Anne Runser

Denis Dujardin

Pauline Ernst

Andrey S Klymchenko

Andreas Reisch

Zwitterionic Stealth Dye-Loaded Polymer Nanoparticles for Intracellular Imaging

Keywords: fluorescent nanoparticles, dye-loaded polymer nanoparticles, zwitterion coating, stealth nanoparticles, single particle tracking, intracellular imaging

come

Introduction

The emergence of single-molecule and super-resolution fluorescence microscopy have opened a new area of sensing and imaging of biological molecules and processes in living cells. [START_REF] Shashkova | Single-Molecule Fluorescence Microscopy Review: Shedding New Light on Old Problems[END_REF][START_REF] Nelson | Localization Microscopy: Mapping Cellular Dynamics with Single Molecules[END_REF][START_REF] Kusumi | Tracking Single Molecules at Work in Living Cells[END_REF] However, these techniques require very bright fluorescent labels and probes, because their spatio-temporal resolution and sensitivity is directly linked to the number of collected photons. [START_REF] Deschout | Precisely and Accurately Localizing Single Emitters in Fluorescence Microscopy[END_REF] Fluorescent nanoparticles (NPs) such as quantum dots, [START_REF] Pinaud | Probing Cellular Events, One Quantum Dot at a Time[END_REF][START_REF] Pierobon | Quantum Dots to Tail Single Bio-Molecules inside Living Cells[END_REF] silica NPs, [START_REF] Bonacchi | Luminescent Silica Nanoparticles: Extending the Frontiers of Brightness[END_REF] conjugated polymer NPs, [START_REF] Liu | Tempo-Spatially Resolved Scattering Correlation Spectroscopy under Dark-Field Illumination and Its Application to Investigate Dynamic Behaviors of Gold Nanoparticles in Live Cells[END_REF] aggregation induced emission NPs [START_REF] Genin | Hyper-Bright" Near-Infrared Emitting Fluorescent Organic Nanoparticles for Single Particle Tracking[END_REF] and dye-loaded NPs 10 can overcome the limited brightness of fluorescent proteins or organic fluorophores. In particular, dye-loaded polymer NPs have recently achieved very high brightness equivalent to 100-1000 organic dyes. [START_REF] Genin | Hyper-Bright" Near-Infrared Emitting Fluorescent Organic Nanoparticles for Single Particle Tracking[END_REF][START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF][START_REF] Gu | Strategies to Overcome the Limitations of AIEgens in Biomedical Applications[END_REF] Achieving such high brightness required the development of new strategies to overcome aggregation-caused quenching of the fluorophores encapsulated at very high concentrations in the polymer matrix of NPs. This was achieved, for example, by using fluorophores that undergo aggregation-induced emission [START_REF] Li | Photostable Fluorescent Organic Dots with Aggregation-Induced Emission (AIE Dots) for Noninvasive Long-Term Cell Tracing[END_REF] or by using salts of fluorophores with hydrophobic bulky counterions to prevent aggregation. [START_REF] Reisch | Collective Fluorescence Switching of Counterion-Assembled Dyes in Polymer Nanoparticles[END_REF] Importantly, the bulky counterions approach enabled generation of ultrabright polymeric NPs with unprecedented energy transfer, [START_REF] Trofymchuk | Giant Light-Harvesting Nanoantenna for Single-Molecule Detection in Ambient Light[END_REF] which is essential for preparation of nanoparticle-based fluorescent biosensors. [START_REF] Melnychuk | DNA-Functionalized Dye-Loaded Polymeric Nanoparticles: Ultrabright FRET Platform for Amplified Detection of Nucleic Acids[END_REF] Furthermore, polymer-based dye-loaded NPs have been shown to be non-toxic, and they can be made from biodegradable polymers, being highly flexible with respect to (co)encapsulation and release of active compounds. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF][START_REF] Li | Polymer-Encapsulated Organic Nanoparticles for Fluorescence and Photoacoustic Imaging[END_REF] However, intracellular applications require small probe sizes and the absence of non-specific interactions in biological environments, which remain key challenges of current fluorescent NPs. [START_REF] Sahl | Fluorescence Nanoscopy in Cell Biology[END_REF] First, in order to increase localization precision, the particle diameter should be comparable to that of the biomolecule (e.g. protein, nucleic acid) it is intended to label/detect. Second, the particle size and the surface coating play a crucial role in defining the intracellular behavior of NPs and impact on the behavior of the labeled biomolecules. [START_REF] Etoc | Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells[END_REF][START_REF] Xu | How Entanglement of Different Physicochemical Properties Complicates the Prediction of in Vitro and in Vivo Interactions of Gold Nanoparticles[END_REF][START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF] Diffusion of NPs can be limited due to structural restrictions and macromolecular crowding effects inside cells. [START_REF] Luby-Phelps | Cytoarchitecture and Physical Properties of Cytoplasm: Volume, Viscosity, Diffusion, Intracellular Surface Area[END_REF][START_REF] Parry | The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity[END_REF] Etoc. et al. determined that particles of ≤50 nm can have Brownian diffusion in the cytosol. [START_REF] Etoc | Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells[END_REF] We showed that the critical core size for NPs to reach almost all parts of the cytosol is around 23 nm. [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF] At the same time, the surface chemistry has a major effect on the behavior of NPs in biological systems [START_REF] Tonga | 25th Anniversary Article: Interfacing Nanoparticles and Biology: New Strategies for Biomedicine[END_REF][START_REF] Walkey | Understanding and Controlling the Interaction of Nanomaterials with Proteins in a Physiological Environment[END_REF] and can affect the diffusion and the motion of NPs in the cytosol. [START_REF] Etoc | Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells[END_REF][START_REF] Xu | How Entanglement of Different Physicochemical Properties Complicates the Prediction of in Vitro and in Vivo Interactions of Gold Nanoparticles[END_REF] One reason for this is that the surface chemistry will determine interactions with proteins, notably their adsorption, which can lead to the formation of a protein corona affecting all aspects of NP behavior from the cellular uptake to their degradation. [START_REF] Monopoli | Biomolecular Coronas Provide the Biological Identity of Nanosized Materials[END_REF][START_REF] Docter | The Nanoparticle Biomolecule Corona: Lessons Learned -Challenge Accepted?[END_REF][START_REF] Ke | A Decade of the Protein Corona[END_REF] Moreover, strong interactions between proteins and the NP surface prevents free movement of NPs in the cytosol. [START_REF] Etoc | Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells[END_REF] Therefore, their surface treatment against protein adsorption is essential. Grafting polyethylene glycol (PEG) is the best known strategy for suppressing protein adsorption on NPs. [START_REF] Jokerst | Nanoparticle PEGylation for Imaging and Therapy[END_REF][START_REF] Suk | PEGylation as a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery[END_REF] However, PEGylation is not fully adapted to protein-sized NPs, because the PEG chains of 2000 to 5000 Da in length, used for this purpose, also significantly increase the particle diameter by ~10 nm. [START_REF] Rabanel | Assessment of PEG on Polymeric Particles Surface, a Key Step in Drug Carrier Translation[END_REF][START_REF] Estephan | Zwitteration As an Alternative to PEGylation[END_REF][START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] An alternative is the use of zwitterionic (ZI) groups, mimicking the surface of the cell membrane. [START_REF] Bangham | A Correlation between Surface Charge and Coagulant Action of Phospholipids[END_REF][START_REF] García | Zwitterionic-Coated "Stealth" Nanoparticles for Biomedical Applications: Recent Advances in Countering Biomolecular Corona Formation and Uptake by the Mononuclear Phagocyte System[END_REF] The mechanism of suppression of protein adsorption in the case of zwitterions is linked to their very hydrophilic nature and the fact that they contain their own counterion, [START_REF] Estephan | Zwitteration As an Alternative to PEGylation[END_REF][START_REF] García | Zwitterionic-Coated "Stealth" Nanoparticles for Biomedical Applications: Recent Advances in Countering Biomolecular Corona Formation and Uptake by the Mononuclear Phagocyte System[END_REF][START_REF] Schlenoff | Coating Surfaces with Zwitterionic Functionality to Reduce Nonspecific Adsorption[END_REF] rather than on steric repulsion as in the case of PEG. [START_REF] Jeon | Protein-surface Interactions in the Presence of Polyethylene Oxide[END_REF] Therefore, zwitterionic coatings can be very thin, having minimal impact on the particle diameter, unlike PEG coatings.

Several approaches have been developed to implement zwitterionic groups, especially carboxybetaine, sulfobetaine and phosphorylcholine, on NP surfaces. Most of these have been developed for inorganic NPs as quantum dots (QDs), [START_REF] Tasso | Sulfobetaine-Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging's Horizons[END_REF][START_REF] Muro | Small and Stable Sulfobetaine Zwitterionic Quantum Dots for Functional Live-Cell Imaging[END_REF][START_REF] Huang | Bioinspired Zwitterionic Surface Coatings with Robust Photostability and Fouling Resistance[END_REF][START_REF] Debayle | Zwitterionic Polymer Ligands: An Ideal Surface Coating to Totally Suppress Protein-Nanoparticle Corona Formation?[END_REF] silica NPs, [START_REF] Rosen | Surface Functionalization of Silica Nanoparticles with Cysteine: A Low-Fouling Zwitterionic Surface[END_REF][START_REF] Estephan | Zwitterion Siloxane to Passivate Silica against Nonspecific Protein Adsorption[END_REF] or Au NPs. [START_REF] Rouhana | Aggregation-Resistant Water-Soluble Gold Nanoparticles[END_REF][START_REF] Moyano | Fabrication of Corona-Free Nanoparticles with Tunable Hydrophobicity[END_REF] Zwitterionic surfaces were achieved in these cases either through the direct grafting of suitable zwitterionic small molecules, [START_REF] Muro | Small and Stable Sulfobetaine Zwitterionic Quantum Dots for Functional Live-Cell Imaging[END_REF][START_REF] Rosen | Surface Functionalization of Silica Nanoparticles with Cysteine: A Low-Fouling Zwitterionic Surface[END_REF][START_REF] Moyano | Fabrication of Corona-Free Nanoparticles with Tunable Hydrophobicity[END_REF][START_REF] Zhan | Multidentate Zwitterionic Ligands Provide Compact and Highly Biocompatible Quantum Dots[END_REF][START_REF] Estephan | Zwitterion-Stabilized Silica Nanoparticles: Toward Nonstick Nano[END_REF] or the grafting or adsorption of zwitterionic polymers. [START_REF] Tasso | Sulfobetaine-Vinylimidazole Block Copolymers: A Robust Quantum Dot Surface Chemistry Expanding Bioimaging's Horizons[END_REF][START_REF] Debayle | Zwitterionic Polymer Ligands: An Ideal Surface Coating to Totally Suppress Protein-Nanoparticle Corona Formation?[END_REF][START_REF] Dong | Phase Behavior of Poly(Sulfobetaine Methacrylate)-Grafted Silica Nanoparticles and Their Stability in Protein Solutions[END_REF][START_REF] Giovanelli | Highly Enhanced Affinity of Multidentate versus Bidentate Zwitterionic Ligands for Long-Term Quantum Dot Bioimaging[END_REF] In the case of polymeric NPs, zwitterionic particles are mainly obtained through the assembly of polymeric micelles, based on block copolymers bearing a polar zwittterionic and a hydrophobic block. [START_REF] Salvage | Novel Biocompatible Phosphorylcholine-Based Self-Assembled Nanoparticles for Drug Delivery[END_REF][START_REF] John | Biomimetic PH/Redox Dual Stimuli-Responsive Zwitterionic Polymer Block Poly(L-Histidine) Micelles for Intracellular Delivery of Doxorubicin into Tumor Cells[END_REF][START_REF] Xie | Biocompatible Zwitterionic Phosphorylcholine Polymers with Aggregation-Induced Emission Feature[END_REF][START_REF] Huang | Zwitterionic Nanoparticles Constructed with Well-Defined Reduction-Responsive Shell and PH-Sensitive Core for "Spatiotemporally Pinpointed" Drug Delivery[END_REF] Statistical copolymers with relatively large amounts of zwitterionic groups (30%->90%), on the other hand, have been studied as water-soluble systems able to translocate through cellular membranes. [START_REF] Ishihara | Polymeric Lipid Nanosphere Consisting of Water-Soluble Poly(2-Methacryloyloxyethyl Phosphorylcholine-Co-n-Butyl Methacrylate)[END_REF][START_REF] Goda | Cell-Penetrating Macromolecules: Direct Penetration of Amphipathic Phospholipid Polymers across Plasma Membrane of Living Cells[END_REF][START_REF] Morimoto | Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres[END_REF] Recently, we introduced a concept of charge-controlled nanoprecipitation of hydrophobic polymers, [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] where a small number of charged groups can tune the size of the obtained NPs down to 7 nm. [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF] To minimize their interaction with proteins and to ensure their free diffusion in the cytosol, they were stabilized by adsorption of PEGylated surfactants. [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF][START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] However, so far, no attempts have been done to develop hydrophobic polymers with limited number of charged and zwitterionic groups for their nanoprecipitation into ultrasmall NPs with stealth shell, compatible with intracellular applications.

In the present work, we developed a novel approach to assemble small fluorescent polymer NPs with a zwitterionic shell in a one-step process. For this, we designed methacrylate-based statistical copolymers, bearing a hydrophobic major monomer (methyl to butyl methacrylate), a small amount of charged monomer to reduce the size, [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF][START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] and a varying amount of zwitterionic sulfobetaine side groups. We then studied the influence of the presence of the zwitterionic groups on the formation of dye-loaded NPs through nanoprecipitation. The resulting NPs were evaluated with respect to their stability in biological media and the capacity of the sulfobetaine groups to prevent protein adsorption. The behavior of the most promising NPs was then monitored directly at the single particle level in the cytosol of living cells in order to access their suitability as nanoprobes for single-particle tracking. In this way we could show that polymers bearing 10% ZI groups and 1 to 2% sulfonate groups formed small (<20 nm) NPs, in which efficient encapsulation of a dye salt was obtained, rendering them brightly fluorescent.

These particles showed stealth behavior with respect to biological media and very high mobility in the cytosol, exceeding that of PEGylated particles of similar core size.

Results and Discussion

Figure 1. Chemical structures of different polymers, dye R18 and its counterion (F5-TPB) used for preparation of zwitterionic dye-loaded NPs through nanoprecipitation.

Methacrylate based copolymers bearing sulfonate and zwitterionic (ZI) groups were synthetized by free radical copolymerization (Figure 1, Scheme S1, and Figure S1). The fraction of sulfonate groups on polymers was 1 or 2 mol%, whereas that of the zwitterionic group was varied from 0 to 10 mol%. The remaining part of the polymers, corresponding to 88 to 99 mol%, was made up of alkyl methacrylates, with varying length of the alkyl groups ranging from methyl to butyl. Increasing the alkyl chain length should thereby increase the hydrophobicity of the polymers and balance the hydrophilicity of the zwitterionic groups. In consequence, the synthesized polymers had an amphiphilic character and a relatively low net negative charge.

Here, the polymers are noted as PXMA-ZI-x%-SO3H-y%, where XMA stands for the corresponding major monomer (MMA for methyl methacrylate, EMA for ethyl methacrylate, PMA for propyl methacrylate, BMA for butyl methacrylate), x and y give the molar percentage of the zwitterionic and sulfonate groups. These polymers were then used to assemble dye-loaded polymer NPs through nanoprecipitation. For this, solutions of the polymers in acetonitrile (with a small amount of methanol) containing the salt of a rhodamine B derivative (R18) with a perfluorinated tetraphenylborate (F5-TPB, typically 10 wt% with respect to the polymer) [START_REF] Reisch | Collective Fluorescence Switching of Counterion-Assembled Dyes in Polymer Nanoparticles[END_REF] were added quickly to a large excess of Milli-Q water. The size of the formed NPs was analyzed through transmission electron microscopy (TEM, Figure 2, Table S1), fluorescence correlation spectroscopy (FCS, Tables S1 andS4), and dynamic light scattering (DLS, Tables S1, S2, S3).

In the case of polymers bearing only sulfonate but no zwitterionic groups, very small particles were obtained as observed previously: [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF] According to TEM, PMMA-SO3H-1% yielded NPs of about 13 nm and the particle size decreased to about 9 nm for 2% sulfonate. The introduction of 10% ZI in the polymers had only a minor influence on the particle size of PMMA-ZI-SO3H based NPs. They reached 11 and 9 nm of diameter for polymers with 1% and 2% sulfonates, respectively. Hence, the presence of the ZI groups did not affect the nanoprecipitation process, while the expected "ZI shell" is thin enough to preserve the small size of NPs. Increasing the hydrophobicity of the alkyl methacrylate monomers, on the other hand, led to increasing particle size (Figure 2, Table S1) in the order PMMA-ZI-10%-SO3H-1% < PEMA-ZI-10%-SO3H-1% < PPMA-ZI-10%-SO3H-1% < PBMA-ZI-10%-SO3H-1%, from 11 to 35 nm. This is in agreement with results obtained previously [START_REF] Rosiuk | Controlling Size and Fluorescence of Dye-Loaded Polymer Nanoparticles through Polymer Design[END_REF] and can be attributed to the fact that increasing hydrophobicity leads to increasing supersaturation, which is the major driving force in nanoprecipitation. [START_REF] Rosiuk | Controlling Size and Fluorescence of Dye-Loaded Polymer Nanoparticles through Polymer Design[END_REF][START_REF] Saad | Principles of Nanoparticle Formation by Flash Nanoprecipitation[END_REF][START_REF] Lepeltier | Nanoprecipitation and the "Ouzo Effect": Application to Drug Delivery Devices[END_REF] DLS and FCS results were in good agreement with the sizes obtained by TEM (Table S1). The slightly larger sizes obtained by TEM in some cases were attributed to a preferred immobilization of larger particles on the grids. Zeta-potential measurements of NPs made from different polymers showed that these were always clearly negatively charged with values in the range -20 to -35 mV (Table S3). The zwitterionic groups did not influence significantly the surface charge.

As we showed earlier, the hydrophobic counterion approach works well for dye encapsulation of cationic R18 into NPs from hydrophobic polymers. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF][START_REF] Reisch | Collective Fluorescence Switching of Counterion-Assembled Dyes in Polymer Nanoparticles[END_REF][START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] As the hydrophobicity of our new polymers is lower because of 10% ZI groups, we evaluated the encapsulation efficiency for several polymers, and notably the least hydrophobic polymer, PMMA-ZI-10%-SO3H-2%, using dialysis against a special -cyclodextrin-based release medium. [START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] This method has been validated for several series of dye-loaded NPs, showing that dyes that are not well encapsulated are removed in this way. [START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF][START_REF] Andreiuk | An Aluminium-Based Fluorinated Counterion for Enhanced Encapsulation and Emission of Dyes in Biodegradable Polymer Nanoparticles[END_REF] Here, dialysis of the dye-loaded NPs over 24 h showed a release of 14% of the used dye for the most hydrophilic polymer, and a release of around 10% for the other tested polymers (Figure S2, Table S5). Hence, the used approach yielded very efficient encapsulation (86-91%) of the dye salt in the particles, in agreement with previous results from dialysis and ultracentrifugation. [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF][START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] The efficient loading has been attributed to the high hydrophobicity of the dye-counterion pair. Furthermore, the quantum yield of the particles remained high, 31 -58% for a 10 wt% loading for all zwitterion bearing polymers (Figure S3, Table S6). A slight decrease was, however, observed upon introduction of the ZI groups, probably due to the lower hydrophobicity of the ZI bearing polymers. [START_REF] Rosiuk | Controlling Size and Fluorescence of Dye-Loaded Polymer Nanoparticles through Polymer Design[END_REF] Taking into account the size, the loading, the encapsulation efficiency, and the quantum yield, this resulted in a per particle brightness of 10 6 to over 10 7 M -1 cm -1 (Table S6). For example, PMMA-ZI-10%-SO3H-1% based NPs loaded with 10 wt% R18/F5-TPB reached a brightness of 2.0×10 6 M -1 cm -1 (for excitation at 565 nm) exceeding nearly tenfold the brightness of corresponding QDs (e.g. ~2.2×10 5 M -1 cm -1 for QD585 from Invitrogen).

The stability of the resulting particles in biological media, and in particular the potential of the zwitterionic groups to improve this, was then investigated (Figure 3, Table S1, S2, S4). [START_REF] Koynov | Fluorescence Correlation Spectroscopy in Colloid and Interface Science[END_REF] The fraction of ZI groups in the polymers was varied from 0 to 10% in order to modify the density of ZI on the particle surface. In a first step, the stability of NPs was evaluated through addition of NaCl (Figure 3A). The size of the particles was monitored by FCS after addition of increasing salt concentrations. The stability limit was defined as the highest tested NaCl concentration showing no aggregation of NPs. Typically, when exceeding this salt concentrations, a marked increase in size was observed. Here we chose an increase of particle size by >20% to define this limit. NPs made from polymers bearing no ZI groups started to aggregate (and precipitate) already at relatively small salt concentration (>20 mM), which is far below that in a physiological medium (~150 mM). Addition of ZI groups to the polymer led to an increase in the particle stability. At 10% of zwitterionic groups the NPs showed neither changes in size nor signs of precipitation up to 1 M NaCl. These ZI bearing NPs also showed an excellent stability over a wide pH range, from 3.6 to 9 as observed by DLS measurements (Table S2). This should be beneficial for their use in different cellular microenvironments. Furthermore, no significant changes in fluorescence brightness were observed in the presence of proteins (Table S4, Figure S4).

The influence of the zwitterionic groups on interactions of the NPs with proteins and other biomolecules was then studied by FCS in the presence of 10% of fetal bovine serum (FBS), a complex mixture containing notably numerous proteins (Figure 3B). In the case of particles without ZI groups a size increase by about 10 nm was observed, which corresponds to the adsorption of at least a monolayer of proteins and thus the formation of a protein corona. [START_REF] Monopoli | Biomolecular Coronas Provide the Biological Identity of Nanosized Materials[END_REF] Starting from 5% of ZI groups the size increase of the NPs upon interaction with proteins was significantly reduced. At 10% of ZI groups no significant increase in particle size was observed, indicating that the surfaces of these NPs resisted to protein adsorption. The stealth properties of NPs with ZI groups were more pronounced for less hydrophobic MMA polymer, whereas more hydrophobic polymers, especially BMA showed a significant size increase in the presence of FBS. These NPs were then directly microinjected in the perinuclear region of the cytosol of living HeLa cells and their behavior was monitored using epifluorescence microscopy (Figure 4 and Figure S5). Direct observation of the cells was conducted over the whole experiment time and showed generally good viability of cells with microinjected NPs (Figure 4A). Microinjection ensures direct delivery of the NPs with minimal modification of their surface and optimal control over the compartment they are delivered to, here the cytosol. The particles were easily visible as bright dots (even when excited with a Xenon lamp), which allowed observing their distribution and movement. In all cases, diffuse staining of cytosolic structures was virtually absent. This confirms the efficient and stable encapsulation of the dyes within the NPs and the absence of dye leaching, that has been shown to lead to a strong and characteristic staining of the cell-interior. [START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF][START_REF] Shulov | Fluorinated Counterion-Enhanced Emission of Rhodamine Aggregates: Ultrabright Nanoparticles for Bioimaging and Light-Harvesting[END_REF][START_REF] Trofymchuk | BODIPY-Loaded Polymer Nanoparticles: Chemical Structure of Cargo Defines Leakage from Nanocarrier in Living Cells[END_REF] Maximum projections of the fluorescence intensities over 60 s gave a general idea of the overall distribution and mobility of the particles throughout the cytosol (Figure 4C, Table S7 for a more quantitative analysis): In the absence of ZI groups, a large part of the particles remained stuck at the injection point, indicating strong interactions of NPs with the cellular constituents. In sharp contrast, NPs bearing 10% ZI groups showed a tendency to distribute throughout the cytosol showing traces of their diffusion trajectories, although their behavior depended on the type of alkyl methacrylate used for the polymer synthesis. PMMA based ZI bearing NPs showed a distribution all over the cytosol, but often also a "crescent"-like signal around the nucleus close to the injection point. This latter might hint to limitations of the physical stability of the PMMA-ZI particles under the conditions experienced during microinjection. PEMA-ZI particles showed a homogeneous distribution over practically the whole cytosol and only a small clustering around the injection point (Figure 4B), which was also observed for PPMA-ZI particles. These results confirmed that 10% ZI groups are sufficient to strongly reduce interactions of the particles with cellular constituents and enable spreading throughout the cytosol. However, in the case of PBMA-based particles a static localization at the injection point was observed. As observed previously, [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF] the particle size has a major influence on intracellular particle diffusion, and only particles below a critical core size of around 23 nm were able to spread throughout the cytosol due to steric hindrance by cellular structures. [START_REF] Luby-Phelps | Cytoarchitecture and Physical Properties of Cytoplasm: Volume, Viscosity, Diffusion, Intracellular Surface Area[END_REF] The size of PBMA-ZI particles used here was clearly above this threshold, which can explain their confinement to the injection site. In a certain measure this can also apply to the PPMA-based zwitterionic particles, for which the size distribution (Figure 2) showed that about 20% of these NPs were larger than 23 nm and thus above this threshold. Observation of NPs microinjected in cells showed that all the NPs made from polymers bearing 10% ZI groups (except PBMA) had a high mobility within the cytosol, in contrast to those made of PMMA-SO3H-1%. At the same time, the high brightness of our particles enabled studying their movement through single-particle tracking, and thus better understanding the influence of the ZI groups and the type of polymer used on their intracellular behavior. An example of a cytoplasmic trajectory of a PEMA-ZI-10%-SO3H-1% NP is represented in Figure 5A. Plotting the mean square displacement (MSD) vs lag time for individual trajectories yields detailed information on their diffusion behavior, notably the diffusion coefficients and the type of diffusion (i.e. free/Brownian, restricted, active transport). [START_REF] Höfling | Anomalous Transport in the Crowded World of Biological Cells[END_REF][START_REF] Rienzo | Fast Spatiotemporal Correlation Spectroscopy to Determine Protein Lateral Diffusion Laws in Live Cell Membranes[END_REF][START_REF] Ruthardt | Single-Particle Tracking as a Quantitative Microscopy-Based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical Nanoparticles[END_REF] Here, for the ZI bearing NPs the MSD showed a linear increase with lag time up to about 10 µm² for over 90% of the particles (Figure 5B). This indicates normal or Brownian diffusion of these particles in the cytosol, which is described, in two dimensions, as MSD = 4D∆t  with D, the diffusion coefficient and  = 1. [START_REF] Ruthardt | Single-Particle Tracking as a Quantitative Microscopy-Based Approach to Unravel Cell Entry Mechanisms of Viruses and Pharmaceutical Nanoparticles[END_REF] The diffusion coefficients of individual NPs were then obtained from the corresponding MSD curves (Figure 5 and Figure S6). Figure 5C shows that for PEMA-ZI NPs the distribution of the diffusion coefficients is centered around 1 µm².s -1 , with a mean D of 0.80 µm².s -1 , in good agreement with values obtained for QDs of similar size. [START_REF] Etoc | Non-Specific Interactions Govern Cytosolic Diffusion of Nanosized Objects in Mammalian Cells[END_REF] Only a small fraction (<10%) of these NPs had diffusion coefficients below 0.2 µm².s -1 . PMMA-ZI NPs had a similar distribution of diffusion coefficients with a slightly lower mean of 0.65 µm².s -1 (Figure 5D, S6), in agreement with their slightly larger hydrodynamic size. PPMA-ZI NPs, on the other hand, showed a clearly lower mean diffusion coefficient of 0.25 µm².s -1 . This is probably due to their larger size leading to a restricted cytoplasmic diffusion (though a less complete suppression of interactions with proteins for these more hydrophobic particles may also contribute). As made PMMA-SO3H-1% NPs without ZI groups remained clustered around the injection point, and hence it was not possible to characterize their diffusion behavior. However, we could show earlier that simple adsorption of Tween 80 (T80), a PEGylated surfactant, on such NPs strongly reduces their interaction with proteins and permits their diffusion in the cytosol. [START_REF] Reisch | Protein-Sized Dye-Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol[END_REF][START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] Interestingly, the mean diffusion coefficient of these NPs was 0.2 µm².s -1 that is 3 to 4-fold lower than those of the PMMA-ZI and PEMA-ZI NPs, even though their core sizes were very close (Figure 2). One reason for this is certainly the fact that the addition of the PEGylated surfactant increases the NP size by > 5 nm (from 13 to 18 nm according to FCS). [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] The large difference in diffusion coefficients further indicates that the ZI groups are probably more effective in reducing non-specific interactions with intracellular biomolecules and structures compared to the adsorbed PEGylated surfactant. Considering the ensemble of these results in view of previous findings can give a better idea of the structure and function of the obtained zwitterionic fluorescent NPs: Here, to develop "stealth" dye-loaded polymer NPs, we designed a series of amphiphilic polymers made up of a majority of hydrophobic monomers, 10% of zwitterionic monomers and 1-2% of negatively charged monomers. These were nanoprecipitated together with a dye salt to assemble the NPs in a single step. The resulting particles combined, on the one hand, efficient dye encapsulation and high fluorescence quantum yields, resulting in high particle brightness with, on the other hand, excellent colloidal stability and resistance to protein adsorption. We have observed previously that high hydrophobicity of the matrix is crucial for achieving good encapsulation and high quantum yields of the encapsulated dyes. [START_REF] Rosiuk | Controlling Size and Fluorescence of Dye-Loaded Polymer Nanoparticles through Polymer Design[END_REF][START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF] In consequence, the particles probably have a hydrophobic core made up predominantly of hydrophobic alkyl-methacrylate units and of the dye salt. The excellent resistance to protein adsorption and the high mobility of the particles in the cytosol, on the other hand, suggest a very hydrophilic surface with a high density of zwitterionic groups. [START_REF] Tonga | 25th Anniversary Article: Interfacing Nanoparticles and Biology: New Strategies for Biomedicine[END_REF][START_REF] García | Zwitterionic-Coated "Stealth" Nanoparticles for Biomedical Applications: Recent Advances in Countering Biomolecular Corona Formation and Uptake by the Mononuclear Phagocyte System[END_REF] We hence suppose that upon mixing of the organic solutions of the polymers and dyes with the aqueous phase, supersaturation of the polymers and the dye salt occurs, which then drives nanoprecipitation. During nucleation and growth, the polymers probably arrange in a way that the sulfonate and zwitterionic groups are primary located at the surface of the particles, while the hydrophobic parts are located in the particle interior, where the highly apolar dye-salt is also embedded. Based on the obtained sizes and a molar mass of the used polymer between 40 000 and 50 000, the number of polymer chains per particle can be estimated to be around 10 per NP, i.e. ~350 ZI groups at the particle surface.

Assuming that the ZI groups are located mainly at the particle surface, their surface density can be estimated based on the particle size to be around 1.1 ZI groups per nm², e.g. for our PMMA-ZI-10%-SO3H-1% NPs with a size of 11 nm. This is close to the density of phosphorylcholine head groups in closely packed lipid monolayers (1.4 -2 per nm²) [START_REF] Zheng | Molecular Simulation Studies of the Structure of Phosphorylcholine Self-Assembled Monolayers[END_REF] and suggests that the ZI groups form a relatively dense layer on the NP surface. The NPs made with MMA and EMA, the least hydrophobic monomers, showed a better resistance to protein adsorption and also the highest mobility. This can be attributed to a better implementation of the ZI groups on the surface and less ZI groups in the particle interior. At the same time, MMA and EMA probably allow for a "softer" particle surface due to dangling polymer loops, contributing steric repulsion to protein adsorption. [START_REF] Jeon | Protein-surface Interactions in the Presence of Polyethylene Oxide[END_REF] The observed high mobility of our ZI bearing particles in the cytosol, notably in comparison with PEGylated NPs, indicates that the zwitterionic groups led to the formation of a very thin shell, capable of resisting protein adsorption.

Conclusions

In this work we showed how the design of hydrophobic statistical copolymers bearing zwitterionic and charged groups can be used to assemble very small and bright dye-loaded polymer nanoparticles. For this, we combined different hydrophobic alkylmethacrylate monomers with sulfobetaine and sulfonate bearing methacrylate monomers. These polymers were then used to assemble dye-loaded polymer NPs, encapsulating high amounts of the salt of a rhodamine derivative with a hydrophobic bulky counterion. The resulting NPs had sizes from 10 to 35 nm, and even 11-nm NPs reached a fluorescence brightness ten times higher than that of corresponding quantum dots. About 10% of zwitterionic groups were sufficient to render these particles stable over a wide range of salt concentrations and pH values as well as resistant to protein adsorption. The high fluorescence brightness of these particles made it possible to monitor their behavior at the single-particle level, showing that the optimized particles diffuse freely within the cytosol of living cells. For equivalent core size the zwitterionic particles showed a higher mobility than corresponding PEGylated NPs. In consequence, the developed zwitterion approach for dye-loaded polymer nanoparticles opens the route to bright labels and probes for sensing and imaging of single biomolecules at work in living cells with very high spatial and temporal resolution.

Materials and Methods

Materials

Methyl methacrylate (99%, M55909), methacrylic acid (99%, 155721), 3-sulfopropyl methacrylate potassium salt (99%, 251658) and 2-(N-3-Sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate (99%, 537284) were purchased from Sigma-Aldrich. Dimethylsulfoxide (DMSO, analytical grade) was obtained from Fisher-Scientific. Milli-Q water (Millipore), acetonitrile (≥ 99.9%, Sigma-Aldrich) and methanol (≥ 99.9%, Carlo Erba reagents) were used for preparation of nanoparticles. dichloromethane (≥99.8%) from CarloErba and methanol (HPLC grade) from VWR. 6-carboxy-tetramethylrhodamine (TMR from Sigma-Aldrich), phosphate buffered saline (PBS, Fisher Scientific), fetal bovine serum (FBS, Lonza) were used for stability study. Monomers were purified using column chromatography or re-crystallization. Azobis isobutyronitrile (Aldrich, ≥ 98%) was recrystallized twice from ethanol. The other compounds were used as received. R18/F5-TPB was synthesized from rhodamine B octadecyl ester perchlorate (Aldrich, >98.0%) and lithium tetrakis(pentafluorophenyl)borate ethyl etherate (AlfaAesar, 97%) through ion exchange followed by purification through column chromatography as described previously. [START_REF] Reisch | Collective Fluorescence Switching of Counterion-Assembled Dyes in Polymer Nanoparticles[END_REF][START_REF] Andreiuk | Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo[END_REF] Polymer synthesis General procedure: The different polymers were synthesized through free radical polymerization. The different monomers were all dissolved in degassed DMSO and mixed at the desired ratio. 0.01 eq. of AIBN were added and the round bottom flask was placed in an oil bath preheated to 70 °C. Once the conversion reached 25 %, the reaction was stopped and the polymers reprecipitated twice in methanol and/or water. After drying, the polymers were characterized through NMR and, where possible, size exclusion chromatography. As an example, the synthesis of PMMA-ZI-SO3H is given:

Poly(methyl methacrylate-co-3-sulfopropyl methacrylate-co-2-(N-3-Sulfopropyl-N,Ndimethyl ammonium)ethyl methacrylate) (PMMA-ZI-SO3H):

Methyl methacrylate, 2-(N-3-Sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate, and 3-sulfopropyl methacrylate potassium salt were dissolved in degased DMSO at a concentration of 2 M. The three solutions were then mixed in a 50 mL two-neck round bottom flask equipped with a stirring bar at the desired ratio to give a total volume of 20 mL. The mixture was degased by bubbling argon for 5 min and placed under argon atmosphere. 0.01 eq. of AIBN in DMSO (40 mg/mL) were added and the round bottom flask was placed in an oil bath preheated to 70 °C. At regular intervals samples were drawn, dissolved in DMSO-d6 and analyzed by NMR. Once the conversion reached 25 %, the reaction was stopped by quickly cooling it to RT. The reaction mixture was then added dropwise to methanol, or, for higher percentages of the charged and zwitterionic monomers, to water. After filtration, the precipitate was redissolved in a small amount of acetonitrile (when needed with a small amount of methanol) and reprecipitated twice in methanol (water for highest percentages). The obtained polymer was dried under vacuum. [START_REF] Shashkova | Single-Molecule Fluorescence Microscopy Review: Shedding New Light on Old Problems[END_REF] H NMR (400 MHz, DMSO-d6, δ): 4.36 (br, 0.04-0.2 H), 3.96 (br. s, 0.02-0.04 H), 3.55 (m, 3.4 H), 3.13 (br, 0.12-0.6 H), 2.45 (br, covered by the solvent peak), 2.04 (br, 0.04-0.2 H), 2.00-0.30 (m, 6 H). Fraction obtained of ZI and SO3H groups based on the peak at 4.36 and 3.97 ppm, respectively (feed: obtained): ZI 10 %: 10.2 % -SO3H 1 %: 1.4%; ZI 10 %: 11 %-SO3H 2 %: 2.8%. Molecular weight by size exclusion chromatography (SEC): 1% SO3H: Mw = 51 200, Mw /Mn = 1.58; 1% ZI: Mw = 43 100, Mw /Mn = 1.14. For polymers with higher amounts of zwitterionic monomers no suitable solvent systems for SEC were available.

Poly(ethyl methacrylate-co-3-sulfopropyl methacrylate-co-2-(N-3-Sulfopropyl-N,Ndimethyl ammonium)ethyl methacrylate) (PEMA-ZI-SO3H):

Preparation of NPs

Stock solutions of copolymers were prepared at a concentration of 10 g.l -1 in acetonitrile (20 vol.% methanol for ZI polymers). These solutions were diluted at 2 g.l -1 in the corresponding solvent containing 1, 10 or 20 wt% of R18/F5-TPB (relative to the polymer). This solution was quickly added to a 10-fold volume excess of water, under shaking (Thermomixer comfort, Eppendorf, 1000 rpm, at 21°C) followed by a second dilution in water.

Characterization of NPs

Absorption and emission spectra were recorded on a Cary 4000 Scan ultraviolet-visible spectrophotometer (Varian) and on a FS5 Spectrofluorometer (Edinburgh Instruments) equipped with a thermostated cell compartment, respectively. The excitation wavelength was set to 530 nm and emission was recorded from 540 to 750 nm. QYs were calculated using rhodamine 101 in ethanol as reference (QY = 0.9). [START_REF] Würth | Critical Review of the Determination of Photoluminescence Quantum Yields of Luminescent Reporters[END_REF] Transmission electron microscopy: 5 µl of nanoparticle solution were deposited onto carboncoated copper-rhodium electron microscopy grids following amylamine glow-discharge. They were then treated for 20 s with a 2% uranyl acetate solution for staining. The obtained grids were observed using a Philips CM120 transmission electron microscope equipped with a LaB6 filament and operating at 100kV.The acquisition of areas of interest was recorded with a Peltier cooled CCD camera (Model 794, Gatan, Pleasanton, CA). Images were analyzed using Fiji software.

Fluorescence correlation spectroscopy: Measurements were performed on a home-built confocal set-up based on a Nikon inverted microscope with a Nikon 60 × 1.2NA water immersion objective. Excitation was provided by a continuous wave (CW) laser diode (532 nm, Oxxius) and photons were detected with a fibered avalanche photodiode (APD SPCM-AQR-14-FC, Perkin Elmer) connected to an online hardware correlator (ALV7000-USB, ALV GmbH, Germany). The solution of NPs containing 1 wt% dyes were diluted 2 times before depositing 200 µl on 96-well optical-bottom plates for measurements. NPs stability in presence of salts and proteins were investigated by adding drop-by-drop 50 vol. % of 10-fold PBS (10x), FBS or water to the solutions of NPs in low-binding 1.5 mL Eppendorf tubes. The data were recorded 5 min after addition and then analyzed using the PyCorrFit software. For FCS measurements, which apply relatively high excitation powers in the focal spot, a reduced dyeloading was used in order to avoid saturation phenomena involving triplet states, which would have made the analysis more complicated.

Cellular experiments

HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM, without phenolred, Gibco-Invitrogen), supplemented with 10% fetal bovine serum (FBS, Lonza), L-glutamine, and 1% antibiotic solution (penicillin-streptomycin, Gibco-Invitrogen) at 37 C in humidified atmosphere containing 5% CO2. Cells were seeded onto round microscope cover glasses (diameter 18 mm) deposited in 6 well plates at a density of 12510 3 cells/well 24 h before the microinjection.

Microinjection of NPs and cellular imaging: For microinjection experiments, subconfluent HeLa cells plated on glass coverslips were mounted in a Ludin Chamber (Life Imaging Services, Basel, Switzerland). The cells were then placed on a Leica DMIRE 2 microscope and solutions of the different nanoparticles at particle concentrations of 0.5 to 2 nM were microinjected into the perinuclear region of the cells, using a Femtojet/InjectMan NI2 microinjector (Eppendorf). Image sequences were then acquired either on the same setup (100x Plan APO 1.4 NA objective, Xenon-source, and Photometrics Prime camera) or on an iMIC microscope (Till Photonics) equipped with a Mutli-LED Spectra X (Lumencor), an Olympus 60x TIRFM (1.45 NA) objective, and a Flash 4 V2+ camera (Hamamatsu) after transfer of the samples. Live cells were maintained at 37°C in a 5% CO2 humidified atmosphere using an environmental control system (Life Imaging Services). Time-lapse movies were recorded over 60 s with a frame rate of 50 ms and binning 2. They were then analyzed using the ImageJ software (National Institutes of Health, USA). For single particle tracking, time-lapse movies were recorded over 30 s with a frame rate of 43 ms for the Imic microscope or 50 ms for the Leica DMIRE 2 microscope and binning 1. The trajectories were obtained using the TrackMate plugin for Fiji. [START_REF] Tinevez | TrackMate: An Open and Extensible Platform for Single-Particle Tracking[END_REF] Then MSD curves were plotted and slopes extracted from the MSD curves with a MATLAB script for particle tracking analysis. At least 50 trajectories from at least 3 different cells were used for analysis.

Figure 2 .

 2 Figure 2. TEM analysis of the sizes of zwitterionic and non zwitterionic sulfonate NPs. NPs

Figure 3 .

 3 Figure 3. Stability of NPs and their interactions with proteins as determined by FCS. (A)

Figure 4 .

 4 Figure 4. Representative micrographs of HeLa cells microinjected with different types of NPs

Figure 5 .

 5 Figure 5. Single-particle tracking. (A) Trajectory (in red) of a PEMA-ZI-10%-SO3H-1% NP

  [START_REF] Shashkova | Single-Molecule Fluorescence Microscopy Review: Shedding New Light on Old Problems[END_REF] H NMR (400 MHz, MeOD-, δ): 4.50 (br, 0.2 H), 4.34-3.92 (m, 2.02 H), 3.81 (br, 0.2 H), 3.70 (br, 0.2 H), 3.29 (br, partial covered by the solvent peak), 2.92 (br, 0.2 H), 2.31 (br, 0.2 H), 2.23-0.5 (m, 9 H). Fraction obtained of ZI groups based on the peak at 4.50 ppm: 10.3 %.

	Poly(propyl methacrylate-co-3-sulfopropyl methacrylate-co-2-(N-3-Sulfopropyl-N,N-
	dimethyl ammonium)ethyl methacrylate) (PPMA-ZI-SO3H): 1 H NMR (400 MHz, MeOD, δ):
	4.50 (br, 0.2 H), 4.29-3.54 (m, 2.42 H), 3.29 (br, partial covered by the solvent peak), 2.92 (br,
	0.2 H), 2.31 (br, 0.2 H), 2.23-0.5 (m, 11 H).
	Fraction obtained of ZI groups based on the peak at 4.50 ppm: 9.4%.
	Poly(butyl methacrylate-co-3-sulfopropyl methacrylate-co-2-(N-3-Sulfopropyl-N,N-
	dimethyl ammonium)ethyl methacrylate) (PBMA-ZI-SO3H): 1 H NMR (400 MHz, CDCl3, δ):
	4.40 (br, 0.2H), 4.30-3.55 (m, 2.42 H), 3.33 (br, 0.6 H), 2.99 (br, 0.2 H), 2.36 (br, 0.2 H), 2.28-
	0.4 (m, 13 H). Fraction obtained of ZI groups based on the peak at 4.40 ppm: 8.3%.
	Poly(methyl methacrylate-co-3-sulfopropyl methacrylate) (PMMA-SO3H): 1 H NMR (400
	MHz, DMSO-d6, δ): 3.97 (br. s, 0.02-0.04 H), 3.57 (s, 3 H), 2.45 (m, partial covered by the
	solvent peak), 2.1 -0.5 (m, 6 H). Fraction of SO3H groups based on the peak at 3.97 ppm (feed:
	obtained): SO3H 1%: 1.1%; SO3H 2%: 2.3%.

Acknowledgements

This work was supported by the Agence National de Recherche JC/JC grant "Supertrack" ANR-16-CE09-0007 and by the European Research Council ERC Consolidator grant BrightSens 648528. The authors thank C. Ruhlmann and C. Crucifix from the FRISBI platform (ANR-10-INBS-05) for help with electron microscopy, M. Legros and C. Foussat for size exclusion chromatography, and Oleksii Dukhno for helpful discussions on single particle tracking analysis.

Funding Sources

Agence National de Recherche JC/JC grant "Supertrack" ANR-16-CE09-0007, European Research Council ERC Consolidator grant BrightSens 648528.

Supporting Information. Results and details of synthesis, fluorescence correlation spectroscopy, dynamic light scattering, quantum yield measurements, dialysis, and particle tracking are given in the Supporting Information.

Corresponding

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.