Octaarginine mediated delivery of fluorescent cargo to human smooth muscle cells

Sweeney, Michele, Watkins, Catherine Louise, Jones, Arwyn Tomos ORCID: https://orcid.org/0000-0003-2781-8905 and Taggart, Michael J. 2010. Octaarginine mediated delivery of fluorescent cargo to human smooth muscle cells. Drug Discovery Today 15 (23-24) , p. 1085. 10.1016/j.drudis.2010.09.367

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Abstract

The high incidence and severity of diseases involving smooth muscle dysfunction, which include cardiovascular diseases and premature labour, dictates the need for our continued search for novel therapeutic strategies to treat these conditions. Cell penetrating peptides (CPP) are a class of non-viral vectors that show considerable promise for drug delivery purposes yet their suitability for uptake, and delivery of biologically active cargo, to human native cells and tissues remains unresolved. For any new drug delivery strategy, including the use of CPPs, to reach fruition this needs to be elucidated. We have begun to explore this issue for CPPs applied to human uterine cells and tissues (including myometrium and blood vessels) obtained from biopsies collected, following LREC-approved written informed consent, from patients undergoing elective Caesarean section at the end of pregnancy. Primary cultured human myometrial cells were prepared on glass-bottomed culture dishes, grown to 80–90% confluence and exposed to serum-reduced conditions overnight before exposure to CPP (or, separately, were methanol-fixed for subsequent immunofluoresence staining of protein localisation). Cellular uptake of fluorescently labelled (Alexa 488) d-Octaarginine (R8, 2 μM) was assessed in the first series of experiments for 24, 48 and 72 hours (n = 2). At each time point, z-section confocal microscopy revealed punctate intracellular fluorescence (indicative of vesicular compartmentalisation) particularly dense in the perinuclear area. A second series of experiments assessed the time-course of intracellular delivery up to 24 hours. Punctate intracellular loading was observed by 4 hours. More dense perinuclear and plasma membrane-localised fluorescence was observed at later time points. Immunofluoresence labelling revealed that human myometrial cells possessed expected cytoskeletal (α-smooth muscle actin, tubulin), plasma membranous and perinuclear localised components of endocytotic pathways (Caveolin-1, Clathrin Heavy Chain, Early Endosomal Antigen-1, Lysosomal Associated Membrane Protein-1 and 2 and Flotillins). Next, small segments of native (non-cultured), human uterine tissue were incubated with 2 μM D-R8 and nuclear dye Hoechst 33342 (1 μM) for 4 hours. Confocal microscopic examination revealed peptide entry into smooth muscle cells of both the myometrium and uterine blood vessels with homogenous intracellular fluorescence in many cells but some with more punctate perinuclear/nuclear fluorescence. In uterine tissues incubated with a similar, putatively cell-impermeant, Alexa488 control peptide (GS)4GC, no intracellular fluorescence was observed. These preliminary investigations illustrate that an octameric cationic CPP can successfully enter primary cultured and native human smooth muscle cells and tissues. This opens up a new avenue for targeted delivery of cellular therapeutics in human tissues and in particular to human smooth muscles.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Subjects:	R Medicine > RS Pharmacy and materia medica
ISSN:	1359-6446
Last Modified:	18 Oct 2022 12:23
URI:	https://orca.cardiff.ac.uk/id/eprint/9649

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