The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS Partikül Preparasyonu <ol><li> 2 saat süre ile 95 ° C &#39;de bir hidrojen peroksit% 30 çözeltisi içinde olmayan işlevselleştirilmiş gözenekli silikon parçacıkları okside eder. % 2 hafifçe karıştırılarak 65 ° C sıcaklıkta 2 gün boyunca izopropil alkol (3-aminopropil) trietoksisilan çözelti içinde oksitlenmiştir parçacıklar aminat. </li><li> Pelet askıya kısa sonikasyon kullanılarak, 18.800 x g&#39;de 30 dakika süre ile çözelti santrifüj ve izopropil alkol ve etanol içinde üç kez iki parçacıkları yıkayın. Adım 1.3 ve 1.4 gerçekleştirirken etanol çözeltisi içinde parçacıkların bırakın. </li><li> 10 ml izoton seyreltici parçacık süspansiyonu (örneğin, 10 ul) olarak bilinen bir hacmi ilave edin ve yedek çözeltide bulunan parçacıkların yoğunluğunu belirlemek için bir parçacık sayma analizörü ile partiküllerin sayısı. </li><li> (3-dimetilaminopropil) - - etilkarbodiimid hidroklorür (EDC, 0.1 nmol) / K, N ile birlikte L-arginin (0.1 nmol) ve asit grubu aktive </em&gt; Hafif karıştırma ile, oda sıcaklığında 2 saat süre ile 20 ml etanol içinde-hidroksisukinimid (NHS, 0.1 nmol). </li><li> Kısaca parçacık stok solüsyonu sonikasyon ve L-arginin solüsyonu 1000000000 parçacıklarının eklenmesi ve yumuşak bir şekilde karıştırma ile, oda sıcaklığında 18 saat reaksiyona bırakın. </li><li> N birinci aspartik asit grubu aktive - (tert-Butoksikarbonil) -L-aspartik asit (Boc-Asp-OH, 1 nmol), 4 saat boyunca 20 ml etanol içerisinde EDC (0.1 nmol) / NHS (0.1 nmol) ile en hafifçe karıştırılarak, 4 ° C. </li><li> 10 ml etanol içindeki, 50 mg polietilenimin içinde çözündürülür ve Boc-Asp-OH, karışıma solüsyonunu ilave edin. Reaksiyon hafifçe karıştırılarak oda sıcaklığında 24 saat boyunca sürmesine izin verin. </li><li> Hafif karıştırma ile 6 saat boyunca 4 ° C sıcaklıkta EDC (0.1 nmol) / NHS (0.1 nmol) ile, Boc-Asp-OH / PEI solüsyonunun ikinci aspartik asit grubu etkinleştirir. </li><li> PCPS parçacıklar elde etmek için, adım 1.8 Boc-Asp-OH / PEI solüsyonu içine 1.5 adım parça çözeltisi ekleyin. Reaksiyon hafif bir st oda sıcaklığında 18 saat devam etmesine izin<html> irring. </li><li> Pelet askıya kısa sonikasyon kullanılarak, 18.800 x g&#39;de 30 dakika süre ile çözelti santrifüj ve etanol ile partikül çözeltisinin üç kez yıkayın. </li></ol> 2. PCPS Parçacık Karakterizasyonu <ol><li> Bir tarama elektron mikroskobu (SEM) ile parçacıkların boyutu ölçülür. <ol><li> Temiz bir silika SEM örnek saplama üzerindeki partikül süspansiyonu bir damla (etanol 10.000 parçacıklar / ul) yerleştirin ve vakum altında oda sıcaklığında kurumaya bırakın. </li><li> Bir de lens dedektörü kullanılarak 3-5 mm çalışma mesafesi ile 8 kV SEM görüntüleri ölçün. </li></ol></li><li> Bir parçacık analiz sistemi kullanılarak parçacıkların zeta potansiyeli ölçülür. <ol><li> 1 mi 10 mM fosfat tamponu (pH 7.4) ile partikül süspansiyonu, 10 ul (etanol içinde 10,000 partikül / ul) karıştırın. </li><li> Katlanmış kılcal hücreler içine yükleyin ve üreticinin talimatlarına uygun olarak zeta potansiyelini ölçer. </li></ol></li></ol>le &quot;&gt; 3. PCPS parçacıklara siRNA Yükleme <ol><li> Vakum O / N altında (PCPS parçacık hazırlama aşamasında 1,9 itibaren) PCPS parçacıkları kurulayın. </li><li> Kurutuldu PCPS parçacıklar ve sonikasyon kısaca siRNA nükleaz içermeyen su (20 ul) (4 ug) ekleyin. SiRNA oranları aşağıdaki parçacık kullanın: 2 x 10 5 parçacıklar / 0.2 ug siRNA, 4 x 10 5 parçacıklar / 0.2 ug siRNA, 6 x 10 5 parçacıklar / 0.2 ug siRNA 8 x 10 5 parçacıklar / 0.2 ug siRNA, 10 x 10 5 parçacıklar / 0.2 ug siRNA ve 12 x 10 5 parçacıklar / 0.2 ug siRNA. </li><li> SiRNA parçacıklar bağlanmaya imkan vermek için bir çalkalayıcı üzerinde 4 ° C de 3 saat karıştırıldı (1000 rpm) inkübe edilir. </li></ol> SiRNA / PCPS Parçacık Oranı 4. Optimizasyon <ol><li> SiRNA farklı oranları parça (PCPS parçacıklar halinde siRNA yükleme bakınız) PCPS / kontrol siRNA parçacıkların 20 ul DNA yükleme boya ekleyin. </li><li> Sam YükAlınan numuneler DNA jel leke ihtiva eden bir% 2 agaroz jeli içinde. </li><li> Çalışan tampon içinde 20 dakika süre ile 120 V sabit gerilimde elektroforez yapın. </li><li> Görüntü elde etme ve analiz yazılımı ile jel analiz. </li></ol> PCPS Taneciklerinden siRNA&#39;nın 5. Sürüm <ol><li> Sodyum dodesil sülfat (SDS,% 2) siRNA oranları (adım 3) farklı bir parçacık ile PCPS / kontrol siRNA parçacıkların 20 ul karıştırın, oda sıcaklığında 1 saat boyunca bekletilir. </li><li> Numune DNA yükleme boya ekleyin. </li><li> DNA jel leke ihtiva eden bir% 2 agaroz jeli yükleyin örnekleri. </li><li> DNA elektroforezi cihaz ve bir güç kaynağı ile çalışan tampon içinde 20 dakika süre ile 120 V sabit gerilimde elektroforez yapın. </li><li> Görüntü elde etme ve analiz yazılımı ile jel analiz. </li></ol> PCPS Parçacıkların 6. Konfokal Mikroskopi <ol><li> PCPS / floresan kontrol siRNA parçacıkların (10 × 10 5 parçacıklar / 0 5 ul ekleyin.Bir cam kapak kayma 2 ug SiRNA / 20 ul). </li><li> Konfokal mikroskobu ile parçacık katmanları gözünüzde canlandırın. </li></ol> Arg-PEI / kontrol siRNA Nanopartiküller 7. Karakterizasyonu <ol><li> Silikon malzemenin indirgeme ve Arg-PEI / kontrol siRNA nanopartiküllerinin oluşması için, fosfat tamponlu tuzlu su, 100 ul PCPS / siRNA parçacıkları (10 x 10 6 PCPS parçacıkları / 2 ug siRNA) ekleyin ve 37 ° C&#39;de (1,000 rpm) sallamak 2 gün. </li><li> 18800 x g&#39;de 30 dakika boyunca örnek santrifüj ve supernatant toplanır. </li><li> Dinamik ışık saçılımı (DLS) ile oluşturulan Arg-PEI / siRNA nanopartiküllerin boyutunu ölçmek. <ol><li> 1 mi, 10 mM fosfat tampon maddesi (pH 7.4) süpernatan 10 ul karıştırın ve bir plastik küvet içine koyun. </li><li> Üreticinin talimatlarına göre bir parçacık analiz sistemi kullanılarak parçacıkların boyutu ölçülür. </li></ol></li><li> Oluşan Arg / PEI-siRNA&#39;nın boyutunu ve morfolojisi belirleyinatomik kuvvet mikroskobu ile nanopartiküller. <ol><li> Bir silikon gofret üzerinde süpernatant ve yerin 10 ul alın. </li><li> Atomik kuvvet mikroskobu (AFM) ile partikülleri gözünüzde canlandırın. </li></ol></li></ol> 8. Hücre Kültürü <ol><li> Hücre kültür ortamı içinde kültür, MDA-MB-231 insan meme kanseri hücreleri,% 10 fetal inek serumu ve% 1 penisilin-streptomisin,% 5 CO2,% 95 nem ve 37 ° C sıcaklıkta ile takviye edilmiştir. </li></ol> PCPS Parçacıklar ile Canlı hücreler 9. Konfokal Mikroskopi <ol><li> 2-çukurlu kültür plakası hücreleri 24 saat / oyuk 3 x 10 5 hücre ilk önce tohum ekme yoğunluğu kayar. </li><li> Hücrelere floresan kontrol siRNA (10 x 10 5 partikülleri / siRNA oranı 0.2 ug parçacık, 50 nM siRNA) ile yüklendiğinde, PCPS parçacıkları ekleyin. </li><li> Parçacık expo aşağıdaki 12 saat (bir odacık,% 5 CO 2,% 95 nem ve 37 ° C ile birlikte) bir konfokal mikroskop ile hücrelerin bir film kaydetmekemin olun. </li></ol> PCPS Parçacıklar Sabit Hücre 10. Konfokal Mikroskopi <ol><li> 2-çukurlu kültür plakası hücreleri 24 saat / oyuk 3 x 10 5 hücre ilk önce tohum ekme yoğunluğu kayar. </li><li> Hücrelere floresan kontrol siRNA (10 x 10 5 partikülleri / siRNA oranı 0.2 ug parçacık, 50 nM siRNA) ile yüklendiğinde, PCPS parçacıkları ekleyin ve 1 gün, 7 gün ve 10 gün boyunca inkübe edilir. </li><li> Fosfat tamponlu salin ile iki kez hücre yıkayın ve daha sonra 10 dakika boyunca% 4 paraformaldehit çözeltisi sabitleyin. </li><li> Fosfat tamponlu tuzlu su ile yıkama hücreleri. </li><li> 10 dakika boyunca% 0.1 oktil fenol etoksilat olan hücreler geçirgenliği ve daha sonra bunları, fosfat tamponlu tuzlu su ile, üç kez yıkayın. </li><li> Hafifçe karıştırılarak oda sıcaklığında 10 dakika boyunca fosfat tamponlu tuz içinde sığır serumu (10 mg / ml) albumin ile hücrelerin bloke eder. </li><li> (1 ul / 40 ul, floresanla işaretlenmiş falloidinle hücreleri inkübe lifli aktin görselleştirmek içinhafifçe karıştırılarak oda sıcaklığında 20 dakika boyunca çözelti) bloke edilmesi ve daha sonra fosfat tamponlu tuzlu su ile yıkayın. </li><li> Çerçeveden slaytlar çıkarın ve çekirdeği görselleştirmek için 4 &#39;, 6-diamidino-2-fenilindol (DAPI) ile antifade reaktif ekleyin. </li><li> Üstüne bir kapak cam ekleyin ve konfokal mikroskobu ile hücrelerin görüntüleri çekmek. </li></ol> PCPS / floresan Kontrol siRNA Parçacıklar ile Hücreleri 11. Sitometrisi <ol><li> 24 saat / oyuk 3 x 10 5 hücre ilk önce tohum ekme yoğunluğu bir 6-yuvalı plaka plaka hücreleri. </li><li> Hücrelere floresan kontrol siRNA (10 x 10 5 partikülleri / siRNA oranı 0.2 ug parçacık, 50 nM siRNA) ile yüklendiğinde, PCPS parçacıkları eklenir ve 24 saat süreyle inkübe edilir. </li><li> Fosfat tamponlu tuzlu su ile yıkanır ve hücrelerin bir hücre kazıyıcı ile kazıyın. </li><li> Analizden önce% 2 fetal sığır serumu ile fosfat tamponlu tuzlu su içinde hücreleri saklayın. Negatif bir kontrol olarak muamele edilmemiş hücreler kullanın. </li><li> Akış cy gerçekleştirintometry. </li></ol> PCPS Parçacıklar ve PCPS / kontrol siRNA Parçacıklar ile Hücreleri 12. Hücre canlılığı <ol><li> 24 saat / oyuk 3 x 10 3 hücrelerinin bir hücre yoğunluğunda 96 oyuklu bir plaka içerisinde Levha hücreleri. </li><li> PCPS parçacıklarla hücreleri tedavi (1.5 x 10 5 / oyuk, 6 x 10 5 / çukur) veya PCPS / kontrol siRNA parçacıkları 48 (10 x 10 5 partikülleri / siRNA oranı 0.2 ug parçacık, 10 nM ve 100 nM siRNA) saat ve 72 saat. Kontrol olarak fosfat tamponlu tuzlu su (ilave parçacıklar ile aynı hacim) ile muamele muamele edilmemiş hücreler ve hücre kullanın. Üç kopya halinde her bir numune deneyi. </li><li> Üreticinin talimatlarına göre bir hücre çoğalması analizi gerçekleştirir. </li><li> Ortalama ± standart sapma olarak verileri temsil. </li></ol> PCPS / ATM Mutated siRNA Parçacıklar ile Hücreleri 13. Western Blot <ol><li> Hücre yoğunluğu 2 x 10 5 hücre / a bir 6-yuvalı plaka Levha hücreleri24 saat süre ile ell. </li><li> PCPS / kontrol siRNA (50 nM), parçacıklar ya da PCPS / ATM siRNA 72 saat boyunca (50 nM), parçacıkların hücreleri inkübe edin. Bir kontrol olarak muamele edilmemiş hücreler kullanın. </li><li> Parçalayıcı bir proteaz inhibitör kokteyli ile takviye edilmiş bir protein ekstraksiyon reaktifi kullanılarak hücreler. </li><li> 14000 x g&#39;de 10 dakika boyunca hücre lizatları santrifüj ve süpernatantı iyileşir. </li><li> Üreticinin talimatlarına uygun olarak, bir protein miktar tayini ile protein konsantrasyonunu belirleyin. </li><li> (5 ul 2-merkaptoetanol / ml tampon) bir numunesi, yükleme tamponu ekleme örnekleri 99 ° C&#39;de 6 dakika boyunca ısıtmaz. </li><li> Çalışan tampon içinde bir% 12 SDS-poliakrilamid jel içinde protein numuneleri (20 ug / ml) yükleyin ve elektroforez cihaz ve bir güç kaynağı ile poliakrilamid jel elektroforez (1 saat, 120 V) gerçekleştirmek. </li><li> Elektroforez ekipmanı kullanılarak bir nitroselüloz zar (1 saat, 100 V) (% 20 metanol), transfer tampon maddesi içinde jel aktarın veBir güç kaynağı. </li><li> 1 saat süreyle% 5 kuru süt ile bloke membran. </li><li> 1000 seyreltme, O / N ile 1: (aşama 13.9 gelen) çözeltiyi engelleme (tavşandan) atm birincil antikor ile membran inkübe edin. </li><li> 1 saat süre ile 2500 seyreltme: 1% 0.1 polietilen glikol, sorbitan monolorat ihtiva eden fosfat tamponlu tuzlu su ile yıkanır ve daha sonra zar (aşama 13.9 gelen) çözeltiyi bloke ikincil antikor (anti-tavşan) ile inkübe edilir. </li><li> % 0.1 polietilen glikol, sorbitan monolorat ihtiva eden fosfat tamponlu tuzlu su ile yıkanır ve zar görüntü elde etme ve analiz yazılımı kullanılarak Batı benek saptama reaktif protein bantları algılar. </li><li> Yükleme kontrolü için, zarın yıkanması ve tekrar (fare, 1: 10,000 seyreltme) bir β-aktin primer antikoru kullanılarak 13,9-13,12 adım (anti-fare 1: 4000 seyrelti) ve bir sekonder antikor. </li></ol>

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The authors declare that they have no competing financial interests.

Bu protokol, hücrelerin içine siRNA gönderinin başarılı olması ve transfeksiyon için bir yöntem tarif eder. Özellikle, siRNA dağıtım polikatyon işlevselleştirilmiş PSI parçacık ihtiva eden çok fonksiyonlu bir platform kullanılarak elde edilir. SiRNA tedavisinin kullanımı çeşitli onkogenler yüksek özgüllük ile hedeflenen edilebilir, örneğin büyük bir potansiyel, kanser tedavisi vardır. Bu nedenle, siRNA&#39;nın tedavisinin zorlukları azaltmak, hangi siRNA teslimat araçları ge...

Küçük, girişim yapan RNA&#39;lar (siRNA&#39;lar), gen ekspresyonunu engellemek çift-şeritli RNA molekülleridir. Son yıllarda, siRNA&#39;lar klinik uygulamada 1-5 gelecekte kullanılmak için terapötik potansiyelini göstermektedir BioDrugs, yeni nesil olarak geliştirilmiştir. Bununla birlikte, siRNA tedavinin başarılı bir şekilde uygulanması nedeniyle nükleazlar, zayıf hücre içi alımı ve düşük transfeksiyon verimi ve endozom / lizozom 5 verimsiz salınması ile bozunmaya, önemli bir sorun teşkil etmektedir. Bu engellerin birçoğu güvenli ve verimli bir şekilde hastalıklı dokuya siRNA sunabilirsiniz teslim platformlarının geliştirilmesi, aşılabilir. Viral taşıyıcılar ile karşılaştırıldığında, viral-olmayan platformları, güvenlik, düşük maliyet ve biçimlendirilmesi kolaylığı gibi çeşitli avantajlar sağlar. Bu polimerlerin ve lipidler gibi, özellikle, katyonik nano-tanecikleri olarak, siRNA taşınması 3 için yararlı olduğu kanıtlanmıştır. Daha önce, bir disk şekilli dr geliştirdikiletim sistemi ug, çok kademeli vektör (MSV) olarak adlandırılan. Bu platform, bir taşıt bir salındığı ardışık aşamaları dayanmaktadır. İkinci aşamada araçlar ilaçlar veya kontrast ajanları 6,7 yüklü nanoparçacıklar ise ilk aşaması bir araç, biyolojik olarak parçalanabilir bir gözenekli silikon (psi) yapılmış bir mikro-parçacık. Si 8 olarak azaldıkça PSI malzeme içine gömülü nanopartiküller, yavaş yavaş serbest bırakılır. Si partikülleri kullanmanın bir avantajı yüzey özelliklerinin, optimum bir biyo dağılıma ve ilaç salımı elde etmek için uygun olmasıdır. Son zamanlarda, tümör dokusuna siRNA lipozomlar verilmesi için MSV platformun başarılı kullanımı, bir yumurtalık ve meme kanseri fare modelinde 9, 10 olarak gösterilmiştir. Bu çalışmada, MSV platformu ilkeleri temelinde siRNA&#39;da için evrensel bir dağıtım sistemi fabrikasyon. Bu taşıyıcı sistemin etkinliği daha önce bize gösterilmiştirFarklı siRNA ing 11 molekülleri. Sistem polietilenimin (PEI) ve arginin (Arg) ile aşılanmış psi oluşan bir polikatyon işlevselleştirilmiş gözenekli silikon (PCPS) taşıyıcıdır. Arg ve PSI PEI toksisitesini azaltmak için hizmet edebilir süre önce 11 demonstarted gibi PEI, siRNA ile elektrostatik etkileşimleri oluşturulmasında yardımcı olabilir. PSI mikro-siRNA koruma ve sürekli salınmasını sağlayacak Buna ek olarak, PEI varlığı, hücre içi alımı ve endozomal kaçış yardımcı olabilir. PSI parçacıkları kademeli olarak ve böylece ayrı bir morfoloji ve dar bir boyut dağılımına sahip 11-Arg-PEI / siRNA nanopartiküller (Şekil 1) oluşması ile sonuçlanan, fizyolojik koşullar altında indirgeme. PCPS / SiRNA sisteminin istikrara ilişkin ayrıntılar için, Shen ve ark. 11 tarafından yapılan çalışmada bakın. İkinci aşama nanopartiküller başlangıçta Prese değil çünkü bu PCPS platformu, geleneksel MSV farklıdırbirinci aşama taşıyıcı 11, 12 olarak azaldıkça nt taşıyıcı içinde, ancak zaman içinde oluşturulur. PCPS sistemi siRNA yükleme verimliliği, sitotoksisite ve gen susturma etkinlik, in vitro olarak değerlendirilmiştir. Transfeksiyon etkililiği, DNA onarımı 10 yer almaktadır (ATM) onkojen mutasyona uğramış ataksi telanjiektazi karşı siRNA kullanılarak ölçülmüştür. Daha önce, ATM bastırılması, bir göğüs kanseri modelinde 10 tümör büyümesini azaltmak için gösterilmiştir.

<table border="0" cellpadding="0" cellspacing="0" width="1812">
	<tbody>
		<tr height="55">
			<td height="55" style="height:55px;width:605px;">Name of the Material/Equipment</td>
			<td style="width:421px;">Company/Institution</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:653px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Polyethylenimine (PEI), branched</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408727</td>
			<td style="width:653px;">Average molecular weight ~25,000 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">L-Arginine</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A5006</td>
			<td style="width:653px;">Reagent grade, &#8805;98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Boc-Asp-OH</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408-468</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">(3-&#8203;Aminopropyl)&#8203;triethoxysilane</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">440140</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Hydrogen peroxide solution</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">216763</td>
			<td style="width:653px;">30&#160;wt. % in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Sulfuric acid</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">339741</td>
			<td style="width:653px;">100.00%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Isopropyl alcohol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">W292907</td>
			<td style="width:653px;">&#8805;99.7%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Ethanol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">459844</td>
			<td>&#8805;99.5%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>03449</td>
			<td>&#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-10G</td>
			<td>Blocking agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Ataxia-telangiectasia mutated siRNA&#160;</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Designed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Tris Acetate-EDTA buffer</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">T9650</td>
			<td style="width:653px;">For DNA agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Penicillin-Streptomycin</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fetal Bovine Serum</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>F2442</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">TWEEN 20</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:653px;">Polyethylene glycol sorbitan monolaurate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">2-Mercaptoethanol</td>
			<td style="width:421px;">Sigma Aldrich</td>
			<td>M6250</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium dodecyl sulfate</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>L3771</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate monobasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71496</td>
			<td style="width:653px;">For making phosphate buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate dibasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71640</td>
			<td style="width:653px;">For making phosphate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A4416</td>
			<td style="width:653px;">Secondary antibody (anti-mouse) for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">N-Hydroxysuccinimide (NHS)</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">130672</td>
			<td style="width:653px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td>
			<td style="width:421px;">Greiner Bio-One</td>
			<td style="width:133px;">655182</td>
			<td style="width:653px;">96-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">10X Tris-Glycine Liquid</td>
			<td style="width:421px;">Li-Cor&#160;</td>
			<td style="width:133px;">928-40010</td>
			<td style="width:653px;">Transfer buffer for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Paraformaldehyde solution 4% in PBS&#160;</td>
			<td style="width:421px;">Santa Cruz</td>
			<td>Sc-281692</td>
			<td style="width:653px;">Fixation of cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td>
			<td style="width:421px;">Promega</td>
			<td style="width:133px;">G5421</td>
			<td style="width:653px;">Proliferation assay</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Phosphate buffered saline</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP399-500</td>
			<td style="width:653px;">10 X Solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Corning cellgro Dulbecco&#39;s Modification of Eagle&#39;s (Mod.)</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">MT-15-017-CM</td>
			<td style="width:653px;">Cell culture media, 1X solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Triton X-100</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>AC21568-2500</td>
			<td style="width:653px;">Octyl phenol ethoxylate, permeabilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Cover glass</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>12-530C&#160;</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Methanol</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td>A412-1</td>
			<td style="width:653px;">For Western blot transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Plastic Cuvettes</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">14-377-010&#160;</td>
			<td style="width:653px;">For size measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Molecular BioProducts RNase AWAY Surface Decontaminant</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">14-754-34</td>
			<td style="width:653px;">Spray for removing RNAse contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Agarose</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP165-25</td>
			<td style="width:653px;">Low melting point, for running RNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td style="width:421px;">Invitrogen</td>
			<td>P36935</td>
			<td style="width:653px;">Antifade reagent with DAPI, nucelus detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">A12379</td>
			<td style="width:653px;">Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">S33102</td>
			<td style="width:653px;">Visualization of RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Negative Control siRNA</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1022076</td>
			<td style="width:653px;">Control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">AllStars Neg. siRNA AF 555</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1027294</td>
			<td style="width:653px;">Fluorescent control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Cell scraper</td>
			<td style="width:421px;">Celltreat</td>
			<td>229310</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite Multidishes and Microwell Plates</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>130184</td>
			<td style="width:653px;">6-well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce LDS Sample Loading Buffer (4X)&#160;</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>84788</td>
			<td style="width:653px;">Sample loading buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Pierce BCS Protein Assay Kit</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">23227</td>
			<td style="width:653px;">Protein quantification assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halt Protease Inhibitor Single-Use Cocktails (100X)</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">78430</td>
			<td style="width:653px;">Protease inhibitor cocktail, use at 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-PER Mammalian Protein Extraction Reagent</td>
			<td style="width:421px;">Thermo Scientific&#160;</td>
			<td style="width:133px;">78501</td>
			<td style="width:653px;">Protein extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sorvall Legend Micro 21R</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Beta Actin Antibody&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>MA1-91399</td>
			<td>&#946;-actin primary antibody (from mouse) for western blor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6X TriTrack DNA Loading Dye</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>R1161</td>
			<td style="width:653px;">DNA loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-Free Water&#160;</td>
			<td style="width:421px;">Life Technologies</td>
			<td>AM9938</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Fat Dry Milk</td>
			<td style="width:421px;">Lab Scientific</td>
			<td>M0841</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well BD Falcon culture slides&#160;</td>
			<td style="width:421px;">BD Biosciences</td>
			<td>354102</td>
			<td style="width:653px;">2-well culture slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Western blot detection reagent.&#160;</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td style="width:133px;">RPN2106</td>
			<td style="width:653px;">Western blot detection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BA Membranes</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td>10402096</td>
			<td style="width:653px;">Nitrocellulose membrane for Wester blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATM (D2E2) Rabbit mAb</td>
			<td style="width:421px;">Cell Signaling</td>
			<td>2873S</td>
			<td style="width:653px;">ATM primary antibody (from rabbit) for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:421px;">Cell Signaling&#160;</td>
			<td>7074</td>
			<td style="width:653px;">Secondary antibody (anti-rabbit) for Western blot&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Folded capillary cells</td>
			<td style="width:421px;">Malvern&#160;</td>
			<td style="width:133px;">DTS 1061</td>
			<td style="width:653px;">For zeta potentail measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">MDA-MB-231 cell line</td>
			<td style="width:421px;">ATCC</td>
			<td style="width:133px;">HTB-26</td>
			<td style="width:653px;">Mammary Gland/Breast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">12% Mini-PROTEAN TGX Gel</td>
			<td>Bio-rad</td>
			<td>456-1043&#160;</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biorad PowerPac HC</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td>Power supply for electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-rad</td>
			<td>161-0732</td>
			<td>Running buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wide Mini-Sub Cell GT Cell</td>
			<td>Bio-rad</td>
			<td>170-4405</td>
			<td>Electrophoresis equipment for DNA agarose gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra cell</td>
			<td>Bio-rad</td>
			<td>165-8000</td>
			<td>Electrophoresis equipment for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc XRS+ System with Image Lab Software</td>
			<td>Bio-rad</td>
			<td>170-8265</td>
			<td>Image acquisition and analysis software for gels and blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; (10cm) dia., 5x7mm diced Silicon Wafer</td>
			<td>Ted Pella&#160;</td>
			<td>16007</td>
			<td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td>Shaker&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoton II diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td>Isoton diluent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td>Particle counting analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Non-functionalized porous silicon particles&#160;</td>
			<td style="width:421px;">Microelectronics Research Center, University of Texas at Austin</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Dicoidal shape. 2.6 &#956;m (diameter) x 0.7 &#956;m (hight), provided in isopropyl alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Zetasizer Nano ZS</td>
			<td style="width:421px;">Malvern</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle analyzer system for size and zeta potential&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Scanning Electron Microscope</td>
			<td style="width:421px;">FEI</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Atomic Force Microscope</td>
			<td style="width:421px;">Bruker</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fluo ViewTM 1000 Confocal Microscope</td>
			<td style="width:421px;">Olympus</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Visualization of fixed and live cells</td>
		</tr>
	</tbody>
</table>

porous silicon microparticles for delivery of sirna therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Bu protokol, güvenli ve etkili bir siRNA transfeksiyon için bir viral-olmayan sağlama sistemi kullanımını tarif eder. SEM sonuçlar PCPS parçacıklar silindir şeklinde olan ve 2.6 mikron (Şekil 2A) bir çapa sahip olduğunu ortaya koymaktadır. partikülleri pozitif ve böylece negatif yüklü nükleotidler ile bağlama elektrostatik sağlayarak, yaklaşık 8,21 bir zeta potansiyeline (Şekil 2B) ile şarj edilmiştir. PCPS parçacıkların farklı katmanlarının Konfokal gör...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Teslimat küçük müdahale RNA (siRNA) terapötik uygulanması için ana sorundur. Bu protokol, arginin ve polietilenimin aşılanmış gözenekli silikon mikro oluşan, çok fonksiyonlu ve biyo-uyumlu bir siRNA taşıyıcı platformun kullanılmasını içerir.

SiRNA Therapeutics teslimi için Gözenekli Silikon Mikropartiküller

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.8K Görüntüleme.  Houston Methodist Research Institute.  Teslimat küçük müdahale RNA (siRNA) terapötik uygulanması için ana sorundur. Bu protokol, arginin ve polietilenimin aşılanmış gözenekli silikon mikro oluşan, çok fonksiyonlu ve biyo-uyumlu bir siRNA taşıyıcı platformun kullanılmasını içerir. 

Video: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

Yumuşak Litografik işlevsellik ve Desenlendirme Oksit Silisyum ve germanyum

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Biyomühendislik

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Kimya ve Biyoalgılayıcı Uygulamaları için Silikon Nanotel Alan etkili Transistör hazırlanması

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Doku Mühendisliği Aljinat mikropartiküllerin ile Thermogelling Poli (N-izopropil) -graft-kondroitin Sülfat Kompozitlerin Sentezi

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Katyonik ve Anyonik lipozomlar kullanılarak merkezi sinir sistemine Terapötik siRNA teslimi

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Gözenekli silisyum filmleri sinir büyüme faktörü taşıyıcı olarak tasarlama

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D gözenekli selüloz Nanocomposite hidrojel iskele baskılı

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

Sitosolik siRNA teslim için Nötrsel olarak şarj edilmiş, pH duyarlı polimerik nanopartiküller hazırlanması

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Silikon Nanowires ve Optik Stimülasyon İç-ve Hücrelerarası Elektrik Kaplin İncelemeler için

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

Silikon Planar İntrakkortikal Mikroelektrotların Yüzey İşlemi için Araçlar

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...