By Ayache Bouakaz and Jean Michel Escoffre
Much research is being directed to targeted drug delivery, where drugs are focused on specific body locations or organs. The combination of ultrasound and microbubbles offer a possible low-cost avenue for moving drugs, genes, or antibodies across tumor cell membranes, increasing treatment efficiency and reducing toxicity side effects.
Over the next decades, the ageing population and the occidental lifestyle will lead to socio-economic and healthcare problems. With increasing proportions of the population becoming inactive, the working population is decreasing while disease prevalence is rising. Since 1999, healthcare expenditures have significantly increased to reach 9.5% in 20101 with an expected faster acceleration in the future. Nowadays, the improvement of the population’s access to advanced medical care, especially in cancer, while keeping healthcare cost reasonable is a major challenge worldwide. Cancer presents the second leading cause of death in the European Union (EU), with 3.45 million new cases of cancer and 1.75 million deaths from cancer in 2012 . Owing to improvements in anticancer therapy, cancer will be managed as a chronic disease rather than through acute intervention. However, this change in therapeutic practice may continuously involve expensive medication and medical care. In the future, the challenge will be to support the healthcare system while keeping the socio-economic impact as reasonable as possible.
Cancer treatment has considerably developed over the past decade, especially in the area of chemotherapy. In clinics, powerful anticancer drugs (e.g., doxorubicin, irinotecan, cetuximab) decreased mortality in patients, either as a monotherapy or associative therapy. However, high or frequent doses are required due to the poor intra-tumoral bioavailability, making the chemotherapy a very expensive option associated with undesirable side effects. Indeed, the early use of such anticancer drugs in clinical application revealed major side effects, such as the development of tumoral resistance and adverse side effects such as the toxicity in healthy tissues. Targeted drug delivery (delivery of a drug to a spatially localized site in the human body) is one of the most ambitious goals of modern therapy against the cancer. The strict localization of the pharmacological activity of a drug to the site of pathology would result in a significant reduction in systemic drug toxicity. This would enable the ability to deliver increased doses of drug to desired tissue, and thus would result in an increase in treatment efficacy and safety. Although a great amount of work is conducted worldwide on the research of various targeted drug delivery systems, clinical applications of site-targeted delivery are still very limited.
In recent years, new promising possibilities for targeted drug delivery have been discovered based on the combination of ultrasound (US) and microbubbles. Ultrasound contrast agents (UCAs) are microbubbles consisting of a gaseous core (e.g., perfluorocarbon) surrounded by a biocompatible shell (e.g., phospholipid, polymer). UCAs are currently used in medical ultrasound diagnostics . They are injected into the bloodstream of patients in order to increase the acoustic contrast between blood and surrounding tissue, thus improving the quality of images and diagnostic confidence (also known as contrast-enhanced ultrasound imaging, CEUs). The main applications of CEUs are the study of organs’ perfusion (e.g., heart) and the characterization of pathological lesions (e.g., liver cancer). The UCA’s shell can be decorated with binding ligands to allow targeted molecular imaging of physiological molecular markers that are over expressed on endothelial cells of the vascular system related to a physiopathological processes . Research in the field of CEUs is increasingly paying attention to fine-tuning pulse sequences and ultrasound equipment towards the acoustic properties of UCAs and vice-versa. Beyond these improvement issues, new promising applications of UCAs will emerge for targeted drug delivery. UCAs can be intravenously co-injected with drugs, genes or antibodies (also known as co-administration approach) or carry these molecules inside or on their encapsulating shell . This capability, in combination with the phenomenon known as sonoporation, provides unprecedented possibilities for a highly selective therapeutic action . The term sonoporation denotes a process in which ultrasonically activated contrast microbubbles result in their pulsation and/or disruption nearby cell membranes or endothelial barriers. This leads to an increase in vascular permeability, thus facilitating extravasation of drugs into tumor tissue and hence an augmented drug bioavailability (Fig.1).
Figure 1: Sonoporation process (Servier Medical Art, www.servier.fr)
The first experiments on sonoporation date back to 1980s, Various US exposure conditions have been tested blindly including frequencies ranging from the kHz to the MHz. Sonoporation has also been evaluated using high pressure amplitude US waves. Since then and with the recent introduction of contrast agents, higher frequency US with cavitation enhanced by microbubbles have been sought to induce a range of effects on cells. Extensive examinations have been carried out to evaluate the efficiency of US in combination with contrast microbubbles in inducing cellular uptake. Although these result and finding were achieved in a controlled in-vitro environment, diagnostic US scanners were also useful for therapeutic applications of sonoporation particularly with the guidance of treatment afforded by the imaging mode.
As an example, we present in the Fig. 2 the result of gene transfection with Vevo Micromarker microbubbles and US by measuring the GFP expression with a flow cytometer 48h after incubation of pmaxFP-Green-C . As shown, the combination of pmaxFP-Green-C incubation with microbubble-assisted ultrasound at 300 kPa induced a transfection level of 17±3% at 48h. The increase of the acoustic pressure from 400 kPa to 600 kPa induced an enhancement of the transfection level compared to that obtained at 300 kPa. At both 600 kPa and 800 kPa, an unprecedented transfection level of approximately 70% was reached. The increase of the transfection level and efficiency with the applied acoustic pressure correlated with a slight increase of the cell mortality but the latter remained below 15%. These results show that microbubble and selected ultrasound settings might induce very high transfection level and efficiency.
Figure 2: Enhancement of gene transfection by ultrasound combined with microbubbles. U-87 MG cells were incubated with pmaxFP-Green-C and the microbubbles and then insonated. Transfection level (left) and mortality level (right) were monitored by flow cytometry 48 h later. Data expressed as mean ± SEM were calculated from five independent experiments. Statistical analysis was performed using the non-parametric Mann-Whitney test. Significance was defined as p<0.05 (NS, non significance, *p<0.05, **p<0.01).
In addition, we recently reported the potential benefit of a co-administration approach for irinotecan delivery in a human subcutaneous glioblastoma mouse model . Hence the delivery of the therapeutic agent in the targeted tissue can be controlled spatially and temporally through US focusing and action. The feasibility of sonoporation concepts for local drug, gene and antibody delivery has been successfully reported . In addition, this delivery system promises to be a low-cost technology.
Today, the mechanisms involved in the sonoporation process and the cell membrane permeabilization remain poorly identified. Although no consensus has been reached, several scenarios have been hypothesized, including the formation of pores, further stimulation of endocytotic pathways and occurrence of membrane wounds. Elucidating the mechanisms responsible for delivery of compounds to the cells and the kinetics of permeabilization are essential in order to improve and control this therapeutic strategy.
For Further Reading
1. J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, S. Rosso, J.W. Coebergh, H. Comber, D. Forman, and F. Bray. “Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012”, Eur. J. Cancer, vol. 49, no. 6, pp. 1374-1403, 2013.
8. J.M. Escoffre, A. Novell, J. Piron, A. Zeghimi, A. Doinikov, and A. Bouakaz. “Microbubble attenuation and destruction: are they involved in sonoporation efficiency?”, IEEE Trans. Ultrason. Ferroelectr. Freq. Control, vol. 60, no. 1, pp. 46-52, 2013.
9. J.M. Escoffre, A. Novell, S. Serriere, T. Lecomte and A. Bouakaz. “Irinotecan Delivery by Microbubble-Assisted Ultrasound: In Vitro Validation and a Pilot Preclinical Study”, Mol. Pharm., vol. 10, no. 7, pp. 2667-2675, 2013.
11. J.M. Escoffre, C. Mannaris, B. Geers, A. Novell, I. Lentacker, M. Averkiou and A. Bouakaz. “Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery”, IEEE Trans Ultrason Ferroelectr Freq Control, vol. 60, no. 1, pp.78-87, 2013.
12. E.J. Park, Y.Z. Zhang, N. Vykhodtseva and N. McDannold. “Ultrasound-mediated blood-brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model”, J. Control. Release, vol. 163, no. 3, pp. 277-284, 2013.
Ayache Bouakaz graduated from the University of Sétif, Algeria, from the Department of Electrical Engineering. He obtained his Ph.D. degree in 1996 from the Department of Electrical Engineering at the Institut National des Sciences Appliquées de Lyon (INSA Lyon), France. In 1998, he joined the Department of Bioengineering at The Pennsylvania State University in State College, PA, where he worked as a postdoc for 1 year. From February 1999 to November 2004, he was employed at the Erasmus University Medical Center, Rotterdam, Netherlands. His research focused on imaging, ultrasound contrast agents and transducer design. Since January 2005, he has held a permanent position as a director of research at the French Institute for Health and Medical Research, INSERM in Tours, France, where he heads the ultrasound laboratory. His research focuses on Imaging and therapeutic applications of ultrasound and microbubbles. Read more
Jean-Michel Escoffre earned his M.Sc. degrees in Vectorology, Gene Therapy, Vaccinology and Cell Biophysics in 2005 and 2006 respectively, at the University Paul Sabatier (Toulouse, France). He obtained his Ph.D. degree in Cell Biophysics in 2010 at the Institute of Pharmacology and Structural Biology (CNRS, UMR 5089) of the University Paul Sabatier. From April 2010 to April 2013, he was employed at the Institute of Imaging and Brain of the French Institute for Health and Medical Science (INSERM, UMR 930) in Tours, France. His research focused on gene and drug delivery using microbubble-assisted ultrasound. He is currently pursuing his postdoc at the University Medical Center Utrecht, Netherlands. His main research interests lie in the fields of the delivery of therapeutic macromolecules by magnetic resonance imaging guided high intensity focused ultrasound. He has authored over 44 journal articles and conference proceedings, and 4 book chapters. Read more