Bu çalışma Sağlık / Ulusal Kanser Enstitüsü (R01CA129822 ve R01CA140995) Ulusal Enstitüleri LKM-K hibe tarafından finanse edildi. Dr Medine-Kauwe sürekli destek C. Rey, M. M-Kauwe ve D. Revetto teşekkür eder.

1. In vivo sistemik olarak verilmesine <ol><li> 6-8 haftalık nu / nu fare deri altı açısı taraflı yan ksenograft tümörler için enjeksiyon başına Herdox bir 0.004 mg / kg dozda, 0.2 ml eşit steril tuzlu su ile yeterli Herdox karıştırın. </li><li> Yavaşça kabarcıklar kaçınarak, 29G iğnesi ile donatılmış bir 3/10 cc şırınga içine insülin Herdox karışımı çizin. </li><li> 0.5-1 L / dk, izofluran konsantrasyonu:% 3-4 (veya daha düşük) Anestezi bir gazı tahliye sistemi (Oksijen akış oranları ile donatılmış bir indüksiyon odasında kısa izofluran maruz indüklenir. </li><li> Bir anestezi fare (enjeksiyon başına 0.2 mi) bir kuyruk damarı içine tüm karışımı enjekte edilir. IV enjeksiyonu da ölçülü, anestezisiz fare gerçekleştirilebilir. </li><li> Günde bir, altı ardışık gün boyunca aynı fare enjeksiyon tekrarlayın. </li></ol> 2. In vivo Floresans Görüntüleme Tümörlerde Herdox floresan birikimi canBir çok modlu kamera kullanarak enjeksiyon son günü (Günlük 7) ile saptanabilir edilebilir. Aşağıdaki işlemleri özel bir makro-aydınlatma ve algılama sistemi (Şekil 1) 8 kullanımını gerektirecektir. <ol><li> In vivo Optik Görüntüleyici olarak Çoklu açın. </li><li> Doksorubisin floresan tespiti için uygun bir emisyon bant geçiren filtre (590 nm ± 30 nm) seçin. </li><li> Argon-Kripton Lazer açın ve lazer optik yolda bir uyarım bant geçiren filtre (488 nm ± 10 nm) yerleştirin. </li><li> Anestezi sistemi açın ve daha sonra anestezi odası (Oksijen akış hızı bir fare yerleştirin: 0.5-1 L / dk, izofluran konsantrasyonu:% 3-4 (veya daha düşük) bir gazı tahliye sistemi ile donatılmıştır. </li><li> Anestezi odasından fare anestezi in vivo Optik Görüntüleyici olarak Çoklu mod ve görüntüleme odasına fare aktarın. </li><li> Fare burun üzerinde bir roket ucu konisi yerleştirin ve sürekli anestezi yönetmek için akış açıkgörüntü alımı sırasında sia (Oksijen akış hızı: 0.5-1 L / dk, izofluran konsantrasyonu:% 2-3 (veya daha düşük). </li><li> 5-15 sn bir pozlama süresi ile floresan görüntüler elde. </li><li> Arka plan düzeltme veya kontrast ayarı da dahil olmak üzere görüntü analizi ve işleme gerçekleştirin. </li></ol> 3. Floresan Görüntüleme ex vivo Herdox floresan tümörler ve belirli organlarda görüntülü olabilir (karaciğer de dahil olmak üzere, böbrek, dalak, kalp ve iskelet kası) Herdox son (Günlük 7) enjeksiyondan sonra 24 saat de ötenazi farelerden alınan hasat. <ol><li> In vivo Optik Görüntüleyici olarak Çoklu açın. </li><li> Doksorubisin floresan tespiti için bir emisyon bant geçiren filtre (580 nm ± 20 nm) seçin. </li><li> Argon-Kripton Lazer açın ve lazer optik yolda bir uyarım bant geçiren filtre (488 nm ± 10 nm) yerleştirin. </li><li> Yeri tümörler ve belirli organları görüntüleme odasına o bir Petri-tabağına düzenlenmişf in vivo Optik Görüntüleyici olarak Multimode. </li><li> 5-15 sn bir pozlama süresi ile dokuların floresan görüntüler elde. Başlangıç ​​floresans görüntü elde etme için bir örnek, Şekil 2a&#39;da gösterilmiştir. Arka Plan (Şekil 2b) olarak görev yapacak olan, Petri-çanak boş kullanarak aynı tekrarlayın. </li><li> Arka plan düzeltme veya kontrast ayarı da dahil olmak üzere görüntü analizi ve işleme gerçekleştirin. Düzeltilmiş bir görüntü örneği Şekil 2a, Şekil 2b çıkarılması sonucu, Şekil 2c&#39;de gösterilmektedir. </li></ol> 4. Tümörler yerinde Konfokal Görüntüleme olarak Yerinde konfokal görüntüleme hücresel düzeyde algılama ve Herdox tümör birikimi analiz sağlar. <ol><li> Leica SPE konfokal mikroskop açın. </li><li> Doksorubisin için doksorubisin ve emisyon dalga boyları (560-620 nm) uyarma için 488 nm lazer ışığı seçinfloresan algılama. </li><li> Objektif lens üzerinde 40X veya 63X objektif ve damla immersiyon seçin. </li><li> Prosedür 2&#39;de tarif edildiği gibi önceden Herdox ve sahte alınan tedaviler ötenazi farenin taze tümörleri Ayıklama. </li><li> Buz üzerinde bir Petri-tabak yerleştirin tümörler doku bozulmasını önlemek ve daha sonra konfokal görüntüleme için Delta T odasına tümörler aktarmak için. </li><li> Sıralı odak derinliği (: 1 mikron, kalınlığı: 20 mikron adım boyutu) de tümörlerin konfokal görüntüler elde. Z ekseni boyunca ardışık olarak-elde edilen görüntü bir örnek Şekil 3, sol panel gösterilmiştir. </li><li> Maksimum yoğunluk görüntüleri z projeksiyon gerçekleştirin. Z-yığılmış görüntülerin maksimum yoğunluk projeksiyonu Şekil 3, sağ panelde gösterilir. </li><li> . Maksimum yoğunluğun floresan yoğunlukları Z ortalama hesaplama - görünüm genel alan üzerinde görüntü floresan yoğunlukları ortalama projeksiyon görüntüleri hesaplamak edildid ImageJ kullanarak. </li></ol> 5. Oranlı metrik Spektral Görüntüleme ve Analiz Oranlı metrik spektral görüntüleme ve analiz Dox floresan ve otofloresans arasında ayrımcılık sağlar. <ol><li> Floresan konfokal mikroskop tarama bir lazer güç. </li><li> Bir adım 10 nm boyutu ve bir Leica SPE konfokal mikroskop kullanılarak 488 nm ışık uyarma ile, 510-650 nm spektral aralığında belirli bir derinlikte Herdox-tedavi edilen ve edilmeyen tümörler 15 görüntüler elde. </li><li> Doksorubisin bir 100 mcM çözüm hazırlayın. </li><li> Dox floresan (: 510-650 nm, bir adım boyutu: 10 nm spektral aralığı) saf spektral imza elde etmek için 100 mcM doksorubisin çözümün spektral görüntüleme gerçekleştirin. Bir grafik Şekil 4&#39;te gösterildiği gibi spektral görüntüleme ve floresan spektrumu çıkan görüntü elde etme tipik sonuçlar çizilmiştir. </li><li> Bir görüntü c otofloresans spektral imza Edinmeube (spektral aralığı: 510-650 nm, bir adım boyutu: 10 nm) tedavi edilmemiş tümörlerin spektral görüntüleme ile elde edilen. Gösteren bir grafiktir, Şekil 5&#39;te gösterildiği gibi spektral görüntüleme ve floresan spektrumu çıkan görüntü elde etme tipik sonuçlar çizilmiştir. </li><li> Biz 9 geliştirilen programı kullanarak dört referans spektral imzaları (saf otofloresansı, 0.1.doxorubin +0.9. Otofloresansı, 0.2. Doksorubisin +0.8. Otofloresansı, 0.3.doxorubin +0.7. Otofloresans) oluşturun. Dört referans spektral imzaları gösteren tipik bir eğrisi Şekil 6&#39;da gösterilmektedir. </li><li> Daha önce 9 geliştirilen program kullanılarak Öklid uzaklık ölçüsü üzerinden referans spektral imzaları tarafından tanımlanan görüntülerin spektral sınıflandırılması gerçekleştirin. </li><li> Rasyometrik spektral görüntüleme ve analiz için karşılaştırma için biz 7 geliştirilmiş bir spektral Karışmama programı (ImageJ plug-in), 10, kullanarak bu görüntülerin doğrusal spektral Karışmama gerçekleştirin. Bir elineer spektral Karışmama göre otofloresans ikinci Herdox floresan ayırma xample Şekil 7 &#39;de gösterilmiştir. </li></ol>

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Yazar, Daniel Farkas, Spektral Moleküler Görüntüleme Başkanıdır. Kalan Yazarlar herhangi bir çıkar çatışması var.

Dox floresan tümörler subkutan olduğunda çoklu görüntüleyicisi kullanılarak in vivo olarak tespit edilebilir. Bununla birlikte, Herdox terapötik olarak etkili doz (0.004 mg / kg) tek bir dozdan sonra aşağıdaki algılama eşiğidir. Buna karşılık, 7 günlük enjeksiyonu (7 gün boyunca 1x/day) sonrasında, parçacığın tümör birikimi ve tutma Dox flöresanının görsellenmesin sağlamak için yeterlidir. Dox veya temiz tekniği kullanılan in vivo görün...

Tümör hedefleme kemoterapi teslim tedavinin daha çok tümör olmayan doku dağıtmak daha da istenilen hedefe birikir, çünkü hedefsiz ilaçlara göre daha düşük dozda kanser hücrelerinin ortadan kaldırmak için bir potansiyele sahiptir. Ikinci durum, ilacın etkinliğinin sulandırmak ve böylece daha yüksek dozlarda etkili olmasını gerektirecektir olarak, tümör hedefleme standart olmayan hedefli tedavi üzerinde her iki tedavi ve güvenlik avantajı vardır. Kendi kendini monte nanopartiküller içine kapsülleme ile, kemoterapi hedefleme ilaç, kimyasal olarak kovalent hedefleme moleküllerine bağlı ilaçların aksine değiştirilmemiş kalmasını sağlar. Bu tür bir bağlantı, uyuşturucu ve hedefleme molekülünün her ikisi de aktivitesini değiştirmek için potansiyele sahiptir; kovalent olmayan bir montaj ilaç kudretini muhafaza edilmesini sağlar. Daha önce yeni üç bileşenli, kendini monte kompleksi, Herdox, HER2 + tümörlerin hedef göstermiştir <em&gt; In vivo ve kalp 1 de dahil olmak üzere, normal doku koruyucu iken tümör büyüme ablasyon ortaya çıkarır. Herdox reseptör bağlanma hücre penetrasyon proteini HerPBK10 ve kemoterapötik ajan arasındaki kovalent olmayan etkileşim yoluyla oluşan küçük bir nükleik asit bağlayıcı yoluyla doksorubisin (Dox). Endozomal membran penetrasyonu, adenovirüs-türetilmiş Penton baz kapsid proteini 4-6 arasında birleşme yoluyla gerçekleştirilir ise HerPBK10, insan epidermal büyüme faktörü reseptörü (HER) bağlar ve 2-4 reseptör aracılı endositoz tetikler. Protein üzerindeki pozitif yüklü bir etki nükleik asit DNA-interkalate Dox hedeflenmiş dağılım için nakledilebileceği üzerinden bağlama 4, 5, sağlar. Elektrofilik, ardalanması, ve muhtemelen protein oligomerizasyonu etkileşimleri farklı sıcaklıklarda 1 kan ve uzun saklama koşulları altında kararlı yuvarlak 10-20 nm parçacıklar halinde kendinden montaj kolaylaştırır. Tercihli HER hedefliyorHER2 yükseldiğinde 2 + tümör hücrelerinin gelişmiş afinite ligandı tarafından kolaylaştırılır. Daha önceki çalışmalar, tümör olmayan doku üzerinde tümörlerde ve karşılaştırma Herdox verim birikmesidir sistemik olarak verilmesine in vivo 7 tümör hücrelerine DOX, 1, ve penetrasyon hedefsiz için göstermiştir. Biz gözlemledim ki tümör hücre girmesinden sonra Herdox bültenleri Dox, çekirdeği 1 içine Dox birikimi sağlar. Tümör-birikimi nispeten düşük HER2-ifade tümörleri nispeten daha yüksek HER2 seviyeleri 1 olanlara göre daha az Herdox biriktikçe, reseptör seviyesi ile uyumlu görünüyor. Dahası, etkin bir şekilde hücre ölümü konsantrasyonu farklı hücre yüzeyi HER2 seviyeleri 1 ifade eden tümör hücre çizgileri üzerinde HER2 ekran ile ters bir ilişki sergiler. Tümör öldürme 10-kat daha düşük dozda üzerinde gerçekleşir olarak Herdox Tar göre, hedefsiz Dox üzerinde tedavi ve güvenlik avantajı sergilergeted uyuşturucu ve hedefsiz Dox 1 aksine, kalp (ekokardiyografi ve histolojik leke ile tespit edilir) ya da karaciğer (leke TÜNEL ile tespit edilir) dokusu üzerinde tespit edilebilir bir yan etki üretmektedir. Bir viral kapsid protein kendi türetme rağmen, HerPBK10 terapötik seviyelerde 2 saptanabilir immünojenitesi sergiler. Bütün adenovirüs için önceden var olan antikorlar HerPBK10 tanıyabilir Oysa, onlar 2 bağlayıcı hücre önlemek mümkün değildir. Zaman içinde ölçülen tümör hacminin hedeflenen tedavi terapötik etkinliğinin değerlendirilmesinde standart bir yöntemdir ve Herdox terapötik etkinliğini değerlendirmek için istihdam edilmiştir. In vivo ve ex vivo floresan görüntüleme ile bu yaklaşım ilave daha iyi verim 7 hedef değerlendirmek için izin verdi. Biz özellikle bu Herdox hiçbir doğrulamak için Dox floresan spektral analizi ile eksize tümörlerin yerinde konfokal görüntüleme entegre vart sadece in vivo tümörlerin biriken ama sitoplazma ve çekirdek 7 içine tümör hücreleri ve teslim Dox nüfuz. Spektral analiz ayrıca bize otofloresansı 7 Dox floresan ayırt sağladı. Burada sistemik doğumdan sonra in vivo Herdox değerlendirilmesi için daha ayrıntılı olarak yaklaşımımızı göstermek, ve en önemlisi, değerlendirmek için çok modlu görüntüleme yöntemleri ve analizleri ile hedef.

<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 + tümör hedefli bir nanoparçacık, Herdox, HER2 + kanseri olan bir hayvan modelinde tümör tercihli birikimi ve tümör büyümesini ablasyon sergiler. Herdox küçük bir nükleik asit bağlayıcı yoluyla, kemoterapi ajanı, ve doksorubisin, bir tümör hücresi hedefli penetrasyon proteininin kovalent olmayan bir montajı ile oluşur. Elektrofilik, ardalanması ve oligomerizasyonu etkileşimleri bir arada turu 10-20 nm parçacıklar halinde kendinden montaj kolaylaştırır. Herdox farklı sıcaklıklarda kan hem de uzun süreli depolama stabilitesi gösterir. Kalp ve karaciğer (hedefsiz doksorubisin ile işaretlenmiş zarar görür olan) da dahil olmak üzere tümör olmayan doku tespit edilebilir bir yan etkileri, tümör-hücre ölümü, tümör taşıyan farelere sonuçları Herdox sistemik olarak verilmesine. HER2 yükseklik ekspres hücreleri ile karşılaştırıldığında bu nedenle yüksek seviyelerde HER2 gösteren tümörlerin Herdox daha fazla birikimi gösteren, insan epidermal büyüme faktörü reseptörü ifade eden hücrelere hedefleme kolaylaştırırin vitro ve in vivo olarak daha düşük seviyelerde, şarkı. Yerinde konfokal ve spektral analiz ile birlikte floresan görüntüleme bizi hedef in vivo tümör ve sistemik doğumdan sonra Herdox tümör hücre nüfuz doğrulamak için izin verdi. Sistemik doğumdan sonra çok modlu görüntüleme ile hedef tümör değerlendirmek için burada detaylı bizim yöntemleri.

Şekil 1 floresan, spektral, ömür boyu, 2-foton, içi hayati konfokal ve biyoparlaklık görüntüleme dahil olmak üzere birden fazla yöntemleri altında görüntü elde etme amacıyla, için inşa edilmiştir in vivo optik görüntüleyici prototip, gösterir. Buna ek olarak, bu sistem dahil soğutmalı yüksek duyarlı kamera ve yüksek güçlü lazer çizgileri özellikle doksorubisin floresan in vivo tespiti için ticari optik görüntüleme sistemleri 11 il...

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

Bu makalede, tümör hedefli nanoparçacık, Herdox optik görüntüleme analizi için prosedürler. Özellikle, hedef ve tümör penetrasyon değerlendirilmesi tümör tespit için çoklu görüntüleme cihazı ayrıntılı kullanımı burada açıklanmıştır.

Hedefli Viral Protein analizi HER2 + tümörler için teslim Nanopartiküller

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 Görüntüleme.  University of Southern California. Bu makalede, tümör hedefli nanoparçacık, Herdox optik görüntüleme analizi için prosedürler. Özellikle, hedef ve tümör penetrasyon değerlendirilmesi tümör tespit için çoklu görüntüleme cihazı ayrıntılı kullanımı burada açıklanmıştır.

Video: 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.

Ultrason Hedeflenen Mikro İmha Tekniği Giriş

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

Biyomühendislik

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.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

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).

Hücresel Lipid Ekstraksiyon Hedefli Kararlı İzotop Seyreltme Sıvı Kromatografi-Kütle Spektrometresi Analizi

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.

İnsan Pankreas Kanseri Hücrelerinde Epidermal Büyüme Faktörü kullanarak Gen Teslimat ve Transfeksiyon Jelatin Tabanlı Engineered Nanovectors Reseptör-hedefli

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.

Viral Nanopartiküller<em&gt; In vivo</em&gt; Tümör Görüntüleme

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.

Polimerik Gen Taşıyıcı Değerlendirilmesi Nanoparçacık İzleme Analizi ve Yüksek throughput Flow Sitometri tarafından Nanopartiküller

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.

Rejeneratif Araştırma harmonik nanopartiküller

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.

Sıvılardaki Darbeli Lazer-Ablasyonuyla Metal Nanopartikül Üretimi: Nanopartiküllerin Antibakteriyel Özelliklerini İncelemek İçin Bir Alet

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.

Cryo-immobilize hücreler yapılarda bağdaşık ışık elektron izleme ve Viral Protein Imaging mikroskobu (CLEM) ilişkili

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.

Protein kinaz C-delta inhibitör peptid formülasyonu altın nano tanecikleri kullanarak

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 ...