Expanding the Palette of SWIR Emitting Nanoparticles Based on Au Nanoclusters for Single-Particle Tracking Microscopy.

Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.

Mixing versus Polymer Chemistry in the Synthesis of Loaded Polymer Nanoparticles through Nanoprecipitation.

Polymer nanoparticles (NPs) loaded with drugs and contrast agents have become key tools in the advancement of nanomedicine, requiring robust technologies for their synthesis. Nanoprecipitation is a particularly interesting technique for the assembly of loaded polymer NPs, which is well-known to proceed under kinetic control, with a strong influence of the assembly conditions. On the other hand, the nature of the used polymer also influences the outcome of nanoprecipitation. Here, we investigated systematically the relative effects of mixing of the organic and aqueous phases and polymer chemistry on the formation of polymer nanocarriers. For this, two mixing schemes, manual mixing and microfluidic mixing using an impact-jet micromixer, were first evaluated, showing mixing times of several tens of milliseconds and a few milliseconds, respectively. Copolymers of ethyl methacrylate with charged and hydrophilic groups and different polyesters (poly(d-l-lactide-co-glycolide) and poly(lactic acid)) were combined with a fluorescent dye salt and tested for particle assembly using these "slow" and "fast" mixing methods. Our results showed that in the case of the most hydrophobic polymers, the speed of mixing had no significant influence on the size and loading of the formed NPs. In contrast, in the case of less hydrophobic polymers, faster mixing led to smaller NPs with better encapsulation. The switch between mixing and polymer-controlled assembly was directly correlated to the solubility limit of the polymers in acetonitrile-water mixtures, with a critical point for solubility limits between 15 and 20 vol % of water. Our results provide simple guidelines on how to evaluate the possible influence of polymer chemistry and mixing on the formation of loaded NPs, opening the way to fine-tune their properties and optimize their large-scale production.

Brightness of fluorescent organic nanomaterials.

Brightness is a fundamental property of fluorescent nanomaterials reflecting their capacity to absorb and emit light. In sensing materials, brightness is crucial for high-sensitivity (bio)molecular detection, while in optical bioimaging it ensures high spatial and temporal resolution. Fluorescent organic nanoparticles (NPs) are particularly attractive because of their superior brightness compared to organic dyes. With the ever-growing diversity of organic nanomaterials, it is important to establish universal principles for measuring and estimating their brightness. This tutorial review provides definitions of brightness and describes the major approaches to its analysis based on ensemble and single-particle techniques. We present the current chemical approaches to fight Aggregation-Caused Quenching (ACQ) of fluorophores, which is a major challenge in the design of bright organic nanomaterials. The main classes of fluorescent organic NPs are described, including conjugated polymer NPs, aggregation-induced emission NPs, and NPs based on neutral and ionic dyes. Their brightness and other properties are systematically compared. Some brightest examples of bulk solid-state emissive organic materials are also mentioned. Finally, we analyse the importance of brightness and other particle properties in biological applications, such as bioimaging and biosensing. This tutorial will provide guidelines for chemists on the design of fluorescent organic NPs with improved performance and help them to estimate and compare the brightness of new nanomaterials with literature reports. Moreover, it will help biologists to select appropriate materials for sensing and imaging applications.

Long-Range Energy Transfer between Dye-Loaded Nanoparticles: Observation and Amplified Detection of Nucleic Acids.

Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics, and biosensing, but its operating range is fundamentally limited by the Förster radius of ≈5 nm. In this work, FRET between fluorescent organic nanoparticles (NPs) is studied in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distances of 15 and 20 nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this long-distance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding the cancer marker survivin, bringing together donor and acceptor NPs at ≈15 nm distance. In this nanoprobe, a single-molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based biosensing.

Biotinylated Fluorescent Polymeric Nanoparticles for Enhanced Immunostaining.

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.

Intracellular accumulation and immunological response of NIR-II polymeric nanoparticles.

Polymeric nanoparticles (NPs) are extremely promising for theranostic applications. However, their interest depends largely on their interactions with immune system, including the capacity to activate inflammation after their capture by macrophages. In the present study, we generated monodisperse poly(ethyl methacrylate) (PEMA) NPs loaded with hydrophobic photoluminescent gold nanoclusters (Au NCs) emitting in the NIR-II optical windows and studied their interaction in vitro with J774.1A macrophages. PEMA NPs showed an efficient time and dose dependent cellular uptake with up to 70 % of macrophages labelled in 24 h without detectable cell death. Interestingly, PEMA and Au-PEMA NPs induced an anti-inflammatory response and a strong down-regulation of nitric oxide level on lipopolysacharides (LPS) activated macrophages, but without influence on the levels of reactive oxygen species (ROS). These polymeric NPs may thus present a potential interest for the treatment of inflammatory diseases.

Engineering Novel 3D Models to Recreate High-Grade Osteosarcoma and its Immune and Extracellular Matrix Microenvironment.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

Assembly of Fluorescent Polymer Nanoparticles Using Different Microfluidic Mixers.

Nanoprecipitation is a facile and efficient approach to the assembly of loaded polymer nanoparticles (NPs) for applications in bioimaging and targeted drug delivery. Their successful use in clinics requires reproducible and scalable synthesis, for which microfluidics appears as an attractive technique. However, in the case of nanoprecipitation, particle formation depends strongly on mixing. Here, we compare 5 different types of microfluidic mixers with respect to the formation and properties of poly(d-l-lactide-co-glycolide) (PLGA) and poly(methyl methacrylate) NPs loaded with a fluorescent dye salt: a cross-shaped mixer, a multilamination mixer, a split and recombine mixer, two herringbone mixers, and two impact jet mixers. Size and fluorescence properties of the NPs obtained with these mixers are evaluated. All mixers, except the cross-shaped one, yield NPs at least as small and fluorescent as those obtained manually. Notably in the case of impact jet mixers operated at high flow speeds, the size of the NPs could be strongly reduced from >50 nm down to <20 nm. Surprisingly, the fluorescence quantum yield of NPs obtained with these mixers also depends strongly on the flow speed, increasing, in the case of PLGA, from 30 to >70%. These results show the importance of precisely controlling the assembly conditions for loaded polymer NPs. The present work further provides guidance for choosing the optimal microfluidic setup for production of nanomaterials for biomedical applications.

Protein-like particles through nanoprecipitation of mixtures of polymers of opposite charge.

HYPOTHESIS: 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. EXPERIMENTS: 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. FINDINGS: 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.

Dynamic tracing using ultra-bright labeling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo.

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 h after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

Amplified Fluorescence in Situ Hybridization by Small and Bright Dye-Loaded Polymeric Nanoparticles.

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.

Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery.

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.

Bulky Barbiturates as Non-Toxic Ionic Dye Insulators for Enhanced Emission in Polymeric Nanoparticles.

Bulky hydrophobic counterions (weakly coordinating anions) can insulate ionic dyes against aggregation-caused quenching (ACQ) and enable preparation of highly fluorescent dye-loaded nanoparticles (NPs) for bioimaging, biosensing and light harvesting. Here, we introduce a family of hydrophobic anions based on fluorinated C-acyl barbiturates with delocalized negative charge and bulky non-polar groups. Similarly to fluorinated tetraphenylborates, these barbiturates prevent ACQ of cationic dye alkyl rhodamine B inside polymer NPs made of biodegradable poly(lactic-co-glycolic acid) (PLGA). Their efficiency to prevent ACQ increases for analogues with higher acidity and bulkiness. Their structure controls dye-dye communication, yielding bright NPs with on/off switching or stable emission. They enhance dye encapsulation inside NPs, allowing intracellular imaging without dye leakage. Compared to fluorinated tetraphenylborates known as cytotoxic transmembrane ion transporters, the barbiturates display a significantly lower cytotoxicity. These chemically available and versatile barbiturate derivatives are promising counterion scaffolds for preparation of bright non-toxic fluorescent nanomaterials.

Enzyme-free amplified detection of cellular microRNA by light-harvesting fluorescent nanoparticle probes.

Detection of cellular microRNA biomarkers is an emerging powerful tool in cancer diagnostics. Currently, it requires multistep tedious protocols based on molecular amplification of the RNA target, e.g. RT-qPCR. Here, we developed a one-step enzyme-free method for microRNA detection in cellular extracts based on light-harvesting nanoparticle (nanoantenna) biosensors. They amplify the fluorescence signal by effective Förster resonance energy transfer (FRET) from ultrabright dye-loaded polymeric nanoparticle to a single acceptor and thus convert recognition of one microRNA copy (through nucleic acid strand displacement) into a response of >400 dyes. The developed nanoprobes of 17-19 nm diameter for four microRNAs (miR-21, let-7f, miR-222 and miR-30a) exhibit outstanding brightness (up to 3.8 × 107 M-1cm-1) and ratiometric sequence-specific response to microRNA with the limit of detection (LOD) down to 1.3 pM (21 amol), equivalent to 24 RT-qPCR cycles. They enable quantitative detection of the four microRNAs in RNA extracts from five cancerous cell lines (human breast cancer - T47D and MCF7, head and neck cancer - CAL33 and glioblastoma - LNZ308 and U373) and two non-cancerous ones (Hek293 and MCF10A), in agreement with RT-qPCR. The results confirmed that let-7f and especially miR-21 are systematically overexpressed in all studied cancerous cell lines. These nanoparticle biosensors are compatible with low-cost portable fluorometers and small detection volumes (11 amol LOD), opening a route to rapid point-of-care cancer diagnostics.

Microcavity-Enhanced Fluorescence Energy Transfer from Quantum Dot Excited Whispering Gallery Modes to Acceptor Dye Nanoparticles.

Whispering gallery mode (WGM) microcavities are emerging as potential candidates in the field of biosensing applications, as their resonance wavelengths shift with changes in the refractive index in the region of their evanescent field. Their high-quality resonance modes and accessible surface functionalities make them promising for molecular assays, but their high sensitivity makes them inherently unstable. Here, we demonstrate that WGM resonances also strongly enhance fluorescence energy transfer between donors placed inside the microcavity and acceptors placed outside. We load colloidal quantum dots (QDs) into polymeric microspheres to provide WGMs that benefit from the QD optical features when used as energy-transfer donors. Spectroscopic analysis of the emission from the microcavities shows that the high quality of WGMs enables a very efficient energy transfer to dye-loaded polymer nanoparticle acceptors placed in their vicinity. Compared to Förster resonance energy transfer, WGM-enabled energy transfer (WGET) occurs over a much more extended volume, thanks to the delocalization of the mode over a typically 105 times larger surface and to the extension of the WGM electromagnetic field to larger distances (>100 nm vs a few nm) from the surface of the microcavity. The resulting sensing scheme combines the sensitivity of WGM spectroscopy with the specificity and simple detection schemes of fluorescence energy transfer, thus providing a potentially powerful class of biosensors.

Counterion-insulated near-infrared dyes in biodegradable polymer nanoparticles for in vivo imaging.

Polymeric nanoparticles (NPs) are highly attractive for biomedical applications due to their potential biodegradability and capacity to encapsulate different loads, notably drugs and contrast agents. For in vivo optical bioimaging, NPs should operate in the near-infrared region (NIR) and exhibit stealth properties. In the present work, we applied the approach of ionic dye insulation with bulky hydrophobic counterions for encapsulation of near-infrared cyanine dyes (Cy5.5 and Cy7 bearing two octadecyl chains) into biodegradable polymer (PLGA) NPs. We found that at high dye loading (20-50 mM with respect to the polymer), the bulkiest fluorinated tetraphenylborate counterion minimized best the aggregation-caused quenching and improved fluorescence quantum yields of both NIR dyes, especially of Cy5.5. In addition, bulky counterions also enabled formation of small 40 nm polymeric NPs in contrast to smaller counterions. To provide them stealth properties, we prepared 40 nm dye-loaded PEGylated NPs through nanoprecipitation of synthetic PLGA-PEG block copolymer with the dye/counterion salt. The obtained NIR NPs loaded with Cy5.5 dye salt allowed in vivo imaging of wild-type mice with a good contrast after IV injection. Compared to the bare PLGA NPs, PLGA-PEG NPs exhibited significantly slower accumulation in the liver. Biodistribution studies confirmed the preferential accumulation in the liver, although PLGA and PLGA-PEG NPs could also be distributed in other organs, with the following tendency: liver > spleen > lungs > kidney > heart > testis > brain. Overall, the present work validated the counterion approach for encapsulation of NIR cyanine dyes into biodegradable polymer NPs bearing covalently attached PEG shell. Thus, we propose a simple and robust methodology for preparation of NIR fluorescent biodegradable polymer NPs, which could further improve the existing optical imaging for biomedical applications.

Ultrabright Fluorescent Polymeric Nanoparticles with a Stealth Pluronic Shell for Live Tracking in the Mouse Brain.

Visualizing single organic nanoparticles (NPs) in vivo remains a challenge, which could greatly improve our understanding of the bottlenecks in the field of nanomedicine. To achieve high single-particle fluorescence brightness, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with a bulky hydrophobic counterion (perfluorinated tetraphenylborate) as a fluorophore insulator to prevent aggregation-caused quenching. To create NPs with stealth properties, we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that pluronics remained at the NP surface after dialysis (at one amphiphile per 5.5 nm2) and prevented NPs from nonspecific interactions with serum proteins and surfactants. In primary cultured neurons, pluronics stabilized the NPs, preventing their prompt aggregation and binding to neurons. By increasing dye loading to 20 wt % and optimizing particle size, we obtained 74 nm NPs showing 150-fold higher single-particle brightness with two-photon excitation than commercial Nile Red-loaded FluoSpheres of 39 nm hydrodynamic diameter. The obtained ultrabright pluronic-coated NPs enabled direct single-particle tracking in vessels of mice brains by two-photon intravital microscopy for at least 1 h, whereas noncoated NPs were rapidly eliminated from the circulation. Following brain injury or neuroinflammation, which can open the blood-brain barrier, extravasation of NPs was successfully monitored. Moreover, we demonstrated tracking of individual NPs from meningeal vessels until their uptake by meningeal macrophages. Thus, single NPs can be tracked in animals in real time in vivo in different brain compartments and their dynamics visualized with subcellular resolution.

Light-Harvesting Nanoparticle Probes for FRET-Based Detection of Oligonucleotides with Single-Molecule Sensitivity.

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20-nm light-harvesting nanoantenna particles, built of a tailor-made hydrophobic charged polymer poly(ethyl methacrylate-co-methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87-fold brighter than quantum dots QDots 605 in single-particle microscopy (with 550-nm excitation), these DNA-functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250-fold signal amplification. The obtained FRET nanoprobes enable single-molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi-fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.

Zwitterionic Stealth Dye-Loaded Polymer Nanoparticles for Intracellular Imaging.

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.

BODIPY-loaded polymer nanoparticles: chemical structure of cargo defines leakage from nanocarrier in living cells.

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.