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

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.