Ce travail a été financé par des subventions du NIH: HL671795, HL048743 et HL108630.

1. Préparation de SDF-1α Glycan nanoparticules En raison de la fin de la livraison in vivo, stériliser tous les récipients, pipettes et embouts utilisés dans la préparation. <ol><li> Préparer les solutions d&#39;achat d&#39;actions suivantes dans UltraPure Eau: 1% de sulfate de dextran; 1 M NaOH (stérile filtrée par une membrane PES); 0,1% de chitosane à 0,2% d&#39;acide acétique glacial (0,8 filtre à travers des filtres de 0,22 um et de façon consécutive et ajuster le pH à 5,5 avec du NaOH après); ZnSO 4 0,1 M; 15% de mannitol; et 0,92 mg / ml SDF-1α (stockée en aliquotes à 80 ° C, et maintenu à 4 ° C, une fois décongelés). </li><li> Stériliser les solutions mères de 0,22 um membranes filtrantes. Évaluer les niveaux d&#39;endotoxines dans la solution préparée avec dosage lysat d&#39;amibocytes de limule (LAL) gel de caillot. Veiller à ce que les niveaux sont &lt;0,06 EU / ml. </li><li> Ajouter 0,18 ml UltraPure eau pour un flacon en verre de 1,5 ml contenant une barre d&#39;agitation magnétique. Réglez la vitesse d&#39;agitation à 800 tpm. Ajouter 0,1 ml 1%le sulfate de dextrane et agiter pendant 2 min. Ajouter 0,08 mg SDF-1α (0,087 ml de 0,92 mg / ml SDF-1α solution) et agiter pendant 20 min. </li><li> Ajouter 0,33 ml 0,1% goutte à goutte de chitosane et remuer pendant 5 min. Modifier la vitesse d&#39;agitation au maximum et ajouter 0,1 ml de 0,1 M de ZnSO 4 lentement avec une seringue 0,1 ml (plus de 1 min). </li><li> La vitesse d&#39;agitation revenir à 800 tpm et remuer pendant 30 min. Ajouter 0,4 ml 15% de mannitol et remuer pendant 5 min. Transférer le mélange réactionnel dans un tube de microcentrifugation de 1,5 ml. Centrifuger à 16 000 xg à 4 ° C pendant 10 min. Remarque: La présence de mannitol facilite la remise en suspension des particules après centrifugation. En fonction de la compacité de la pastille, la concentration en mannitol peut varier de 0% à 5%. Remise en suspension des particules après chaque centrifugation est essentielle pour éviter des agrégats dans la suspension finale. </li><li> Aspirer le surnageant et utiliser une pipette pour enlever la dernière goutte de liquide lentement. Ajouter 0,2 ml 5% de mannitol. Suspendre le culot avec 0,5 ml, 29 G neeseringue dle. Ajouter 1 ml 5% de mannitol. Centrifuger à 16 000 g pendant 15 min. </li><li> Répétez l&#39;étape 1.6. </li><li> Aspirer le surnageant. Reprendre le culot dans 0,2 ml 5% de mannitol. Conservez la suspension de particules à 4 ° C. Remarque: La suspension de particules peut également être conservé congelé à -80 ° C ou lyophilisé. Dont 5% de mannitol dans la suspension est essentiel pour empêcher l&#39;agrégation des particules après congélation et décongélation ou après lyophilisation et réhydratation. Le mannitol peut être éliminé par centrifugation de la suspension après le stockage. </li></ol> 2. Mesure de la taille des particules et potentiel Zeta La taille des particules et le potentiel zêta sont analysées par diffusion de lumière dynamique et électrophorétique diffusion de la lumière, respectivement, avec un analyseur de particules indiqué dans la liste de matériaux. <ol><li> Définissez les paramètres suivants pour la mesure de la taille des particules: temps d&#39;accumulation: 70; Répéter fois: 4; Température: 25 ° C;Diluant: eau; réglage de l&#39;intensité: auto. </li><li> Diluer l&#39;échantillon SDF-1α-DS-CS avec de l&#39;eau (10 dilution). Charger 100 pl de l&#39;échantillon dans une cuvette UV jetable tel qu&#39;une cuvette Eppendorf. Insérer la cuve dans le porte-cellule. Attendez que le réglage de l&#39;intensité pour atteindre &quot;Optimum&quot; (~ 10 000 cps). Lancer l&#39;acquisition de données. </li><li> Une fois la mesure terminée, enregistrer les résultats de cumulants diamètre (nm) et l&#39;indice de polydispersité. La moyenne des résultats obtenus à partir de chacune des quatre lectures répétées et calculer l&#39;écart-type. </li><li> Charge 500 ul de l&#39;échantillon de particules dilué 10 fois à une cellule d&#39;écoulement pour la mesure du potentiel zêta. Définissez les paramètres suivants pour la mesure: temps d&#39;accumulation: 10; Répéter fois: 5; Température: 25 ° C; Diluant: eau; réglage de l&#39;intensité: auto. Consigner les résultats de la potentiel zêta (mV). Moyenne, le résultat obtenu par chacune des cinq lectures répétées, et calculer la norme deviation. </li></ol> 3. Quantification des SDF-1α dans les particules <ol><li> Diluer forme libre SDF-1α à des concentrations de 0,01, 0,02, 0,03, 0,04 et 0,05 mg / ml dans un tampon d&#39;échantillon Laemmli 1.3x. Diluer 6 pi échantillons SDF-1α-DS-CS avec 40 ul 1.3x tampon d&#39;échantillon Laemmli. (Préparer 4x Laemmli stocks tampon contenant 0,25 M Tris-HCl, pH 7,5, 8% de SDS, 40% de glycérol, 0,05 mg / ml de bleu de bromophénol, et 8% de 2-mercaptoéthanol. Divisez la solution mère dans de petites fractions et de garder à -20 ° C à usage unique.) </li><li> Chauffer les échantillons à 100 ° C pendant 10 min. Vortex les échantillons deux fois (chacun pendant 10 secondes à la vitesse maximale) pendant le temps de chauffage de 10 min pour dissocier les particules complètement. Refroidir les échantillons à température ambiante pendant 2 min. Centrifuger à 10 000 g pendant 0,5 à 1 min pour faire baisser le condensat de l&#39;eau. Vortex à nouveau pour bien mélanger. Remarque: À ce stade, la solution de l&#39;échantillon doit être clair sans précipité visible présente. </li><li> Charge 10 &amp;# 181; l &#39;échantillon / puits d&#39;un gel de SDS à 4-20%. Exécutez électrophorèse à 200 V pendant 20 min. Colorer le gel avec Coomassie protéines bleuissement. </li><li> Examiner la densité de la bande de protéine de SDF-1α en utilisant un logiciel d&#39;imagerie et d&#39;analyse de densitométrie moléculaire. Calculer la quantité de SDF-1α dans les particules contre une courbe standard construite aux normes SDF-1α gratuits. </li></ol> 4. Dosage de libération in vitro <ol><li> Mélanger des particules de glycane SDF-1α avec une solution saline tamponnée au phosphate de Dulbecco sans calcium et magnésium (D-PBS) à un rapport 1: 1 (v / v). </li><li> Divisez la suspension de particules dans 0,05 ml aliquotes dans 1,5 ml tubes. Charger les échantillons au fond des tubes. Éviter l&#39;introduction de bulles d&#39;air ou de troubler la surface du liquide. Sceller le haut des tubes avec du parafilm. Remarque: Ce faisant, le liquide reste au fond lors de la rotation de haut en bas sur le mélangeur ultérieur du tube. </li><li> Tournez les tubes à 37 ans &amp;# 176; C sur une rotation Micro-Tube Mixer. Enlever des parties aliquotes des tubes aux moments désignés et centrifuger les échantillons immédiatement à 16 000 xg pendant 10 min à 4 ° C. </li><li> Séparer les surnageants et granulés, et reconstituer le culot avec 0,05 ml de D-PBS. Conserver les échantillons à -20 ° C. Après que tous les échantillons sont prélevés, examiner les surnageants et culots sur un gel de SDS comme décrit ci-dessus. </li></ol> 5. Migration Assay Ce test mesure l&#39;activité chimiotactique des SDF-1α. Interaction de SDF-1α avec son récepteur (CXCR4) sur la surface de la cellule provoque la migration de la cellule en direction du gradient SDF-1α. Dans ce dosage, les cellules sont chargées dans un puits supérieur (séparés par une membrane semi-perméable à partir d&#39;un puits inférieur) solution SDF-1α et dans une bien moindre. <ol><li> Diluer SDF-1α (libre ou liée à la particule forme) avec le tampon de migration (milieu RPMI-1640 contenant 0,5% d&#39;albumine de sérum bovin) dans un trois-fold dilution en série à des concentrations finales de 100, 33, 11, 3,7, 1,2, 0,41, 0,14, et 0,05 ng / ml. </li><li> Ajouter 0,6 ml de la solution SDF-1α dilué ou tampon de migration uniquement (contrôle négatif) à un puits dans la plaque de 24 puits. Ajouter 0,57 ml de tampon de migration vers un puits (contrôle de la cellule d&#39;entrée). Incuber à 37 ° C pendant 30 min. Placer un perméable insert de culture cellulaire tel que Transwell (taille des pores 5 um, diamètre 6,5 mm) au-dessus du puits inférieur. Charge 0,1 ml des cellules de Jurkat (5 × 10 5) dans l&#39;insert Transwell. Chargez 0,03 ml de cellules directement à la commande de cellules d&#39;entrée ainsi. Incuber la plaque à 37 ° C pendant 2 heures dans un incubateur à CO2 à 5%. </li><li> Retirez les inserts Transwell. Transférer les cellules qui ont migré vers les puits inférieurs à un tube 4 ml de polystyrène. Comptez le nombre de cellules ayant migré avec un cytomètre de flux. </li><li> Calculer la migration en pourcentage du nombre de cellules d&#39;entrée (nombre de cellules dans le contrôle de la cellule d&#39;entrée et x 100/30) après soustraction des nombres dans negative contrôles (cellules ayant migré dans les puits contenant tampon de migration uniquement). </li></ol>

<ol>
	<li>Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+polyelectrolyte+complexes+of+chitosan+and+dextran+sulfate+towards+versatile+nanocarriers+of+bioactive+molecules.">Colloidal polyelectrolyte complexes of chitosan and dextran sulfate towards versatile nanocarriers of bioactive molecules.</a> Eur J Pharm Biopharm. 78 (1), 10-18 (2011).</li><li>Morachis, J. M., Mahmoud, E. A., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+strategies+for+therapeutic+delivery+by+using+polymeric+nanoparticles.">Physical and chemical strategies for therapeutic delivery by using polymeric nanoparticles.</a> Pharmacol Rev. 64 (3), 505-519 (2012).</li><li>Yue, Z. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+charge+affects+cellular+uptake+and+intracellular+trafficking+of+chitosan-based+nanoparticles.">Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles.</a> Biomacromolecules. 12 (7), 2440-2446 (2011).</li><li>Ghadge, S. K., Muhlstedt, S., Ozcelik, C., Bader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDF-1alpha+as+a+therapeutic+stem+cell+homing+factor+in+myocardial+infarction.">SDF-1alpha as a therapeutic stem cell homing factor in myocardial infarction.</a> Pharmacol Ther. 129 (1), 97-108 (2011).</li><li>Sharma, M., Afrin, F., Satija, N., Tripathi, R. P., Gangenahalli, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-derived+factor-1/CXCR4+signaling:+indispensable+role+in+homing+and+engraftment+of+hematopoietic+stem+cells+in+bone+marrow.">Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow.</a> Stem Cells Dev. 20 (6), 933-946 (2011).</li><li>Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+stromal+cell-derived+factor-1alpha-heparin+complex.">Characterization of the stromal cell-derived factor-1alpha-heparin complex.</a> J Biol Chem. 276 (11), 8288-8296 (2001).</li><li>Amara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell-derived+factor-1alpha+associates+with+heparan+sulfates+through+the+first+beta-strand+of+the+chemokine.">Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine.</a> J Biol Chem. 274 (34), 23916-23925 (1999).</li><li>Sadir, R., Imberty, A., Baleux, F., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate/heparin+oligosaccharides+protect+stromal+cell-derived+factor-1+(SDF-1)/CXCL12+against+proteolysis+induced+by+CD26/dipeptidyl+peptidase+IV.">Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV.</a> J Biol Chem. 279 (42), 43854-43860 (1074).</li><li>Yin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDF-1alpha+in+glycan+nanoparticles+exhibits+full+activity+and+reduces+pulmonary+hypertension+in+rats.">SDF-1alpha in glycan nanoparticles exhibits full activity and reduces pulmonary hypertension in rats.</a> Biomacromolecules. 14 (11), 4009-4020 (2013).</li><li>Huang, M., Vitharana, S. N., Peek, L. J., Coop, T., Berkland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+complexes+stabilize+and+controllably+release+vascular+endothelial+growth+factor.">Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor.</a> Biomacromolecules. 8 (5), 1607-1614 (2007).</li><li>McCall, R. L., Sirianni, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA+nanoparticles+formed+by+single-+or+double-emulsion+with+vitamin+E-TPGS.">PLGA nanoparticles formed by single- or double-emulsion with vitamin E-TPGS.</a> J Vis Exp. (82), 51015 (2013).</li><li>Carrillo-Conde, B. R., Roychoudhury, R., Chavez-Santoscoy, A. V., Narasimhan, B., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+synthesis+of+carbohydrates+and+functionalization+of+polyanhydride+nanoparticles.">High-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles.</a> J Vis Exp. (65), 3967 (2012).</li><li>Xu, J., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+gene+delivery+and+transfection+in+human+pancreatic+cancer+cells+using+epidermal+growth+factor+receptor-targeted+gelatin+nanoparticles.">Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles.</a> J Vis Exp. (59), e3612 (2012).</li><li>Lauten, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoglycan+complex+formulation+extends+VEGF+retention+time+in+the+lung.">Nanoglycan complex formulation extends VEGF retention time in the lung.</a> Biomacromolecules. 11 (7), 1863-1872 (2010).</li><li>Schatz, C., Domard, A., Viton, C., Pichot, C., Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+and+efficient+formation+of+colloids+of+biopolymer-based+polyelectrolyte+complexes.">Versatile and efficient formation of colloids of biopolymer-based polyelectrolyte complexes.</a> Biomacromolecules. 5 (5), 1882-1892 (2004).</li></ol>

The authors declare that they have no competing financial interests.

Comme mentionné ci-dessus, les nanoparticules DS-CS sont formés par neutralisation de la charge entre le polyanion (DS) et de polycation (CS) molécules. Étant donné que l&#39;interaction de charge se produit facilement pendant la collision moléculaire, la concentration des solutions de polymère et la vitesse d&#39;agitation au cours du mélange est critique pour la taille des particules résultantes. Une tendance générale est que plus dilué DS et CS solutions 15 et le résultat de la vitesse d&#39;a...

Sulfate de dextran (DS) et le chitosan (CS) sont des polysaccharides avec plusieurs groupes sulfate chargés négativement substitués (dans DS) ou des groupes aminés chargés positivement (désacétylés CS). Lorsqu&#39;il est mélangé à une solution aqueuse, les deux polysaccharides forment des complexes polyélectrolytiques par des interactions électrostatiques. Les complexes résultants peuvent former de gros agrégats qui seront phases séparées de la solution aqueuse (précipités), ou de petites particules qui sont dispersables dans l&#39;eau (colloïdes). Les conditions spécifiques qui contribuent à ces résultats ont été largement étudiées et ont été résumés et illustrés en détail dans une étude récente 1. Parmi ces conditions, deux conditions de base pour la production de particules dispersibles dans l&#39;eau sont les polymères de charges opposées doivent 1) ont une masse molaire sensiblement différente; et 2) être mélangés dans un rapport non stoechiométrique. Ces conditions permettront aux segments polymères complexés charge neutre produites par la chargeneutralisation de séparer et de former le noyau de la particule, et le polymère en excès pour former la coque externe 1. Les particules glycanes décrites dans ce protocole sont destinés à une administration pulmonaire, et sont conçus pour être nette chargé négativement, et de dimensions nanométriques. La charge de surface négative réduit la probabilité d&#39;absorption cellulaire des particules 2,3. Les particules de dimension nanométrique de faciliter le passage à travers les voies aériennes distales. Pour atteindre cet objectif, la quantité de DS utilisé dans cette préparation est supérieure à CS (rapport pondéral 3: 1); et de haute masse moléculaire DS (moyenne en poids MW 500 000) et de faible poids moléculaire CS (gamme de 50 à 190 MW kDa, 75-85% désacétylé) sont utilisés. SDF-1α est un facteur de ralliement de cellules souches, qui exerce la fonction de référencement à travers son activité chimiotactique. SDF-1α joue un rôle important dans la prise d&#39;origine et le maintien des cellules souches hématopoïétiques dans la moelle osseuse, et dans le recrutement de progecellules Nitor à tissus périphériques à la réparation des lésions de 4,5. SDF-1α a un site de liaison à l&#39;héparine dans la séquence de la protéine, ce qui permet à la protéine de se lier à l&#39;héparine / sulfate d&#39;héparane, former des dimères, être protégés contre la protéase (CD26 / DPPIV) inactivation, et d&#39;interagir avec des cellules cibles par l&#39;intermédiaire des récepteurs de surface cellulaire 6-8. DS a des propriétés structurelles similaires à celles de l&#39;héparine / héparane-sulfate; Ainsi, la liaison du SDF-1α pour DS serait similaire à celui de ses ligands polymères naturels. Dans le protocole suivant, nous décrivons la préparation de nanoparticules SDF-1α-DS-CS. Les procédures représentent l&#39;une des formules qui ont été précédemment étudiés 9. Le protocole est à l&#39;origine adapté d&#39;une enquête de nanoparticules VEGF-DS-CS 10. Une petite préparation à grande échelle est décrite, qui peut être facilement mise à l&#39;échelle avec les mêmes solutions d&#39;achat d&#39;actions et les conditions de préparation. Après la préparation, les particules sont caractérisées by examiner leur taille, le potentiel zêta, degré d&#39;incorporation SDF-1α, in vitro temps de libération, et l&#39;activité du SDF-1α incorporé.

<table border="0" cellpadding="0" cellspacing="0" width="489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Name</td>
			<td style="width:136px;">Company</td>
			<td style="width:99px;">Catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulfate</td>
			<td>Fisher</td>
			<td>BP1585-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, low molecular weight&#160;</td>
			<td>Sigma</td>
			<td>448869</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>204986</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure water&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>10977-023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>Prepared according to reference 8.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, PES membrane 0.22 um.&#160;&#160;&#160;</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic Micro Stirring Bars (2 x 7 mm)</td>
			<td>Fisher&#160;</td>
			<td>14-513-63</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass vial Kit; SUN-SRi</td>
			<td>Fisher&#160;</td>
			<td>14-823-182</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Delsa Nano C Particle Analyzer&#160;</td>
			<td>Backman Coulter</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf UVette Cuvets</td>
			<td>Eppendorf</td>
			<td>952010069</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4&#8211;20% Mini-PROTEAN TGX Gel</td>
			<td>Bio-Rad</td>
			<td>456-1096</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GelCode Blue Safe Protein Stain</td>
			<td>Fisher&#160;</td>
			<td>PI-24592</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Imager VersaDoc MP 4000 System</td>
			<td>BioRad</td>
			<td>170-8640</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Transwell Permeable Supports</td>
			<td>Corning</td>
			<td>3421</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accuri C6 Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>D8537</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyrogent plus kit</td>
			<td>Fisher</td>
			<td>NC9753738</td>
		</tr>
	</tbody>
</table>

preparation and characterization of sdf-1&#945;-chitosan-dextran sulfate nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

La taille et le potentiel zéta des particules SDF-1α-DS-CS préparés sont déterminées avec un analyseur de particules. La figure 1 montre l&#39;analyse de la mesure de la taille. A partir des résultats obtenus à partir des cumulants quatre mesures répétées, le diamètre hydrodynamique moyen des particules SDF-1α-DS-CS est 661 ± 8,2 (nm) et la polydispersité est de 0,23 ± 0,02. Le résultat de la mesure du potentiel zêta est représenté sur la Figure 2. A partir des cinq ...

Watch this Scientific Journal Video about Preparation and Characterization of SDF-1Î±-Chitosan-Dextran Sulfate Nanoparticles at JoVE.com

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Préparation et caractérisation des SDF-1α-chitosane-Sulfate de Dextran nanoparticules

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

preparation-characterization-sdf-1-chitosan-dextran-sulfate

Research

JoVE Journal

Chemistry

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

12.3K vues.  Brigham and Women's Hospital and Harvard Medical School. The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

Vidéo: Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Préparation de nanoparticules de silice Grâce assistée par micro-ondes acide catalyse

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Recherche

Chimie

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Préparation et caractérisation de la personne et de multi-drogue Loaded Physiquement piégé micelles polymériques

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Synthèse, caractérisation et fonctionnalisation des hybrides Au / CdS et Au / ZnS de base / Shell Nanoparticules

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Synthèse et caractérisation de supramoléculaires Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

Séparation Sulfate par Selective Cristallisation avec un Ligand Bis-iminoguanidinium

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Préparation et<em&gt; In vitro</em&gt; Caractérisation des agents de contraste à base de dendrimères pour imagerie par résonance magnétique

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Préparation et caractérisation de Nanostructures hybrides de C60/Graphene

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

Induite par le ligand de nucléation et croissance de nanoparticules métalliques de Palladium

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Caractérisation physique à haute résolution des nanoparticules métalliques uniques

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Synthèse et caractérisation des nanoparticules d'or amphiphiles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...