Doxorubicin release and uptake from Trastuzumab and Folic acid liposomes with ultrasound

A Doctor of Philosophy Dissertation in Materials Science and Engineering by Nour Majdi AlSawaftah entitled, “Doxorubicin release and uptake from Trastuzumab and Folic acid liposomes with ultrasound”, submitted in December 2024. Dissertation advisor is Dr. Ghaleb Husseini and dissertation co-advisor is Dr. William Pitt. Soft copy is available (Dissertation, Completion Certificate, Approval Signatures, and AUS Archives Consent Form).

Abstract

Smart drug delivery in cancer therapy, using surface-functionalized liposomes and triggers like ultrasound (US), helps minimize the side effects and enhance drug accumulation at tumor sites. This research investigated the effects of physically mixing Doxorubicin (DOX)-loaded liposomes decorated with folic acid (FA) and decorated with Trastuzumab (TRA) to treat breast cancer. It examined the US-mediated release of DOX from control, FA-modified, TRA-modified, and mixed FA and TRA liposomes at three different volume fractions. The size of the synthesized liposomes was found to be well within the range for the enhanced permeability and retention (EPR) effect to take place (<200 nm in diameter). The liposomes had a uniform lipid content and were stable under physiological conditions and at 4 °C. Moreover, the TRA conjugation was confirmed using the bicinchoninic acid (BCA) assay while the FA conjugation was established using nuclear magnetic resonance (HNMR). Then, DOX release using low-frequency ultrasound (LFUS) at 20 kHz and three power densities (6.2, 9, and 10 mW/cm²) and high-frequency ultrasound (HFUS) at 1 MHz and a power density of 2.5 W/cm² was examined. Mixed liposomes showed the best release performance in both LFUS- and HFUS-triggered conditions, with M75 liposomes showing the highest release using both LFUS and HFUS sonication. The LFUS and HFUS release data were fitted to three different kinetic models (zero-, first-, and second-order). The LFUS data was best fit by the zero-order model while the HFUS data was best fitted by the first-order model. In vitro uptake was studied using flow cytometry in FRα+ and HER2+ HCC-1954 breast cancer cells. The cells treated with the M25 group showed the highest cellular uptake with an 8.5-times increase compared to the control liposomes group when sonicated with LFUS. Similarly, the M25 group exposed to HFUS with microbubbles (MBs) enhanced DOX uptake by 10-times compared to the control liposomes. Finally, the cell viability of the developed formulations was assessed using the MTT assay. Physically mixed liposomes reduced the viability of sonicated cells more than individually targeted liposomes, especially the M25 group. These findings highlighted the potential of combining physically mixed liposomes and US to treat breast cancer.