JP4933569B2 - Method for forming nanobubbles - Google Patents
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- JP4933569B2 JP4933569B2 JP2009007241A JP2009007241A JP4933569B2 JP 4933569 B2 JP4933569 B2 JP 4933569B2 JP 2009007241 A JP2009007241 A JP 2009007241A JP 2009007241 A JP2009007241 A JP 2009007241A JP 4933569 B2 JP4933569 B2 JP 4933569B2
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- 239000002101 nanobubble Substances 0.000 title claims description 82
- 238000000034 method Methods 0.000 title claims description 55
- 239000002105 nanoparticle Substances 0.000 claims description 48
- 229920000642 polymer Polymers 0.000 claims description 37
- 239000002904 solvent Substances 0.000 claims description 28
- 239000007789 gas Substances 0.000 claims description 25
- 239000010931 gold Substances 0.000 claims description 22
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 20
- 229910052737 gold Inorganic materials 0.000 claims description 20
- 239000002961 echo contrast media Substances 0.000 claims description 18
- 230000015572 biosynthetic process Effects 0.000 claims description 17
- 238000000576 coating method Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000011246 composite particle Substances 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 9
- 239000010954 inorganic particle Substances 0.000 claims description 8
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- QZPSXPBJTPJTSZ-UHFFFAOYSA-N aqua regia Chemical group Cl.O[N+]([O-])=O QZPSXPBJTPJTSZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000003759 clinical diagnosis Methods 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 229910010272 inorganic material Inorganic materials 0.000 claims description 3
- 239000011147 inorganic material Substances 0.000 claims description 3
- 229910052742 iron Inorganic materials 0.000 claims description 3
- 229910052754 neon Inorganic materials 0.000 claims description 3
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- 229910052724 xenon Inorganic materials 0.000 claims description 3
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 claims description 3
- OLBVUFHMDRJKTK-UHFFFAOYSA-N [N].[O] Chemical compound [N].[O] OLBVUFHMDRJKTK-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 33
- 238000002604 ultrasonography Methods 0.000 description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 235000010339 sodium tetraborate Nutrition 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 239000002872 contrast media Substances 0.000 description 5
- XRWMGCFJVKDVMD-UHFFFAOYSA-M didodecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCC XRWMGCFJVKDVMD-UHFFFAOYSA-M 0.000 description 5
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 229910021538 borax Inorganic materials 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- -1 for example Chemical group 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 4
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000008051 TBE buffer Substances 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005266 casting Methods 0.000 description 3
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 3
- 238000002296 dynamic light scattering Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 101710106714 Shutoff protein Proteins 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
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- CDMADVZSLOHIFP-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane;decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 CDMADVZSLOHIFP-UHFFFAOYSA-N 0.000 description 2
- JRBPAEWTRLWTQC-UHFFFAOYSA-N dodecylamine Chemical compound CCCCCCCCCCCCN JRBPAEWTRLWTQC-UHFFFAOYSA-N 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical compound Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000009022 nonlinear effect Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 229910003771 Gold(I) chloride Inorganic materials 0.000 description 1
- 229910003803 Gold(III) chloride Inorganic materials 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010304 firing Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical group O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
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- 230000000638 stimulation Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
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- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、ナノバブルの形成方法に係り、特に超音波造影剤を用いるナノ級バブルの形成方法に関する。 The present invention relates to a method for forming nanobubbles, and more particularly to a method for forming nanoclass bubbles using an ultrasonic contrast agent.
超音波造影剤（Ultrasound Contrast Agent:UCA）は超音波画像形成において画像のコントラスト強化をする為の物質である。一般にはミクロン級直径の包膜マイクロバブルであり、静脈注射が血液循環システムに進入し超音波の反射強度を増強することによって、超音波造影によって形成される画像の解析度を向上させる目的を達成する。 Ultrasound Contrast Agent (UCA) is a substance for enhancing image contrast in ultrasonic imaging. Generally, micron-sized enveloped microbubbles, which achieve the purpose of improving the resolution of images formed by ultrasound contrast, by intravenous injection entering the blood circulation system and enhancing the reflection intensity of ultrasound. To do.
超音波造影剤を血管に注入した後、組織の超音波特性（背部方向散乱係数、減衰係数、音声スピード、及び非線形効果）が変化し造影効果が生まれる。増強効果は超音波造影剤の濃度、サイズ、及び超音波の発射周波率によって決まる。それの最も基本的性質は組織のエコー能力を増強できることにあり、超音波画像形成において画像の明晰度とコントラストを高めることが可能となる。前記非線形効果は一定エネルギーの調波量を生じさせ、調波画像形成技術は体内の微小血管血流と組織灌流を測定し、超音波造影剤を含まない組織がベースバンド上で運動し生成されるクラッタ信号を抑制することによりS/N比を大幅に向上させることが可能である。 After injecting the ultrasound contrast agent into the blood vessel, the ultrasound properties of the tissue (backward direction scattering coefficient, attenuation coefficient, sound speed, and non-linear effect) change to produce a contrast effect. The enhancement effect depends on the concentration and size of the ultrasound contrast agent and the ultrasound firing frequency rate. Its most basic property is that it can enhance the echo ability of the tissue, and it is possible to increase the clarity and contrast of the image in ultrasonic imaging. The non-linear effect produces constant energy harmonics, harmonic imaging technology measures microvascular blood flow and tissue perfusion in the body, and tissue that does not contain ultrasound contrast agents is generated by motion on the baseband. By suppressing the clutter signal, the S / N ratio can be greatly improved.
超音波造影剤の研究と応用は1968年Gramiakらによる心臓内への塩水注入後の大動脈根部において雲状コントラストエコー効果が得られた研究に遡る。８０年代後期、超音波組織はある困難に遭遇した。それは、ある組織は病理上区別されているが、それらの超音波特性が類似していることにあった。これに対して、組織と血液のエコー能力を増強できる超音波造影剤は非常に大きな注目を浴びている。 The study and application of ultrasound contrast agents go back to 1968 by Gramiak et al., Where a cloud-like contrast echo effect was obtained in the aortic root after infusion of saline into the heart. In the late 80s, ultrasound tissue encountered certain difficulties. It was that some tissues were pathologically distinct but their ultrasound characteristics were similar. In contrast, ultrasonic contrast agents that can enhance the ability of tissue and blood to echo have received much attention.
造影剤は主にマイクロバブル内に含まれる気体の種類によって分類される。第一世代造影剤はマイクロバブル内に空気を含み、包膜は一般にアルブミンまたはガラクトース等の重合体である。第二世代超音波造影剤のマイクロバブルは主に高密度惰性気体（水や血液に溶け難い）を含むもので、その外膜は薄くて柔軟である。前記第一世代造影剤のマイクロバブルの包膜は比較的厚く、弾性に劣り、含まれる空気は水に溶け易い等から、前記第一世代造影剤は破裂し易くライフタイムも短く、臨床応用における観察及び診断時間が制限される。しかし第二世代造影剤のマイクロバブルの直径は2-5um前後にまで縮小することができ、安定時間を延長し、振動及びエコー特性を更に良好にする。 Contrast agents are classified mainly by the type of gas contained in the microbubbles. First generation contrast agents contain air in microbubbles and the envelope is generally a polymer such as albumin or galactose. The microbubbles of the second generation ultrasound contrast agent mainly contain a high-density gas (it is difficult to dissolve in water and blood), and its outer membrane is thin and flexible. The microbubble envelope of the first generation contrast agent is relatively thick, inferior in elasticity, and the contained air is easily dissolved in water, etc., so the first generation contrast agent is easy to rupture and has a short lifetime. Observation and diagnosis time is limited. However, the diameter of the microbubbles of the second generation contrast agent can be reduced to around 2-5um, extending the stabilization time and further improving the vibration and echo characteristics.
近年来、超音波造影剤は既に超音波治療領域で広く応用されている。超音波造影剤中のマイクロバブルにキャビテーション効果が強化され、更に超音波生物効果が促進される故、超音波造影剤は超音波血栓溶解療法、介在遺伝子転移、薬物デリバリー、高密度焦点式超音波療法（ＨＩＦＵ）等への応用にも既に研究が始められている。超音波造影剤の応用範囲は拡大を続け、応用価値もまた向上し続けている。よって、安定性が高く、サイズコントロール性を備え、生物分解性と生物適合性を備えるマイクロバブル超音波造影剤は業界において発展が大きく期待されるものである。 In recent years, ultrasonic contrast agents have already been widely applied in the field of ultrasonic therapy. Because the cavitation effect is enhanced by the microbubbles in the ultrasound contrast agent and the ultrasound biological effect is further promoted, the ultrasound contrast agent is used for ultrasound thrombolysis, intervening gene transfer, drug delivery, high-intensity focused ultrasound Research has already begun on applications to therapy (HIFU) and the like. The application range of ultrasound contrast agents continues to expand, and the application value continues to improve. Therefore, the microbubble ultrasonic contrast agent having high stability, size controllability, biodegradability and biocompatibility is highly expected in the industry.
本発明はナノバブルの形成方法を提供することにあり、前記形成方法は公知の油水乳化反応とは異なるものである。本発明のナノバブルの形成方法は、無機粒子を中心核として、前記中心核の表面に最低一つの第一高分子をコーティングして有機／無機複合粒子を形成する。次に、第一溶剤によって前記有機／無機複合粒子の中心核を溶解して除去し溶剤型ナノ粒子を形成する。続いて冷凍乾燥プロセスにおいて前記第一溶剤を除去し前記溶剤型ナノ粒子に中空ナノ粒子を形成させる。最後に前記中空ナノ粒子を第二溶剤に溶かして前記ナノバブルを形成する。 The present invention provides a method for forming nanobubbles, which is different from a known oil-water emulsification reaction. In the method of forming nanobubbles of the present invention, organic / inorganic composite particles are formed by coating inorganic particles as central nuclei and coating at least one first polymer on the surface of the central nuclei. Next, the central core of the organic / inorganic composite particles is dissolved and removed with a first solvent to form solvent-type nanoparticles. Subsequently, in the freeze-drying process, the first solvent is removed to form hollow nanoparticles in the solvent-type nanoparticles. Finally, the hollow nanoparticles are dissolved in a second solvent to form the nanobubbles.
請求項１の発明は、無機粒子を中心核として、高分子層コーティングプロセスを行い、前記中心核の表面に最低一つの第一高分子を被覆して有機／無機複合粒子を形成し、
前記有機／無機複合粒子は第一溶剤に接触し、前記第一溶剤で前記有機／無機複合粒子の中心核を溶解して除去し溶剤型ナノ粒子を形成し、前記溶剤型ナノ粒子の内部には前記第一溶剤を内含し、
冷凍乾燥プロセスにおいて前記第一溶剤を除去して前記溶剤型ナノ粒子に中空ナノ粒子を形成させ、
溶解プロセスにおいて前記中空ナノ粒子を第二溶剤に溶かしてナノバブルを形成することを特徴とするナノバブルの形成方法としている。
請求項２の発明は、請求項１記載のナノバブルの形成方法において、前記無機粒子は、金、銀、鉄、及びその他無機材料のナノ粒子のグループの内から何れか一つを選択することを特徴とするナノバブルの形成方法としている。
請求項３の発明は、請求項１記載のナノバブルの形成方法において、前記第一高分子は、ポリ（イソブチレン−アルト−無水マレイン酸）−グラフト−ドデシルアミン（poly(isobutylene-alt-maleic anhydride)-graft-dodecylamine）及びそれに類似する高分子であることを特徴とするナノバブルの形成方法としている。
請求項４の発明は、請求項１記載のナノバブルの形成方法において、前記第一溶剤は王水であることを特徴とするナノバブルの形成方法としている。
請求項５の発明は、請求項１記載のナノバブルの形成方法において、前記第二溶剤は、純水及び各種バッファ溶液のグループの何れか一つとすることを特徴とするナノバブルの形成方法としている。
請求項６の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルの粒径範囲は３０ｎｍ〜１００００ｎｍとすることを特徴とするナノバブルの形成方法としている。
請求項７の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルの粒径は無機粒子のサイズによりコントロールされることを特徴とするナノバブルの形成方法としている。
請求項８の発明は、請求項１記載のナノバブルの形成方法において、前記第一高分子層コーティングプロセスを行った後、更にバイオコンジュゲーションプロセスを行い、生物適応性を備える第二高分子を第一高分子層の表面に接合することを特徴とするナノバブルの形成方法としている。
請求項９の発明は、請求項８記載のナノバブルの形成方法において、前記第二高分子はＮＨ2官能基グループを含む分子であることを特徴とするナノバブルの形成方法としている。
請求項１０の発明は、請求項１記載のナノバブルの形成方法において、前記溶解プロセスには、更に希望する気体気流を中空ナノ粒子上に施用し、並びに中空ナノ粒子を第二溶剤に溶かして希望する気体のナノバブルを形成することを特徴とするナノバブルの形成方法としている。
請求項１１の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルを真空にしてナノバブルの包覆する気体を除去し、希望の気体を充填することを特徴とするナノバブルの形成方法としている。
請求項１２の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルに含まれる気体は、フッ素化気体、酸素、窒素、アルゴン、ネオン、キセノン、及び空気のグループから何れか一つを選択することを特徴とするナノバブルの形成方法としている。
請求項１３の発明は、請求項１２記載のナノバブルの形成方法において、前記ナノバブルに含まれる気体は、ＣＦ4、Ｃ2Ｆ6、Ｃ2Ｆ4、Ｃ3Ｆ8、Ｃ4Ｆ8、Ｃ4Ｆ10、Ｃ3Ｆ6、ＳＦ6のグループから何れか一つを選択することを特徴とするナノバブルの形成方法としている。
請求項１４の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルのライフタイムは２５〜３０分間であることを特徴とするナノバブルの形成方法としている。
請求項１５の発明は、請求項１記載のナノバブルの形成方法において、前記ナノバブルは、超音波造影剤、薬物キャリア、遺伝子キャリア、及び臨床における診断と治療に応用することを特徴とするナノバブルの形成方法としている。
The invention of claim 1 performs an organic layer coating process using inorganic particles as a central core, and coats at least one first polymer on the surface of the central core to form organic / inorganic composite particles.
The organic / inorganic composite particles come into contact with the first solvent, and the central core of the organic / inorganic composite particles is dissolved and removed with the first solvent to form solvent-type nanoparticles, and inside the solvent-type nanoparticles. Contains the first solvent,
Removing the first solvent in a freeze-drying process to form hollow nanoparticles in the solvent-type nanoparticles;
In the dissolution process, a nanobubble is formed by dissolving the hollow nanoparticles in a second solvent to form nanobubbles.
According to a second aspect of the present invention, in the nanobubble forming method according to the first aspect, the inorganic particles are selected from the group of nanoparticles of gold, silver, iron, and other inorganic materials. This is a characteristic nanobubble formation method.
The invention of claim 3 is the method of forming nanobubbles according to claim 1, wherein the first polymer is poly (isobutylene-alt-maleic anhydride) -poly (isobutylene-alt-maleic anhydride). -graft-dodecylamine) and a polymer similar thereto.
According to a fourth aspect of the present invention, there is provided the nanobubble forming method according to the first aspect, wherein the first solvent is aqua regia.
A fifth aspect of the present invention is the nanobubble forming method according to the first aspect, wherein the second solvent is one of a group of pure water and various buffer solutions.
A sixth aspect of the present invention is the nanobubble forming method according to the first aspect, wherein the nanobubbles have a particle size range of 30 nm to 10,000 nm.
A seventh aspect of the invention is the nanobubble forming method according to the first aspect, wherein the nanobubbles have a particle size controlled by the size of the inorganic particles.
The invention according to claim 8 is the nanobubble formation method according to claim 1, wherein after the first polymer layer coating process is performed, a bioconjugation process is further performed to obtain a second polymer having biocompatibility. The nanobubble formation method is characterized by bonding to the surface of one polymer layer.
The invention of claim 9 is the nanobubble formation method according to claim 8, wherein the second polymer is a molecule containing an NH 2 functional group.
According to a tenth aspect of the present invention, in the nanobubble formation method according to the first aspect, the dissolution process further includes applying a desired gas stream on the hollow nanoparticles and dissolving the hollow nanoparticles in the second solvent. The nanobubble formation method is characterized by forming a gas nanobubble.
The invention of claim 11 is the method of forming nanobubbles according to claim 1, wherein the nanobubbles are evacuated to remove the gas encapsulated by the nanobubbles and filled with a desired gas. Yes.
The invention of claim 12 is the method for forming nanobubbles of claim 1, wherein the gas contained in the nanobubbles is selected from the group consisting of fluorinated gas, oxygen, nitrogen, argon, neon, xenon, and air. The nanobubble formation method is characterized by selection.
According to a thirteenth aspect of the present invention, in the nanobubble formation method according to the twelfth aspect, the gas contained in the nanobubbles is CF 4 , C 2 F 6 , C 2 F 4 , C 3 F 8 , C 4 F 8 , C One of the groups of 4 F 10 , C 3 F 6 , and SF 6 is selected.
The invention of claim 14 is the nanobubble forming method according to claim 1, wherein the nanobubble has a lifetime of 25 to 30 minutes.
The invention of claim 15 is the nanobubble formation method according to claim 1, wherein the nanobubble is applied to an ultrasound contrast agent, a drug carrier, a gene carrier, and clinical diagnosis and treatment. It's a way.
本発明のナノバブルは超音波造影剤、薬物キャリア、遺伝子キャリア、及び臨床における診断と治療に応用できることを特徴とする。 The nanobubbles of the present invention are characterized by being applicable to ultrasound contrast agents, drug carriers, gene carriers, and clinical diagnosis and treatment.
本発明はナノバブルの形成方法を開示する。本発明を徹底的に御理解戴く為に、下記の説明においてステップの詳細及びその構成を提供する。本発明の実施においては当該領域の技術者の熟知する特殊内容に制限されないことは顕著である。また、本発明が不必要に制限されるのを防ぐ為に、周知の組成もしくはステップを詳細内容中に説明しないこととする。本発明の実施例の詳細は次の通りであり、これらの詳細説明の他、本発明はその他実施例に広く応用することが可能であり、しかも本件の発明範囲は制限を受けることはなく、その範囲は下記の特許登録申請の範囲を基準とする。 The present invention discloses a method for forming nanobubbles. In order to provide a thorough understanding of the present invention, the details of the steps and their construction are provided in the following description. It is remarkable that the implementation of the present invention is not limited to special contents familiar to engineers in the relevant area. In other instances, well known compositions or steps will not be described in detail to prevent the invention from being unnecessarily limited. Details of the embodiments of the present invention are as follows. Besides these detailed descriptions, the present invention can be widely applied to other embodiments, and the scope of the present invention is not limited. The scope is based on the scope of application for patent registration below.
本発明の実施例はナノバブルの形成方法を開示するものであり、前記形成方法は、まず、無機ナノ粒子を中心核として、高分子層コーティングプロセス（polymer coating process）を行い、前記中心核の表面に最低一つの第一高分子を被覆して有機／無機複合粒子を形成する。次に、前記第一高分子はポリ（イソブチレン−アルト−無水マレイン酸）−グラフト−ドデシルアミン（poly(isobutylene-alt-maleic anhydride)-graft-dodecylamine）及びそれに類似する両性高分子とし、次に前記有機／無機複合粒子は第一溶剤に接触し、前記第一溶剤で前記有機／無機複合粒子の中心核を溶解して除去し溶剤型ナノ粒子を形成する。前記溶剤型ナノ粒子には前記第一溶剤を内含する。続いて冷凍乾燥プロセスにおいて前記第一溶剤を除去し前記溶剤型ナノ粒子に中空ナノ粒子を形成させる。前記中空ナノ粒子には空気を内含する。更に前記中空ナノ粒子を第二溶剤に溶かして前記ナノバブルを形成する。 An embodiment of the present invention discloses a method of forming nanobubbles, and the forming method first performs a polymer coating process using inorganic nanoparticles as a central core, and then the surface of the central core. The organic / inorganic composite particles are formed by coating at least one first polymer. Next, the first polymer is poly (isobutylene-alt-maleic anhydride) -graft-dodecylamine and a similar amphoteric polymer, The organic / inorganic composite particles are in contact with a first solvent, and the central core of the organic / inorganic composite particles is dissolved and removed with the first solvent to form solvent-type nanoparticles. The solvent-type nanoparticles include the first solvent. Subsequently, in the freeze-drying process, the first solvent is removed to form hollow nanoparticles in the solvent-type nanoparticles. The hollow nanoparticles include air. Further, the nanobubbles are formed by dissolving the hollow nanoparticles in a second solvent.
また、前記ナノバブルの形成方法には更に、前記第一高分子層コーティングプロセスの後、バイオコンジュゲーションプロセス（bioconjugation process）を含み、生物適合性を備える第二高分子を前記第一高分子層の表面に接合する。前記第二高分子はＮＨ2官能基グループを含む分子であり、例えば１−エチル基３−ジメチル基−カルボジイミド塩酸塩（ＥＤＣ）である。
前記無機ナノ粒子は、金、銀、鉄、及びその他無機材料のナノ粒子のグループから何れか一つを選択し、しかも第一溶剤は王水とし第一溶剤は純水と各種バッファ溶液とする。
前記ナノバブルの粒径範囲は３０ｎｍ〜１００００ｎｍとし、その粒径の大きさは無機粒子のサイズによりコントロールする。
The nanobubble formation method further includes a bioconjugation process after the first polymer layer coating process, and a second polymer having biocompatibility is added to the first polymer layer. Bond to the surface. The second polymer is a molecule containing an NH 2 functional group, for example, 1-ethyl group 3-dimethyl group-carbodiimide hydrochloride (EDC).
The inorganic nanoparticles are selected from a group of nanoparticles of gold, silver, iron, and other inorganic materials, and the first solvent is aqua regia and the first solvent is pure water and various buffer solutions. .
The particle size range of the nanobubbles is 30 nm to 10000 nm, and the particle size is controlled by the size of the inorganic particles.
本発明の実施例において前記溶解プロセスには更に、希望する気体気流を前記中空ナノ粒子上に施用し、並びに中空ナノ粒子を前記第二溶剤で溶かし、希望する気体を含む前記ナノバブルの形成を便利にする。
本発明のもう一つの実施例において前記のナノバブルを真空にし前記ナノバブルが包覆する気体を取り除き、並びに希望する気体を充填する。
In an embodiment of the present invention, the dissolution process further includes applying a desired gas stream onto the hollow nanoparticles, and dissolving the hollow nanoparticles with the second solvent to form the nanobubbles containing the desired gas. To.
In another embodiment of the invention, the nanobubbles are evacuated to remove the gas covered by the nanobubbles and filled with the desired gas.
前記ナノバブルの含む気体は下記グループから何れの一つを選択する。即ち、フッ素化気体、酸素、窒素、アルゴン、ネオン、キセノン、及び空気である。その内良好であるのは、ＣＦ4、Ｃ2Ｆ6、Ｃ2Ｆ4、Ｃ3Ｆ8、Ｃ4Ｆ8、Ｃ4Ｆ10、Ｃ3Ｆ6、ＳＦ6である。本発明に開示するナノバブルは、超音波造影剤、薬物キャリア、遺伝子キャリア、及び臨床における診断と治療に応用することが可能である。並びに、前記ナノバブルは安定したライフタイムを備え、そのライフタイムは２５〜３０分に到達する。前記のナノバブルのライフタイムは、ナノバブルが特定超音波パラメータによる刺激の下、超音波エコー信号の時間を延長できることを指している。 The gas included in the nanobubble is selected from the following group. That is, fluorinated gas, oxygen, nitrogen, argon, neon, xenon, and air. Among them, CF 4 , C 2 F 6 , C 2 F 4 , C 3 F 8 , C 4 F 8 , C 4 F 10 , C 3 F 6 , and SF 6 are preferable. Nanobubbles disclosed in the present invention can be applied to ultrasonic contrast agents, drug carriers, gene carriers, and clinical diagnosis and treatment. In addition, the nanobubbles have a stable lifetime, which reaches 25 to 30 minutes. The lifetime of the nanobubble indicates that the nanobubble can extend the time of the ultrasonic echo signal under stimulation by a specific ultrasonic parameter.
下記に、ナノバブル合成方法を説明する。
[実施例１] 金ナノ粒子の合成
予め以下の溶液を配置する。
（ａ）ジドデシルジメチルアンモニウムブロミド（ＤＤＡＢ）溶液：微量用電子天秤を使いＤＤＡＢ（ＭＷ=462.63）を4.63ｇ量り、それを１００ｍＬトルエンに加え、振動器で均等に混合する。
（ｂ）塩化金（AuCl3）溶液：微量用電子天秤を使いＡｕＣｌ３（ＭＷ=303.33）を0.15ｇ量り、それを２０ｍＬのＤＤＡＢに加え、振動器で均等に混合する。
（ｃ）ドデカニック酸（Dodecanic acid）溶液：微量用電子天秤を使いドデカニック酸（ＭＷ=172.26）を1.376ｇ量り、それを80ｍＬのトルエンに加え、振動器で均等に混合する。
（ｄ）テトラブチルアンモニウムブロマイド（ＴＢＡＢ）溶液：微量用電子天秤を使いテトラブチルアンモニウムブロマイド（ＭＷ=257.31）を0.15ｇ量り、それを1.08ｍＬのＤＤＡＢに加え、振動器で均等に混合する。
（ｅ）トリオクチルホスフィン（trioctylpyosphine：ＴＯＰ）溶液：0.17ｍＬのＴＯＰを3.8ｍＬのトルエンに加え振動器で均等に混合する。ＴＢＡＢ溶液は金ナノ粒子を合成した活性化剤である故、合成が必要な時にその場で配合する。
The nanobubble synthesis method will be described below.
[Example 1] Synthesis of gold nanoparticles The following solutions are arranged in advance.
(A) Didodecyldimethylammonium bromide (DDAB) solution: Using a micro balance electronic balance, weigh 4.63 g of DDAB (MW = 462.63), add it to 100 mL toluene, and mix evenly with a vibrator.
(B) Gold chloride (AuCl 3) solution: 0.15 g of AuCl 3 (MW = 303.33) is weighed using an electronic balance for a minute amount, added to 20 mL of DDAB, and mixed evenly with a vibrator.
(C) Dodecanic acid solution: Weigh 1.376 g of dodecanic acid (MW = 172.26) using an electronic balance for a minute amount, add it to 80 mL of toluene, and mix evenly with a vibrator.
(D) Tetrabutylammonium bromide (TBAB) solution: Weigh 0.15 g of tetrabutylammonium bromide (MW = 257.31) using a microbalance, add it to 1.08 mL DDAB, and mix evenly with a vibrator.
(E) Trioctylpyosphine (TOP) solution: Add 0.17 mL of TOP to 3.8 mL of toluene and mix evenly with a vibrator. Since TBAB solution is an activator that synthesizes gold nanoparticles, it is blended in-situ when synthesis is required.
磁石を２０ｍＬの試料フラスコ中に入れ、2.5ｍＬのドデカニック酸溶液と１ｍＬのＴＢＡＢ溶液を２０ｍＬの試料フラスコ中に入れ、磁石攪拌器を使い適当な回転速度で持続的に攪拌し、続いて、0.8ｍＬのＡｕＣｌ３溶液を加え、２時間反応させる（先ず１０ｕＬ加え、更に素早く７９０ｕＬ加える）。次に、７０℃水浴で５分間加熱し、温度のバランスが取れた後４０ｕＬのＴＯＰ溶液を加えて５分間反応させる。その後、磁石を取り出し、メチルアルコールを溶液に混濁が出現するまで加える。更に、3000ｒｐｍの回転速度で５分間遠心し、小分子と小さな雑質を除去する。その後、上澄み液を取り除いた後にクロロホルム（chloroform）で沈殿した金ナノ粒子を再懸濁する。最後に、更に3000rpmの回転速度で５分間遠心させて比較的大きな金ナノ粒子と大きな雑質を除去し、並びに、上澄み液をきれいな試料フラスコ中に回収する。 A magnet is placed in a 20 mL sample flask, 2.5 mL dodecanic acid solution and 1 mL TBAB solution are placed in a 20 mL sample flask and continuously stirred with a magnetic stirrer at an appropriate rotational speed, followed by 0.8 Add mL of AuCl3 solution and react for 2 hours (add 10 uL first, then add 790 uL more quickly). Next, it is heated in a 70 ° C. water bath for 5 minutes, and after balancing the temperature, 40 uL of a TOP solution is added and reacted for 5 minutes. Thereafter, the magnet is removed and methyl alcohol is added until turbidity appears in the solution. Furthermore, it is centrifuged for 5 minutes at a rotational speed of 3000 rpm to remove small molecules and small impurities. Thereafter, after removing the supernatant, the gold nanoparticles precipitated with chloroform are resuspended. Finally, it is further centrifuged for 5 minutes at a rotational speed of 3000 rpm to remove relatively large gold nanoparticles and large contaminants, and the supernatant is collected in a clean sample flask.
[実施例２] 高分子コーティング（polymer coating）
先ず下記薬品を配置する。
（ａ）高分子溶液：3.084ｇ（20mmole）のポリ（イソブチレン−アルト−無水マレイン酸）（Ｍｗ〜6,000）粉末を取り、丸底フラスコ中に置く。2.78ｇ（15mmole）のドデシルアミン（dodecylamine）を100ｍＬのトテラヒドロフラン（Tetrahydrofuran:ＴＨＦ）溶液中に溶かす。ドデシルアミン溶液とポリ（イソブチレン−アルト−無水マレイン酸）の粉末を迅速に均等混合して混合溶液を作る。次に減圧濃縮器の温度を60℃に設定し、適当な回転速度で１０分間反応させて、前記の混合溶液を透明状態にする。続いて、圧力を２００ｍｂａｒに設定し２４時間反応させる。溶液を完全に抜き取った後、２５ｍＬのクロロホルムを加え、前記で合成した高分子溶液を溶解する。前記高分子溶液（monomer）濃度は0.8Ｍであり、各高分子単体は25％の無水マレイン酸グループ（maleic anhydride groups）及び75％の疎水性炭化水素鎖を含む。
（ｂ）ホウ酸ナトリウムバッファ液（Sodium borate buffer:SBB）：微量用電子天秤を使い3.09ｇのホウ酸（Boric Acid, ＭＷ＝61.83Ｄａ, 50ｍＭ）と19.07ｇの四ホウ酸ナトリウム十水和物（Sodium Tetraborate Decahydrate, Ｗ＝381.37Ｄａ, 50ｍＭ）を量り、続いて1000ｍＬの二次水（ｐＨ＝9.0）に溶解し室温下に保存する。
（ｃ）５×ＴＢＥバッファ液：微量用電子天秤を使い54ｇのトリス塩基（Tris Base）と27.5ｇのホウ酸を量った後、980ｍＬの二次水に溶かし、続いて20ｍＬのエチレンジアミン四酢酸（EDTA, 0.5M, pH=8.0）を加え、並びに磁石で均等攪拌した後、室温下で保存する。使用時は0.5×希釈した後に使用する。
（ｄ）2％のアガロースゲル（agarose gel）：ゲル製作トレイをゲルキャスティングトレイに入れ、並びに電気泳動コームをゲルキャスティングトレイ上に架設する。微量用電子天秤を使い2.4ｇのアガロースゲルを量り250ｍＬの三角フラスコに入れ、120ｍＬの0.5×ＴＢＥバッファを加え、続いてマイクロウェーブでアガロースが完全に溶解するまで加熱し、均等に揺らし、温度が若干下がった後、ゆっくり溶液を架設したゲルキャスティング槽に流し込み、その後、常温でアガロースが冷却凝固するまで静置し、最後に、電気泳動コームを抜き取り電気泳動槽に入れる。
（ｅ）ローディングバッファ（Loading buffer）：グリセリンと二次水を混合する（ｖ：ｖ＝1：10）。
[Example 2] Polymer coating
First, the following chemicals are arranged.
(A) Polymer solution: 3.084 g (20 mmole) of poly (isobutylene-alt-maleic anhydride) (Mw˜6,000) powder is taken and placed in a round bottom flask. 2.78 g (15 mmole) of dodecylamine is dissolved in 100 mL of a solution of Tetrahydrofuran (THF). A dodecylamine solution and a poly (isobutylene-alt-maleic anhydride) powder are rapidly and uniformly mixed to form a mixed solution. Next, the temperature of the vacuum concentrator is set to 60 ° C., and the mixture solution is reacted for 10 minutes at an appropriate rotation speed to make the mixed solution transparent. Subsequently, the pressure is set to 200 mbar and reacted for 24 hours. After completely removing the solution, 25 mL of chloroform is added to dissolve the polymer solution synthesized above. The polymer solution concentration is 0.8M, and each polymer element contains 25% maleic anhydride groups and 75% hydrophobic hydrocarbon chains.
(B) Sodium borate buffer (SBB): 3.09 g of boric acid (Boric Acid, MW = 61.83 Da, 50 mM) and 19.07 g of sodium tetraborate decahydrate using a microbalance. (Sodium Tetraborate Decahydrate, W = 381.37 Da, 50 mM) is weighed and then dissolved in 1000 mL of secondary water (pH = 9.0) and stored at room temperature.
(C) 5 × TBE buffer solution: 54 g of Tris Base and 27.5 g of boric acid were weighed using an electronic balance for trace, and then dissolved in 980 mL of secondary water, followed by 20 mL of ethylenediaminetetraacetic acid. (EDTA, 0.5M, pH = 8.0) is added, and the mixture is stirred uniformly with a magnet and then stored at room temperature. Use after diluting 0.5X.
(D) 2% agarose gel: Place the gel production tray in the gel casting tray and lay the electrophoresis comb on the gel casting tray. Using a microbalance, weigh 2.4 g of agarose gel into a 250 mL Erlenmeyer flask, add 120 mL of 0.5 × TBE buffer, then heat until the agarose is completely dissolved in the microwave, shake evenly, After slightly lowering, the solution is slowly poured into a gel-casting tank in which the solution is installed, and then allowed to stand at room temperature until the agarose cools and solidifies. Finally, the electrophoresis comb is extracted and placed in the electrophoresis tank.
(E) Loading buffer: Mix glycerin and secondary water (v: v = 1: 10).
光吸収スペクトル測定器を使い合成後の６ｎｍ金ナノ粒子の備える520ｎｍの吸収ピーク値を測定する。ベールの法則（Beer’s Law）：Ａ＝εｂｃを使って金ナノ粒子の濃度（６ｎｍの金ナノ粒子のモル吸収係数は2.905E7）を算出する。前記のＡは吸収値、εはモル吸収係数（L/mol cm）、ｂは光路径厚さ（cm）、ｃは容積モル濃度（mol/L）である。 The absorption peak value at 520 nm provided for the synthesized 6 nm gold nanoparticles is measured using an optical absorption spectrometer. Beer's Law: A = εbc is used to calculate the concentration of gold nanoparticles (the molar absorption coefficient of 6 nm gold nanoparticles is 2.905E7). A is the absorption value, ε is the molar absorption coefficient (L / mol cm), b is the optical path diameter thickness (cm), and c is the molar volume concentration (mol / L).
金ナノ粒子外層の有機分子の厚さが1.1ｎｍであるなら、全金ナノ粒子の表面積が推測できる。１ｎｍ２が200個の高分子単体（monomer units）を必要とするのなら、高分子溶液に加える必要のある体積を算出可能である。実験の始めに計算後の高分子溶液と５ｍＬのクロロホルムを三角フラスコ中で均等混合し、続いて金ナノ粒子溶液を加えて手で５分間揺らし、金ナノ粒子溶液と高分子溶液を均等混合する。減圧濃縮器（200millibar 回転速度100rpm）を使用して溶液を全て抜き取る。溶液を全て抜き取った後、大気圧に戻し更に１ｍｂａｒまでエアを抜き取る。溶液を完全に抽出したことが確定した後、更に0.1Ｎの水酸化ナトリウム（NaOH）を加え、高分子改質を経た金ナノ粒子を迅速に溶解する。続いて、５ｍＬの注射器を使って0.2ｕｍの濾過膜に通し、更に100ＫＤａの分子ストッパーを使って小さい分子を除去し、溶剤をホウ酸ナトリウムバッファ液（ＳＢＢ）に置き換える。 If the thickness of the organic molecule in the outer layer of gold nanoparticles is 1.1 nm, the surface area of all gold nanoparticles can be estimated. If 1 nm2 requires 200 polymer units, the volume that needs to be added to the polymer solution can be calculated. At the beginning of the experiment, the calculated polymer solution and 5 mL of chloroform are mixed evenly in an Erlenmeyer flask, and then the gold nanoparticle solution is added and shaken by hand for 5 minutes to evenly mix the gold nanoparticle solution and the polymer solution. . Remove all solution using a vacuum concentrator (200 millibar rotation speed 100 rpm). After all the solution has been extracted, it is returned to atmospheric pressure and air is further extracted to 1 mbar. After it is determined that the solution is completely extracted, 0.1N sodium hydroxide (NaOH) is further added to rapidly dissolve the gold nanoparticles that have undergone polymer modification. Subsequently, a 5 mL syringe is used to pass through a 0.2 um filtration membrane, a small molecule is removed using a 100 KDa molecular stopper, and the solvent is replaced with sodium borate buffer (SBB).
アガロース純化によって高分子コーティングを経た金ナノ粒子をローディングバッファと混合をする（ｖ：ｖ＝10：1）。その内、2％のアガロースゲルを電気泳動槽のＴＢＥバッファ中に置いて純化を行い（電圧100Ｖ、所要時間１時間）、未反応の高分子を除去する。続いて金ナノ粒子をアガロースゲルから取り出し、透析膜中に置き、電圧100Ｖの電気泳動槽内で電場を使って金ナノ粒子とアガロースゲルを分離させる。試料を回収した後、0.22ｕｍの濾過膜で濾過し、更に100ＫＤａ分子ストッパーで試料を濃縮し、並びに純化した金ナノ粒子をホウ酸ナトリウムバッファ液中で保存する。最後に電気泳動で純化した両性高分子コーティングナノ金粒子を超高速の遠心機50000rpmで20分間遠心した後、上澄み液を除去し、更にホウ酸ナトリウムバッファ液で沈殿したナノ金粒子を再懸濁する（３回繰り返し）。 Gold nanoparticles subjected to polymer coating by agarose purification are mixed with a loading buffer (v: v = 10: 1). Among them, 2% agarose gel is placed in the TBE buffer of the electrophoresis tank for purification (voltage 100 V, required time 1 hour) to remove unreacted polymer. Subsequently, the gold nanoparticles are removed from the agarose gel, placed in a dialysis membrane, and the gold nanoparticles and the agarose gel are separated using an electric field in an electrophoresis tank with a voltage of 100V. After the sample is collected, it is filtered through a 0.22 um filter membrane, the sample is further concentrated with a 100 KDa molecular stopper, and the purified gold nanoparticles are stored in a sodium borate buffer solution. Finally, the amphoteric polymer-coated nanogold particles purified by electrophoresis were centrifuged for 20 minutes at 50000 rpm in an ultra-high speed centrifuge, the supernatant was removed, and the nanogold particles precipitated with sodium borate buffer were resuspended. (Repeat three times).
[実施例３] バイオコンジュゲーション（Biocompatible layer conjugation）
高分子コーティングを経た金ナノ粒子とポリエチレングリコール（Polyethyleneglycol:PEG）及び１−エチル基３−ジメチル基−カルボジイミド塩酸塩（ＥＤＣ）を混合して（[Au]/[PEG]=1/200,Au:PEG:EDC=v:v:v=1:1:1）２時間反応させる。その後100ＫＤａタンパク質濾過管（ＭＣＷＯ）で未反応のＰＥＧを除去し、並びに試料を１ｕＭまで濃縮し、ＳＢＢ（ｐＨ＝９）で保存する。
[Example 3] Biocompatible layer conjugation
Gold nanoparticles after polymer coating were mixed with polyethylene glycol (Polyethyleneglycol: PEG) and 1-ethyl 3-dimethyl group-carbodiimide hydrochloride (EDC) ([Au] / [PEG] = 1/200, Au : PEG: EDC = v: v: v = 1: 1: 1) React for 2 hours. The unreacted PEG is then removed with a 100 KDa protein filter tube (MCWO) and the sample is concentrated to 1 uM and stored in SBB (pH = 9).
[実施例４]
実施例２、実施例３を経た試料中に溶液が無色透明状態になるまで王水を落とし、100ＫＤａタンパク質濾過管で溶液中の王水をＳＢＢ（ｐＨ＝９）に置き換え、更にＳＢＢを脱イオン水に置き換える。最後に冷凍乾燥機で溶液を完全に抜き取り、試料を白色固体にする。超音波で刺激する場合は、脱イオン水を加えて前記白色固体を直接溶解すればよく、これによりナノバブルが完成する。前記ナノバブルの合成図は図１に示す通りであり、エアコア（Air Core）中の気体は空気と各種惰性気体とし、良好なのはＣ３Ｆ８、Ｃ４Ｆ１０、ＳＦ６である。
[Example 4]
The aqua regia is dropped into the sample obtained in Example 2 and Example 3 until the solution becomes colorless and transparent, and the aqua regia in the solution is replaced with SBB (pH = 9) in a 100 KDa protein filter tube, and SBB is further deionized. Replace with water. Finally, the solution is completely extracted with a freeze dryer, and the sample is turned into a white solid. When stimulating with ultrasonic waves, deionized water may be added to dissolve the white solid directly, thereby completing nanobubbles. The synthesis diagram of the nanobubble is as shown in FIG. 1, and the gas in the air core is air and various kinds of inertial gases, and C3F8, C4F10, and SF6 are good.
超音波でナノバブルを刺激した超音波画像は図２に示す通りであり、バブル粒径は10μｍである。図３に示す通り、前記バブルは動的光散乱（Dynamic Light Scattering: DLS）を使用して測定したものである。更に、図４に示す通り、前記バブルのライフタイムは20分間である。超音波は25ＭＨｚのディテクターを使用してＢモード画像を形成し5900によって自動的に送受信する。 An ultrasonic image obtained by stimulating nanobubbles with ultrasonic waves is as shown in FIG. 2, and the bubble particle size is 10 μm. As shown in FIG. 3, the bubbles are measured using dynamic light scattering (DLS). Furthermore, as shown in FIG. 4, the lifetime of the bubble is 20 minutes. Ultrasound forms a B-mode image using a 25 MHz detector and is automatically transmitted and received by the 5900.
超音波パラメータ
PRT: EXT-BNC Gain: 40dB Energy:32μJ
Damping:50 Ohm High Pass: 10MHz
Low Pass:50 MHz Lab View インターフェースパラメータ
Sample Number:512 Scan
Sequence:512PRF:512
Mode: B-scan
Sample rate: 120M Vectors:4mm Dynamic range:1V Speed:slow
Delay time:0.0147 C scan Trig Type: positive swept
Depth: 1mm interval:1μm
Ultrasonic parameters
PRT: EXT-BNC Gain: 40dB Energy: 32μJ
Damping: 50 Ohm High Pass: 10MHz
Low Pass: 50 MHz Lab View interface parameters
Sample Number: 512 Scan
Sequence: 512PRF: 512
Mode: B-scan
Sample rate: 120M Vectors: 4mm Dynamic range: 1V Speed: slow
Delay time: 0.0147 C scan Trig Type: positive swept
Depth: 1mm interval: 1μm
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