この作品は、プリンセスマーガレット病院財団、カナダの国立科学と工学研究評議会、健康研究のためのカナダの研究所、イノベーションカナダ財団、ジョーイとトビータネンバウム/前立腺癌研究におけるブラジルのボールチェア、によってサポートされていましたニューヨーク州立大学研究財団からの助成金やバッファロー大学からのサポートを起動します。

1。ピロ - 脂質の合成<ol><li> 4、98.7 mgの16時00分lysophophatidylcholine（1 -パルミトイル-2 -ヒドロキシ-sn-グリセロ、（。Zhengら 、Bioconj頁、2002年、13 -392前述のように藻 ​​類スピルリナパシフィカから調製した）200ミリモルピロコンバイン-3 - ホスホコリン、アバンティ極性脂質＃855675）、EDCの76.3ミリグラム、48.7ミリグラムのDMAP（4 - （ジメチルアミノ）ピリジン）5ミリリットルアミレン中1:1:2:2パイロの比率クロロホルム安定化：脂質：EDC ：DMPA。 </li><li> 24時間アルゴン下、室温で反応混合物を撹拌した。 40℃に加熱しながらロータリーエバポレーターを用いて減圧下でクロロホルムを蒸発させる（ピロのsn-1またはsn-2位で結合）ピロ - 脂質異性体の混合物が生成される。 </li><li> 1mMのCaCl 2、50 mMのトリス（pH 8）、0.5％トリトンX-100および5 mg / mlの濃度で10％メタノールに再懸濁パイロ脂質異性体混合物。ミツバチ毒ATからホスホリパーゼA2（PLA2）を追加0.1 mg / mlの濃度およびsn-1ピロ脂質を消化すると、純粋なsn-2位ピロ脂質異性体を生成するために24時間、37℃で溶液をインキュベートする。 </li><li>追加のクロロホルムの2ボリュームおよびメタノール抽出物1.25ボリュームを追加し、40℃で減圧下で回転蒸発を用いて溶媒を除去DCM中の1％MeOH中の切断生成物を再懸濁し、異性体的に純粋なピロ - 脂質のための小さなジオールシリカカラム上の精製。 </li></ol> 2。 Porphysomesの調製<ol><li>別途クロロホルム中のピロフェオホルビド - 脂質、ジステアロイル-sn-グリセロ-3 - ホスホエタノールアミン-N-メトキシ（ポリエチレングリコール）（PEG-2000-PE）、およびコレステロールを溶解し、それぞれの濃度を記録する。 </li><li> 65モル％のポルフィリン脂質、5モル％のPEG-2000-PEおよび30モル％コレステロールを組み合わせることにより、5ミリグラム/チューブの総脂質重量が12×75mmのホウケイ酸試験管内porphysome脂質フィルムを準備します。 </li><li>ニトロの流れの下で脂質フィルムを乾燥させる世代ガス及びそれらをさらに1時間真空下で乾燥させてみましょう。将来の水和および押出しまでアルゴン下で-20℃で脂質フィルムを保管してください。 </li><li>各脂質膜チューブに1ミリリットルのリン酸緩衝生理食塩水（PBS、150mMのNaCl、10mMリン酸塩、pH7.4）に入れる。完全に凍結するまで液体窒素中で試験管を凍結し、その後、試料は全て溶融するまで65℃の水浴中で解凍管。 30秒（各ボルテックスの間に65℃の水浴中で5秒再加熱して10秒ごとに3回）の計ボルテックスチューブ。 </li><li>大きな凝集体は、水和溶液中で観察することができないまで、必要に応じ5サイクル以上、凍結融解を繰り返します。完了したら、氷の上porphysomeサスペンションのままにしておきます。 </li><li>二つの循環水浴65℃ためのサーモスタットサーキュレーターにサーモ押出機の接続ピンを設置100nmのポリカーボネートフィルターを積層し、高圧力窒素タンクに押出機システムを接続して、高圧押出機（10ml）にアセンブル必ず電源。 </li><li> 9インチのガラス移送ピペットを用いて押出機室にporphysome懸濁液を移す。 200と300 psigの間の読み取りは、最初の押出圧力として観察されるまで、窒素タンクレギュレーターのダイヤルを回して圧力を設定します。十分porphysome流量が達成されるか、圧力800psigの（5440キロパスカル）の最大値に達するまで徐々に圧力調整器の弁を開く。 </li><li>最初の押出パスが完了した後、圧力制御弁を閉じて、押出機の上部に圧力逃がし弁を開くことにより、押出機の圧力を解放する。新鮮に押し出されporphysome溶液で押出機を補充し、最終的な押し出された溶液が均一であることを確認するために、少なくとも10回押し出しを繰り返します。 </li><li>紫外可視分光計により、メタノールで希釈した試料の吸収を測定することにより、最終的なporphysome溶液の濃度を決定する。次式のウィットを使用して濃度を計算するH 97,000 Mの吸光係数-1 cm -1の410 nmにおけるピロ-脂質のため。 </li></ol><img alt="式（1）" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li>動的光散乱によってマルバーンサイザーZS90を使用してporphysomesの最終的なサイズ分布を測定する。 PBSにporphysome溶液を希釈し、平均の結果を得るために15の実行ごとに3回の測定を行う。 </li><li>分光蛍光光度計を使用して（1％トリトンX-100で1 mM）を蛍光急冷porphysomeの排出量（PBS中1 mM）および非消光porphysomeを比較することにより、porphysomeの蛍光消光を決定します。洗剤の前後のスペクトルを取る（Tirton X-100）を添加し、最大蛍光に読ん信号を正規化されている。 600〜800 nmと410 nmであり、発光波長範囲であることが励振を設定する。 </li><li>将来使用するまで4℃でアルゴン下でporphysomesソリューションを保ち、アルミfで光から保護するオイル。 </li></ol> 3。動物異種移植モデルの作製<ol><li>培養KBのRPMI-1640中の細胞（10％FBSを有する）、および37℃、5％CO 2および加湿雰囲気中で細胞を維持する</li><li>標準的な細胞培養技術を用いて収穫KB細胞。注射時まで、短期保存のために氷上でリン酸緩衝生理食塩水（PBS）中の細胞懸濁液（20,000細胞/ ml）で保つ。 </li><li>ヌードマウス上のKBの異種移植片を準備します。照射LM-485、：食事;標準食や寝具（ハーランラボラトリーズ与え、：彼らは20グラム（NU（NCR） - FOXN1 NU、6〜7週齢のカテゴリ＃CRL）のときのヌードマウスはチャールズ·リバー·ラボラトリーズから購入＃7912;寝具：トウモロコシの穂軸寝具、¼ &quot;、＃7097）。実験する前に、すべての動物取扱ツール（注射器、ピンセット、および動物パッド）を殺菌する。 100％酸素キャリアガス中の2％イソフルランv / vのでマウスを麻酔。全ての動物プロトコルは機関（大学健康NEによって承認されているtworkこの場合）動物管理使用委員会。 </li><li>マウスの右脇腹に、PBSを皮下に懸濁した腫瘍細胞を注入する。マウスは、腫瘍4-5 mmで2ないし3mmの厚さの直径に達したときに、10日後に使用する準備ができている。 </li></ol> 4。 インビボ光熱療法<ol><li>尾静脈を介して静脈内porphysomes（200μL容量で750ナノモルのパイロを含む）を注入。 porphysomesのレーザー照射24時間後の注入を行っています。 </li><li>このセクションでは、囲まれたレーザー安全室ですべての手順を実施しています。レーザー照射時にレーザーの安全ゴーグルを着用する。 </li><li>レーザー照射装置を設定します。スタンドにクランプによってレーザーファイバーとディフューザーを開催しています。低レベルのレーザーパワーを設定し、レーザをオンにします。直径9mm（DPSSレーザー、LaserGlow Technologies社、トロント、カナダ）の照射領域を達成するために、レーザーファイバーの高さを調整すると、pを用いて、750ミリワット（1.18 W / cm 2）と 、レーザパワーを較正その後、文章理解メートルや動物を準備するために、レーザーをオフにしてください。 </li><li> NO）捕捉明らかイソフルランのノーズコーンを通じて100％の酸素キャリアガス中の2％のイソフルランV / Vヒトの健康リスクの危険で動物を麻酔。動物は、動物の後肢に鉗子や指で足指のピンチを行うことで十分に深く麻酔をかけていることを確認します。彼らは、この手順に応答しないときの動物は、レーザー照射のための準備が整いました。 </li><li>赤外線熱カメラ（Mikroshot、LUMASENSE技術）によって腫瘍の温度を監視します。 、近い腫瘍にカメラを置きだけでなく、それを集中して、PTT照射が始まる前に、事前のPTT画像だけでなく、通常の白色光画像を取る。 </li><li>再びレーザーの電源を入れ、750ミリワットに光パワーを増加させる。完全にカバーされた腫瘍を得るために、レーザビームの中心部に腫瘍を配置し、照射開始タイミング。 1分間のレーザー治療のための腫瘍の温度画像ごとに5秒を取る。 1分でレーザーをオフにします。 STOP 2％のイソフルランのガス供給、動物が麻酔から回復し、ケージに戻すまで待ってください。 </li><li>大学健康ネットワークによって承認された動物のプロトコールに従って12時12時間の明暗サイクルの下で正常体温の動物室でマウスを保管してください。ブプレノルフィン（0.05 mg / kgで、瘢痕によって引き起こされる疼痛を軽減するレーザー照射後の週の皮下に1日2回注射する。2日毎に、カリパスを用いて腫瘍径を測定し、写真を撮る。用いて腫瘍体積を計算する以下の式：V =π/ 6···B 2、aは長径であり、bが短いものである。 </li><li>それが私たちの研究室で定義されたエンドポイントであるように、腫瘍の長径は、10ミリメートルに達したときに、CO 2室でそれらを置くことによって、動物を生け贄に捧げる。 CO2の治療後に頸椎脱臼を実行します。生存曲線をプロットします。 </li></ol>

<ol>
	<li>Lovell, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

我々は、開示することは何もありません。

高い薬物ペイロードを維持しながら、薬物送達技術の開発においては、多官能性ナノ粒子は、薬物送達ビヒクルの正確な追跡調査のための広い下現在。 5,6リポソームから血漿タンパク質へのポルフィリン導入されたリポソームは、より良好な薬物動態特性及びポルフィリンの直接投与よりも効率的な送達のために開発されてきたが、障害物が存在し、制限されたポルフィリンの装填?...

Porphysomesはマルチモーダルイメージングおよび治療 ​​の1が可能である我々は最近開発された新たな多機能ナノベシクルです。それらは、自己組織化ポルフィリン二重層から形成され、独特の構造に依存する蛍光の自己消光大きな吸光係数と結果を生成する（000/porphysome粒子、83を超える）ポルフィリンの極めて高い密度を含有する。 Porphysomesは、 インビボで良好な薬物動態および生体内分布特性を有する：それらは全身投与後12時間の血中半減期を示し、受動的に24時間後に注射2で7.5％ID / gの異種移植片腫瘍において蓄積する。 彼らのユニークな構造と物理化学的性質は、マルチモーダルイメージングと画像誘導療法のporphysome良い候補を作る。まず第一に、ポルフィリンを含む、porphysomesは、腫瘍蓄積の際に1腫瘍の蛍光イメージングのために本質的に適している。さらに、各ポルフィリンしたがって、porphysomesを容易PETイメージングのためのそのような3は64 Cu等の放射性同位体で標識することができるキレート化放射性同位体のための安定した部位を有している。 porphysome構造が損なわれていないときporphysomesにもユニークな音響イメージングと、PTT機能を発揮するので、さらに、吸収された光エネルギーは、レーザー照射の曝露下に熱放散される。それはporphysomesの静脈内注射後24時間、porphysome蓄積さ腫瘍のレーザ照射急速な温度上昇と強い光熱腫瘍切除を誘導したことが示された。これはporphysomesは金ナノ粒子（AuNPs）と高い吸光係数を有する効率的な光熱エンハンサーであることが実証さ1。一方、AuNPsを含む他の無機光熱剤と比較して、porphysomesは、その有機性に生物学的安全性で優れた利点を示す。 Porphysomesは、酵素的に生分解性であり、最小の急性toxicitを誘導千mg / kgの1という高い静脈内投与したマウスでは、Y。さらに、リポソームと同様に、porphysomesの大水性コアは、受動的または能動的に治療薬または造影剤を装填することができた。 porphysomesの光学的性質と生体適合性がバイオフォトニックイメージングおよび治療のための有機ナノ粒子の多峰性を示しています。 本論文では、ピロ-脂質複合体の合成方法、製造、高圧押し出しを使用してporphysomesの特性評価方法をご紹介します。マウスでのインビボのPTTは、腫瘍内porphysome対応のPTTの効率を実証するためにも実施される皮下異種移植腫瘍モデルを用いた治療。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

我々は最近、本質的に多機能ナノベシクルとしてporphysomesを開発しました。光増感剤は、αピロ、リン脂質に結合させた後、リポソーム様の球状の小胞への自己組織化。によりポルフィリン - 脂質二重層中のポルフィリンの非常に高い密度、porphysomes、大きな吸光係数、構造に依存する蛍光自己消光し、優れた光熱効果を生成した。我々の製剤において、porphysomes高圧押し出しを使用して合成し、120nmの周りの平均粒径を示した。 porphysomesの二十四時間後、静脈内注射、腫瘍の局所的な温度は急速に750 MW（1.18 W / cm 2）での1分間の曝露、671 nmのレーザー照射により62℃まで30℃から上昇した。腫瘍の完全な熱切除に続いて、形成され、癒さ2週間以内に、対照群の腫瘍は成長を続けている間、すべてが定義されている専用に達した焼痂3週間以内に、D点。これらのデータは、porphysomesが強力な光熱療法（PTT）剤として使用することができる方法を示す。

ピロは、安定な脂質モノマー（ 図1a）などのリン脂質とコンジュゲートの自己集合高圧押出機を用いて膜押し出し法によってporphysomesを形成するために結合される。これは、比較的遅い流速で押し出しの第3サイクルの間porphysome - 脂質懸濁液を押し出すことは通常困難である。より押し出しが繰り返されるように、押し出された液の流量が​​徐々に増加し、必要に応じて圧力が...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

そこで我々は、強力な光熱療法剤機能、構造依存的な蛍光の自己消光及び光熱ユニークな特性を有するporphysomesと呼ばれる新規真性多官能ナノベシクルを開発した。我々は、高圧押し出しを使用してporphysomesを策定し、異種移植腫瘍モデルにおけるそれらの光熱治療の有効性を検討した。

1分Porphysomesを使用してサブワンワット光熱腫瘍焼灼、真性多機能ナノベシクル

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

one-minute-sub-one-watt-photothermal-tumor-ablation-using-porphysomes

Research

JoVE Journal

Bioengineering

10.7K ビュー.  University of Toronto. そこで我々は、強力な光熱療法剤機能、構造依存的な蛍光の自己消光及び光熱ユニークな特性を有するporphysomesと呼ばれる新規真性多官能ナノベシクルを開発した。我々は、高圧押し出しを使用してporphysomesを策定し、異種移植腫瘍モデルにおけるそれらの光熱治療の有効性を検討した。 

ビデオ: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

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

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

の光周波数領域イメージング<em&gt;エクスビボ</em&gt;肺切除標本：組織病理相関へのOne to Oneイメージの入手

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

のためのウイルス粒子<emインビボ&gt;</em&gt;腫瘍イメージング

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

腫瘍サイズ測定における品質保証のために組織模倣材料を用いた前臨床マルチモダリティファントムの構築

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

マイクロウェルプレートを用いてサイズ制御の腫瘍球、多数の生産

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

粒子追跡マイクロレオロジーを用いて3次元腫瘍モデルにおける細胞外マトリックス剛性の縦測定

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高強度のリアルタイムモニタリングの超音波（HIFU）アブレーションを重視しました<em&gt;インビトロ</em集束超音波のための調和運動イメージングの使用&gt;犬の肝臓（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

中に組み込み血管新生による組織工学<em&gt;インビボ</em&gt;ティッシュ・エンジニアリング商工会議所

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

光熱角度光散乱ラピッド・化学・フリーヘモグロビンアッセイ

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...