Conference Agenda

Colloidal Nano- and Microparticles: Towards Sensing Applications

Wolfgang Parak

Philipps Universität Marburg, Germany

Nanomedicine nowadays is a popular key word in the media, though everyone seems to associate it with different visions, hopes, and even fears. From the point of view of a materials scientist it will be pointed out what new materials will be possible, how they will be designed, and which properties they could offer for diagnosis and treatment. It will be critically discussed that though sophisticated materials with advanced novel properties will be available in the future, they do not automatically match the requirements and demands of clinicians. The discussion is centred around one example, multifunctional polyelectrolyte capsules which might act as a "nano-submarine" for in vivo sensing and delivery, which is used to highlight promising interfaces between both disciplines.

_C. Röcker, M. Pötzl, F. Zhang, W. J. Parak, G. U. Nienhaus, "A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles", Nature Nanotechnology 4, 577-580 (2009).

P. Rivera Gil, S. de Koker, B. G. de Geest, W. J. Parak, "Intracellular processing of proteins mediated by biodegradable polyelectrolyte capsules", Nanoletters 9, 4398-4402 (2009).

J. Peteiro-Cartelle, M. Rodríguez-Pedreira, F. Zhang, P. Rivera Gil, L. L. del Mercato, W. J. Parak, "How colloidal nano- and microparticles could contribute to medicine - a personal perspective both from the eyes of physicians and materials scientists", Nanomedicine 4, 967-979 (2009)._

Interactions between cells and colloidal nanoparticles: From receptor-mediated interactions to laser hyperthermia.

Antonios Kanaras

University of Southampton, United Kingdom

Manipulating the interactions of biological cells using nanotechnology is of great importance for the development of new diagnostic and therapy methods, drug delivery, and imaging. An important step to manipulate biological processes is to understand how the engineering, in terms of size, shape and functionality, of advanced colloidal nanoparticles can be employed to our benefit in order to control cellular functions.

In this presentation we demonstrate the interactions of a specific type of peptide-coated gold nanospheres with the angiogenic receptors VEGF and NRP-1 in the membrane of endothelial cells.[1-3] The particles containing the peptides bind to the receptors and promote an angiogenic response. We found that angiogenic genes are over-expressed in the case of pro-angiogenic-coated gold nanospheres and down-regulated in the case of anti-angiogenic-coated gold nanoparticles. The striking observation is that when the anti-angiogenic or pro-angiogenic molecules are attached to the gold nanospheres, the particle are 500 times more reactive in up-regulating or down-regulating the expression of angiogenic genes than the unbound angiogenic molecules.

Furthermore, we employ a low intensity laser-induced technique to manipulate the damage and repair of endothelial cells.[4] We will show that the degree of damage and repair of the cells is influenced by laser illumination in the presence of gold nanoparticles of various morphologies, which target the cellular membrane. Thus, we illustrate that plasmon-mediated mild laser hyperthermia, combined with specific targeting of cellular membranes, can enable new routes for controlling cell permeability and gene regulation in endothelial cells.

References:

1. Bartczak D.; Sanchez-Elsner T.; Louafi F., Millar T.; Kanaras, A. G. Small 2011, 7, 388–394.

2. Bartczak D.; Kanaras A. G. Langmuir 2011, 27 (16), 10119–10123

3. Kanaras, A. G.; Bartczak, D.; Millar, T. M.; Sanchez-Elsner, T.; Muskens, O. L. Proc. of SPIE 2011, Vol. 7909 79090J-1 doi: 10.1117/12.871602.

4. Bartczak, D.; Muskens, O. L.; Millar, T. M.; Sanchez-Elsner, T. Kanaras, A. G. NanoLett. 2011, 11, 1358–1363.

Internalization of gold nanoparticles in spermatozoa can be promoted by distinct bio-functionalization

Annette Barchanski¹, Ulrike Taylor², Csaba Laszlo Sajti¹, Stephan Barcikowski³, Detlef Rath²

¹Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany; ²Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Höltystrasse 10, 31535 Neustadt-Mariensee, Germany; ³University of Duisburg-Essen, Institute for Technical Chemistry, Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CeNIDE), Universitaetsstraße 7, 45141 Essen, Germany

Directed targeting and strong labeling of specific cell populations with known phenotype, using long-term stable markers, is essential for their detection and separation from a heterogeneous cell suspension. Currently, an efficient method for cell sorting is based on high-speed flow cytometry, mostly after co-incubation of cells with fluorophore-tagged targeting moieties like DNA, antibodies or aptamers. However, photostability, cytotoxicity and membrane permeability are limiting fluorophore-specific attributes.

Gold nanoparticles (AuNP) as novel, fluorophore-alternative labeling markers will improve high-throughput screening, since the particles display high quantum yield without photo-bleaching and the possibility of non-destructive membrane penetration when coupled to penetrating agents additionally. Especially, AuNP fabricated by pulsed laser ablation in liquids are highly interesting for this application, due to an excellent biocompatibility, fabrication-dependent, tunable particle size and ease of efficient (bio)-functionalization in a one-step process with strong ligand binding.

We have recently demonstrated the penetration of bi-functionalized, laser-generated AuNP loaded with a targeting DNA fragment and various membrane-penetrating agents in distinct combinations and ligand ratios into sperm. A clear trend was observed, starting with sporadic cell membrane attachment of the conjugates, via membrane penetration and accumulation inside sperm up to explicit nucleus internalization, depending on attached cargo and nanoparticle size.

Moreover, first results on AuNP online detection by advanced flow cytometry were obtained, supporting the high potential of specific cell population labeling with gold nanoparticles for improved high-throughput sorting.

Exposure to gold nanoparticles affects sperm functionality and early embryogenesis

Ulrike Taylor¹, Annette Barchanski², Stephan Barcikowski³, Wilfried Kues¹, Detlef Rath¹

¹Institute of Farm Animal Genetics, Friedrich Loeffler Institut, Germany; ²Laserzentrum Hannover e.V., Hannover, Germany; ³University of Duisburg-Essen, Institute for Technical Chemistry, Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CeNIDE), Essen, Germany

Due to their unique optical properties and easy modification with functional molecules, gold nanoparticles (AuNP) are considered to be valuable new tools for bio-imaging and therapeutic applications. However, it has been shown that nanoparticles can cross the blood-testicular-barrier. Therefore, the present work aimed to investigate the effect of AuNP on the fertilising ability of spermatozoa, as well as on the developmental capacity of the resulting embryo. Nanoparticles were produced by laser ablation in water and coincubated (1 and 10 µg/ml final concentration) with bovine spermatozoa for 2 hours. In parallel a negative control was run. Subsequently nanoparticles were removed via centrifugation. Immediately after treatment, the spermatozoa were used for in vitro fertilisation of in vitro matured bovine oocytes. For the experiments a total of 1406 oocytes was equally divided between treatment groups. In vitro culture was stopped either after 19h to investigate sperm fertilizing ability by assessment of pronucleus formation, or after 8 days to measure blastocyst development. Both parameters were significantly influenced by the presence of AuNP at a concentration of 10 µg/ml. Pronucleus formation dropped from 66.6±7.5% (untreated control) to 36.7±9.3% (p<0.05) (treated group), while blastocyst development decreased from 32.6±1.0% to 21.1±1.1% (p<0.001), respectively. In conclusion, the results show that low doses of gold nanoparticles (1µg/ml) left the spermatozoa unimpaired. However, concentrations of 10 μg/ml AuNP resulted already after a exposition time of 2 hours in detrimentral effects on sperm function and early embryogenesis. These results request a more detailed analysis on the interaction of nanoparticles with germ cells and early embryos. Further research will focus on the understanding of the mechanisms behind the observed phenomenon.