Polarization-resolved single-molecule tracking reveals strange dynamics of individual fluorescent tracers through a plasticized (rubbery) polymer network

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization-error (LE) of SM trajectories (~30-50 nm) is considerably larger than size of molecular tracers (~1-3 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicator for underlying anomalous-transport mechanisms, remains obscured within the realms of SMT. Since translation of passive tracers in an isotropic network is associated with fast dipolar rotation, we propose authentic pauses within LE can be revealed upon probing SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission-anisotropy at each super-localized position, thereby revealing tumbling propensity of SMs during random walk. For Rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analyses of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in hydrated (plasticized) PVP films reveal a wide distribution of dwell-times and pause-frequencies, which demonstrate that majority of probes intermittently experience complete translational and rotational immobilization. This indicates tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a redominantly deep-rubbery polymer network far above the glass transition. PR-SMT is simple to implement and opens up alternate avenues to interrogate transient (bio)molecular interactions leading to anomalous transport in inhomogeneous media.</div

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide₂₀-<i>b</i>-poly-l-lysine₁₀₀ and PEG_2k-<i>b</i>-poly-l-glutamate₁₀₀ [PEG = poly­(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of <i>N</i>-carboxyanhydrides and “click” chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide₂₀-<i>b</i>-poly-l-lysine₁₀₀ and PEG_2k-<i>b</i>-poly-l-glutamate₁₀₀ [PEG = poly­(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of <i>N</i>-carboxyanhydrides and “click” chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes