Ultrasound-triggered herceptin liposomes for breast cancer therapy

Abstract

The functionalization of liposomes with monoclonal antibodies is a potential strategy to increase the specificity of liposomes and reduce the side-effects associated with chemotherapeutic agents. The active targeting of the Human Epidermal growth factor Receptor 2 (HER2), which is overexpressed in HER2 positive breast cancer cells, can be achieved by coating liposomes with an anti-HER2 monoclonal antibody. In this study, we synthesized calcein and Doxorubicin-loaded immunoliposomes functionalized with the monoclonal antibody Trastuzumab (TRA). Both liposomes were characterized for their size, phospholipid content and antibody conjugation. Exposing the liposomes to low-frequency ultrasound (LFUS) triggered drug release which increased with the increase in power density. Trastuzumab conjugation resulted in enhancing the sensitivity of the liposomes to LFUS. Compared to the control liposomes, TRA-liposomes showed higher cellular toxicity and higher drug uptake by the HER2 + cell line (SKBR3) which was further improved following sonication with LFUS. Combining immunoliposomes with LFUS is a promising technique in the field of targeted drug delivery that can enhance efficiency and reduce the cytotoxicity of antineoplastic drugs.

Figure 1

Protein concentrations (per mg of lipids) for the control and TRA-liposomes in both calcein and DOX-loaded liposome.

Figure 2

Normalized release profiles of the control and TRA-liposomes loaded with calcein at 6.2 mW/cm², 9 mW/cm² and 10 mW/cm². Results are the average of three batches of liposomes with three replicates each.

Figure 3

Flow cytometry analysis of calcein uptake by SKBR3 and MDA-MB-231 cells following their incubation with control liposomes or TRA-liposomes followed by ultrasound (US) sonication (35-kHz) for 5 min at a power density of 20 mW/cm². Untreated cells served as a negative control for background fluorescence. An average of 1 × 10⁴ cells was analyzed from each sample with calcein fluorescence intensity showed on a four-decade log scale.

Figure 4

Flow cytometry analysis showing calcein fluorescence intensity inside SKBR3 cells incubated with the either the control or TRA-liposomes with or without prior incubation with free Trastuzumab (1 mg/ml). An average of 1 × 10⁴ cells was analyzed from each sample with calcein fluorescence intensity showing on a four-decade log scale.

Figure 5

Fluorescence microscopy images of SKBR3 cells following 4 h incubation with control and TRA-liposomes encapsulating calcein in addition to TRA-liposomes exposed to LFUS (35-kHz) for 5 min.

Figure 6

MTT results for (A) SKBR3 cells and (B) MDA-MB-231 cells treated with free DOX, Control-DOX liposomes and TRA-DOX liposomes (DOX concentration: 8 μM) in the absence and presence of LFUS exposure. Sonicated cells were exposed to continuous LFUS in a 35-kHz water bath for 5 min. Data are representative of three independent experiments (mean ± SD %, n = 3).