Gli autori desiderano ringraziare l&#39;US Army Medical Research e Materiel Command (Grant # W81XWH-10-1-0806) e il National Institutes of Health (Grant # AI091031 U19-01 e Grant # 1R01GM090280) per il sostegno finanziario. BN riconosce la cattedra Balloun in Ingegneria chimica e biologica e NLBP riconosce la cattedra di Ingegneria Interdisciplinare Wilkinson. Ringraziamo Julia Vela per la sua assistenza nello svolgimento degli esperimenti funzionalizzazione delle nanoparticelle.

1. High-throughput carboidrati Sintesi <ol><li> Prima della sintesi automatizzata di dimannoside, un donatore di zucchero opportunamente protetta, tipicamente tricloroacetimmidato, e accettore, principalmente un alcool alchenile fluorous, sono sintetizzati in banco. </li><li> Un programma è stato scritto per la sintesi automatica di dimannoside. Una rappresentazione schematica della procedura automatizzata di base è presentata in Figura 2. Nel programma, è garantito che prima dell&#39;aggiunta del promotore, la miscela di donatore e accettore viene agitata per almeno 30 min. </li><li> Soluzioni del donatore sintetico, accettore e trimethylsilyltrifluoromethanesulfonate sono realizzati in diclorometano. Toluene e diclorometano sono usati più frequentemente per le reazioni di glicosilazione. </li><li> Inoltre, preparare soluzioni di reagenti per la deprotezione di gruppi di protezione temporaneo in 80% metanolo e 100% di metanolo. </li><li> Prima dell&#39;inizio del programma, in modo che il relaive di umidità nella stanza è del 30% o inferiore nella camera di automazione. Alto tasso di umidità è dannoso per le reazioni di glicosilazione. </li><li> Quando il programma viene avviato, il braccio robotico trasferisce le soluzioni di donatore e accettore nella fiala di reazione sequenziale. Quindi la miscela viene agitata per 30 min. </li><li> Poi il braccio robotico trasferisce 0,2 e 0,3 equivalenti di trimethylsilyltrifluoromethanesulfonate nella miscela, tipicamente a temperatura ambiente sebbene basse temperature come -20 ° C può essere raggiunto. La miscela di reazione viene agitata per 30 min. </li><li> Dopo 30 minuti, la reazione viene fermata e una piccola aliquota rimosso per monitorare il progresso della reazione. Se non è completa, la reazione può essere continuata e infine il tempo richiesto può essere modificato. </li><li> Una volta che la reazione è completa, la miscela di reazione viene trasferita alle fluorous estrazione in fase solida (FSPE) cartucce contenenti C 8 F 17-modificato gel di silice per la purificazione. </li><li> Il carrellocreste vengono prima lavati con un 80% miscela metanolo-acqua (8 mL) per eliminare la frazione non fluorous. </li><li> Poi le cartucce vengono lavate con 100% metanolo per ottenere il prodotto desiderato fluorous-etichettato. Se si desidera ulteriore purificazione, la macchina può essere interrotta e il prodotto di reazione (s) rimosso per la purificazione mediante ulteriore. </li><li> Dopo il ciclo di depurazione, il braccio robotico dispensa metilato di sodio nel flaconcino di reazione. La reazione viene agitata per 2 h. Se non è completa, la reazione può nuovamente essere continuata per un periodo più lungo e infine il tempo programmato richiesto può essere modificato. </li><li> Dopo il completamento della reazione, il prodotto viene purificato mediante FSPE e quindi sottoposta a dissoluzione in toluene anidro seguita da evaporazione per rimuovere l&#39;acqua residua. </li><li> Quindi il ciclo (da passaggio 6 a 13) si ripete fino alla lunghezza di catena desiderata è ottenuta per la molecola bersaglio. </li><li> Il prodotto ottenuto dalla protetto automazione °en ulteriormente purificati e interamente caratterizzati mediante tecniche come la risonanza magnetica nucleare (NMR). Deprotezione completa (rimozione di tutti i restanti gruppi di protezione) della molecola bersaglio finale viene poi completato all&#39;esterno della piattaforma di automazione di regola perché comporta di solito idrogeno esplosivo e palladio. La fase di deprotezione finale è stata effettuata su banco di fuori della piattaforma di automazione. Il primo passo è stato ozonolisi del doppio legame in tag fluorous seguita da ossidazione dell&#39;aldeide prodotto di un acido carbossilico. Il prodotto è stato purificato mediante cromatografia su colonna. La fase finale era deprotezione di gruppi eterei benzilici da palladio-catalizzata idrogenazione. Il prodotto è stato fatto passare attraverso celite pad per sbarazzarsi di palladio per ottenere puro prodotto finale. </li></ol> 2. High-throughput nanoparticelle funzionalizzazione di superfici <ol><li> High-throughput sintesi del polimero e la fabbricazione di nanoparticelle viene effettuata seguendo lo stesso protocol e robotica istituito descritto da Petersen et al 19. I sistemi copolimero utilizzato per la fabbricazione di particelle sono a base di acido sebacico (SA) e 1,6-bis (para-carbossifenossi) esano (CPH), e 1,8-bis ( para-carbossifenossi) -3,6-dioxaoctane (CPTEG) e CPH. Una rappresentazione schematica dell&#39;apparato di deposizione robotico utilizzati è presentato in figura 1b. </li><li> A seguito di fabbricazione di nanoparticelle, il titolare contenere i tubi con la libreria nanoparticelle viene riattaccata alla fase di attuatore lineare. </li><li> Per il fissaggio di carboidrati alla superficie delle particelle polianidride, una ammina-acido carbossilico reazione di accoppiamento 20 costituito da due reazioni consecutive viene eseguita. </li><li> Per la prima reazione, la siringa nella prima pompa programmabile siringa è riempita con 10 equivalenti (eq.) (equivalenti di media concentrazione molare di acido carbossilico sulla superficie delle particelle) di 1-etil-3-(3-dimetilamminopropil)-carbodiimide cloridrato (EDC) e 10 eq. di etilendiammina in una soluzione acquosa, mentre la siringa nella seconda pompa siringa programmabile viene caricato con 12 eq. di N-idrossisuccinimmide (NHS) in soluzione acquosa. </li><li> Utilizzando il programma LabVIEW, sospensioni reagenti sono depositati nella biblioteca * nanoparticelle. </li><li> Successivamente, ciascun campione viene sonicata (30 s a 40 Hz) e il supporto tubo è staccato dalla piattaforma robotica. </li><li> Sospensioni di nanoparticelle sono incubate per 9 h ** con rotazione costante a 4 ° C. </li><li> Dopo il tempo di reazione è completata, i tubi vengono centrifugati (12000 xg per 5 min) e ritorno alla stazione robotizzata di effettuare due fasi di lavaggio. </li><li> Per il lavaggio, una siringa rimane vuoto e caricati nella prima pompa siringa programmabile mentre la siringa nella pompa seconda siringa è riempita con acqua fredda. Il sovranatante in ciascun tubo è ritirato nella siringa vuota ed i depositi della pompa secondo acqua fredda. </li><li> Omogeneizzazione del nanosospensione di particelle viene eseguita come descritto nel passaggio 2,6. I tubi sono poi centrifugato (12000 xg per 5 min) e una seconda fase di lavaggio viene eseguita come descritto nel passaggio 2,9. </li><li> Per la seconda reazione, due fasi di deposizione vengono utilizzati. Nella fase di prima deposizione, 12 eq. di EDC vengono caricati con una pompa e 12 eq. di NHS sono caricati con la seconda pompa. </li><li> La fase di deposizione secondo comprende 10 eq. di un saccaride specifico sulle pompe prima e seconda (cioè, galattosio, lattosio o di-mannosio) *** ed una terza pompa con 10 eq. di acido glicolico (usato come controllo ****). </li><li> Sospensioni di nanoparticelle viene omogeneizzata come descritto a passo 2,6 e incubate per 9 h con rotazione costante a 4 ° C. </li><li> Dopo il tempo di reazione è completa, una fase di lavaggio viene eseguita come descritto in fasi 2.8, 2.9 e 2.10. </li><li> La libreria nanoparticelle funzionalizzato viene quindi posto in una camera a vuoto asciugare per almeno 2 ore. </li><li> Le nanoparticelle funzionalizzate sono quindi carattereized raggi X spettroscopia di fotoelettroni e un elevato rendimento fenolo-acido solforico saggio per determinare la composizione di superficie e la concentrazione del saccaride rispettivamente. Microscopia elettronica a scansione e dinamico dispersione della luce vengono utilizzati per determinare la dimensione delle particelle, la distribuzione delle dimensioni, e la carica superficiale. </li></ol> Note: * I volumi di deposizione variano con la massa di nanoparticelle contenute in ciascun tubo. I tempi di reazione ** per reazioni prima e seconda può essere modificato per regolare la concentrazione finale saccaride. *** Ogni saccaride viene depositato in provette a seconda del gruppo desiderato. **** Per la reazione specifico impiegato in questo studio per il fissaggio di carboidrati, acido glicolico viene utilizzato come controllo linker dal saccaridi deprotetti già questa molecola legata covalentemente, che consente di fissaggio ulteriore superficie delle nanoparticelle. 3. Risultati rappresentativi Il fuldimannoside ly protetto illustrato nella figura 2 è stato sintetizzato utilizzando la piattaforma di automazione. Il composto sintetizzato è stata caratterizzata da 1 H NMR in uno spettrometro VXR 400 MHz usando CDCl 3 come solvente. Lo spettro NMR è mostrato in Figura 3. Utilizzando l&#39;elevata produttività fabbricazione nanoparticelle e funzionalizzazione di polianidride nanoparticelle qui descritto, attaccamento di dimannose, lattosio e galattosio è stata effettuata con successo 10, 11. Usando questa configurazione, condizioni di reazione ottimali (cioè, temperatura di reazione e il tempo) sono stati identificati per ottenere nanoparticelle funzionalizzazione desiderata e morfologia. Quando la reazione è stata condotta a 4 ° C invece di temperatura ambiente, una riduzione aggregazione delle nanoparticelle è stato osservato mediante SEM (dati non mostrati). La tabella 1 mostra i risultati rappresentativi della caratterizzazione di funzionalizzato CPTEG 50:50: nanoparticelle CPH sia con di-mannosio olattosio, sintetizzato a 4 ° C. I dati indicano un piccolo aumento del diametro medio delle nanoparticelle per la funzionalizzazione. Mentre i non funzionalizzati nanoparticelle aveva un potenziale negativo zeta di ca. -20 MV, le particelle funzionalizzate mostrato un valore positivo potenziale zeta, dimostrando funzionalizzazione successo della superficie delle nanoparticelle. Lattosio e di-mannosio sono entrambi zuccheri neutri, tuttavia, i gruppi amminici liberi dal etilendiammina linker utilizzato per fissare i saccaridi può essere responsabile della potenziale positivo zeta. Il tempo di reazione è un&#39;altra variabile che potrebbe influire sia la morfologia finale delle nanoparticelle e il grado di attaccamento zucchero raggiunto. Regolando il tempo di reazione, la concentrazione finale di zucchero attaccato alla superficie nanoparticelle può essere controllato come mostrato nella Figura 4A. Come previsto, la concentrazione di dimannose sulla superficie di 50:50 CPTEG: CPH nanoparticelle aumentatoil tempo totale di reazione e raggiunsero un massimo dopo 18 ore. Nanoparticelle funzionalizzate con il tempo di 24 ore di reazione totale sono stati utilizzati per valutare la loro capacità di indirizzare CLR sul mouse cellule di midollo osseo derivate dendritiche (DC). Citometria di flusso è stato usato per valutare l&#39;espressione di due recettori CL (cioè, CIRE (CD209, DC-SIGN) e recettore del mannosio (CD206)) dopo stimolazione con non-funzionalizzato, e lattosio e nanoparticelle funzionalizzate di-mannosio (Figura 4B). Una più alta espressione di entrambi i recettori, che è un indicativo di un orientamento efficace, è stato ottenuto quando le cellule sono state stimolate con sia lattosio e nanoparticelle funzionalizzate di-mannosio. Tuttavia, di-mannosio-funzionalizzati particelle mostrato un alto livello di espressione indica una specificità di questo ligando per i recettori che sono stati studiati. <table border="1"><tbody><tr><td> Tipo di nanoparticelle </td><td> Diametro particellare medio (nm) </td><td> Averabbia particelle ζ-potenziale (mV) </td></tr><tr><td> Non-funzionalizzato </td><td> 162 ± 43 </td><td> -20 ± 0,6 </td></tr><tr><td> Lattosio </td><td> 235 ± 34 </td><td> 26 ± 2,4 </td></tr><tr><td> Di-mannosio </td><td> 243 ± 32 </td><td> 30 ± 4,2 </td></tr></tbody></table> Tabella 1. Caratterizzazione delle nanoparticelle. Non-funzionalizzato e funzionalizzati sono stati caratterizzati da quasi elastico light scattering e misurazioni potenziale zeta. Dimensioni delle particelle dati rappresentano la media ± valore di deviazione standard (SD) di dati dinamici diffusione di luce raccolti in tre esperimenti indipendenti. Dati potenziale zeta rappresentano la media ± SD valore di tre letture indipendenti. Cambiare nel segno del potenziale zeta dimostra che lo zucchero era efficiente, coniugato con l&#39;50:50 CPTEG: CPH superficie delle nanoparticelle. <img alt="Figura 1" src<html> = &quot;/ Files/ftp_upload/3967/3967fig1.jpg&quot; /&gt; Figura 1. (A) Rappresentazione grafica della approccio seguito con funzionalizzazione carboidrati di nanoparticelle polianidride e un esempio delle librerie nanoparticelle funzionalizzate che possono essere progettati con l&#39;descritto ad elevata capacità approccio. (B) Rappresentazione schematica dell&#39;apparato di deposizione automatizzata utilizzata per funzionalizzazione particelle, che consiste di (i) tre NE 1000 pompe, (ii) una fase robotico integrato da due attuatori (Zaber): uno per il movimento nella direzione x e l&#39;altra per il movimento nella direzione y, (iii) una seconda fase robotico con due cremagliere adiacenti (appropriata per tubi e le cuvette) costituito da tre attuatori, uno per ciascuna direzione (x, y, z). Le pompe e un totale di cinque attuatori sono collegati in serie. Attuatori e le pompe sono gestiti da un computer utilizzando il software LabVIEW. Questo schema non è in scala.arge.jpg &quot;target =&quot; _blank &quot;&gt; Clicca qui per ingrandire la figura. <img alt="Figura 2" src="/files/ftp_upload/3967/3967fig2.jpg" /> Figura 2. Rappresentazione grafica della automatizzato iterativo sintesi di carboidrati usando mannosio come esempio. <img alt="Figura 3" src="/files/ftp_upload/3967/3967fig3.jpg" /> Figura 3. 1 H NMR del dimannoside protetto. <img alt="Figura 4" src="/files/ftp_upload/3967/3967fig4.jpg" /> Figura 4. (A) Effetto del tempo di reazione sulla concentrazione superficie delle nanoparticelle di saccaride. Nei dati riportati, 50:50 CPTEG: nanoparticelle CPH stati funzionalizzati con dimannose a differenti tempi di reazione e la reazione è stata condotta a 4 ° C. L&#39;errore medio e standard di due esperimenti indipendenti funzionalizzazione viene mostrato. (B) nanoparticelle Lattosio e di-mannosio funzionalizzatiefficace recettore bersaglio DC-SIGN (CIRE, CD209) e mannosio (CD206) su midollo osseo cellule dendritiche, come dimostrato dalla espressione di questi due marcatori dopo stimolazione con funzionalizzato CPTEG 50:50: nanoparticelle CPH se confrontato con l&#39;espressione ottenuta con i non-funzionalizzate particelle.

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	<li>Zepp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+vacine+design-lessons+from+nature.">Principles of vacine design-lessons from nature.</a> Vaccine. 28, C14-C24 (2010).</li><li>Ulery, B. D., Phanse, Y., Sinha, A., Wannemuehler, M. J., Narasimhan, B., Bellaire, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+chemistry+influences+monocytic+uptake+of+polyanhydride+nanospheres.">Polymer chemistry influences monocytic uptake of polyanhydride nanospheres.</a> Pharm. Res. 26, 683-690 (2009).</li><li>Torres, M. P., Wilson-Welder, J. H., Lopac, S. K., Phanse, Y., Carrillo-Conde, B., Ramer-Tait, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyanhydride+microparticles+enhance+dendritic+cell+antigen+presentation+and+activation.">Polyanhydride microparticles enhance dendritic cell antigen presentation and activation.</a> Acta Biomater. 7, 2857-2864 (2011).</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>Petersen, L. K., Ramer-Tait, A. E., Broderick, S. R., Kong, C. S., Ulery, B. D., 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=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>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>Lopac, S. K., Torres, M. P., Wilson-Welder, J. H., 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=Effect+of+polymer+chemistry+and+fabrication+method+on+protein+release+and+stability+from+polyanhydride+microspheres.">Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres.</a> J. Biomed. Mater. Res. B. 91, 938-947 (2009).</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>Determan, A. S., Lin, V. S. Y., 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. Controlled Release. 100, 97-109 (2004).</li><li>Chavez-Santoscoy, A., Roychoudhury, R., Ramer-Tait, A. E., Pohl, N. L. B., 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=Tailoring+the+immune+response+of+alveolar+macrophages+by+targeting+different+C-type+lectin+receptors+using+"pathogen-like"+amphiphilic+polyanhydride+nanoparticles.">Tailoring the immune response of alveolar macrophages by targeting different C-type lectin receptors using "pathogen-like" amphiphilic polyanhydride nanoparticles.</a> Biomaterials. , (2011).</li><li>Carrillo-Conde, B., Song, E. -. H., Chavez-Santoscoy, A., Phanse, Y., Ramer-Tait, A., 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=Mannose-functionalized+"pathogen-like"+polyanhydride+nanoparticles+target+C-type+lectin+receptors+on+dendritic+cells.">Mannose-functionalized "pathogen-like" polyanhydride nanoparticles target C-type lectin receptors on dendritic cells.</a> Mol. Pharmaceutics. 8, 1877-1886 (2011).</li><li>Carrillo-Conde, B., Schiltz, E., Torres, M. P., Yu, J., Phillips, G., Minion, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphipilic+polyanhydrides+for+stabilization+of+Yersinia+pestis+antigens.">Amphipilic polyanhydrides for stabilization of Yersinia pestis antigens.</a> Acta. Biomater. 6, 3110-3119 (2010).</li><li>Reddy, S. T., Swartz, M. A., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+dendritic+cells+with+biomaterials:+developing+the+next+generation+of+vaccines.">Targeting dendritic cells with biomaterials: developing the next generation of vaccines.</a> Trends Immunol. 27, 573-580 (2006).</li><li>Higashi, N., Fujioka, K., Denda-Nagai, K., Hashimoto, S., Nagai, S., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+C-type+lectin+specific+for+galactose/N-acetylgalactosamine+is+an+endocytic+receptor+expressed+on+monocyte-derived+immature+dendritic+cells.">The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.</a> J. Biol. Chem. 277, 20686 (2002).</li><li>Geijtenbeek, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+through+C-type+lectin+receptors:+shaping+immune+responses.">Signalling through C-type lectin receptors: shaping immune responses.</a> Nat. Rev. Immunol. 9, 465-479 (2009).</li><li>Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+oligosaccharide+synthesis.">Automated oligosaccharide synthesis.</a> Chem. Soc. Rev. 37, 19-28 (2008).</li><li>Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Carbohydrate+Synthesis+as+Platform+to+Address+Fundamental+Aspects+of+Glycobiology-Current+Status+and+Future+Challenges.">Automated Carbohydrate Synthesis as Platform to Address Fundamental Aspects of Glycobiology-Current Status and Future Challenges.</a> Carb. Res. 343, 1889-1896 (2008).</li><li>Jaipuri, F. A., 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=Toward+solution-phase+automated+iterative+synthesis:+fluorous-tag+assisted+solution-phase+synthesis+of+linear+and+branched+mannose+oligomers.">Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers.</a> Org. Biomol. Chem. 6, 2686-2691 (2008).</li><li>Petersen, L. K., Chavez-Santoscoy, A., 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+synthesis+of+and+high-throughput+protein+release+from+polymer+film+and+nanoparticle+libraries.">Combinatorial synthesis of and high-throughput protein release from polymer film and nanoparticle libraries.</a> J. Vis. Exp. , (2011).</li><li>Song, E. -. H., Osanya, A. O., Petersen, C. A., Pohl, N. L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multivalent+tuberculosis+and+Leishmania-associated+capping+carbohydrates+reveals+structure-dependent+responses+allowing+immune+evasion.">Synthesis of multivalent tuberculosis and Leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion.</a> J. Am. Chem. Soc. 132, 11428-11430 (2010).</li><li>Hakamori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+glycosylation+in+tumor+and+tumor+associated+carbohydrate+antigens.">Aberrant glycosylation in tumor and tumor associated carbohydrate antigens.</a> Adv. Cancer Res. 59, 257-331 (1989).</li><li>Atherton, T., Sheppard, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Solid-phase peptide synthesis: a practical approach. , (1999).</li><li>Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+synthesis+machines:+DNA+chemistry+and+the+uses.">Gene synthesis machines: DNA chemistry and the uses.</a> Science. 230, 281-285 (1985).</li><li>Plante, O. J., Palmacci, E. R., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solid-+phase+synthesis+of+oligosaccharides.">Automated solid- phase synthesis of oligosaccharides.</a> Science. 291, 1523-1527 (2001).</li><li>Ko, K. -. S., Park, G., Yu, Y., 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=Protecting+group-based+colorimetric+monitoring+of+fluorous-phase+and+solid-phase+synthesis+of+oligoglucosamines.">Protecting group-based colorimetric monitoring of fluorous-phase and solid-phase synthesis of oligoglucosamines.</a> Org. Lett. 10, 5381-5384 (2008).</li><li>Pohl, N. L., Chen, X. H. R., Wang, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+solution-phase+oligosaccharide+synthesis+and+carbohydrate+microarrays:+development+of+fluorous-based+tools+for+glycomics.">Automated solution-phase oligosaccharide synthesis and carbohydrate microarrays: development of fluorous-based tools for glycomics.</a> Chemical Glycobiology. , 272-287 (2008).</li></ol>

NLBP è cofondatore e detiene partecipazioni in società carboidrati LuCella Biosciences, Inc.

L&#39;efficacia di carboidrati come agenti per il targeting nanoparticelle interazioni dirette alle cellule immunitarie è stato precedentemente dimostrato 10, 11. Precedente ricerca nei nostri laboratori hanno mostrato che gli zuccheri specifici collegati polianidride nanoparticelle sono in grado di indirizzare CLR differenti sulle cellule presentanti l&#39;antigene (APC), migliorando così l&#39;attivazione di cellule immunitarie che può essere importante per l&#39;ulteriore attivazione delle cellule T <su...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Motorizzato XYZ Stage: 3x T-LSM050A, corsa 50 mm per asse </td><td> Zaber Technologies </td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000 pompa a siringa singola </td><td> New Era pompa Sistemi </td><td> NE-1000 </td></tr><tr><td> Pyrex * Vista * Rimless riutilizzabili in vetro Cultura Tubes </td><td> Corning </td><td> 07-250-125 </td></tr><tr><td> ASW 1000 </td><td> Chemspeed Technologies </td><td></td></tr><tr><td> LabVIEW </td><td> National Instruments </td><td> 776671-35 </td></tr><tr><td> SGE Siringhe a tenuta di gas Luer Loc </td><td> Sigma Aldrich </td><td> 509507 </td></tr><tr><td> XL-2000 Sonicator </td><td> Qsonica </td><td> Q55 </td></tr><tr><td> Mini-tube dei rotatori </td><td> Fisher Scientific </td><td> 05-450-127 </td></tr></tbody></table>

high-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles

Approcci multidisciplinari che coinvolgono settori come la progettazione di materiale, le nanotecnologie, la chimica, immunologia e devono essere utilizzate per progettare razionalmente efficaci vettori vaccini. Nanoparticelle basate su piattaforme può prolungare la persistenza di antigeni del vaccino, che potrebbero migliorare vaccino immunogenicità 1. Vari polimeri biodegradabili sono stati studiati come veicoli di mandata del vaccino 1, in particolare, polianidride particelle hanno dimostrato la capacità di fornire il rilascio prolungato di antigeni proteici stabili e di attivare le cellule presentanti l&#39;antigene e modulare le risposte immunitarie 2-12. Il disegno molecolare di questi vettori vaccinali deve integrare la selezione razionale delle proprietà dei polimeri così come l&#39;inserimento di opportuni agenti di targeting. Fabbricazione elevato throughput automatizzato di mira leganti e particelle funzionalizzate è un potente strumento che migliorerà la capacità di studiare una gamma range di proprietà e porterà alla progettazione di dispositivi riproducibili somministrazione del vaccino. L&#39;aggiunta di mira ligandi in grado di essere riconosciuto da specifici recettori sulle cellule immunitarie è stato dimostrato per modulare e adattare le risposte immunitarie di tipo C 10,11,13 recettori lectina (CLR) sono recettori di riconoscimento di pattern (PRRS) che riconoscono carboidrati presenti sul superficie di agenti patogeni. La stimolazione di cellule immunitarie attraverso CLR consente di internalizzazione maggiore di antigene e successiva esposizione per ulteriore attivazione delle cellule T 14,15. Pertanto, molecole di carboidrati svolgono un ruolo importante nello studio di risposte immunitarie, tuttavia, l&#39;uso di queste biomolecole soffre spesso dalla mancanza di disponibilità di strutturalmente ben definiti e carboidrati puri. Una piattaforma di automazione basato sulla soluzione iterativa fase reazioni possono consentire rapida sintesi e controllato di queste molecole sinteticamente impegnativi utilizzando b significativamente più bassoOSTRUIRE quantità di blocchi rispetto ai tradizionali metodi in fase solida 16,17. Qui riportiamo un protocollo per la soluzione automatizzata di sintesi in fase di oligosaccaridi come ligandi destinate mannosio-based con fluorous estrazione in fase solida per la purificazione intermedia. Dopo lo sviluppo di metodi automatici per rendere la base di carboidrati agente di targeting, si descrivono metodi per il loro fissaggio sulla superficie di polianidride nanoparticelle impiegando un set up robotizzato azionato da LabVIEW come descritto in precedenza 10. Funzionalizzazione superficiale con carboidrati ha dimostrato l&#39;efficacia in termini di orientamento CLR 10,11 e aumentare il throughput del metodo di fabbricazione per portare alla luce le complessità associate a un sistema multi-parametrico sarà di grande valore (Figura 1a).

Watch this Scientific Journal Video about High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles at JoVE.com

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

In questo articolo, un metodo ad alta velocità viene presentato per la sintesi di oligosaccaridi e fissaggio alla superficie delle nanoparticelle polianidride per ulteriore uso in mira specifici recettori sulle cellule presentanti l&#39;antigene.

High-throughput sintesi dei carboidrati e funzionalizzazione di nanoparticelle polianidride

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

high-throughput-synthesis-carbohydrates-functionalization

Research

JoVE Journal

Bioengineering

11.3K Views.  Iowa State University. In questo articolo, un metodo ad alta velocità viene presentato per la sintesi di oligosaccaridi e fissaggio alla superficie delle nanoparticelle polianidride per ulteriore uso in mira specifici recettori sulle cellule presentanti l'antigene.

Video: High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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 polimerici per Discovery High Throughput di Biomateriali

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

Ricerca

Bioingegneria

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

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Sintesi combinatoria di e High-throughput di rilascio di proteine ​​da film polimerico e Biblioteche nanoparticelle

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

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Raccolta murini macrofagi alveolari e Valutare l&#39;attivazione cellulare indotta da nanoparticelle polianidride

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

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 singola cellula e cellula Multiple-Micro-incapsulamento

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.

Valutazione della consegna del gene polimerici Nanoparticelle per l&#39;analisi delle nanoparticelle Monitoraggio e High-throughput Citometria a Flusso

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

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

Forno a microonde-driven sintesi di ossido di ferro Nanoparticelle per il rilevamento rapido di aterosclerosi

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificazione di batteri Polimeri repellente per dispositivi medici

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Funzionalizzazione superficiale delle nanoparticelle di Virus di epatite E utilizzando metodi di coniugazione chimica

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...