Liposomes—self-assembled spherical nanoparticles possessing an aqueous core surrounded by phospholipid bilayers—have gained considerable attention as delivery vehicles for a variety of therapeutics. Researchers have made numerous efforts to develop liposome-based pharmaceutical products that offer the multiple benefits of liposome encapsulation [1,2]. However, the first generation of liposomal drugs carrying doxorubicin (DOX) failed to achieve translation into clinical development, mainly owing to premature drug release and extensive uptake by the reticuloendothelial system (RES) after intravenous administration [3]. Subsequent efforts of researchers to overcome these limitations have led to the development of liposome-based products that have received approval for clinical use. These include surface coating of liposomes with the hydrophilic polymer, polyethylene glycol (PEG), which prolongs systemic circulation and averts RES uptake of liposomes, and a remote drug-loading method, which enables highly efficient, stable encapsulation of hydrophilic small chemical drugs. In addition, the use of rigid phosphatidylcholine (PC) with a high phase transition temperature (>50 °C) and cholesterol as components of liposome membranes has contributed to the generation of stable liposomes with a robust membrane [3,4].

Caelyx (Doxil), a liposomal preparation of DOX, provides considerable benefits to cancer patients, including prolonged drug circulation, reduced cardiotoxic side effects, and increased drug accumulation in the tumor [5]. Nonetheless, its antitumor efficacy is comparable to that of free DOX solution, a disappointing finding given the tumor-delivery efficacy it has shown in preclinical studies [6,7]. This limited efficacy could be attributable to overly stable retention of DOX in PEGylated liposomes composed of robust bilayers (rigid PC and cholesterol). Since the cellular uptake of intact liposomes is limited at the tumor site [3,8], strong DOX entrapment in liposomes would hinder cellular uptake and subsequent nuclear transport of DOX. Hence, despite the preferential accumulation of liposomal DOX at the tumor site, if DOX is not released from liposomes, its bioavailability within the tumor interstitium is limited [5].

Designing liposomes such that the release of entrapped drugs is triggered by external stimuli, such as heat or ultrasound, has been an attractive strategy for achieving drug release at the right time and place [[9], [10], [11]]. In this approach, a component capable of responding to a given stimulus (e.g., thermoresponsive lipids) is incorporated in liposomes. However, these specialized liposomes are prone to exhibit decreased storage stability and/or shortened systemic circulation time compared with conventional liposomes and require complicated treatment processes utilizing medical devices (e.g., heat generator). One example is ThermoDox, the most clinically advanced thermosensitive liposomal formulation, which is provided in frozen solid form [12,13] and is cleared more quickly from the circulation compared with thermo-insensitive liposomes [14].

A sequential administration strategy, in which surfactant-based vehicles are delivered after liposomal drug administration, is another strategy that has been attempted to trigger the release of drugs from liposomes at the tumor site without affecting the sustained circulation of drug-loaded liposomes [15,16]. In this strategy, a second injection of surfactant-based vehicles (micelles composed of Pluronic P85 or niosomes composed of Span 80) made 24–48 h after liposomal drug (Doxil) administration (a delay chosen based on the time required for liposomes to accumulate at the tumor site) increased localized DOX release, thereby enhancing the therapeutic efficacy of liposomal DOX. This enhanced DOX release appears to reflect increased membrane permeabilization and/or membrane fusion between two nano-vehicles, mediated mainly by surfactants incorporated into liposomal membranes. Moreover, tumor accumulation of the surfactant vehicles themselves is achieved through the enhanced permeability and retention (EPR) effect, as is the case for Caelyx, contributing to higher vesicle-vesicle interactions in tumor tissue [16]. Nevertheless, a two-dose regimen in which doses are separated by >1 day is a complicated schedule that requires prolonged patient hospitalization. In addition, because a significant dose percent of liposomal DOX is retained in the systemic circulation even 24–48 h after injection [14,17], there is still a risk of drug release prior to reaching the tumor site. On the other hand, because of the rapid dilution of both types of vesicles immediately after injection, as well as possible interactions with biological components, there may be insufficient membrane interaction between two vehicles in vivo to lead to efficient drug release, even at the tumor site. These speculations prompted us to explore an alternative and simple strategy for improving the pharmacological availability of liposomal anticancer drugs at the tumor site.

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with 22 carbons and six double bonds, is the most unsaturated fatty acid commonly found in biological systems. The multiple double bonds in DHA allow its acyl chains to twist at various angles; as a result, DHA is readily incorporated into the phospholipids that form cell membranes [18,19]. Notably, there is substantial evidence indicating that DHA and DHA-bound phospholipids impact phospholipid membrane properties, including membrane packing, fluidity, permeability, and fusion [20]. On the basis of these properties of DHA, together with its excellent biodegradability/biocompatibility, we here sought to explore the potential of liposomes based on DHA-containing phospholipids as a release-booster for anticancer-drug–loaded liposomes composed of rigid membranes. Since the incorporation of DHA in liposomes composed of rigid PC decreased the storage stability of liposomes by inducing time-dependent aggregation of liposomes in our earlier work [21], an approach in which liposomes composed of DHA-containing phospholipids are separately prepared and premixed with drug-loaded liposomes prior to administration was investigated as a strategy for improving the pharmacological availability of liposomal anticancer drugs at the tumor site. Our work herein demonstrates that premixing of DHA-containing-PC-based liposomes (Omega-L) with Caelyx induces in vitro release of drug from anticancer drug-loaded liposomes in a mixing ratio- and Omega-L composition-dependent manner. As proof-of-concept, we performed an in vivo study using a Caelyx and Omega-L mixture (Caelyx:Omega-L), which demonstrated that pre-mixing enhanced the therapeutic efficacy of DOX-loaded liposomes (Caelyx) while having a modest impact on the pharmacokinetic parameters of DOX.