Published: May 05, 2011 r2011 American Chemical Society 748 dx.doi.org/10.1021/mp100360g | Mol. Pharmaceutics 2011, 8, 748–757 ARTICLE pubs.acs.org/molecularpharmaceutics Development of Polymeric Microbubbles Targeted to Prostate-Specific Membrane Antigen as Prototype of Novel Ultrasound Contrast Agents Vanna Sanna,* ,† Gianfranco Pintus,* ,‡ Pasquale Bandiera, ‡ Roberto Anedda, † Stefania Punzoni, ‡ Bastiano Sanna, ‡ Vincenzo Migaleddu, § Sergio Uzzau, †,‡ and Mario Sechi* ,|| † Porto Conte Ricerche, Localit  a Tramariglio, 07041, Alghero, Sassari, Italy ‡ Department of Biomedical Sciences, Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, 07100 Sassari, Italy § Sardinian Mediterranean Imaging Research Group—no profit foundation, 07100, Sassari, Italy ) Dipartimento di Scienze del Farmaco, University of Sassari, 07100 Sassari, Italy b S Supporting Information ’ INTRODUCTION Ultrasonography is a widely used noninvasive diagnostic medical imaging technique, which is cost-effective and easy to use and provides real-time imaging avoiding the use of hazardous ionizing radiation. 1,2 Ultrasound (US) contrast agents, such as microbubbles (MBs), are providing new opportunities to en- hance the diagnostic capabilities of US imaging, because specia- lized contrast-specific US techniques are able to reveal their nonlinear responses while the linear static ultrasound signals coming from tissues are suppressed. This allows clinicians to obtain information regarding the perfusion behavior of the organs and their diffuse or focal diseases. 3,4 MBs consist of a specific gas surrounded by a stabilizing shell, with a typical diameter of 18 μm, which, intravenously injected in the bloodstream, have the capability to function as blood pool- enhancing agents and to assess tissue blood flow at a micro- vascular level. 5 With the gas content, shell composition is a key determinant for MBs' physical properties as well as for their acoustic behavior and imaging time. 6 Proposed shell materials, used to prevent gas escaping from the core and to avoid the microbubbles’ coalescence, 7 include proteins, 8 lipids, 9 biocom- patible polymers 10 and surfactants. 11 The generation of targeted MBs to a specific pathological tissue (for example, neoangiogen- esis or tumor antigens) constitutes an emerging field of US contrast agents, as it allows increase of their overall diagnostic potential and, if MBs are loaded with a drug, delivery locally. 12,13 Active targeting usually requires a proper modification of the MB shell to enable selective binding to cellular epitopes or receptors of interest. Ligands belong to various classes of molecules including monoclonal antibodies, polysaccharides and specific peptide sequences that recognize disease antigens. 1416 Targeting ligands can be either coincorporated in the shell during MB preparation (i.e., albumin), 15 or attached to the surface of preformulated MBs by covalent or noncovalent methods. 17 In the covalent coupling, the ligand is attached to Received: October 21, 2010 Accepted: May 5, 2011 Revised: April 11, 2011 ABSTRACT: Ultrasound-targeted microbubbles (MBs) offer new opportunities to enhance the capabilities of diagnostic ultrasound (US) imaging to specific pathological tissue. Herein, we report on the design and development of a novel prototype of US contrast agent based on polymeric MBs targeted to prostate- specific membrane antigen (PSMA) for use in the diagnosis of prostate cancer (PCa). First, a set of air-filled MBs by a variety of biocompatible polymers were prepared and characterized in terms of morphology and echogenic properties after exposure to US. MBs derived from poly(D,L-lactic-co-glycolic acid) (PLGA)- poly(ethylene glycol) (PEG) copolymer resulted as the most effective in terms of reflectivity. Such polymer was therefore preconjugated with a urea-based PSMA inhibitor molecular probe (DCL), and the obtained MBs were investigated in vitro for their targeting efficacy toward PSMA positive PCa (LNCaP) cells. Fluorescence microscopy proved a specific and efficient adhesion of targeted MBs to LNCaP cells. To our knowledge, this work reports the first model of polymeric MBs appropriately engineered to target PSMA, which might be further optimized and used for PCa diagnosis and potential carriers for selective drug delivery. KEYWORDS: targeted microbubbles, biodegradable polymers, ultrasound, contrast agents, prostate cancer, prostate-specific membrane antigen (PSMA)