Search

The performance of fluorescence immunostaining is physically limited by the brightness of organic dyes, whereas fluorescence labeling with multiple dyes per antibody can lead to dye self-quenching. The present work reports a methodology of antibody labeling by biotinylated zwitterionic dye-loaded polymeric nanoparticles (NPs). A rationally designed hydrophobic polymer, poly(ethyl methacrylate) bearing charged, zwitterionic and biotin groups (PEMA-ZI-biotin), enables preparation of small (14 nm) and bright fluorescent biotinylated NPs loaded with large quantities of cationic rhodamine dye with bulky hydrophobic counterion (fluorinated tetraphenylborate). The biotin exposure at the particle surface is confirmed by Förster resonance energy transfer with dye-streptavidin conjugate. Single-particle microscopy validates specific binding to biotinylated surfaces, with particle brightness 21-fold higher than quantum dot-585 (QD-585) at 550 nm excitation. The nanoimmunostaining method, which couples biotinylated antibody (cetuximab) with bright biotinylated zwitterionic NPs through streptavidin, significantly improves fluorescence imaging of target epidermal growth factor receptors (EGFR) on the cell surface compared to a dye-based labeling. Importantly, cetuximab labeled with PEMA-ZI-biotin NPs can differentiate cells with distinct expression levels of EGFR cancer marker. The developed nanoprobes can greatly amplify the signal from labeled antibodies, and thus become a useful tool in the high-sensitivity detection of disease biomarkers., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

Detection and imaging of RNA at the single-cell level is of utmost importance for fundamental research and clinical diagnostics. Current techniques of RNA analysis, including fluorescence in situ hybridization (FISH), are long, complex, and expensive. Here, we report a methodology of amplified FISH (AmpliFISH) that enables simpler and faster RNA imaging using small and ultrabright dye-loaded polymeric nanoparticles (NPs) functionalized with DNA. We found that the small size of NPs (below 20 nm) was essential for their access to the intracellular mRNA targets in fixed permeabilized cells. Moreover, proper selection of the polymer matrix of DNA-NPs minimized nonspecific intracellular interactions. Optimized DNA-NPs enabled sequence-specific imaging of different mRNA targets (survivin, actin, and polyA tails), using a simple 1 h staining protocol. Encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green-, red-, and far-red-emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously, showing distinctive mRNA expression profiles in three cancer cell lines. Image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of the method. AmpliFISH was found to be semiquantitative, correlating with RT-qPCR. In comparison with the commercial locked nucleic acid (LNA)-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) and requires only three steps vs ∼20 steps together with a much shorter time. Thus, combination of bright fluorescent polymeric NPs with FISH yields a fast and sensitive single-cell transcriptomic analysis method for RNA research and clinical diagnostics.

Efficient and safe delivery of nanoparticles (NPs) into the cytosol of living cells constitutes a major methodological challenge in bio-nanotechnology. Electroporation allows direct transfer of NPs into the cytosol by forming transient pores in the cell membrane, but it is criticized for invasiveness, and the applicable particle sizes are not well defined. Here, in order to establish principles for efficient delivery of NPs into the cytosol with minimal cytotoxicity, the influence of the size of NPs on their electroporation and intracellular behavior is investigated. For this study, fluorescent dye-loaded polymer NPs with core sizes between 10 and 40 nm are prepared. Optimizing the electroporation protocol allows minimizing contributions of endocytosis and to study directly the effect of NP size on electroporation. NPs of <20 nm hydrodynamic size are efficiently delivered into the cytosol, whereas this is not the case for NPs of >30 nm. Moreover, only particles of core size <15 nm diffuse freely throughout the cytosol. While electroporation at excessive electric fields induces cytotoxicity, the use of small NPs <20 nm allows efficient delivery at mild electroporation conditions. These results give clear methodological and design guidelines for the safe delivery of NPs for intracellular applications., (© 2020 Wiley-VCH GmbH.)

Intracellular applications of fluorescent nanoparticles (NPs) as probes and labels are currently limited by significant molecular crowding and the high level of complexity encountered inside living cells. The solution is to develop very small, bright, and noninteracting (stealth) NPs. Combining these properties requires implementing the stealth behavior through the thinnest possible hydrophilic shell. Here, we propose a one-step process for preparing ultrasmall and bright stealth NPs based on a zwitterionic (ZI) methacrylate-based copolymer. Dye-loaded polymer NPs are assembled through nanoprecipitation of the copolymer together with the salt of a rhodamine B derivative and a bulky hydrophobic counterion to achieve high particle brightness. We found that 10 mol % ZI groups in the polymer yield NPs of less than 15 nm that are stable in physiological salt conditions and practically resistant to protein adsorption, as suggested by fluorescence correlation spectroscopy. The combination of the very small size with the nonfouling nature of these particles enables spreading of ZI polymer NPs in the whole cytosol after their microinjection into living cells. In addition, single-particle tracking showed up to four times faster diffusion of ZI NPs in the cytosol compared to PEGylated NPs. The obtained dye-loaded ZI polymer NPs open the route to intracellular single-particle tracking and biosensing applications.

Uncontrolled release of encapsulated drugs and contrast agents from biodegradable polymer nanoparticles (NPs) is a central problem in drug delivery and bioimaging. In particular, it concerns polymeric NPs prepared by nanoprecipitation, where this release (so-called burst release) can be very significant, leading to side effects and/or bioimaging artifacts. Here, we systematically studied the effect of the chemical structure of cargo molecules, BODIPY dye derivatives, on their capacity to be loaded into ∼50 nm PLGA NPs without leakage in biological media. Absorption and fluorescence spectroscopy suggested that all the dyes, except the most polar BODIPY derivative, formed blended structures with polymer NPs. Fluorescence correlation spectroscopy of dye-loaded NPs in the presence of serum proteins revealed that only the most hydrophobic BODIPY dyes, bearing one octadecyl chain or two octyl chains, remain inside the NPs, while all other derivatives are released into the serum medium. The time-lapse absorption and fluorescence studies confirmed this result, suggesting the release kinetics for the leaky NPs on the time scale of hours. Fluorescence microscopy of living cells incubated with BODIPY-loaded NPs showed that most of them exhibit strong dye leakage observed as a homogeneous distribution of fluorescence all over the cytoplasm. Importantly, NPs loaded with the most hydrophobic dyes exhibited high stability showing a dotted pattern in the perinuclear region, typical for endosomes and lysosomes. Our results highlight the significance of the cargo hydrophobicity for efficient encapsulation inside polymeric NPs prepared by nanoprecipitation, which enables designing stable cargo-loaded nanomaterials for bioimaging and drug delivery.

The key challenge in the field of fluorescent nanoparticles (NPs) for biological applications is to achieve superior brightness for sizes equivalent to single proteins (3-7 nm). We propose a concept of shell-cross-linked fluorescent micelles, in which PEGylated cyanine 3 and 5 bis-azides form a covalently attached corona on micelles of amphiphilic calixarene bearing four alkyne groups. The fluorescence quantum yield of the obtained monodisperse NPs, with a size of 7 nm, is a function of viscosity and reached up to 15 % in glycerol. In the on-state they are circa 2-fold brighter than quantum dots (QD-585), which makes them the smallest PEGylated organic NPs of this high brightness. FRET between cyanine 3 and 5 cross-linkers at the surface of NPs suggests their integrity in physiological media, organic solvents, and living cells, in which the NPs rapidly internalize, showing excellent imaging contrast. Calixarene micelles with a cyanine corona constitute a new platform for the development of protein-sized ultrabright fluorescent NPs., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Speed, resolution and sensitivity of today's 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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

Photoactive transition‐metal complexes are luminophores combining high photostability and long luminescence lifetimes. However, reduced optical performance in aqueous solutions has limited their use in biological systems. Herein, the physicochemical and photophysical properties and bioimaging compatibility of Re diimine complexes and near‐infrared (NIR) emitting Cy5 dyes coencapsulated in polymer nanoparticles (NPs) are investigated. By varying the polymers, NPs with sizes from 20 to 70 nm and encapsulating ≤ 40 wt.% of Re complexes, i.e., ≈11 000 Re complexes per NP, are obtained. The photoluminescence (PL) quantum yields of the Re complexes increase eightfold to ≈50% upon encapsulation (vs 6–7% in acetonitrile), resulting in PL brightness up to 108 m−1 cm−1 and PL lifetimes of 3–4 µs. Coencapsulation of Cy5 yields very bright NIR emission upon Re complex excitation. Very close Re‐to‐Cy5 donor–acceptor distances down to ≤2 nm and FRET efficiencies over 90% are confirmed by PL lifetime measurements. The Re‐Cy5 NPs enter mammalian cells for high‐contrast PL imaging in both visible and NIR. This detailed characterization provides a better understanding of the photophysical properties of the transition‐metal‐dye FRET NPs and presents a vital step toward the efficient design of a new class of bright luminescent NP probes. [ABSTRACT FROM AUTHOR]

Dye‐loaded polymer nanoparticles (NPs) emerge as a powerful tool for bioimaging applications, owing to their exceptional brightness and controlled small size. However, aggregation‐caused quenching (ACQ) and leakage of dyes at high loading remain important challenges of these nanomaterials. The use of bulky hydrophobic counterions has been recently proposed as an effective approach to minimize ACQ and dye leakage, but the role of counterion structure is still poorly understood. Here, a systematic study based on ten counterions, ranging from small hydrophilic perchlorate up to large hydrophobic tetraphenylborate derivatives, reveals how counterion nature can control encapsulation and emission of a cationic dye (rhodamine B octadecyl ester) in NPs prepared by nanoprecipitation of a biodegradable polymer, poly‐lactide‐co‐glycolide (PLGA). We found that increase in counterion hydrophobicity enhances dye encapsulation efficiency and prevents dye adsorption at the particle surface. Cellular imaging studies revealed that ≥95 % encapsulation efficiency, achieved with most hydrophobic counterions (fluorinated tetraphenylborates), is absolutely required because non‐encapsulated dye species at the surface of NPs are the origin of dye leakage and strong fluorescence background in cells. The size of counterions is found to be essential to prevent ACQ, where the largest species, serving as effective spacer between dyes, provide the highest fluorescence quantum yield. Moreover, we found that the most hydrophobic counterions favor dye–dye coupling inside NPs, leading to ON/OFF fluorescence switching of single particles. By contrast, less hydrophobic counterions tend to disperse dyes in the polymer matrix favoring stable emission of NPs. The obtained structure‐property relationships validate the counterion‐based approach as a mature concept to fight ACQ and dye leakage in the development of advanced polymeric nanomaterials with controlled optical properties. Counterion‐enhanced encapsulation and emission: A systematic study based on ten counterions revealed that their size and hydrophobicity is essential to prevent aggregation‐caused quenching and ensure efficient encapsulation of a cationic rhodamine dye in polymeric nanoparticles. Here, the use of counterions is established as mature concept for preparation of bright and leakage‐free fluorescent nanomaterials for bioimaging applications. [ABSTRACT FROM AUTHOR]

[Display omitted] Polymer nanoparticles (NPs) have a very high potential for applications notably in the biomedical field. However, synthetic polymer NPs cannot yet concurrence the functionalities of proteins, their natural counterparts, notably in terms of size, control over internal structure and interactions with biological environments. We hypothesize that kinetic trapping of polymers bearing oppositely charged groups in NPs could bring a new level of control and allow mimicking the surfaces of proteins. Here, the assembly of mixed-charge polymer NPs through nanoprecipitation of mixtures of oppositely charged polymers is studied. Two series of copolymers made of ethyl methacrylate and 1 to 25 mol% of either methacrylic acid or a trimethylammonium bearing methacrylate are synthesized. These carboxylic acid or trimethylammonium bearing polymers are then mixed in different ratios and nanoprecipitated. The influence of the charge fraction, mixing ratio of the polymers, and precipitation conditions on NP size and surface charge is studied. Using this approach, NPs of less than 25 nm with tunable surface charge from +40 mV to −40 mV are assembled. The resulting NPs are sensitive to pH and certain NP formulations have an isoelectric point allowing repeated charge reversal. Encapsulation of fluorescent dyes yields very bright fluorescent NPs, whose interactions with cells are studied through fluorescence microscopy. The obtained results show the potential of nanoprecipitation of oppositely charged polymers for the design of NPs with precisely tuned surface properties. [ABSTRACT FROM AUTHOR]