engleski

Combined vectorization of chemotherapeutic drug and nucleic acids by gas microbubbles for a targeted delivery by ultrasound

Ultrasound is widely used in the medical imaging field for diagnostic purposes. Twenty years ago, their use has turned to therapeutic app lications for musculoskeletal disorders mainly. The invention of gas microbubble as ultrasound contrast agent was the trigger of a large number of studies on the delivery of molecules into a cell by coupling ultrasound and gas microbubbles. This technique called sonoporation has the advantage to deliver locally active drugs through the incorporation of molecules to be delivered around or in the membrane of the microbubble. Microbubble gas will therefore serve as a drug reservoir triggered by ultrasound. We developed gas microbubbles capable of delivering both a cytotoxic agent (Paclitaxel) and RNA interference to promote tumour regression. Microbubbles were developed with original cationic lipids capable of binding nucleic acids by electrostatic interactions . On the other hand, these microbubbles may also be loaded paclitaxel. The concentration of loaded paclitaxel significantly changes their size and their acoustic properties. In vitro measurements have been done to depict the behaviour of these microbubbles in the presence of tumour cells after ultrasound activation. It follows that the ultrasounds enable paclitaxel loaded microbubbles to interact specifically with the surrounding cells. Confocal microscopy and flow cytometry experiments demonstrated that ca tionic microbubbles were able to complex siRNA molecules. The ultrasound parameters were optimized in vitro for siLuc delivery in 4T1 cells stably expressing luciferase. Ultrasound at 1MHz, 180kPa, 40% duty cycle, 10kHz pulse repetition frequency during 60 seconds in combination with 10μl of cationic microbubbles complexing 100μM of siLuc led to 53.6% specific inhibition of luciferase expression, 2 days post - sonoporation. In vivo, a proof of principle has been validated using 4T1 orthotopic murine mammary t umoral model. Our siRNA formulations were injected either inside the tumour or via tail vein followed by ultrasound application at the site of the tumour. The ultrasound settin gs were similar to those previously depicted with a stimulation time of 3 to 10 minutes. The luciferase expression was inhibited by 69% 48 hours after sonoporation. When using Paclitaxel loaded microbubbles a weekly treatment repeated three times has led to 51% tumour growth inhibition. In conclusion, this project has demonstrated the ability to exploit original cationic lipids to produce stable gas microbubbles that can be simultaneously loaded with Paclitaxel and bind to RNA interference molecules. The continuation of this work will aim to perform perfusion studies of microbubble ult rasound contrast to fine - tune the delivery protocol in the tumour and to study the delivery of siRNA having a therapeutic interest to enhance the effect of chemotherapy.

ultrasound, cationic microbubble, siRNA, paclitaxel

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