JP5476620B2 - Cell containing magnetic fine particles and method for producing the same - Google Patents
Cell containing magnetic fine particles and method for producing the same Download PDFInfo
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Description
本発明は、免疫細胞療法などの医学的治療、磁気共鳴画像診断（ＭＲＩ）などの医療診断などに用いることができる磁気微粒子包含細胞及びその製造方法に関する。 The present invention relates to a magnetic microparticle-containing cell that can be used for medical treatment such as immune cell therapy, medical diagnosis such as magnetic resonance imaging (MRI), and the like, and a method for producing the same.
近年、磁気微粒子を、ドラッグデリバリーシステム（ＤＤＳ）、核磁気共鳴画像診断（ＭＲＩ）、温熱療法などの医療に応用することが提案されている。そのなかでも、免疫細胞等の細胞に磁気粒子を導入又は結合させ、これを体内に導入し、免疫細胞の作用によりがん細胞等のターゲットに到達した磁気粒子に、外部から磁気を与えて、磁気誘導による発熱させてがん細胞等のターゲットを治療する手法や、体内に導入した免疫細胞の位置を、磁気粒子を利用してＭＲＩにより特定する手法などが考えられている。 In recent years, it has been proposed to apply magnetic fine particles to medicines such as drug delivery systems (DDS), nuclear magnetic resonance imaging (MRI), and thermotherapy. Among them, magnetic particles are introduced or bound to cells such as immune cells, this is introduced into the body, the magnetic particles that have reached the target such as cancer cells by the action of immune cells, give magnetism from the outside, A method of treating a target such as a cancer cell by generating heat by magnetic induction, a method of specifying the position of an immune cell introduced into the body by MRI using magnetic particles, and the like are considered.
このような手法に用いる磁気粒子は、体内での拒絶反応を避けるため細胞に内包される必要があり、細胞より小さいサイズであることが必要である。また、磁気誘導や検出に有効な磁気を有するサイズ以上であることも必要である。このようなサイズ範囲の中でより小さいものを用いることが、免疫細胞の利用効率や、体内での負荷の点からも好ましい。 Magnetic particles used in such a technique need to be encapsulated in cells in order to avoid rejection in the body, and must be smaller in size than the cells. It is also necessary to have a size larger than that of magnetism effective for magnetic induction and detection. It is preferable to use a smaller one in such a size range from the viewpoint of utilization efficiency of immune cells and load in the body.
従来、このような手法において用いることを想定した磁気微粒子包含細胞の磁気粒子としては、例えば、走磁性細菌が有するマグネトソームと呼ばれる磁気微粒子鎖を利用する方法（特開昭６２−２７５６７９号公報（特許文献１））などが報告されているが、このような生物学的手法による磁気微粒子の製造は、製造効率の点で劣っている。 Conventionally, as a magnetic particle of a magnetic particle-containing cell assumed to be used in such a method, for example, a method using a magnetic particle chain called a magnetosome possessed by a magnetotactic bacterium (Japanese Patent Laid-Open No. 62-275679 ( Patent Document 1)) has been reported, but the production of magnetic fine particles by such a biological technique is inferior in terms of production efficiency.
一方、化学的合成手法により磁気粒子を製造する手法によれば、磁気粒子の大量製造は可能であるが、ミクロンからサブミクロンサイズの粒子を導入しようとすると、細胞内に効率よく分散させることができないという問題があるため、より小さい合成磁気粒子を利用することが望まれるが、分散性の高い小粒径の磁気微粒子を、粒径を制御して効率よく製造することができていなかった。 On the other hand, according to the method of producing magnetic particles by a chemical synthesis method, mass production of magnetic particles is possible. However, when particles of micron to submicron size are introduced, they can be efficiently dispersed in cells. Since there is a problem that it is impossible, it is desired to use smaller synthetic magnetic particles. However, magnetic particles having a small particle size with high dispersibility cannot be efficiently produced by controlling the particle size.
本発明は、上記事情に鑑みなされたもので、免疫細胞療法などの医学的治療、磁気共鳴画像診断（ＭＲＩ）などの医療診断などにおいて有効に用いることができる磁気微粒子包含細胞、及びこのような磁気微粒子包含細胞を効率よく製造することができる方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and magnetic particle-containing cells that can be used effectively in medical treatment such as immune cell therapy, medical diagnosis such as magnetic resonance imaging (MRI), and such It is an object of the present invention to provide a method capable of efficiently producing magnetic particle-containing cells.
本発明者は、上記目的を達成するため鋭意検討を重ねた結果、平均粒径が１０〜４０ｎｍの合成磁気微粒子と、免疫細胞等の細胞とを混合して培養することによって、上記合成磁気微粒子を細胞内に導入することにより、合成磁気微粒子が効率よく、また分散性よく細胞内に内包され、平均粒径が１０〜４０ｎｍの合成磁気微粒子及び／又は該合成磁気微粒子の凝集粒子を内包した磁気微粒子包含細胞を製造することが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 to achieve the above object, the present inventor mixed and cultured synthetic magnetic fine particles having an average particle diameter of 10 to 40 nm and cells such as immune cells, and thus the above synthetic magnetic fine particles. Is incorporated into cells efficiently and with good dispersibility, and encapsulates synthetic magnetic fine particles having an average particle size of 10 to 40 nm and / or aggregated particles of the synthetic magnetic fine particles. It has been found that magnetic particle-containing cells can be produced, and the present invention has been made.
従って、本発明は、下記の磁気微粒子包含細胞及びその製造方法を提供する。
請求項１：
細胞内に平均粒径が１０〜４０ｎｍのアミン化合物で表面が修飾された合成磁気微粒子及び／又は該合成磁気微粒子の凝集粒子を内包してなることを特徴とする磁気微粒子包含細胞。
請求項２：
上記細胞が、免疫細胞であることを特徴とする請求項１記載の磁気微粒子包含細胞。
請求項３：
上記アミン化合物がアルキルジアミンであることを特徴とする請求項１又は２記載の磁気微粒子包含細胞。
請求項４：
上記合成磁気微粒子が、水溶性鉄塩を、アミン化合物を含む水溶液中で加水分解させることにより生成したマグネタイトであることを特徴とする請求項１〜３のいずれか１項記載の磁気微粒子包含細胞。
請求項５：
平均粒径が１０〜４０ｎｍのアミン化合物で表面が修飾された合成磁気微粒子と、細胞とを混合して培養することにより、上記合成磁気微粒子を細胞内に導入することを特徴とする請求項１記載の磁気微粒子包含細胞の製造方法。
請求項６：
上記細胞が、免疫細胞であることを特徴とする請求項５記載の磁気微粒子包含細胞の製造方法。
請求項７：
上記アミン化合物がアルキルジアミンであることを特徴とする請求項５又は６記載の磁気微粒子包含細胞の製造方法。
請求項８：
上記合成磁気微粒子が、水溶性鉄塩を、アミン化合物を含む水溶液中で加水分解させることにより生成したマグネタイトであることを特徴とする請求項５〜７のいずれか１項記載の磁気微粒子包含細胞の製造方法。
Accordingly, the present invention provides the following magnetic particle-containing cells and a method for producing the same.
Claim 1:
A magnetic particle-containing cell comprising cells containing synthetic magnetic fine particles whose surface is modified with an amine compound having an average particle size of 10 to 40 nm and / or aggregated particles of the synthetic magnetic fine particles.
Claim 2:
2. The magnetic particle-containing cell according to claim 1, wherein the cell is an immune cell.
Claim 3:
3. The magnetic fine particle-containing cell according to claim 1, wherein the amine compound is an alkyl diamine.
Claim 4 :
The magnetic fine particle-containing cell according to any one of claims 1 to 3, wherein the synthetic magnetic fine particle is a magnetite produced by hydrolyzing a water-soluble iron salt in an aqueous solution containing an amine compound. .
Claim 5 :
2. The synthetic magnetic fine particles are introduced into cells by mixing and culturing the cells with synthetic magnetic fine particles whose surface is modified with an amine compound having an average particle size of 10 to 40 nm. A method for producing the magnetic particle-containing cell according to the description.
Claim 6 :
6. The method for producing magnetic microparticle-containing cells according to claim 5 , wherein the cells are immune cells.
Claim 7:
The method for producing cells containing magnetic fine particles according to claim 5 or 6, wherein the amine compound is an alkyl diamine.
Claim 8 :
The magnetic fine particle-containing cell according to any one of claims 5 to 7, wherein the synthetic magnetic fine particle is a magnetite produced by hydrolyzing a water-soluble iron salt in an aqueous solution containing an amine compound. Manufacturing method.
本発明によれば、免疫細胞療法などの医学的治療、磁気共鳴画像診断（ＭＲＩ）などの医療診断などにおいて有効に用いることができる磁気微粒子包含細胞として、小粒径の微粒子及び／又はその凝集粒子が分散性よく内包された磁気微粒子包含細胞を提供することができ、また、このような磁気微粒子包含細胞を効率よく製造することができる。本発明の磁気微粒子包含細胞は、磁気誘導を利用した温熱療法等にも応用が可能である。 According to the present invention, small particles and / or aggregates thereof can be used as magnetic particle-containing cells that can be used effectively in medical treatment such as immune cell therapy, medical diagnosis such as magnetic resonance imaging (MRI), and the like. Magnetic fine particle-containing cells in which particles are encapsulated with good dispersibility can be provided, and such magnetic fine particle-containing cells can be efficiently produced. The magnetic particle-containing cells of the present invention can be applied to hyperthermia using magnetic induction.
以下、本発明につき更に詳しく説明する。
本発明の磁気微粒子包含細胞は、細胞内に合成磁気微粒子を内包するものであり、合成磁気微粒子は、平均粒径が１０〜４０ｎｍの合成磁気微粒子の単一粒子として、また上記合成磁気微粒子の凝集粒子、例えば、平均長さが１μｍ以下、好ましくは０．１〜１μｍの鎖状等の凝集体として内包されている。
Hereinafter, the present invention will be described in more detail.
The magnetic fine particle-containing cell of the present invention encapsulates synthetic magnetic fine particles in the cell. The synthetic magnetic fine particle is a single particle of synthetic magnetic fine particles having an average particle size of 10 to 40 nm, and Aggregated particles, for example, encapsulated as aggregates such as chains having an average length of 1 μm or less, preferably 0.1 to 1 μm.
本発明において対象とする細胞としては、例えば免疫細胞が好適であり、免疫細胞としては、腫瘍抗原特異的な細胞傷害性Ｔ細胞（ＣＴＬ細胞）、活性化リンパ球、正常リンパ球等のリンパ球細胞、樹状細胞、Ｂ細胞などを挙げることができる。このような免疫細胞にマグネタイト等の磁気微粒子を取り込ませることにより、例えば、マグネタイトを内包した高機能性がん特異的キラー細胞を製造し、磁力によりがん局所へ集積させることによる治療効果が期待できる。また磁気微粒子を取り込んだキラー細胞のがん局所への集積により、磁気微粒子をがん局所へ到達させ、磁気誘導を利用した誘導加熱により、局所的にがん細胞のみを死滅させる高効率ながん免疫細胞療法も可能となる。また、磁気微粒子を内包したキラー細胞が到達したがん部位を、磁性微粒子をマーカーとして、ＭＲＩによりモニタリングすることが可能であり、これにより発見が難しい微小ながんの早期発見も可能である。 As the cells targeted in the present invention, for example, immune cells are suitable, and examples of immune cells include lymphocytes such as tumor antigen-specific cytotoxic T cells (CTL cells), activated lymphocytes, and normal lymphocytes. Examples thereof include cells, dendritic cells, and B cells. By incorporating magnetic fine particles such as magnetite into such immune cells, for example, a highly functional cancer-specific killer cell encapsulating magnetite is produced, and a therapeutic effect is expected by accumulating locally in the cancer by magnetic force. it can. In addition, high-efficiency is effective in that killer cells that have taken in magnetic particles accumulate in the local cancer area, so that the magnetic fine particles reach the local cancer area, and induction heating using magnetic induction locally kills only the cancer cells. Cancer cell therapy is also possible. In addition, it is possible to monitor the cancer site reached by the killer cells encapsulating the magnetic fine particles by MRI using the magnetic fine particles as a marker, thereby enabling early detection of minute cancers that are difficult to find.
本発明において、磁性微粒子としては、酸化鉄、特に、マグネタイト（Ｆｅ3Ｏ4）が好適である。平均粒径が１０〜４０ｎｍのマグネタイトは、例えば、水溶性鉄塩を、アミン化合物を含む水溶液中で加水分解させることにより製造することができる。 In the present invention, iron oxide, particularly magnetite (Fe 3 O 4 ) is suitable as the magnetic fine particles. A magnetite having an average particle size of 10 to 40 nm can be produced, for example, by hydrolyzing a water-soluble iron salt in an aqueous solution containing an amine compound.
この場合、水溶性鉄塩としては、硫酸鉄［ＩＩ］（ＦｅＳＯ4・７Ｈ2Ｏなど）、硫酸鉄［ＩＩＩ］（Ｆｅ2（ＳＯ4）・ｎＨ2Ｏ）、塩化鉄［ＩＩ］（ＦｅＣｌ2・４Ｈ2Ｏなど）、塩化鉄［ＩＩＩ］（ＦｅＣｌ3・６Ｈ2Ｏなど）などを用いることができる。 In this case, as the water-soluble iron salt, iron sulfate [II] (FeSO 4 .7H 2 O etc.), iron sulfate [III] (Fe 2 (SO 4 ) · nH 2 O), iron chloride [II] (FeCl such as 2 · 4H 2 O), iron chloride [III] (FeCl 3 · 6H 2 O , etc.) or the like can be used.
一方、加水分解反応においてアルカリとして作用するアミン化合物としては、１，６−ヘキサンジアミン等のアルキルジアミンなどを用いることができる。アミン化合物は、生成したマグネタイト微粒子の表面を修飾して粒子同士の凝集を抑制する作用も有し、これにより、マグネタイト粒子をより分散性よく生成させることが可能となる。 On the other hand, alkyldiamines such as 1,6-hexanediamine can be used as the amine compound that acts as an alkali in the hydrolysis reaction. The amine compound also has an action of modifying the surface of the generated magnetite fine particles to suppress aggregation of the particles, thereby making it possible to generate magnetite particles with better dispersibility.
本発明においては、水溶性鉄塩とアミン化合物とを混合した水溶液を、必要に応じて攪拌しながら２０〜２５℃程度の温度で２０〜２４時間程度熟成することによりマグネタイトを生成させることが可能である。例えば、水溶性鉄塩水溶液の濃度を０．０４〜０．０６ｍｏｌ／Ｌ、アミン化合物水溶液の濃度を０．２４〜０．２６ｍｏｌ／Ｌとして、水溶性鉄塩に対してアミン化合物を５当量程度となるように両水溶液を混合すればよい。 In the present invention, it is possible to generate magnetite by aging an aqueous solution in which a water-soluble iron salt and an amine compound are mixed at a temperature of about 20 to 25 ° C. for about 20 to 24 hours while stirring as necessary. It is. For example, the concentration of the water-soluble iron salt aqueous solution is 0.04 to 0.06 mol / L, the concentration of the amine compound aqueous solution is 0.24 to 0.26 mol / L, and the amine compound is about 5 equivalents to the water-soluble iron salt. What is necessary is just to mix both aqueous solution so that it may become.
生成したマグネタイト粒子は、必要に応じて、ろ過、洗浄、乾燥等の工程を経て、回収される。このような方法でマグネタイトを製造すれば、平均粒径が１０〜４０ｎｍのマグネタイトを分散性よく製造することができ、また、例えば、水溶性鉄塩（Ｆｅイオン）の濃度を調整することにより、所望の平均粒径のマグネタイト微粒子を製造することが可能である。 The produced magnetite particles are recovered through steps such as filtration, washing, and drying as necessary. If magnetite is produced by such a method, magnetite having an average particle size of 10 to 40 nm can be produced with good dispersibility. For example, by adjusting the concentration of water-soluble iron salt (Fe ion), It is possible to produce magnetite fine particles having a desired average particle diameter.
このような方法でマグネタイト微粒子を製造すれば、４０〜９０ｅｍｕ／ｇ（Ａ・ｍ2／ｋｇ）、特に７０〜９０ｅｍｕ／ｇ（Ａ・ｍ2／ｋｇ）の飽和磁化、１０〜１００Ｏｅ、特に５０〜１００Ｏｅの保磁力を示す良好な磁気特性を示すマグネタイト微粒子を得ることができる。 When magnetite fine particles are produced by such a method, saturation magnetization of 40 to 90 emu / g (A · m 2 / kg), particularly 70 to 90 emu / g (A · m 2 / kg), 10 to 100 Oe, especially 50 Magnetite fine particles having good magnetic properties exhibiting a coercive force of ˜100 Oe can be obtained.
次に、磁性微粒子を細胞に導入する方法について説明する。例えば、上述した方法により製造した平均粒径が１０〜４０ｎｍの合成磁気微粒子及び／又は該合成磁気微粒子の凝集粒子を用い、この合成磁気微粒子（凝集粒子）を、リン酸緩衝生理食塩水等の生理食塩水中に、必要に応じて超音波等を使用して、分散させて分散液を調製し、この分散液と、従来公知の方法で採取、調製したリンパ球細胞等の免疫細胞と、必要に応じて生理食塩水とを混合し、磁気微粒子が１００〜２００μｇ／ｍｌ程度、免疫細胞が１×１０6〜２×１０6個／ｍｌ程度の混合液とし、これを例えばヒトの免疫細胞であればその体温前後の温度、例えば３７℃程度の温度で、磁性微粒子の内包に必要な所定時間、例えば４時間程度培養すれば、磁性微粒子を細胞に導入することが可能である。特に、上述した方法により製造した合成磁気微粒子（凝集粒子）は、合成磁気微粒子（凝集粒子）であるが、免疫細胞との適合性がよく、粒子の内包がスムーズに進行すると共に、粒子の内包によって免疫細胞自身の機能（活性）が損なわれることがない。 Next, a method for introducing magnetic fine particles into cells will be described. For example, synthetic magnetic fine particles having an average particle diameter of 10 to 40 nm produced by the above-described method and / or aggregated particles of the synthetic magnetic fine particles are used, and the synthetic magnetic fine particles (aggregated particles) are converted into phosphate buffered saline or the like. Disperse in physiological saline using ultrasound as necessary to prepare a dispersion, and this dispersion, immune cells such as lymphocyte cells collected and prepared by a conventionally known method, and necessary Depending on the condition, the mixture is mixed with physiological saline to obtain a mixed solution of about 100 to 200 μg / ml of magnetic fine particles and about 1 × 10 6 to 2 × 10 6 cells / ml of immune cells. If present, the magnetic fine particles can be introduced into the cells by culturing at a temperature around the body temperature, for example, about 37 ° C., for a predetermined time required for inclusion of the magnetic fine particles, for example, about 4 hours. In particular, the synthetic magnetic fine particles (aggregated particles) produced by the above-described method are synthetic magnetic fine particles (aggregated particles), but have good compatibility with immune cells, and the inclusion of the particles proceeds smoothly and the inclusion of the particles. Thus, the function (activity) of the immune cell itself is not impaired.
このような方法により、本発明の磁気微粒子包含細胞を製造することが可能であるが、培養の条件を変更、例えば、濃度を変更したり、培養温度や培養時間を選択したりすることにより、得られた磁気微粒子包含細胞の細胞内に、平均粒径が１０〜４０ｎｍの合成磁気微粒子が単一で分散したもの、平均粒径が１０〜４０ｎｍの合成磁気微粒子が、平均長さが１μｍ以下、特に０．１〜１μｍの鎖状等の凝集粒子として分散したもの、双方が分散したものを製造することが可能である。 By such a method, it is possible to produce the magnetic particle-containing cells of the present invention, but by changing the culture conditions, for example, by changing the concentration, selecting the culture temperature and culture time, A single synthetic magnetic fine particle having an average particle diameter of 10 to 40 nm dispersed within the cells of the obtained magnetic fine particle-containing cells, or an average length of synthetic magnetic fine particles having an average particle diameter of 10 to 40 nm is 1 μm or less. In particular, it is possible to produce a dispersion in which the particles are dispersed as 0.1-1 μm chain aggregated particles, or in which both are dispersed.
以下、実施例を示し、本発明を具体的に説明するが、本発明は下記実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to the following Example.
［実施例１］
水溶性鉄塩として、硫酸鉄［ＩＩ］を０．０５ｍｏｌ／Ｌで含有する水溶液１００ｍｌに、１，６−ヘキサンジアミンを０．２５ｍｏｌ／Ｌで含有する水溶液１００ｍｌを加え、２５℃で２４時間強攪拌することにより、黒色の沈殿物を得た。得られた沈殿物をろ過により分離し、水洗後、室温で乾燥して微粒子を得た。
[Example 1]
As a water-soluble iron salt, 100 ml of an aqueous solution containing 1,6-hexanediamine at 0.25 mol / L was added to 100 ml of an aqueous solution containing iron sulfate [II] at 0.05 mol / L. A black precipitate was obtained by stirring. The obtained precipitate was separated by filtration, washed with water, and dried at room temperature to obtain fine particles.
得られた微粒子は、Ｘ線回折法により酸化鉄（マグネタイト）であることが確認された。また、微粒子の粒径を透過型電子顕微鏡像から計測したところ、平均粒子径が４０ｎｍのナノ粒子であることが確認された。更に、得られたマグネタイトの飽和磁化は８７Ａ・ｍ2／ｋｇ、保磁力は６６Ｏｅであり、良好な磁気特性を有する磁気粒子であることが確認された。 The obtained fine particles were confirmed to be iron oxide (magnetite) by an X-ray diffraction method. Moreover, when the particle size of the fine particles was measured from a transmission electron microscope image, it was confirmed that the particles were nanoparticles having an average particle size of 40 nm. Further, the obtained magnetite had a saturation magnetization of 87 A · m 2 / kg, a coercive force of 66 Oe, and was confirmed to be a magnetic particle having good magnetic properties.
次に、得られた酸化鉄（マグネタイト）ナノ粒子を、リン酸緩衝生理食塩水中に１０〜２０ｍｇ／ｍｌの濃度となるように混合し、超音波処理により分散させた分散液を調製した。その後、２×１０6個／ｍｌのヒト由来正常リンパ球細胞に対して、マグネタイトの添加量が２００μｇ／ｍｌとなるように混合（リン酸緩衝生理食塩水により希釈）し、緩やかに撹拌しながら、３７℃で４時間培養することによって、リンパ球細胞に酸化鉄（マグネタイト）ナノ粒子を導入する処理を実施した。 Next, the obtained iron oxide (magnetite) nanoparticles were mixed in phosphate buffered saline so as to have a concentration of 10 to 20 mg / ml, and a dispersion was prepared by dispersing by ultrasonication. Thereafter, 2 × 10 6 / ml human-derived normal lymphocyte cells were mixed (diluted with phosphate buffered saline) so that the amount of magnetite added was 200 μg / ml, and gently stirred. Incubation was carried out at 37 ° C. for 4 hours to carry out treatment for introducing iron oxide (magnetite) nanoparticles into lymphocyte cells.
培養後の培養液よりリンパ球細胞のみを遠心分離によって分離し、細胞内に取り込まれた鉄イオン濃度より、マグネタイトナノ粒子の取り込み量を見積もったところ、添加量に対して１０〜２０質量％のマグネタイトナノ粒子がリンパ球細胞中に取り込まれていることが確認された。 Only lymphocyte cells were separated from the culture broth after culturing by centrifugation, and the amount of magnetite nanoparticles incorporated was estimated from the concentration of iron ions incorporated into the cells. It was confirmed that magnetite nanoparticles were taken up into lymphocyte cells.
更に、リンパ球細胞中へのマグネタイトナノ粒子の取り込みを確認するために、透過型電子顕微鏡による細胞観察を行った。その結果、図１に示されるように、リンパ球中へベシクルの形成（図中の矢印で示した部分）を伴って、マグネタイトナノ粒子が数マイクロメートルのベシクル中に凝集体として取り込まれている様子が確認された。 Furthermore, in order to confirm the uptake of the magnetite nanoparticles into the lymphocyte cells, the cells were observed with a transmission electron microscope. As a result, as shown in FIG. 1, magnetite nanoparticles are incorporated as aggregates into vesicles of several micrometers with the formation of vesicles in lymphocytes (portions indicated by arrows in the figure). The situation was confirmed.
［実施例２］
実施例１と同様の方法により得た酸化鉄（マグネタイト）ナノ粒子を用い、ヒト由来正常リンパ球細胞の代わりに、メラノーマ抗原であるｇｐ１００−Ａ２ペプチド（ｇｐ１００209-217:ＩＭＤＱＶＰＦＳＶ）に特異的なＣＴＬ ｌｉｎｅ（ＣＴＬ細胞株：ｇｐ１００ペプチド ＨＬＡ−Ａ２ ｔｅｔｒａｍｅｒ ４４％）を用いて、ＣＴＬ細胞に酸化鉄（マグネタイト）ナノ粒子を導入する処理を実施した。
[Example 2]
Using iron oxide (magnetite) nanoparticles obtained by the same method as in Example 1, instead of human-derived normal lymphocyte cells, it is specific to the melanoma antigen gp100-A2 peptide (gp100 209-217 : IMDQVPFSV) Using CTL line (CTL cell line: gp100 peptide HLA-A2 tetramer 44%), treatment for introducing iron oxide (magnetite) nanoparticles into CTL cells was performed.
培養後の培養液よりリンパ球細胞のみを遠心分離によって分離し、細胞内に取り込まれた鉄イオン濃度より、マグネタイトナノ粒子の取り込み量を見積もったところ、添加量に対して３回の平均で２７．６質量％のマグネタイトナノ粒子がＣＴＬ細胞中に取り込まれていることが確認された。 Only lymphocyte cells were separated from the culture broth after culturing by centrifugation, and the amount of magnetite nanoparticles incorporated was estimated from the concentration of iron ions incorporated into the cells. It was confirmed that .6% by mass of magnetite nanoparticles was taken up into CTL cells.
更に、リンパ球細胞中へのマグネタイトナノ粒子の取り込みを確認するために、透過型電子顕微鏡による細胞観察を行った。その結果、ＣＴＬ細胞中へベシクルの形成を伴って、マグネタイトナノ粒子が数マイクロメートルのベシクル中に凝集体として取り込まれている様子が確認された。 Furthermore, in order to confirm the uptake of the magnetite nanoparticles into the lymphocyte cells, the cells were observed with a transmission electron microscope. As a result, it was confirmed that the magnetite nanoparticles were incorporated as aggregates in the vesicles of several micrometers with the formation of vesicles in CTL cells.
次に、マグネタイトナノ粒子を取り込んだＣＴＬ細胞株をｇｐ１００−Ａ２ペプチド（５０μｇ／ｍｌ）にて処理したＴ２細胞（ヒトＴ／Ｂ細胞のハイブリッドであり、ＨＬＡ−Ａ２分子のみ発現する細胞株）と２４時間インキュベートした。細胞数は、１×１０5ずつに調整（９６−ｗｅｌｌマイクロプレート）し、培地はＲＰＭＩ１６４０＋１０％ＦＢＳを用いた。培養後、上澄みを回収し産生されたＩＦＮ−γの量を測定した。 Next, a T2 cell (a cell line that is a hybrid of human T / B cell and expresses only HLA-A2 molecule) obtained by treating a CTL cell line incorporating magnetite nanoparticles with gp100-A2 peptide (50 μg / ml) Incubated for 24 hours. The number of cells was adjusted to 1 × 10 5 (96-well microplate), and RPMI1640 + 10% FBS was used as the medium. After culturing, the supernatant was recovered and the amount of IFN-γ produced was measured.
マグネタイトナノ粒子を取り込んだＣＴＬ細胞株は、ｇｐ１００−Ａ２ペプチドに特異的に反応し、高濃度のＩＦＮ−γを産生した。マグネタイトナノ粒子を取り込ませていない細胞との比較では、明らかな差は認められなかった。結果を図２に示す。これによりマグネタイトナノ粒子を取り込んだＣＴＬ細胞において、ペプチド特異的な細胞障害活性は損なわれないことが確認された。なお、ペプチドの陰性コントロールとして同じＨＬＡ−Ａ２拘束性をもつインフルエンザＭＰペプチド（Ｆｌｕ−ＭＰ58-66:ＧＩＬＧＦＶＦＴＬ）を使用して評価した。 CTL cell lines that incorporated magnetite nanoparticles specifically reacted with the gp100-A2 peptide and produced high concentrations of IFN-γ. A clear difference was not observed in comparison with cells not incorporating magnetite nanoparticles. The results are shown in FIG. This confirmed that peptide-specific cytotoxic activity was not impaired in CTL cells incorporating magnetite nanoparticles. Note that the influenza MP peptide having the same HLA-A2-restricted as a negative control peptide (Flu-MP 58-66: GILGFVFTL) were assessed using.
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