Escherichia coli Biofilm Formation, Motion and Protein Patterns on Hyaluronic Acid and Polydimethylsiloxane Depend on Surface Stiffness.

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9-574 kPa) and HA (hyaluronic acid gels, 44 Pa-2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth.

Non-covalent small molecule partnership for redox-active films: Beyond polydopamine technology.

HYPOTHESIS: The possibility to use hexamethylenediamine (HMDA) to impart film forming ability to natural polymers including eumelanins and plant polyphenols endowed with biological activity and functional properties has been recently explored with the aim to broaden the potential of polydopamine (PDA)-based films overcoming their inherent limitations. 5,6-dihydroxyindole-2-carboxylic acid, its methyl ester (MeDHICA) and eumelanins thereof were shown to exhibit potent reducing activity. EXPERIMENTS: MeDHICA and HMDA were reacted in aqueous buffer, pH 9.0 in the presence of different substrates to assess the film forming ability. The effect of different reaction parameters (pH, diamine chain length) on film formation was investigated. Voltammetric and AFM /SEM methods were applied for analysis of the film redox activity and morphology. HPLC, MALDI-MS and 1HNMR were used for chemical characterization. The film reducing activity was evaluated in comparison with PDA by chemical assays and using UV stressed human immortalized keratinocytes (HaCat) cells model. FINDINGS: Regular and homogeneous yellowish films were obtained with moderately hydrophobic properties. Film deposition was optimal at pH 9, and specifically induced by HMDA. The film consisted of HMDA and monomeric MeDHICA accompanied by dimers/small oligomers, but no detectable MeDHICA/HMDA covalent conjugation products. Spontaneous assembly of self-organized networks held together mainly by electrostatic interactions of MeDHICA in the anion form and HMDA as the dication is proposed as film deposition mechanism. The film displayed potent reducing properties and exerted significant protective effects from oxidative stress on HaCaT.

Optimization of the Elasticity and Adhesion of Catechol- or Dopamine-Loaded Gelatin Gels under Oxidative Conditions.

The synthesis of surgical adhesives is based on the need to design glues that give rise to strong and fast bonds without cytotoxic side effects. A recent trend in surgical adhesives is to use gel-forming polymers modified with catechol groups, which can undergo oxidative crosslinking reactions and are strongly adhesive to all kinds on surfaces in wet conditions. We previously showed that blending gelatin with catechol can yield strong adhesion when the catechol is oxidized by a strong oxidant. Our previous work was limited to the study of the variation in the sodium periodate concentration. In this article, for an in-depth approach to the interactions between the components of the gels, the influence of the gelatin, the sodium periodate and dopamine/(pyro)catechol concentration on the storage (G') and loss (G″) moduli of the gels, as well as their adhesion on steel, have been studied by shear rheometry. The hydrogels were characterized by infrared and UV-Vis spectroscopy and the size of their pores visualized by digital microscopy and SEM after freeze drying but without further additives. In terms of adhesion between two stainless steel plates, the optimum was obtained for a concentration of 10% w/v in gelatin, 10 mM in sodium periodate, and 20 mM in phenolic compounds. Below these values, it is likely that crosslinking has not been maximized and that the oxidizing environment is weakening the gelatin. Above these values, the loss in adhesiveness may result from the disruption of the alpha helixes due to the large number of phenolic compounds as well as the maintenance of an oxidizing environment. Overall, this investigation shows the possibility to design strongly adhesive hydrogels to metal surfaces by blending gelatin with polyphenols in oxidative conditions.

Impacts of Resveratrol and Pyrogallol on Physicochemical, Mechanical and Biological Properties of Epoxy-Resin Sealers.

This study aimed at evaluating the physicochemical and biological properties of experimental epoxy-resin sealers containing polyphenols such as resveratrol and pyrogallol. A conventional epoxy resin (OB) was modified by adding different concentrations of resveratrol (RS) or pyrogallol (PY) to its composition. Antibacterial and antioxidant activities, mechanical properties, along with wettability and morphological changes were investigated. The results were statistically analyzed using ANOVA and multiple comparison tests (α = 0.05). The incorporation of the tested polyphenols into the epoxy resin enhanced its mechanical properties. PY demonstrated much better antioxidant and antibacterial activities than RS, which were associated with a higher release of PY. In contrast, PY showed a higher cytotoxicity than OB and OB doped with RS. OB containing PY presented a rougher surface and higher water absorption than OB doped with RS. Both tested polyphenols caused no notable changes to the overall porosity of OB. Resveratrol and pyrogallol may not only influence the morphology and mechanical properties of epoxy-resin sealers, but could also enhance antioxidant activity and antibacterial effects against Enterococcus faecalis. Most epoxy-resin sealers currently available in the market can be considered as "passive" materials. Thus, doping their composition with specific polyphenols may be a suitable strategy to confer some antibacterial properties, antioxidant potential, along with improvement of some mechanical properties.

Antibacterial and Bonding Properties of Universal Adhesive Dental Polymers Doped with Pyrogallol.

This study investigated the antibacterial activity, bond strength to dentin (SBS), and ultra-morphology of the polymer-dentin interface of experimental adhesive systems doped with pyrogallol (PY), which is a ubiquitous phenolic moiety that is present in flavonoids and polyphenols. A universal adhesive containing 4-META and 10-MDP was used in this study. PY behaves as an antioxidant and anti-cancerogenic agent and it was incorporated into the adhesive at different concentrations (0.5 and 1 wt.%). The antibacterial activity and SBS were analyzed and the results were statistically analyzed. The ultra-morphology of the polymer-dentin interface was assessed using scanning electron microscopy (SEM). At 24 h, a lower antibacterial activity was observed for the control adhesive compared to those with 0.5% and 1% PY. No difference was seen in SBS between the three groups at 24 h. After 6 months, the SBS of the 0.5% PY adhesive was significantly lower than the other tested adhesives. The specimens created with 1% PY adhesive presented a higher bond strength at six months compared with that found at 24 h. No morphological differences were found at the polymer-dentin interfaces of the tested adhesives. Pyrogallol may be incorporated into modern universal adhesive systems to preserve the polymer-dentin bonding interface and confer a certain degree of antibacterial activity.

In vitro comparison of the surface roughness of polymethyl methacrylate and bis-acrylic resins for interim restorations before and after polishing.

STATEMENT OF PROBLEM: Polymethyl methacrylate and bis-acrylic based resins are widely used for interim restorations. Their initial surface roughness is important because it determines their aesthetic properties and the potential for biofilm adhesion. PURPOSE: The purpose of this in vitro study was to assess the surface roughness and morphology of 6 bis-acrylic and 2 polymethyl methacrylate resins widely used for interim dental restorations, both before and after polishing. MATERIAL AND METHODS: Specimens made of different bis-acrylic resins (Protemp 4, Luxatemp Star, Systemp, Telio, Structur Premium, Structur 3) or of polymethyl methacrylate (Unifast Trad, Unifast 3) were polished using a 2-step polishing system (Diatech). The average surface roughness before and after polishing (10 seconds at low speed in dry conditions) was measured by optical profilometry. Atomic force microscopy and scanning electron microscopy were used to analyze surface morphology. The Mann-Whitney and Kruskal-Wallis tests were used to evaluate the differences in roughness among specimens (α=.05), and the Pearson r correlation was computed to assess the relationship between fillers and average surface roughness. RESULTS: In the 8 groups evaluated, the roughness significantly increased (P<.001) for Protemp 4 (from 0.12 to 0.50 µm), Luxatemp Star (0.17 to 1.19 µm), Unifast 3 (0.40 to 1.00 µm), Systemp (0.46 to 1.51 µm), Structur 3 (0.85 to 1.06 µm), Structur Premium (1.00 to 1.74 µm), and Telio (1.13 to 1.21 µm), except for Unifast Trad (9.20 to 2.59 µm). Pairwise multiple comparisons identified Protemp 4 as having the smoothest surface before and after polishing, while Unifast Trad was the roughest in both. Atomic force microscopy and scanning electron microscopy observations showed that the surface roughness of bis-acrylic resins was related to their surface morphology and average filler sizes. A positive relation between fillers and roughness was assessed (r=0.345, P<.001). CONCLUSIONS: For the bis-acrylic interim resins, the surface roughness after polishing was correlated to the material used and its filler sizes. Nanofiller-based resins showed the smoothest surfaces. For the polymethyl methacrylate-based resins, the recently marketed Unifast 3 had the lowest overall roughness values.

Physicochemical and Antibacterial Properties of Novel, Premixed Calcium Silicate-Based Sealer Compared to Powder-Liquid Bioceramic Sealer.

The aim of this study was to compare the physicochemical properties, filling ability, and antibacterial activity of a premixed calcium silicate-based sealer to those of a powder-liquid bioceramic sealer. Ceraseal (CS) and BioRoot (BR) materials were analyzed using scanning electron microscopy and energy-dispersive X-ray spectroscopy at 7 and 14 d of immersion in distilled water. The filling ability of the two sealers as well as the water contact angle, solubility, flow, roughness, crystalline microstructure, pH, and compressive strength were also evaluated. The antibacterial activity was assessed through an agar diffusion as well as through direct tests. All the results were statistically analyzed using one-way or two-way analysis of variance tests. Statistically significant lower void percentages were observed for CS at 2 and 8 mm from the working length (WL) compared to those for the BR group, whilst no significant difference was observed at 5 mm from the WL. BR sealer showed higher alkaline pH, rougher surface, lower water contact angle values, lower flowability, and higher solubility compared to CS. BR showed globular and needle-like crystalline microstructure, whilst CS had globular and flower-like crystalline microstructure up to 72 h. No statistical difference was found for the compressive strength between the two sealers. BR and CS showed no antibacterial effect against Enterococcus faecalis after 3 h, whilst both sealers showed antibacterial capacity after 24 and 72 h. BR demonstrated higher antibacterial activity after 24 h. In conclusion, the use of bioceramic sealers may play an important role in controlling bacterial growth. Moreover, CS may have superior filling ability and lower solubility than the BioRoot sealer due to its specific chemical composition and mixing method.

A Clean and Tunable Mussel-Inspired Coating Technology by Enzymatic Deposition of Pseudo-Polydopamine (ψ-PDA) Thin Films from Tyramine.

The tyrosinase-catalyzed oxidation of tyramine, leading to the deposition of pseudo-polydopamine (ψ-PDA) thin films, is disclosed herein as a superior technology for surface functionalization and coating at a neutral pH and at a low substrate concentration, compared to the standard autoxidative PDA coating protocols. Smooth ψ-PDA thin films of variable thickness up to 87 nm were obtained from 1 mM tyramine by varying tyrosinase concentrations (5-100 U/mL). Compared to the PDA films obtained by the similar enzymatic oxidation of 1 mM dopamine with tyrosinase (T-PDA), ψ-PDA displayed slower deposition kinetics, lower water contact angles in the range of 11°-28°, denoting higher hydrophilicity but similar UV-vis absorption profiles, as well as electrochemical properties and antioxidant activity. MALDI-MS analysis indicated for ψ-PDA a well defined pattern of peaks compatible with dopamine tetrameric structures degraded to a variable extent. The exposure to a tyramine solution of tyrosinase-loaded alginate spheres, or films deposited on glass or polyethylene, resulted in a rapid gel-confined ψ-PDA formation with no leakage or darkening of the solution, allowing the complete recovery and re-utilization of the unreacted tyramine. In contrast, an abundant PDA precipitation outside the gel was observed with dopamine under the same conditions. The ψ-PDA deposition by tyrosinase-catalyzed tyramine oxidation is thus proposed as a controllable and low-waste technology for selective surface functionalization and coating or for clean eumelanin particle production.

Does Etching of the Enamel with the Rubbing Technique Promote the Bond Strength of a Universal Adhesive System?

AIM: The aim of this in vitro research was to study the effect of etching by phosphoric acid with rubbing technique on the shear bond strength (SBS) of adhesive universal to enamel. MATERIALS AND METHODS: Sixty extracted teeth were obtained. Three application methods (self-etch, etch-and-rinse, and etch-and-rinse with rubbing technique) were performed to bond the enamel surfaces by a universal adhesive. After 24 hours of immersion in water at 37°C, the specimens were prepared for the SBS test. Scanning electron microscopy was performed to observe the adhesive-enamel interfaces. Optical numeric microscope was used to observe the failure style. Statistical analyses were done with one-way analysis of variance test. RESULTS: Statistically significant higher bond strength values were observed for etch-and-rinse mode with rubbing technique (25.98 ± 5.70) MPa then for the etch-and-rinse without rubbing (22.07 ± 5.27) MPa and self-etch modes (9.96 ± 2.98) MPa. CONCLUSION: Enamel etched by 37% phosphoric acid with rubbing technique for 20 seconds showed an increase in the SBS of the universal adhesive to enamel surfaces. The tags of the adhesive can be presented more efficiently by rubbing the acid before the bonding process, consequently, an optimal interface for the bonding. CLINICAL SIGNIFICANCE: According to the results of this in vitro study, the selective enamel etching mode with rubbing technique is advisable when using the universal adhesive, as it significantly increased the bond strength of this adhesive to enamel surfaces. The clinician should etch the enamel using phosphoric acid with rubbing technique for 20 seconds to promote the bond strength of the universal adhesive system.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed 'labeling occupancy' (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Extending Single Molecule Imaging to Proteome Analysis by Quantitation of Fluorescent Labeling Homogeneity in Complex Protein Samples.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications.

In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2, and its two analogues, 4-MeOxyLH and 4,6'-DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in vitro biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (5,5-Cpr-OxyLH) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.

Protein-Sized Bright Fluorogenic Nanoparticles Based on Cross-Linked Calixarene Micelles with Cyanine Corona.

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.

Non-coordinating anions assemble cyanine amphiphiles into ultra-small fluorescent nanoparticles.

A non-coordinating anion, fluorinated tetraphenylborate, assembles specially designed cationic cyanine amphiphiles into 7-8 nm fluorescent nanoparticles that are >40-fold brighter than a single cyanine dye. This kind of anion, combining hydrophobic and electrostatic forces in aqueous media, constitutes promising building blocks in the self-assembly of functional nanomaterials.

Fluorinated counterion-enhanced emission of rhodamine aggregates: ultrabright nanoparticles for bioimaging and light-harvesting.

The key to ultrabright fluorescent nanomaterials is the control of dye emission in the aggregated state. Here, lipophilic rhodamine B derivatives are assembled into nanoparticles (NPs) using tetraphenylborate counterions with varied fluorination levels that should tune the short-range dye ordering. Counterion fluorination is found to drastically enhance the emission characteristics of these NPs. Highly fluorinated counterions produce 10-20 nm NPs containing >300 rhodamine dyes with a fluorescence quantum yield of 40-60% and a remarkably narrow emission band (34 nm), whereas, for other counterions, aggregation caused quenching with a weak broad-band emission is observed. NPs with the most fluorinated counterion (48 fluorines) are ∼40-fold brighter than quantum dots (QD585 at 532 nm excitation) in single-molecule microscopy, showing improved photostability and suppressed blinking. Due to exciton diffusion, revealed by fluorescence anisotropy, these NPs are efficient FRET donors to single cyanine-5 acceptors with a light-harvesting antenna effect reaching 200. Finally, NPs with the most fluorinated counterion are rather stable after entry into living cells, in contrast to their less fluorinated analogue. Thus, the present work shows the crucial role of counterion fluorination in achieving high fluorescence brightness and photostability, narrow-band emission, efficient energy transfer and high intracellular stability of nanomaterials for light harvesting and bioimaging applications.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

Charge-controlled nanoprecipitation as a modular approach to ultrasmall polymer nanocarriers: making bright and stable nanoparticles.

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

The HIV-1 nucleocapsid protein recruits negatively charged lipids to ensure its optimal binding to lipid membranes.

UNLABELLED: The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (∼ 10(7) M(-1)) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM. IMPORTANCE: The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.

Disassembly-driven fluorescence turn-on of polymerized micelles by reductive stimuli in living cells.

Stimuli-response nanoparticles have emerged as powerful tools for imaging and therapeutic applications. Ideally, they should be assembled from biodegradable materials featuring small size and cooperative response to biological stimuli that trigger particle disassembly and release of an active molecule that could be readily monitored in situ. A concept is developed that consists of organic nanoparticles, assembled from fluorescent amphiphiles and polymerized with a redox-cleavable cross-linker. We obtained 20 nm nanoparticles bearing self-quenched Nile Red dye residues, which can disassemble in living cells into highly fluorescent molecular units owing to an external or internal reductive stimulus. The obtained results pave the way to new stimuli-responsive nanomaterials for applications in background-free imaging as well as in drug delivery, as the concept can be further extended to other active molecules including drugs and to cross-linkers cleavable by other biological stimuli.

Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles.

The current challenge in the field of fluorescent nanoparticles (NPs) for bioimaging is to achieve extreme brightness and external control of their emission using biodegradable materials. Here we propose a new concept of fluorescent polymer NPs, doped with ionic liquid-like salts of a cationic dye (octadecyl rhodamine B) with a bulky hydrophobic counterion (fluorinated tetraphenylborate) that serves as spacer minimizing dye aggregation and self-quenching. The obtained 40-nm poly(D,L-lactide-co-glycolide) NPs containing up to 500 dyes are brighter than quantum dots and exhibit photo-induced reversible on/off fluorescence switching, never reported for dye-doped NPs. We show that this collective switching of hundreds of dyes is due to ultrafast excitation energy transfer and can be used for super-resolution imaging. These NPs, being spontaneously endocytosed by living cells, feature high signal-to-noise ratio and absence of toxicity. The counterion-based concept opens the way to a new class of nanomaterials for sensing, imaging and light harvesting.