この作品は、健康/国立がん研究所の国立研究所（R01CA129822とR01CA140995）からLKM-Kへの補助金によって賄われていた。継続的なサポートのためにメディナ-Kauweのおかげでレイ、M. M-KauweとD. Revetto博士。

1。 生体内で全身送達<ol><li> 6-8週齢NU / NUマウスベアリング皮下二国間のフランク異種移植腫瘍に対する注射当たりHerDoxの0.004 mg / kgで投与量の0.2ミリリットルを同一視するように滅菌生理食塩水で十分にHerDox混ぜる。 </li><li>優しく泡を避けて、29G針を装着し3/10 ccのインスリン注射器にHerDox混合物を描画します。 </li><li> 0.5〜1リットル/分、イソフルラン濃度：3〜4％（又は低い）麻酔ガスを掃気システム（酸素流量を備えた誘導室にイソフルラン短時間の曝露によって誘導される。 </li><li>麻酔マウス（注射当たり0.2 ml）を尾静脈内に混合物全体を注入。 IV注入はまた、拘束、無麻酔マウスで行うことができる。 </li><li> 1日1回、6以上の連続日間同じマウスに注射を繰り返します。 </li></ol> 2。 インビボ蛍光イメージング腫瘍におけるHerDox蛍光の蓄積ができマルチイメージャを用いた注射の最終日（7日）によって検出される。以下の手順は、カスタマイズされたマクロ照明および検出システム（ 図1）8の使用を伴う。 <ol><li> 生体内光学イメージャでマルチモードをオンにします。 </li><li>ドキソルビシン蛍光検出に適した排出バンドパスフィルタ（590nmの±30 nm）を選択します。 </li><li>アルゴンクリプトンレーザーをオンにして、レーザー光路の励起バンドパスフィルタ（488nmの±10 nm）を配置します。 </li><li>麻酔システムの電源を入れた後、麻酔（酸素流量にマウスを置き：0.5 L /分、イソフルラン濃度：3〜4％（以下）ガス掃気システムを装備。 </li><li>麻酔からマウスが麻酔である生体内光学イメージャでマルチモードの撮影室にマウスを移す。 </li><li>マウスの鼻の上のノーズコーンを置き、連続anestheを管理する流れを開く画像取得時にSIA（酸素流量：0.5〜L /分、イソフルラン濃度：2〜3％（以下）。 </li><li> 5-15秒の露光時間を用いて蛍光画像を取得。 </li><li>バックグラウンド補正、コントラスト調整など​​の画像解析処理を行う。 </li></ol> 3。蛍光イメージングエクスビボ HerDox蛍光はHerDoxの最終（第7日）注射後24時間後に安楽死させたマウスから採取した腫瘍と特定の臓器（肝臓、腎臓、脾臓、心臓、骨格筋を含む）で撮像することができます。 <ol><li> 生体内光学イメージャでマルチモードをオンにします。 </li><li>ドキソルビシン蛍光検出用の放射帯域通過フィルタ（580nmの±20 nm）を選択します。 </li><li>アルゴンクリプトンレーザーをオンにして、レーザー光路の励起バンドパスフィルタ（488nmの±10 nm）を配置します。 </li><li>場所の腫瘍および特定の臓器は、イメージングチャンバーOにペト​​リ皿上に配置されfの生体光学イメージャでマルチ。 </li><li> 5-15秒の露光時間を使用して組織の蛍光画像を取得。初期蛍光画像取得の一例を図2aに示されている。背景（図2b）となる空のペトリ皿を使用して同じように繰り返します。 </li><li>バックグラウンド補正、コントラスト調整など​​の画像解析処理を行う。補正された画像の例を図2aから図2bの減算に起因する、 図2cに示されている。 </li></ol> （4）腫瘍の in situ共焦点イメージング 現場共焦点イメージングで細胞レベルでHerDox腫瘍蓄積の検出と分析を可能にします。 <ol><li>ライカSPE共焦点顕微鏡をオンにします。 </li><li>ドキソルビシンためのドキソルビシン及び発光波長（560から620 nm）での励起のために488nmのレーザ光を選択蛍光検出。 </li><li>対物レンズ上の40Xまたは63X客観アンドドロップイマージョンオイルを選択します。 </li><li>手順2で説明したように以前にHerDoxとモックの治療を受けた安楽死させたマウスからの新鮮な腫瘍を抽出します。 </li><li>氷の上でペトリ皿の上に置いて腫瘍は組織の劣化を避けるために、次に共焦点イメージングのためのデルタT室に腫瘍を転送します。 </li><li>シーケンシャル焦点深度（1ミクロン、厚さ：20μmのステップサイズ）で腫瘍の共焦点画像を取得する。 z軸に沿って順次取得された画像の例を図3、左側のパネルに示されている。 </li><li>最大強度画像のz投影を実行します。 z軸積層画像の最大強度の投影は、図3、右のパネルに示されている。 </li><li> 最大強度Zの蛍光強度を意味計算- 。投影画像をビューの全体のフィールドの上に画像の蛍光強度の平均値を計算したImageJのを使用します。d。 </li></ol> 5。レシオメトリックスペクトルイメージングと解析レシオメトリックスペクトルイメージングと分析Doxを蛍光と自家蛍光の判別を可能にします。 <ol><li>蛍光共焦点レーザ走査型顕微鏡の電源をオンにします。 </li><li>ステップ10nmのサイズ、およびライカSPE共焦点顕微鏡を用いて488 nmの光で励起で、510から650ナノメートルのスペクトル範囲内で指定された深さでHerDox処理と未処理の腫瘍の15の画像を取得する。 </li><li>ドキソルビシン100μMのソリューションを準備します。 </li><li> Doxを蛍光（：510から650 nmであり、ステップサイズ：10 nmのスペクトル範囲）の純粋なスペクトルの署名を取得するために100μMのドキソルビシンソリューションの分光イメージングを実行します。グラフは、図4に示すように、得られた分光イメージング蛍光スペクトルからの画像取得の典型的な結果をプロットした。 </li><li>画像Cからの自家蛍光スペクトルの署名を取得未処理の腫瘍の分光イメージングによって得られたUBE（10 nmのスペクトル範囲：510から650 nmであり、ステップサイズ）。グラフは、図5に示すように、得られた分光イメージング蛍光スペクトルからの画像取得の典型的な結果をプロットした。 </li><li>我々は9を開発したプログラムを使用して、4つの基準スペクトルシグネチャを（純粋自家蛍光、0.1.doxorubin +0.9。自家、0.2。ドキソルビシン+0.8。自家、0.3.doxorubin +0.7。自家蛍光）を生成します。 4つの基準スペクトル特性を示す典型的な曲線を図6に示す。 </li><li>我々は以前に9を開発したプログラムを使用したユークリッド距離尺度を通して基準スペクトルシグネチャで定義されている画像のスペクトル分類を実行します。 </li><li>レシオメトリックスペクトルイメージングと解析との比較のために、我々は7を開発したスペクトルアンミキシングプログラム（ImageJの中でプラグイン）、10を使用して、それらの画像の線形スペクトルアンミキシングを行います。電子線形スペクトルアンミキシングすることにより自家蛍光からHerDox蛍光を分離するXAMPLEは、 図7に示されている。 </li></ol>

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著者、ダニエルFarkasのは、スペクトル分子イメージングの会長である。残りの著者は全く競合する利害関係はありません。

腫瘍は皮下あるときDoxの蛍光はマルチイメージャを用いたin vivoで検出することができます。しかし、HerDoxの治療有効用量（0.004 mg / kg体重）を単回投与した後、検出しきい値を下回っている。対照的に、7毎日注射（7日間1x/day）の後、粒子の蓄積および腫瘍保持はDoxの蛍光の可視化を可能にするのに十分である。 ドキシサイクリンまたはクリーン技術が使用されて?...

腫瘍ターゲティング化学療法の、配信療法のよりは、その目的の宛先に蓄積ではなく、非腫瘍組織に配布することができるので、関連性の低い薬に比べ低用量でがん細胞を排除する可能性を秘めている。後者の状況は、薬物の有効性を希釈し、より高い用量が効果的であることが必要となるので、腫瘍標的は、標準的な非標的治療上の両方の治療と安全性の利点があります。 自己組織化ナノ粒子にカプセル化することにより化学療法を標的とする薬剤が化学的に共有結合する分子を標的にリンクされている薬とは対照的に変更されていないままにすることができます。などの結合は、薬物および標的分子の両方の活性を変化させる可能性を有し、非共有結合アセンブリは、薬物の効力を保持することができる。 我々は以前に新規な三成分、自己組織化錯体、HerDoxは、HER2 +腫瘍を標的とすることが示されている<em&gt; in vivoおよび心臓1を含む、正常組織を温存しながら腫瘍成長アブレーションを引き出す。 HerDoxは、小さな核酸リンカーを介して結合受容体細胞侵入タンパク質、HerPBK10、及び化学療法剤、ドキソルビシン（ドキシサイクリン）の間の非共有結合性相互作用を介して形成されている。 HerPBK10は、ヒト上皮成長因子受容体（HER）と結合し、エンドソーム膜の浸透は、アデノウイルス由来のペントンベースキャプシドタンパク質4-6の取り込みを介して行われている間、受容体を介したエンドサイトーシス2-4をトリガします。タンパク質上の正に帯電したドメインは、DNAインターカレードキシサイクリンを標的送達のために輸送することができるような核酸結合4,5を可能にする。求電子性、インターカレーション、及びおそらくはオリゴマー化タンパク質の相互作用は、血液や異なる温度1に長期貯蔵で安定である平均10〜20 nmの粒子に自己組織化を促進する。彼女にターゲティング優遇HER2が上昇したとき2 +腫瘍細胞を増強リガンド親和性によって促進される。 我々の以前の研究では、非腫瘍組織上および非標的Doxを1、およびin vivo 7 中の腫瘍細胞への浸透と比較して、腫瘍におけるHerDox利回り優遇蓄積のその全身送達を示している。我々は核​​1にDoxの蓄積を可能にし、腫瘍細胞侵入後HerDoxリリースDoxのことを観察した。腫瘍蓄積は比較的低HER2発現腫瘍は比較的高いHER2レベル1のものに比べてより少ないHerDox蓄積として、受容体レベルと相関することが表示されます。また、効果的な細胞死濃度が異なる細胞表面のHER2のレベル1を発現する腫瘍細胞株に対するHER2ディスプレイと逆相関を示す。腫瘍殺害解凍に比べて10倍も低い用量で発生するとHerDoxは、非標的ドックス以上の治療と安全優位性を発揮geted薬とは関連性の低いDoxの1とは対照的に、心臓（心エコー検査および組織学的染色によって検出された）や肝臓（染色TUNELによって検出された）組織には検出可能な悪影響をもたらしません。ウイルスキャプシドタンパク質からの派生にもかかわらず、HerPBK10は治療レベル2での検出可能な免疫原性を示さない。全体アデノウイルスに既存の抗体がHerPBK10を認識することができる一方で、彼らは、2細胞結合を防止することができない。 経時的に測定した腫瘍体積は、標的治療の治療効果を評価するための標準的な方法であり、HerDoxの治療効果を評価するために採用されている。 生体とex vivoでの蛍光強度イメージングでこのアプローチを補完する、私たちはより良い効率7をターゲットに評価することができました。我々は、特にそのHerDoxないことを確認しないようにDoxを蛍光のスペクトル分析で切り出した腫瘍の in situ共焦点イメージングに統合されているtは唯一のin vivoで腫瘍に蓄積されますが、細胞質と核の7への腫瘍細胞と配信ドキシサイクリンに浸透。スペクトル解析はさらに自家7からDoxの蛍光を区別することができました。 ここでは、全身送達後にin vivoで HerDoxを評価するための、より詳細に我々のアプローチを実証し、そして最も重要なのは、評価するためのマルチモードイメージング法と分析を通してターゲティング。

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

HER2 +腫瘍標的ナノ粒子、HerDoxは、HER2 +癌の動物モデルにおいて腫瘍優先的な蓄積および腫瘍増殖アブレーションを示す。 HerDoxは、小さな核酸リンカーを介して、化学療法剤、ドキソルビシン、腫瘍標的細胞透過タンパク質の非共有結合性の自己集合によって形成される。求電子性、インターカレーション、及びオリゴマーの相互作用の組み合わせは、ラウンド10〜20 nmの粒子に自己組織化を促進する。 HerDoxは異なる温度で血液中と同様、拡張ストレージに安定性を示す。心臓と肝臓（非標的ドキソルビシンによってマークされた被害を受けている）を含む非腫瘍組織には検出副作用、腫瘍細胞死における担癌マウス結果のHerDoxの全身送達。 HER2上昇が上昇したHER2のレベルを表示する腫瘍細胞の発現と比較して、よりHerDox蓄積を示すため、ヒト上皮成長因子受容体を発現する細胞を標的容易インビトロおよびインビボの両方で 、より低いレベルを歌う。 現場共焦点とスペクトル解析にと組み合わせる蛍光強度イメージングは、私たちが標的生体内腫瘍と全身送達後HerDoxの腫瘍細胞の浸透に確認することができました。全身送達後のマルチモードイメージングを介して標的の腫瘍を評価するためここでは詳細私たちの方法を。

図1は、蛍光強度、スペクトル、寿命、2光子内、重要な焦点、および生物発光イメージングを含む複数のモダリティ、下の画像の取得を目的として建設された生体内光学イメージャプロトタイプに示しています。さらに、このシステムに組み込まれた冷却された高感度カメラと高出力レーザー線は、特にドキソルビシン蛍光のインビボでの検出のための商業?...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

この記事の詳細腫瘍標的ナノ粒子、HerDoxの光学イメージング解析のための手続き。特に、ターゲティングおよび腫瘍浸透性を評価する腫瘍を検出するためのマルチモードの撮像装置の詳細な使用がここで記載されている。

標的ウイルスタンパク質の分析はHER2 +腫瘍に配信ナノ粒子

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

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

Research

JoVE Journal

Bioengineering

10.9K ビュー.  University of Southern California. この記事の詳細腫瘍標的ナノ粒子、HerDoxの光学イメージング解析のための手続き。特に、ターゲティングおよび腫瘍浸透性を評価する腫瘍を検出するためのマルチモードの撮像装置の詳細な使用がここで記載されている。

ビデオ: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

超音波標的マイクロバブル破壊テクニック入門

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

生物工学

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

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

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

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

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

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

上皮成長因子受容体をターゲットとしたゼラチンベースのエンジニアNanovectorsを使用してヒト膵臓癌細胞における遺伝子デリバリーとトランスフェクション

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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 ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

ナノ粒子トラッキング分析とハイスループットフローサイトメトリーによる高分子遺伝子デリバリーナノ粒子の評価

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

回生研究ハーモニックナノ粒子

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中のパルスレーザアブレーションによる金属ナノ粒子の生成：ナノ粒子の抗菌特性を調べるためのツール

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

相関光電子顕微鏡 (クレム) 追跡のウイルス蛋白質のイメージング関連するクライオ固定化細胞の構造

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

金ナノ粒子を用いた蛋白質キナーゼ C デルタ阻害ペプチド製剤

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...