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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.

Hypothesis: Polymer nanoparticles (NPs) have a very high potential for applications notably in the biomedical field. However, synthetic polymer NPs cannot yet concurrence the functionalities of proteins, their natural counterparts, notably in terms of size, control over internal structure and interactions with biological environments. We hypothesize that kinetic trapping of polymers bearing oppositely charged groups in NPs could bring a new level of control and allow mimicking the surfaces of proteins., Experiments: Here, the assembly of mixed-charge polymer NPs through nanoprecipitation of mixtures of oppositely charged polymers is studied. Two series of copolymers made of ethyl methacrylate and 1 to 25 mol% of either methacrylic acid or a trimethylammonium bearing methacrylate are synthesized. These carboxylic acid or trimethylammonium bearing polymers are then mixed in different ratios and nanoprecipitated. The influence of the charge fraction, mixing ratio of the polymers, and precipitation conditions on NP size and surface charge is studied., Findings: Using this approach, NPs of less than 25 nm with tunable surface charge from +40 mV to -40 mV are assembled. The resulting NPs are sensitive to pH and certain NP formulations have an isoelectric point allowing repeated charge reversal. Encapsulation of fluorescent dyes yields very bright fluorescent NPs, whose interactions with cells are studied through fluorescence microscopy. The obtained results show the potential of nanoprecipitation of oppositely charged polymers for the design of NPs with precisely tuned surface properties., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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

Fluorescent polymer nanoparticles for long-term labeling and tracking of living cells with any desired color code are developed. They are built from biodegradable poly(lactic-co-glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions, which minimize aggregation-caused quenching. At the single particle level, these particles are ≈20-fold brighter than quantum dots of similar color. Due to their identical 40 nm size and surface properties, these nanoparticles are endocytosed equally well by living cells. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding is validated on 7 cell lines (HeLa, KB, embryonic kidney (293T), Chinese hamster ovary, rat basophilic leucemia, U97, and D2A1), 13 color codes, and it enables simultaneous tracking of co-cultured barcoded cell populations for >2 weeks. It is also applied to studying competition among drug-treated cell populations. This technology enabled six-color imaging in vivo for (1) tracking xenografted cancer cells and (2) monitoring morphogenesis after microinjection in zebrafish embryos. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co-localized inside cells by combining structurally close nanoparticles carrying different functions., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Speed, resolution and sensitivity of today's fluorescence bioimaging can be drastically improved by fluorescent nanoparticles (NPs) that are many-fold brighter than organic dyes and fluorescent proteins. While the field is currently dominated by inorganic NPs, notably quantum dots (QDs), fluorescent polymer NPs encapsulating large quantities of dyes (dye-loaded NPs) have emerged recently as an attractive alternative. These new nanomaterials, inspired from the fields of polymeric drug delivery vehicles and advanced fluorophores, can combine superior brightness with biodegradability and low toxicity. Here, we describe the strategies for synthesis of dye-loaded polymer NPs by emulsion polymerization and assembly of pre-formed polymers. Superior brightness requires strong dye loading without aggregation-caused quenching (ACQ). Only recently several strategies of dye design were proposed to overcome ACQ in polymer NPs: aggregation induced emission (AIE), dye modification with bulky side groups and use of bulky hydrophobic counterions. The resulting NPs now surpass the brightness of QDs by ≈10-fold for a comparable size, and have started reaching the level of the brightest conjugated polymer NPs. Other properties, notably photostability, color, blinking, as well as particle size and surface chemistry are also systematically analyzed. Finally, major and emerging applications of dye-loaded NPs for in vitro and in vivo imaging are reviewed., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 H2O2 and 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.

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 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.

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion., (© 2011 American Chemical Society)

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

Reduced scattering and autofluorescence in the second near‐infrared window (NIR‐II, 1000–1700 nm) hold the promise of high‐resolution photoluminescence (PL) in vivo imaging at depths well beyond the µm scale. Yet, contrast agents with bright emission in this spectral region remain limited. Ultrasmall gold nanoclusters (AuNCs) are of particular interest for NIR‐II PL imaging. However, they exhibit limited brightness. Here, high amounts of NIR‐II emitting AuNCs are encapsulated in small polymer nanoparticles (NPs) made of poly(ethyl methacrylates). Using nanoprecipitation, loadings of up to 50 wt% can be reached, corresponding to over 10 000 AuNCs per 60 nm NP. PL quantum yields up to 1.3% and NP brightnesses up to 3.8 × 106 M−1 cm−1 are accomplished. Increasing inter‐AuNC interactions at higher loading result in a bathochromic shift of the PL emission. According to time‐resolved PL measurements, this can be attributed to distinct inter‐AuNC energy transfer pathways between the core and surface‐centered PL modes. Evaluation of the nanohybrids in tissue‐mimicking models for in vivo blood vessel imaging shows that the ultrabright and red‐shifted NIR‐II PL results in significantly improved detection sensitivity and resolution, which makes AuNC‐loaded polymer NPs highly promising candidates for advanced in vivo imaging. [ABSTRACT FROM AUTHOR]