polymer is more preferably in the range of 1.0 mM or more and 50 mM or less. When the content of gold nanoparticles is 1.0 mM or more, the effect of killing cancer cells is high. The content of the gold nanoparticles in the bioabsorbable polymer is more preferably in the range of 3.0 mM or more and 40.0 mM or less. Within this range, cancer cells can be reliably killed. For example, as shown in Examples described later, it was found that if the content of gold nanoparticles is in the range of 3.0 mM or more and 6.0 mM or less, cancer cells can be reliably killed even with a small amount of gold nanoparticles. .. Since it is difficult to determine the content of gold nanoparticles in the composite porous material with high accuracy, it can be simply regarded as synonymous with the concentration of gold nanoparticles used in the production of the composite porous material. can.
上記生体吸収性高分子を溶かす溶媒には、純水、純水とエタノールの混合溶媒、ｐＨを調整した酢酸水溶液、塩酸水溶液、酢酸／水／エタノール混合溶媒、及び塩酸／エタノール混合溶媒、クロロホルム、四塩化炭素、ジオキサン、トリクロロ酢酸、ジメチルホルムアミド、塩化メチレン、酢酸エチル、アセトン、ヘキサフルオロイソプロパノール、ジメチルアセトアミド、ヘキサフルオロ−２−プロパノールなどが挙げられる。望ましい溶媒は純水、純水とエタノールとの混合溶媒、ｐＨを調整した酢酸水溶液、ｐＨを調製した塩酸水溶液、酢酸／水／エタノール混合溶媒、及び塩酸／エタノール混合溶媒である。ｐＨを調整した溶液のｐＨは１〜６．８で、望ましいｐＨは２．５から６までである。エタノールと水との体積比は１：９９から５０：５０でよいが、望ましくは１：９９から２０：８０である。 The solvent for dissolving the bioabsorbable polymer includes pure water, a mixed solvent of pure water and ethanol, an acetic acid aqueous solution with adjusted pH, a hydrochloric acid aqueous solution, an acetic acid / water / ethanol mixed solvent, and a hydrochloric acid / ethanol mixed solvent, chloroform. Examples thereof include carbon tetrachloride, dioxane, trichloroacetic acid, dimethylformamide, methylene chloride, ethyl acetate, acetone, hexafluoroisopropanol, dimethylacetamide, hexafluoro-2-propanol and the like. Desirable solvents are pure water, a mixed solvent of pure water and ethanol, an aqueous acetic acid solution having an adjusted pH, an aqueous solution of hydrochloric acid having an adjusted pH, an acetic acid / water / ethanol mixed solvent, and a hydrochloric acid / ethanol mixed solvent. The pH of the adjusted pH is 1 to 6.8, with a preferred pH of 2.5 to 6. The volume ratio of ethanol to water may be 1:99 to 50:50, but is preferably 1:99 to 20:80.
生体吸収性高分子の溶液を調製する温度はその生体吸収性高分子が分解、ゲル化しない温度で行われる。通常は１〜６０℃であるが、望ましくは４〜５０℃である。 The temperature at which the solution of the bioabsorbable polymer is prepared is a temperature at which the bioabsorbable polymer does not decompose or gel. It is usually 1 to 60 ° C, but preferably 4 to 50 ° C.
多孔質材料の生体吸収性高分子原料と金ナノ粒子とを混合してから多孔質化する（多孔質化工程）方法では、まず生体吸収性高分子の溶液に金ナノ粒子を添加した後、超音波あるいは機械的な攪拌により、金ナノ粒子を生体吸収性物質によく分散させる。この分散溶液を用いて金ナノ粒子と生体吸収性高分子との複合多孔質材料を作製することができる。 In the method of mixing the bioabsorbable polymer raw material of the porous material and the gold nanoparticles and then making them porous (porousization step), first, the gold nanoparticles are added to the solution of the bioabsorbable polymer, and then the gold nanoparticles are added. The gold nanoparticles are well dispersed in the bioabsorbable material by ultrasonic or mechanical agitation. This dispersion solution can be used to prepare a composite porous material of gold nanoparticles and a bioabsorbable polymer.
ここで、金ナノ粒子の濃度は、生体吸収性高分子の溶液に対して、好ましくは、０．１ｍＭ以上１００ｍＭ以下となるように調製される。これにより、０．１ｍＭ未満である場合、近赤外光の照射による発熱が十分でなく、がん細胞の死滅の効果が小さくなり得る。金ナノ粒子の含有量が１００ｍＭを超えると、金ナノ粒子が過剰にあり、すべての金ナノ粒子を多孔質材料に保持するのは困難で、所定の空孔を有する複合多孔質材料を得られない場合があり得る。金ナノ粒子の濃度は、さらに好ましくは、１．０ｍＭ以上５０ｍＭ以下の範囲に調製される。金ナノ粒子の濃度が１．０ｍＭ以上であれば、がん細胞の死滅の効果が高くなる。金ナノ粒子の濃度は、さらに好ましくは、３．０ｍＭ以上４０．０ｍＭ以下の範囲である。この範囲であれば、がん細胞を確実に死滅できる複合多孔質材料を提供できる。例えば、後述する実施例に示されるように、金ナノ粒子の濃度が３．０ｍＭ以上６．０ｍＭ以下の範囲であれば、少ない金ナノ粒子によっても確実にがん細胞を死滅できることが分かった。 Here, the concentration of the gold nanoparticles is adjusted to be preferably 0.1 mM or more and 100 mM or less with respect to the solution of the bioabsorbable polymer. As a result, if it is less than 0.1 mM, the heat generated by irradiation with near-infrared light is not sufficient, and the effect of killing cancer cells may be reduced. When the content of gold nanoparticles exceeds 100 mM, it is difficult to retain all the gold nanoparticles in the porous material due to the excess of gold nanoparticles, and a composite porous material having predetermined pores can be obtained. It may not be. The concentration of gold nanoparticles is more preferably adjusted in the range of 1.0 mM or more and 50 mM or less. When the concentration of gold nanoparticles is 1.0 mM or more, the effect of killing cancer cells is high. The concentration of gold nanoparticles is more preferably in the range of 3.0 mM or more and 40.0 mM or less. Within this range, it is possible to provide a composite porous material capable of reliably killing cancer cells. For example, as shown in Examples described later, it was found that if the concentration of gold nanoparticles is in the range of 3.0 mM or more and 6.0 mM or less, cancer cells can be reliably killed even with a small amount of gold nanoparticles.
前記金ナノ粒子と生体吸収性高分子との複合多孔質材料を作製する方法としては、例えば、前記金ナノ粒子と生体吸収性高分子からなる混合溶液をそのまま凍結乾燥する方法と、あらかじめ作製した氷微粒子を前記金ナノ粒子と生体吸収性高分子からなる混合溶液に添加し、多孔質化する方法とが挙げられる。そのまま凍結乾燥する方法では、空孔径や空孔の形状は凍結速度や凍結温度に依存し、あらかじめ作製した氷微粒子を用いる方法では、空孔径や空孔の形状は氷微粒子の大きさや形状に依存する。 Examples of the method for producing the composite porous material of the gold nanoparticles and the bioabsorbable polymer include a method of freeze-drying the mixed solution of the gold nanoparticles and the bioabsorbable polymer as it is, and a method prepared in advance. Examples thereof include a method in which ice particles are added to a mixed solution composed of the gold nanoparticles and a bioabsorbable polymer to make them porous. In the method of freeze-drying as it is, the pore diameter and the shape of the pores depend on the freezing rate and the freezing temperature, and in the method using the ice fine particles prepared in advance, the pore diameter and the shape of the pores depend on the size and shape of the ice fine particles. do.
前記金ナノ粒子と生体吸収性高分子からなる混合溶液をそのまま凍結乾燥する方法では、金ナノ粒子と生体吸収性高分子の混合溶液を予備凍結する。その方法としては、例えば、生体吸収性高分子の溶液に金ナノ粒子を添加し、超音波あるいは機械的な攪拌により、金ナノ粒子を生体吸収性高分子によく分散させる。金ナノ粒子を均一に分散させた金ナノ粒子と生体吸収性高分子からなる混合溶液をフリーザーに数時間静置し、凍結する。フリーザーの温度は−１〜−１００℃で、望ましい温度は−５〜−８０℃である。凍結時間は１〜２４時間で、望ましい凍結時間は２〜８時間である。 In the method of freeze-drying the mixed solution of gold nanoparticles and the bioabsorbable polymer as it is, the mixed solution of the gold nanoparticles and the bioabsorbable polymer is pre-frozen. As a method, for example, gold nanoparticles are added to a solution of a bioabsorbable polymer, and the gold nanoparticles are well dispersed in the bioabsorbable polymer by ultrasonic waves or mechanical stirring. A mixed solution consisting of gold nanoparticles in which gold nanoparticles are uniformly dispersed and a bioabsorbable polymer is allowed to stand in a freezer for several hours and then frozen. The temperature of the freezer is -1 to -100 ° C, and the desired temperature is -5 to -80 ° C. The freezing time is 1 to 24 hours, and the desired freezing time is 2 to 8 hours.
あるいは、空孔形成剤としてあらかじめ作製した氷微粒子を前記金ナノ粒子と生体吸収性高分子からなる混合溶液に添加し、多孔質化する方法を使用してもよい。この場合は、まず純水を容器に満たした液体窒素中に噴霧し、氷微粒子を作製する。形成した氷微粒子を低温チャンバー（−１５℃）に容器ごと移し、容器内の液体窒素が気化して消失するまで、容器を静置する。その後、大きな目開きの篩と小さな目開きの篩とを用いて所定の粒径の氷を篩い分ける。何れの篩もその目開きは公称１〜１０００μｍで、望ましいのは公称１０〜８００μｍである。篩い分けた氷微粒子を−４℃の低温チャンバー内に１〜６時間静置し、氷微粒子の温度を−４℃で平衡化させる。そして、前記金ナノ粒子と生体吸収性高分子からなる混合溶液を−４℃の低温チャンバーに移し、数十分間静置することによって温度平衡化させる。温度を−４℃にした金ナノ粒子と生体吸収性高分子からなる混合溶液と前記の温度を−４℃にした篩い分けた氷微粒子を一定の体積ｍＬ対重量ｇの比率で−４℃の低温チャンバーで混合する。金ナノ粒子と生体吸収性高分子からなる混合溶液と氷微粒子との比率は、体積ｍＬ対重量ｇで９９：１〜１０：９０で良いが、望ましい比率（体積ｍＬ対重量ｇ）は８０：２０〜３０：７０である。氷微粒子が金ナノ粒子と生体吸収性高分子からなる混合溶液に均一に分散するようによく攪拌する。この混合物を−２０℃で１２時間静置した後、さらに−８０℃で４時間静置することにより、混合物を凍結する。 Alternatively, a method of adding ice fine particles prepared in advance as a pore-forming agent to a mixed solution of the gold nanoparticles and a bioabsorbable polymer to make them porous may be used. In this case, first, pure water is sprayed into liquid nitrogen filled in a container to prepare ice fine particles. The formed ice fine particles are transferred to a low temperature chamber (-15 ° C.) together with the container, and the container is allowed to stand until the liquid nitrogen in the container is vaporized and disappears. Then, ice having a predetermined particle size is sieved using a sieve having a large opening and a sieve having a small opening. The mesh size of each sieve is nominally 1 to 1000 μm, preferably 10 to 800 μm. The sieved ice particles are allowed to stand in a low temperature chamber at -4 ° C for 1 to 6 hours to equilibrate the temperature of the ice particles at -4 ° C. Then, the mixed solution composed of the gold nanoparticles and the bioabsorbable polymer is transferred to a low temperature chamber at -4 ° C. and allowed to stand for several tens of minutes to achieve temperature equilibrium. A mixture of gold nanoparticles at a temperature of -4 ° C, a bioabsorbable polymer, and sieved ice particles at a temperature of -4 ° C were placed at a constant volume of mL to weight g at a ratio of -4 ° C. Mix in a cold chamber. The ratio of the mixed solution consisting of gold nanoparticles and the bioabsorbable polymer to the ice fine particles may be 99: 1 to 10:90 in volume mL to weight g, but the desirable ratio (volume mL to weight g) is 80: It is 20 to 30:70. Stir well so that the ice particles are uniformly dispersed in a mixed solution consisting of gold nanoparticles and a bioabsorbable polymer. The mixture is frozen at −20 ° C. for 12 hours and then at −80 ° C. for 4 hours.
上記２つの方法の何れにおいても、その過程で準備した凍結物を室温、５Ｐａ以下の減圧下で３日間凍結乾燥を行うことにより、金ナノ粒子と生体吸収性高分子との複合多孔質構造を形成させる。 In either of the above two methods, the frozen material prepared in the process is freeze-dried at room temperature under a reduced pressure of 5 Pa or less for 3 days to obtain a composite porous structure of gold nanoparticles and a bioabsorbable polymer. To form.
前記の金ナノ粒子と生体吸収性高分子の複合多孔質材料を架橋することで多孔質材料の構造を安定させ、複合多孔質材料とする。ゼラチンやコラーゲンに代表される水溶性生体吸収性高分子を用いた場合には、架橋は特に好ましいが、ポリ乳酸やポリグリコール酸に代表される水不溶性生体吸収性高分子を用いた場合には、架橋は必ずしも必要ではない。 By cross-linking the composite porous material of the gold nanoparticles and the bioabsorbable polymer, the structure of the porous material is stabilized to obtain a composite porous material. Crosslinking is particularly preferable when a water-soluble bioabsorbable polymer typified by gelatin or collagen is used, but when a water-insoluble bioabsorbable polymer typified by polylactic acid or polyglycolic acid is used, cross-linking is particularly preferable. , Cross-linking is not always necessary.
用いられる架橋方法としては、従来公知のものが何れも使用できる。一般的に蒸気法や溶液法を用いることができる。蒸気法で用いられる架橋剤としては、従来公知のものが何れも使用できる。好ましく使用される架橋剤は、グルタルアルデヒド、ホルムアルデヒド、パラホルムアルデヒドのようなアルデヒド類、特にグルタルアルデヒドである。 As the cross-linking method used, any conventionally known cross-linking method can be used. Generally, the vapor method or the solution method can be used. As the cross-linking agent used in the steam method, any conventionally known cross-linking agent can be used. Preferred cross-linking agents are aldehydes such as glutaraldehyde, formaldehyde, paraformaldehyde, especially glutaraldehyde.
前記の蒸気法は、上記の架橋剤をガス状にして用いるのが好ましい。具体的には、上記金ナノ粒子と生体吸収性高分子との複合多孔質材料を一定温度で一定濃度の架橋剤又はその水溶液で飽和した架橋剤蒸気の雰囲気下で一定時間架橋を行う。架橋温度は、上記金ナノ粒子と生体吸収性高分子との複合多孔質材料が溶解せず、且つ架橋剤の蒸気が形成できる範囲内であればよく、通常、２０℃〜５０℃に設定される。架橋時間は、架橋剤の種類や架橋温度にもよるが、上記金ナノ粒子と生体吸収性高分子との複合多孔質材料の生体吸収性を阻害せず、かつ生体への移植時にこのものが溶解しないような架橋固定化が行われる範囲で行うのが望ましい。好ましい架橋時間は１０分から１２時間程度である。架橋反応後の金ナノ粒子と生体吸収性高分子との複合多孔質材料を室温で純水に浸漬して洗浄し、これを１回の洗浄として４回以上繰り返す。洗浄後、未反応の活性官能基をブロッキングするため、グリシン水溶液に金ナノ粒子と生体吸収性高分子との複合多孔質材料を室温で数時間浸漬する。グリシン水溶液の濃度は０．０１〜１．０Ｍで、望ましいのは０．０５〜０．３Ｍである。温度は４℃〜３７℃で、望ましいのは４℃〜３０℃である。時間は１〜２４時間で、望ましいのは４〜１２時間である。 In the steam method, it is preferable to use the above-mentioned cross-linking agent in the form of a gas. Specifically, the composite porous material of the gold nanoparticles and the bioabsorbable polymer is crosslinked at a constant temperature for a certain period of time in an atmosphere of a crosslinker saturated with a constant concentration of a crosslinker or an aqueous solution thereof. The cross-linking temperature may be within a range in which the composite porous material of the gold nanoparticles and the bioabsorbable polymer does not dissolve and vapor of the cross-linking agent can be formed, and is usually set to 20 ° C. to 50 ° C. NS. The cross-linking time depends on the type of cross-linking agent and the cross-linking temperature, but does not inhibit the bioabsorbability of the composite porous material of the above-mentioned gold nanoparticles and the bioabsorbable polymer, and this product is used at the time of transplantation into a living body. It is desirable to carry out in the range where cross-linking and immobilization are performed so as not to dissolve. The preferred cross-linking time is about 10 minutes to 12 hours. The composite porous material of the gold nanoparticles and the bioabsorbable polymer after the cross-linking reaction is washed by immersing it in pure water at room temperature, and this is repeated 4 times or more as one washing. After washing, a composite porous material of gold nanoparticles and a bioabsorbable polymer is immersed in an aqueous solution of glycine for several hours at room temperature in order to block unreacted active functional groups. The concentration of the aqueous glycine solution is 0.01 to 1.0 M, preferably 0.05 to 0.3 M. The temperature is 4 ° C. to 37 ° C., preferably 4 ° C. to 30 ° C. The time is 1 to 24 hours, preferably 4 to 12 hours.
溶液架橋法では、カルボジイミド、アルデヒド類、或いはエポキシ類などの架橋剤とＮ−ヒドロキシコハク酸イミドなどの活性化剤を用いて架橋する。未架橋の金ナノ粒子と生体吸収性高分子との複合多孔質材料は水に溶解してしまうので、これらの架橋剤をエタノールと水の混合溶媒に溶解させ、数段階にかけて架橋する。各段階の混合溶媒のエタノール対水の割合は異なり、最初の段階から最終段階までエタノール対水の割合は高いほうから低いほうに変える。エタノール対水の割合は９９／１から１／９９までである。架橋温度は４℃から４０℃で、望ましくは室温である。架橋剤の濃度は５ｍＭ〜５００ｍＭで、望ましくは１０ｍＭ〜１００ｍＭである。活性化剤の濃度は５ｍＭ〜５００ｍＭで、望ましくは１０ｍＭ〜１００ｍＭである。最後の架橋反応後の金ナノ粒子と生体吸収性高分子との複合多孔質材料を室温で純水に浸漬して洗浄し、これを１回の洗浄として４回以上繰り返す。洗浄後未反応の活性官能基を失活させるため、グリシン水溶液に金ナノ粒子と生体吸収性高分子との複合多孔質材料を室温で数時間浸漬する。グリシン水溶液の濃度は０．０１〜１．０Ｍで、望ましいのは０．０５〜０．３Ｍである。温度は４℃〜３７℃で、望ましくは４〜３０℃である。時間は１〜２４時間で、望ましくは４〜１２時間である。 In the solution cross-linking method, cross-linking is performed using a cross-linking agent such as carbodiimide, aldehydes or epoxies and an activator such as N-hydroxysuccinimide. Since the composite porous material of uncrosslinked gold nanoparticles and bioabsorbable polymer dissolves in water, these crosslinkers are dissolved in a mixed solvent of ethanol and water and crosslinked in several steps. The ratio of ethanol to water in the mixed solvent of each stage is different, and the ratio of ethanol to water is changed from high to low from the first stage to the final stage. The ratio of ethanol to water is from 99/1 to 1/99. The cross-linking temperature is 4 ° C to 40 ° C, preferably room temperature. The concentration of the cross-linking agent is 5 mM to 500 mM, preferably 10 mM to 100 mM. The concentration of the activator is 5 mM to 500 mM, preferably 10 mM to 100 mM. The composite porous material of the gold nanoparticles and the bioabsorbable polymer after the final cross-linking reaction is washed by immersing it in pure water at room temperature, and this is repeated 4 times or more as one washing. In order to inactivate unreacted active functional groups after washing, a composite porous material of gold nanoparticles and a bioabsorbable polymer is immersed in an aqueous glycine solution at room temperature for several hours. The concentration of the aqueous glycine solution is 0.01 to 1.0 M, preferably 0.05 to 0.3 M. The temperature is 4 ° C. to 37 ° C., preferably 4 to 30 ° C. The time is 1 to 24 hours, preferably 4 to 12 hours.
上記の複合多孔質材料を３０分間純水での洗浄を３回以上繰り返す。洗浄後、５Ｐａ以下の減圧下で４８時間凍結乾燥を行い、目的の金ナノ粒子と生体吸収性高分子との複合多孔質材料を得る。 Washing the above composite porous material with pure water for 30 minutes is repeated 3 times or more. After washing, freeze-drying is carried out under a reduced pressure of 5 Pa or less for 48 hours to obtain a composite porous material of the target gold nanoparticles and a bioabsorbable polymer.
このような複合多孔質材料に対して外部から近赤外光を照射すると、金ナノ粒子は発熱し、がん細胞とがん組織を死滅させることができる。必要に応じて前記の外部刺激を繰り返し与え、金ナノ粒子を繰り返し発熱させることにより、がん細胞やがん組織への殺傷効果を高められると期待される。また、複合多孔質材料の基材である生体吸収性高分子は、多孔質であることから体内で急速に分解・吸収され、それにともなって金ナノ粒子が放出される。そして、金ナノ粒子は周囲の細胞に取り込まれ、周囲のがん細胞を死滅させることも可能である。これにより、がん組織とがん細胞を効率よく死滅させることが期待される。また、このような複合多孔質材料は、外部からの電気的な配線やあるいは加熱流体などの供給のための配管の接続なしで、複合体内部やその近傍部位を温熱療法に適した温度まで昇温させ、またその温度を所望の時間だけ維持することができる。更に、本発明に係る複合多孔質材料では、その周囲に存在する細胞を孔の中に効率よく取り込み、またそれを加熱することができる。従って、例えば手術後に切除箇所の近傍に残留したがん細胞を捕捉・死滅させることもできる。 When such a composite porous material is irradiated with near-infrared light from the outside, the gold nanoparticles generate heat and can kill cancer cells and cancer tissues. It is expected that the killing effect on cancer cells and cancer tissues can be enhanced by repeatedly applying the above-mentioned external stimulus as needed to repeatedly generate heat of the gold nanoparticles. In addition, since the bioabsorbable polymer, which is the base material of the composite porous material, is porous, it is rapidly decomposed and absorbed in the body, and gold nanoparticles are released accordingly. Then, the gold nanoparticles are taken up by the surrounding cells and can kill the surrounding cancer cells. This is expected to efficiently kill cancer tissues and cancer cells. In addition, such a composite porous material raises the temperature inside or near the composite to a temperature suitable for hyperthermia without connecting electrical wiring from the outside or piping for supplying a heating fluid or the like. It can be heated and maintained at that temperature for a desired period of time. Further, in the composite porous material according to the present invention, cells existing around the composite porous material can be efficiently taken into the pores and heated. Therefore, for example, cancer cells remaining in the vicinity of the excision site after surgery can be captured and killed.
以下では、本発明に係るナノ粒子を生体吸収性高分子と複合化した多孔質材料を各種作製して、その表面形状などを調べた結果を示す。以下では複合多孔質材料の製造方法と、その結果得られる特異な構造についての例を示す。また、本発明に係る多孔質材料が外部からの刺激により実際に発熱すること、発熱によってがん細胞を死滅させることを実証する。以下の実施例では、ナノ粒子として金ナノロッドおよび金ナノスターを、生体吸収性高分子としてゼラチンを用いたが、これに限定されない。 The following shows the results of preparing various porous materials in which the nanoparticles according to the present invention are composited with a bioabsorbable polymer and examining the surface shape and the like thereof. The following shows an example of a method for producing a composite porous material and a unique structure obtained as a result. In addition, it will be demonstrated that the porous material according to the present invention actually generates heat due to an external stimulus and kills cancer cells by the heat generation. In the following examples, gold nanorods and gold nanostars were used as nanoparticles, and gelatin was used as a bioabsorbable polymer, but the present invention is not limited thereto.
［実施例１］金ナノロッド（長軸長さ３５ｎｍ）／架橋ゼラチン複合多孔質材料を作製するために、原料ゼラチンの濃度が０．１（ｗ／ｗ）％〜５０．０（ｗ／ｗ）％の範囲内のものを用いた。Ａｕ濃度０〜１０．０ｍＭの範囲内で作製した。なお、後述するように、実施例１では比較のために、金ナノ粒子を添加しない架橋ゼラチン多孔質材料を作製した。
[Example 1] In order to prepare a gold nanorod (
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノロッド（長軸長さ３５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノロッド／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤（ポローゲンとも呼ぶ）として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
A gold nanorod / crosslinked gelatin composite porous material was prepared using a 4.0 (w / v)% gelatin solution containing gold nanorods (
まず、０．８ｇのブタ由来ゼラチンに７０（ｖ／ｖ）％ 酢酸水溶液１０ｍＬを加えて、４５℃で２時間、つづいて室温で４時間撹拌し、８．０（ｗ／ｖ）％ゼラチン溶液を調製した。 First, 10 mL of a 70 (v / v)% acetic acid aqueous solution was added to 0.8 g of porcine-derived gelatin, and the mixture was stirred at 45 ° C. for 2 hours and then at room temperature for 4 hours to obtain an 8.0 (w / v)% gelatin solution. Was prepared.
一方、平均の長軸長さ３５ｎｍをもつ金ナノロッド（ＡｕＮＲ３５）はシード媒介成長法を用いて合成した。まず、０．０１ Ｍ ＨＡｕＣｌ４ ２５０μＬを０．１Ｍ ヘキサデシルトリメチルアンモニウムブロミド（ＣＴＡＢ）溶液７．５ ｍＬに添加し、５ｍｉｎ、室温で撹拌した。さらに、０．０１ Ｍ ＮａＢＨ４ ０．６ ｍＬを添加し、１０ｍｉｎ、室温で撹拌したものをＡｕシード溶液とした。本Ａｕシード溶液は用時調製とし、調製後２時間以内に結晶成長反応に使用した。 On the other hand, gold nanorods (AuNR35) having an average major axis length of 35 nm were synthesized using the seed-mediated growth method. First, 250 μL of 0.01 M HAuCl 4 was added to 7.5 mL of a 0.1 M hexadecyltrimethylammonium bromide (CTAB) solution, and the mixture was stirred for 5 minutes at room temperature. Further, 0.01 M NaBH 4 0.6 mL was added, and the solution was stirred for 10 minutes at room temperature to prepare an Au seed solution. This Au seed solution was prepared at the time of use and used for the crystal growth reaction within 2 hours after the preparation.
成長溶液は、６００ ｍＬの０．１ Ｍ ＣＴＡＢ溶液中に０．０１ Ｍ ＨＡｕＣｌ４を３０ ｍＬ、１．０ Ｍ ＨＣｌを１２ ｍＬ、１０ｍＭ ＡｇＮＯ３溶液を１．３２ ｍＬ、０．１ Ｍ アスコルビン酸（ＡＡ）を６．６ ｍＬ添加することによって調製した。つづいて、前記Ａｕシード溶液 １．４４ ｍＬを成長溶液に加え、室温で１２ｈインキュベートした。結晶成長反応によって生成したＡｕＮＲは遠心分離によって回収し、フリーのＣＴＡＢを除去するために純水で洗浄した。再び遠心分離を行った後、上澄みを除去し、０．５（ｗｔ／ｖ）％ゼラチン水溶液を添加して室温で攪拌した。ゼラチンによって分散安定化した金ナノロッド（ＡｕＮＲ）は遠心分離によって回収し、純水で洗浄後、複合多孔質材料を作製するために水に再分散させた。 The growth solution was 30 mL of 0.01 M HAuCl 4 and 12 mL of 1.0 M HCl in 600 mL of 0.1 M CTAB solution, 1.32 mL of 10 mM AgNO 3 solution, 0.1 M ascorbic acid. Prepared by adding 6.6 mL of (AA). Subsequently, 1.44 mL of the Au seed solution was added to the growth solution, and the mixture was incubated at room temperature for 12 hours. AuNR produced by the crystal growth reaction was recovered by centrifugation and washed with pure water to remove free CTAB. After centrifuging again, the supernatant was removed, a 0.5 (wt / v)% gelatin aqueous solution was added, and the mixture was stirred at room temperature. Gold nanorods (AuNR) dispersed and stabilized with gelatin were recovered by centrifugation, washed with pure water, and then redispersed in water to prepare a composite porous material.
ポローゲンである氷微粒子を作製するため、純水３００ｍＬを液体窒素１０Ｌの入った容器に噴霧し、水滴を凍結させた。凍結物を−１５℃の低温チャンバーに容器ごと移した。そのまま低温チャンバー内で約２時間静置することによって、液体窒素を気化させた。次に、低温チャンバー内で公称５００μｍの目開きをもつ篩と公称４２５μｍの目開きをもつ篩を用いて大きさ４２５μｍ〜５００μｍ氷を篩い分けた。低温チャンバー内の温度を−４℃に変更し、前記の氷微粒子を２時間静置し、氷微粒子の温度を−４℃に調製した。 In order to prepare ice fine particles which are pologens, 300 mL of pure water was sprayed on a container containing 10 L of liquid nitrogen, and water droplets were frozen. The frozen product was transferred to a low temperature chamber at −15 ° C. together with the container. Liquid nitrogen was vaporized by allowing it to stand in a low temperature chamber for about 2 hours. Next, ice having a size of 425 μm to 500 μm was sieved using a sieve having a nominal opening of 500 μm and a sieve having a nominal opening of 425 μm in a low temperature chamber. The temperature in the low temperature chamber was changed to -4 ° C., the ice fine particles were allowed to stand for 2 hours, and the temperature of the ice fine particles was adjusted to -4 ° C.
前記の８．０（ｗ／ｖ）％ゼラチン溶液１０ｍＬと濃度が２．０ｍＭ、４．０ｍＭ、８．０ｍＭの金ナノロッド懸濁液１０ｍＬのそれぞれとを室温で混合し、超音波処理により金ナノロッドをゼラチン溶液に均一に分散させた。調製した混合溶液のゼラチンの濃度は４．０（ｗ／ｖ）％で、金ナノロッドの濃度はそれぞれ１．０ｍＭ、２．０ｍＭ、４．０ｍＭであった。これらの金ナノロッド／ゼラチン混合溶液を−４℃の低温チャンバーに移し、４０分間静置することによって温度平衡化させた。温度を−４℃に冷却した金ナノロッド／ゼラチン混合溶液２０ｍＬと前記の大きさ４２５μｍ〜５００μｍの氷微粒子を３：７（体積ｍＬ対重量ｇ）の比率で−４℃の低温チャンバーで混合した。氷微粒子が金ナノロッド／ゼラチン混合溶液に均一に分散するようによく攪拌した。続いて、このゼラチン／ＡｕＮＲ溶液と氷微粒子の混合物を、あらかじめ冷却しておいたシリコーン型枠内に充てんした。この混合物を−２０℃で１２時間静置した後、さらに−８０℃で４時間静置することにより、混合物を凍結させた。凍結物を室温、５Ｐａ以下の減圧下で３日間凍結乾燥を行うことにより、金ナノロッド／ゼラチン複合多孔質材料を形成させた。なお、以降では、分かり易さのために、架橋反応前の中間生成物を「金ナノロッド／ゼラチン複合多孔質材料」と称し、架橋反応後の最終生成物を「金ナノロッド／架橋ゼラチン複合多孔質材料」と称する。 10 mL of the above 8.0 (w / v)% gelatin solution and 10 mL of gold nanorod suspensions having concentrations of 2.0 mM, 4.0 mM, and 8.0 mM were mixed at room temperature, and gold nanorods were subjected to ultrasonic treatment. Was uniformly dispersed in the gelatin solution. The concentration of gelatin in the prepared mixed solution was 4.0 (w / v)%, and the concentration of gold nanorods was 1.0 mM and 2.0 mM, 4.0 mM, respectively. These gold nanorod / gelatin mixed solutions were transferred to a low temperature chamber at -4 ° C. and allowed to stand for 40 minutes for temperature equilibration. 20 mL of the gold nanorod / gelatin mixed solution cooled to -4 ° C. and the above-mentioned ice fine particles having a size of 425 μm to 500 μm were mixed in a low temperature chamber at -4 ° C. at a ratio of 3: 7 (volume mL to weight g). The ice particles were stirred well so as to be uniformly dispersed in the gold nanorod / gelatin mixed solution. Subsequently, this mixture of gelatin / AuNR solution and ice fine particles was filled in a pre-cooled silicone mold. The mixture was allowed to stand at −20 ° C. for 12 hours and then at −80 ° C. for 4 hours to freeze the mixture. A gold nanorod / gelatin composite porous material was formed by freeze-drying the frozen product at room temperature under a reduced pressure of 5 Pa or less for 3 days. Hereinafter, for the sake of clarity, the intermediate product before the cross-linking reaction will be referred to as "gold nanorod / gelatin composite porous material", and the final product after the cross-linking reaction will be referred to as "gold nanorod / cross-linked gelatin composite porous material". It is called "material".
次に、金ナノロッド／ゼラチン複合多孔質材料を９９．５％のエタノールで洗浄（３０分間×１０回）した。その後、次の３段階の工程に分けて逐次的に架橋反応を行った。ここで、架橋反応の工程でゼラチンが溶解するのを防ぎつつ、架橋反応の効率を高めるために、エタノールの濃度を段階的に下げた３種類のエタノール／水混合溶媒（エタノール／水（ｖ／ｖ）＝ ９５／５、９０／１０、８５／１５）を用いた。 Next, the gold nanorod / gelatin composite porous material was washed with 99.5% ethanol (30 minutes × 10 times). Then, the cross-linking reaction was carried out sequentially in the following three steps. Here, in order to increase the efficiency of the cross-linking reaction while preventing the gelatin from dissolving in the step of the cross-linking reaction, three kinds of ethanol / water mixed solvents (ethanol / water (v /) in which the concentration of ethanol is gradually lowered. v) = 95/5, 90/10, 85/15) was used.
第１段階の架橋反応工程では、エタノール／水（９５／５、ｖ／ｖ）３０ｍＬに０．０３ｇの２−モルホリノエタンスルホン酸（ＭＥＳ、終濃度０．１ｗｔ％）を撹拌しながら加え、そのまま１〜２ｈ撹拌した。このＭＥＳ溶液に０．２８８ｇの１−（３−ジメチルアミノプロピル）−３−エチルカルボジイミド塩酸塩（ＥＤＣ、終濃度５０ｍＭ）及び０．０６９ｇのＮ−ヒドロキシコハク酸イミド（ＮＨＳ、終濃度２０ｍＭ）を加えて室温で１０分間撹拌することにより、第１段階の架橋反応溶液を調製した。この第１段階の架橋反応溶液に前記の金ナノロッド／ゼラチン複合多孔質材料を室温で８時間浸漬することによって、第１段階の架橋反応を行った。
In the first-step cross-linking reaction step, 0.03 g of 2-morpholinoetan sulfonic acid (MES, final concentration 0.1 wt%) was added to 30 mL of ethanol / water (95/5, v / v) with stirring, and the mixture was added as it was. The mixture was stirred for 1 to 2 hours. 0.288 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC,
第２段階の架橋反応工程では、エタノール／水（９０／１０、ｖ／ｖ）３０ｍＬに０．０３ｇのＭＥＳを撹拌しながら加えた。このＭＥＳ溶液に０．２８８ｇのＥＤＣ及び０．０６９ｇのＮＨＳを加えて１０分間撹拌することにより、第２段階の架橋反応溶液を調製した。この第２段階の架橋反応溶液に第１段階の架橋反応後の金ナノロッド／ゼラチン複合多孔質材料を室温で８時間浸漬し、第２段階の架橋反応を行った。 In the second step of the cross-linking reaction, 0.03 g of MES was added to 30 mL of ethanol / water (90/10, v / v) with stirring. 0.288 g of EDC and 0.069 g of NHS were added to this MES solution, and the mixture was stirred for 10 minutes to prepare a second-stage cross-linking reaction solution. The gold nanorod / gelatin composite porous material after the first-stage cross-linking reaction was immersed in the second-stage cross-linking reaction solution at room temperature for 8 hours to carry out the second-stage cross-linking reaction.
第３段階の架橋反応工程では、エタノール／水（８５／１５、ｖ／ｖ）３０ｍＬに０．０３ｇのＭＥＳを撹拌しながら加え、そのまま１〜２ｈ撹拌した。このＭＥＳ溶液に０．２８８ｇのＥＤＣ及び０．０６９ｇのＮＨＳを加えて１０分間撹拌することにより、第３段階の架橋反応溶液を調製した。この第３段階の架橋反応溶液に第２段階の架橋反応後の金ナノロッド／ゼラチン複合多孔質材料を室温で８時間浸漬して、第３段階の架橋反応を行った。最後の架橋反応後の金ナノロッド／ゼラチン複合多孔質材料を室温で１時間超純水に浸漬し、これを１回の洗浄として６回繰り返した。ついで、得られた生成物を０．１Ｍのグリシン水溶液に室温で８時間浸漬した後、１時間、純水での洗浄を６回繰り返した。洗浄後、５Ｐａ以下の減圧下で４８時間凍結乾燥を行い、最終製造物の金ナノロッド／架橋ゼラチンの複合多孔質材料を得た。 In the third step of the cross-linking reaction step, 0.03 g of MES was added to 30 mL of ethanol / water (85/15, v / v) with stirring, and the mixture was stirred as it was for 1 to 2 hours. 0.288 g of EDC and 0.069 g of NHS were added to this MES solution, and the mixture was stirred for 10 minutes to prepare a third-stage cross-linking reaction solution. The gold nanorod / gelatin composite porous material after the second-stage cross-linking reaction was immersed in the third-stage cross-linking reaction solution at room temperature for 8 hours to carry out the third-stage cross-linking reaction. The gold nanorod / gelatin composite porous material after the final cross-linking reaction was immersed in ultrapure water for 1 hour at room temperature, and this was repeated 6 times as one washing. Then, the obtained product was immersed in a 0.1 M aqueous solution of glycine at room temperature for 8 hours, and then washed with pure water for 1 hour was repeated 6 times. After washing, freeze-drying was carried out under reduced pressure of 5 Pa or less for 48 hours to obtain a composite porous material of gold nanorods / crosslinked gelatin as a final product.
上述したように、比較のため金ナノ粒子を添加しない架橋ゼラチン多孔質材料（コントロール）も作製した。詳細には、上述の４．０（ｗ／ｖ）％のゼラチン溶液１０ｍＬを−８０℃の低温で６時間静置することにより、ゼラチン溶液を凍結させた。凍結物を室温、５Ｐａ以下の減圧下で３日間凍結乾燥を行うことにより、多孔質構造を形成した。次に、実施例１と同様の洗浄、架橋、ブロッキング処理、凍結の各工程を経て、架橋ゼラチン多孔質材料を得た。 As described above, a crosslinked gelatin porous material (control) to which gold nanoparticles were not added was also prepared for comparison. Specifically, the gelatin solution was frozen by allowing 10 mL of the above 4.0 (w / v)% gelatin solution to stand at a low temperature of −80 ° C. for 6 hours. A porous structure was formed by freeze-drying the frozen product at room temperature under a reduced pressure of 5 Pa or less for 3 days. Next, a crosslinked gelatin porous material was obtained through the same washing, cross-linking, blocking treatment, and freezing steps as in Example 1.
得られた金ナノロッド（長軸長さ３５ｎｍ）／架橋ゼラチンの複合多孔質材料、および架橋ゼラチン多孔質材料（コントロール）を走査電子顕微鏡で観察し、図１（ａ）〜（ｄ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノロッドに相当する粒子状のものが観察された。コントロールとして作製した架橋ゼラチン多孔質材料も氷微粒子を反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有した。
The obtained gold nanorods (
［実施例２］金ナノロッド（長軸長さ６５ｎｍ）／架橋ゼラチン複合多孔質材料
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノロッド（長軸長さ６５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノロッド／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
[Example 2] Gold nanorod (
平均の長軸長さ６５ｎｍをもつ金ナノロッド（ＡｕＮＲ６５）は、実施例１と同様に、シード媒介成長法を用いて合成した。Ａｕシード溶液は、実施例１と同様にして調製した。成長溶液は、ＡｇＮＯ３溶液を６．６ ｍＬとした以外、実施例１と同様にして調製した。このようにして得られた金ナノロッド懸濁液を用い、以降の手順は、実施例１と同様にして、最終製造物の金ナノロッド／架橋ゼラチンの複合多孔質材料を得た。 Gold nanorods (AuNR65) having an average major axis length of 65 nm were synthesized using the seed-mediated growth method as in Example 1. The Au seed solution was prepared in the same manner as in Example 1. The growth solution was prepared in the same manner as in Example 1 except that the AgNO 3 solution was 6.6 mL. Using the gold nanorod suspension thus obtained, the following procedure was carried out in the same manner as in Example 1 to obtain a final product, a composite porous material of gold nanorod / crosslinked gelatin.
得られた金ナノロッド（長軸長さ６５ｎｍ）／架橋ゼラチンの複合多孔質材料を走査電子顕微鏡で観察し、図２（ａ）〜（ｃ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノロッドに相当する粒子状のものが観察された。
The obtained composite porous material of gold nanorods (
［実施例３］金ナノロッド（長軸長さ１１５ｎｍ）／架橋ゼラチン複合多孔質材料
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノロッド（長軸長さ１１５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノロッド／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
[Example 3] Gold nanorod (major axis length 115 nm) / crosslinked gelatin composite porous material 4.0 (major axis length 115 nm) containing gold nanorod (major axis length 115 nm) with Au concentration of 1.0 mM, 2.0 mM, 4.0 mM A gold nanorod / crosslinked gelatin composite porous material was prepared using a w / v)% gelatin solution. As the pore-forming agent for the porous material, ice fine particles having a size of 425 μm to 500 μm were used.
平均の長軸長さ１１５ｎｍをもつ金ナノロッド（ＡｕＮＲ１１５）は、ワンポット法によって合成した。まず、０．０１ Ｍ ＨＡｕＣｌ４を２２．８ ｍＬ、０．０２ Ｍ ＡｇＮＯ３を６ ｍＬ、０．６２ Ｍヒドロキノン４．８ ｍＬを５３４ ｍＬの０．１１ Ｍ ＣＴＡＢ溶液に順番に添加し、無色の溶液を得た。さらに、０．５ Ｍ ＮａＢＨ４溶液を３７８ μＬ添加し、３０℃で２４ ｈ静置した。得られたＡｕＮＲは遠心分離によって回収し、フリーのＣＴＡＢを除去するために水で洗浄した。遠心後上澄みを除去し、０．５（ｗｔ／ｖ）％ゼラチン水溶液を添加して室温で攪拌した。ゼラチンで分散安定化させた金ナノ粒子は遠心分離によって回収し、純水で洗浄後、複合多孔質材料を作製するために水に再分散させた。このようにして得られた金ナノロッド懸濁液を用い、以降の手順は実施例１と同様にして、最終製造物の金ナノロッド／架橋ゼラチンの複合多孔質材料を得た。 Gold nanorods (AuNR115) with an average major axis length of 115 nm were synthesized by the one-pot method. First, 0.01 M HAuCl 4 was added to 22.8 mL, 0.02 M AgNO 3 was added to 6 mL, and 0.62 M hydroquinone 4.8 mL was added to 534 mL of 0.11 M CTAB solution in this order, and the color was colorless. Solution was obtained. Further, 378 μL of 0.5 M NaBH 4 solution was added, and the mixture was allowed to stand at 30 ° C. for 24 hours. The resulting AuNR was collected by centrifugation and washed with water to remove free CTAB. After centrifugation, the supernatant was removed, a 0.5 (wt / v)% aqueous gelatin solution was added, and the mixture was stirred at room temperature. The gold nanoparticles dispersed and stabilized with gelatin were recovered by centrifugation, washed with pure water, and then redispersed in water to prepare a composite porous material. Using the gold nanorod suspension thus obtained, the following procedure was the same as in Example 1 to obtain a final product, a composite porous material of gold nanorod / crosslinked gelatin.
得られた金ナノロッド（長軸長さ１１５ｎｍ）／架橋ゼラチンの複合多孔質材料を走査電子顕微鏡で観察し、図３（ａ）〜（ｃ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノロッドに相当する粒子状のものが観察された。 The obtained composite porous material of gold nanorods (major axis length 115 nm) / crosslinked gelatin was observed with a scanning electron microscope to obtain the images shown in FIGS. 3 (a) to 3 (c). (A) Au concentration 1.0 mM, gelatin concentration 4.0 (w / v)%, (b) Au concentration 2.0 mM, gelatin concentration 4.0 (w / v)%, (c) Au concentration 4.0 mM , The composite porous material of gold nanorod / cross-linked gelatin prepared by using each raw material solution having a gelatin concentration of 4.0 (w / v)% has a spherical pore structure and a spherical shape reflecting the size of ice fine particles. It has a porous structure consisting of voids communicating with the pores, and particles corresponding to gold nanorods were observed on the wall surface of the pores.
［実施例４］金ナノスター（直径３５ｎｍ）／架橋ゼラチン複合多孔質材料
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノスター（長軸長さ３５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノスター／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
[Example 4] Gold nanostar (
最大径３５ｎｍをもつ金ナノスター（ＡｕＮＳ３５）もシード媒介成長法を用いて合成した。Ａｕシード溶液は、０．１２ｍＭクエン酸三ナトリウム４ ｍＬの存在下で、０．０８７ｍＭ ＮａＢＨ４１ ｍＬによる０．１ ｍｇ／ｍＬ ＨＡｕＣｌ４ ９０ ｍＬの還元反応により調製した。その後、ＨＡｕＣｌ４溶液（３０．０ ｍｇ ｍＬ−１， ２．２５ ｍＬ）、 Ａｕシード溶液（７５ ｍＬ）、 ＡｇＮＯ３溶液（５．０ ｍｇ ｍＬ−１、 ０．９ ｍＬ）、ＡＡ溶液（３８．０ ｍｇ ｍＬ−１， １．５ ｍＬ）は、１０分間穏やかに撹拌しながら７５ ｍＬの純水に連続して滴下した。Ａｕシード溶液の添加量を変えることによって、異なる直径をもつ金ナノスター（ＡｕＮＳ）が得られた。ここでは、結晶成長過程における金ナノスターの分散安定性を高めるために、金のナノ粒子の表面にクエン酸三ナトリウムを物理吸着させた。 Gold nanostars (AuNS35) with a maximum diameter of 35 nm were also synthesized using the seed-mediated growth method. Au seed solution, in the presence of 0.12mM trisodium citrate 4 mL, was prepared by the reduction reaction of 0.1 mg / mL HAuCl 4 90 mL by 0.087mM NaBH 4 1 mL. Then, HAuCl 4 solution (30.0 mg mL -1 , 2.25 mL), Au seed solution (75 mL), AgNO 3 solution (5.0 mg mL -1 , 0.9 mL), AA solution (38). .0 mg mL- 1 , 1.5 mL) was continuously added dropwise to 75 mL of pure water with gentle stirring for 10 minutes. By varying the amount of Au seed solution added, gold nanostars (AuNS) with different diameters were obtained. Here, in order to enhance the dispersion stability of gold nanostars in the crystal growth process, trisodium citrate was physically adsorbed on the surface of gold nanoparticles.
遠心後上澄みを除去し、０．５（ｗｔ／ｖ）％ゼラチン水溶液を添加して室温で攪拌した。ゼラチンで分散安定化させた金ナノ粒子（金ナノスター）は遠心分離によって回収し、純水で洗浄後、複合多孔質材料を作製するために水に再分散させた。以降の手順は、実施例１と同様にして、氷微粒子をポローゲンに用いて、金ナノスター／架橋ゼラチンの複合多孔質材料を作製した。 After centrifugation, the supernatant was removed, a 0.5 (wt / v)% aqueous gelatin solution was added, and the mixture was stirred at room temperature. The gold nanoparticles (gold nanoparticles) dispersed and stabilized with gelatin were recovered by centrifugation, washed with pure water, and then redispersed in water to prepare a composite porous material. In the following procedure, ice fine particles were used as pologens in the same manner as in Example 1 to prepare a composite porous material of gold nanostar / crosslinked gelatin.
得られた金ナノスター（最大径３５ｎｍ）／架橋ゼラチンの複合多孔質材料を走査電子顕微鏡で観察し、図４（ａ）〜（ｃ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノスターが観察された。
The obtained composite porous material of gold nanostar (
［実施例５］金ナノスター（直径６５ｎｍ）／架橋ゼラチン複合多孔質材料
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノスター（長軸長さ６５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノスター／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
[Example 5] Gold nanostar (
最大径６５ｎｍをもつ金ナノスター（ＡｕＮＳ６５）は、Ａｕシード溶液を２５ｍＬとした以外は、実施例４と同様にして、シード媒介成長法を用いて合成された。このようにして得られた金ナノスター（ＡｕＮＳ）を用い、以降の手順は、実施例１と同様にして、金ナノスター／架橋ゼラチンの複合多孔質材料を得た。 Gold nanostars (AuNS65) having a maximum diameter of 65 nm were synthesized using the seed-mediated growth method in the same manner as in Example 4 except that the Au seed solution was 25 mL. Using the gold nanostar (AuNS) thus obtained, a composite porous material of gold nanostar / crosslinked gelatin was obtained in the same manner as in Example 1 thereafter.
得られた金ナノスター（最大径６５ｎｍ）／架橋ゼラチンの複合多孔質材料を走査電子顕微鏡で観察し、図５（ａ）〜（ｃ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノスターを示す粒子状のものが観察された。
The obtained composite porous material of gold nanostar (
［実施例６］金ナノスター（直径１１５ｎｍ）／架橋ゼラチン複合多孔質材料
Ａｕ濃度１．０ｍＭ、２．０ｍＭ、４．０ｍＭの金ナノスター（長軸長さ１１５ｎｍ）を含む４．０（ｗ／ｖ）％ゼラチン溶液を用いて金ナノスター／架橋ゼラチン複合多孔質材料を作製した。多孔質材料の空孔形成剤として、大きさが４２５μｍ〜５００μｍの氷微粒子を用いた。
[Example 6] Gold nanostar (115 nm in diameter) / crosslinked gelatin composite porous material 4.0 (w / v) containing gold nanostar (major axis length 115 nm) having Au concentrations of 1.0 mM, 2.0 mM and 4.0 mM. A gold nanostar / crosslinked gelatin composite porous material was prepared using a% gelatin solution. As the pore-forming agent for the porous material, ice fine particles having a size of 425 μm to 500 μm were used.
最大径１１５ｎｍをもつ金ナノスター（ＡｕＮＳ１１５）は、Ａｕシード溶液を３．３ｍＬとした以外は、実施例４と同様にして、シード媒介成長法を用いて合成された。このようにして得られた金ナノスター（ＡｕＮＳ）を用い、以降の手順は、実施例１と同様にして、金ナノスター／架橋ゼラチンの複合多孔質材料を得た。 Gold nanostars (AuNS115) having a maximum diameter of 115 nm were synthesized using the seed-mediated growth method in the same manner as in Example 4 except that the Au seed solution was 3.3 mL. Using the gold nanostar (AuNS) thus obtained, a composite porous material of gold nanostar / crosslinked gelatin was obtained in the same manner as in Example 1 thereafter.
得られた金ナノスター（最大径１１５ｎｍ）／架橋ゼラチンの複合多孔質材料を走査電子顕微鏡で観察し、図６（ａ）〜（ｃ）に示す像を得た。（ａ）Ａｕ濃度１．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｂ）Ａｕ濃度２．０ｍＭ、ゼラチン濃度４．０（ｗ／ｖ）％、（ｃ）Ａｕ濃度４．０ｍＭ、ゼラチン濃度が４．０（ｗ／ｖ）％の各原料溶液を用いて作製した金ナノロッド／架橋ゼラチンの複合多孔質材料は、いずれも氷微粒子のサイズを反映した球状の空孔構造及び球状の空孔を連通する空隙からなる多孔質構造を有し、空孔壁面には金ナノスターを示す粒子状のものが観察された。 The obtained composite porous material of gold nanostar (maximum diameter 115 nm) / crosslinked gelatin was observed with a scanning electron microscope to obtain the images shown in FIGS. 6 (a) to 6 (c). (A) Au concentration 1.0 mM, gelatin concentration 4.0 (w / v)%, (b) Au concentration 2.0 mM, gelatin concentration 4.0 (w / v)%, (c) Au concentration 4.0 mM , The composite porous material of gold nanorod / cross-linked gelatin prepared by using each raw material solution having a gelatin concentration of 4.0 (w / v)% has a spherical pore structure and a spherical shape reflecting the size of ice fine particles. It has a porous structure consisting of voids communicating with the pores, and particles showing gold nanostars were observed on the wall surface of the pores.
［実施例７］近赤外レーザーの照射による複合多孔質材料の発熱効果
本実施例では、実施例１〜６で作製した金ナノロッド／架橋ゼラチン複合多孔質材料および金ナノスター／架橋ゼラチン複合多孔質材料の光熱効果を調べた。実施例１〜６で作製した金ナノロッドまたは金ナノスターである金ナノ粒子／架橋ゼラチン複合多孔質材料と、金ナノ粒子を含有しない架橋ゼラチン多孔質材料（コントロール）をそれぞれ５．０ｍｍ×３．０ｍｍ×１．０ｍｍに切断した。次に、各多孔質材料をそれぞれ２０μＬの細胞培養用培地浸漬し、波長８０５ｎｍ、出力密度１．６Ｗｃｍ−２の近赤外レーザー光を１８０ｓ間照射した。レーザー光照射の間、サンプルの温度はデジタル温度計を用いて１０ｓおきに測定した。結果を図７に示す。図７において、Ｇｅｌはコントロールを示し、Ｇｅｌ／Ｒ３５、Ｇｅｌ／Ｒ６５、Ｇｅｌ／Ｒ１１５、Ｇｅｌ／Ｓ３５、Ｇｅｌ／Ｓ６５およびＧｅｌ／Ｓ１１５は、それぞれ、実施例１〜６で作製した金ナノ粒子／架橋ゼラチンの複合多孔質材料を示す。
[Example 7] Heat-generating effect of the composite porous material by irradiation with a near-infrared laser In this example, the gold nanorod / crosslinked gelatin composite porous material and the gold nanostar / crosslinked gelatin composite porous prepared in Examples 1 to 6 are used. The photothermal effect of the material was investigated. The gold nanoparticles / crosslinked gelatin composite porous material which is the gold nanorod or gold nanostar prepared in Examples 1 to 6 and the crosslinked gelatin porous material (control) which does not contain gold nanoparticles are 5.0 mm × 3.0 mm, respectively. It was cut to × 1.0 mm. Next, each porous material was immersed in 20 μL of a cell culture medium, and irradiated with near-infrared laser light having a wavelength of 805 nm and an output density of 1.6 Wcm-2 for 180 s. During the laser irradiation, the temperature of the sample was measured every 10 seconds using a digital thermometer. The results are shown in FIG. In FIG. 7, Gel indicates control, and Gel / R35, Gel / R65, Gel / R115, Gel / S35, Gel / S65 and Gel / S115 are the gold nanoparticles / crosslinks prepared in Examples 1 to 6, respectively. The composite porous material of gelatin is shown.
図７（ａ）〜（ｃ）から、近赤外レーザー光照射の間の複合多孔質材料の温度は、照射時間、Ａｕ濃度の増加とともに上昇することが確認された。照射開始１８０ｓ後には、いずれの金ナノ粒子と複合化した架橋ゼラチン多孔質材料においても、金ナノ粒子不含架橋ゼラチン多孔質材料よりも高い温度に到達することがわかった。実施例１〜６で作製したいずれの金ナノ粒子でも架橋ゼラチン多孔質材料への含有率を調整することにより、がんの温熱療法で必要とされる温度である４２．５℃以上に１８０ｓ以内に加熱することができた。これに対して、金ナノ粒子と複合化していない架橋ゼラチン多孔質材料（コントロール）では、温度はほとんど上昇せず、近赤外レーザー光を１８０ｓ照射しても４２．５℃には到達しなかった。以上のことから、金ナノロッド／架橋ゼラチン複合多孔質材料、金ナノスター／架橋ゼラチン複合多孔質材料の光熱効果が確認された。生体内で前記複合多孔質材料を加熱する方法として近赤外レーザー光を用いることは可能なので、本発明の複合多孔質材料はがんの温熱療法に好適である。 From FIGS. 7 (a) to 7 (c), it was confirmed that the temperature of the composite porous material during the irradiation with the near-infrared laser light increased as the irradiation time and the Au concentration increased. It was found that 180 s after the start of irradiation, the crosslinked gelatin porous material composited with any of the gold nanoparticles reached a higher temperature than the gold nanoparticles-free crosslinked gelatin porous material. By adjusting the content of all the gold nanoparticles prepared in Examples 1 to 6 in the crosslinked gelatin porous material, the temperature is 42.5 ° C. or higher, which is the temperature required for hyperthermia for cancer, within 180 seconds. Could be heated to. On the other hand, in the crosslinked gelatin porous material (control) that is not composited with gold nanoparticles, the temperature hardly rises, and even if it is irradiated with near-infrared laser light for 180 s, it does not reach 42.5 ° C. rice field. From the above, the photothermal effect of the gold nanorod / crosslinked gelatin composite porous material and the gold nanostar / crosslinked gelatin composite porous material was confirmed. Since it is possible to use near-infrared laser light as a method for heating the composite porous material in a living body, the composite porous material of the present invention is suitable for hyperthermia for cancer.
［実施例８］複合多孔質材料の光熱効果によるがん細胞の殺傷効果
本実施例では、実施例１〜６で作製した金ナノロッド／架橋ゼラチン複合多孔質材料および金ナノスター／架橋ゼラチン複合多孔質材料によるがん細胞殺傷効果を調べた。まず、実施例１〜６で作製した金ナノ粒子／架橋ゼラチン複合多孔質材料と、コントロールの架橋ゼラチン多孔質材料をそれぞれ５．０ｍｍ×３．０ｍｍ×１．０ｍｍに切断した。次に、各多孔質材料に子宮頸がん由来のＨｅＬａ細胞を１×１０５個ずつ播種し、１日間培養した。波長８０５ｎｍ、出力密度１．６Ｗｃｍ−２の近赤外レーザー光を１８０ｓ間照射した後、公知のＷＳＴ−１法を用いて細胞生存率を測定した。結果を図８に示す。図８において、Ｇｅｌはコントロールを示し、Ｇｅｌ／Ｒ３５、Ｇｅｌ／Ｒ６５、Ｇｅｌ／Ｒ１１５、Ｇｅｌ／Ｓ３５、Ｇｅｌ／Ｓ６５およびＧｅｌ／Ｓ１１５は、それぞれ、実施例１〜６で作製した金ナノ粒子／架橋ゼラチンの複合多孔質材料を示す。
[Example 8] Killing effect of cancer cells by the photothermal effect of the composite porous material In this example, the gold nanorod / crosslinked gelatin composite porous material and the gold nanostar / crosslinked gelatin composite porous prepared in Examples 1 to 6 were prepared. The cancer cell killing effect of the material was investigated. First, the gold nanoparticles / crosslinked gelatin composite porous material prepared in Examples 1 to 6 and the crosslinked gelatin porous material of the control were cut into 5.0 mm × 3.0 mm × 1.0 mm, respectively. Next, 1 × 10 5 HeLa cells derived from cervical cancer were seeded on each porous material and cultured for 1 day. After irradiating with near-infrared laser light having a wavelength of 805 nm and an output density of 1.6 Wcm- 2 for 180 s, the cell viability was measured using a known WST-1 method. The results are shown in FIG. In FIG. 8, Gel indicates control, and Gel / R35, Gel / R65, Gel / R115, Gel / S35, Gel / S65 and Gel / S115 are the gold nanoparticles / crosslinks prepared in Examples 1 to 6, respectively. The composite porous material of gelatin is shown.
図８（ａ）に示すように、ナノ粒子の含有率が増加するにつれて、金ナノ粒子不含架橋ゼラチン多孔質材料よりも細胞生存率が低下し、殺傷効果が示された。一方、図８（ｂ）に示すように、近赤外レーザー光を照射しなかった場合も同様に細胞生存率を測定した。その結果、架橋ゼラチン多孔質材料が金ナノ粒子を含むか否かにかかわらず、同程度の非常に高い細胞生存率を示した。よって、本発明の金ナノ粒子／架橋ゼラチン複合多孔質材料は近赤外レーザー光照射による光熱効果によりがん細胞を殺傷する効果を持つことが実証された。 As shown in FIG. 8A, as the nanoparticle content increased, the cell viability decreased as compared with the gold nanoparticle-free crosslinked gelatin porous material, and a killing effect was shown. On the other hand, as shown in FIG. 8B, the cell viability was measured in the same manner when the near-infrared laser light was not irradiated. As a result, the cross-linked gelatin porous material showed the same very high cell viability regardless of whether or not it contained gold nanoparticles. Therefore, it was demonstrated that the gold nanoparticle / crosslinked gelatin composite porous material of the present invention has an effect of killing cancer cells by the photothermal effect of near-infrared laser irradiation.
上述してきたように、ナノ粒子として金ナノ粒子について説明してきたが、金ナノ粒子と同様に近赤外線を照射することにより発熱するナノ粒子であれば、本発明に適用できることは言うまでもない。 As described above, gold nanoparticles have been described as nanoparticles, but it goes without saying that nanoparticles that generate heat when irradiated with near-infrared rays, like gold nanoparticles, can be applied to the present invention.
本発明の複合多孔質材料は、がん組織の切除部位に移植される、または、がん組織を覆うことにより、近赤外光の照射によってがん組織およびがん細胞を死滅させることができるだけでなく、がん細胞を多孔質内に侵入させることができるので、がん細胞を効率的に死滅させることができる。しかも、ナノ粒子は多孔質材料に担持されているため、ナノ粒子がすばやく拡散して光熱効率が低下してしまうことを防ぎ、繰り返しがん組織の切除部位やがん組織を局所的に加熱することが可能である。よって、本発明の複合多孔質材料は、がんの治療に極めて有効である。 The composite porous material of the present invention can kill cancer tissue and cancer cells by irradiation with near-infrared light by being transplanted to the excision site of the cancer tissue or covering the cancer tissue. Instead, the cancer cells can be invaded into the porous medium, so that the cancer cells can be killed efficiently. Moreover, since the nanoparticles are supported on a porous material, they prevent the nanoparticles from diffusing quickly and reducing the photothermal efficiency, and repeatedly heat the excision site of the cancer tissue and the cancer tissue locally. It is possible. Therefore, the composite porous material of the present invention is extremely effective in treating cancer.
Claims (14)
生体吸収性高分子と、近赤外線照射で発熱するナノ粒子とを含み、
前記ナノ粒子は金ナノ粒子であり、
前記生体吸収性高分子に対する前記ナノ粒子の含有量は、３．０ｍＭ以上６．０ｍＭ以下の範囲であり、
前記生体吸収性高分子は、ゼラチン、コラーゲン、ヒアルロン酸、コンドロイチン硫酸、フィブロネクチン、ビトロネクチン、ラミニン、細胞成長因子、細胞分化制御因子、ポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体、ポリ（ε−カプロラクトン）、ポリ（グリセロールセバシン酸）及びこれらの共重合体からなる群から選択され、
気孔率は、２０％〜９９．９％の範囲である、複合多孔質材料。 Because it is implanted in the site where the cancer tissue has been removed by surgery, or it covers the cancer tissue directly, and when it is irradiated with near-infrared light from the outside, it generates heat and kills the cancer cells inside or around it. It is a composite porous material of
Contains bioabsorbable polymers and nanoparticles that generate heat when irradiated with near infrared rays.
The nanoparticles are gold nanoparticles and
The content of the nanoparticles to the bioabsorbable polymer, Ri 6.0mM the range der least 3.0 mM,
The bioabsorbable polymer includes gelatin, collagen, hyaluronic acid, chondroitin sulfate, fibronectin, bitronectin, laminin, cell growth factor, cell differentiation regulator, polylactic acid, polyglycolic acid, copolymer of lactic acid and glycolic acid, and poly. Selected from the group consisting of (ε-caprolactone), poly (glycerol sebacic acid) and copolymers thereof.
A composite porous material having a porosity in the range of 20% to 99.9%.
前記ナノ粒子の長軸長さは、６５ｎｍである、請求項３に記載の複合多孔質材料。The composite porous material according to claim 3, wherein the nanoparticles have a major axis length of 65 nm.
孔質材料。 The composite porous material according to claim 1, wherein the nanoparticles are a dense body, a porous body, or a cage body.
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