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Abstract:
Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble–cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Keywords:
Ultrasound, Microbubble, Sonoporation, Gene delivery, Endocytosis, Clathrin-mediated endocytosis pathway

Abstract:
Purpose
Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50 % are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer.

Experimental Design
An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound.

Results
Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10 % demonstrated in comparison to the control groups.

Conclusion
Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation.

Keywords:
Sonoporation, Pancreatic cancer, Ultrasound, Chemotherapy, 3D ultrasound, Bioluminescence

Abstract:
Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Keywords:
Ultrasound, Microbubbles, Physical gene delivery method, Gene therapy

Abstract:
Having rst been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble–cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Keywords:
Ultrasound, Gene delivery, Sonoporation

Abstract:
The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5 × 10^5 BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Keywords:
Gene delivery, Sonoporation, Tendon

Abstract:
The purpose of this study was to investigate the physical mechanisms of sonoporation, in order to understand and improve ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation and resealing of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. The authors studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6?6 MHz ultrasound with a mechanical index of 0?2 and observed with a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell while entering. The authors have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic agent is added to the shell of the bubble or inside the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. In addition, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Keywords:
Sonoporation, Low mechanical index, Microbubbles, Ultrasound contrast agent, HeLa cells, Cell penetration

Abstract:
The purpose of this study was to investigate the physical mechanisms of sonoporation, to understand and ameliorate ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation of a cell membrane with help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. We studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6.6-MHz ultrasound with a mechanical index of 0.2 and observed with a high- speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell whilst entering. We have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic load is added to the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. However, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Keywords:
Sonoporation, Ultrasound, Microbubbles, HeLa Cancer cells, Drug delivery, Low Mechanical Index

Abstract:
Ultrasound as a therapeutic means is increasing in popularity, owing to its non-invasiveness and low cost. Biomedical applications include surgery, cyanobacteria eradication, and drug and gene delivery. Recent advances have been associated with the ultrasound-induced formation of transient porosities in cell membranes. These and other applications have been associated with the behaviour of medical microbubbles near cells.
Common methods of studying the dynamic behaviour of microbubbles are high-speed photography and optical microscopy.
Here, we give a brief overview of our fields of study in biomedical ultrasonics, concentrating on bubble–cell interactions.
There are three standards for the safe use of biomedical sound. When introducing bubbles, these safety guidelines may be misleading.

Keywords:
Ultrasound, Sonoporation, Safety guidelines

Abstract:
Ultrasound that is routinely used for imaging is now exploited for therapeutic applications including drug delivery or gene transfer. Today, ultrasound imaging is an established and confident technique for diagnosis. It is mainly based on the development of contrast imaging methods that aim to identify and display the echo from contrast agent as well as rejecting the echo from surrounding tissue offering thus a more resolutive detection. Ultrasound contrast agents or microbubbles (MB) are small gas bubbles encapsulated by a stabilizing shell, with a typical diameter of micron range. Ultrasound pulses are typically applied with a frequency near the resonance frequency of the gas bubble and the bubbles oscillations produce strong echoes from regions of perfused tissue [1-2]. Activation of microbubbles (MB) under specific ultrasound (US) beams induces a transient cell membrane permeabilization with a process known as sonoporation [3-4]. This work aims at evaluating the use of ultrasound and microbubbles for gene transfer in Achilles tendons.

Keywords:
Ultrasound, Sonoporation, Gene transfer