Polymeric microspheres as protein transduction reagents


Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.

Publication DOI: https://doi.org/10.1074/mcp.O113.034900
Divisions: College of Health & Life Sciences > School of Biosciences
College of Engineering & Physical Sciences > School of Infrastructure and Sustainable Engineering > Chemical Engineering & Applied Chemistry
College of Health & Life Sciences > Chronic and Communicable Conditions
College of Health & Life Sciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences > School of Biosciences > Cell & Tissue Biomedical Research
College of Engineering & Physical Sciences > Aston Institute of Materials Research (AIMR)
Additional Information: For content in ASBMB journals – The Journal of Biological Chemistry (JBC), Molecular & Cellular Proteomics (MCP) and the Journal of Lipid Research (JLR) – that was published under ASBMB journals’ paid open access publishing option, Author’s Choice, the article was distributed under the CC-BY license which automatically grants all commercial and noncommercial use of the article to all, as long as appropriate attribution is given to the original work. This work was funded by EPSRC Grant No. EP/D038057/1 to A.V.H. and A.J.S. This article contains supplemental material.
Uncontrolled Keywords: Biochemistry,Molecular Biology,Analytical Chemistry
Publication ISSN: 1535-9484
Last Modified: 19 Apr 2024 07:11
Date Deposited: 19 Aug 2019 10:06
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
https://www.sci ... 53594762033084X (Publisher URL)
PURE Output Type: Article
Published Date: 2014-06-01
Published Online Date: 2014-04-01
Authors: Nagel, David (ORCID Profile 0000-0002-9055-1775)
Behrendt, Jonathan M.
Chimonides, Gwen F.
Torr, Elizabeth E.
Devitt, Andrew (ORCID Profile 0000-0002-4651-6761)
Sutherland, Andy J. (ORCID Profile 0000-0003-3651-1809)
Hine, Anna V. (ORCID Profile 0000-0003-4065-831X)



Version: Published Version

License: Creative Commons Attribution

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