Este trabalho foi financiado pela Golfers Contra o Câncer, Carolina Centro de Nanotecnologia Excelência Pilot concessão, Cancer Research Fund e Universidade Nacional de Saúde Instituto K-12 Prêmio de Desenvolvimento de Carreira.

1. Síntese de PLGA-b-polímero PEG <ol><li> Poli (D, L-ácido lático-co-ácido glicólico) (PLGA) com terminal grupos carboxilato (PLGA-carboxilato) é dissolvido em qualquer solvente para PLGA (como mencionado na seção de materiais) na concentração de 5mM. PLGA pode ser dissolvido nesta concentração com agitação suave. </li><li> Ambos NHS (peso molecular 115,09) e EDC (peso molecular 191,7) são dissolvidos na solução PLGA em uma concentração de 25mM. (Ambos EDC e NHS são adicionados em um excesso estequiométrico de 5 vezes comparado ao PLGA). PLGA-carboxilato é convertida em PLGA-NHS adicionando EDC e NHS para PLGA-carboxilato solução com agitação suave por cerca de 1 hora. </li><li> O produto da reação PLGA-NHS é precipitado pela adição de metanol a solução de lavagem. Aproximadamente 10 vezes excesso de volume de metanol é adicionado à solução. A solução é centrifugada a 2000 xg para precipitar o PLGA-NHS e desprezar o sobrenadante (remove os traços de EDC e NHS. Este procedimento de lavagem com metanol é repetido pelo menos três vezes. </li><li> O pellet PLGA-NHS é seco sob vácuo por 30 minutos para remover qualquer vestígio da solução desengraxante. </li><li> O pellet PLGA-NHS é agora re-dissolvido no mesmo solvente na mesma concentração que foi usado inicialmente para dissolver PLGA. O PEG heterobifunctional (PEG-amina-carboxilato) é então adicionado à solução PLGA na concentração de 5mM (relação estequiométrica de 1:1). A solução mistura é incubada por 24 horas, com agitação constante. </li><li> Após 24 horas, o produto da reação PLGA-b-PEG copolímero em bloco é precipitado pela adição de metanol a solução de lavagem em excesso. Repita o processo de lavagem e centrifugação, tal como mencionado acima três vezes. Isto irá remover todos os PEG em excesso que não reagiu. </li><li> O PLGA-b-PEG copolímero em bloco é seco sob vácuo. </li></ol> 2. PLGA-b-PEG preparação de nanopartículas Nanopartículas de PLGA com núcleo coberto com PEG na superfície podem ser preparados com estes copolímeros dibloco. Uma variedade de diferentes drogas hidrofóbicas podem ser encapsulados em nanopartículas tal. Compostos fluorescentes podem ser encapsulados em nanopartículas de ou pode ser conjugado com PLGA e, portanto, essas nanopartículas podem ser usadas para imagens de fluorescência. Nanoprecipitation método é usado para fazer as nanopartículas especialmente quando a carga desejada para ser encapsulado é altamente hidrofóbico na natureza. <ol><li> O PLGA-b-PEG copolímero em bloco ea droga / carga (a ser encapsulados) são dissolvidos em qualquer solvente que dissolve PLGA. PLGA pode ser dissolvido por muitos solventes comuns, incluindo acetonitrila, DCM, tetrahidrofurano, acetona ou acetato de etila. A escolha do solvente é fundamental, pois influencia as propriedades de nanopartículas. Assim, solvente apropriado deve ser usado nesta etapa. </li><li> A mistura de polímero / medicamento é então adicionado gota a gota a 3-5 volumes de água mexendo, dando uma concentração de polímero final de cerca de 3 mg / mL. (Fig. 2) </li><li> A agitação é mantida por 2 horas sob pressão reduzida para permitir que as nanopartículas para formar por auto-montagem e remover vestígios do solvente orgânico. </li><li> Colheita e purificação: As nanopartículas são, então, concentrada por centrifugação a 2.700 x g por 10 min, utilizando um filtro Amicon (MWCO 20KDa), lavado e reconstituída em PBS. Isso remove todas as cargas não-entrapped droga /. Caracterizações básicas biofísicos, tais como tamanho, carga superficial, e eficiência de carregamento de drogas podem ser realizados para melhor compreender as propriedades das nanopartículas. </li></ol> 3. Armazenamento Liofilização é um método comumente usado para armazenar nanopartículas 11. Liofilização vai preservar as características físicas e químicas das nanopartículas para a estabilidade a longo prazo 12. O processo de liofilização pode causar estresse sobre as partículas e desestabilizar a formulação, por isso crio-protetores (proteção contra o estresse de congelamento) e lyo-protetores (proteção contra o estresse de secagem) são comumente usados. A escolha destes protetores é determinada pelo comprimento desejado do tempo de armazenamento 13. <ol><li> Na liofilização, há a solidificação total da amostra por congelamento abaixo de sua Tg. </li><li> Na etapa de secagem, o gelo é removido por sublimação. Temperatura e pressão devem ser otimizados para atingir um processo de liofilização eficiente. </li></ol> 4. Resultados representativos: Caracterização de Copolímero Di-bloco PLGA-b-PEG Diferentes técnicas podem ser usadas para confirmar a conjugação de sucesso de polímeros. A composição do PLGA-PEG-b podem ser caracterizadas usando um 400 MHz 1H de ressonância magnética nuclear (RMN). Peso molecular do produto formado (PLGA-b-PEG) pode ser verificada por cromatografia de permeação em gel (GPC). O PLGA-b - PEG repartição do peso molecularn curva e tempo de eluição deve ser diferente do PLGA e sozinho PEG. Em conjunto, essas técnicas devem caracterizar o produto formado e determinar se a reação de conjugação foi bem sucedida. Caracterização de PLGA-b-PEG nanopartículas Tamanho das partículas e distribuição de tamanho pode ser medido por espalhamento de luz dinâmico. Diferentes parâmetros no processo nanoprecipitation afetar o tamanho das partículas. Peso molecular dos polímeros usados ​​inicialmente (ambos PLGA e PEG) também efeito da distribuição de tamanho de partícula. Microscopia eletrônica de transição (TEM) também pode ser usado para confirmar a distribuição do tamanho e estrutura das nanopartículas como visto na figura 3. A faixa de tamanho de partícula é geralmente na faixa de nm. Tamanhos de partículas grandes, com distribuição de tamanho irregular pode indicar um erro na reação de conjugação ou o método de otimização nanoprecipitation necessidades. Além disso, o potencial zeta da superfície pode ser medida por ZetaPALS. A eficiência de carregamento de drogas / carga pode ser quantificado com o padrão HPLC. As partículas são dissolvidos em solvente orgânico e HPLC pode ser realizada para medir a absorvância da droga / carga (Fig. 4). O estudo de liberação do fármaco cinética pode ser feito quando conhecidos quantidades fixas das nanopartículas são dialisada em 30 Slide-A-Lyzer unidades de diálise MINI. Em intervalos fixos, o conteúdo na unidade de diálise é coletado e volume igual de solvente orgânico é adicionado para dissolver as nanopartículas. HPLC é feito sobre essas amostras para quantificar o conteúdo de drogas / carga. <img alt="Figura 1" src="/files/ftp_upload/3398/3398fig1.jpg" /> Figura 1. EDC / NHS química <img alt="Figura 2" src="/files/ftp_upload/3398/3398fig2.jpg" /> Figura 2. Nanoprecipitation método para a preparação de nanopartículas poliméricas. A solução orgânica de um solvente (acetonitrila ou DCM) que contém o dibloco PEG-PLGA ea droga ou carga a ser carregada para a partícula é adicionado gota a gota a 3-5 mL de H 2 O. mexendo <img alt="Figura 3" src="/files/ftp_upload/3398/3398fig3.jpg" /> Figura 3. Microscopia eletrônica de transmissão de nanopartices. A imagem TEM de PEG-PLGA contendo nanopartículas wortamin. Ácido fosfotúngstico foi usado como um agente de contraste. <img alt="Figura 4" src="/files/ftp_upload/3398/3398fig4.jpg" /> Figura 4. Liberação controlada de drogas a partir de nanopartículas. Liberação de paclitaxel a partir de nanopartículas após a diálise em PBS. No momento observado, as partículas foram retirados a partir de cassetes de diálise e solublized em acetonitrila. A solução foi medido por HPLC. Dois lotes separados de nanopartículas foram comparados. <div id="myEventWatcherDiv"></div><div id="myEventWatcherDiv"></div>

<ol>
	<li>Drotleffa, S., Lungwitz, U., Breuniga, M., Dennis, A., Blunk, T., Tessmarc, J., Go&#235;pferich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+polymers+in+pharmaceutical+and+biomedical+sciences.">Biomimetic polymers in pharmaceutical and biomedical sciences.</a> European Journal of Pharmaceutics and Biopharmaceutics. 58, 385-407 (2004).</li><li>Bulte, J. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanoparticles in Biomedical Imaging. 3, (2008).</li><li>Omid, C., Farokhzad, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Nanotechnology+on+Drug+Delivery.">Impact of Nanotechnology on Drug Delivery.</a> ACS NANO. 3, 16-20 (2009).</li><li>Li, Y. -. P., Pei, Y. -. Y., Xian-Ying, Z., Zhou-Hui, G., Zhao-Hui, Z., Wei-Fang, Y., Jian-Jun, Z., Jian-Hua, Z., Xiu-Jian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+PLGA+nanoparticles+as+protein+carriers:+synthesis,+preparation+and+biodistribution+in+rats.">PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats.</a> Journal of Controlled Release. 71, 203-211 (2011).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (2008).</li><li>Jeong, B., Bae, Y. H., Lee, D. S., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+block+copolymers+as+injectable+drug-delivery+systems.">Biodegradable block copolymers as injectable drug-delivery systems.</a> Nature. 388, 860-862 (1997).</li><li>Yoo, H. S., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate+receptor+targeted+biodegradable+polymeric+doxorubicin+micelles.">Folate receptor targeted biodegradable polymeric doxorubicin micelles.</a> Journal of Controlled Release. 96, 273-283 (2004).</li><li>Cheng, J., Teply, B. A., Sherifi, I., Sung, J., Luther, G., Gu, F. X., Levy-Nissenbaum, E., Radovic-Moreno, A. F., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+Functionalized+PLGA-PEG+Nanoparticles+for+In+Vivo+Targeted+Drug+Delivery.">Formulation of Functionalized PLGA-PEG Nanoparticles for In Vivo Targeted Drug Delivery.</a> Biomaterials. 28, 869-876 (2007).</li><li>Gu, F., Zhang, L. F., Teply, B. A., Mann, N., Wang, A., Radovic-Moreno, A. F., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+engineering+of+targeted+nanoparticles+by+using+self-assembled+biointegrated+block+copolymers.">Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers.</a> Proceedings of the National Academy of Science. 105, 2586-2591 (2008).</li><li>Sanna, V., Pintus, G., Roggio, A. M., Punzoni, A., Posadino, A. M., Arca, A., Marceddu, S., Bandiera, P., Uzzau, S., Sechi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Biocompatible+Nanoparticles+for+the+Delivery+of+(-)-Epigallocatechin+3-Gallate+to+Prostate+Cancer+Cells.">Targeted Biocompatible Nanoparticles for the Delivery of (-)-Epigallocatechin 3-Gallate to Prostate Cancer Cells.</a> J. Med. Chem. 54, 1321-1332 (2011).</li><li>Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-drying+of+nanoparticles:+Formulation,+process+and+storage+considerations.">Freeze-drying of nanoparticles: Formulation, process and storage considerations.</a> Advanced Drug Delivery Reviews. 58, 1688-1713 (2006).</li><li>Holzer, M., Vogel, V., M&#228;ntele, W., Schwartz, D., Haase, W., Langer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physico-chemical+characterisation+of+PLGA+nanoparticles+after+freeze-drying+and+storage.">Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage.</a> European Journal of Pharmaceutics and Biopharmaceutics. 72, 428-437 (2009).</li><li>Lee, M. K., Kim, M. Y., Kim, S., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoprotectants+for+Freeze+Drying+of+Drug+Nano-Suspensions:+Effect+of+Freezing+Rate.">Cryoprotectants for Freeze Drying of Drug Nano-Suspensions: Effect of Freezing Rate.</a> Journal of Pharmaceutical Sciences. 98, 4808-4817 (2009).</li><li>Wang, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalized+targeted+nanoparticles+for+therapeutic+applications.">Biofunctionalized targeted nanoparticles for therapeutic applications.</a> Expert opinion on biological therapy. 8, 1063-1070 (2008).</li><li>Jeong, B., Bae, Y. H., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+release+from+biodegradable+injectable+thermosensitive+hydrogel+of+PEG-PLGA-PEG+triblock+copolymers.">Drug release from biodegradable injectable thermosensitive hydrogel of PEG-PLGA-PEG triblock copolymers.</a> J. Control Release. 63, 155-163 (2000).</li><li>Gref, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+long-circulating+polymeric+nanospheres.">Biodegradable long-circulating polymeric nanospheres.</a> Science. 263, 1600-1603 (1994).</li></ol>

Não há conflitos de interesse declarados.

O método nanoprecipitation usando dibloco co-polímeros representa um método simples e rápida de engenheiro nanopartículas poliméricas. As nanopartículas resultantes são compostos por um núcleo hidrofóbico que pode ser utilizado para a entrega de compostos pouco solúveis. A camada de superfície hidrofílica permite excelente solubilidade em água, proporcionando uma porção de conjugação mais potencial para um ligante alvo. Existem plataformas de nanopartículas muitos, incluin...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> EDC </td><td> Thermo Scientific </td><td> 22980 </td><td> Reagente conjugação </td></tr><tr><td> NHS </td><td> Thermo Scientific </td><td> 24500 </td><td> Reagente conjugação </td></tr><tr><td> aminas PEG-carboxilato </td><td> Laysan Bio Inc. </td><td> NH2-PEG-CM-5000 </td><td> Polímero (Pode usar qualquer MW PEG, 5000 está listado aqui) </td></tr><tr><td> PLGA-carbxylate </td><td> Lactel </td><td> B6013-2 </td><td> Polímero </td></tr><tr><td> Diclorometano (DCM) </td><td> Sigma-Aldrich </td><td> 34856 </td><td> Solvente </td></tr><tr><td> Acetonitrila&gt; pureza de 99% </td><td> Sigma-Aldrich </td><td> 34851 </td><td> Solvente </td></tr><tr><td> Metanol&gt; pureza de 99% </td><td> Sigma-Aldrich </td><td> 34860 </td><td> Lavar </td></tr></tbody></table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

A nanotecnologia é um ramo relativamente novo da ciência que envolve aproveitar as propriedades únicas de partículas que são nanômetros em escala (nanopartículas). Nanopartículas podem ser projetados de forma precisa onde a química tamanho, composição e superfície pode ser cuidadosamente controlada. Isto permite uma liberdade sem precedentes para modificar algumas das propriedades fundamentais da sua carga, tais como solubilidade, de difusividade, biodistribuição características, liberação e imunogenicidade. Desde a sua criação, as nanopartículas têm sido utilizados em muitas áreas da ciência e da medicina, incluindo a entrega da droga, imagem e biologia celular 1-4. No entanto, não foi totalmente utilizado fora de &quot;laboratórios de nanotecnologia&quot;, devido à percepção barreira técnica. Neste artigo, descrevemos um método simples para sintetizar uma plataforma de nanopartículas à base de polímeros que tem uma vasta gama de aplicações potenciais. O primeiro passo é sintetizar uma dibloco co-polímero que tem tanto domínio de um domínio hidrofóbico e hidrofílico. Utilizando PLGA e PEG como polímeros de modelo, descrevemos uma reação de conjugação usando EDC / NHS química 5 (Fig. 1). Discutimos também o processo de purificação de polímeros. O dibloco sintetizado co-polímero pode auto-organizar em nanopartículas no processo nanoprecipitation através hidrofóbico-hidrofílico interações. A nanopartícula de polímero descrito é muito versátil. O núcleo hidrofóbico da nanopartícula pode ser utilizado para transportar drogas pouco solúveis para a entrega da droga experiments6. Além disso, as nanopartículas podem superar o problema de solventes tóxicos para pouco solúveis reagentes de biologia molecular, tais como wortmannin, o que exige um solvente como o DMSO. No entanto, DMSO pode ser tóxico para as células e interferir com o experimento. Estes fármacos pouco solúveis e reagentes podem ser efetivamente entregue usando nanopartículas poliméricas com toxicidade mínima. Nanopartículas poliméricas também pode ser carregado com corante fluorescente e utilizados para estudos de tráfico intracelular. Por fim, essas nanopartículas de polímeros pode ser conjugado com a segmentação ligantes através PEG superfície. Tais nanopartículas alvo pode ser utilizada para rotular epítopos específicos em células ou em 10/07.

Watch this Scientific Journal Video about Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique at JoVE.com

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Este artigo descreve um método para sintetizar nanoprecipitation polímero baseado em nanopartículas usando dibloco co-polímeros. Vamos discutir a síntese de dibloco co-polímeros, a técnica nanoprecipitation e aplicações potenciais.

Formulação de nanopartículas poliméricas através da técnica de dibloco Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

formulation-diblock-polymeric-nanoparticles-through-nanoprecipitation

Research

JoVE Journal

Bioengineering

37.1K visualizações.  University of North Carolina School of Medicine. Este artigo descreve um método para sintetizar nanoprecipitation polímero baseado em nanopartículas usando dibloco co-polímeros. Vamos discutir a síntese de dibloco co-polímeros, a técnica nanoprecipitation e aplicações potenciais.

Vídeo: Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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 ...

Bioengenharia

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Células-tronco de programação para Therapeutic angiogênese Usando nanopartículas poliméricas biodegradáveis

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricação microfluida de fibras poliméricas e biohibridas com tamanho e forma predesigned

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

Polimérico Fabrication matriz Microneedle por fotolitografia

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Fabricação de super-hidrofóbicas Materiais Poliméricos para aplicações biomédicas

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

Predicando silenciamento de genes através do controle Spatiotemporal da liberação de siRNA de Nanocarriers poliméricos com fotos

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Montagem rápida, escalável e carregamento de proteínas bioativas e Imunoestimuladores em diversas Nanocarriers sintéticas através de Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Proteína quinase C-delta inibidor do Peptide formulação utilizando nanopartículas de ouro

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

Flash NanoPrecipitation para o encapsulamento de compostos hidrofóbicos e hidrofílicos em nanopartículas poliméricas

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

Preparação de nanopartículas poliméricos neutralmente carregadas e responsivas ao pH para entrega de siRNA citosólica

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...