Les auteurs remercient le prix ONR-MURI (NN00014-06-1-1176) et le US Army Medical Research et du matériel de commande (Grant No. W81XWH-10-1-0806) pour le soutien financier. Ce matériel est basé sur des travaux appuyés par le National Science Foundation Grant n ° 0552584 et 0851519 CEE.

1. Combinatoire Polymer Bibliothèque de synthèse (variant en chimie des polymères) - voir Figure 1 pour l&#39;installation robotisée <ol><li> Dissoudre chaque monomère dans le solvant approprié (concentration = 25 mg / ml) et charger chaque gaz dans une seringue étanche à 10cc. </li><li> Fixer les tubes de verrouillage résistant aux solvants leurres capillaires à la fin de chaque seringue. </li><li> Placez les seringues sur les pompes à seringues (New Era Pompes seringues programmables) et la verrouiller en position. </li><li> Définir les actionneurs linéaires (Zaber) à la position de départ. </li><li> Grâce aux pinces support universel, positionner l&#39;extrémité des deux tubes capillaires dans le flacon de départ / puits pour le dépôt de monomère. </li><li> Lancer le programme LabVIEW, qui pompe des volumes variables de chaque monomère dans chaque puits en fonction de la composition copolymère désirée. Ce résultat est obtenu en chargeant le programme de l&#39;actionneur de l&#39;axe Z pour abaisser les tubes capillaires dans le flacon / puits, puis chaque pompe pour distribuer le volume désiré. Next, le programme ordonne la Z-actionneur pour revenir à sa position d&#39;origine et les X et Y actionneurs pour déplacer la position de la fiole suivante / puits. Ceci est effectué jusqu&#39;à ce que chacun d&#39;eux a bien le volume souhaité de monomère qui y sont déposées. </li><li> Après le dépôt de monomère, la bibliothèque monomère multi-puits ou multi-flacon est transféré dans un four pré-chauffé sous vide et on incube sous vide pendant la durée de la réaction de polymérisation par condensation. Pour CPH: synthèse SA, la réaction est effectuée à 180 ° C, 0,3 torr, pendant 1,5 heures, mais ces conditions de réaction varient entre différents systèmes polymères. </li></ol> 2. Blank combinatoire et en protéines chargé de nanoparticules de polymère et de fabrication Cinémathèque - voir Figure 1 pour l&#39;installation robotisée <ol><li> La seringue dans la pompe à seringue programmable première est rempli avec un solvant (bibliothèque vide) ou avec un solvant protéine dispersée dans celui-ci (protéine chargée en bibliothèque), tandis que la seringue dans la seconde seringuepompe reste vide. Des tubes pour la fabrication de nanoparticules dans le porte-échantillon adjacent sont remplies avec le non-solvant (rapport du solvant au non-solvant est de 1 à 100). Pour la fabrication de film d&#39;un vide plaque multi-puits est utilisée à la place des tubes dans le porte-échantillon adjacent. </li><li> En utilisant le programme LabVIEW, le solvant est déposé dans tous les flacons polymère / puits de la bibliothèque (= concentration de 20 mg / ml) et incubé pendant 1-5 min. Une étape de sonication en option (30 s à 40 Hz) peut être introduit pour assurer la dissolution complète de polymère. </li><li> Ensuite, en lançant un programme distinct LabVIEW, l&#39;échantillon est retiré dans la seringue vide, et déposé dans son tube correspondant de puits non-solvant (nanoparticules) ou vide (films) dans le porte-échantillon adjacent. </li><li> Ce procédé est effectué pour chaque composition de polymère discrète de la bibliothèque. </li><li> La nanoparticule ou de la bibliothèque de film est alors placé dans une chambre à vide pour l&#39;élimination du solvant et de non-solvant (la balance des nanoparticulesry peut aussi être récupéré par filtration sous vide). </li></ol> 3. À haut débit cinétique de libération de protéines <ol><li> Pour étudier haut débit cinétique de libération de protéines à partir d&#39;une bibliothèque polymère, un 96 profond jailli (2 ml / puits), plaque de polypropylène est modifiée de sorte que le 1/3 supérieur de la paroi du puits dans les colonnes voisines (ex: A et B, C et D, E et F, G et H) a été retirée pour se raccordent les puits. Les films de protéines chargées doivent être fabriqués dans des nanoparticules ou transférés dans cette plaque pour effectuer à haut débit libération études cinétiques. Échantillons de protéines chargé de nanoparticules ou de film ne doit être placé dans un puits des colonnes adjacentes (ex: A, C, E et G). Voir la figure 4 pour plus de détails. </li><li> Après le transfert des nanoparticules à la plaque à 96 libération profonde jailli, les particules peuvent se déposer. Tampon PBS (0,1 mM, pH 7,4) est ajouté lentement à chaque puits d&#39;échantillon (colonnes A, C, E et G), puis à chaque puits voisin (colonnes B, D, F et H) jusqu&#39;à ce que les puits sont pleins et le tampon est à écoulement libre entre les puits adjacents. Pour les nanoparticules, ce processus doit être effectué avec prudence pour s&#39;assurer que les particules restent au bas de l&#39;échantillon et colonnes (A, C, E et G) et non transférées dans la version attenante bien (colonnes B, D, F et H). Dans certains cas, la centrifugation est nécessaire pour localiser des échantillons de nanoparticules au fond du puits d&#39;échantillon (colonnes A, C, E et G). </li><li> Ensuite, la plaque de séparation est fermée par un couvercle et placé à la température souhaitée (par exemple 37 ° C) sous agitation pendant la durée de l&#39;expérience. </li><li> À certains moments supplémentaires (soit 0,04, 1, 2, 3, 5, 7, 9, 12, 15, 19, 24 et 30 jours) le montant des libérés conjugué à un fluorochrome protéine est quantifié à l&#39;aide d&#39;un Typhoon 9400 Scanner à plat fluorescent ( GE Healthcare). La plaque de libération est placé sur la surface du scanner, numériser à l&#39;aide du laser d&#39;excitation et d&#39;émission appropriés filters, et l&#39;intensité de la fluorescence de la protéine libérée dans le puits de sortie (colonnes B, D, F et H) est quantifiée (Quant image). Il est suggéré que les normes de protéines de concentrations connues être inclus dans la plaque et pour le calcul de la quantité de protéines et de comptabilité pour la trempe fluorochrome. </li></ol> 4. Les résultats représentatifs Lors de la fabrication de la bibliothèque de polymère, la caractérisation a été réalisée avec 1 H RMN, GPC, et FTIR pour valider cette méthode combinatoire 1,7,8,11. Moléculaire gamme de poids 10,000-20,000 g / mol, gammes indice de polydispersité de 1,5 à 3,0, et la composition chimique a été montré pour être précis et en accord avec les méthodes conventionnelles de 12-15 synthèse polyanhydride. De même, les images MEB des bibliothèques nanoparticules a montré une morphologie de surface similaire, la taille et distribution de taille que celle des nanoparticules fabriquées traditionnellement 2. Protéines communiqué kinetics à partir de nanoparticules de polyanhydride ou de films est effectué dans une plaque de puits à jour comme décrit précédemment 1. Les résultats ont révélé une libération d&#39;ordre zéro approximative avec ou sans une rafale dépend de la charge en protéines et chimie des polymères (figures 2 et 3) 1,12,14,16. <img alt="Figure 1" src="/files/ftp_upload/3882/3882fig1.jpg" /> Figure 1. Combinatoire film polymère et un appareil de fabrication de nanoparticules. <img alt="Figure 2" src="/files/ftp_upload/3882/3882fig2.jpg" /> Figure 2 haut-débit libération de Texas Red sérum albumine bovine (TRBSA) à partir d&#39;un CPH:. Bibliothèque SA nanoparticules de polymère. SA riches en chimies de polymère encapsulé TRBSA libérer le plus rapidement, alors que CPH-riches chimies de polymère libérer le plus lent. Les barres d&#39;erreur représentent écart typetion et n = 4. Reproduit avec la permission de Petersen et al. al. 1. Copyright 2010 American Chemical Society. <img alt="Figure 3" src="/files/ftp_upload/3882/3882fig3.jpg" /> Figure 3 à haut débit libération de Texas Red sérum albumine bovine (TRBSA) à partir d&#39;un CPTEG:. Bibliothèque CPH film polymère. CPTEG riches en chimies de polymère encapsulé TRBSA libérer le plus rapidement, alors que CPH-riches chimies de polymère libérer le plus lent. Les barres d&#39;erreur représentent l&#39;écart type et n = 3. <img alt="Figure 4" src="/files/ftp_upload/3882/3882fig4.jpg" /> Figure 4. Image montrant deux puits voisins &quot;avant&quot; et &quot;après&quot; modification de la plaque 96 en polypropylène profonde jailli. L&#39;image &quot;après&quot; modification de droite représente également l&#39;addition d&#39;un film polymère (en bas à gauche du puits) avec une molécule fluorescente encapsuléed&#39;être libéré entre les deux puits dans une solution tampon. La molécule libérée fluorescente est ensuite détectée dans le droit bien.

<ol>
	<li>Petersen, L. K., Sackett, C. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+high-throughput+method+to+study+in+vitro+protein+release+from+polymer+nanospheres.">A novel, high-throughput method to study in vitro protein release from polymer nanospheres.</a> J. Comb. Chem. 12, 51-56 (2010).</li><li>Petersen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+innate+immune+responses+in+a+pathogen-mimicking+manner+by+amphiphilic+polyanhydride+nanoparticle+adjuvants.">Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants.</a> Biomaterials. 32, 6815-6822 (2011).</li><li>Vogel, B. M., Cabral, J. T., Eidelman, N., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+synthesis+and+high-throughput+dissolution+testing+of+biodegradable+polyanhydride+copolymers.">Parallel synthesis and high-throughput dissolution testing of biodegradable polyanhydride copolymers.</a> J. Comb. Chem. 7, 921-928 (2005).</li><li>Petersen, L. K. <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+evaluation+of+in+vivo+biodistribution+of+polyanhydride+nanoparticles.">High-throughput evaluation of in vivo biodistribution of polyanhydride nanoparticles.</a> Adv. Healthcare Mater. , (2012).</li><li>Petersen, L. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+design+of+biomaterials+for+drug+delivery:+opportunities+and+challenges.">Combinatorial design of biomaterials for drug delivery: opportunities and challenges.</a> Expert Opin. Drug Deliv. 5, 837-846 (2008).</li><li>Petersen, L. K., Oh, J., Sakaguchi, D. S., Mallapragada, S. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiphilic+polyanhydride+films+promote+neural+stem+cell+adhesion+and+differentiation.">Amphiphilic polyanhydride films promote neural stem cell adhesion and differentiation.</a> Tissue Eng. 17, 2533-2541 (2011).</li><li>Petersen, L. K., Sackett, C. K., Narasimhan, B. <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+analysis+of+protein+stability+in+polyanhydride+nanoparticles.">High-throughput analysis of protein stability in polyanhydride nanoparticles.</a> Acta Biomater. 6, 3873-3881 (2010).</li><li>Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+simultaneous+effect+of+polymer+chemistry+and+device+geometry+on+the+in+vitro+activation+of+murine+dendritic+cells.">The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells.</a> Biomaterials. 30, 5131-5142 (2009).</li><li>Thorstenson, J. B., Petersen, L. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial/high-throughput+methods+for+the+determination+of+polyanhydride+phase+behavior.">Combinatorial/high-throughput methods for the determination of polyanhydride phase behavior.</a> J. Comb. Chem. 11, 820-828 (2009).</li><li>Xue, L., Petersen, L., Broderick, S., Narasimhan, B., Rajan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+factors+controlling+protein+release+from+combinatorial+biomaterial+libraries+via+hybrid+data+mining+methods.">Identifying factors controlling protein release from combinatorial biomaterial libraries via hybrid data mining methods.</a> ACS Comb. Sci. 13, 50-58 (2011).</li><li>Adler, A. F. <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+cell-based+screening+of+biodegradable+polyanhydride+libraries.">High-throughput cell-based screening of biodegradable polyanhydride libraries.</a> Comb. Chem. High Through. Screen. 12, 634-645 (2009).</li><li>Determan, A. S., Trewyn, B. G., Lin, V. S., Nilsen-Hamilton, M., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation,+stabilization,+and+release+of+BSA-FITC+from+polyanhydride+microspheres.">Encapsulation, stabilization, and release of BSA-FITC from polyanhydride microspheres.</a> J. Control. Release. 100, 97-109 (2004).</li><li>Determan, A. S., Wilson, J. H., Kipper, M. J., Wannemuehler, M. J., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+stability+in+the+presence+of+polymer+degradation+products:+consequences+for+controlled+release+formulations.">Protein stability in the presence of polymer degradation products: consequences for controlled release formulations.</a> Biomaterials. 27, 3312-3320 (2006).</li><li>Torres, M. P., Determan, A. S., Anderson, G. L., Mallapragada, S. K., Narasimhan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiphilic+polyanhydrides+for+protein+stabilization+and+release.">Amphiphilic polyanhydrides for protein stabilization and release.</a> Biomaterials. 28, 108-116 (2007).</li><li>Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+novel+polyanhydrides+with+tailored+erosion+mechanisms.">Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms.</a> J. Biomed. Mater. Res. A. 76, 102-110 (2006).</li><li>Carrillo-Conde, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+into+amphiphilic+polyanhydride+microparticles+stabilizes+Yersinia+pestis+antigens.">Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens.</a> Acta Biomater. 6, 3110-3119 (2010).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Connaissance des conditions de synthèse nécessaires et les températures de transition vitreuse (Tg) des polymères étant synthétisés sont indispensables pour la fabrication de la bibliothèque. Si la Tg est inférieure à la température ambiante, l&#39;étape de fabrication de nanoparticules peuvent avoir besoin d&#39;être effectuée dans un environnement à température contrôlée en dessous de la T g des polymères. En outre, des précautions doivent être prises pour s&#39;as...

<table border="1"><tbody><tr><td> Nom </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Motorisée XYZ étape: 3x T-LSM050A, course 50 mm par axe </td><td> Technologies Zaber </td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000 Pompe à seringue unique </td><td> Nouveaux systèmes de pompage Era </td><td> NE-1000 </td></tr><tr><td> Pyrex * Vista * Rimless réutilisables Tubes de culture en verre </td><td> Corning </td><td> 07-250-125 </td></tr><tr><td> Cuves en verre </td><td> Stratégies scientifiques </td><td> G102 </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> 776671-35 </td></tr><tr><td> Seringues étanches aux gaz SGE, Luer Loc </td><td> Sigma Aldrich </td><td> 509507 </td></tr><tr><td> U96 DeepWell plaques 1,3 ml et 2,0 ml </td><td> Thermo Scientific: Nunc </td><td> 278743 </td></tr><tr><td> Tapis de capitalisation ainsi </td><td> Thermo Scientific: Nunc </td><td> 276000 </td></tr><tr><td> Typhoon 9400 </td><td> GE Healthcare </td><td> 63-0055-79 </td></tr><tr><td> Whatman Grade 50 cercles de 90 mm </td><td> Whatman </td><td> 1450-090 </td></tr></tbody></table>

combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries

Polyanhydrides sont une classe de biomatériaux avec une excellente biocompatibilité et les capacités d&#39;administration de médicaments. Même si elles ont été largement étudiés avec conventionnelle d&#39;un échantillon-à-la-fois des techniques de synthèse, une étude plus récente à haut débit approche a été développée permettant la synthèse et le test des grandes bibliothèques de polyanhydrides 1. Cela facilitera l&#39;optimisation plus efficace et processus de conception de ces biomatériaux pour la délivrance de médicaments et de vaccins. La méthode présentée dans ce travail décrit la synthèse combinatoire du film polyanhydride biodégradable et bibliothèques nanoparticules et la détection à haut débit de la libération de protéines à partir de ces bibliothèques. Dans ce procédé robot actionné (figure 1), des actionneurs linéaires et les pompes à seringue sont commandés par LabVIEW, qui permet à un protocole automatisé mains libres, ce qui élimine une erreur d&#39;utilisateur. De plus, cette méthode permet la fabrication rapide de bibliothèques polymères micro-échelle, rougepremière utilisation de la taille du lot tout en conduisant à la création de systèmes de polymères multivariée. Cette approche combinatoire pour la synthèse de polymères facilite la synthèse de jusqu&#39;à 15 différents polymères en une quantité équivalente de temps il faudrait pour synthétiser un polymère classique. En outre, la banque combinatoire de polymère peut être fabriqué dans des géométries vides ou protéine-chargé notamment des films ou des nanoparticules lors de la dissolution de la bibliothèque de polymère dans un solvant et précipitation dans un non-solvant (nanoparticules) ou par séchage sous vide (pour les films). Lors du chargement d&#39;une protéine conjugué à un fluorochrome dans les bibliothèques de polymères, cinétique de libération de protéines peut être évalué à haut débit en utilisant une méthode de détection basée sur la fluorescence (figures 2 et 3) comme décrit précédemment 1. Cette plate-forme combinatoire a été validé avec des méthodes conventionnelles 2 et le film polyanhydride et les bibliothèques des nanoparticules ont été caractérisées par <sup&gt; 1 H RMN et FTIR. Les bibliothèques ont été sélectionnés pour la cinétique de libération de protéines, de stabilité et d&#39;antigénicité, la toxicité cellulaire in vitro, la production de cytokines, marqueurs expression à la surface, l&#39;adhérence, la prolifération et de la différenciation et de biodistribution in vivo et mucoadhésion 1-11. La méthode combinatoire développée ici permet la synthèse de polymères à haut débit et la fabrication de protéines chargé de nanoparticules et de cinémathèques, qui peut, à son tour, être examinés in vitro et in vivo pour l&#39;optimisation de la performance biomatériau.

Watch this Scientific Journal Video about Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries at JoVE.com

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Cette méthode décrit la synthèse combinatoire du film polyanhydride biodégradable et bibliothèques nanoparticules et la détection à haut débit de la libération de protéines à partir de ces bibliothèques.

Synthèse combinatoire des protéines et de décharge à haut débit à partir du film polymère et bibliothèques nanoparticules

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

combinatorial-synthesis-high-throughput-protein-release-from-polymer

Research

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Bioengineering

10.3K vues.  Iowa State University. Cette méthode décrit la synthèse combinatoire du film polyanhydride biodégradable et bibliothèques nanoparticules et la détection à haut débit de la libération de protéines à partir de ces bibliothèques.

Vidéo: Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Synthèse, l&#39;Assemblée, et caractérisation des nanoparticules Monocouche protégées Films d&#39;or pour Electrochimie monocouche de protéines

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

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Bio-ingénierie

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Un Based Chitosan, Laser adhésif activé Thin Film chirurgicaux, «SurgiLux &#39;: Préparation et démonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polymères pour la découverte à haut débit des biomatériaux

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

À haut débit Générateur expression des protéines utilisant une plate-forme microfluidique

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

À haut débit la synthèse des glucides et fonctionnalisation de nanoparticules polyanhydride

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

High Throughput unicellulaire et multiples cellules de micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Évaluation de livraison Gene polymère nanoparticules d&#39;analyse de suivi par des nanoparticules et de cytométrie en flux à haut débit

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Synthèse de l&#39;or nanoparticules liposomes intégré photosensible et mesurer leur microbulles cavitation sur Pulse Laser excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Film Extrusion de<em&gt; Crambe abyssinica</em&gt; / Gluten de blé Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Poly magnétique et thermosensible (<em&gt; N</em&gt; Microgels à base de isopropylacrylamide) pour une libération contrôlée magnétiquement déclenchée

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...