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Liposomes are the most successful nanoparticles used to date to load and deliver chemotherapeutic agents to cancer cells. They are nano-sized vesicles made up of phospholipids, and targeting moieties can be added to their surfaces for the active targeting of specific tumors. Furthermore, Ultrasound can be used to trigger the release of the loaded drugs by disturbing their phospholipid bilayer structure. In this study, we have prepared pegylated liposomes using four types of phospholipids with similar saturated hydrocarbon tails including a phospholipid with no head group attached to the phosphate head (DPPA) and three other phospholipids with different head groups attached to their phosphate heads (DPPC, DPPE and DPPG). The prepared liposomes were conjugated to the monoclonal antibody trastuzumab (TRA) to target the human epidermal growth factor receptor 2 (HER2) overexpressed on HER2-positive cancer cells (HER2+). We have compared the response of the different formulations of liposomes when triggered with low-frequency ultrasound (LFUS) and their cellular uptake by the cancer cells. The results showed that the different formulations had similar size, polydispersity, and stability. TRA-conjugated DPPC liposomes showed the highest sensitivity to LFUS. On the other hand, incubating the cancer cells with TRA-conjugated DPPA liposomes triggered with LFUS showed the highest uptake of the loaded calcein by the HER2+ cells., (© 2023. Springer Nature Limited.)

Targeted liposomes enable the delivery of encapsulated chemotherapeutics to tumours by targeting specific receptors overexpressed on the surfaces of cancer cells; this helps in reducing the systemic side effects associated with the cytotoxic agents. Upon reaching the targeted site, these liposomes can be triggered to release their payloads using internal or external triggers. In this study, we investigate the use of low-frequency ultrasound as an external modality to trigger the release of a model drug (calcein) from non-targeted and targeted pegylated liposomes modified with cyclic arginine-glycine-aspartate (cRGD). Liposomes were exposed to sonication at 20-kHz using three different power densities (6.2, 9, and 10 mW/cm 2 ). Our results showed that increasing the power density increased calcein release from the sonicated liposomes. Moreover, cRGD conjugation to the surface of the liposomes rendered cRGD-liposomes more susceptible to ultrasound compared to the non-targeted liposomes. cRGD conjugation was also found to increase cellular uptake of calcein by human colorectal carcinoma (HCT116) cells which were further enhanced following sonicating the cells with low-frequency ultrasound (LFUS).

Targeted liposomes are designed to target specific receptors overexpressed on the surfaces of cancer cells. This technique ensures site-specific drug delivery to reduce undesirable side effects while enhancing the efficiency of the encapsulated therapeutics. Upon reaching the tumor site, these liposomes can be triggered to release their content in a controlled manner using ultrasound (US). In this study, drug release from pegylated calcein-loaded liposomes modified with transferrin (Tf) and triggered with US was evaluated. Low-frequency ultrasound at 20-kHz using three different power densities (6.2 mW/cm 2 , 9 mW/cm 2 and 10 mW/cm 2 ) was found to increase calcein release. In addition, transferrin-conjugated pegylated liposomes (Tf-PEG liposomes) were found to be more sonosensitive compared to the non-targeted (control) liposomes. Calcein uptake by HeLa cells was found to be significantly higher with the Tf-PEG liposomes compared to the non-targeted control liposomes. This uptake was further enhanced following the exposure to low-frequency ultrasound (at 35 kHz). These findings show that targeted liposomes triggered with US have promising potential as a safe and effective drug delivery platform.

The successful targeting of tumors can be achieved by conjugating targeting moieties to nanoparticles. These modifications allow nannocarriers to achieve greater targeting specificity through binding to specific receptors overexpressed on the surface of the tumor cells. In this study, pegylated liposomes encapsulating the model drug/dye calcein and conjugated to hyaluronic acid (HA) molecules were successfully synthesized, and their ability to target HA receptors overexpressed on a breast cancer cell line was investigated in vitro . Low-frequency ultrasound (LFUS), applied at three different power densities (6.2, 9, and 10 mW/cm²) were used to trigger the release of the entrapped calcein. Both the control and HAconjugated liposomes showed similar release profiles. HA conjugation to the liposomes resulted in a significant increase in calcein uptake by the breast cancer cell line MDA-MB-231 known for its CD44 (HA receptor) overexpression, while such an effect was not recorded with NIH-3T3, an embryonic mouse fibroblast, with low levels of CD44 expression. The application of low LFUS showed a significant enhancement of calcein uptake by MDA-MB-231 cells from our liposome compared to calcein uptake without cell exposure to ultrasound. These findings suggest that combining HA-conjugated liposomes with ultrasound is a promising drug delivery platform in breast cancer treatment.

The use of targeted liposomes encapsulating chemotherapy drugs enhances the specific targeting of cancer cells, thus reducing the side effects of these drugs and providing patient-friendly chemotherapy treatment. Targeted pegylated (stealth) liposomes have the ability to safely deliver their loaded drugs to the cancer cells by targeting specific receptors overly expressed on the surface of these cells. Applying ultrasound as an external stimulus will safely trigger drug release from these liposomes in a controlled manner. In this study, we investigated the release kinetics of the model drug "calcein" from targeted liposomes sonicated with low-frequency ultrasound (20 kHz). Our results showed that pegylated liposomes were more sonosensitive compared to nonpegylated liposomes. A comparison of the effect of three targeting moieties conjugated to the surface of pegylated liposomes, namely human serum albumin (HSA), transferrin (Tf) and arginylglycylaspartic acid (RGD), on calcein release kinetics was conducted. The fluorescent results showed that HSA-PEG and Tf-PEG liposomes were more sonosensitive (showing higher calcein release following the exposure to pulsed LFUS) compared to the control pegylated liposomes, thus adding more acoustic benefits to their targeting efficacy.

Targeted liposomes have high potentials in the specific and effective delivery of their loaded therapeutic agents to the tumour site. Once at the tumour site, it is important that these liposomes are triggered to release their load in a controlled and effective manner. In this study, pegylated (stealth) liposomes conjugated to human serum albumin (HSA) were investigated for the delivery of a model drug (calcein) to breast cancer cells. The fluorescent results showed that calcein uptake by the two breast cancer cell lines (MDA-MB-231 and MCF-7) was significantly higher with the HSA-PEG liposomes compared to the non-targeted control liposomes. Furthermore, the exposure to low-frequency ultrasound (LFUS) resulted in a statistically significant uptake of calcein compared to the uptake without ultrasound. The described drug delivery (DD) system, which involves combining the targeted liposomal formulation with ultrasonic triggering techniques, promises a safe, effective and site-specific breast cancer therapy.