Delivery of Encapsulated Drugs to Cancer Cells and Tissue: The Impact of Ultrasound
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- Institutt for fysikk 
Encapsulated drugs have improved tumor to normal tissue uptake compared to free drugs, however, the concentration of drugs at the tumor site is still low and heterogeneous due to the tumor microenvironment which serves as barriers for the delivery to the target site. Combining ultrasound (US) with encapsulated drugs might enhance the transport of the encapsulated drug across the vasculature and into tumor tissues. US can also increase local drug release and the uptake of the drug into cancer cells. In this thesis, we combined US with encapsulated drug delivery to improve cancer therapy. We studied the effect of US exposure parameters that maximizes the release of dierucoylphosphatidylcholine (DEPC)-based liposomes in vitro using low (300 kHz) and medium (1 MHz) frequency US. The mechanism of US-enhanced cellular uptake of nanoparticles (NPs) (DEPC-based liposomes and polymeric NPs) and dextrans was also investigated using low frequency US and microbubbles (MBs, commercial and a novel drug delivery system – airfilled MBs stabilized by polymeric NPs). An in vivo study was conducted to investigate the effect of 300 kHz and 1 MHz US on distribution of liposomal doxorubicin and released drug in tumor tissues using tumor bearing nude mice. Lastly, the effect of NPs PEGylation, surfactant and size on cellular uptake and cell viability was studied. In vitro drug release was measured with a spectrophotometer whereby flow cytometry was used to measure cellular uptake of released drug and NPs. Confocal laser scanning microscopy was used to image the distribution and internalization of NPs and released drug. Drug release was demonstrated in vitro and in vivo with both frequencies. In vitro drug release was shown to be caused by inertial cavitation, whereas in vivo drug release was suspected to be cavitation, although it is still unclear. Mechanical index and exposure time were found to determine the total drug release from DEPC-based liposomes in vitro. The data also suggests that the duty cycle may be used to control the amount of energy deposited and heat generated in tissue during US-mediated drug delivery. The data from the in vivo studies showed increased levels of released doxorubicin in US (for both frequencies) exposed tumors compared to control tumors. Also, we observed higher penetration of liposomes and released doxorubicin from blood vessels in tumors exposed to 1 MHz US as compared to 300 kHz US exposure. This might be attributed to the acoustic radiation force generated during US exposure. In vitro data shows the dependency of MBs to obtain efficient intracellular uptake of NPs and dextrans, suggesting the mechanism of the improved cellular uptake to be sonoporation and enhanced endocytosis. Although, the percentage of cells internalizing dextran was size-independent (up to 2 MDa), the 4-kDa dextran was internalized in higher quantities than the larger dextrans. Low frequency US did not enhance the cellular uptake of polymeric NPs; neither in the presence nor absence of MBs stabilized by polymeric NPs. Cellular uptake of polymeric NPs was largely dependent on the surface properties (PEGylation and surfactant) of the particles. Thus, type and length of PEG molecules as well as the type of surfactant used for emulsification of the particles had effect on the cellular uptake of the particles. Polymeric NPs exhibited dose-response toxicity on PC3 cell line and the toxicity was dependent on the type of surfactant. Altogether, the results show that US can increase the local drug concentration, enhance the penetration depth of drugs for the drug to reach more cancer cells and increase the permeability of the cells for more drugs to enter the cell thereby improving cancer therapy.