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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.

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.

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 situhybridization (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.

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 105times larger surface and to the extension of the WGM electromagnetic field to larger distances (>100 nm vsa 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.

Visualizing single organic nanoparticles (NPs) in vivoremains 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 vivoin different brain compartments and their dynamics visualized with subcellular resolution.

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.

Nanoprecipitation is a straightforward yet powerful technique to synthesize polymer nanoparticles loaded with various biologically active compounds or contrast agents. Particle formation in this approach is kinetically controlled, and various assembly parameters have been used to control the size distribution and properties of the formed nanoparticles. Here, the influence of the nature of the polymer on the formation of nanoparticles in nanoprecipitation is studied systematically by varying its hydrophobicity and charge over a broad range. For this, methacrylate copolymers with different types and fractions of hydrophobic, hydrophilic, and charged side groups are synthesized. Nanoprecipitation of these polymers shows that particle size increases with increasing global hydrophobicity of the polymers. At the same time, both hydrophilic and charged groups reduce particle size. In this way, we achieve control over particle size from ∼10 to 200 nm. Furthermore, the effect of the polymer nature on the photophysical properties of nanoparticles loaded with a fluorescent dye, a rhodamine B derivative with a bulky hydrophobic counterion (fluorinated tetraphenylborate), is studied. It is found that the hydrophobic/hydrophilic balance of the polymer modulates to a large extent the spectral properties and fluorescence quantum yield of the dye encapsulated at high concentration, which reflects changes in the dye aggregation within the polymer matrix. Thus, we show how polymer chemistry can tune kinetically controlled formation of nanoparticles and encapsulation of the load. The concepts introduced here should be valuable tools for the design of nanoparticles for imaging and drug-delivery applications.

Imaging single molecules and nanoparticles in complex biological media is highly challenging notably due to autofluorescence of cells and tissues. Lanthanides and lanthanide complexes, with their particularly long luminescence lifetimes, offer the possibility to perform time-gated imaging and thus to strongly reduce the autofluorescence background. However, their very low brightness and photon flux have limited their use in single-molecule imaging. Here, we encapsulate high amounts of Europium complexes into poly(methyl methacrylate)-based particles of 10, 20, and 30 nm size. The resulting particles contain up to 5000 copies of the complex with a quantum yield of ≥0.2, resulting in a per particle brightness of up to 4 × 107M–1cm–1. They can be imaged at the single-particle level using low illumination intensities (0.24 W cm–2) and low acquisition times (300 ms) and internalize well into living cells, where they can be monitored through time-gated imaging at illumination conditions compatible with living specimen. These Eu-complex-loaded nanoparticles can thus be applied for highly sensitive and autofluorescence-free imaging and have the potential to become very performant probes for fast intracellular tracking of single biomolecules.

Fluorescent nanoparticles (NPs) help to increase spatial and temporal resolution in bioimaging. Advanced microscopy techniques require very bright NPs that exhibit either stable emission for single-particle tracking or complete on/off switching (blinking) for super-resolution imaging. Here, ultrabright dye-loaded polymer NPs with controlled switching properties are developed. To this aim, the salt of a dye (rhodamine B octadecyl ester) with a hydrophobic counterion (fluorinated tetraphenylborate) is encapsulated at very high concentrations up to 30 wt % in NPs made of poly(lactic-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), and polycaprolactone (PCL) through nanoprecipitation. The obtained 35 nm NPs are nearly 100 times brighter than quantum dots. The nature of the polymer is found to define the collective behavior of the encapsulated dyes so that NPs containing thousands of dyes exhibit either whole particle blinking, for PLGA, or stable emission, for PMMA and PCL. Fluorescence anisotropy measurements together with small-angle X-ray scattering experiments suggest that in less hydrophobic PLGA, dyes tend to cluster, whereas in more hydrophobic PMMA and PCL, dyes are dispersed within the matrix, thus altering the switching behavior of NPs. Experiments using a perylene diimide derivative show a similar effect of the polymer nature. The resulting fluorescent NPs are suitable for a wide range of imaging applications from tracking to super-resolution imaging. The findings on the organization of the load innside NPs will have impact on the development of materials for applications ranging from photovoltaics to drug delivery.

Here, we explore the enhancement of single-molecule emission by a polymeric nanoantenna that can harvest energy from thousands of donor dyes to a single acceptor. In this nanoantenna, the cationic dyes are brought together, in very close proximity, using bulky counterions, thus enabling ultrafast diffusion of excitation energy (≤30 fs) with minimal losses. Our 60 nm nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1–2 acceptors, resulting in an antenna effect of ~1,000. Therefore, single Cy5-based acceptors become 25-fold brighter than quantum dots QD655. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers (1–10 mW cm−2) that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic set-up, which includes a ×20 air objective and a scalable complementary metal-oxide–semiconductor camera, we could detect single Cy5 molecules by simply shining divergent light on the sample at powers equivalent to sunlight. Donor dye nanoparticles have been used to realize structures that are 25 times brighter than quantum dots. This enabled single-molecule imaging using ambient light.

Curcumin, a neuroprotective agent with promising therapeutic approach has poor brain bioavailability. Herein, we demonstrate that curcumin-encapsulated poly(lactide-co-glycolide) (PLGA) 50:50 nanoparticles (NPs-Cur 50:50) are able to prevent the phosphorylation of Akt and Tau proteins in SK-N-SH cells induced by H2O2and display higher anti-inflammatory and antioxidant activities than free curcumin. PLGA can display various physicochemical and degradation characteristics for controlled drug release applications according to the matrix used. We demonstrate that the release of curcumin entrapped into a PLGA 50:50 matrix (NPs-Cur 50:50) is faster than into PLGA 65:35. We have studied the effects of the PLGA matrix on the expression of some key antioxidant- and neuroprotective-related genes such as APOE, APOJ, TRX, GLRX, and REST. NPs-Cur induced the elevation of GLRX and TRX while decreasing APOJ mRNA levels and had no effect on APOE and REST expressions. In the presence of H2O2, both NPs-Cur matrices are more efficient than free curcumin to prevent the induction of these genes. Higher uptake was found with NPs-Cur 50:50 than NPs-Cur 65:35 or free curcumin. By using PLGA nanoparticles loaded with the fluorescent dye Lumogen Red, we demonstrated that PLGA nanoparticles are indeed taken up by neuronal cells. These data highlight the importance of polymer composition in the therapeutic properties of the nanodrug delivery systems. Our study demonstrated that NPs-Cur enhance the action of curcumin on several pathways implicated in the pathophysiology of Alzheimer’s disease (AD). Overall, these results suggest that PLGA nanoparticles are a promising strategy for the brain delivery of drugs for the treatment of AD.

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. Accelerating cures for osteosarcoma (OS) poor outcome patients remain a challenge. So, there is an urgent need to develop relevant preclinical 2D and 3D modelization able to mirror pathophysiologically cell‐to‐cell interactions impacting OS cell behavior and engineer extrinsic heterogeneity able to modulate response to therapies like the bone extrinsic mechanical properties, which are taking part to OS development.

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.

Tough, dense polyelectrolyte complexes (PECs) with well‐defined cross‐sections are prepared using a laboratory extruder and plasticizing the complexes with salt water. Stoichiometric starting materials yield stoichiometric complexes of poly(diallyldimethylammonium) (PDADMA) and poly(styrene sulfonate) (PSS). As an example of this enabling technology, macroscopic tubes of PEC are produced. Microscopy images of cross‐sections of rods, tape, and tubes show a pore volume of less than 10% in the bulk of the extruded complex and fully dense material towards the surface, where the shear is greatest. Thermal gravimetric analysis reveals the expected salt content for PECs doped with NaCl, and a lack of salt for PECs rinsed in water. The fact that doped PECs are transparent suggests they are supersaturated with salt. Residual stress following extrusion is relieved by exposure to solutions of NaCl. Stress relaxation experiments show decreasing equilibrium moduli as a function of increasing salt doping, consistent with prior results on multilayers of the same polymers.

We report on the modification of the polyelectrolytes poly(L‐glutamic acid) (PGA), poly(acrylic acid) (PAA), and poly(L‐lysine) (PLL) with side groups bearing phosphorylcholine (PC) groups. Two different side chains were synthesized from monoamino tri‐ or tetraethylene glycol, both with PC group linked to the OH end: the tert‐butyloxycarbonyl protected compounds BOCNH(EO)nPC (n= 2, 3) and the succinylated compound SucNH(EO)3PC. BOCNH(EO)nPC was deprotected and the resulting amine was condensed with PGA or PAA and SucNH(EO)3PC was directly coupled to PLL. Degrees of substitution as high as 80% were obtained with the method described. These polyelectrolytes are potential candidates to fabricate multilayers with protein repellent properties.

Owing to a technical error, the wrong version of the Supplementary Information was published for this Article; the equation E = xcoupled donors × ElocalFRET related to Supplementary Table 6 appeared incorrectly. This error has now been corrected.