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Study of the chicken chorioallantoic membrane (CAM) as an in vivo-model for ultrasound-mediated delivery of drugs using nanoparticle-stabilized- and standard ultrasound microbubbles

Salomonsen, May Lise
Master thesis
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URI
http://hdl.handle.net/11250/2615599
Date
2018
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  • Institutt for fysikk [2164]
Abstract
The chick chorioallantoic membrane (CAM) model has been widely used in the fields of biology and medicine, and it provides a good in vivo model for studying vascularisation properties

of a developing chicken embryo. The aim of this project has been to design and establish

an experimental setup to study the effect of ultrasound in combination with microbubbles

on the delivery of drugs and nanoparticles in cancer treatment of tumours growing on the

CAM and the effect the treatment has on normal tissue. The experiment was conducted by

cracking the eggs open and creating an ex ovo culture after having incubated the eggs for

three days. Cancer cells were inoculated onto the surface of the CAMs at day 6 in their development. At day 13 or 14 fluorescent labelled dextran and microbubbles were injected into

the CAM s blood vessels to image the vessels by an epi-fluorescent widefield microscope. The

microbubbles were injected to increase the effect of the ultrasound treatment. The CAMs

were exposed to ultrasound bursts of either mechanical index 0.4 and 0.8 for a total of five

minutes. The images of the blood vessels were acquired before, during insonification of the

CAMs, and some time after, to visualize the extravasation of fluorescent dyes as a function of time due to ultrasound. One CAM was used as a control and was not treated with ultrasound.

Two different types of microbubbles were used, both Sonazoid microbubbles - a standard

ultrasound contrast agent, and polymeric nanoparticle-stabilized microbubbles developed

by SINTEF. The polymeric nanoparticles were visualized using the fluorescent dye modified

Nile red (NR668) encapsulated inside the nanoparticles. The blood vessels and extravasation

due to ultrasound was visualized using 2 MDa fluorescein isothiocyanate (FITC) dextran. In

an effort to quantify the extravasation, a pilot measurement was done using MATLAB for one

of the CAMs in which extravasation had been observed visually.

The ultrasound treatment with microbubbles was observed to significantly increase the extravasation of fluorescent dye from the blood vessels of most CAMs, both in both normal and cancer tissue, and for both types of microbubbles. The increased leakage induced by the insonification of the microbubbles could increase the amount of drugs reaching the tumour tissue in the area near the blood vessels and thereby improve cancer treatment. The setup shows much promise for further investigation on the effect of ultrasound with microbubbles on cancer treatment in the CAM model.
Publisher
NTNU

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