助成金は、NHLBI HL080532、NHLBI HL073449、NCRR RR16453、およびAHAナショナル費補助金援助賞（RVSに）含まれています。特別な感謝は、ビデオ制作ととDCDCを設立した教育グラント第P336C050047の米国務省への支援のため、距離のコースデザインとコンサルティング（DCDC）グループ、dcdcgroup.orgに拡張されます。

1。マイクロバブル株式の準備<ol><li> 10にPBSのMLSは、1gのグルコースと200 mgの1,2 - ジパルミトイル- sn -グリセロ-3 - ホスファチジルコリンと50mgの1,2 - ジパルミトイル- sn -グリセロ-3 - ホスファチジルエタノールアミンを混ぜる。 </li><li>沸騰水浴中で混合物20〜30分、5分ごとに混合ピペットを加熱する。 </li><li>ソリューションは、最大6ヶ月までは4℃で保存することができます。 </li></ol> 2。マイクロバブルの準備<ol><li> 40で準備したマイクロバブルストック溶液とインキュベート℃で15分間の250μlを取る。 </li><li>予め温めておいたマイクロバブル溶液は、次いで、グリセロール50μlを含む1.5mlチューブに転送されます。 </li><li>発現をコードするプラスミドDNAを精製1-2 mgのは、目的の遺伝子（4mg/mlの最適な濃度でキアゲンエンドトキシンフリーMegaPrepキット、キアゲン、メリーランド州ジャーマンタウン、で、この例で精製された）のために構築する。 2.4）リン酸緩衝生理食塩水は、500μlの最終容量に追加されます。エンドトキシンフリーキアゲンmaxiprepsは、無菌性を保証するために使用するだけでなく、滅菌PBSされています。 </li><li>マイクロチューブ内の空気は、オクタフルオロプロパンガスで置換されます。 </li><li>マイクロチューブを20秒間歯科融合する人で激しく振盪されています。 </li><li>マイクロバブルにバインドされていないsubnatant含む残存DNAとバッファを注意深く除去され、マイクロバブル層がアタッチされていないDNAを除去する滅菌PBSで3回洗浄し、各洗浄サイクルの間に氷の上に配置されます。我々は通常、30〜40％の結合効率を実現。すべての試薬は無菌であり、注意が汚染を最小限に抑えるために行われます。 </li><li>プラスミドDNA結合マイクロバブルは、次に使用するまで2時間のために氷の上に配置されます。 </li><li>ミキシングとPBS洗浄後のマイクロチューブから除去subnatantは、使用して260nmの波長で、このソリューションの光学密度を測定することによって、未結合のDNAの濃度、および同様に既知の初期濃度に基づいて結合量を決定するために使用することができます。分光光度計。 </li></ol> 3。機器の校正<ol><li>最初の使用に先立ち、1 MHzのキャビテーションのトランスデューサは、適切な機械的なインデックスとパルス繰り返しを確保するためにキャリブレーションする必要があります。 submergible 1 MHzの、13ミリメートル、焦点の合っていないトランスデューサーはパワーアンプを介して20MHzファンクション/任意波形発生器に接続されています。 </li><li>トランスデューサは、チャージアンプを経由して500 MHzのオシロスコープに接続されているハイドロフォン、で直接目的とした、水の完全なプラスチック容器に入れています。 </li><li>波形、周波数、振幅、バーストサイクル、および電力増幅は、すべての適切なデューティサイクルとマイクロバブルの空洞を作るために最適な機械的なインデックスを取得するように変更することができます。この特定の実験では、我々は1 MHzで〜1.3と同等の機械的なインデックスにシステムをキャリブレーションしている。 </li></ol> 4。マイクロバブルデリバリー＆UTMD <ol><li>前にマイクロバブル配信しUTMDに、C57BL / 6マウスでは、IP注射を通じてケタミン及び5mg/kgキシラジンを100mg/kgで麻酔した。 </li><li>マイクロバブルの配信は、心臓の左心室にまたは尾静脈にカテーテルを通して直接注入によって静脈内投与した。直接心臓注射の場合は、上のプラスミドDNAにロードされたマイクロバブル溶液100μlにボリュームが心臓の左心室に超音波可視化の下前歯4 番目の肋間腔に挿入された30ゲージの針を通して注入される。 </li><li>左心室の注入に起因するマイクロバブル溶液のボーラスは、VEVO 2100イメージングシステムのVisual SonicsのVisualSonics使用して長軸像でマウスの胸部上の静止位置に配置された38 MHzの高い周波数の超音波トランスデューサー&quot;を用いて可視化です。すべての注射器と注射針は、これらの注射のため、可能な限り無菌環境を保証する、無菌である。 </li><li>すぐに注射後、マイクロバブルの破壊は、この地域に破壊を標的に、目的の臓器の上に直接保持していた2番目の、より小さいサイズの低周波数1.0 MHzの振動子を用いて〜5分間行われます。この例では、超音波検査は、機械的に約1.3から1.5へのインデックスと同等、およびすべての20サイクルの100 msのパルス繰り返し周期で、1.0 MHzのパルス繰り返し周波数で肝臓に投与した。また、パルスは、超音波の3フレーム、すべての4から6の心臓周期のバーストにマウスのECG（この実験で示されていない）にゲートすることができます。我々は超音波のバーストの間に気泡を補給する毛細血管床を可能にするプロトコルとトランスフェクションの効率を得る。 </li></ol> 5。代替配信方法我々は、このようなマイクロバブルの長時間注入として、多くのインスタンスでの尾静脈を手続きの複雑さに起因する心室間注射を強調するために選択したが、jectionが好ましい方法である。マイクロバブル配信の尾静脈方式の場合、マウスは、同じ方法で麻酔です。プラスミドDNA結合マイクロバブルを含む注射器は、27ゲージの針/尾静脈カテーテルに接続されています。尾静脈カテーテルは、そのマウスの尾部に沿って右または左横静脈の遠位3分の1に挿入されます。マイクロバブルを含む注射器は、自動的に一定時間以上の解の一様なプリセットボリュームを管理する輸液ポンプに配置されます。我々は一般的に3ml/hourの速度で200 -300μlを注入する。 動物の使用 すべての動物は、関連する国内及び/またはローカルの動物福祉団体によって定義された適切な動物の習慣に従って処理され、そしてすべての動物の作品は（ハワイ動物実験用の委員会の大学、​​承認番号07から100に適切な委員会によって承認された-3）。適切な麻酔（ケタミン/ zylazine）を使用し、鎮痛薬（Bupivicaineとブプレノルフィンは）は、必須ではありません、利用可能であったれました。 6。代表的な結果： UTMDを介したプラスミドDNAの配信の有効性は、構造、などでエンコードする遺伝子に応じて、さまざまな方法で評価したが、これらに限定されないが、in vivoイメージング、B - galをex vivoでの染色、及び/ またはルシフェラーゼ免疫組織学。特に、 生体内生物発光イメージングの一つをコードするプラスミド生物発光レポーター遺伝子（ルシフェラーゼ）をトランスフェクションしたマウスのシリアル遺伝子の発現の有無と持続時間を監視することができます。 in vivoイメージングシステム（IVIS）（キャリパーライフサイエンス、ホプキントン、マサチューセッツ州） では Xenogenは、生物発光イメージングに使用されます。画像は通常、すべてのマウスのUTMD媒介トランスフェクション後の最初の日を取られるし、生物発光遺伝子の発現は、システム（図1）によって、もはや視覚的に検出不可能になるまで、3〜4日に繰り返されます。生物発光イメージングのためのマウスを準備するには、マウスは、最初にルシフェラーゼレポータープローブD -ルシフェリン（キャリパーライフサイエンス）のIP注射を受信し、麻酔〜3分後。 D -ルシフェリン基質の生体内分布は、動物がIVISイメージングチャンバー内に設置され、フルボディイメージのスキャンが取られる前に〜10分間進行させる。買収時には、ホタルルシフェラーゼ/ D -ルシフェリン光化学反応から放出された光子が測定されます。図1は、またUTMD以下の肝臓の同様のIVIS生物発光イメージングを示し、そして図2は、抗ルシフェラーゼ一次抗体（Sigma - Aldrich社）とAlexFluor - 568標識二次抗体（Invitrogen）を用いてトランスフェクトされた肝臓の落射蛍光（100倍）の画像です。 。それはUTMDを介した肝トランスフェクションは、内皮細胞ではないだけに影響を与えていることを確認することは明らかですが、hepatocyes同様。 <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="図1" /> 図1心臓のXenogen / IVISイメージングは、治療したマウスをUTMD（A）ネガティブコントロールマウス：。。プラスミド+ PBSは、心臓の指向性UTMD続いて、（B）治療のマウス：プラスミド+心臓が続くマイクロバブルはUTMD指示、および（C）治療のマウス：肝臓に続くプラスミド+のマイクロバブルはUTMD監督。 <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="図2" /> 図せずUTMD 2。肝臓UTMDの免疫組織化学（赤の抗ルシフェラーゼ）。（A）プラスミド、及びUTMDと（B）プラスミド。共焦点画像（100X）、核はDAPI染色した青です。

<ol>
	<li>Bekeredjian, R., Chen, S., Frenkel, P. A., Grayburn, P. A., Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart.">Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.</a> Circulation. 108, 1022-1026 (2003).</li><li>Bekeredjian, R., Katus, H. A., Kuecherer, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+ultrasound+targeted+microbubble+destruction:+a+review+of+non-cardiac+applications.">Therapeutic use of ultrasound targeted microbubble destruction: a review of non-cardiac applications.</a> Ultraschall Med. 27, 134-140 (2006).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy.">Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.</a> Gene Ther. 17, 1411-1420 (2010).</li><li>Miao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid.">Ultrasound enhances gene delivery of human factor IX plasmid.</a> Hum Gene Ther. 16, 893-905 (2005).</li><li>Shimoda, M., Chen, S., Noguchi, H., Matsumoto, S., Grayburn, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+non-viral+gene+delivery+of+human+vascular+endothelial+growth+factor+improves+revascularisation+and+restoration+of+euglycaemia+after+human+islet+transplantation+into+mouse+liver.">In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.</a> Diabetologia. 53, 1669-1679 (2010).</li><li>Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium.">Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.</a> Circulation. 101, 2554-2556 (2000).</li><li>Sirsi, S., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+Compositions,+Properties+and+Biomedical+Applications.">Microbubble Compositions, Properties and Biomedical Applications.</a> Bubble Sci Eng Technol. 1, 3-17 (2009).</li><li>Li, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo.">Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.</a> Gene Ther. 16, 1146-1153 (2009).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Newman, C. M., Bettinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+ultrasound+for+gene+transfer.">Gene therapy progress and prospects: ultrasound for gene transfer.</a> Gene Ther. 14, 465-475 (2007).</li><li>Vancraeynest, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction:+evidence+for+the+contribution+of+myocardial+ischemia.">Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.</a> Ultrasound Med Biol. 35, 672-679 (2009).</li></ol>

利害の衝突は宣言されません。

UTMDは、遺伝子デリバリーへの新しいアプローチを表しています。プラットフォーム技術としては、組織特異性の高度が望まれている生理活性分子の無数を提供するために、多くの潜在的な遺伝子治療の戦略のいずれかと組み合わせることができます。技法の主な生物学的限界は、トランスフェクションの効率が低いことです。もう一つの重要な考慮事項は、著しく骨や空気を介在すること?...

<table border="1"><tbody><tr><th>試薬/装置の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> 1,2 - ジパルミトイル- sn -グリセロ-3 - ホスファチジルコリン</td><td>シグマアルドリッチ</td><td> P - 5911 </td><td>マイクロバブル脂質の殻の成分</td></tr><tr><td> 1,2 - ジパルミトイル- sn -グリセロ-3 - ホスファチジルエタノールアミン</td><td>シグマアルドリッチ</td><td> P - 3275 </td><td>マイクロバブル脂質の殻の成分</td></tr><tr><td>グルコース</td><td>シグマアルドリッチ</td><td> G5400 </td><td>マイクロバブルを安定化すると考えられて</td></tr><tr><td>リン酸緩衝食塩水</td><td>シグマアルドリッチ</td><td> P5368 </td><td></td></tr><tr><td>グリセロール</td><td>シグマアルドリッチ</td><td> G5516 </td><td>合体からマイクロバブルを防止するためと考えられ</td></tr><tr><td>オクタフルオロプロパンガス</td><td> Airgasの</td><td> N / </td><td>臨床アプリケーションで使用される不活性ガス</td></tr><tr><td> VialMix歯科融合する人</td><td>ブリストルマイヤーズスクイブ</td><td> N / </td><td></td></tr><tr><td> 1 MHzの、13ミリメートル、焦点の合っていない変換器</td><td>オリンポス</td><td> A303S - SU </td><td></td></tr><tr><td> 20MHzファンクション/任意波形発生器</td><td>アジレント</td><td> 33220A </td><td></td></tr><tr><td>パワーアンプ</td><td>クルーン-ハイト（株） </td><td>モデル7500 </td><td></td></tr><tr><td>ハイドロフォン</td><td>ブリュエルケアーと</td><td>タイプ1803 </td><td></td></tr><tr><td>チャージアンプ</td><td>ブリュエルケアーと</td><td>タイプ2634 </td><td></td></tr><tr><td> 500 MHzのオシロスコープ</td><td>レクロイ</td><td> 9354L </td><td></td></tr><tr><td> 34 MHzのトランスデューサとVisualSonics&quot;VEVO 2100イメージングシステム</td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G 1インチ尾静脈カテーテル</td><td> VisualSonics </td><td> N / </td><td></td></tr><tr><td>精霊プラス輸液ポンプ</td><td>ケントサイエンティフィック</td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

そのような目的の遺伝子をコードして負に荷電したプラスミドDNAベクターとしてUTMD、生理活性分子、で、脂質マイクロバブル造影剤7-9のカチオン性シェルに追加されます。マウスではこれらのベクトルを媒介するマイクロバブルは、心臓の左心室に静脈内投与、または直接投与することができます。大型動物ではそれらはまた冠動脈内カテーテルを通して注入することができます。循環から標的臓器へのその後の送達は、マイクロバブルの共振周波数で音響キャビテーションが発生します。それは力学的エネルギーがでたり、標的領域10の微小血管の内皮細胞との間の一時的な細孔形成におけるマイクロバブルの破壊の結果によって生成される可能性が高いようです。このソノポレーションの効果の結果として、内皮細胞へと全体のトランスフェクション効率が強化され、導入遺伝子をコードするベクターは、周囲の組織に堆積される。循環に残っているプラ​​スミドDNAは、さらに、非超音波組織への配信の可能性を低減し、非常に特異的な標的器官のトランスフェクションにつながる血、でヌクレアーゼによる急速に分解される。

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

マイクロバブル破壊（UTMD）ターゲット超音波検査は、心臓や肝臓などの超音波にアクセス可能な臓器を標的とする治療用遺伝子を含む生理活性分子の部位特異的送達を、、、指示するために使用することができます。<sup&gt; 1月6日</sup&gt;。

超音波標的マイクロバブル破壊テクニック入門

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.4K ビュー.  University of Hawaii. マイクロバブル破壊（UTMD）ターゲット超音波検査は、心臓や肝臓などの超音波にアクセス可能な臓器を標的とする治療用遺伝子を含む生理活性分子の部位特異的送達を、、、指示するために使用することができます。 1月6日。

ビデオ: Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

コントラストの超音波は、薬剤ベアリングナノ粒子デリバリーと微小血管アブレーションを経由してマウスの神経膠腫の治療を対象と

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

標的ウイルスタンパク質の分析はHER2 +腫瘍に配信ナノ粒子

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

濃縮された高せん断均質化およびシリアル濃度によって脂質系酸素マイクロバブルエマルジョンの製造

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

マイクロ流体技術は、Cell変形能の探査

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMSサンプル導入のためのマイクロ流体チップ

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

パルスレーザー励起時の金ナノ粒子の統合フォト応答性リポソームとそのマイクロバブルキャビテーションの測定の合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

脳イメージングへの応用による CMUT 技術に基づく新しい超音波プローブの性能を評価するための実験プロトコル

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 つの補完的な手法を使用して胎盤をターゲットとした薬剤投与の安全性の総合評価

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...