Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm 2 , 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (480%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.

No abstract

Gene therapy clinical trials are limited due to several hurdles concerning the type of vector used, particularly, the viral vectors, and transfection efficacy when nonviral vectors are used. Therapeutic ultrasound is a promising non -viral technology that can be used in the clinical setting. Here, for the first time, we show the efficacy of therapeutic ultrasound to deliver genes encoding for hemopexin-like domain fragment (PEX), an inhibitor of angiogenesis, to prostate tumors in vivo. Moreover, the addition of an ultrasound contrast agent (Optison) to the transfection process was evaluated. Prostate cancer cells and endothelial cells (EC) were transfected in vitro with cDNA-PEX using therapeutic ultrasound alone (TUS + pPEX) or with Optison (TUS + pPEX + Optison). The biological activity of the expressed PEX was assessed using proliferation, migration, and apoptosis assays done on EC and prostate cancer cells. TUS + pPEX + Optison led to the inhibition of EC and prostate cancer cell proliferation (<65%), migration (<50%), and an increase in apoptosis. In vivo, C57/black mice were inoculated s.c. with prostate cancer cells. The tumors were treated with TUS + pPEX and TUS + pPEX + Optison either once or repeatedly. Tumor growth was evaluated, after which histology and immunohistochemistry analyses were done. A single treatment of TUS + pPEX led to a 35% inhibition in tumor growth. Using TUS + PEX + Optison led to an inhibition of 50%. Repeated treatments of TUS + pPEX + Optison were found to significantly (P < 0.001) inhibit prostate tumor growth by 80%, along with the angiogenic indices, with no toxicity to the surrounding tissues. These results depict the efficacy of therapeutic ultrasound as a non -viral technology to efficiently deliver genes to tumors in general, and to deliver angiogenic inhibitors to prostate cancer in particular. [Mol Cancer Ther 2007;6(8):2371 -82]

No abstract

Therapeutic ultrasound (TUS) is a promising non-viral clinical approach for the delivery of genes. This study demonstrates the efficient delivery and localization of DNA in subcutaneous tumors facilitated by TUS application and examines the contribution of ultrasound contrast-agent (USCA) addition on transfection. The study addresses the importance of in vivo optimization when using long-term TUS and USCA based on data achieved in vitro. In vitro results showed that transfection of TrampC2 prostate cancer (Pca) cells using genes encoding for luciferase and green fluorescent protein was enhanced when DNA and Optison were added together and TUS was applied for 20 or 30 min. In vivo results showed that the highest transfection was achieved when Optison and DNA were co-injected intratumoraly, and TUS was applied for 20 min. Using Optison significantly increased protein distribution in the tumor. However, in vivo expression level was decreased by two and four fold at 7 and 14 days, respectively, post-TUS. The study establishes the potential of intratumoral delivery of DNA-Optison, followed by TUS as an effective, non-toxic, gene delivery method that could provide a safe, clinical alternative to current viral gene delivery approaches where short-term gene expression is needed.