Generación de Scalable, metálico de alta Aspect Ratio nanocompuestos en una Biológica Medio Líquido
Summary
Aquí se presenta un protocolo para sintetizar nuevos, biocomposites elevada relación de aspecto en condiciones biológicas y en medios líquidos. Los biocomposites escala de nanómetros a micrómetros de diámetro y longitud, respectivamente. Nanopartículas de cobre (CNP) y sulfato de cobre en combinación con cistina son los componentes clave para la síntesis.
Abstract
El objetivo de este protocolo es describir la síntesis de dos nuevos biocomposites con estructuras de elevada relación de aspecto. Los biocomposites consisten en cobre y cistina, ya sea con nanopartículas de cobre (CNP) o sulfato de cobre contribuye el componente metálico. La síntesis se llevó a cabo en un líquido en condiciones biológicas (37 ° C) y la forma composites autoensambladas después de 24 hr. Una vez formados, estos compuestos son muy estables tanto en medios líquidos y en una forma seca. Los materiales compuestos escala de la nano- a micro gama de longitud, y desde unos pocos micrómetros a 25 nm de diámetro. Emisión de campo microscopía electrónica de barrido con espectroscopia de rayos X de energía dispersiva (EDX) demostró que el azufre estaba presente en las estructuras lineales derivados de NP, mientras que estaba ausente de material de partida CNP, confirmando así la cistina como la fuente de azufre en los nanocompuestos finales . Durante la síntesis de estos nano y micro-compuestos lineales, una amplia gama de longitudes de structures se forma en el recipiente de síntesis. La sonicación de la mezcla líquida después de la síntesis se demostró para ayudar a controlar el tamaño promedio de las estructuras por la disminución de la longitud media con un aumento del tiempo de sonicación. Dado que las estructuras formadas son muy estables, no se aglomeran, y están formados en fase líquida, centrifugación también puede ser usada para ayudar en la concentración y la segregación de los materiales compuestos formados.
Introduction
Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 °C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called “urchin” and “nanoflower” type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 °C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described.
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells.
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described “coffee ring effect” 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.
Protocol
Representative Results
Discussion
Mientras que la evaluación de potenciales efectos tóxicos de los nanomateriales incluyendo CNP, se observó que en el largo plazo, CNP fueron transformados de una distribución de partículas más dispersa inicialmente a una forma más grande, agregada (Figura 2). En algunos casos, estas formaciones altamente agregados que se produjeron en la placa de cultivo de células, en condiciones biológicas, formaron proyecciones altamente lineales del agregado central, que recuerda cobre descrito anteriorment…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).
Materials
| Mini Vortexer | VWR (https://us.vwr.com) | 58816-121 | |
| CO2 Incubator Model # 2425-2 | VWR (https://us.vwr.com) | Contact vendor | Current model calalog # 98000-360 |
| Eppendorf Centrifuge (Refrigerated Microcentrifuge) | Labnet (http://labnetinternational.com/) | C2500-R | Model Prism R |
| Cell Culture Centrifuge Model Z323K | Labnet (http://labnetinternational.com/) | Contact vendor | Current model Z206A catalog # C0206-A |
| Sonicator (Ultrasonic Cleaner) | Branson Ultrasonics Corporation (http://www.bransonic.com/) | 1510R-MTH | |
| Balance | Sartorius (http://dataweigh.com) | Model CP225D similar model CPA225D | |
| Olympus IX51 Inverted Light Microscope | Olympus (http://olympusamerica.com | Contact vendor | |
| Olympus DP71 microscope digital camera | Olympus (http://olympusamerica.com | Contact vendor | |
| external power supply unit- white light for Olympus microscope | Olympus (http://olympusamerica.com | TH4-100 | |
| 10x, 20, and 40x microscope objectives | Olympus (http://olympusamerica.com | Contact vendor | |
| Scanning Electron Microscope | Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html) | model S-4800 | |
| Transmission Electron Microscope | Zeiss (http://zeiss.com/microscopy/en_de/products.html) | model Libra 120 | |
| Table Top Work Station Unidirectional Flow Clean Bench | Envirco (http://envirco-hvac.com) | model PNG62675 | Used for sterile cell culture technique |
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