Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation

Controlled aqueous polymerization of acrylamides and acrylates and “in situ” depolymerization in the presence of dissolved CO2

Aqueous copper-mediated radical polymerization of acrylamides and acrylates in carbonated water resulted in high monomer conversions (t t > 10 min). The regenerated monomer was characterized and repolymerized following deoxygenation of the resulting solutions to reyield polymers in high conversions that exhibit low dispersities

Critical Dependence of Molecular Weight on Thermoresponsive Behavior of Diblock Copolymer Worm Gels in Aqueous Solution

Reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization of 2-hydroxypropyl methacrylate was used to prepare three poly(glycerol monomethacrylate)x–poly(2-hydroxypropyl methacrylate)y (denoted Gx-Hy or PGMA-PHPMA) diblock copolymers, namely G37-H80, G54-H140, and G71-H200. A master phase diagram was used to select each copolymer composition to ensure that a pure worm phase was obtained in each case, as confirmed by transmission electron microscopy (TEM) and small-angle x-ray scattering (SAXS) studies. The latter technique indicated a mean worm cross-sectional diameter (or worm width) ranging from 11 to 20 nm as the mean degree of polymerization (DP) of the hydrophobic PHPMA block was increased from 80 to 200. These copolymer worms form soft hydrogels at 20 °C that undergo degelation on cooling. This thermoresponsive behavior was examined using variable temperature DLS, oscillatory rheology, and SAXS. A 10% w/w G37-H80 worm dispersion dissociated to afford an aqueous solution of molecularly dissolved copolymer chains at 2 °C; on returning to ambient temperature, these chains aggregated to form first spheres and then worms, with the original gel strength being recovered. In contrast, the G54-H140 and G71-H200 worms each only formed spheres on cooling to 2 °C, with thermoreversible (de)gelation being observed in the former case. The sphere-to-worm transition for G54-H140 was monitored by variable temperature SAXS: these experiments indicated the gradual formation of longer worms at higher temperature, with a concomitant reduction in the number of spheres, suggesting worm growth via multiple 1D sphere–sphere fusion events. DLS studies indicated that a 0.1% w/w aqueous dispersion of G71-H200 worms underwent an irreversible worm-to-sphere transition on cooling to 2 °C. Furthermore, irreversible degelation over the time scale of the experiment was also observed during rheological studies of a 10% w/w G71-H200 worm dispersion. Shear-induced polarized light imaging (SIPLI) studies revealed qualitatively different thermoreversible behavior for these three copolymer worm dispersions, although worm alignment was observed at a shear rate of 10 s–1 in each case. Subsequently conducting this technique at a lower shear rate of 1 s–1 combined with ultra small-angle x-ray scattering (USAXS) also indicated that worm branching occurred at a certain critical temperature since an upturn in viscosity, distortion in the birefringence, and a characteristic feature in the USAXS pattern were observed. Finally, SIPLI studies indicated that the characteristic relaxation times required for loss of worm alignment after cessation of shear depended markedly on the copolymer molecular weight

Structure and Properties of Nanocomposites Formed by the Occlusion of Block Copolymer Worms and Vesicles Within Calcite Crystals

This article describes an experimentally versatile strategy for producing inorganic/ organic nanocomposites, with control over the microstructure at the nano- and meso-scales. Taking inspiration from biominerals, CaCO3 is co-precipitated with anionic diblock copolymer worms or vesicles to produce single crystals of calcite occluding a high density of the organic component. This approach can also be extended to generate complex structures in which the crystals are internally patterned with nano-objects of differing morphologies. Extensive characterization of the nanocomposite crystals using high resolution synchrotron powder XRD and vibrational spectroscopy demonstrates how the occlusions affect the short and long-range order of the crystal lattice. By comparison with nanocomposite crystals containing latex particles and copolymer micelles, we show that the effect of these occlusions on the crystal lattice is dominated by the interface between the inorganic crystal and the organic nano-objects, rather than the occlusion size. This is supported by in situ AFM studies of worm occlusion in calcite, which reveal flattening of the copolymer worms on the crystal surface, followed by burial and void formation. Finally, the mechanical properties of the nanocomposite crystals are determined using nanoindentation techniques, which reveals that they have hardnesses approaching those of biogenic calcites

Poly(methacrylic acid)-based AB and ABC block copolymer nano-objects prepared via RAFT alcoholic dispersion polymerization

International audienceA series of well-defined amphiphilic poly(methacrylic acid)-poly(benzyl methacrylate) (PMAA-PBzMA) diblock copolymers are synthesized via polymerization-induced self-assembly using an alcoholic dispersion polymerization formulation. Chain growth is mediated via reversible addition-fragmentation chain transfer polymerization (RAFT) chemistry using a trithiocarbonate-based chain transfer agent (CTA) at 70 degrees C. The poly(methacrylic acid) block is soluble in ethanol and acts as a steric stabilizer for the growing insoluble PBzMA chains, resulting in the in situ generation of diblock copolymer nano-objects in the form of spheres, worms or vesicles, depending on the precise reaction conditions. Copolymer morphologies can be covalently stabilized via cross-linking to prevent their dissociation when transferred into aqueous solution, which leads to the formation of highly anionic nano-objects due to ionization of the PMAA stabilizer chains. ABC triblock copolymer nanoparticles can also be prepared using this approach, where the third block is based on the semi-fluorinated monomer, 2,2,2-trifluoroethyl methacrylate (TFEMA). GPC studies confirm that chain extension is efficient and high TFEMA conversions can be achieved. Microphase separation between the mutually incompatible PBzMA and semi-fluorinated PTFEMA core-forming blocks occurs, producing a range of remarkably complex semi-fluorinated triblock copolymer morphologies

Efficient Synthesis of Amine-Functional Diblock Copolymer Nanoparticles via RAFT Dispersion Polymerization of Benzyl Methacrylate in Alcoholic Media

Benzyl methacrylate (BzMA) is polymerized via reversible addition–fragmentation chain transfer (RAFT) chemistry under alcoholic dispersion polymerization conditions in ethanol using a poly­(2-(dimethylamino)­ethyl methacrylate) (PDMA) chain transfer agent (CTA) at 70 °C. In principle, polymerization-induced self-assembly can lead to the formation of either spherical micelles, worm-like micelles, or vesicles, with the preferred morphology being dictated by the hydrophilic–hydrophobic balance of the PDMA–PBzMA diblock copolymer chains. Very high monomer conversions (>99%) are routinely obtained within 24 h as judged by ¹H NMR studies. Moreover, THF GPC analyses confirmed that relatively low polydispersities (<i>M</i>_w/<i>M</i>_n < 1.30) are achieved, indicating reasonably good pseudoliving character. A detailed phase diagram was constructed using a PDMA₃₁ macro-CTA by systematically varying both the target degree of polymerization of the PBzMA block and the total solids concentration of the reaction solution. This phase diagram can be used to reliably predict the synthesis conditions required to produce pure phases, rather than merely mixed phases (e.g., spheres plus worms or worms plus vesicles). Finally, these PDMA–PBzMA diblock copolymer nanoparticles remain colloidally stable when transferred from ethanol into water; aqueous electrophoresis studies confirmed that the particles acquire appreciable cationic character below pH 7 due to protonation of the PDMA stabilizer chains

Low-Viscosity Route to High-Molecular-Weight Water-Soluble Polymers: Exploiting the Salt Sensitivity of Poly(<i>N</i>‑acryloylmorpholine)

We report a new one-pot low-viscosity synthetic route to high molecular weight non-ionic water-soluble polymers based on polymerization-induced self-assembly (PISA). The RAFT aqueous dispersion polymerization of N-acryloylmorpholine (NAM) is conducted at 30 °C using a suitable redox initiator and a poly­(2-hydroxyethyl acrylamide) (PHEAC) precursor in the presence of 0.60 M ammonium sulfate. This relatively low level of added electrolyte is sufficient to salt out the PNAM block, while steric stabilization is conferred by the relatively short salt-tolerant PHEAC block. A mean degree of polymerization (DP) of up to 6000 was targeted for the PNAM block, and high NAM conversions (>96%) were obtained in all cases. On dilution with deionized water, the as-synthesized sterically stabilized particles undergo dissociation to afford molecularly dissolved chains, as judged by dynamic light scattering and 1H NMR spectroscopy studies. DMF GPC analysis confirmed a high chain extension efficiency for the PHEAC precursor, but relatively broad molecular weight distributions were observed for the PHEAC–PNAM diblock copolymer chains (Mw/Mn > 1.9). This has been observed for many other PISA formulations when targeting high core-forming block DPs and is tentatively attributed to chain transfer to polymer, which is well known for polyacrylamide-based polymers. In fact, relatively high dispersities are actually desirable if such copolymers are to be used as viscosity modifiers because solution viscosity correlates closely with Mw. Static light scattering studies were also conducted, with a Zimm plot indicating an absolute Mw of approximately 2.5 × 106 g mol–1 when targeting a PNAM DP of 6000. Finally, it is emphasized that targeting such high DPs leads to a sulfur content for this latter formulation of just 23 ppm, which minimizes the cost, color, and malodor associated with the organosulfur RAFT agent

Sterilizable Gels from Thermoresponsive Block Copolymer Worms

Biocompatible hydrogels have many applications, ranging from contact lenses to tissue engineering scaffolds. In most cases, rigorous sterilization is essential. Herein we show that a biocompatible diblock copolymer forms wormlike micelles via polymerization-induced self-assembly in aqueous solution. At a copolymer concentration of 10.0 w/w %, interworm entanglements lead to the formation of a free-standing physical hydrogel at 21 °C. Gel dissolution occurs on cooling to 4 °C due to an unusual worm-to-sphere order–order transition, as confirmed by rheology, electron microscopy, variable temperature ¹H NMR spectroscopy, and scattering studies. Moreover, this thermo-reversible behavior allows the facile preparation of sterile gels, since ultrafiltration of the diblock copolymer nanoparticles in their low-viscosity spherical form at 4 °C efficiently removes micrometer-sized bacteria; regelation occurs at 21 °C as the copolymer chains regain their wormlike morphology. Biocompatibility tests indicate good cell viabilities for these worm gels, which suggest potential biomedical applications