Os autores gostariam de agradecer os EUA Army Medical Research e Material Command (Grant # W81XWH-10-1-0806) e os Institutos Nacionais de Saúde (Grant # U19 AI091031-01 e Grant # 1R01GM090280) pelo apoio financeiro. BN reconhece a Professorship Balloun em Engenharia Química e Biológica e NLBP reconhece a Professorship Wilkinson de Engenharia Interdisciplinar. Agradecemos Julia Vela por sua assistência na realização dos experimentos de funcionalização de nanopartículas.

1. Alta capacidade de síntese de carboidratos <ol><li> Antes da síntese automatizada de dimannoside, um dador de açúcar adequadamente protegido, tipicamente tricloroacetimidato, e aceitador, principalmente álcool uma alcenilo fluorosa, são sintetizados no banco-top. </li><li> Um programa é escrito para a síntese automatizada de dimannoside. Uma representação esquemática do procedimento básico automatizado é apresentada na Figura 2. No programa, é assegurado que, antes da adição do promotor, a mistura de dador e aceitador é agitada durante pelo menos 30 min. </li><li> As soluções do sintética doador aceitador, e trimethylsilyltrifluoromethanesulfonate são feitas em diclorometano. Tolueno e diclorometano, são usados ​​mais freqüentemente para as reações de glicosilação. </li><li> Além disso, preparar soluções de reagentes para a desprotecção de grupos protectores temporários em metanol a 80% e metanol a 100%. </li><li> Antes do início do programa, assegurar que o Relative humidade da sala é de 30% ou inferior na câmara de automação. A alta umidade é prejudicial para as reações de glicosilação. </li><li> Uma vez que o programa é iniciado, o braço robótico transfere as soluções de dador e aceitador para dentro do frasco de reacção sequencialmente. Em seguida a mistura é agitada durante 30 min. </li><li> Em seguida, o braço robótico transfere 0,2 a 0,3 equivalentes de trimethylsilyltrifluoromethanesulfonate na mistura, tipicamente à temperatura ambiente, embora temperaturas mais baixas como -20 ° C pode ser alcançada. A mistura de reacção é agitada durante 30 min. </li><li> Após 30 min, a reacção é parada e uma pequena alíquota removido para monitorizar o progresso da reacção. Se não está completa, a reacção pode ser continuada e, finalmente, o tempo necessário pode ser modificado. </li><li> Uma vez que a reacção está completa, a mistura de reacção é transferida para os fluorosas extracção de fase sólida (FSPE) cartuchos contendo C 8 F 17-modificado de gel de sílica para a purificação. </li><li> O carrinhocristas são primeiro lavado com uma mistura de 80% de metanol-água (8 mL) para se livrar da fracção não-fluorosa. </li><li> Em seguida, os cartuchos são lavados com metanol a 100% para se obter o produto desejado fluorosa-etiquetado. Se purificação adicional for desejado, a máquina pode ser interrompido eo produto da reacção (s) removido para purificação por meios adicionais. </li><li> Após o ciclo de purificação, o braço robótico dispensa metóxido de sódio para dentro do frasco de reacção. A reacção é agitada durante 2 hr. Se não está completa, a reacção pode novamente ser continuado durante um período mais longo e, finalmente, o tempo programado necessário pode ser modificado. </li><li> Após a conclusão da reacção, o produto é purificado por FSPE e, em seguida, submetido a dissolução em tolueno anidro seguido de evaporação para remover a água residual. </li><li> Em seguida, o ciclo (do passo 6 a 13) é repetido até que o comprimento da cadeia desejada é obtida para a molécula alvo. </li><li> O produto protegido obtido a partir de automação é then ainda purificado e caracterizados por técnicas tais como a espectroscopia de ressonância magnética nuclear (RMN). Desprotecção completa (remoção de todos os grupos protectores restantes) da molécula alvo final é então completado fora da plataforma de automação como uma regra, pois que normalmente envolve o gás de hidrogénio explosivo e paládio. O passo de desprotecção final foi realizada no banco de cima para fora da plataforma de automação. O primeiro passo foi ozonólise da ligação dupla em tag fluorosa seguido por oxidação do aldeído produzido para um ácido carboxílico. O produto foi purificado por cromatografia em coluna. O passo final foi desprotecção de grupos éter benzílico por paládio catalisada-hidrogenação. O produto foi passado através de celite almofada para se livrar do paládio para obter produto final puro. </li></ol> 2. Alta capacidade funcionalização da superfície de nanopartículas <ol><li> Alta capacidade de síntese de polímeros e fabricação de nanopartículas é realizada após a mesma protocol e robótico configurar descrito por Petersen et al 19. Os sistemas de copolímero utilizado para a fabricação de partículas são à base de ácido sebácico (SA) e 1,6-bis (para-carboxifenoxi)-hexano (CPH), e 1,8-bis ( para-carboxifenoxi) -3,6 dioxaoctane-(CPTEG) e CPH. Uma representação esquemática do aparelho de deposição robótico utilizada é apresentada na Figura 1b. </li><li> Após a fabricação de nanopartículas, o detentor contendo os tubos com a biblioteca de nanopartículas é recolocado para a fase do actuador linear. </li><li> Para a fixação de hidratos de carbono para a superfície de partículas de polianidrido, uma amina-carboxílico reacção de acoplamento ácido 20 constituído por duas reacções consecutivas é executada. </li><li> Para a reacção em primeiro lugar, a seringa na bomba de seringa primeiro programável é preenchido com 10 equivalentes (eq.) (equivalentes de concentração de ácido carboxílico molar média sobre a superfície da partícula) de 1-etil-3-(3-dimetilaminopropil)-cacloridrato rbodiimide (EDC) e 10 eq. de etilenodiamina em uma solução aquosa, enquanto que a seringa na bomba de seringa segundo programável é carregado com 12 eq. de N-hidroxissuccinimida (NHS) em solução aquosa. </li><li> Usando o programa LabVIEW, suspensões reagentes são depositadas na * biblioteca de nanopartículas. </li><li> Em seguida, cada amostra é sonicada (30 s em 40 Hz) e do suporte do tubo é separada a partir da plataforma robótico. </li><li> As suspensões de nanopartículas são incubadas durante 9 ** h com rotação constante a 4 ° C. </li><li> Após o tempo de reacção é completado, os tubos são centrifugados (12000 xg durante 5 min) e voltou para a estação de robótico para realizar duas etapas de lavagem. </li><li> Para a lavagem, uma seringa permanece vazio e carregado na bomba de seringa primeiro programável enquanto a seringa na bomba de seringa segundo é enchido com água fria. O sobrenadante de cada tubo é retirada para a seringa vazia e os depósitos segunda bomba de água fria. </li><li> Homogeneização de nanosuspensão de partículas é realizada como descrito no passo 2.6. Os tubos são em seguida centrifugada (12000 xg durante 5 min) e um segundo passo de lavagem é realizada como descrito no passo 2.9. </li><li> Para a reacção segundo, dois passos de deposição são utilizados. Na etapa de deposição em primeiro lugar, 12 eq. de EDC são carregados com uma bomba e 12 eq. NHS de são carregados com a segunda bomba. </li><li> O passo de deposição segundo inclui 10 eq. de um sacárido específica sobre as bombas primeiro e segundo (isto é, galactose, lactose ou di-manose) *** e uma terceira bomba com 10 eq. de ácido glicólico (usado como controlo ****). </li><li> As suspensões de nanopartículas é homogeneizada como descrito na etapa 2,6 e incubou-se durante 9 h, com rotação constante a 4 ° C. </li><li> Após o tempo de reacção é completada, um passo de lavagem é realizada como descrito nos passos 2.8, 2.9 e 2.10. </li><li> A biblioteca de nanopartículas funcionalizado é então colocado numa câmara de vácuo para secar durante pelo menos 2 horas. </li><li> As nanopartículas são funcionalizados em seguida carácterzada por espectroscopia de raios X de fotoelétrons e um ensaio de ácido elevada taxa de transferência de fenol-sulfúrico para determinar a composição da superfície e da concentração do sacárido respectivamente. Microscopia electrónica de varrimento e de dispersão de luz dinâmica são utilizados para determinar o tamanho de partícula, distribuição de tamanho, e carga de superfície. </li></ol> Notas: * Os volumes de deposição variar de acordo com a massa de nanopartículas contidas em cada tubo. Vezes ** reaccionais para reacções primeiro e segundo pode ser alterado para ajustar a concentração de sacárido final. Cada *** sacárido é depositado em tubos de ensaio, dependendo do grupo desejado. **** Para a reacção específico empregue neste estudo para a ligação de hidratos de carbono, o ácido glicólico é usado como um controlo linker desde sacáridos desprotegidos já têm esta molécula ligada covalentemente, que permite a fixação adicional para a superfície de nanopartículas. 3. Os resultados representativos O fuldimannoside ly protegido mostrado na Figura 2 foi sintetizado utilizando a plataforma de automação. O composto sintetizado foi caracterizado por 1 H RMN num espectrómetro de VXR 400 MHz usando CDCI3 como solvente. O espectro de RMN é mostrado na Figura 3. Utilizando a fabricação de nanopartículas de alto rendimento e funcionalização de polianidrido nanopartículas aqui descrito, a ligação de dimannose, lactose e galactose foi realizada com sucesso 10, 11. Utilizando esta configuração, as condições reaccionais óptimas (isto é, temperatura de reacção e tempo) foram identificados para atingir funcionalização de nanopartículas desejado e morfologia. Quando a reacção foi realizada a 4 ° C em vez de temperatura ambiente, uma redução na agregação de nanopartículas foi observada por SEM (dados não mostrados). A Tabela 1 mostra os resultados representativos da caracterização de CPTEG 50:50 funcionalizado: nanopartículas CPH quer com di-manose oulactose, sintetizado a 4 ° C. Os dados indicam um pequeno aumento no diâmetro médio de nanopartículas devido à funcionalização. Enquanto as nanopartículas não-funcionalizados tinha um potencial zeta negativo de aprox. -20 MV, as partículas funcionalizadas mostrou um valor de potencial zeta positivo, demonstrando funcionalização bem sucedida da superfície de nanopartículas. Lactose e di-manose são ambos açúcares neutros, no entanto, os grupos amina livres do etileno diamina ligante utilizado para fixar os sacáridos podem ser responsáveis ​​do potencial zeta positivo. Tempo de reacção é outra variável que pode afectar tanto morfologia do final das nanopartículas e do grau de ligação alcançados açúcar. Ao ajustar o tempo de reacção, a concentração final de açúcar ligada à superfície nanopartículas podem ser controladas, como mostrado na Figura 4A. Como esperado, a concentração de dimannose sobre a superfície de 50:50 CPTEG: CPH nanopartículas aumentou como tempo total de reacção e atingiu um máximo após 18 h. As nanopartículas funcionalizados com o tempo de reacção total de 24 hr foram utilizados para avaliar a sua capacidade para atingir CLRs no rato derivadas da medula óssea células dendríticas (DC). A citometria de fluxo foi utilizado para avaliar a expressão de dois receptores de CL (isto é, CIRE (CD209, DC-SIGN) e receptor de manose (CD206)) após estimulação com não-funcionalizado, e lactose, e di-manose nanopartículas funcionalizadas (Figura 4B). Uma maior expressão de ambos os receptores, que é um indicativo de direccionamento eficaz, foi obtido quando as células foram estimuladas com ambos lactose e di-manose nanopartículas funcionalizados. No entanto, di-manose-funcionalizados partículas mostraram um maior nível de expressão indicando uma especificidade deste ligando para os receptores que foram estudados. <table border="1"><tbody><tr><td> Tipo de nanopartícula </td><td> Diâmetro médio de partícula (nm) </td><td> AvePartículas raiva ζ-potencial (mV) </td></tr><tr><td> Non-funcionalizado </td><td> 162 ± 43 </td><td> -20 ± 0,6 </td></tr><tr><td> Lactose </td><td> 235 ± 34 </td><td> 26 ± 2,4 </td></tr><tr><td> Di-manose </td><td> 243 ± 32 </td><td> 30 ± 4,2 </td></tr></tbody></table> Tabela 1. Caracterização de nanopartículas. Non-funcionalizado e funcionalizado foram caracterizados por quasi-elástica de dispersão de luz e as medições de potencial zeta. Dados de tamanho de partículas representam o valor médio ± desvio padrão (SD) de dados dinâmicos de espalhamento de luz coletados em três experimentos independentes. Dados de potencial zeta representam o valor médio ± DP de três leituras independentes. Mudança no sinal do potencial zeta demonstra que o açúcar era eficiente conjugado com o CPTEG 50:50: superfície de nanopartículas CPH. <img alt="A Figura 1" src<html> = &quot;/ Files/ftp_upload/3967/3967fig1.jpg&quot; /&gt; Figura 1. (A) representação gráfica da abordagem prosseguido com funcionalização hidrato de carbono de nanopartículas polianidrido e um exemplo das bibliotecas de nanopartículas funcionalizados que poderiam ser concebidos com o descrito abordagem de alto rendimento. (B) representação esquemática do aparelho de deposição automatizado utilizado para funcionalização de partícula, que consiste de (i) três NE 1000 bombas, (ii) uma fase robótico integrado por dois actuadores (Zaber): um para o movimento na direcção X ea outra para o movimento na direcção y, (iii) um segundo estágio robótico com duas cremalheiras adjacentes (adequado para os tubos e tinas) que consiste em três actuadores, um para cada direcção (x, y, ez). As bombas e um total de cinco actuadores estão ligados em série. Atuadores e bombas são operados por um computador utilizando o software LabVIEW. Este diagrama não está em escala.arge.jpg &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver maior figura. <img alt="A Figura 2" src="/files/ftp_upload/3967/3967fig2.jpg" /> Figura 2. Representação gráfica da automatizado iterativo síntese de hidratos de carbono, usando manose como um exemplo. <img alt="A Figura 3" src="/files/ftp_upload/3967/3967fig3.jpg" /> Figura 3. 1 H RMN do dimannoside protegido. <img alt="A Figura 4" src="/files/ftp_upload/3967/3967fig4.jpg" /> Figura 4. (A) Efeito do tempo de reacção sobre a concentração de nanopartículas de superfície de sacárido. Nos dados mostrados, 50:50 CPTEG: nanopartículas CPH foram funcionalizados com dimannose em diferentes tempos de reacção ea reacção foi realizada a 4 ° C. O erro médio e padrão de duas experiências independentes de funcionalização é mostrado. (B) nanopartículas lactose e di-manose funcionalizadoseficazmente receptor alvo DC-SIGN (CIRE, CD209) e manose (CD206) em derivadas da medula óssea células dendríticas, como demonstrado pelo aumento da expressão destes dois marcadores após estimulação com CPTEG 50:50 funcionalizado: nanopartículas CPH, quando comparado com a expressão obtida com não-funcionalizados partículas.

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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 é co-fundador e detém no capital da empresa de carboidratos LuCella Biosciences, Inc.

A eficácia de hidratos de carbono como agentes de segmentação a interacções de nanopartículas directos para células do sistema imunológico foi anteriormente demonstrada 10, 11. Uma pesquisa anterior nos nossos laboratórios têm mostrado que os açúcares específicas associadas a nanopartículas polianidrido são capazes de atingir CLRs diferentes em células apresentadoras de antigénio (APCs), aumentando assim a activação de células imunitárias que pode ser importante para a activação das cé...

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Motorizado Stage XYZ: 3x T-LSM050A, 50 viagens mm por eixo </td><td> Zaber Technologies </td><td> T-XYZ-LSM050A-KT04 </td></tr><tr><td> NE-1000 Bomba de seringa </td><td> Sistemas de bomba nova Era </td><td> NE-1000 </td></tr><tr><td> Pyrex * Vista * sem aro tubos de cultura de vidro reutilizáveis </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> Seringas SGE estanque aos gases Luer Loc </td><td> Sigma Aldrich </td><td> 509507 </td></tr><tr><td> XL-2000 sonicador </td><td> Qsonica </td><td> Q55 </td></tr><tr><td> Mini-tubo rotador </td><td> Fisher Scientific </td><td> 05-450-127 </td></tr></tbody></table>

high-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles

Abordagens transdisciplinares envolvendo áreas como design de materiais, nanotecnologia, química e imunologia tem que ser utilizado para projetar racionalmente portadores eficazes vacinas. Nanopartículas baseadas em plataformas pode prolongar a persistência de antígenos vacinais, que poderia melhorar a imunogenicidade da vacina 1. Vários polímeros biodegradáveis ​​têm sido estudados como veículos de entrega de vacinas 1, em particular, as partículas de polianidrido demonstraram a capacidade para proporcionar uma libertação sustentada de antigénios de proteínas estáveis ​​e para activar as células apresentadoras de antigénio e modulam as respostas imunes 2-12. A concepção molecular destes transportadores de vacina necessita de integrar a selecção racional de propriedades do polímero, bem como a incorporação de agentes adequados de segmentação. Fabricação de alta taxa de transferência automatizado de segmentação ligantes e partículas funcionalizados é uma poderosa ferramenta que irá melhorar a capacidade de estudar uma variedade rAnge de propriedades e irá conduzir à concepção de dispositivos de entrega de vacinas reprodutíveis. A adição de segmentação ligandos capazes de serem reconhecidos por receptores específicos em células imunes tem sido demonstrado que modulam e adaptar as respostas imunes 10,11,13 receptores de tipo C-lectina (CLRs) são receptores de reconhecimento de padrões (PRRS) que reconhecem hidratos de carbono presentes no superfície de agentes patogénicos. A estimulação de células do sistema imunológico através de CLRs permite para a internalização melhorada do antigénio ea apresentação subsequente para a activação das células T adicional 14,15. Portanto, as moléculas de hidratos de carbono têm um papel importante no estudo das respostas imunitárias, no entanto, a utilização destas biomoléculas frequentemente sofre da falta de disponibilidade de estruturalmente bem definida e hidratos de carbono puros. Uma plataforma de automação com base na solução iterativa de fase-reacções podem permitir a síntese rápida e controlada destas moléculas sinteticamente desafiantes utilizando b significativamente menoruilding quantidades de blocos do que as tradicionais métodos de fase sólida 16,17. Descrevemos aqui um protocolo para a síntese de solução de fase-automatizado de oligossacáridos, tais como manose baseados ligandos de segmentação com extracção de fase sólida para a purificação fluorosa intermédia. Após o desenvolvimento de métodos automatizados para tornar o agente de hidrato de carbono com base de segmentação, que descrevem métodos para a sua fixação sobre a superfície do polianidrido nanopartículas empregando um conjunto automatizado robótico até operado por LabVIEW como descrito anteriormente 10. Funcionalização da superfície com hidratos de carbono tem demonstrado eficácia na segmentação CLRs 10,11 e aumentando o rendimento do método de fabricação para descobrir as complexidades associadas com um sistema de multi-paramétricos será de grande valor (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

Neste artigo, um método de produção elevada é apresentada para a síntese de oligossacáridos e à sua fixação à superfície de nanopartículas de polianidrido para utilização posterior em alvejar receptores específicos em células apresentadoras de antigénio.

Alta capacidade de síntese de hidratos de carbono e funcionalização de nanopartículas polianidrido

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 visualizações.  Iowa State University. Neste artigo, um método de produção elevada é apresentada para a síntese de oligossacáridos e à sua fixação à superfície de nanopartículas de polianidrido para utilização posterior em alvejar receptores específicos em células apresentadoras de antigénio.

Vídeo: 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 de polímeros para Discovery High Throughput de Biomateriais

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

Bioengenharia

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.

Síntese combinatória de alta capacidade e liberação de proteínas a partir de polímero de Cinema e Bibliotecas Nanopartículas

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.

Colheita Murino macrófagos alveolares e Avaliação de ativação celular induzida por nanopartículas polianidrido

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.

Alta taxa de transferência de célula única e múltipla de células Micro-encapsulamento

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.

Avaliação de Entrega Gene Polimérico Nanopartículas por Análise Nanoparticle Rastreamento e alta capacidade de Citometria de Fluxo

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

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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.

Síntese orientada por microondas de óxido de ferro nanopartículas para detecção rápida de Aterosclerose

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

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 identificação de bactérias Polymers Repelente para dispositivos médicos

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

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.

Functionalization superfície de nanopartículas de vírus de hepatite E usando métodos de conjugação química

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

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

Síntese de nanotubos de carbono de paredes Multi modificados com nanopartículas de prata e avaliação das suas actividades antibacterianas e propriedades citotóxicas

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...