RESEARCH PAPER Ultrasound Enhanced PEI-Mediated Gene Delivery Through Increasing the Intracellular Calcium Level and PKC-δ Protein Expression Jyun-Lin Lee & Chia-Wen Lo & Claude Inserra & Jean-Christophe Béra & Wen-Shiang Chen Received: 16 October 2013 /Accepted: 8 February 2014 # Springer Science+Business Media New York 2014 ABSTRACT Purpose Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expres- sion. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. Methods Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis path- ways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. Results Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6 h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3 h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. Conclusions Our results suggest that US enhanced PEI- mediated gene transfection specifically by increasing PKC-δ relat- ed fluid phase endocytosis, which was induced by increasing the intracellular calcium levels. KEY WORDS calcium . fluid phase endocytosis . PKC-δ . polyethylenimine . ultrasound INTRODUCTION Non-viral gene transfer methods such as electroporation, ul- trasound (US), cationic polymer or cationic lipid, have recent- ly been reported as being potentially applicable to gene ther- apy (1–6). Gene transfer mediated by non-viral vectors ex- hibits many of the desired characteristics for future human gene therapy applications, including low immunogenicity, treatment convenience, and site specificity (7–9). US has been shown to enhance the delivery efficiency of drugs or genes into cells (10–14), achieving safe and local delivery of drugs or genes to the target organs by focusing the US beam on the specific target site. The use of skeletal muscle in our in vivo study has unique advantages. Muscle constitutes about 30% of adult human body mass and has an abundant blood vascular supply, lending itself to use as target tissue for the production of transgene proteins to act as systemic therapeutic agents ( 14,15). For example, a US-based systemic gene therapy method allows for the US-mediated delivery of anti-angiogenic genes into skeletal muscles, Electronic supplementary material The online version of this article (doi:10.1007/s11095-014-1332-4) contains supplementary material, which is available to authorized users. J.<L. Lee : C.<W. Lo : W.<S. Chen (*) Department of Physical Medicine and Rehabilitation National Taiwan University Hospital National Taiwan University College of Medicine Taipei, Taiwan, Republic of China e-mail: wenshiang@gmail.com C. Inserra : J.<C. Béra INSERM, U1032, LabTAU, Université Claude Bernard Lyon 1 151 Cours Albert Thomas, 69424 Lyon, France W.<S. Chen Division of Medical Engineering Research National Health Research Institutes Miaoli, Taiwan, Republic of China Pharm Res DOI 10.1007/s11095-014-1332-4