Conjugation of DNA to Silanized Colloidal Semiconductor Nanocrystalline Quantum Dots

Parak, Wolfgang J.; Gerion, Daniele; Zanchet, Daniela; Waerz, Anke S.; Micheel, Christine; Williams, Shara C.; Seitz, Markus; Bruehl, Richard E.; Bryant, Zev; Bustamante, Carlos; Bertozzi, Carolyn R.; Alivisatos, Paul

doi:10.1021/cm0107878

Cited by 322 publications

(252 citation statements)

References 23 publications

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“…We used silica-coated CdSe/ZnS Qdots terminated with both thiol and PEG functional groups. 28 While the core/shell Qdots are only ~4-5 nm in size, the silane shell adds 2-3 nm in thickness and thus silanized Qdots are ~8-10 nm in diameter. 29 Such Qdot chemistry was observed to pose minimal toxicity to breast cancer cells when the cells were exposed to a solution containing 2-10 nM of PEG-silane-Qdots.…”

Section: Methodsmentioning

confidence: 99%

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

et al. 2006

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Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd 2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silaneQdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEGsilane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/ necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing ~22,000 total probe sets, containing 18,400 probe sets from known genes. Only ~50 genes (~0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater than 2-fold. Genes activated in treated cells included NIH Public Access

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Section: Methodsmentioning

confidence: 99%

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

et al. 2006

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“…In this approach the velocity of NPs in an applied electric filed [52] is a parameter related to the mean NP charge [53,54], if other hydrodynamic features are not modified. As in capillary electrophoresis no matrix as in gel electrophoresis is used increase in hydrodynamic diameters has less effect than in case of gel electrophoresis, in which conjugates of biological molecules and NPs may be sorted predominantly by size [55][56][57][58]. The charge change due to protein binding to NPs hence modifies their electrophoretic mobility, which in this way reflects the amount the amount of bound proteins protein.…”

Section: Description Of Experimental Techniquesmentioning

confidence: 99%

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

Hühn

Fedeli

Zhang

et al. 2016

The International Journal of Biochemistry & Cell Biology

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“…The electrical charges on the surface of the nanoparticles could also play an important role in determining the transfection and localization properties. The charges on the surface of the silanized qdots can be altered 30 , and the proteins that carry the NLS-qdots to the nucleus may have different levels of affinity to qdots with a different surface electrostatic charge. We are also actively investigating whether the same techniques can be used to target other organelles in the cell system, such as ER, Golgi, membrane, proteasome, perixisome, or mitochondria, with qdots bearing the proper signal peptides.…”

Section: An Intriguing Question Remains As To Why Certain Nls-qdots Cmentioning

confidence: 99%

Fluorescent CdSe/ZnS Nanocrystal−Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells

2004

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One of the biggest challenges in cell biology is the imaging of living cells. For this purpose, the most commonly used visualization tool is fluorescent markers. However, conventional labels, such as organic fluorescent dyes or green fluorescent proteins (GFP), lack the photostability to allow the tracking of cellular events that happen over a period from minutes to days. In addition, they are either toxic to cells (dyes) or difficult to construct and manipulate (GFP). We report here the use of a new class of fluorescent labels, silanized CdSe/ZnS nanocrystal−peptide conjugates, for imaging the nuclei of living cells. CdSe/ZnS nanocrystals, or so-called quantum dots (qdots), are extremely photostable, and have been used extensively in cellular imaging of fixed cells. Most of the studies about living cells so far have been concerned only with particle entry into the cytoplasm or the localization of receptors on the cell membrane. Specific targeting of qdots to the nucleus of living cells has not been reported in previous studies, due to the lack of a targeting mechanism and proper particle size. Here we demonstrate for the first time the construction of a CdSe/ZnS nanocrystal−peptide conjugate that carries the SV40 large T antigen nuclear localization signal (NLS) and the transfection of the complex into living cells. By a novel adaptation for qdots of a commonly used cell transfection technique, we were able to introduce and retain the NLS−qdots conjugate in living cells for up to a week without detectable negative cellular effects. Moreover, we can visualize the movement of the CdSe/ZnS nanocrystal−peptide conjugates from the cytoplasm to the nucleus, as well as the accumulation of the complex in the cell nucleus, over a long observation time period. This report opens the door for using qdots to visualize long-term biological events that happen in the cell nucleus and provides a new nontoxic, long-term imaging platform for observing nuclear trafficking mechanisms and cell nuclear processes.

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Conjugation of DNA to Silanized Colloidal Semiconductor Nanocrystalline Quantum Dots

Cited by 322 publications

References 23 publications

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

Fluorescent CdSe/ZnS Nanocrystal−Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells

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