title: Enhanced gene transfection using calcium phosphate co-precipitates and low-intensity pulsed ultrasound.
abstract: The capability to controllably disrupt the cell membrane by ultrasound (US), thus facilitating entry of exogenous species, has now reached a state of some maturity. However, a compelling question asks whether there is a residual role for US in enhancing transfection: that is, once the genetic material has been delivered to the cytosol, can US assist in its transport into the nucleus? The present experiment was designed with a view to addressing this question. As such, our experimental setup discriminates between: (i) the precursor cell membrane permealization step, and (ii) any subsequent intracellular trafficking into the nucleus. In this study, calcium phosphate co-precipitates (CaP) were used to internalize plasmid DNA encoding for luciferase (pDNA-Luc) (>90%) in HeLa cells. After 2h incubation with the CaP-pDNA-Luc, cells were washed and insonated for varying durations. The results showed that US can indeed enhance the intracellular trafficking of previously internalized genes when longer insonation periods are implemented, culminating with an increased probability for successful nuclear localization, as inferred from an enhanced luciferase expression. Moreover, the results suggest that the intracellular role of US might be mediated through a pathway that appears not to be limited to destabilizing the endosomal vesicles. The study thus provides new information regarding the intracellular effects of US, and in effect represents a new modality combining US and CaP carriers for improved efficiency in gene delivery.
authors.affiliation: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan.
authors.forename: Mariame A
authors.initials: MA
authors.lastname: Hassan
authors.forename: Iman S
authors.initials: IS
authors.lastname: Ahmed
authors.forename: Paul
authors.initials: P
authors.lastname: Campbell
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
mesh_terms: D001692:Biological Transport; D002130:Calcium Phosphates / Q000737:chemistry*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D022161:Transport Vesicles / Q000378:metabolism; D014465:Ultrasonics / Q000379:methods*
chemical_list: D002130:Calcium Phosphates; D004247:DNA; C020243:calcium phosphate; D008156:Luciferases