Name: cell labeling and targeting with superparamagnetic iron oxide nanoparticles
Uploaded: 2015-10-19T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles at JoVE.com

The authors wish to acknowledge funding from the European Regional Development Fund &#8211; FNUSA-ICRC (no. CZ.1.05/ 1.1.00/ 02.0123), the American Heart Association Scientist Development Grant (AHA #06-35185N), and the National Institutes of Health (NIH #T32HL007111).

マグネタイトゲルの合成1。 <ol><li>エチルアルコールが続く脱イオン水、続いて濃塩酸を使用して、すべてのガラス器具を洗ってください。好ましくは乾燥炉中で、O / Nを乾燥することができます。 注意！塩酸は危険です - ドラフト内で個人用保護具や作業を着用。エチルアルコールは有害である - 個人用保護具を着用します。 </li><li>脱ガス、脱イオンH 2 Oを 500ミリリットルを静かに30分間N 2ガ ​​スをバブリングすることによりDreschelボトルを使用してください。 </li><li>セットアップマグネタイト合成装置を化学ヒュームフード内。 <ol><li> isomantleヒーター内500mlの三口丸底フラスコを置き、クランプを使用して、中央の口を確保し、スタンド。 </li><li>丸底フラスコの側面口の1つ、残りの側の首にゴム隔壁を還流冷却器にゴム隔膜を取り付けます。連続還流冷却器を通して冷たい水を実行します。 </li><li>パンク番目N 2ガ ​​スラインに接続された針を用いて電子丸底フラスコのゴム隔壁とガス流出を可視化するためにバブラー（水とのすなわち、フラスコ）に実行されているガスラインに接続された針で還流冷却器のゴム隔膜を穿刺。 </li><li>パドルアダプタを介して丸底フラスコの中央の口にブレードパドルを取り付けます。スタンドに取り付けられたオーバーヘッドスターラーをブレードパドルのシャフトを取り付けます。 </li></ol></li><li> N 2ガ ​​スを用いた丸底フラスコをパージし、低いが検出速度で流れるN 2ガ ​​スを残します。 </li><li>丸底フラスコから還流冷却器を取り外し、塩化鉄（III）、鉄の0.6125グラム（II）塩化物水和物の1.000グラムを追加し、脱気H 2 Oの50ミリリットル 注意！塩化鉄（III）および塩化鉄（II）四水和物は、有害である - 個人用保護具を着用します。 </li><li> 50に加熱しながら還流冷却器を取り付け、1000rpmで攪拌°C。これらの条件下で攪拌すると、10 nmの直径のマグネタイトナノ粒子を生成します。 </li><li>一旦、50℃で、さらに攪拌しながら丸底フラスコにゴム隔膜を介して注入することにより、28％水酸化アンモニウム溶液10mlを加えます。 注意！水酸化アンモニウムは有害である - 個人用保護具を着用します。 注：水酸化アンモニウム溶液は、マグネタイトを沈殿させるために使用され、溶液が黒くなるべきです。 </li><li>まだ攪拌しながらアンモニアガスを沸騰する90℃に丸底フラスコと熱からゴム隔壁および N 2ガ ​​スラインを外します。 注：これは、還流冷却器のゴム隔壁を穿刺することにより、丸底フラスコに、N 2の流れを維持するために、任意である、しかし、マグヘマイトへの磁鉄鉱 ​​の酸化は、このステップの間に無視できるものです。 </li><li>いったん90℃でまだ攪拌しながら、丸底フラスコにオレイン酸の1ミリリットルを追加します。オレイン酸は、マグネタイトNA被覆するために使用されていますマグネタイトのゲルを形成するnoparticles。 注意！オレイン酸は有害である - 個人用保護具を着用します。 </li><li>丸底フラスコにゴム隔壁および N 2ガ ​​スラインを交換し、還流冷却器を取り外します。 </li><li>熱をオフにして、2時間、500rpmで攪拌​​します。 </li><li> isomantleヒーターからの丸底フラスコを取り外し、マグネタイトゲルを保持するために、フラスコの底に対して保持強力な磁石を使用している間、残りの液体をデカント。 注意！損傷または傷害を避けるために、細心の注意を持つ強力な磁石を処理します。 </li><li>空気乾燥O / N（オプション）にマグネタイトゲルを許可します。 </li></ol>マグネタイトゲルの2精製<ol><li>マグネタイトゲルを溶解させた丸底フラスコに、ヘキサン40 mlを加え 注意！ヘキサンは有害である - ドラフト内で個人用保護具と仕事を着用してください。 </li><li>残留H 2 Oあちこち除去するために、脱気の H 2 O 40mLで分液漏斗を使用してくださいマグネタイト溶液メートル。 <ol><li>ゆっくりと分液漏斗内のH 2 Oにマグネタイト液を注ぎ、軽く5分間二相液体を旋回。 </li><li>外排出し、下部の水部分を破棄します。 </li><li>ゆっくりマグネタイトソリューションの下に、それが落ち着くように、分液漏斗に脱気したH 2 Oの40ミリリットルを加え、穏やかに旋回し、以前のようにドレイン。 </li><li>三度目の洗浄するために繰り返します。 </li></ol></li><li> 、三角フラスコにマグネタイト液を移し、無水硫酸ナトリウムの価値はいくつかへらを追加し、マグネタイト溶液から、残りの残留H 2 Oを除去するための渦。 </li><li>硫酸ナトリウム及び残留H 2 Oを除去するための濾過漏斗で1ミクロンの濾紙マグネタイト溶液を濾過 注：真空支援が推奨されます。 </li><li> 50ミリリットル蒸発フラスコにマグネタイト液を移し、ヘキサンを蒸発させるために、ロータリーエバポレーターを使用以下の条件で2時間：中程度の回転速度、真空度は50℃の水浴でフラスコを蒸発させ、塗布し、24℃の水が凝縮器を循環します。 注：必要に応じて、PLGAでコーティングする前にマグネタイトゲルを保存します。 </li></ol> PLGAシェルを使用したマグネタイトナノ粒子の3コーティング<ol><li> 1.5％（M / V）のソリューションを作成するために、酢酸エチル240mlの中のPLGAの3.60グラム（75/25ブレンド）を溶解します。注意：酢酸エチルは有害である - ドラフト内で個人用保護具と仕事を着用してください。 </li><li> 5.0％（M / V）溶液を作成するために、磁気スターラーを用いて脱気H 2 O 500ml中のPluronic F-127の25.00グラムを溶解します。 注：プルロニックF-127は、生体適合性界面活性剤として作用する非イオン性両親媒性ブロック共重合体です。なお、ステップ3.3.2で水中油型エマルジョンを安定化するのに役立ちます。 </li><li>ミクロを使用して、重み付けされたガラスバイアル内で6 0.040グラムのアリコートにマグネタイトゲルを収集します。 PERF各アリコートについては、以下のコーティング及び洗浄工程をORM。 注：一定分量は、ステップ4で劣化する前に凍結乾燥を最小限に抑えながら、純度と収率を最大れる、効率的な処理および磁気デカンテーションを確保するために必要です。 <ol><li> 10分間超音波洗浄器で0.040グラムのマグネタイトゲルのアリコートおよびプラスチックビーカー超音波処理にPLGA溶液の40ミリリットルを追加します。 </li><li>プラスチックビーカーのプルロニック溶液80 mlを加え、すぐに水中油型エマルジョンとしてマグネタイトナノ粒子上のPLGAコーティングを形成するために7分間最高設定で実験室用ミキサーで乳化します。 </li><li>すぐに酢酸エチルを蒸発させ、化学換気フード中で1時間脱イオンH 2 O及び超音波処理を1 LにSPION溶液を希釈します。 </li><li> SPION溶液に次の強力な磁石を置き、静かに磁石で茶色がかっSPIONsを収集するためにかき混ぜます。 注：これは、断続的に、数時間攪拌する必要があるかもしれません溶液前SはSPIONsのほとんどが収集されたことを示す白っぽくなります。 </li><li>磁石をビーカーにSPIONsを維持しながら水溶液をデカント。 </li><li>次のようにSPIONsを3回洗浄します。 <ol><li>脱イオンH 2 O 1LにSPIONsサスペンド</li><li> 20分間SPION液を超音波処理。 </li><li> SPION溶液に次の強力な磁石を置き、静かに磁石で茶色がかっSPIONsを収集するためにかき混ぜます。これは、溶液がSPIONsの大部分が回収されたことを示す明確なターン前に断続的に数時間撹拌することが必要であり得ます。 </li><li>磁石をビーカーにSPIONsを維持しながら水溶液をデカント。 </li></ol></li></ol></li><li>水性懸濁液のような単一の加重ガラス瓶に6マグネタイトゲルアリコートのそれぞれから合成SPIONsを収集します。必要に応じて、必要に応じて磁気的に余分な水分をデカント。 </li></ol> 4.フリーズSPIONsの-drying <ol><li> SPIONソリューションをフリーズします。 </li><li>凍結乾燥した凍結乾燥機でSPION液O / Nに。 </li><li>凍結乾燥SPIONsを計量。細胞標識に使用するまで凍結乾燥SPIONsを-20℃で保存することができます。 注：-20°C劇的での保存は、分解速度を減少させ、貯蔵寿命を向上させます。 </li></ol> SPIONsによる細胞の5ラベリング<ol><li> 40 mg / mlの濃度でリン酸緩衝生理食塩水（PBS）中SPIONsのアリコートを一時停止し、30分間超音波処理します。 </li><li>細胞培養培地を5μl/ mlの濃度で細胞のほぼコンフルエントなフラスコにSPION溶液を加えます。静かにフラスコを揺動することにより均一な分布を確認してください。 </li><li> 37℃で16時間細胞をインキュベートします。 </li><li>穏やかに培地を吸引し、PBSで細胞を2回洗浄します。 </li><li>磁気標識細胞を収集し、実験に使用します。 </li><li>未使用SPION溶液は​​4℃で保存することができ、米国でなければなりません数ヶ月以内にエド。各使用前に30分間超音波処理。 </li></ol>

<ol>
	<li>Burgess, A., 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=Targeted+delivery+of+neural+stem+cells+to+the+brain+using+MRI-guided+focused+ultrasound+to+disrupt+the+blood-brain+barrier.">Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier.</a> PLoS One. 6 (11), e27877 (2011).</li><li>Nguyen, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cells+as+Targeted+Cell+Vehicles+to+Deliver+Drug-loaded+Nanoparticles+for+Cancer+Therapy.">Mesenchymal Stem Cells as Targeted Cell Vehicles to Deliver Drug-loaded Nanoparticles for Cancer Therapy.</a> J Nanomed Nanotechol. 4 (1), e128 (2013).</li><li>Kean, T. J., Lin, P., Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSCs:+Delivery+Routes+and+Engraftment,+Cell-Targeting+Strategies,+and+Immune+Modulation.">MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation.</a> Stem Cells Int. , 732742 (2013).</li><li>Suzuki, K., 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=Targeted+cell+delivery+into+infarcted+rat+hearts+by+retrograde+intracoronary+infusion:+distribution,+dynamics,+and+influence+on+cardiac+function.">Targeted cell delivery into infarcted rat hearts by retrograde intracoronary infusion: distribution, dynamics, and influence on cardiac function.</a> Circulation. 110 (11 Suppl 1), II225-II230 (2004).</li><li>Garbern, J. C., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+stem+cell+therapy+and+the+promise+of+heart+regeneration.">Cardiac stem cell therapy and the promise of heart regeneration.</a> Cell Stem Cell. 12 (6), 689-698 (2013).</li><li>Duckers, H. J., 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=Accelerated+vascular+repair+following+percutaneous+coronary+intervention+by+capture+of+endothelial+progenitor+cells+promotes+regression+of+neointimal+growth+at+long+term+follow-up:+final+results+of+the+Healing+II+trial+using+an+endothelial+progenitor+cell+capturing+stent+(Genous+R+stent).">Accelerated vascular repair following percutaneous coronary intervention by capture of endothelial progenitor cells promotes regression of neointimal growth at long term follow-up: final results of the Healing II trial using an endothelial progenitor cell capturing stent (Genous R stent).</a> EuroIntervention. 3 (3), 350-358 (2007).</li><li>Pan, Y., Du, X., Zhao, F., Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticles+for+the+manipulation+of+proteins+and+cells.">Magnetic nanoparticles for the manipulation of proteins and cells.</a> Chem Soc Rev. 41 (7), 2912-2942 (2012).</li><li>Huang, Z. Y., 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=Deep+magnetic+capture+of+magnetically+loaded+cells+for+spatially+targeted+therapeutics.">Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics.</a> Biomaterials. 31 (8), 2130-2140 (2010).</li><li>Shen, Y., 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=Comparison+of+Magnetic+Intensities+for+Mesenchymal+Stem+Cell+Targeting+Therapy+on+Ischemic+Myocardial+Repair:+High+Magnetic+Intensity+Improves+Cell+Retention+but+Has+No+Additional+Functional+Benefit.">Comparison of Magnetic Intensities for Mesenchymal Stem Cell Targeting Therapy on Ischemic Myocardial Repair: High Magnetic Intensity Improves Cell Retention but Has No Additional Functional Benefit.</a> Cell Transplant. , (2014).</li><li>Cheng, K., 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=Magnetic+antibody-linked+nanomatchmakers+for+therapeutic+cell+targeting.">Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting.</a> Nat Commun. 5, 4880 (2014).</li><li>Vandergriff, A. C., 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=Magnetic+targeting+of+cardiosphere-derived+stem+cells+with+ferumoxytol+nanoparticles+for+treating+rats+with+myocardial+infarction.">Magnetic targeting of cardiosphere-derived stem cells with ferumoxytol nanoparticles for treating rats with myocardial infarction.</a> Biomaterials. 35 (30), 8528-8539 (2014).</li><li>Huang, Z., 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=Magnetic+targeting+enhances+retrograde+cell+retention+in+a+rat+model+of+myocardial+infarction.">Magnetic targeting enhances retrograde cell retention in a rat model of myocardial infarction.</a> Stem Cell Res Ther. 4 (6), 149 (2013).</li><li>Chaudeurge, A., 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=Can+magnetic+targeting+of+magnetically+labeled+circulating+cells+optimize+intramyocardial+cell+retention.">Can magnetic targeting of magnetically labeled circulating cells optimize intramyocardial cell retention.</a> Cell Transplant. 21 (4), 679-691 (2012).</li><li>Cheng, K., 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=Magnetic+targeting+enhances+engraftment+and+functional+benefit+of+iron-labeled+cardiosphere-derived+cells+in+myocardial+infarction.">Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction.</a> Circ Res. 106 (10), 1570-1581 (2010).</li><li>Yanai, A., 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=Focused+magnetic+stem+cell+targeting+to+the+retina+using+superparamagnetic+iron+oxide+nanoparticles.">Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles.</a> Cell Transplant. 21 (6), 1137-1148 (2012).</li><li>Ordidge, K. L., 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=Coupled+cellular+therapy+and+magnetic+targeting+for+airway+regeneration.">Coupled cellular therapy and magnetic targeting for airway regeneration.</a> Biochem Soc Trans. 42 (3), 657-661 (2014).</li><li>El Haj, A. J., 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=An+in+vitro+model+of+mesenchymal+stem+cell+targeting+using+magnetic+particle+labelling.">An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling.</a> J Tissue Eng Regen Med. , (2012).</li><li>Vanecek, V., 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=Highly+efficient+magnetic+targeting+of+mesenchymal+stem+cells+in+spinal+cord+injury.">Highly efficient magnetic targeting of mesenchymal stem cells in spinal cord injury.</a> Int J Nanomedicine. 7, 3719-3730 (2012).</li><li>Sasaki, H., 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=Therapeutic+effects+with+magnetic+targeting+of+bone+marrow+stromal+cells+in+a+rat+spinal+cord+injury+model.">Therapeutic effects with magnetic targeting of bone marrow stromal cells in a rat spinal cord injury model.</a> Spine (Phila Pa 1976). 36 (12), 933-938 (2011).</li><li>Oshima, S., 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=Enhancement+of+bone+formation+in+an+experimental+bony+defect+using+ferumoxide-labelled+mesenchymal+stromal+cells+and+a+magnetic+targeting+system.">Enhancement of bone formation in an experimental bony defect using ferumoxide-labelled mesenchymal stromal cells and a magnetic targeting system.</a> J Bone Joint Surg Br. 92 (11), 1606-1613 (2010).</li><li>Luciani, A., 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=Magnetic+targeting+of+iron-oxide-labeled+fluorescent+hepatoma+cells+to+the+liver.">Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver.</a> Eur Radiol. 19 (5), 1087-1096 (2009).</li><li>Oshima, S., Kamei, N., Nakasa, T., Yasunaga, Y., Ochi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+muscle+repair+using+human+mesenchymal+stem+cells+with+a+magnetic+targeting+system+in+a+subchronic+muscle+injury+model.">Enhancement of muscle repair using human mesenchymal stem cells with a magnetic targeting system in a subchronic muscle injury model.</a> J Orthop Sci. 19 (3), 478-488 (2014).</li><li>Ohkawa, S., 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=Magnetic+targeting+of+human+peripheral+blood+CD133++cells+for+skeletal+muscle+regeneration.">Magnetic targeting of human peripheral blood CD133+ cells for skeletal muscle regeneration.</a> Tissue Eng Part C Methods. 19 (8), 631-641 (2013).</li><li>Tefft, B. J., 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=Magnetizable+Duplex+Steel+Stents+Enable+Endothelial+Cell+Capture.">Magnetizable Duplex Steel Stents Enable Endothelial Cell Capture.</a> Ieee T Magn. 49 (1), 463-466 (2013).</li><li>Uthamaraj, S., 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=Design+and+validation+of+a+novel+ferromagnetic+bare+metal+stent+capable+of+capturing+and+retaining+endothelial+cells.">Design and validation of a novel ferromagnetic bare metal stent capable of capturing and retaining endothelial cells.</a> ABME. 42 (12), 2416-2424 (2014).</li><li>Polyak, B., 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=High+field+gradient+targeting+of+magnetic+nanoparticle-loaded+endothelial+cells+to+the+surfaces+of+steel+stents.">High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents.</a> Proc Natl Acad Sci U.S.A. 105 (2), 698-703 (2008).</li><li>Pislaru, S. V., 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=Magnetically+targeted+endothelial+cell+localization+in+stented+vessels.">Magnetically targeted endothelial cell localization in stented vessels.</a> J Am Coll Cardiol. 48 (9), 1839-1845 (2006).</li><li>Pislaru, S. V., 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=Magnetic+forces+enable+rapid+endothelialization+of+synthetic+vascular+grafts.">Magnetic forces enable rapid endothelialization of synthetic vascular grafts.</a> Circulation. 114 (1 Suppl), 314-318 (2006).</li><li>Wang, Y. X., Xuan, S., Port, M., Idee, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+superparamagnetic+iron+oxide+nanoparticles+for+cellular+imaging+and+targeted+therapy+research.">Recent advances in superparamagnetic iron oxide nanoparticles for cellular imaging and targeted therapy research.</a> Curr Pharm Des. 19 (37), 6575-6593 (2013).</li><li>Yellen, B. B., 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=Targeted+drug+delivery+to+magnetic+implants+for+therapeutic+applications.">Targeted drug delivery to magnetic implants for therapeutic applications.</a> J Magn Magn Mater. 293 (1), 647-654 (2005).</li><li>Granot, D., 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=Clinically+viable+magnetic+poly(lactide-co-glycolide)+particles+for+MRI-based+cell+tracking.">Clinically viable magnetic poly(lactide-co-glycolide) particles for MRI-based cell tracking.</a> Magn Reson Med. , (2013).</li><li>Levy, M., 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=Long+term+in+vivo+biotransformation+of+iron+oxide+nanoparticles.">Long term in vivo biotransformation of iron oxide nanoparticles.</a> Biomaterials. 32 (16), 3988-3999 (2011).</li><li>Mirshafiee, V., Mahmoudi, M., Lou, K., Cheng, J., Kraft, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+corona+significantly+reduces+active+targeting+yield.">Protein corona significantly reduces active targeting yield.</a> Chem Commun (Camb). 49 (25), 2557-2559 (2013).</li><li>Salvati, A., 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=Transferrin-functionalized+nanoparticles+lose+their+targeting+capabilities+when+a+biomolecule+corona+adsorbs+on+the+surface.">Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.</a> Nat Nanotechnol. 8 (2), 137-143 (2013).</li><li>Landazuri, N., 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=Magnetic+targeting+of+human+mesenchymal+stem+cells+with+internalized+superparamagnetic+iron+oxide+nanoparticles.">Magnetic targeting of human mesenchymal stem cells with internalized superparamagnetic iron oxide nanoparticles.</a> Small. 9 (23), 4017-4026 (2013).</li><li>Sun, J. H., 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=In+vitro+labeling+of+endothelial+progenitor+cells+isolated+from+peripheral+blood+with+superparamagnetic+iron+oxide+nanoparticles.">In vitro labeling of endothelial progenitor cells isolated from peripheral blood with superparamagnetic iron oxide nanoparticles.</a> Mol Med Rep. 6 (2), 282-286 (2012).</li><li>Zhang, B., 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=Detection+of+viability+of+transplanted+beta+cells+labeled+with+a+novel+contrast+agent+-+polyvinylpyrrolidone-coated+superparamagnetic+iron+oxide+nanoparticles+by+magnetic+resonance+imaging.">Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.</a> Contrast Media Mol Imaging. 7 (1), 35-44 (2012).</li><li>Song, M., 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=Labeling+efficacy+of+superparamagnetic+iron+oxide+nanoparticles+to+human+neural+stem+cells:+comparison+of+ferumoxides,+monocrystalline+iron+oxide,+cross-linked+iron+oxide+(CLIO)-NH2+and+tat-CLIO.">Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.</a> Korean J Radiol. 8 (5), 365-371 (2007).</li></ol>

The authors declare that they have no competing financial interests.

任意のナノ粒子合成プロトコルと同様に、反応物質の化学物質の純度は、最小限の細胞毒性効果を持つことになり、高品質のSPIONsを達成するために重要です。これは、オレイン酸（≥99％）、塩化鉄（II）四水和物（≥99.99％）、塩化鉄（III）（≥99.99％）、酢酸エチルなどの非常に純粋な試薬を購入することが重要である（HPLCグレード、≥99.9％ ）、ヘキサン（HPLCグレード、≥97.0％）、水酸化アンモニウム（≥99.99％）、及び硫酸ナトリウム（≥99.0％）。これは、比較的高価であり、非常に純粋な、高品質のPLGAを、購入するために特に重要です。また、すべてのガラス製品は、完全に塩酸、脱イオン水、エチルアルコールで洗浄し、使用前に乾燥させなければなりません。 同様に、プロトコル内の精製工程および洗浄工程は、最終的なSPIONsが高品質であること、最小限の細胞毒性作用を持つことになります確保するために重要です。マグネタイトゲルはあってはなりませんPLGAで被覆する前に可能な限り多くの水酸化アンモニウム、水、及びヘキサン等。したがって、プロトコルの多くはマグネタイトゲルの純度を確保することに専念しています。その後、PLGA-マグネタイトSPIONsは、酢酸エチル、プルロニック、過剰PLGAの自由でなければなりません。最終SPIONの洗浄工程は、プロトコルの中で最も時間のかかる部分ですが、高純度を確保するために完了する必要があります。具体的には、各洗浄工程の間に粒子の磁気回収は非常に時間がかかる可能性があります。溶液を攪拌して大幅に粒子収集の速度を上げることができるが、磁気攪拌棒を使用することができません。低速で動作するオーバーヘッド攪拌機は、急速な粒子収集のための最も有効な手段です。確認SPIONsの大きな茶色がかったコレクションが磁石に表示され、その溶液をデカント前に白またはクリア表示されます。これは多くの場合、攪拌の数時間を必要とすることができますが、より高い最終収量になります。磁気デカンテーションステップはまた、唯一のMAGNを確保するのに役立ちますすべての非磁性材料が廃棄されている間エティックな粒子が保持されます。 過剰な鉄レベルは、細胞傷害性であることができるので、この技術を用いて細胞に付与することができる磁気質量の量が制限されます。鉄の濃度は、特に敏感な細胞型で減少または特に弱い磁場で増加する必要があるかもしれませんが、ここで説明されたプロトコルは、安全性と有効性のバランスをとるための実証済みの出発点を提供します。このプロトコルによって合成SPIONsはPLGAのマグネタイトの質量比が1時15分を有する溶液から作られ、SPIONsは、細胞培養培地に200μg/ mlの濃度で細胞に導入されます。これらのパラメータのいずれかが必要に応じて、各細胞によってエンドサイトーシス、鉄の量を変化させるように調整することができます。 SPIONsは、人間の移植のための安全であり、時間（約40〜50日の半減期）31の上に生分解されます。マグネタイトおよびPLGAの両方が無害degradを形成エーションの製品とは、自然の経路32を介して体内から消去されます。 SPIONsの生分解性の性質は、任意の細胞毒性効果は時間とともに減少することを意味するだけでなく、数ヶ月を超えて、その磁気特性を維持するために、細胞を必要としないものへの潜在的なアプリケーションを制限します。 SPIONsはまた、細胞を標識し、表面タンパク質を必要とせず、その磁気効果を付与も生物学的環境33,34に暴露されると、タンパク質のコロナの形成を受けやすいリガンドを標的にするという利点を有します。 細胞に磁気特性を付与することは、標的細胞の配信を必要とするか、29をソートする生物医学的用途の幅広いするのに便利です。種々の細胞型が安全に間葉系幹細胞35、内皮前駆細胞36、β島細胞37、及び神経幹細胞38を含むSPIONsをエンドサイトーシスする能力を実証しました。磁気CEL配信条件の制御の高度が必要である場合、Lは、標的化技術を標的とする他​​の細胞よりも好ましいです。

Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke1. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction4,5. Vascular stents have been developed with CD34 antibodies to capture circulating progenitor cells6. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.
The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects7. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body8. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart9-14, retina15, lung16, skin17, spinal cord18,19, bone20, liver21, and muscle22,23 in order to improve regeneration outcomes.
Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents24-27 and vascular grafts28 have both been fabricated and tested for this purpose.
Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types29. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe3O4) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.

<table><tbody><tr><td>Ammonium Hydroxide solution, 28% NH&lt;sub&gt;3&lt;/sub&gt; in H&lt;sub&gt;2&lt;/sub&gt;O, &amp;ge;99.99% trace metal basis</td><td>Sigma-Aldrich</td><td>338818-100ML&amp;nbsp;</td><td>Harmful reagent - wear personal protective equipment</td></tr><tr><td>Dreschel bottle, 500 ml</td><td>Ace Glass</td><td>5516-16</td><td></td></tr><tr><td>Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>650528-1L</td><td>Harmful reagent - wear personal protective equipment &amp;amp; work in fume hood</td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Harmful reagent - wear personal protective equipment</td></tr><tr><td>Evaporating flask, 50 ml, 24/40 joint</td><td>Sigma-Aldrich</td><td>Z515558</td><td>For use with rotoevaporator</td></tr><tr><td>Filter paper, 3 cm dia, grade 1</td><td>Fisher</td><td>09-805P</td><td>For use with glass filter funnel</td></tr><tr><td>Glass beakers, 1 L</td><td>Fisher</td><td>FB-101-1000</td><td>For washing SPIONs</td></tr><tr><td>Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mL</td><td>Fisher</td><td>K954100-0344&amp;nbsp;</td><td></td></tr><tr><td>Glass vial caps</td><td>Fisher</td><td>03-391-46</td><td>For use with glass vials</td></tr><tr><td>Glass vials, 2 ml</td><td>Fisher</td><td>03-391-44</td><td>For collecting magnetite gel &amp;amp; SPIONs</td></tr><tr><td>Hexane, CHROMASOLVR, for HPLC, &amp;ge;97.0% (GC)</td><td>Sigma-Aldrich</td><td>34859-1L&amp;nbsp;</td><td>Harmful reagent - wear personal protective equipment &amp;amp; work in fume hood</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td>Harmful reagent - wear personal protective equipment &amp;amp; work in fume hood</td></tr><tr><td>Iron(II) chloride tetrahydrate, &amp;ge;99.99% trace metals basis&amp;nbsp;</td><td>Sigma-Aldrich</td><td>380024-5G</td><td>Harmful reagent - wear personal protective equipment</td></tr><tr><td>Iron(III) chloride anhydrous, powder, &amp;ge;99.99% trace metals basis</td><td>Sigma-Aldrich</td><td>451649-1G</td><td>Harmful reagent - wear personal protective equipment</td></tr><tr><td>Isomantle heater, 500 mL</td><td>Voight Global</td><td>EM0500/CEX1</td><td></td></tr><tr><td>Laboratory mixer</td><td>Silverson</td><td>L5M-A</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>7670520</td><td></td></tr><tr><td>Microspatulas</td><td>Fisher</td><td>21-401-25A</td><td>For transfering magnetite gel</td></tr><tr><td>NdFeB magnet, 1 in x 1 in x 1 in</td><td>Amazing Magnets</td><td>C1000H-M</td><td>Very strong magnet, handle with care</td></tr><tr><td>Oleic acid, &amp;ge;99% (GC)</td><td>Sigma-Aldrich</td><td>O1008-5G&amp;nbsp;</td><td>Store in freezer; Harmful reagent - wear personal protective equipment</td></tr><tr><td>Overhead stirrer</td><td>IKA</td><td>2572201</td><td></td></tr><tr><td>Overhead stirrer clamp</td><td>IKA</td><td>2664000</td><td>For use with overhead stirrer</td></tr><tr><td>Overhead stirrer H-stand</td><td>IKA</td><td>1412000</td><td>For use with overhead stirrer</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Plastic beakers, 250 ml</td><td>Fisher</td><td>02-591-28</td><td></td></tr><tr><td>PLGA PURASORB PDLG (75/25 blend)</td><td>Purac</td><td>PDLG 7502</td><td>PDLG 7502A may be used as well; Store in freezer</td></tr><tr><td>Pluronic F-127 powder, BioReagent, suitable for cell culture</td><td>Sigma-Aldrich</td><td>P2443-250G&amp;nbsp;</td><td></td></tr><tr><td>PTFE expandable blade paddle, 8 mm dia</td><td>SciQuip</td><td>SP4018</td><td></td></tr><tr><td>PTFE vessel adapter, fits 24/40, 8 mm dia paddle</td><td>Monmouth Scientific</td><td>PTFE Vessel Adaptor A480</td><td>For use with PTFE expandable blade paddle</td></tr><tr><td>Recirculating chiller</td><td>Clarkson</td><td>696613</td><td>For use with rotoevaporator</td></tr><tr><td>Reflux condenser, fits 24/40, 250 mm</td><td>Ace Glass</td><td>5997-133</td><td></td></tr><tr><td>Rotoevaporator</td><td>Clarkson</td><td>216949</td><td></td></tr><tr><td>Rubber septa, fits 24/40</td><td>Ace Glass</td><td>9096-56</td><td></td></tr><tr><td>Separatory funnel with stopper, 250 ml</td><td>Fisher</td><td>10-438E</td><td></td></tr><tr><td>Sodium sulfate ACS reagent, &amp;ge;99.0%, anhydrous, granular</td><td>Sigma-Aldrich</td><td>239313-500G&amp;nbsp;</td><td></td></tr><tr><td>Three neck round bottom flask, angled, 24/40 joints, 500 ml</td><td>Ace Glass</td><td>6948-16</td><td></td></tr><tr><td>Ultrasonic cleaner perforated pan</td><td>Fisher</td><td>15-335-20A</td><td>For use with ultrasonic cleaner</td></tr><tr><td>Ultrasonic cleaner, 2.8 L</td><td>Fisher</td><td>15-335-20</td><td></td></tr><tr><td>Vacuum controller</td><td>Clarkson</td><td>216639</td><td>For use with rotoevaporator (optional)</td></tr><tr><td>Vacuum pump</td><td>Clarkson</td><td>219959</td><td>For use with rotoevaporator</td></tr></tbody></table>

cell labeling and targeting with superparamagnetic iron oxide nanoparticles

細胞および治療剤の標的化送達は、オフターゲット部位への悪影響を最小限に抑えながら、標的部位で治療効果を集中することにより、生物医学的用途の広い範囲に利益をもたらします。磁気細胞標的化は、効率的で、安全で、簡単な配信技術です。超常磁性酸化鉄ナノ粒子（SPION）は、生体適合性、生分解性であり、および磁界に応答してレンダリングするために細胞内に取り込ませることができます。合成プロセスは、マグネタイト鉄（Fe 3 O 4）を作成することを含む、ポリ（乳酸-コ-グリコール酸）（PLGA）コーティングを形成するために、高速乳化続いナノ粒子。 PLGA-マグネタイトSPIONsは約10 nmの直径のマグネタイトコアを含む直径約120nmです。培養培地中に置かれたとき、SPIONsは、天然細胞によってエンドサイトーシスおよび細胞質エンドソーム内の小さなクラスターとして記憶されます。これらの粒子は、細胞に十分な磁気質量を与え磁場内にターゲットを可能にします。多数の細胞選別とターゲットアプリケーションは、磁場に反応する様々な細胞型をレンダリングすることで有効になっています。 SPIONsは、同様に、腫瘍の治療または組織はんだ付けのための医療用イメージング造影剤として使用する、標的薬物や遺伝子デリバリー、診断アッセイ、およびローカル温熱療法の生成を含む他の生物医学的用途の多様性を持っています。

マグネタイトナノ粒子は、50℃、1000回転（図1）での鉄（III）、塩化鉄（II）塩化物水和物の水溶液を攪拌した結果、直径が約10nmです。これらの結果は、マグネタイトナノ粒子の合成に成功を実証します。これは、最初に合成しようとすると、バッチの少量の試料から採取したマグネタイトナノ粒子のサイズおよび形状を確認することが重要です。透過型電子顕微鏡（TEM）は、これらの粒子を可視化するための好ましい方法です。バッチは破棄されるべきであり、 図1に示すように、マグネタイトナノ粒子は直径および球形で10nm程度でない場合、合成が再試行する必要があります。 120nmで（図2）の直径を有するPLGA-マグネタイトSPIONsの高速乳化剤結果を用いて、PLGAとマグネタイトナノ粒子をコーティングします。これらの結果は、成功した実証しますPLGA-マグネタイトSPIONsの合成。これは、初めて合成をしようとしたときにバッチの小さなサンプルから採取したPLGA-マグネタイトSPIONsの大きさや形状を確認することが重要です。走査型電子顕微鏡（SEM）は、これらの粒子を可視化するための好ましい方法です。バッチは廃棄されるべきであり、 図2に示すように、PLGA-マグネタイトSPIONsがより大きいまたはより小さい粒子は、特定の用途のために望ましいかもしれないが、組成が不明である。直径の球状で120nm程度でない場合、合成を再度試みるべきしたがって、細胞標識、磁化率、および細胞毒性は予測できません。 ナノ粒子のエンドサイトーシスにおける16時間の結果（図3）のためのSPIONsと血液伸長内皮細胞のインキュベーション。これらの結果は、SPIONsでの細胞の成功したラベルを示しています。それから採取された細胞内SPIONsの存在を確認することが重要です初めての標識をしようとバッチの小さなサンプル。透過型電子顕微鏡では、これらのSPION標識細胞を可視化するための好ましい方法です。細胞は廃棄すべきであり、PLGA-mangetite SPIONsは、図3に示すように、細胞質エンドソーム内で一緒にクラスタ化された黒色粒子丸として表示されない場合は、ラベルを再度試みるべきである。また、SPIONsのより低い濃度は、磁気細胞標的化を可能にするために失敗し、 SPIONsのより高い濃度は、細胞傷害性であり得ます。必要に応じて、細胞を標識するために使用されるSPIONsの濃度に応じて調整することができます。 細胞内にロードされた鉄の量は、強磁性植え込み可能な医療装置（図4）と生存細胞の磁気捕捉を達成するのに十分です。これらの結果は、成功したSPION媒介磁気細胞標的化を実証します。効果をターゲットに強力な細胞が所望される場合、好ましい戦略はincreaことです生成または印加磁界8,30の強度や勾配をSE。細胞を標識するために使用さSPIONsの濃度を増加することのみによる細胞毒性の懸念への最後の手段として試みられるべきです。改善された細胞生存率が所望される場合、細胞を標識するために使用されるSPIONsの濃度を減少させるべきです。 <img alt="図1" src="/files/ftp_upload/53099/53099fig1.jpg" /> マグネタイトナノ粒子の図1のTEM像。マグネタイトナノ粒子は、透過電子顕微鏡（TEM）によって示されるように、直径が約10nmです。粒子のサイズは球状で均一です。スケールバー= 100 nmの。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53099/53099fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53099/53099fig2.jpg" /><br<html> PLGA-マグネタイトSPIONsの /&gt; 図2のSEM像。PLGA被覆されたマグネタイトSPIONs走査電子顕微鏡（SEM）により見られるように、直径が約120nmです。粒子のサイズは球状で均一です。スケールバー=500μmの。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53099/53099fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53099/53099fig3.jpg" /> 磁気的に標識された内皮細胞の図3のTEM像。透過型電子顕微鏡（TEM）によって可視化し、磁気標識された血液伸長内皮細胞。 SPIONsは自然に細胞によってエンドサイトーシスおよび細胞質エンドソーム内の小さなクラスターに格納されています。左スケールバー=2μmで、右のスケールバー= 0.5ミクロン。 24から許可を得て再印刷します。/files/ftp_upload/53099/53099fig3large.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図4" src="/files/ftp_upload/53099/53099fig4.jpg" /> 磁気細胞捕獲の図4蛍光顕微鏡画像は、磁気的に標識された内皮細胞は、非磁 ​​性ステント（左）よりも有意に高い率で強磁性血管ステント（右）に引き寄せられます。スケールバー=100μmです。再印刷24からの許可を得て。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53099/53099fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles at JoVE.com

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

細胞標識および超常磁性酸化鉄ナノ粒子とターゲティング

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

cell-labeling-targeting-with-superparamagnetic-iron-oxide

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

12.0K ビュー.  Mayo Clinic. Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

ビデオ: Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

診断と治療モニタリングのためのナノ材料の登録バイオイメージング

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

生物工学

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Ferumoxytol、FDA承認酸化鉄ナノ粒子で幹細胞のラベリング

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

医学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

生物学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

神経科学

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

細胞の超高速磁化のためのカチオン化Magnetoferritinの合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

<meta charset="utf8"></head><body>マウス神経膠芽腫における画像誘導治療送達のための酸化鉄ナノ粒子

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>著者スポットライト:膠芽腫治療のための酸化鉄ナノ粒子による革新的ながん治療

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

神経膠芽腫多形動物モデルにおけるオリゴヌクレオチドペイロードのナノ粒子送達

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

がん研究

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

磁気測定を用いて細胞の組み込みとそれに続く化学的合成酸化鉄ナノ粒子の生分解をモニタリングする

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

磁性ナノ材料の生体機能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

細胞標識および超常磁性酸化鉄ナノ粒子とターゲティング

要約

さらに動画を探す

この動画の章

診断と治療モニタリングのためのナノ材料の登録バイオイメージング

Ferumoxytol、FDA承認酸化鉄ナノ粒子で幹細胞のラベリング

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

細胞の超高速磁化のためのカチオン化Magnetoferritinの合成

神経膠芽腫多形動物モデルにおけるオリゴヌクレオチドペイロードのナノ粒子送達

神経膠芽腫多形動物モデルにおけるオリゴヌクレオチドペイロードのナノ粒子送達

神経膠芽腫多形動物モデルにおけるオリゴヌクレオチドペイロードのナノ粒子送達

磁気測定を用いて細胞の組み込みとそれに続く化学的合成酸化鉄ナノ粒子の生分解をモニタリングする

磁性ナノ材料の生体機能化