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. Préparation de particules PCPS <ol><li> Oxyder les particules de silicium poreux non-fonctionnalisés dans une solution à 30% de peroxyde d&#39;hydrogène à 95 ° C pendant 2 heures. Aminer les particules oxydées dans 2% (3- aminopropyl) triéthoxysilane solution dans l&#39;alcool isopropylique pendant 2 jours à 65 ° C sous agitation douce. </li><li> Centrifuger la solution pendant 30 min à 18 800 xg et laver les particules deux fois dans de l&#39;alcool isopropylique et trois fois dans de l&#39;éthanol, en utilisant brève sonication de suspendre le culot. Laisser les particules dans une solution d&#39;éthanol quand on effectue l&#39;étape 1.3 et 1.4. </li><li> Ajouter un volume connu de suspension de particules (par exemple, 10 ul) au diluant de 10 ml et compter les particules avec un comptage de particules analyseur pour déterminer la concentration de particules dans la solution mère. </li><li> Activer le groupe acide de L-arginine (0,1 nmol) de N - (3-diméthylaminopropyl) - chlorhydrate -ethylcarbodiimide N &#39;(EDC, 0,1 nmol) / N </em&gt; Hydroxysuccinimide (NHS, 0,1 nmol) dans 20 ml d&#39;éthanol pendant 2 heures à température ambiante avec agitation douce. </li><li> Soniquer brièvement la solution particules de stock et ajouter 1 milliard de particules à la solution de L-arginine et de laisser la réaction pendant 18 heures à température ambiante avec agitation douce. </li><li> Activer le premier groupe de l&#39;acide aspartique de la N - (tert-butoxycarbonyl) -L-aspartique (Boc-Asp-OH, 1 nmol) avec du EDC (0,1 nmol) / NHS (0,1 nmol) dans 20 ml d&#39;éthanol pendant 4 heures à 4 ° C avec agitation douce. </li><li> Dissoudre 50 mg de polyéthylène-imine dans 10 ml d&#39;éthanol et ajouter la solution au mélange de Boc-Asp-OH. Que la réaction se déroule pendant 24 heures à température ambiante avec agitation douce. </li><li> Activez le deuxième groupe d&#39;acide aspartique de la solution Boc-Asp-OH / PEI avec EDC (0,1 nmol) / NHS (0,1 nmol) à 4 ° C pendant 6 heures sous agitation douce. </li><li> Pour obtenir des particules, PCP, ajouter la solution de particules de l&#39;étape 1.5 dans la solution de Boc-Asp-OH / PEI de l&#39;étape 1.8. Laisser la réaction se dérouler pendant 18 heures à température ambiante avec douceur st<html> irring. </li><li> Centrifuger la solution pendant 30 min à 18 800 xg et laver la solution de particules à trois reprises avec de l&#39;éthanol, en utilisant brève sonication à suspendre la pastille. </li></ol> 2. PCPS caractérisation des particules <ol><li> Mesure de la taille des particules en utilisant un microscope électronique à balayage (MEB). <ol><li> Placez une goutte de suspension de particules (10 000 particules / ul dans de l&#39;éthanol) sur un échantillon de silice propre SEM talon et laisser sécher à température ambiante sous vide. </li><li> Mesurer images MEB à 8 kV avec une distance de travail de 3-5 mm en utilisant un détecteur de lentille. </li></ol></li><li> Mesurer le potentiel zêta des particules en utilisant un système d&#39;analyse de particules. <ol><li> Mélanger 10 pl de suspension de particules (10 000 particules / ul) dans de l&#39;éthanol avec 1 ml de 10 mM de tampon phosphate (pH 7,4). </li><li> Chargez l&#39;échantillon dans les cellules capillaires plié et en mesurer le potentiel zêta selon les instructions du fabricant. </li></ol></li></ol>le &quot;&gt; 3. Chargement de siRNA dans des particules PCPS <ol><li> Sécher les particules PCPS (PCPS de particules étape de préparation 1,9) sous vide O / N. </li><li> Ajouter siRNA (4 pg) dans l&#39;eau sans nucléase (20 pi) pour les particules PCPS secs et brièvement sonicat. Utilisez la particule rapports suivants siARN: 2 × 10 5 particules / 0,2 pg siRNA, 4 × 10 5 particules / 0,2 pg siARN, 6 × 10 5 particules / 0,2 pg siRNA, 8 × 10 5 particules / 0,2 pg siARN, 10 × 10 5 particules / 0,2 ug siRNA et 12 × 10 5 particules / 0,2 ug siARN. </li><li> Incuber pendant 3 heures à 4 ° C sur un agitateur (1000 rpm) pour permettre la liaison des particules de siRNA. </li></ol> 4. Optimisation du ratio des particules siRNA / PCPS <ol><li> Ajouter ADN colorant de charge à 20 pi de particules de siARN PCPS / de contrôle avec particules différents rapports siARN (voir Chargement de siRNA en particules PCPS). </li><li> Chargez le sampes dans un gel d&#39;agarose à 2% contenant du gel de l&#39;ADN tache. </li><li> Effectuer l&#39;électrophorèse à une tension constante de 120 V pendant 20 min dans un tampon en cours d&#39;exécution. </li><li> Analyser le gel avec l&#39;acquisition de l&#39;image et des logiciels d&#39;analyse. </li></ol> 5. Libération de siRNA de PCPS particules <ol><li> Mélanger 20 pi de particules de siARN PCPS / de contrôle avec particules différents rapports siRNA (voir l&#39;étape 3) en dodécylsulfate de sodium (SDS, 2%) et laisser reposer pendant 1 heure à température ambiante. </li><li> Ajouter ADN colorant de charge pour les échantillons. </li><li> Charger les échantillons dans un gel agarose à 2% contenant du gel de l&#39;ADN tache. </li><li> Effectuer l&#39;électrophorèse à une tension constante de 120 V pendant 20 min dans un tampon en cours d&#39;exécution en utilisant un équipement d&#39;électrophorèse de l&#39;ADN et une alimentation électrique. </li><li> Analyser le gel avec l&#39;acquisition de l&#39;image et des logiciels d&#39;analyse. </li></ol> 6. microscopie confocale de PCPS particules <ol><li> Ajouter 5 ul de particules fluorescentes de siARN de contrôle PCPS / (10 × 10 5 particules / 0.2 pg siRNA / 20 pi) à une lamelle de verre. </li><li> Visualiser couches de particules par microscopie confocale. </li></ol> 7. Caractérisation de Arg-PEI / siRNA contrôle nanoparticules <ol><li> À dégrader le matériau de silicium et former Arg-PEI nanoparticules / contrôle de siARN, ajouter des particules PCPS / siRNA (10 × 10 6 particules PCPS / 2 pg siARN) à 100 pi de tampon phosphate salin et secouer (1000 rpm) à 37 ° C pendant 2 jours. </li><li> Centrifuger l&#39;échantillon pendant 30 min à 18 800 xg et récupérer le surnageant. </li><li> Mesurer la taille des nanoparticules formées Arg-PEI / siARN avec diffusion de la lumière dynamique (DLS). <ol><li> Mélanger 10 pi du surnageant avec 1 ml de tampon phosphate 10 mM (pH 7,4) et le placer dans une cuvette en matière plastique. </li><li> Mesure de la taille des particules en utilisant un système d&#39;analyse de particules selon les instructions du fabricant. </li></ol></li><li> Déterminer la taille et la morphologie du Arg / PEI-siRNA forménanoparticules avec la microscopie à force atomique. <ol><li> Prenez 10 pi du surnageant et déposer sur une plaquette de silicium. </li><li> Visualisez les particules avec la microscopie à force atomique (AFM). </li></ol></li></ol> 8. Culture Cellulaire <ol><li> Culture MDA-MB-231 cellules de cancer du sein humain dans un milieu de culture cellulaire supplémenté avec 10% de sérum bovin fœtal et 1% de pénicilline-streptomycine à 5% de CO2, 95% d&#39;humidité et 37 ° C. </li></ol> 9. microscopie confocale de cellules vivantes avec PCPS particules <ol><li> cellules de la plaque dans la culture deux puits diapositives à une densité de semis de 3 x 10 5 cellules / puits pendant 24 heures. </li><li> Ajouter particules PCPS chargés de siRNA contrôle fluorescent (10 × 10 5 particules / 0,2 pg particules rapport siRNA, 50 nM siRNA) pour les cellules. </li><li> Enregistrer un film des cellules avec un microscope confocal (fourni avec une chambre, 5% de CO 2, 95% d&#39;humidité et 37 ° C) pendant 12 heures suivant expo de particulesassurer. </li></ol> 10. microscopie confocale de cellules fixes avec PCPS particules <ol><li> cellules de la plaque dans la culture deux puits diapositives à une densité de semis de 3 x 10 5 cellules / puits pendant 24 heures. </li><li> Ajouter PCPS particules chargées avec l&#39;ARNsi de contrôle fluorescent (10 x 10 5 particules / particules de 0,2 ug rapport siRNA, 50 nM de siRNA) aux cellules et incuber pendant 1 jour, 7 jours et 10 jours. </li><li> Laver les cellules deux fois avec une solution saline tamponnée au phosphate, puis les fixer avec une solution de paraformaldehyde à 4% pendant 10 min. </li><li> Laver les cellules avec du tampon phosphate salin. </li><li> Perméabiliser les cellules avec 0,1% d&#39;octyl phénol éthoxylate pendant 10 min, puis les laver trois fois avec du tampon phosphate salin. </li><li> Bloquer les cellules avec de l&#39;albumine de sérum bovin (10 mg / ml) dans une solution saline tamponnée au phosphate pendant 10 min à température ambiante avec agitation douce. </li><li> Pour visualiser l&#39;actine filamenteuse, incuber les cellules avec fluorescence phalloïdine marquée (1 pi / 40 piune solution de blocage) pendant 20 min à température ambiante avec agitation douce, puis lavez en tampon phosphate salin. </li><li> Retirer les glissières du cadre et ajouter antifade réactif avec 4 &#39;, 6-diamidino-2-phénylindole (DAPI) pour visualiser le noyau. </li><li> Ajouter un verre de couverture sur le dessus et prendre des images de cellules avec la microscopie confocale. </li></ol> 11. cytométrie en flux des cellules avec PCPS / fluorescentes contrôle Particules siARN <ol><li> cellules de la plaque dans une plaque à 6 puits à une densité d&#39;ensemencement de 3 x 10 5 cellules / puits pendant 24 h. </li><li> Ajouter PCPS particules chargées avec l&#39;ARNsi de contrôle fluorescent (10 x 10 5 particules / particules de 0,2 ug rapport siRNA, 50 nM de siRNA) aux cellules et incuber pendant 24 heures. </li><li> Laver les cellules avec une solution saline tamponnée au phosphate et les gratter avec un grattoir à cellules. </li><li> Stocker les cellules dans une solution saline tamponnée au phosphate avec 2% de sérum bovin foetal avant l&#39;analyse. Utilisation des cellules non traitées comme témoin négatif. </li><li> Effectuer cy de fluxtometry. </li></ol> 12. viabilité cellulaire des cellules avec des particules et PCPS PCPS / commande particules siARN <ol><li> cellules de la plaque dans une plaque à 96 puits à une densité cellulaire de 3 × 10 3 cellules / puits pendant 24 heures. </li><li> Traiter les cellules avec des particules PCPS (1,5 x 10 5 / puits et 6 × 10 5 / puits) ou des particules de siARN PCPS / de commande (10 x 10 5 particules / 0,2 ug particules rapport siRNA, 10 nM et 100 nM d&#39;ARNsi) de 48 h et 72 h. Utilisation des cellules non traitées et des cellules traitées avec une solution saline tamponnée phosphate (même volume que les particules ajoutées) comme témoins. Doser chaque échantillon en triple exemplaire. </li><li> Effectuer un essai de prolifération de cellules selon les instructions du fabricant. </li><li> Représenter des données comme la moyenne ± écart-type. </li></ol> 13. Western Blot de cellules avec PCPS / ATM mutées particules siARN <ol><li> cellules de la plaque dans une plaque à 6 puits à une densité de cellules de 2 x 10 5 cellules / well pendant 24 heures. </li><li> Incuber les cellules avec siRNA PCPS / de commande (50 nM) de particules ou PCPS / ATM siRNA (50 nM) particules pour 72 h. Utilisation des cellules non traitées comme témoin. </li><li> Lyse des cellules en utilisant un réactif d&#39;extraction de protéine additionné d&#39;un cocktail d&#39;inhibiteurs de protease. </li><li> Centrifuger les lysats cellulaires pendant 10 min à 14 000 xg et récupérer le surnageant. </li><li> Déterminer la concentration en protéine par un dosage de protéine de quantification selon les instructions du fabricant. </li><li> Ajouter tampon de charge d&#39;échantillon (avec 5 pi 2-mercaptoéthanol / ml de tampon) pour les échantillons et les chauffer pendant 6 min à 99 ° C. </li><li> Charger les échantillons de protéine (20 ug / ul) dans un gel de SDS-polyacrylamide 12% en tampon en cours d&#39;exécution et exécuter une électrophorèse sur gel de Polyacrylamide (1 h, 120 V) en utilisant un équipement d&#39;électrophorèse et une alimentation électrique. </li><li> Transvaser le gel dans un tampon de transfert (avec 20% de methanol) à une membrane de nitrocellulose (1 h, 100 V) en utilisant un équipement d&#39;électrophorèse etune alimentation électrique. </li><li> Bloquer la membrane avec 5% de lait en poudre pendant 1 h. </li><li> Incuber la membrane avec l&#39;anticorps primaire ATM (de lapin) dans une solution de blocage (à partir de l&#39;étape 13.9) à une dilution de 1: O / N 1000. </li><li> Laver la membrane avec du tampon phosphate salin contenant 0,1% de polyethylene glycol sorbitan monolaurate, puis incuber avec l&#39;anticorps secondaire (anti-lapin) dans une solution de blocage (à partir de l&#39;étape 13.9) à une dilution de 1: 2500 pendant 1 heure. </li><li> Laver la membrane avec du tampon phosphate salin contenant 0,1% de polyéthylène glycol monolaurate de sorbitan et de détecter les bandes de protéines par Western blot avec un réactif de détection à l&#39;aide de l&#39;acquisition d&#39;image et un logiciel d&#39;analyse. </li><li> Pour le contrôle de chargement, laver la membrane et répétez les étapes de 13,9 à 13,12 utilisant un anticorps primaire β-actine (de la souris, 1: 10,000 dilution) et un anticorps secondaire (anti-souris 1: 4000 dilution). </li></ol>

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The authors declare that they have no competing financial interests.

Ce protocole décrit un procédé pour le succès de livraison de siRNA et la transfection dans les cellules. En particulier, la délivrance de siRNA est obtenue en utilisant une plate-forme multifonctionnelle constituée de particules de l&#39;ISP polycation-fonctionnalisé. L&#39;utilisation de la thérapie siRNA a un grand potentiel, par exemple, le traitement du cancer, que divers oncogènes peuvent être ciblées avec une grande spécificité. Par conséquent, il existe une demande pour développer des vé...

Les petits ARN interférents (siRNA) sont des molécules d&#39;ARN double brin qui peuvent supprimer l&#39;expression des gènes. Au cours des dernières années, siRNA ont été développés comme une nouvelle génération de biomédicaments qui montrent un potentiel thérapeutique pour une utilisation future dans les applications cliniques 1-5. Cependant, la mise en œuvre réussie de la thérapie siRNA reste un défi considérable, en raison de la dégradation par les nucléases, une mauvaise absorption intracellulaire, la faible efficacité de transfection et la libération inefficace de l&#39;endosome / 5 lysosome. Beaucoup de ces obstacles peuvent être surmontés par le développement de plates-formes de livraison, qui peuvent en toute sécurité et efficacement offrir siRNA pour les tissus malades. Comparé aux transporteurs virales, les plates-formes non-virales offrent plusieurs avantages, tels que la sécurité, le faible coût et la facilité d&#39;adaptation. En particulier, les nanoparticules cationiques, tels que des polymères et des lipides, se sont révélés utiles pour la délivrance de siRNA 3. Auparavant, nous avons développé un dr discoïdeug système de distribution, appelé le vecteur à plusieurs étages (MSV). Cette plate-forme est basée sur les étapes séquentielles, dans lequel un véhicule est libéré à partir de l&#39;autre. Le premier véhicule de l&#39;étape est un microparticules biodégradables fabriqués à partir de silicium poreux (PSI), tandis que les secondes véhicules de scène sont des nanoparticules chargées de médicaments ou d&#39;agents de contraste 6,7. Les nanoparticules qui sont noyés dans le matériau de PSI sont progressivement libérés comme le Si dégrade 8. Un avantage d&#39;utiliser des particules de Si est que les caractéristiques de morphologie et de surface peuvent être facilement adaptées pour réaliser la biodistribution et la libération du médicament optimal. Récemment, l&#39;utilisation réussie de la plate-forme MSV pour la fourniture de liposomes à siARN tissu tumoral a été démontré dans un modèle de souris de l&#39;ovaire et le cancer du sein 9, 10. Dans ce travail, nous avons fabriqué un système de distribution universel pour siRNA basée sur les principes de la plate-forme de MSV. L&#39;efficacité de ce système de livraison a déjà été démontré nousment différente des molécules de siRNA 11. Le système est un silicium poreux (PCPS) porte-polycation-fonctionnalisé, comprenant des ISP greffé avec la polyéthylèneimine (PEI) et l&#39;arginine (Arg). PEI peut aider à former des interactions électrostatiques avec des siRNA, tandis que Arg et psi peuvent servir à réduire la toxicité de PEI, comme précédemment demonstarted 11. En outre, la présence de PEI peut aider à l&#39;absorption intracellulaire et endosomal évasion, tandis que les microparticules de l&#39;ISP permettent la protection de siRNA et la libération prolongée. Les particules de l&#39;ISP dégradent peu à peu dans des conditions physiologiques, ce qui conduit à la formation de nanoparticules Arg-PEI / siRNA (figure 1), qui ont une morphologie distincte et une distribution de taille étroite 11. Pour plus de détails au sujet de la stabilité du système PCPS / siRNA, se il vous plaît se référer à l&#39;étude de Shen et al. 11. Cette plate-forme de PCPS diffère de la MSV classique, puisque les secondes nanoparticules de scène ne sont pas initialement Present dans le support, mais sont formées au fil du temps que le support de premier étage dégrade 11, 12. L&#39;efficacité siRNA chargement efficacité, la cytotoxicité et silençage génique du système PCPS a été évaluée in vitro. L&#39;efficacité de transfection a été mesurée en utilisant le siRNA contre l&#39;ataxie télangiectasie mutée (ATM) oncogene, qui est impliqué dans la réparation de l&#39;ADN 10. Auparavant, la suppression de l&#39;ATM a été montré pour diminuer la croissance tumorale dans un modèle de cancer du sein 10.

<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.

Ce protocole décrit l&#39;utilisation d&#39;un système de distribution non viral sûr et efficace pour la transfection de siRNA. Les résultats de SEM montrent que les particules PCPS sont de forme cylindrique et ont un diamètre de 2,6 um (figure 2A). Les particules sont chargées positivement à un potentiel zêta d&#39;environ 8,21 (figure 2B), ce qui permet la liaison électrostatique avec des nucleotides chargés négativement. Les images confocales de différentes couches de par...

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

Livraison reste le principal défi pour la mise en œuvre thérapeutique de petits ARN interférents (siRNA). Ce protocole implique l&#39;utilisation d&#39;une plate-forme de délivrance de siRNA biocompatible et multifonctionnel, constitué par l&#39;arginine et polyéthylènimine greffé microparticules de silicium poreux.

Silicium poreux microparticules pour la livraison de 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 ...

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

Research

JoVE Journal

Bioengineering

10.8K vues.  Houston Methodist Research Institute.  Livraison reste le principal défi pour la mise en œuvre thérapeutique de petits ARN interférents (siRNA). Ce protocole implique l'utilisation d'une plate-forme de délivrance de siRNA biocompatible et multifonctionnel, constitué par l'arginine et polyéthylènimine greffé microparticules de silicium poreux. 

Vidéo: 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.

La fonctionnalisation douce lithographiques et les motifs sans oxyde silicium et de germanium

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

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Bio-ingénierie

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.

Les échafaudages auto-déclaration pour les 3-Dimensional Culture cellulaire

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.

Préparation de la Silicon Nanowire à effet de champ Transistor pour chimiques et les biocapteurs Applications

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.

Synthèse de thermogélifiant Poly (N-isopropylacrylamide) -graft-chondroïtine Composites Sulfate avec Alginate Microparticules pour le génie tissulaire

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.

Livraison de siRNA thérapeutique du SNC Utilisation cationique et anionique Liposomes

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.

Conception de silicium poreux Films comme porteurs du facteur de croissance nerveuse

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 imprimés Cellulose poreuse Nanocomposite Hydrogel échafaudages

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.

Préparation de nanoparticules polymériques à charge neutralement et sensibles au pH pour la livraison de siRNA cytosoliques

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.

Nanofils de silicium et stimulation optique pour les études de couplage électrique intra- et intercellulaire

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.

Outils pour le traitement de surface des microélectrodes intracorticales planes au silicium

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