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Dye-loaded polymeric nanoparticles

Mise à jour 17 janvier 2018

In 2014, we proposed a new concept of fluorescent polymer NPs, doped with ionic liquid-like salts of a cationic dye (octadecyl rhodamine B) with a bulky hydrophobic counterion (fluorinated tetraphenylborate) 1. The latter serves as spacer between cationic dyes minimizing dye aggregation and self-quenching (Figure 1). Remarkably, obtained ultra-bright nanoparticles undergo due to efficient dye-dye communication, the particles undergo photo-induced reversible on/off fluorescence switching (blinking) 1. This implies collective on off behavior of >100 dyes coupled by fast excitation energy transfer. The size of particles can be tuned down to 15 nm using single negative charge in polymer (PMMA, PLGA or PCL) 2. The obtained 15-nm PMMA-based NPs loaded with rhodamine based dyes are >10-fold brighter than quantum dots (Figure 1) 2. Moreover, by tuning dye loading and polymer matrix we can generate nanoparticles presenting stable emission or complete on/off switching (blinking) 3. Using photochromic dyes, these NPs can be effectively photo-switched 4.

Figure 1. Design of dye-doped fluorescent NPs using bulky counterions (left) ; fluorescence microscopy images of quantum dots QD585 and 15 nm PMMA-MA NPs containing R18/F5-TPB immobilized on a glass surface (middle) : our NPs are >10-fold brighter than QDs ; on/off switching (blinking) of single PLGA NPs containing >100 dyes (R18/F5-TPB) per particle. Data adapted from 1,2.

These NPs, being spontaneously endocytosed by living cells, feature high signal-to-noise ratio and absence of toxicity. The counterion-based concept opens the way to a new class of ultrabright nanomaterials for sensing, imaging and light harvesting. This class of nanoparticles has been recently reviewed by us 5.

Giant light-harvesting nanoantenna for single molecule detection

Nanoparticles encapsulating large number of dyes could serve as light harvesting antenna that collect light energy by ensemble of donor dyes and transfer to single dye acceptors, as realized by nature in photosynthetic centers (chloroplasts). Recently, we showed than our 60 nm PMMA polymer nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1–2 acceptors 6. As a result the emission of Cy5-based acceptor dye is amplified 1,000 (Figure 2) and it becomes 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1–10 mW cm−2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic set-up, which includes a ×20 air objective and a sCMOS camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight 6.

Figure 2. Scheme of nanoparticle antenna containing >10,000 donor dyes and 1-2 acceptor dyes, which can be detected by simple imaging setup using illumination equivalent to sunlight. Right : electron microscopy image of 60-nm nanoantennas and fluorescence photographs of single acceptor dye molecules inside nanoantenna using sunlight mimicking conditions. Representative single-particle emission at the donor and acceptor channels recorded at the excitation power of 1 mW/cm2 at 532 nm . Data adapted from 6.

Cell barcoding

An important application of fluorescence nanoparticles is long-term cell tracking. Recently we developed nanoparticles of three different color built from biodegradable poly(lactic-co-glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions 5. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations (Figure 3). Cell barcoding has already been validated on 7 cell lines, 13 color codes, and it enables simultaneous tracking of co-cultured barcoded cell populations for >2 weeks 7. This technology enabled six-color imaging in vivo for tracking xenografted cancer cells and monitoring morphogenesis after microinjection in zebrafish embryos (Figure 3) 7. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co-localized inside cells by combining structurally close nanoparticles carrying different functions.

Figure 3. Cell barcoding in vitro and in vivo using combination of cyanine-loaded polymer NPs of different color. The large micrograph shows a confocal image of six mixed cell types labeled with different RGB barcodes. Imaging of the development of zebrafish embryos labeled by intracellular injection of fluorescent NPs. Data adapted from 7.


1) Reisch, A., P. Didier, L. Richert, S. Oncul, Y. Arntz, Y. Mely and A.S. Klymchenko, Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nature Communications, 2014, 5, 4089.

2) Reisch, A., A. Runser, Y. Arntz, Y. Mely and A.S. Klymchenko, Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers : Making Bright and Stable Nanoparticles. ACS Nano, 2015, 9, 5104-5116.

3) Reisch, A., Trofymchuk, K., Runser, A., Fleith, G., Rawiso, M., and Klymchenko, A.S. Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix ACS Appl. Mater. Interfaces, DOI : 10.1021/acsami.7b12292.

4) Trofymchuk K., Prodi L., Reisch A., Mély Y., Altenhöner K., Mattay J., Klymchenko A.S. Exploiting fast exciton diffusion in dye-doped polymer nanoparticles to engineer efficient photoswitching, J. Phys. Chem. Lett. 2015, 6, 2259−2264.

5) Reisch, A. and A.S. Klymchenko, Fluorescent Polymer Nanoparticles Based on Dyes : Seeking Brighter Tools for Bioimaging. Small, 2016, 12, 1968-1992.

6) Trofymchuk, K. ; Reisch, A. ; Didier, P. ; Fras, F. ; Gilliot, P. ; Mely, Y. ; Klymchenko, A. S. : Giant light-harvesting nanoantenna for single-molecule detection in ambient light. Nature Photonics 2017, 11, 657.

7) Andreiuk, B., A. Reisch, M. Lindecker, G. Follain, N. Peyrieras, J.G. Goetz and A.S. Klymchenko, Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo. Small, 2017, 13(38).