[5] H. Tamura, A. Tanaka, K. Mita, R. Furuichi, Surface hydroxyl site densities on metal oxides as a measure for the ion-exchange capacity, J. Colloid Interface Sci. 209 (1999) 225–231. doi:10.1016/j.jconrel.2010.07.021 Adeno-associated virus loaded microbubbles as a tool for targeted gene delivery Bart Geers 1, ⁎, Ine Lentacker 1 , Angelika Alonso 3 , Stephen Meairs 3 , Joseph Demeester 1 , Stefaan C. De Smedt 1 , Niek N. Sanders 2 1 Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Gent, Belgium 2 Laboratory of Veterinary Gene therapy, Faculty of Veterinary Sciences, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium 3 Univ. Klinikum Mannheim, Department of Neurology, Heidelberg University, D-68167 Mannheim, Germany ⁎Corresponding author. E-mail: Bart.geers@ugent.be. Abstract summary Ultrasound responsive microbubbles can be a useful platform for targeted gene delivery. Although extensively used for the delivery of pDNA and siRNA, the knowledge on the use of this platform for viral gene delivery is poor. This study evaluated the use of these ultrasound responsive microbubbles for viral gene delivery. Introduction Adeno-associated virus is a promising gene therapy vector because of its high transfection efficiency. However, the use of AAVs in humans is limited by their high toxicity and their risk of infecting non-target cells. To obtain a safe viral vector, the immunogenic capsid of the AAV should be shielded from the immune system and the vector should be targeted to specific tissues. The objective of this study is to couple PEGylated AAVs with avidin–biotin chemistry to lipid microbubbles in order to obtain an ultrasound targeted release and transfection [1]. This study will evaluate whether lipid micro- bubbles release PEG-biotinylated AAVs upon ultrasound exposure and evaluate the transfection efficiency of AAV loaded microbubbles in vitro. Hence AAV-loaded microbubbles may provide a solution for ultrasound targeted viral gene delivery. Experimental methods PEGylation of EGFP expressing AAV2 was done using NHS-PEG- Biotin (Iris Biotech GmbH, Marktredwitz, Germany). Lipid micro- bubbles containing 85 mol.% DPPC and 15 mol.% of DSPE-PEG-Biotin were prepared. In vitro experiments were performed on BLM melanoma cells in Opticell plates and EGFP expression was measured with an FC500 Flow Cytometer. Ultrasound experiments were performed using a Sonitron sonoporator with an ultrasound frequency of 1 MHz, 2 W/cm 2 intensity and a 10% duty cycle. Result and discussion Zeta-potential measurements confirmed AAV PEGylation using NHS chemistry. Flow cytometry revealed that PEGylation of the AAV surface lowers their biological activity. We could prove microbubble coupling of AAVs through NHS-PEG-Biotin chemistry, by confocal microscopy. Flow cytometry analysis showed that melanoma cells can be infected by AAV loaded microbubbles and ultrasound, provided that the AAVs are not too strongly PEGylated. Confocal microscopy uptake studies however, showed an increased uptake after ultrasound treatment, possibly due to sonoporation after bubble implosion. Although enhanced uptake was observed, an increased transfection rate is not seen; possibly due to the fact sonoporation evades receptor mediated endocytosis. Conclusion This study shows that loading of softly-PEGylated AAV vectors to microbubbles by avidin–biotin linkers is possible and ultrasound targeted delivery is possible using these constructs. Targeted delivery of fully-PEGylated AAVs however is shown to be impossible, although more vectors are taken up after ultrasound exposure. We believe that this enhanced uptake is due to sonoporation, a mechanism which is believed to evade receptor mediated uptake [2]. Adenoviral vectors are mostly dependent of this mechanism, hereby limiting the functionality of ultrasound targeted drug delivery of viral vectors. Acknowledgements This project is funded by the European Commission via the FP7- project ARISE. Ine Lentacker is a post-doctoral fellow of the FWO-flanders. References [1] O.J. Muller, S. Schinkel, J.A. Kleinschmidt, H.A. Katus, R. Bekeredjian, Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats, Gene Ther. 15 (23) (2008) 1558–1565. [2] I. Lentacker, N. Wang, R.E. Vandenbroucke, J. Demeester, S.C. De Smedt, N.N. Sanders, Ultrasound exposure of lipoplex loaded microbubbles facilitates direct cytoplasmic entry of the lipoplexes, Mol. Pharm. (2009). doi:10.1016/j.jconrel.2010.07.022 Ultrasound responsive doxorubicin-loaded microbubbles; towards an easy applicable drug delivery platform Bart Geers 1, ⁎, Ine Lentacker 1 , Steven Cool 2 , Joseph Demeester 1 , Stefaan C. De Smedt 1 , Niek N. Sanders 2 1 Lab of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium 2 Lab of Veterinary Gene Therapy, Faculty of Veterinary Sciences, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium ⁎Corresponding author. E-mail: Bart.geers@ugent.be. Abstract summary The use of ultrasound responsive microbubbles for chemother- apeutic drug delivery has been extensively evaluated by different research groups. To obtain optimal drug release, we believe the drug, encapsulated in a nanoparticle, should be attached to the micro- bubble. Although significant results could be achieved using this strategy, it remains unapplicable. This study will provide a solution to these problems by presenting an easy to prepare and clinically applicable drug-loaded microbubble. Introduction Chemotherapy is nowadays one of the most widespread anti-cancer therapies. However, the incidence of severe side-effects due to undesired toxicity in non-target tissue is very high. A possible solution is to target the chemotherapeutic molecule to the tumor tissue. Literature provides a plethora of targeting strategies. But a very elegant way of targeting is the use of gas filled ultrasound responsive microbubbles [1–3], which can be monitored by means of ultrasound imaging. Providing a platform for image guided drug delivery [4]. These microbubbles have the ability of releasing a chemotherapeutic drug when located in the focus of an ultrasound beam. If this beam is located in the vicinity of a tumor, specific tumor targeting can be achieved. We believe the best way to deliver drugs using this strategy is when the drug, encapsulated in a nanoparticle, is coupled on the surface of these microbubbles. Up to now, all proposed drug-loaded microbubble systems had a hampered clinical application, due to the incorporation of immunogenic proteins or due to their limited loading capacity. This study proposes an easy to Abstracts / Journal of Controlled Release 148 (2010) e57–e73 e59