TY - JOUR
T1 - Cellular uptake of ribonuclease A-functionalised core-shell silica microspheres
AU - Chimonides, G.F
AU - Behrendt, J.M.
AU - Chundoo, E.
AU - Bland, C.
AU - Hine, A.V.
AU - Devitt, A.
AU - Nagel, D.A.
AU - Sutherland, A.J.
N1 - This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
Funding: EPSRC Industrial CASE (GFC) grant number EP/D038057/1
Electronic supplementary information: see DOI: 10.1039/c4tb01130a
PY - 2014/11/14
Y1 - 2014/11/14
N2 - Analysis of protein function in a cellular context ideally requires physiologically representative levels of that protein. Thus conventional nucleic acid-based transfection methods are far from ideal owing to the over expression that generally results. Likewise fusions with protein transduction domains can be problematic whilst delivery via liposomes/nanoparticles typically results in endosomal localisation. Recently polymer microspheres have been reported to be highly effective at delivering proteins into cells and thus provide a viable new alternative for protein delivery (protein transduction). Herein we describe the successful delivery of active ribonuclease A into HeLa cells via novel polymer core-silica shell microspheres. Specifically, poly(styrene-co-vinylbenzylisothiouronium chloride) core particles, generated by dispersion polymerisation, were coated with a poly(styrene-co-trimethoxysilylpropyl methacrylate) shell. The resultant core-shell morphology was characterised by transmission electron, scanning electron and fluorescence confocal microscopies, whilst size and surface charge was assessed by dynamic light scattering and zeta-potential measurements, respectively. Subsequently ribonuclease A was coupled to the microspheres using simple carbodiimide chemistry. Gel electrophoresis confirmed and quantified the activity of the immobilised enzyme against purified HeLa RNA. Finally, the polymer-protein particles were evaluated as protein-transduction vectors in vitro to deliver active ribonuclease A to HeLa cells. Cellular uptake of the microspheres was successful and resulted in reduced levels of both intracellular RNA and cell viability.
AB - Analysis of protein function in a cellular context ideally requires physiologically representative levels of that protein. Thus conventional nucleic acid-based transfection methods are far from ideal owing to the over expression that generally results. Likewise fusions with protein transduction domains can be problematic whilst delivery via liposomes/nanoparticles typically results in endosomal localisation. Recently polymer microspheres have been reported to be highly effective at delivering proteins into cells and thus provide a viable new alternative for protein delivery (protein transduction). Herein we describe the successful delivery of active ribonuclease A into HeLa cells via novel polymer core-silica shell microspheres. Specifically, poly(styrene-co-vinylbenzylisothiouronium chloride) core particles, generated by dispersion polymerisation, were coated with a poly(styrene-co-trimethoxysilylpropyl methacrylate) shell. The resultant core-shell morphology was characterised by transmission electron, scanning electron and fluorescence confocal microscopies, whilst size and surface charge was assessed by dynamic light scattering and zeta-potential measurements, respectively. Subsequently ribonuclease A was coupled to the microspheres using simple carbodiimide chemistry. Gel electrophoresis confirmed and quantified the activity of the immobilised enzyme against purified HeLa RNA. Finally, the polymer-protein particles were evaluated as protein-transduction vectors in vitro to deliver active ribonuclease A to HeLa cells. Cellular uptake of the microspheres was successful and resulted in reduced levels of both intracellular RNA and cell viability.
UR - http://www.scopus.com/inward/record.url?scp=84907942482&partnerID=8YFLogxK
UR - http://pubs.rsc.org/en/content/articlelanding/2014/tb/c4tb01130a#!divAbstract
U2 - 10.1039/C4TB01130A
DO - 10.1039/C4TB01130A
M3 - Article
AN - SCOPUS:84907942482
SN - 2050-750X
VL - 2
SP - 7307
EP - 7315
JO - Journal of Materials Chemistry B
JF - Journal of Materials Chemistry B
IS - 42
ER -