Andreas Reisch

Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging

Speed, resolution and sensitivity of todays fluorescence bioimaging can be drastically improved by fluorescent nanoparticles (NPs) that are many-fold brighter than organic dyes and fluorescent proteins. While the field is currently dominated by inorganic NPs, notably quantum dots (QDs), fluorescent polymer NPs encapsulating large quantities of dyes (dye-loaded NPs) have emerged recently as an attractive alternative. These new nanomaterials, inspired from the fields of polymeric drug delivery vehicles and advanced fluorophores, can combine superior brightness with biodegradability and low toxicity. Here, we describe the strategies for synthesis of dye-loaded polymer NPs by emulsion polymerization and assembly of pre-formed polymers. Superior brightness requires strong dye loading without aggregation-caused quenching (ACQ). Only recently several strategies of dye design were proposed to overcome ACQ in polymer NPs: aggregation induced emission (AIE), dye modification with bulky side groups and use of bulky hydrophobic counterions. The resulting NPs now surpass the brightness of QDs by ≈10-fold for a comparable size, and have started reaching the level of the brightest conjugated polymer NPs. Other properties, notably photostability, color, blinking, as well as particle size and surface chemistry are also systematically analyzed. Finally, major and emerging applications of dye-loaded NPs for in vitro and in vivo imaging are reviewed.

Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles

The current challenge in the field of fluorescent nanoparticles (NPs) for bioimaging is to achieve extreme brightness and external control of their emission using biodegradable materials. Here we propose 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) that serves as spacer minimizing dye aggregation and self-quenching. The obtained 40-nm poly(D,L-lactide-co-glycolide) NPs containing up to 500 dyes are brighter than quantum dots and exhibit photo-induced reversible on/off fluorescence switching, never reported for dye-doped NPs. We show that this collective switching of hundreds of dyes is due to ultrafast excitation energy transfer and can be used for super-resolution imaging. 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 nanomaterials for sensing, imaging and light harvesting.

On the Benefits of Rubbing Salt in the Cut: Self‐Healing of Saloplastic PAA/PAH Compact Polyelectrolyte Complexes

The inherent room temperature mending and self-healing properties of saloplastic PAA/PAH CoPECs are studied. After ultracentrifugation of PAA/PAH polyelectrolyte complexes, tough, elastic materials are obtained that undergo self-healing facilitated by salt. At intermediate salt concentrations the CoPECs remain elastic enough to recover their original shape while the chains are mobile enough to repair the cut, thus leading to actual self-healing behavior.

Polyelectrolyte multilayers capped with polyelectrolytes bearing phosphorylcholine and triethylene glycol groups: parameters influencing antifouling properties.

In this paper, we investigate the design of antifouling surfaces by the deposition of polyelectrolytes modified by grafting of antifouling groups onto a (PSS/PAH)n precursor multilayer film [PSS, poly(styrenesulfonate); PAH, poly(allylamine)]. Different polyelectrolytes and different antifouling moieties are investigated, in particular, (EO)3 and (EO)3PC moieties (EO, ethylene oxide; PC, phosphorylcholine group). We find that protein adsorption can strongly be reduced and even practically suppressed through the deposition of only one layer of polyelectrolyte modified with PC and/or (EO)3 groups. We discuss the influence of various parameters such as the nature of the polyelectrolyte backbone, the nature of the antifouling moiety, and the grafting ratio on the reduction of protein adsorption. We find in particular that (EO)3 and (EO)3PC moieties grafted on poly(acrylic acid) (PAA) totally prevent protein adsorption for grafting ratios of 25% or more, at least within the detection limits of the used quartz crystal microbalance and optical waveguide light mode spectroscopy devices. The mechanism that leads to the antifouling property is discussed and compared to that leading to the antifouling properties of ethylene oxide self-assembled monolayers. Finally, by incorporating biotin on top of the precursor film, we show that one layer of PAA-(EO)3PC is not sufficient to prevent interaction with streptavidin but a PAA-(EO)3PC/PAH/PAA-(EO)3PC multilayer largely protects the biotin from interacting with streptavidin.

Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles

Ultrasmall polymer nanoparticles are rapidly gaining importance as nanocarriers for drugs and contrast agents. Here, a straightforward modular approach to efficiently loaded and stable sub-20-nm polymer particles is developed. In order to obtain ultrasmall polymer nanoparticles, we investigated the influence of one to two charged groups per polymer chain on the size of particles obtained by nanoprecipitation. Negatively charged carboxylate and sulfonate or positively charged trimethylammonium groups were introduced into the polymers poly(d,l-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA). According to dynamic light scattering, atomic force and electron microscopy, the presence of one to two charged groups per polymer chain can strongly reduce the size of polymer nanoparticles made by nanoprecipitation. The particle size can be further decreased to less than 15 nm by decreasing the concentration of polymer in the solvent used for nanoprecipitation. We then show that even very small nanocarriers of 15 nm size preserve the capacity to encapsulate large amounts of ionic dyes with bulky counterions at efficiencies >90%, which generates polymer nanoparticles 10-fold brighter than quantum dots of the same size. Postmodification of their surface with the PEG containing amphiphiles Tween 80 and pluronic F-127 led to particles that were stable under physiological conditions and in the presence of 10% fetal bovine serum. This modular route could become a general method for the preparation of ultrasmall polymer nanoparticles as nanocarriers of contrast agents and drugs.

Cyto-mechanoresponsive Polyelectrolyte Multilayer Films

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion.

Anti-fouling phosphorylcholine bearing polyelectrolyte multilayers: Cell adhesion resistance at rest and under stretching

We investigate the anti-fouling properties of polyelectrolyte multilayers bearing phosphorylcholine and triethylene glycol moieties and their adhesive response under stretching towards mammalian cells and fungi. More precisely we use a precursor multilayer deposited on glass and on an elastomeric silicone sheet and onto which one or two layers of polyacrylic acid modified with triethylene glycol or phosphorylcholine groups are added. In previous studies, these architectures proved to be resistant to protein adsorption (A. Reisch, J. C. Voegel, E. Gonthier, G. Decher, B. Senger, P. Schaaf and P. J. Mesini, Langmuir, 2009, 25, 3610; A. Reisch, J. Hemmerle, J. C. Voegel, E. Gonthier, G. Decher, N. Benkirane-Jessel, A. Chassepot, D. Mertz, P. Lavalle, P. Mesini and P. Schaaf, J. Mater. Chem., 2008, 18, 4242.). Here we investigate the adhesion of mammalian cells (fibroblasts) and of fungi (Candida albicans) both at rest and under uniaxial stretching of the substrate. Two layers of these polyelectrolytes yield surfaces that are practically resistant to the adhesion of fungi and mammalian cells at rest. Under stretching of the substrate, fungi adhesion remains almost totally prevented at least up to a stretching degree of 1.5, while fibroblast adhesion remains only prevented up to a stretching degree of 1.2. Fibroblast adhesion starts to take place and increases when the substrate is further stretched. The onset of fibroblast adhesion under stretching is retarded for phosphorylcholine containing films compared to those that contain triethylene glycol. These systems thus provide a first example of surfaces that present excellent anti-fouling properties at rest and become specifically adhesive under stretching.

Exploiting Fast Exciton Diffusion in Dye-Doped Polymer Nanoparticles to Engineer Efficient Photoswitching

Photoswitching of bright fluorescent nanoparticles opens new possibilities for bioimaging with superior temporal and spatial resolution. However, efficient photoswitching of nanoparticles is hard to achieve using Förster resonance energy transfer (FRET) to a photochromic dye, because the particle size is usually larger than the Förster radius. Here, we propose to exploit the exciton diffusion within the FRET donor dyes to boost photoswitching efficiency in dye-doped polymer nanoparticles. To this end, we utilized bulky hydrophobic counterions that prevent self-quenching and favor communication of octadecyl rhodamine B dyes inside a polymer matrix of poly(D,L-lactide-co-glycolide). Among tested counterions, only perfluorinated tetraphenylborate that favors the exciton diffusion enables high photoswitching efficiency (on/off ratio ∼20). The switching improves with donor dye loading and requires only 0.1-0.3 wt % of a diphenylethene photochromic dye. Our nanoparticles were validated both in solution and at the single-particle level. The proposed concept paves the way to new efficient photoswitchable nanomaterials.

Electrochemically addressed cross-links in polyelectrolyte multilayers: cyclic duravoltammetry.

In situ nanoindentation was performed on a multilayer of poly(acrylic acid) and a high molecular weight, pendant chain polyviologen under controlled electrochemical potential. The modulus of the thin film of polyelectrolyte complex was reversibly modulated, by about an order of magnitude, upon changing the state of charge within the material using the electrochemically active and addressable viologen repeat units. The applied potential, under aqueous conditions, is believed to control the extent of cross-link formation. Simultaneous quartz crystal microbalance measurements revealed the flux of ions into or out of the multilayer during redox cycling. Apparent film modulus also depends on the identity of the last layer.

Giant light-harvesting nanoantenna for single-molecule detection in ambient light

Here, we explore the enhancement of single-molecule emission by a polymeric nanoantenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nanoantenna, the cationic dyes are brought together, in very close proximity, using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60 nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1–2 acceptors, resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 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 scalable complementary metal-oxide–semiconductor camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight.Donor dye nanoparticles have been used to realize structures that are 25 times brighter than quantum dots. This enabled single-molecule imaging using ambient light.