Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

Microbubbles Engineering for Drug Delivery-assisted by Ultrasound – MEDDU

Ultrasound and microbubbles assisted DNA delivery, a step toward theranostic approach

Recent gene therapy clinical studies indicate that its application is not anymore a myth but a reality. Our project aims at developing targeted delivery system based on the use of gaseous microbubbles capable of binding DNA which oscillate by ultrasound on demand. Microbubbles oscillation improve their delivery in tissue located in insonated area. Ultrasound offer the advantage to reach deep organs in non invasive manner.

New formulations of gas microbubbles for targeted gene delivery by ultrasound

Despite tremendous efforts to develop drug delivery systems, therapeutic index and selectivity toward diseased tissue remain very low. The lack of selectivity often results in high toxicity. The use of ultrasounds coupled to the gaseous microbubbles is promising approach to deliver and reach with noninvasive manner deep torgans. It is important to develop new microbubbles adapted to the drug and deepen knowledge of the extra-and intracellular behavior of these bubbles and their contents for extentive applications. <br />The project's objective is to develop gaseous microbubbles capable of transporting drugs and specifically deliver genes in a given location of the body under ultrasound application on demand. The challenge is to provide particles capable of binding DNA and to deliver it to the nucleus of target cells where it can be expressed. In addition, these particles must be stable in the body, acoustically active and echogenic enough to be imaged by ultrasound. <br />

Microbubbles are prepared by mechanical agitation of original lipids formulations made of cationic lipids, fusogenic colipids and surfactants. New microbubbles are analyzed by optical microscopy, flow cytometry and confocal fluorescence microscopy. The microbubbles acoustic activity is analyzed by the measuring acoustic attenuation and by high speed imaging. The gene transfer efficiency is determined using a reporter gene as read-out and with a custom-build sonoporator.

Optimized formulations have enabled the production of microbubble size and concentration compatible with our application. These microbubbles have a good ability to bind to the plasmid DNA. Complexes made of microbubbles and DNA were activated by ultrasound with a resonance peak of between 1 and 1.5 MHz. The complexes are stable enough over time to achieve efficient gene transfer. High speed imaging experiments showed preferential interactions of particles to some area of cell membranes. Interactions of these particles with cell membranes wereobserved using high speed imaging camera. In vitro gene transfer protocol was established allowing a specific gene delivery after ultrasound activation. The optimal parameters for an efficient gene transfer were determined. In vivo preliminary experiments gave promising results showing a gene transfer in superficial as well as deep organs.

Our project aims at developing gas filled microbubbles that can act as platforms for drug delivery to targeted cells using ultrasound to guide the bubbles and control the delivery process. Ultrasound platforms allow areal-time monitoring of the drug delivery with the added advantage that ultrasound imaging is the cheapest, safest and most available imaging technique. New developments are improving the sensitivity of the ultrasound technology whilst making the devices smaller, meaning that the development of ultrasound-based techniques will continue to be used and improved in the future.The potential value of our bubbles is promising since we earn patents of molecules that used in their production.

1. « Ultrasound and microbbule-assisted gene delivery :recent advances and ongoing challenges, Delalande, Postema, Mignet, Midoux, Pichon. Therapeutic Delivery 2012;3(10):1199-215.
2. Sonoporation: Mechanistics insights and ongoing challenges for gene transfer, Delalande, Kotopoulis, Postema, Midoux, Pichon. Gene 2013; 525(2):191-9.

The main aim of the project is to develop original liposome-based bubbles formulations for safe, non invasive and controllable ultrasound (US) and microbubbles (MB) targeted gene delivery (UMTD). Compared to other physical delivery methods, it allows the possibility to reach deeper organs with a non-invasive manner. Under US activation, MB oscillations near a plasma membrane increase cell permeability attributed to transient pores formation (sonoporation). However, recent data including ours suggest that endocytosis mechanism could also occur. The type of mechanism(s) involved could be both dependent both on the size and on the microbubble chemical composition and on the type of insonified tissue or cells. Our preliminary data from real time experiments showing a better entry of small bubbles under specific acoustic conditions strengthen this hypothesis.
Thus, a key to success of this system lies in understanding of mechanisms governing the interactions of MB and cells. This can be addressed by controlling the size of MB and tailoring the chemical composition of the MB to assess their impact on interactions with cell membranes.
Compared to available MB, these original bubbles will have specific acoustic properties and surface modifications to increase their interactions and/or fusion with cellular membranes and therefore the delivery of their payload.
This project comprises two parts: a fundamental one consisting of smart liposomes bubbles preparation, the study of their acoustic and physicochemical properties, as well as the understanding of all mechanisms governing ultrasound-microbubble-cell interactions; and a second one consisting of their experimental development for in vivo gene delivery.
Lipids that will be used are original and have been developed by the partners. They possess specific activities towards cell membranes, especially in terms of pH sensitivity.
New bubbles will be characterized by their physico-chemical and acoustic parameters. We will also study internalization and intracellular routing of both MB and their cargo. In the literature, no clear knowledge on intracellular routing of MB is known.
For in vivo application, we will investigate the relationship between physicochemical properties of MB and their ability to accumulate in a specific area of the body after systemic administration. Finally, MB will be exploited for gene delivery. Especially, we will focus on their capacity to deliver genes in tendons and liver following local and systemic administration respectively.
Undertaking this project will allow us to fine tune MB chemical composition for an efficient ultrasound and microbubbles targeted delivery. Furthermore, our original MB could serve as platforms to develop targeted MB for both imaging and delivery applications in the future.

Project coordinator

Madame Chantal PICHON (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE POITOU-CHARENTES) – chantal.pichon@cnrs-orleans.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION REGIONALE DE PARIS V
UPCGI CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A
CNRS-CBM CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE POITOU-CHARENTES

Help of the ANR 400,000 euros
Beginning and duration of the scientific project: - 36 Months

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