Name: combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries
Uploaded: 2012-09-06T12:00:00
Description: Watch this Scientific Journal Video about Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries at JoVE.com

Forfatterne anerkender ONR-Muri Award (NN00014-06-1-1176) og den amerikanske Army Medical Research og Materiel Kommando (Grant No W81XWH-10-1-0806) om økonomisk støtte. Dette materiale er baseret på arbejde, støttet af National Science Foundation under Grant No EØF 0552584 og 0.851.519.

1. Kombinatorisk Polymer Library Synthesis (Varierende i Polymer Chemistry) - se figur 1 for Robotic Setup <ol><li> Opløs hver monomer i det passende opløsningsmiddel (koncentration = 25 mg / ml) og indlæsning hver i en 10cc gastæt sprøjte. </li><li> Fastgør modstandsdygtige over for opløsningsmidler lokke lås kapillarrør til slutningen af ​​hver sprøjte. </li><li> Placer sprøjterne på sprøjtepumper (New Era Programmerbare sprøjtepumper) og lås på plads. </li><li> Indstille de line...

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	<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>

Kendskab til de nødvendige syntesebetingelserne og glasovergangstemperaturer (Tg s) af polymererne, der syntetiseres, er afgørende for biblioteket fabrikation. Hvis Tg s er under stuetemperatur, kan nanopartikel fremstillingstrin skal udføres i et temperaturkontrolleret miljø under Tg af polymererne. Desuden bør der udvises forsigtighed for at sikre, at alt udstyr, der kommer i kontakt med høje temperaturer og de opløsningsmidler, skal være egnet til at håndtere disse betingelse...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Motoriseret XYZ Stage: 3x T-LSM050A, 50 mm rejser hver akse </td><td> Zaber Technologies </td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000 Single Sprøjtepumpe </td><td> New Era Pumpesystemer </td><td> NE-1000 </td></tr><tr><td> Pyrex * Vista * Uindfattede Genbrugelige Glas Kultur Tubes </td><td> Corning </td><td> 07-250-125 </td></tr><tr><td> Glaskuvetter </td><td> Videnskabelige strategier </td><td> G102 </td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> 776.671-35 </td></tr><tr><td> SGE gastætte Sprøjter, Luer Loc </td><td> Sigma Aldrich </td><td> 509.507 </td></tr><tr><td> U96 Deepwell Plates 1,3 ml &amp; 2,0 ml </td><td> Thermo Scientific: Nunc </td><td> 278.743 </td></tr><tr><td> Well cap måtter </td><td> Thermo Scientific: Nunc </td><td> 276.000 </td></tr><tr><td> Typhoon 9400 </td><td> GE Healthcare </td><td> 63-0055-79 </td></tr><tr><td> Whatman Grade 50 Circles 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

Polyanhydrider er en klasse af biomaterialer med fremragende biokompatibilitet og drug delivery kapaciteter. Mens de er blevet omfattende undersøgt med konventionelle en-prøve-at-a-time synteseteknikker, har en nyere high-throughput fremgangsmåde blevet udviklet muliggør syntese og afprøvning af store biblioteker af polyanhydrider 1. Dette vil lette en mere effektiv optimering og design processen i disse biomaterialer for stof-og vaccine levering applikationer. Fremgangsmåden i dette arbejde beskriver den kombinatoriske syntese af bionedbrydelige polyanhydrid film og nanopartikler biblioteker og high-throughput påvisning af protein frigivelse fra disse biblioteker. I denne robot betjent fremgangsmåde (fig. 1), er lineære aktuatorer og sprøjtepumper kontrolleret af LabVIEW, der muliggør en håndfri automatiseret protokol, eliminerer brugerfejl. Desuden er denne metode giver mulighed for hurtig fremstilling af mikro-skala polymer biblioteker, røducing batchstørrelsen samtidig resulterer i skabelsen af ​​multivariant polymersystemer. Denne kombinatoriske strategi for polymersyntese letter syntesen af ​​op til 15 forskellige polymerer i en ækvivalent mængde tid, det ville tage at syntetisere en polymer konventionelt. Desuden kan det kombinatoriske bibliotek polymer fremstilles til blanke eller protein-loaded geometrier, herunder film eller nanopartikler ved opløsning af polymeren biblioteket i et opløsningsmiddel og udfældning i et ikke-opløsningsmiddel (for nanopartikler) eller ved vakuumtørring (for film). Ved indlæsning af et fluorochrom-konjugeret protein i polymeren biblioteker, kan protein-frigivelseskinetik vurderes til high-throughput under anvendelse af en fluorescens-baseret påvisningsfremgangsmåde (figur 2 og 3) som beskrevet tidligere 1. Denne kombinatoriske platform er valideret med konventionelle metoder 2 og polyanhydridet film og nanopartikler biblioteker er blevet karakteriseret med <sup&gt; 1H-NMR og FTIR. Bibliotekerne er blevet screenet for protein frigivelseskinetik, stabilitet og antigenicitet, in vitro cellulær toksicitet, produktion cytokin, overflademarkør udtryk, adhæsion, spredning og differentiering, og in vivo biodistribution og mucoadhæsion 1-11. Det kombinatoriske metode udviklet heri muliggør high-throughput polymersyntese og fremstilling af protein-loaded nanopartikler og filmarkiver, der kan på sin side blive screenet in vitro og in vivo til optimering af biomateriale ydeevne.

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

Denne metode beskriver kombinatorisk syntese af bionedbrydelige polyanhydrid film og nanopartikler biblioteker og high-throughput påvisning af protein frigivelse fra disse biblioteker.

Kombinatorisk syntese af og Højkapacitetsforskning Protein Udgivelse fra Polymer Film og nanopartikel Biblioteker

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

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