Simple conjugated polymer nanoparticles as biological labels

Green, Mark; Howes, Philip D.; Berry, Catherine C.; Argyros, Orestis; Thanou, Maya

doi:10.1098/rspa.2009.0181

Cited by 47 publications

(43 citation statements)

References 28 publications

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“…Larger (50–250 nm) CP nanospheres referred to as semiconducting polymer nanospheres have also been prepared using an alternative miniemulsion process 28, 29. Preliminary investigations have been made on their potential cellular uptake mechanisms and cytotoxicity30–32.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles

et al. 2010

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Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.

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Section: Introductionmentioning

confidence: 99%

Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles

et al. 2010

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“…19 More recently, conjugated-polymer nanoparticles (polymer dots or Pdots) were generated for fluorescence imaging. 19−22 Exceptional brightness of Pdots (greater than QDs), photostability thousands of times greater than dyes, and cellular labeling were demonstrated. 23−25 Since fluorescence of conjugated polymers can be quenched by long-range intrachain and interchain energy transfer 26 and Pdots have heterogeneous brightness, 27 indicating that all fluorescent polymer chains do not emit with equal efficiency, it is important to test if there are Pdots which are fully quenched, that is, dark.…”

mentioning

confidence: 99%

Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol-Dependence of IGF1R Nanodomains

et al. 2013

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Fluorescent nanoparticles have enabled many discoveries regarding how molecular machines function. Quantum dots have been the dominant class of fluorescent nanoparticles but suffer from blinking and from a substantial dark fraction—particles where the fluorescence is never seen—complicating any analysis of biological function. Nanoparticles composed of conjugated fluorescent polymers (Pdots) have recently been shown to have high brightness and no blinking. Here we develop a robust and efficient means to measure the dark fraction of Pdots, conjugating Atto dyes to the nanoparticles and testing fluorescence colocalization of dye and Pdot puncta. This established that the Pdots we generated had minimal dark fraction: ∼3%. The application of nanoparticles in biological environments is highly sensitive to surface functionalization. For Pdots we found that passivation with uncharged hydroxy-terminated polyethylene glycol caused a dramatic reduction in nonspecific cell binding and aggregation compared to a charged coating. Using carbonyl di-imidazole the hydroxy-Pdots were functionalized efficiently with streptavidin for high stability targeting, allowing specific labeling of mammalian cells. Type I insulin-like growth factor receptor (IGF1R) regulates cell survival and development, with roles in aging, heart disease, and cancer. We used hydroxy-Pdots to track the dynamics of IGF1R on a breast cancer cell-line, determining the diffusion characteristics and showing cholesterol-containing membrane nanodomains were important for receptor mobility at the plasma membrane. The near-unity bright fraction and low nonspecific binding of hydroxy-Pdots, combined with Pdot photostability and lack of blinking, provides many advantages for investigations at the single molecule level.

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“…Today, fluorescence sensing and microscopy can be performed by labelling a sample with fluorescent dyes, fluorescent proteins, quantum dots (Resch-Genger et al, 2008) or other nanoparticles (Green et al, 2009;Howes et al, 2010Howes et al, , 2014, including nanodiamonds (Faklaris et al, 2009;Kuo et al, 2013;Mohan et al, 2010;Neugart et al, 2007) and nano-ruby (Edmonds et al, 2013), as reviewed recently , as well as imaging intrinsically fluorescent molecules naturally occurring within the sample -autofluorescence. In addition to fluorescence dyes, quantum dots and other nanoparticles have also recently found favour in cell imaging applications due to their high fluorescence quantum yield, low photobleaching susceptibility and narrow, size-dependent emission spectra which can be excited with a single wavelength (Grecco et al, 2004;Green, 2004;Howes et al, 2010;Michalet et al, 2005;Resch-Genger et al, 2008).…”

Section: Fluorescence Probesmentioning

confidence: 98%

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

et al. 2015

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Simple conjugated polymer nanoparticles as biological labels

Cited by 47 publications

References 28 publications

Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles

Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles

Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol-Dependence of IGF1R Nanodomains

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

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