Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic.

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.

Close-packed silane nanodot arrays by capillary nanostamping coupled with heterocyclic silane ring opening.

We report the parallel generation of close-packed ordered silane nanodot arrays with nanodot diameters of few 100 nm and nearest-neighbor distances in the one-micron range. Capillary nanostamping of heterocyclic silanes coupled with ring-opening triggered by hydroxyl groups at the substrate surfaces yields nanodots consisting of silane monolayers with exposed terminal functional groups. Using spongy mesoporous silica stamps with methyl-terminated mesopore walls inert towards the heterocyclic silanes, we could manually perform multiple successive stamping cycles under ambient conditions without interruptions for ink refilling. Further functionalizations include the synthesis of polymer nanobrushes on the silane nanodots by surface-initiated atom-transfer radical polymerization. Proteins-of-interest fused to the HaloTag were site-specifically captured to silane nanodots functionalized by copper-free reactions with azide derivatives. Thus, bioorthogonal functionalization for bioanalytics with a spatial resolution in the one-micron range may be realized on solid supports compatible with fluorescence-based optical microscopy. The feature sizes of the silane nanodot arrays match well the length scales characteristic of a variety of biomolecular submicroscopic organizations in living cells, thus representing a compromise between miniaturization and the resolution limit of optical microscopy for sensitive high-throughput bioanalytics.

Publisher Correction: Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells.

In the version of this Article originally published, Supplementary Videos 3-5 were incorrectly labelled; 3 should have been 5, 4 should have been 3 and 5 should have been 4. This has now been corrected.

Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells.

The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model.

Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells.

Magnetogenetics is emerging as a novel approach for remote-controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP-nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote-controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine.

Magnetogenetic control of protein gradients inside living cells with high spatial and temporal resolution.

Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells.

On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane.

Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells.

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine-receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane.

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.