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 Particle Vorbereitung <ol><li> Oxidieren nicht-funktionalisierten porösen Siliciumpartikel in einer 30% igen Wasserstoffperoxidlösung bei 95 ° C für 2 Std. Aminieren die oxidierten Teilchen in 2% (3-Aminopropyl) triethoxysilan-Lösung in Isopropylalkohol für 2 Tage bei 65 ° C unter leichtem Rühren. </li><li> Zentrifugieren Sie die Lösung für 30 Minuten bei 18.800 xg und waschen Sie die Partikel zweimal in Isopropanol und dreimal in Ethanol, mit kurzen Ultraschallbehandlung, um das Pellet zu suspendieren. Lassen Sie die Partikel in Ethanol-Lösung bei der Durchführung von Schritt 1.3 und 1.4. </li><li> Hinzufügen eines bekannten Volumens Teilchensuspension (beispielsweise 10 &amp; mgr; l) auf 10 ml isotone Verdünnungsmittel und zählt die Teilchen mit einem Analysator Partikelzählung, um die Konzentration der Teilchen in der Stammlösung zu bestimmen. </li><li> Aktivieren der Säuregruppe von L-Arginin (0,1 nmol) mit N - (3-dimethylaminopropyl) - N &#39;-ethylcarbodiimide Hydrochlorid (EDC, 0,1 nmol) / N </em&gt; Hydroxysuccinimid (NHS, 0,1 nmol) in 20 ml Ethanol 2 h bei RT unter leichtem Rühren. </li><li> Kurz beschallen die Partikelstammlösung und fügen eine Milliarde Teilchen an der L-Arginin-Lösung und lassen Sie die Reaktion für 18 Stunden bei Raumtemperatur unter leichtem Rühren. </li><li> Aktiviere die erste Asparaginsäure Gruppe N - (tert-Butoxycarbonyl) -L-asparaginsäure (Boc-Asp-OH, 1 nmol) mit EDC (0,1 nmol) / NHS (0,1 nmol) in 20 ml Ethanol 4 Stunden bei 4 ° C unter leichtem Rühren. </li><li> Man löst 50 mg Polyethylenimin in 10 ml Ethanol und Hinzufügen der Lösung zu der Boc-Asp-OH-Mischung. Lassen Sie die Reaktion ablaufen zu 24 Stunden bei Raumtemperatur unter leichtem Rühren. </li><li> Aktivieren des zweiten Asparaginsäure Gruppe der Boc-Asp-OH / PEI-Lösung mit EDC (0,1 nmol) / NHS (0,1 nmol) bei 4 ° C für 6 h unter leichtem Rühren. </li><li> Um PCPS Partikel zu erhalten, fügen Sie die Partikellösung aus Schritt 1.5 in die Boc-Asp-OH / PEI-Lösung aus Schritt 1.8. Die Reaktion kann für 18 Stunden bei Raumtemperatur unter leichtem st fortzufahren<html> Irring. </li><li> Zentrifugieren Sie die Lösung für 30 Minuten bei 18.800 xg und waschen die Partikellösung dreimal mit Ethanol, mit kurzen Ultraschallbehandlung, um das Pellet zu suspendieren. </li></ol> 2. PCPS Partikelcharakterisierung <ol><li> Messung der Größe der Teilchen unter Verwendung eines Rasterelektronenmikroskops (SEM). <ol><li> Geben Sie einen Tropfen Partikelsuspension (10.000 Partikel / &amp; mgr; l in Ethanol) auf eine saubere Kieselsäure SEM Proben Stub und unter Vakuum bei Raumtemperatur trocken lassen. </li><li> Messen Sie REM-Aufnahmen bei 8 kV mit einer 3-5 mm Arbeitsabstand mit Hilfe eines in-lens-Detektor. </li></ol></li><li> Messung des Zetapotentials der Teilchen mit einer Partikelanalysesystem. <ol><li> Mix 10 ul Partikelsuspension (10.000 Teilchen / &amp; mgr; l in Ethanol) mit 1 ml 10 mM Phosphatpuffer (pH 7,4). </li><li> Laden Sie die Probe in gefalteten Kapillarzellen und messen Sie die Zetapotential nach den Anweisungen des Herstellers. </li></ol></li></ol>le &quot;&gt; 3. Laden von siRNA in PCPS Partikel <ol><li> Trocknen Sie die PCPS Partikel (aus PCPS Partikelvorbereitungsschritt 1.9) unter Vakuum O / N. </li><li> Fügen siRNA (4 ug) in Nuklease-freies Wasser (20 ul) zu den getrockneten PCPS Partikeln und kurz sonifizieren. Verwenden Sie die folgende Partikel siRNA Verhältnisse: 2 x 10 5 Partikel / 0,2 ug siRNA, 4 x 10 5 Partikel / 0,2 ug siRNA, 6 x 10 5 Partikel / 0,2 ug siRNA, 8 x 10 5 Partikel / 0,2 ug siRNA, 10 × 10 5 Teilchen / 0,2 ug siRNA und 12 × 10 5 Teilchen / 0,2 ug siRNA. </li><li> Inkubieren für 3 Stunden bei 4 ° C auf einem Schüttler (1.000 rpm) siRNA Bindung an die Teilchen zu ermöglichen. </li></ol> 4. Optimierung der siRNA / PCPS Partikel-Verhältnis <ol><li> Fügen DNA Loading Dye zu 20 ul der PCPS / Kontroll-siRNA-Partikel mit unterschiedlichen Partikel siRNA Verhältnisse (siehe Laden von siRNA in PCPS Teilchen). </li><li> Laden Sie das samsätze in ein 2% Agarosegel, das DNA-Gel-Färbung. </li><li> Führen der Elektrophorese bei einer konstanten Spannung von 120 V für 20 min in Laufpuffer. </li><li> Analysieren Sie das Gel mit der Bildaufnahme und Analyse-Software. </li></ol> 5. Freisetzung von siRNA PCPS Partikel <ol><li> Mischen Sie 20 ul der PCPS / Kontroll-siRNA-Partikel mit unterschiedlichen Partikel siRNA Verhältnisse (siehe Schritt 3) in Natriumdodecylsulfat (SDS, 2%) und stehen lassen für 1 h bei RT. </li><li> Hinzufügen DNA Ladefarbstoff zu den Proben. </li><li> Belastungsproben werden in ein 2% Agarosegel, das DNA-Gelfärbung. </li><li> Führen der Elektrophorese bei einer konstanten Spannung von 120 V für 20 min in Laufpuffer unter Verwendung von DNA-Apparatur und eine Stromversorgung. </li><li> Analysieren Sie das Gel mit der Bildaufnahme und Analyse-Software. </li></ol> 6. konfokale Mikroskopie von PCPS Partikel <ol><li> In 5 ul PCPS / fluoreszierende Kontroll-siRNA-Partikel (10 x 10 5 Partikel / 0.2 ug siRNA / 20 ul) auf einem Deckglas. </li><li> Visualisieren Partikelschichten durch konfokale Mikroskopie. </li></ol> 7. Charakterisierung von Arg-PEI / Kontroll-siRNA-Nanopartikel <ol><li> Um die Silizium-Material abgebaut wird und Arg-PEI / Kontroll-siRNA-Nanopartikel, fügen PCPS / siRNA-Partikel (10 × 10 6 Teilchen PCPS / 2 ug siRNA) zu 100 ul phosphatgepufferter Kochsalzlösung und Schütteln (1000 UpM) bei 37 ° C 2 Tagen. </li><li> Zentrifugieren Sie die Probe für 30 min bei 18.800 xg und sammeln Sie den Überstand. </li><li> Messen Sie die Größe der gebildeten Arg-PEI / siRNA-Nanopartikel mit der dynamischen Lichtstreuung (DLS). <ol><li> Nimm 10 &amp; mgr; l des Überstandes mit 1 ml 10 mM Phosphatpuffer (pH 7,4) und in einen Plastik Küvette. </li><li> Messung der Größe der Partikel mit einer Partikelanalysesystem nach den Anweisungen des Herstellers. </li></ol></li><li> Bestimmen Sie die Größe und die Morphologie des gebildeten Arg / PEI-siRNANanopartikel mit Rasterkraftmikroskopie. <ol><li> Nimm 10 &amp; mgr; l des Überstands und auf ein Siliziumwafer ist. </li><li> Visualisieren Sie die Partikel mit Rasterkraftmikroskopie (AFM). </li></ol></li></ol> 8. Zellkultur <ol><li> Kultur MDA-MB-231-Brustkrebszellen in Zellkulturmedium mit 10% fötalem Rinderserum und 1% Penicillin-Streptomycin bei 5% CO 2, 95% Luftfeuchtigkeit und 37 ° C ergänzt. </li></ol> 9. konfokale Mikroskopie von lebenden Zellen mit PCPS Partikel <ol><li> Platten Zellen in 2-Well-Kultur gleitet bei einer Aussaatdichte von 3 x 10 5 Zellen / Vertiefung für 24 h. </li><li> Fügen PCPS Partikel mit fluoreszierenden Kontroll-siRNA (10 x 10 5 Partikel / 0,2 ug Teilchens siRNA-Verhältnis, 50 nM siRNA) zu den Zellen geladen. </li><li> Eine Videoaufnahme der Zellen mit einem konfokalen Mikroskop 12 h (mit einer Kammer, 5% CO 2, 95% Luftfeuchtigkeit und 37 ° C zugeführt wird) folgende Korn Exposicher. </li></ol> 10. konfokale Mikroskopie der fixierten Zellen mit PCPS Partikel <ol><li> Platten Zellen in 2-Well-Kultur gleitet bei einer Aussaatdichte von 3 x 10 5 Zellen / Vertiefung für 24 h. </li><li> Fügen PCPS Partikel mit fluoreszierenden Kontroll-siRNA (10 x 10 5 Partikel / 0,2 ug Teilchens siRNA-Verhältnis, 50 nM siRNA) geladen, um Zellen und Inkubation für 1 Tag, 7 Tage und 10 Tage. </li><li> Waschen Sie die Zellen zweimal mit phosphatgepufferter Kochsalzlösung und dann befestigen Sie sie mit 4% Paraformaldehydlösung für 10 min. </li><li> Mit Phosphat gepufferte Kochsalzlösung waschen der Zellen. </li><li> Permeabilisierung der Zellen mit 0,1% Octylphenolethoxylat für 10 min und dann wasche sie dreimal mit phosphatgepufferter Kochsalzlösung. </li><li> Blockieren der Zellen mit Albumin aus Rinderserum (10 mg / ml) in phosphatgepufferter Salzlösung für 10 min bei RT unter leichtem Rühren. </li><li> Um filamentösen Aktin visualisieren, Inkubation der Zellen mit fluoreszenzmarkierten Phalloidin (1 ul / 40 &amp; mgr;Blockierungslösung) für 20 min bei RT unter leichtem Rühren und dann in phosphatgepufferter Salzlösung zu waschen. </li><li> Entfernen Sie die Folien aus dem Rahmen, und fügen Antifade Reagenz mit 4 &#39;, 6-Diamino-2-phenylindol (DAPI), um den Kern zu visualisieren. </li><li> Fügen Sie ein Deckglas oben und nehmen Bilder der Zellen mit der konfokalen Mikroskopie. </li></ol> 11. Durchflusszytometrie von Zellen mit PCPS / Fluoreszenzkontrolle siRNA Partikel <ol><li> Platten Zellen in einer 6-Well-Platte bei einer Aussaatdichte von 3 x 10 5 Zellen / Vertiefung für 24 h. </li><li> Fügen PCPS Partikel mit fluoreszierenden Kontroll-siRNA (10 x 10 5 Partikel / 0,2 ug Teilchens siRNA-Verhältnis, 50 nM siRNA) in die Zellen geladen und Inkubation für 24 Stunden. </li><li> Waschen der Zellen mit phosphatgepufferter Salzlösung und kratzen, mit einem Zellschaber. </li><li> Lagern Sie die Zellen in phosphatgepufferter Salzlösung mit 2% fötalem Rinderserum vor der Analyse. Verwenden unbehandelten Zellen als Negativkontrolle. </li><li> Führen Fluss cytrie. </li></ol> 12. Zelllebensfähigkeit von Zellen mit PCPS Partikel und PCPS / Kontroll-siRNA Partikel <ol><li> Platten Zellen in einer Platte mit 96 Vertiefungen bei einer Zelldichte von 3 × 10 3 Zellen / Vertiefung für 24 h. </li><li> Behandlung der Zellen mit PCPS-Teilchen (1,5 × 10 5 / Vertiefung und 6 × 10 5 / Vertiefung) oder PCPS / Steuer siRNA Partikel (10 × 10 5 Teilchen / 0,2 ug Teilchens siRNA-Verhältnis, 10 nM und 100 nM siRNA) 48 h und 72 h. Verwenden unbehandelten Zellen und Zellen mit phosphatgepufferter Salzlösung (das gleiche Volumen wie die hinzugefügten Teilchen) als Kontrolle behandelt. Assay jede Probe in dreifacher Ausfertigung. </li><li> Durchführen einer Zellproliferations-Assay gemß den Herstelleranweisungen. </li><li> Repräsentieren Daten als Mittelwert ± Standardabweichung. </li></ol> 13. Western Blot von Zellen mit PCPS / ATM Mutierte siRNA Partikel <ol><li> Platten Zellen in einer Platte mit 6 Vertiefungen bei einer Zelldichte 2 × 10 5 Zellen / well für 24 Stunden. </li><li> Inkubieren der Zellen mit PCPS / Kontroll-siRNA (50 nM) Teilchen oder PCPS / ATM siRNA (50 nM) Teilchen für 72 Stunden. Verwenden Sie unbehandelten Zellen als Kontrolle. </li><li> Lyse der Zellen unter Verwendung einer Protein-Extraktionsreagenz mit einem Protease-Inhibitor-Cocktail ergänzt. </li><li> Zentrifugieren Zelllysate für 10 min bei 14.000 × g und Gewinnen des Überstandes. </li><li> Bestimmung der Proteinkonzentration mit einem Proteinquantifizierungsassay gemäß den Anweisungen des Herstellers. </li><li> Hinzufügen Probenbeladungspuffer (5 &amp; mgr; l 2-Mercaptoethanol / ml Puffer) zu den Proben und erhitzen sie für 6 min bei 99 ° C. </li><li> Laden die Proteinproben (20 ug / ul) in einer 12% SDS-Polyacrylamid-Gel mit Laufpuffer und führen Polyacrylamidgelelektrophorese (1 h, 120 V) unter Verwendung von Elektrophorese Ausrüstung und eine Stromversorgung. </li><li> Übertragen des Gels in Transferpuffer (mit 20% Methanol) auf eine Nitrocellulosemembran (1 h, 100 V) unter Verwendung von Elektrophorese Ausrüstung undein Netzteil. </li><li> Blockieren Sie die Membran mit 5% Trockenmilch für 1 Stunde. </li><li> Inkubieren der Membran mit der ATM-Primär-Antikörpers (aus Kaninchen) in Blocking-Lösung (aus Stufe 13.9) bei einer 1: 1.000-Verdünnung O / N. </li><li> Waschen der Membran mit phosphatgepufferter Kochsalzlösung, die 0,1% Polyethylenglykol-Sorbitan-Monolaurat und dann inkubiert mit dem sekundären Antikörper (anti-Kaninchen) in Blocking-Lösung (aus Stufe 13.9) bei einer 1: 2.500-Verdünnung für 1 Stunde. </li><li> Waschen der Membran mit phosphatgepufferter Kochsalzlösung, die 0,1% Polyethylenglykol-Sorbitan-Monolaurat und detektieren die Proteinbanden mit Western-Blot-Nachweis-Reagens unter Verwendung von Bilderfassung und -analyse-Software. </li><li> Für die Ladesteuerung, Waschen der Membran und die Schritte 13,9-13,12 Verwendung eines β-Actin primären Antikörper (von der Maus, 1: 10.000-Verdünnung) und einem Sekundärantikörper (anti-Maus-1: 4.000-Verdünnung). </li></ol>

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The authors declare that they have no competing financial interests.

Dieses Protokoll beschreibt ein Verfahren zur erfolgreichen und Transfektion von siRNA in Zellen. Insbesondere ist die Verabreichung von siRNA durch Verwendung eines multifunktionellen Plattform, bestehend aus Polykation-funktionalisierten pSi Partikel erreicht. Die Verwendung von siRNA-Therapie hat ein großes Potenzial, zum Beispiel Krebsbehandlung, da verschiedene Onkogene können mit hoher Spezifität ausgerichtet sein. Daher besteht ein Bedarf an siRNA Lieferfahrzeugen zu entwickeln, die die Herausforderun...

Small interfering RNAs (siRNAs) sind doppelsträngige RNA-Moleküle, die die Expression von Genen unterdrücken kann. In den letzten Jahren haben siRNAs als eine neue Generation von biodrugs die therapeutisches Potential für die zukünftige Verwendung in klinischen Anwendungen 1-5 zeigen entwickelt. Die erfolgreiche Umsetzung der siRNA-Therapie bleibt jedoch eine große Herausforderung, durch Abbau durch Nukleasen, schlechte intrazelluläre Aufnahme, geringe Transfektionseffizienz und ineffizient Freisetzung aus dem Endosom / Lysosom 5. Viele dieser Hindernisse können durch die Entwicklung der Delivery-Plattformen, die sicher und effizient liefern kann siRNA auf erkranktes Gewebe zu überwinden. Im Vergleich zu Virusträgern nicht viralen Plattformen bieten mehrere Vorteile, wie Sicherheit, geringe Kosten und Einfachheit des Zuschneidens. Insbesondere kationischen Nanopartikeln, wie Polymere und Lipide, haben sich als nützlich für die siRNA-3 bewiesen. Bisher haben wir eine scheibenförmige dr entwickeltug Liefersystem, der sogenannten mehrstufigen Vektor (MSV). Diese Plattform ist an aufeinanderfolgenden Stufen, in denen ein Fahrzeug, das von einem anderen veröffentlicht basiert. Die erste Stufe Fahrzeug ein Mikroteilchen aus biologisch abbaubaren porösen Silicium (psi) hergestellt, während die Fahrzeuge zweiten Stufe sind Nanopartikel mit Arzneimitteln oder Kontrastmittel 6,7 geladen. Die Nanopartikel, die im PSI Material eingebettet sind, werden nach und nach freigesetzt, wie die Si verschlechtert 8. Ein Vorteil der Verwendung von Si-Partikel ist, dass die Morphologie und Oberflächeneigenschaften können leicht angepasst, um eine optimale biologische Verteilung und Wirkstofffreisetzung erreicht werden. Kürzlich wurde die erfolgreiche Anwendung des MSV-Plattform für die Bereitstellung von siRNA Liposomen an Tumorgewebe in einer Eierstock- und Brustkrebs Mausmodell 9, 10 gezeigt. In dieser Arbeit haben wir einen universellen Träger für siRNA, basierend auf den Prinzipien der MSV Plattform hergestellt. Die Wirksamkeit dieses Verabreichungssystem hat uns früher gezeigting unterschiedlichen siRNA-Moleküle 11. Das System ist ein Polykation-funktionalisierten porösen Silizium (PCPS) Träger, bestehend aus pSi mit Polyethylenimin (PEI) und Arginin (Arg) gepfropft. PEI kann bei der Bildung elektrostatische Wechselwirkungen mit siRNA unterstützen, während Arg und pSi kann dazu dienen, um die Toxizität von PEI zu reduzieren, wie zuvor demonstarted 11. Darüber hinaus kann die Anwesenheit von PEI in die intrazelluläre Aufnahme und endosomalen Flucht zu unterstützen, während die PSI-Mikropartikel ermöglichen siRNA Schutz und anhaltende Freisetzung. Die PSI-Partikel nach und nach unter physiologischen Bedingungen abgebaut werden, was zu der Bildung von Arg-PEI / siRNA-Nanopartikel (Abbildung 1), die einen ausgeprägten Morphologie und einer engen Größenverteilung 11 zu haben. Für detaillierte Angaben zur Stabilität der PCPS / siRNA-System finden Sie in der Studie beziehen von Shen et al. 11. Diese PCPS Plattform unterscheidet sich von der herkömmlichen MSV, da die Stufe Nanopartikel zweiten anfänglich nicht present in dem Träger, sondern werden über die Zeit gebildet wird wie das erste-Stufe-Träger abbaut 11, 12. Die siRNA Beladungseffizienz, Zytotoxizität und Gen-Silencing-Effizienz der PCPS System wurde in vitro untersucht. Transfektionseffizienz wurde mit siRNA gegen das Ataxia telangiectasia mutiert (ATM) Onkogen, das in der DNA-Reparatur beteiligt 10 gemessen. Früher war die Unterdrückung der ATM ist gezeigt worden, das Tumorwachstum in einer Brustkrebsmodell 10 zu verringern.

<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.

Dieses Protokoll beschreibt die Verwendung eines nicht-viralen Zufuhrsystem für einen sicheren und effizienten siRNA-Transfektion. Die SEM-Ergebnisse zeigen, dass die PCPS Teilchen eine zylindrische Form und haben einen Durchmesser von 2,6 &amp; mgr; m (2A). Die Partikel sind positiv mit einem Zetapotential von ungefähr 8,21 (2B) geladen, wodurch elektrostatische Bindung mit negativ geladenen Nukleotiden. Konfokale Bilder von verschiedenen Schichten der PCPS Teilchen zeigen, daß fluo...

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

Lieferung bleibt die größte Herausforderung für die therapeutische Anwendung der small interfering RNA (siRNA). Dieses Protokoll schließt die Verwendung eines multifunktionellen und biokompatibel siRNA Abgabeplattform, bestehend aus Arginin und Polyethylenimin gepfropft porösen Siliciummikropartikel.

Poröses Silizium Mikropartikel für die Lieferung von siRNA-Therapeutika

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 Aufrufe.  Houston Methodist Research Institute.  Lieferung bleibt die größte Herausforderung für die therapeutische Anwendung der small interfering RNA (siRNA). Dieses Protokoll schließt die Verwendung eines multifunktionellen und biokompatibel siRNA Abgabeplattform, bestehend aus Arginin und Polyethylenimin gepfropft porösen Siliciummikropartikel. 

Serie: 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.

Soft-Lithographie Funktionalisierung und Strukturierung Oxydfreie Silizium und Germanium

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

Forschung

Biotechnik

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.

Selbstberichts Gerüste für 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 ...

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.

Herstellung von Silizium-Nanodraht-Feldeffekttransistor für chemische und Biosensorik Anwendungen

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.

Synthese von thermogelierenden Poly (N-isopropylacrylamid) -Pfropf-Chondroitinsulfat Composites mit Alginate Mikroteilchen für 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 ...

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.

Abgabe von therapeutischen siRNA an das ZNS mit kationischen und anionische Liposome

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.

Gestaltung von porösem Silizium Filme als Träger von Nerve Growth Factor

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-Druck poröse Zellulose Nanocomposites Hydrogel Gerüste

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.

Vorbereitung von Neutral-aufgeladenen, pH-responsiven polymischen Nanopartikeln für zytosolische 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 ...

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.

Silizium-Nanodrähte und optische Stimulation für Untersuchungen der intra- und interzellulären elektrischen Kopplung

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.

Werkzeuge zur Oberflächenbehandlung von siliziumplanaren intrakortikalen Mikroelektroden

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