title: The Application of Clinical Lithotripter Shock Waves to RNA Nucleotide Delivery to Cells.
abstract: The delivery of genes into cells through the transfer of ribonucleic acids (RNAs) has been found to cause a change in the level of target protein expression. RNA-based transfection is conceptually more efficient than commonly delivered plasmid DNA because it does not require division or damage of the nuclear envelope, thereby increasing the chances of the cell remaining viable. Shock waves (SWs) have been found to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanying SW bio-effects include dose-dependent irreversible cell injury and cytotoxicity. Here, the effect of SWs generated by a clinical lithotripter on the viability and permeabilisation of three different cell lines in vitro was investigated. Comparison of RNA stability before and after SW exposure revealed no statistically significant difference. Optimal SW exposure parameters were identified to minimise cell death and maximise permeabilisation, and applied to enhanced green fluorescent protein (eGFP) messenger RNA (mRNA) or anti-eGFP small interfering RNA delivery. As a result, eGFP mRNA expression levels increased up to 52-fold in CT26 cells, whereas a 2-fold decrease in GFP expression was achieved after anti-eGFP small interfering RNA delivery to MCF-7/GFP cells. These results indicate that SW parameters can be employed to achieve effective nucleotide delivery, laying the foundation for non-invasive and high-tolerability RNA-based gene therapy.
authors.affiliation: Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom. Electronic address: sandra.nwokeoha@eng.ox.ac.uk.
authors.forename: Sandra
authors.initials: S
authors.lastname: Nwokeoha
authors.affiliation: Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Carlisle
authors.affiliation: Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, United Kingdom.
authors.forename: Robin O
authors.initials: RO
authors.lastname: Cleveland
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000628:therapy*; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D007680:Kidney Neoplasms / Q000628:therapy*; D008096:Lithotripsy / Q000379:methods*; D008325:Mammary Neoplasms, Experimental / Q000628:therapy*; D051379:Mice; D009711:Nucleotides; D010539:Permeability; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014162:Transfection / Q000379:methods
chemical_list: D009711:Nucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: Drug delivery; Gene therapy; High-amplitude acoustic waves; Messenger RNA; Shock waves; Small interfering RNA; Ultrasound