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Targeted intracellular delivery of proteins with spatial and temporal control.

ABSTRACT: While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

SUBMITTER: Morales DP

PROVIDER: S-EPMC4319691 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Targeted intracellular delivery of proteins with spatial and temporal control.

Morales Demosthenes P DP Braun Gary B GB Pallaoro Alessia A Chen Renwei R Huang Xiao X Zasadzinski Joseph A JA Reich Norbert O NO

Molecular pharmaceutics 20141223 2

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer ...[more]

PMID: 25490248

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Project description:BackgroundSmall extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules.ResultsIn this study, we proposed a membrane-associated protein, milk fat globule-epidermal growth factor 8 protein (MFG-E8), to produce αvβ3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)-like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvβ3 and αvβ5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs' target to integrin αvβ3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvβ3 positive cells and αvβ3 negative cells. Analysis showed higher target protein could transfect into αvβ3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvβ3 positive cells.ConclusionThis study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvβ3 positive cells.

Project description:Objective.The goal of this work is to identify the spatio-temporal facets of state-of-the-art electroencephalography (EEG)-based continuous neurorobotics that need to be addressed, prior to deployment in practical applications at home and in the clinic.Approach.Nine healthy human subjects participated in five sessions of one-dimensional (1D) horizontal (LR), 1D vertical (UD) and two-dimensional (2D) neural tracking from EEG. Users controlled a robotic arm and virtual cursor to continuously track a Gaussian random motion target using EEG sensorimotor rhythm modulation via motor imagery (MI) commands. Continuous control quality was analyzed in the temporal and spatial domains separately.Main results.Axis-specific errors during 2D tasks were significantly larger than during 1D counterparts. Fatigue rates were larger for control tasks with higher cognitive demand (LR, left- and right-hand MI) compared to those with lower cognitive demand (UD, both hands MI and rest). Additionally robotic arm and virtual cursor control exhibited equal tracking error during all tasks. However, further spatial error analysis of 2D control revealed a significant reduction in tracking quality that was dependent on the visual interference of the physical device. In fact, robotic arm performance was significantly greater than that of virtual cursor control when the users' sightlines were not obstructed.Significance.This work emphasizes the need for practical interfaces to be designed around real-world tasks of increased complexity. Here, the dependence of control quality on cognitive task demand emphasizes the need for decoders that facilitate the translation of 1D task mastery to 2D control. When device footprint was accounted for, the introduction of a physical robotic arm improved control quality, likely due to increased user engagement. In general, this work demonstrates the need to consider both the physical footprint of devices, the complexity of training tasks, and the synergy of control strategies during the development of neurorobotic control.

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Targeted intracellular delivery of proteins with spatial and temporal control.

Publications

Targeted intracellular delivery of proteins with spatial and temporal control.

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