Preparation, Physicochemical Properties, and In Vitro Toxicity towards Cancer Cells of Novel Types of Arsonoliposomes

Abstract

Arsonoliposomes (ARSL) are liposomes that incorporate arsonolipids (ARS) in their membranes. They have demonstrated significant toxicity towards cancer cells, while being less toxic towards normal cells. In this study, we sought to investigate the possibility to prepare novel types of arsonoliposomes (ARSL) by incorporating a lipidic derivative of curcumin (TREG) in their membrane, and/or by loading the vesicles with doxorubicin (DOX). The final aim of our studies is to develop novel types of ARSL with improved pharmacokinetics/targeting potential and anticancer activity. TREG was incorporated in ARSL and their integrity during incubation in buffer and serum proteins was studied by monitoring calcein latency. After evaluation of TREG-ARSL stability, the potential to load DOX into ARSL and TREG-ARSL, using the active loading protocol, was studied. Loading was performed at two temperatures (40 °C and 60 °C) and different time periods of co-incubation (of empty vesicles with DOX). Calculation of DOX entrapment efficiency (%) was based on initial and final drug/lipid ratios. The cytotoxic activity of DOX-ARSL was tested towards B16F10 cells (mouse melanoma cells), LLC (Lewis Lung carcinoma cells), and HEK-293 (Human embryonic kidney cells). Results show that TREG-ARSL have slightly larger size but similar surface charge with ARSL and that they are both highly stable during storage at 4 °C for 56 d. Interestingly, the inclusion of TREG in ARSL conferred increased stability to the vesicles towards disruptive effects of serum proteins. The active-loading protocol succeeded to encapsulate high amounts of DOX into ARSL as well as TREG-LIP and TREG-ARSL, while the release profile of DOX from the novel liposome types was similar to that demonstrated by DOX-LIP. The cytotoxicity study results are particularly encouraging, since DOX-ARSL were less toxic towards the (normal) HEK cells compared to the two cancer cell-types. Furthermore, DOX-ARSL demonstrated lower toxicities (at all concentrations tested) for HEK cells, compared to that of the corresponding mixtures of free DOX and empty ARSL, while the opposite was true for the cancer cells (in most cases). The current results justify further in vivo exploitation of DOX-ARSL, as well as TREGARSL as anticancer therapeutic systems.

Rapid Production and Purification of Dye-Loaded Liposomes by Electrodialysis-Driven Depletion

“…Imaging was completed with an Olympus IX71 fluorescence microscope (Olympus Scientific Solutions Americas Corp, Waltham, MA, USA) equipped with filter sets specific for the used dyes. The release of encapsulated dyes was monitored from the fluorescence changes elicited by membrane permeabilization with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) [41,42]. A similar procedure was utilized to assess the unilamellarity of EDD-produced liposomes by inducing permeabilization with the pore-forming toxin lysenin (Sigma-Aldrich).…”

Novel TNBC-Targeted DOX-Arsonoliposomes

“…Cholesterol (Chol) was purchased from Sigma-Aldrich (Darmstadt, Germany). The rac-2,3-dipalmitoyl-oxypropylarsonic acid [ARS] (C16), was synthesized as described [1]. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] [DSPE-PEG2000-FA] was synthesized in our laboratory.…”

“…Both liposomes were prepared by the thin-film-hydration method followed by probe sonication for size reduction. [1] The thin film is hydrated with 1 mL of ammonium sulfate solution (120 mM, pH 5.5, 300 mOsm) at 60 o C, and the ARSL suspension was sonicated with probe sonicator (Sonics and Materials, Leicestershire, UK) for approximately 10 min, until the dispersion became completely clear. ARSL were purified from non-encapsulated ammonium sulfate by repeated ultracentrifugations for 1 h (each) at 60,000 rpm (Sorvall WX90 Ultra, Thermo Scientific, Waltham, MA, USA), and resuspended in PBS pH 7.4, for exchange of the dispersion media.…”

“…At a lipid concentration of 1.4 mg/mL (in PBS) they were then incubated with 0.2 mg/mL DOX solution (in PBS) (corresponding to a lipid/DOX ratio equal to 7:1 [w/w]) for 60min at 60 o C, for acive loading of DOX. [1] Afterwards, the ARSL were purified from non-encapsulated drug by two repeated ultracentrifugation steps (60,000 rpm for 1 h, each) and both the supernatants containing nonencapsulated DOX. The lipid content of the samples was routinely determined using a colorimetric technique, which is widely applied for phospholipids, the Stewart assay.…”

“…It has been confirmed through in-vivo and in-vitro studies, that specific toxicity towards cancer is shown, and that ARSL are more toxic towards cancer cells, compared to normal cells. Recently, the loading of DOX into ARSL revealed that interesting synergistc effects may be demonstrated [1].…”