Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exo κ-GFP ) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo κ-GFP were also characterized. Unlike skin exosome, wound-edge Exo κ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by woundmacrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exo κ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo κ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP κ ) were designed with 94.3% encapsulation *

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle’s mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.

Photoswitchable fluorescent polymeric nanoparticles with AIE-based fluorogens as FRET donors were prepared for rewritable fluorescence patterning and intracellular dual-color imaging.

No abstract

Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.