(316a) Ultrasound Enhanced Release of Targeted Liposomes in Cancer Treatment

Introduction: Site-specific drug delivery is an attractive approach in cancer treatment because it can reduce the undesirable side-effects of anticancer therapeutics and increase the accumulation of drugs at tumor sites. To further improve the selectivity of the drug carrier, the surface of these nanoparticles, such as liposomes, are modified with targeting moieties specific to the receptors on the surface of tumor cells. In addition, external triggers such as ultrasound can be used to enhance the release of liposomal contents. Our research focuses on the synthesis of targeted-poly-ethylene glycol (PEG)-liposomes encapsulating the model drug calcein and studying the effects of low- frequency ultrasound (LFUS), applied at different power densities, on calcein release. Three targeting moieties were used in this research, namely, human serum albumin (HSA), arginyl glycyl aspartic acid (RGD), and estrone (ES).

Methods: Unconjugated liposomes were prepared using the thin-film hydration method. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG(2000)-NH₂), and cholesterol were dissolved in chloroform at a molar ratio of 65:5:30. The thin film was formed by the slow evaporation of the organic solvent under vacuum using a rotary evaporator. The dried lipid film was then hydrated with 2 mL of 50 mM calcein disodium salt solution (pH~7.4). The liposomes were then sonicated and extruded to obtain small, unilamellar liposomes. Any external, unencapsulated calcein was removed using size exclusion chromatography. ES was attached to the lipids prior to the formation of liposomes, while RGD and HSA were conjugated to the liposomes post-formation. ES-modified liposomes were prepared by conjugating ES to DSPE-PEG2000-NH₂. Cyanuric chloride (2,4,6 trichloro-1,3,5 triazine (CC)) was used as a linking molecule to attach ES to DSPE-PEG2000-NH₂. ES was reacted with CC in a 1:1 molar ratio, in the presence of two molar equivalents of triethyl-amine (TEA), a chloride acceptor. The ES-modified lipids were then used to formulate liposomes using the procedure detailed above. RGD and HAS, on the other hand, were conjugated to liposomes via the PEG terminus modified with CC. The liposomes were characterized for size by dynamic light scattering (DLS), estimation of total phospholipid content by the Stewart method, the quantitation of the protein using the bicinchoninic acid (BCA) assay, and finally the release of the model drug by measuring fluorescence using a phosphorescence/fluorescence spectrofluorometer. Low-frequency ultrasound (LFUS) induced release was triggered via 20-kHz pulsed US (20s on 10s off) at three different power densities (6, 7, and 12 W/cm²). Finally, the surfactant Triton X-100 was added to the sample to lyse the liposomes (and obtain the maximum release). The recorded fluorescent intensities were used to calculate the percentage of the released calcein using the following equation:

Results: The sizes of 3 batches of control and conjugated liposomes were measured using DLS. For HSA and RGD liposomes, the radius of the nontargeted pegylated liposomes (control) was 83.77 ± 0.91 nm. The average radius of the HSA-PEG liposomes was 84.86 ± 1.81 nm, while the RGD-PEG liposomes showed an average radius of 84.42 ± 1.50 nm. The radius of ES-conjugated, calcein liposomes was found to be 92.9 ± 15.8 nm, while a radius of 96.8 ± 3.8 nm was obtained for their corresponding control liposomes. This confirmed that both types of synthesized liposomes fell within the size category of small unilamellar vesicles (SUV) expected to be efficient for drug delivery as they can make use of the enhanced permeability and retention (EPR) effect. The Stewart assay was used to confirm that both the control and targeted liposomes have similar lipid concentrations before measuring their protein content. The results of the BCA assay showed that HSA and RGD liposomes had a higher protein content compared to control liposomes with no moieties conjugated. HSA-PEG liposomes showed a 3-fold increase in protein content (1.05 ± 0.43 μg/mL) compared to the control liposomes (0.35 ± 0.006 μg/mL); whereas the protein content of RGD-PEG liposomes (2.1 ± 0.114 μg/mL) showed a 5-fold increase compared to the control liposomes (0.41 ± 0.008 μg/mL). As for the online low-frequency US (LFUS) induced release, the normalized-averaged release profiles of control and conjugated liposomes are shown in Figure 1. As can be seen, the curve started with a baseline that measured the fluorescence level in the sample; upon sonication, the level increased due to calcein release. The 20s on 10s off-cycle mentioned earlier was continued until a plateau was reached, and no more calcein release was observed. Moreover, upon the addition of Triton X-100, the fluorescence level increased slightly above the plateau, indicating that the liposomes have released most of their encapsulated calcein contents. Following the first three pulses, calcein release from HSA-PEG liposomes and ES-PEG liposomes was significantly higher than that of the control liposomes at the three power densities investigated. However, for RGD-PEG liposomes, the calcein release following the sonication at the highest investigated power density (i.e., 12 W/cm²) showed no significant difference compared to the control liposomes after the first three pulses. This, however, was not the case following sonication with the lower power intensities. When RGD-PEG liposomes were sonicated at 7 W/cm², they showed more calcein release compared to the control following the first and the third pulses, but no significant change in calcein release was recorded following the second pulse. Sonication at the lowest power density (6 W/cm²) showed that RGD-PEG liposomes were more susceptible to US compared to the control liposomes releasing significantly more of the encapsulated calcein, following the first three pulses.

Conclusions: In this study, control and modified liposomes were tested for size, lipid content, conjugation of proteins, and calcein release triggered by LFUS. The DLS findings showed that the modification of pegylated liposomes with HSA, ES, and RGD had no significant effect on the size of the liposomes. Furthermore, HSA-PEG and ES-PEG liposomes were more sonosensitive compared to the control liposomes at the three power densities, showing significantly higher calcein release following the exposure to pulsed LFUS. Using this data, we inch closer to developing a drug delivery system that uses ultrasound as a modality to reduce the side effects of conventional chemotherapy, hence improving the quality of life of patients around the world.