Partner: Patrick Midoux

CNRS (FR)

Ostatnie publikacje
1.Delalande A., Leduc C., Midoux P., Postema M., Pichon C., Efficient gene delivery by sonoporation is associated with microbubble entry into cells and the Clathrin-dependent endocytosis pathway, ULTRASOUND IN MEDICINE AND BIOLOGY, ISSN: 0301-5629, DOI: 10.1016/j.ultrasmedbio.2015.03.010, Vol.41, No.7, pp.1913-1926, 2015

Streszczenie:

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.

Słowa kluczowe:

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

Afiliacje autorów:

Delalande A.-CNRS (FR)
Leduc C.-CNRS (FR)
Midoux P.-CNRS (FR)
Postema M.-other affiliation
Pichon C.-CNRS (FR)
35p.
2.Delalande A., Kotopoulis S., Postema M., Midoux P., Pichon C., Sonoporation: Mechanistic insights and ongoing challenges for gene transfer, Gene, ISSN: 0378-1119, DOI: 10.1016/j.gene.2013.03.095, Vol.525, pp.191-199, 2013

Streszczenie:

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.

Słowa kluczowe:

Ultrasound, Microbubbles, Physical gene delivery method, Gene therapy

Afiliacje autorów:

Delalande A.-CNRS (FR)
Kotopoulis S.-Haukeland University Hospital (NO)
Postema M.-other affiliation
Midoux P.-CNRS (FR)
Pichon C.-CNRS (FR)
20p.
3.Delalande A., Postema M., Mignet N., Midoux P., Pichon Ch., Ultrasound and microbubble-assisted gene delivery: recent advances and ongoing challenges, Therapeutic Delivery, ISSN: 2041-5990, DOI: 10.4155/TDE.12.100, Vol.3, No.10, pp.1199-1215, 2012

Streszczenie:

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.

Słowa kluczowe:

Ultrasound, Gene delivery, Sonoporation

Afiliacje autorów:

Delalande A.-CNRS (FR)
Postema M.-other affiliation
Mignet N.-other affiliation
Midoux P.-CNRS (FR)
Pichon Ch.-CNRS (FR)
4.Delalande A., Bouakaz A., Renault G., Tabareau F., Kotopoulis S., Midoux P., Arbeille B., Uzbekov R., Chakravarti S., Postema M., Pichon C., Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype, Journal of Controlled Release, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2011.08.020, Vol.156, pp.223-230, 2011

Streszczenie:

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.

Słowa kluczowe:

Gene delivery, Sonoporation, Tendon

Afiliacje autorów:

Delalande A.-CNRS (FR)
Bouakaz A.-Université François Rabelais (FR)
Pichon C.-CNRS (FR)
Renault G.-CNRS (FR)
Tabareau F.-CHR, Service d'anatomie et cytologie pathologiques (FR)
Kotopoulis S.-Haukeland University Hospital (NO)
Midoux P.-CNRS (FR)
Arbeille B.-Université François Rabelais (FR)
Uzbekov R.-Université François Rabelais (FR)
Chakravarti S.-Johns Hopkins School of Medicine (US)
Postema M.-other affiliation
32p.

Lista rozdziałów w ostatnich monografiach
1.
457
Delalande A., Kotopoulis S., Midoux P., Postema M., Pichon C., Micro-acoustics in marine and medical research, rozdział: Ultrasound and microbubble-assisted gene delivery: insights for intracellular mechanism, pp.119-130, 2012

Prace konferencyjne
1.Delalande A., Bouakaz A., Midoux P., Postema M., Pichon C., Ultrasound-activated microbubbles for tendon gene transfer: in vivo efficiency and confocal microscopy real time intracellular investigations, ICA 2010, 20th International Congress on Acoustics, 2010-08-23/08-27, Sydney (AU), pp.#524-1-3, 2010

Streszczenie:

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.

Słowa kluczowe:

Ultrasound, Sonoporation, Gene transfer

Afiliacje autorów:

Delalande A.-CNRS (FR)
Bouakaz A.-Université François Rabelais (FR)
Midoux P.-CNRS (FR)
Postema M.-other affiliation
Pichon C.-CNRS (FR)