Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy Anthony Delalande a,c , Michel-Francis Bureau b , Patrick Midoux a , Ayache Bouakaz c , Chantal Pichon a, * a Centre de Biophysique Moléculaire, UPR 4301 CNRS, 45071 Orléans, France b Laboratoire de Pharmacologie Chimique et Génétique, UMR 8151 CNRS, 75270 Paris, France c INSERM U930, CNRS ERL 3106, Université François-Rabelais, Tours, France article info Article history: Received 3 July 2009 Received in revised form 28 September 2009 Accepted 30 September 2009 Available online 7 October 2009 Keywords: Ultrasound Microbubbles Tendon Gene transfer Gene therapy abstract Our study aimed at evaluating the use of ultrasound-assisted microbubbles gene transfer in mice Achilles tendons. Using a plasmid encoding luciferase gene, it was found that an efficient and stable gene expres- sion for more than two weeks was obtained when tendons were injected with 10 lg of plasmid in the presence of 5  10 5 BR14 microbubbles with the following acoustic parameters: 1 MHz, 200 kPa, 40% duty cycle and 10 min of exposure time. The rate of gene expression was 100-fold higher than that obtained with naked plasmid injected alone without ultrasound or with ultrasound in absence of micro- bubbles. The long term expression of transgene makes ultrasound-assisted microbubble a suitable method for gene therapy in tendons. Ó 2009 Elsevier B.V. All rights reserved. 1. Introduction Tendons are axial connective tissue that ensures the transmis- sion of the traction exerted by muscle to bone resulting to joint movement. Tissue degeneration is the common clinical feature found in tendon disorders due to tendon injuries caused by their overuse, the loss of elasticity due to ageing or as a consequence of inherited rare connective diseases like Ehlers–Danlos syndrome type III and Marfan syndrome [1]. Spontaneous tendon ruptures occur very often in these patients due to over elasticity of connec- tive tissues. Absence of basal reparative ability and repetitive mi- cro-traumatic processes overwhelm tendon cells to repair the damaged tissue. Tendons heal poorly because of their hypocellular- ity and hypovascularity [2,3]. Tendon regeneration is difficult and requires a very long period. Moreover, the return at the initial state of biomechanical parameters is never reached. To date, the mechanism of tendons remodelling is known [3]. This process is controlled by growth factors acting at precise phases of regenera- tion. The development of non-invasive efficient therapy to enhance tendon regeneration as in situ delivery of active molecules by gene therapy could be useful [4–7]. Ultrasound that is routinely used for imaging is now exploited for therapeutic applications including drug delivery or gene transfer [8–13]. Activation of microbub- bles (MB) under specific ultrasound (US) beams induces a transient cell membrane permeabilization with a process known as sonoporation [14–16]. Physiotherapy ultrasound is used for the acceleration of wound healing and softening of scar tissue [17]. The aim of this study is to evaluate ultrasound and microbub- bles-mediated gene transfer as an alternative physical method for gene transfer in tendon. In addition to deliver genes that posi- tively affects tendon healing; it will also offer the ultrasound stim- ulation effect reported as a physical therapy for tendinopathy. 2. Experimental methods 2.1. Microbubbles and ultrasound set-up BR14 microbubbles were generously provided by Bracco (Bracco Research, Geneva, Switzerland). They are composed of per- fluorobutan gas encapsulated in a phospholipid shell. BR14 micro- bubbles have a mean diameter of 2.6 lm. The microbubbles suspension was prepared by injecting sodium chloride (0.9%) on BR14 powder giving approximately 2  10 8 microbubbles per mil- liliter. The experimental set-up for tendon insonation is shown in Fig. 1. Ultrasound waves were generated from a 0.5 in. transducer with 1 MHz frequency (IBMF-014, Sofranel, Sartrouville, France). The transducer was driven with an electrical signal generated by an arbitrary waveform generator (Agilent 33220A, Agilent technol- ogies) and amplified with a power amplifier (ADECE, Artannes, France). Pulsed ultrasound (10 kHz) with a duty cycle of 40% were used in this study. The negative peak acoustic pressures investi- gated were measured in a separate set-up with a calibrated PVDF 0041-624X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ultras.2009.09.035 * Corresponding author. Tel.: +33 238255595; fax: +33 238631517. E-mail address: chantal.pichon@cnrs-orleans.fr (C. Pichon). Ultrasonics 50 (2010) 269–272 Contents lists available at ScienceDirect Ultrasonics journal homepage: www.elsevier.com/locate/ultras