Bu çalışma NIH / NIBIB hibe R00 EB009105 (NFS) ve P30 EB011317 (NFS), bir NIH / NIBIB eğitim bursu T32 EB007509 (AMW için), bir Case Western Reserve Üniversitesi Disiplinlerarası Birlik Yatırım Hibe (NFS), tarafından desteklenen ve Bir Olgu Kapsamlı Kanser Merkezi&#39;nde hibe P30 CA043703 (NFS). Biz onların için Steinmetz Lab lisans öğrencisi araştırmacıların teşekkür hands-on desteği: Nadia Ayat, Kevin Chen, Sourav (Sid) Dey, Alice Yang, Sam Alexander, Craig D&#39;Cruz, Stephen Hern, Lauren Randolph, Brian Yani, ve Paul Chariou .

1. VNP (CPMV, BMV, PVX ve TMV) Yayılım <ol><li> Kapalı bitki odasının günün 15 saat (% 100 ışık, 25 ° C,% 65 nem) ve gece 9 saat (% 0 ışık, 22 ° C,% 60 nem) kontrollerini ayarlayın. </li><li> Tablo 1&#39;de çizelgesine göre bitki inoküle edin. </li></ol><table border="1"><tbody><tr><td> CPMV </td><td> PVX, TMV ve BMV </td></tr><tr><td rowspan="2"> Gün 0: Bitki 3 börülce tohum / saksı. </td><td> Gün 0: Bitki ~ 30 N. benthamiana tohum / saksı. 1 çorba kaşığı gübre / 5 L su ile haftada bir kez gübreleyin. </td></tr><tr><td> Gün 14: 1 Bitki / pot Re-pot N. benthamiana. </td></tr><tr><td> Gün 10: Infect korindon tozunu bir ışık kullanarak mekanik inokulasyon tarafından CPMV (5 μg/50 ul / yaprak) ile primer yapraklar bırakır. </td><td> Gün 28: Infect üç five korindon tozunu bir ışık kullanarak mekanik inokulasyon yoluyla PVX, TMV veya BMV (5 μg/50 ul / yaprak) ile bırakır. </td></tr><tr><td> Gün 20: Hasat yaprak ve -80 mağaza ° C. </td><td> Gün 42: Hasat yaprak ve -80 mağaza ° C. </td></tr></tbody></table> , Büyüyen bulaşmasını ve yaprakları hasat için Tablo 1. Timeline. Not: Sadece CPMV yayma, örnek olarak gösterilmiştir. 2. VNP (CPMV, BMV, PVX, ve TMV) saflaştırılması Not: Tüm adımlar buz üzerinde veya 4 yürütülmektedir ° C <ol><li> Soğuk tampon 2 cilt (bkz. Tablo 2) kullanılarak standart bir blender dondurulmuş yaprakları 100 g homojenize. Tülbent 2-3 katmanları süzülür. </li><li> PVX için, 1 M HCl ile pH 6.5 &#39;e ayarlayın. % 0.2 (w / v) askorbik asit ve% 0.2 (w / v) sodyum sulfi eklemete. </li><li> 20 dakika boyunca 5.500 xg&#39;de ham bitki Homojenat santrifüjleyin. Süpernatant toplayın. </li><li> BMV için, tabaka% 10 (w / v) sakaroz çözeltisi, 5 ml süpernatant üzerine, 25 ml. % 38.5 CsCl çözeltisi (w / v) içinde 3 saat ve pelet tekrar süspansiyon 9,000 x g&#39;de santrifüjleyin. 5 saat sallayarak karıştırın, ardından adım 2.12 ile devam edin. </li><li> 1:1 (v / v) kloroform :1-bütanol 0.7 hacim ekleme yoluyla bitki materyali ayıklamak. 30-60 dakika boyunca karışımı karıştırın. </li><li> 20 dakika boyunca 5,500 x g çözelti santrifüjleyin. Üst sulu faz toplanır. </li><li> M 0.2 ve% 8 (ağırlık / hacim) PEG (MW 8000) ile NaCl ekleyin. TMV için, aynı zamanda (v / v) Triton X-100,% 1 ekleyin. En az 1 saat süre ile karıştırınız, sonra en az 1 saat boyunca bekletilir. </li><li> 15 dakika boyunca 15.000 xg&#39;de çözümü santrifüjleyin. 10 ml tampon içinde yeniden süspanse pelet. PVX için, 0.5 M için% 0.1 β-merkaptoetanol ve üre ekleyin </li><li> 30 dakika boyunca 8.000 xg&#39;de santrifüjleyin ve süpernatant toplamak. </li><li> 3 saat boyunca 160,000 x g süpernatant Ultrasantrifüj. 5 ml tampon o da süspanse peletvernight. </li><li> Eşit hacimde% 10,% 20,% 30, ve tampon içinde% 40 sakaroz (ağır ilk) ile bir% 10-40 oranında sakaroz eğim hazırlayın. Gradyan oda sıcaklığında gece boyunca dengelenmeye bırakın. </li><li> Ultrasantrifüj 2 saat (BMV için 24 saat) için 100.000 xg&#39;de sukroz gradient üzerinde pelet yeniden süspanse. </li><li> Işık saçılması bandı toplayın ve tampon karşı dialyze. </li><li> 4&#39;te VNPs (aşağıda) ve mağaza Karakterize ° C. Uzun süreli depolama için, -80 ° C&#39;de saklayın </li></ol><table border="1"><tbody><tr><td> CPMV ve TMV </td><td> 0.1 M potasyum fosfat tamponu (pH 7.0) 38.5 mM KH 2 PO 4 61.5 mM K 2 HPO 4 </td></tr><tr><td> PVX </td><td> 0.5 M borat tamponu (pH 7.8) 0.5 M borik asit NaOH ile pH ayarlama </td></tr><tr><td> BMV </td><td> SAMA tamponu (pH 4.5) 250 mM sodyum asetat 10 mM MgCl2 2 mM β-merkaptoetanol (taze ekleyin) </td></tr></tbody></table> Tablo 2. Her VNP için Tamponlar ve kendi tarifleri. Not: Sadece CPMV yayma, örnek olarak gösterilmiştir. 3. VNP (CPMV, BMV, PVX ve TMV) Karakterizasyonu <ol><li> VNPs konsantrasyonunu belirlemek için UV / Görünür Bölge spektroskopisi gerçekleştirin. <ol><li> Bir Nanodrop spektrofotometre kullanılarak numunenin 2 ul absorbansı ölçülür. </li><li> Beer-Lambert yasası kullanılarak partikül ve boyaların konsantrasyonu belirleyin (A absorbans A = εcl, ε sönüm katsayısı, c konsantrasyon ve l yol uzunluğu). Yol uzunluğu Nanodrop için 0,1 cm dir. VNP özgü nesli katsayıları şunlardır: CPMV: 8.1 cm -1 mg -1 ml (260 nm) PVX: 2.97 cm -1 mg -1 ml (260 nm) TMV: 3,0 cm -1 mg -1 ml (260 nm) BMV: 5.15 cm -1 mg -1 ml (260 nm) </li></ol></li><li> Boyut dışlama hızlı protein sıvı kromatografisi (FPLC) parçacıkları analiz edin. <ol><li> Bir Superpoze 6 boyut dışlama kolon ve AKTA Explorer, 0.1 M potasyum fosfat tamponu (pH 7.0) ve 200 ul içinde VNPs yük 50-100 ug kullanılması. </li><li> 260 nm (nükleik asit), 280 nm (protein) ve bağlı herhangi boyaların uyarma dalga boyu dedektörü olarak ayarlayın. </li><li> 72 dakika boyunca 0.5 ml / dak &#39;lık bir akış hızında çalışırlar. </li><li> Elüsyon profili ve A260: A280 nm VNP hazırlık saf olup olmadığını ve parçacıklar sağlam ve monte olup olmadığını gösterir. Aşağıdaki A260: 280 oranlarında saf VNP hazırlık göstermektedir: CPMV: 1,8 ± 0,1 PVX: 1.2 ± 0.1 TMV:1.1 ± 0.1 BMV: 1.7 ± 0.1 </li></ol></li><li> Bireysel kat proteinlere hazırlanması ve konjugasyon saflığını analiz (prekast NuPAGE) Bis-Tris poliakrilamid 4-12% gradyan jel elektroforezi denatüre gerçekleştirin. <ol><li> Potasyum fosfat tampon 9 ul içinde 10 ug parçacıkların için 4x LDS numune tamponu 3 ml ilave edilir. Disülfid bağlarının yüksek sayısını azaltmak için BMV 4x LDS örnek tampon ve β-merkaptoetanol 3 ul ilave 1 ul ekleyin. </li><li> 100, 5 dakika süreyle ısıtma bloğu içinde inkübe ° C. </li><li> SDS jel üzerine örnekler yükleyin. </li><li> Tampon çalıştıran 1x MOPS 1 saat için 200 V örnekleri çalıştırın. </li><li> Floresan ceket proteinleri görselleştirmek için UV ışık altında jel belgeleyin. </li><li> Floresan olmayan protein için, 1 saat için Coomassie mavisi (% 0.25 (w / v) Coomassie Brillant Blue R-250,% 30 (v / v) metanol,% 10 (v / v) asetik asit) ile leke. </li><li> Gece boyunca% 30 metanol,% 10 asetik asit ile Destain. Change çözüm isteniyorsa. </li><li> Beyaz ışık altında jel belgeleyin. </li></ol></li><li> Transmisyon elektron mikroskobu (TEM) tarafından parçacıkların bütünlüğünü analiz. <ol><li> 0.1-1 mg / DI su 20 ul ml numune sulandırınız. </li><li> Parafilm ile ilgili örnekler 20 ul damla yerleştirin. </li><li> TEM ızgara ile damla örtün ve 2 dakika bekletin. Filtre kağıdı ile ızgara üzerinde aşırı Solüsyon Wick. </li><li> Kuru esneklik ardından DI bir damla su üzerine koyarak ızgara yıkayın. </li><li> % 2&#39;lik bir 20 ul damla (w / v) 2 dk için uranil asetat ile ilgili yerleştirilmesi ile ızgara Leke. Filtre kağıdı ile leke aşırı kapalı Wick. </li><li> Suda bir kez daha ızgara yıkayın. </li><li> Bir transmisyon elektron mikroskobu altında ızgara gözlemleyin. </li></ol></li></ol> 4. PEG ve fluorophores, Saflaştırılması, ve karakterizasyonu ile VNPs Kimyasal Konjugasyon <ol><li> Aşağıdaki reaksiyonlar için hesaplamalar için, VNPs molar kütlesi vardır: CPMV: 5.6x 10 6 g / mol PVX: 35 x 10 6 g / mol TMV: 41 x 10 6 g / mol BMV: 4.6 x 10 6 g / mol </li><li> Tek adımlı bir N-hidroksi süksinimit bağlama reaksiyonu kullanılarak CPMV ve PVX yüzey lysines ile boyalar ve PEG konjugatı: Alexa Fluor 647 süksinimidil ester ve 4.500, 2.500 eşdeğer molar eşdeğerleri (tüm molar aşırı VNP göre molar fazlalıkta bakınız) ekleyin NHS PEG (MW 5,000) 0.1 M potasyum fosfat tamponu içinde CPMV için DMSO içinde çözülür. PVX çalışırken, NHS-boya ve NHS-PEG 10.000 molar aşırı ekleyin. CPMV ve PVX nihai konsantrasyon 2 mg / ml DMSO ve içeriği toplam reaksiyon hacmi% 10 olacağı şekilde DMSO ve tampon hacim ayarlayın. Işıktan koruyarak oda sıcaklığında, reaksiyon karışımı gece boyunca inkübe edin. CPMV ve PVX, sırasıyla, 300 ve 1,270 adreslenebilir lysines sahiptir. (Okuyucu üzerine daha ileri okumalar için aşağıdaki referanslar denirCPMV ve PVX kimyasal modifikasyonu: 13-15). </li><li> Diazonyum kaplin ile TMV tirozinlerin konjugat boyalar ve PEG bakır (I) katalizli azit-alkin siklo izledi. <ol><li> 0.3 M p-tolüensülfonik asit monohidrat, 3.0 M sodyum nitritin 25 ul, ve 0.68 M damıtık, 1 saat boyunca 4 ° C &#39;de, asetonitril içerisinde çözündürüldü 3-ethynylaniline için 75 ul 400 ul karıştırılarak diazonyum tuzu (alkin) hazırlayın. </li><li> TMV 1.25 ml (20 mg / ml stok çözeltisi) için NaCl 100 mM borat tamponu, pH 8.8, 3.3 ml ilave edilir. </li><li> Bir alkin ligasyon diazonyum bağlanması ile TMV işlemek için eklemek için 3 saat boyunca bir buz banyosu içinde diazonyum tuzu (alkin) çözeltisi 450 ul TMV React. Çözüm açık kahverengi bir renk dönüşecektir. TMV konjugasyon için 2.140 kullanılabilir tirozinlerin vardır. </li><li> Adım 4.4 &#39;de tarif edildiği gibi bir sükroz tampon kullanarak nihai ürün üzerine koyulmuştur. </li><li> Azid fonksiyonlu Alexa Fluor 647 ve PEG-azid (MW 5000) istimal takıng bakır (I) katalizli azit-alkin siklo (CuAAC). 1 mM CuSO 4, 2 mM AMG, ve 15 dakika süreyle oda sıcaklığında 2 mM sodyum askorbat ile kılıf proteini ve inkübe başına boya ve PEG-azit 2 eşdeğer ekleyin. TMV nihai konsantrasyon tepki 2 mg / ml &#39;dir ki bu tür tampon hacim ayarlama. (: 16,17 okuyucu TMV kimyasal modifikasyonu üzerine daha ileri okumalar için aşağıdaki referanslar denir). </li></ol></li><li> Lysines ve BMV sistein mutant (cBMV) ve sistein ile PEG boyalar Dualı: <ol><li> 0.1 M TNKM tampon maddesi (50 mM Tris baz, 50 mM NaCI, 10 mM KCI, 5 mM MgCl2, pH 7.4) içinde cBMV için DMSO içinde çözülür Oregon Green 488 süksinimidil ester, 2.000 molar eşdeğer ekleyin. BMV nihai konsantrasyon 1 mg / ml DMSO ve içeriği toplam reaksiyon hacmi% 10 olacağı şekilde DMSO ve tampon hacim ayarlayın. 4. gece boyunca reaksiyon karışımı inkübe ° C&#39;de ışıktan korunur. </li><li> Santrifüje kullanarak Purify parçacıklarolarak fugal filtreleri adım 4.4 &#39;de tanımlanmıştır. </li><li> Daha önce olduğu gibi aynı reaksiyon koşulları kullanılarak, PEG-maleimit (MW 2,000), 2.000 molar fazlası ilave edin ve 4 de 2 saat boyunca, reaksiyon karışımı inkübe ° C cBMV 180 reaktif lysines ve sistein sahiptir. (: 18 okuyucu BMV kimyasal modifikasyonu üzerine daha ileri okumalar için aşağıdaki referanslar denir). </li></ol></li><li> Saflaştırma: 2.5 saat boyunca 160,000 x g bir% 40 (w / v) sakaroz yastığı çözelti geçirir. Tampon pelet yeniden çözülür. Alternatif olarak, 10 kDa kesme spin-off filtreleri kullanarak uygun tampon karşı dialyze. </li><li> Karakterizasyon: Pegile ve floresan-etiketli VNPs yukarıda tarif edilen yöntemler kullanılarak analiz edilmiştir: görünür / UV spektroskopi, SDS jel elektroforezi, FPLC ve TEM (gösterilmemiştir, ancak, Şekil 6 ve 7&#39;ye bakın). </li></ol> 5. Bir Fare Xenograftlarında Modeli kullanılarak Tümör Hedefleme ve Görüntüleme<ol><li> Kültür HT-29 RPMI medyumu içinde insan kolon kanser hücreleri 175 cm2 hücre kültür şişeleri ile% 5 CO2 içinde% 5 FBS, 37% 1 penisilin-streptomisin,% 1 L-glutamin ° C ile desteklenmiştir. </li><li> 5 dk için 37 ° C &#39;de tripsin-EDTA 5 ml steril PBS ile inkübe ederek ve hasat ile iki kere yıkayın hücrelerini. RPMI ortam 5 ml tripsin inaktive eder. 5 dakika boyunca 500 xg&#39;de santrifüj hücreleri toplayın. 4 at 5x10 6 cells/50 ul orta (tripan mavi ve bir hemasitometre kullanarak toplam hücre sayısını belirlemek) taze RPMI ° C ve tekrar süspansiyon. Enjeksiyonu (tüm çözeltiler ve reaktifler steril tutmak) öncesinde Matrigel eşit hacmi ile karıştırın. </li><li> Procure altı hafta eski NCR nu / nu farelerine ve 2 hafta boyunca bir yonca ücretsiz diyet onları korumak. [Not:. Bütün hayvan prosedürleri IACUC onaylanmış olmalıdır] subkutan enjeksiyon yoluyla tümör xenografts øndükle 5x10 6 cells/100 steril bir 18 1/2 ölçü kullanarak yamaçlarında ul / tümör (2 tümörler / fare)iğne. Düzenli hayvanları izleyin. Pergel ile ölçülür ve tümör boyutu tümör 20 mm 3 (bir sonraki 12 gün içinde) bir ortalama hacim büyümesine izin verir. PBS ve VNP (n = 3 hayvan / grup / zaman noktası): rastgele iki farklı grup fareler atayın. Bir 1 ml 28 gauge insülin şırıngası kullanılarak, damar içi steril PBS kuyruk damarından enjeksiyon 100 ul ya da 10 mg / kg VNP formülasyon tarafından idare. </li></ol> Not: Canlı hayvanlar ile Doku kültürü deneyleri ve çalışmalar göstermiştir olmayacaktır. Hands-on gösteri doku işleme ve veri toplama ile sınırlı olacaktır. HT-29 tümör ksenogreft modelinde bir referans için, okuyucunun ref adlandırılır. 19 Üç teknikleri VNPs yuvalandırma tümörü değerlendirmek için kullanılır: <ol start="4"><li> CO 2 kullanarak farklı zaman noktalarında (2, 24 ve 72 saat) de Kurban fareler: Maestro Görüntüleme Sistemi kullanarak Floresans görüntülemegaz. Kanatlarda tümörleri ile birlikte hayvan ve tüketim tüm önemli organları (beyin, kalp, akciğer, dalak, böbrek ve karaciğer) teşrih, parafilm üzerindeki dokular yerleştirin ve (800 ms sarı eksitasyon ve emisyon filtreleri kullanarak floresan görüntüleme cihazı ile analiz dokularda floresan sinyalleri (VNPs konjuge A647 etiket türetilen) varlığını tespit maruz kalma). Görüntüleri kaydedin ve ImageJ 1.44o yazılımını kullanarak (floresan yoğunlukları analiz <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a> ). Süre ile diğer dokularda önemli olan tümörlerde VNPs alımını paterni karşılaştırın. </li><li> Görüntüleme sonra yarıda her doku kesilmiş ve kriyo-kesit ve konfokal analizi için Ekim bileşik bir buçuk gömün. Önceden tartılmış kriyo-flakonlarda diğer yarısı toplayın ve bunları hemen sıvı N 2 kullanarak dondurma. Mağaza -80 ° C daha fazla işlem için hazır olana kadar. </li><li> Floresans ölçümü: Rekord dokuları ağırlıkları. Oda sıcaklığında dondurulmuş doku çözülme ve PBS 1 ml içeren ayrı 50 ml Falcon tüpleri yerleştirin. Bir el dokusu homojenizatör kullanarak, PBS içinde 2-3 dakika süreyle doku homojenize sonra mikrofuge&#39;de tüplere homojenat aktarın. Non-homojenize doku kaldırmak için 13.000 g&#39;de 10 dakika süreyle homojenatlarında santrifüjleyin. </li><li> 384 de siyah UV plaka içine her grup (PBS ve VNP formülasyonları / zaman puan) dokulardan süpernatant pipetleyin 100 ul. Bir plaka okuyucu kullanarak floresan yoğunluğu (Ex / Em dalga boylarında 600/665) değerlendirin. Doku ağırlıkları ile elde edilen floresan değerlerini normalleştirmek. </li><li> İmmünohistokimya: -20 ° C&#39;de kriyo-mikrotom bölümleri (10 mikron) ve mağaza hazırlayın Hücre çekirdeğinin (DAPI) ve endotel hücre işaretleyici (CD31 antikoru anti-fare FITC-etiketli) için doku kesitleri Leke. Floresan-etiketli VNP ve vasküler ve intra-tümöral yerelleştirme haritasına konfokal mikroskopi analizi yapıns. </li></ol> Not: Bu işlem ortaya olmayacak, temsilci veriler Şekil 8 &#39;de gösterilmiştir. Immünohistokimya ve anlatılan boyama yöntemleri ile ilgili bir başvuru için okuyucu ref adlandırılır. 19

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+human+prostate+cancer+using+viral+nanoparticles+targeted+to+gastrin-releasing+Peptide+receptors.">Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors.</a> Small. 7, 1664-1672 (2011).</li><li>Wu, C., Barnhill, H., Liang, X., Wang, Q., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+probe+using+hybrid+virus-dye+nanoparticles+for+near-infrared+fluorescence+tomography.">A new probe using hybrid virus-dye nanoparticles for near-infrared fluorescence tomography.</a> Optics Communications. 255, 366-374 (2005).</li><li>Steinmetz, N. F., Cho, C. F., Ablack, A., Lewis, J. D., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cowpea+mosaic+virus+nanoparticles+target+surface+vimentin+on+cancer+cells.">Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells.</a> Nanomedicine (Lond). 6, 351-364 (2011).</li><li>Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability+and+the+EPR+effect+in+macromolecular+therapeutics:+a+review.">Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.</a> Journal of Controlled Release. 65, 271-284 (2000).</li><li>Chatterji, A., Ochoa, W., Paine, M., Ratna, B. R., Johnson, J. E., Lin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+addresses+on+an+addressable+virus+nanoblock:+uniquely+reactive+Lys+residues+on+cowpea+mosaic+virus.">New addresses on an addressable virus nanoblock: uniquely reactive Lys residues on cowpea mosaic virus.</a> Chem. Biol. 11, 855-863 (2004).</li><li>Steinmetz, N. F., Mertens, M. E., Taurog, R. E., Johnson, J. E., Commandeur, U., Fischer, R., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+novel+platform+for+potential+biomedical+applications.">Potato virus X as a novel platform for potential biomedical applications.</a> Nano Lett. 10, 305-312 (2010).</li><li>Wang, Q., Lin, T., Tang, L., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Icosahedral+virus+particles+as+addressable+nanoscale+building+blocks.">Icosahedral virus particles as addressable nanoscale building blocks.</a> Angew. Chem. Int. Ed. 41, 459-462 (2002).</li><li>Bruckman, M. A., Kaur, G., Lee, L. A., Xie, F., Sepulveda, J., Breitenkamp, R., Zhang, X., Joralemon, M., Russell, T. P., Emrick, T., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+tobacco+mosaic+virus+with+"click"+chemistry.">Surface modification of tobacco mosaic virus with "click" chemistry.</a> Chembiochem. 9, 519-523 (2008).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+of+the+tobacco+mosaic+virus.">Dual-surface modification of the tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Yildiz, I., Tsvetkova, I., Wen, A. M., Shukla, S., Masarapu, M. H., Dragnea, B., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+Brome+mosaic+virus+for+biomedical+applications.">Engineering of Brome mosaic virus for biomedical applications.</a> RSC Advances. , (2012).</li><li>Brunel, F. M., Lewis, J. D., Destito, G., Steinmetz, N. F., Manchester, M., Stuhlmann, H., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano Lett. 10, 1093-1097 (2010).</li><li>Shukla, S., Ablack, A., Wen, A., Lee, K., Lewis, J., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tumor+homing+and+tissue+penetration+of+the+filamentous+plant+viral+nanoparticle+Potato+virus+X.">Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.</a> Molecular Pharmaceutics. , (2012).</li><li>Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S. P., Wong, S. M., Clinton, G., Ghosh, P., Lin, T., Johnson, 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=Chemical+conjugation+of+heterologous+proteins+on+the+surface+of+Cowpea+mosaic+virus.">Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.</a> Bioconjug. Chem. 15, 807-813 (2004).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol vivo tümör görüntüleme için VNPs ve uygulamaları kimyasal modifikasyonu için bir yaklaşım sağlar. Burada sunulan hayvan floresan görüntüleme, floresans ölçme ve immunohistokimyasal teknikler biyodağılımını okuyan ve tümör homing değerlendirmek için yararlıdır. Bu teknikler EPR etkisi ile tümörün nanopartiküller erişim konusunda değerli bilgiler sağlamaktadır. Çeşitli analitik yöntemlerle elde edilen sonuçları birleştirerek, VNPs lokalizasyonu ve biyo...

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>VNP production</td>
		</tr>
		<tr>
			<td>Indoor plant chamber</td>
			<td>Percival Scientific</td>
			<td>E-41L2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>V. unguiculata seeds (California black-eye no. 5)</td>
			<td>Burpee</td>
			<td>51771A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N. benthamiana seeds</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.</td>
		</tr>
		<tr>
			<td>Carborundum</td>
			<td>Fisher</td>
			<td>C192-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pro-mix BX potting soil</td>
			<td>Premier Horticulture</td>
			<td>713400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Jack's Professional 20-10-20 Peat-Lite Fertilizer</td>
			<td>JR Peters</td>
			<td>77860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>50.2 Ti rotor</td>
			<td>Beckman</td>
			<td>337901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW 32 Ti rotor</td>
			<td>Beckman</td>
			<td>369694</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optima L-90K ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SLA-3000 rotor</td>
			<td>Thermo Scientific</td>
			<td>07149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SS-34 rotor</td>
			<td>Thermo Scientific</td>
			<td>28020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sorvall RC-6 Plus centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene bottle</td>
			<td>Beckman</td>
			<td>355607</td>
			<td>For SLA-3000 rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>For SS-34 rotor</td>
		</tr>
		<tr>
			<td>Ultra-Clear tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>For sucrose gradient and SW 32 Ti rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>355618</td>
			<td>For pelleting and 50.2 Ti rotor</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop2000c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PowerEase 500 pre-cast gel system</td>
			<td>Invitrogen</td>
			<td>EI8675EU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL (24 ml) size-exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&Auml;KTA Explorer 100 Chromatograph</td>
			<td>GE Healthcare</td>
			<td>28-4062-66</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra X-12R</td>
			<td>Beckman</td>
			<td>392302</td>
			<td>Benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maestro 2</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>Tissue-Tearor</td>
			<td>Biospec Products</td>
			<td>985370-395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>ZEISS</td>
			<td>Libra 200FE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoView laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Chemicals and Reagents</td>
		</tr>
		<tr>
			<td>3-ethynylaniline</td>
			<td>Sigma Aldrich</td>
			<td>498289-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>384 well black plate</td>
			<td>BD Biosciences</td>
			<td>353285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-12% Bis-Tris NuPAGE SDS gel</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X LDS sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher</td>
			<td>A385-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>271004-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 azide</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ml Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC501096</td>
			<td>10 kDa cut-off</td>
		</tr>
		<tr>
			<td>Aminoguanidine hydrochloride</td>
			<td>Acros Organics</td>
			<td>36891-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>A74-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Fisher</td>
			<td>BP101-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Acros Organics</td>
			<td>42285-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MP Biomedicals</td>
			<td>157574</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>09-801K</td>
			<td>P5 grade</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			<td>BioLegend</td>
			<td>102406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Invitrogen</td>
			<td>16210-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid sodium salt</td>
			<td>Acros Organics</td>
			<td>35268-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>VWR</td>
			<td>48366-277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Invitrogen</td>
			<td>NP0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-2k</td>
			<td>MW 2000</td>
		</tr>
		<tr>
			<td>mPEG-N3</td>
			<td>Nanocs</td>
			<td>PG1-AZ-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>mPEG-NHS</td>
			<td>Nanocs</td>
			<td>PG1-SC-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 succinimidyl ester *6-isomer*</td>
			<td>Invitrogen</td>
			<td>O-6149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-toluenesulfonic acid monohydrate</td>
			<td>Acros Organics</td>
			<td>13902-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher</td>
			<td>BP363-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher</td>
			<td>BP362-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher</td>
			<td>BP333-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Acros Organics</td>
			<td>42435-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Amresco</td>
			<td>0628-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S6-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>Ted Pella</td>
			<td>FCF-400Cu</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>TX1568-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>O3446I-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HT-29 cells</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Invitrogen</td>
			<td>31800-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Scientific</td>
			<td>SV30084.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal Studies</td>
		</tr>
		<tr>
			<td>18% Protein Rodent Diet</td>
			<td>Harlan Teklad</td>
			<td>Teklad Global 2018S</td>
			<td>Alfalfa free diet</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>329410</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AHN3637</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NCR nu/nu mice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>CWRU School 
			of Medicine Athymic Animal and Xenograft Core Facility</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>18 1/2 gauge</td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Andwin Scientific</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral nanoparticles for in vivo tumor imaging

Nanomalzemelerin kullanımı malzeme bilimi ve tıp devrim potansiyeline sahiptir. Şu anda değişik nanopartiküller bir dizi görüntüleme ve tedavi uygulamaları için araştırılmaktadır. Bitkilerden elde edilen viral nanopartikül (VNPs) belirlenmiş boyutlarda ve şekillerde ile kendini monte bionanomaterials olarak kabul edilebilir. Steinmetz laboratuarda inceleme altına Bitki virüsleri çapı 30 nm her ikisi de Börülce mozaik virüsü (CPMV) ve Brom mozaik virüsü (BMV) oluşan icosahedral parçacıkları içerir. Biz de şu bitki virüsleri türetilen çubuk şeklinde ve ipliksi yapılar geliştiriyoruz: 18 nm 300 nm boyutlarında ve ipliksi parçacıklar 515 oluştururlar Patates X virüsü (PVX), rijit çubuklar oluşturur Tütün mozaik virüsü (TMV), uzunluk ve genişlik olarak 13 nm olarak nm (okuyucu Refs ifade edilir. VNPs hakkında daha fazla bilgi için 1 ve 2). planta büyük ölçekte kolaylıkla üretilebilir, son derece istikrarlı ve biyouyumlu vardır. Ayrıca, VNPs özellikle genetik modifikasyon veya kimyasal bioconjugation yöntemleri 3 kullanılarak tasarlanmış olabilir &quot;programlanabilir&quot; birimlerdir. VNPs yapısı atomik çözüm bulunmakta olduğu bilinmektedir, ve modifikasyonlar atomik seviyede 4, mevcut teknoloji teknolojileri ile sentetik nano kullanılarak elde edilemeyen bir kontrol seviyesinde uzaysal hassasiyet ile gerçekleştirilebilir. Bu yazıda CPMV, PVX, TMV ve Vigna ungiuculata ve Nicotiana benthamiana bitkilerde BMV yayılımı tanımlar. Her VNP için Ekstraksiyon ve saflaştırma protokolleri verilmektedir. Saflaştırılmış ve kimyasal-etiketli VNPs karakterizasyonu için yöntemler tarif edilmiştir. Bu çalışmada, ch odaklanmakemical florofor ile VNPs etiketlenmesi (örneğin Alexa Fluor 647) ve polietilen glikol (PEG). Boyalar izleme ve 5-10 VNPs tespiti kolaylaştırmak ve onların farmakokinetiği 8,11 artırırken PEG protein nanopartiküller immünojenite azaltır. Biz bir fare ksenogreft tümör modeli kullanılarak Pegile VNPs yuvalandırma tümör ortaya. Maestro Görüntüleme Sistemi kullanılarak dokuların ex vivo floresan görüntüleme, homojenize dokularda floresans ölçme ve konfokal mikroskopi kombinasyonu biyodağılımını incelemek için kullanılır. VNPs retiküloendotelyal sistem (RES) ile temizlenir; homing tümör geliştirilmiş geçirgenlik ve saklama (EPR) etkisi 12 ile pasif olarak elde edilir. VNP nanoteknoloji güçlü bir plug-and-play görüntü teknolojisi ve in vivo hastalık siteleri tedavi. Bu daha fazla ilaç için yük ve klinik olarak-ilgili görüntüleme kısımları gibi doku-spesifik ligandlar taşımak için VNPs geliştirmektedirkanser ve kardiyovasküler hastalık aşırı eksprese moleküler reseptörleri hedef.

<img alt="Figure 1" src="/files/ftp_upload/4352/4352fig1.jpg" /> 
 Figure 1. Plant virus-infected plants. Vigna unguiculata plants infected with CPMV (A). Nicotiana benthamiana plants infected with PVX (B), TMV (C), and BMV (D). The pictures were taken about 10 days post infection by mechanical inoculation.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4352...

Watch this Scientific Journal Video about Viral Nanoparticles for In vivo Tumor Imaging at JoVE.com

Viral Nanoparticles for In vivo Tumor Imaging

Bitki viral nanopartiküller (VNPs) biyomedikal uygulamalar için platformlar umut vericidir. Burada, biz bitki VNP yayılımı, saflaştırma, karakterizasyon ve bioconjugation ilişkin prosedürler anlatılmaktadır. Son olarak, biz bir fare ksenogreft model ve floresan görüntüleme kullanılarak tümör homing ve görüntüleme için VNPs uygulama göstermektedir.

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 ...

viral-nanoparticles-for-in-vivo-tumor-imaging

Research

JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

17.1K Görüntüleme.  Case Western Reserve University. Bitki viral nanopartiküller (VNPs) biyomedikal uygulamalar için platformlar umut vericidir. Burada, biz bitki VNP yayılımı, saflaştırma, karakterizasyon ve bioconjugation ilişkin prosedürler anlatılmaktadır. Son olarak, biz bir fare ksenogreft model ve floresan görüntüleme kullanılarak tümör homing ve görüntüleme için VNPs uygulama göstermektedir.

Video: Viral Nanoparticles for In vivo Tumor Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Için basit Mikroakışkan Cihazlar<em&gt; In vivo</emVe&gt; Görüntüleme<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Ve Zebra balığı

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

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

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

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. ...

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

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

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

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

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 ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Modlu Moleküler Görüntüleme Uygulamaları için Biofunctionalized Prusya Mavisi Nanopartikülleri

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

Ateroskleroz Hızlı Algılama Demir Oksit Nanopartiküller Mikrodalga odaklı sentezi

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Angiogenez Multiphoton mikroskobu In Vivo genetiği değiştirilmiş 3D-PLGA/nHAp İskele tarafından grefti kritik Kemik defekti tamir için görselleştirme

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antikor conjugates ilk değerlendirilmesi ile Viral kaynaklı peptidler hücresel birikimi artan ve tümör hedefleme artırmak için değiştirilmiş

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

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 ...