Journal of Environmental Pathology, Toxicology and Oncology, 35(1):91-98 (2016) 91 0731-8898/16/$35.00 © 2016 Begell House, Inc. www.begellhouse.com Comparing the Effects of Light- or Sonic-Activated Drug Delivery: Photochemical/Sonochemical Internalization Steen J Madsen, a,* Jonathan Gonzales, b Genesis Zamora, b Kristian Berg, c Rohit Kumar Nair, b & Henry Hirschberg a,b a Department of Health Physics and Diagnostic Sciences, University of Nevada, Las Vegas, NV 89154; b Beckman Laser Institute and Medical Clinic, University of California, Irvine, CA 92612; c Oslo University Hospital, The Norwegian Radium Hospital, Dept. of Radiation Biology, Oslo, 0379, Norway *Address all correspondence to: Steen Madsen, University of Nevada, Las Vegas, Department of Health Physics and Diagnostic Sciences, 4505 Maryland Pkwy, Box 453037, Las Vegas, NV 89154-3037; Tel.: 702-895-1805; Fax: 702-895-4819; steen.madsen@unlv.edu ABSTRACT: Photochemical internalization (PCI) is a technique that uses the photochemical properties of pho- todynamic therapy (PDT) for the enhanced delivery of endolysosomal-trapped macromolecules into the cell cy- toplasm. The released agent can therefore exert its full biological activity, in contrast to being degraded by lyso- somal hydrolases. Activation of photosensitizers via ultrasound (US), called sonodynamic therapy (SDT), has been proposed as an alternative to light-activated PDT for the treatment of cancerous tumors. The use of focused US (FUS) to activate photosensitizers allows treatment at tumor sites buried deep within tissues, overcoming one of the main limitations of PDT/PCI. We have examined ultrasonic activation of photosensitizers together with the anticancer agent bleomycin (BLM) using sonochemical internalization (SCI), as an alternative to light-activat- ed PCI. Our results indicate that, compared to drug or US treatment alone, US activation of the photosensitizer AlPcS 2a together with BLM signifcantly inhibits the ability of treated glioma cells to form clonogenic colonies. KEY WORDS: sonodynamic therapy, sonochemical internalization, photodynamic therapy, photochemical internal- ization, bleomycin, aluminum phthalocyanine disulfonate, glioma I. INTRODUCTION PCI is a technique that uses the photochemical properties of PDT for the enhanced and site-spe- cifc delivery of drugs into the cell cytoplasm. 1–5 Drugs that are internalized into cells via endocyto- sis end up trapped in intracellular endosomes and lysosomes. The concept of PCI is based on using photosensitizers that localize in the cell membrane and are carried into the cell during the endocytotic event. The photosensitizer remains in the endosome membrane while the macromolecule is localized within the lumen. Specifc amphiphilic photosensi- tizers (e.g., AlPcS 2a and TPPS 2a ) preferentially accu- mulate in the membranes of endosomes, and upon light exposure the photosensitizer interacts with ambient oxygen to produce singlet oxygen. Singlet oxygen has a very short range of action (<20 nm), so only the area of the vesicular membrane where the photosensitizer is localized will be damaged by singlet oxygen–mediated reactions with amino ac- ids, unsaturated fatty acids, and cholesterol in the membrane bilayer. The released agent can therefore exert its full biological activity, in contrast to be- ing degraded by lysosomal hydrolases. Originally, the PCI method was shown to be effective for the liberation of drugs that have already been endocy- tosed and trapped in endocytic vesicles. This was based on illumination after drug incubation, the so- called “light after” strategy, as explained above. In contrast, a number of studies have demonstrated ef- fcacy when photochemical disruption of endocytic vesicles occurs before delivery of macromolecules or the “light before” strategy. 6 Sonodynamic therapy (SDT) has been proposed as an alternative to photodynamic therapy (PDT) for the treatment of cancerous tumors. 7–12 Whereas PDT uses light to activate the sensitizing drug, SDT uses US. The advantage of US versus light activa- tion is that US has a much lower tissue attenuation