Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia

Abstract

Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG₂₀₀₀ (DSPE-PEG₂₀₀₀) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG₂₀₀₀ from 1 to 10 mol%, around 80 nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG₂₀₀₀ density. However, 6 mol% and higher DSPE-PEG₂₀₀₀ caused CF leakage at physiological temperature. TSL with 5 mol% DSPE-PEG₂₀₀₀ were stable at 37 °C, while released 60% CF in 1 min and almost 100% CF in 1 h at 42 °C. In vivo optical intravital imaging showed immediate massive CF release above 41 °C. In conclusion, incorporation of 5 mol% DSPE-PEG₂₀₀₀ optimized stealth TSL content release triggered by HT.

Introduction

The major obstacle of current available chemotherapy is the inability to deliver adequate concentration of drugs to tumors, but causes considerable systemic toxicity which limits their applicable dose. Most chemotherapeutic drugs are delivered intravenously and rapidly cleared from circulation, e.g. doxorubicin (Dox) has a five to ten minutes half-life in plasma [1]. Therefore, only a fraction of the administered dose can reach tumors. Long-circulating drug-carrying nanoparticles, e.g. pegylated liposomal Dox (Doxil or Caelyx), reduce toxicity and augment intratumoral delivery compared to free drugs [2], [3], [4], [5]. Despite the prolonged circulation time and increased tumor accumulation [2], [6], slow and passive drug release from these liposomes hinders an optimal antitumor effect. Thus, it is critical to actively trigger liposomal drug release. Therefore, thermosensitive liposomes (TSL) [7] and local hyperthermia (HT) are used. This approach relies on features of tumor microvasculature and TSL. Relative leakiness of partial tumor vessels can cause extravasation of small liposomes (i.e. < 400 nm) [8], [9], [10], [11], [12]. Permeability of tumor microvasculature can be further enhanced by heat [13], causing more liposomes to accumulate intratumorally [14].

Intratumoral liposome accumulation does not guarantee drug bioavailability. The entrapped drugs need to be released from the liposomes to reach tumor cells for a therapeutic effect. TSL are phospholipid-based vesicles with a large capacity to encapsulate drugs and to release them upon heat. This release depends on temperature and lipid composition of TSL [15]. When a TSL lipid membrane undergoes a gel-to-liquid crystalline phase transition, it becomes more permeable towards water and solutes, because trans to gauche conformational changes increase the mobility of phospholipids [16]. In this way, the entrapped hydrophilic drugs can be released.

For phospholipids with saturated hydrocarbon chains, lengths of fatty acid chains primarily determine the T_m, the main gel-to-liquid crystalline phase transition temperature of lipid membranes. For stealth non-thermosensitive liposomes [17], [18], the concentration of grafted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG₂₀₀₀ (DSPE-PEG₂₀₀₀) in lipid membrane influences membrane permeability [19]. Surface-grafted polymers can disturb membrane integrity by inducing steric repulsion between opposing polymers, e.g. DSPE-PEG₂₀₀₀ and DSPE-PEG₅₀₀₀ [20]. Molecular structures of PEG-polymers can be distinguished as interdigitated mushroom, mushroom and brush regimes [21]. Polymer regimes are determined by distance between grafting sites, and by polymer sizes [21]. Kenworthy et al. suggested that 4 mol% DPPE-PEG₂₀₀₀ induced maximum membrane permeability [21], [22].

For stealth non-thermosensitive liposomes in general, 1–5 mol% DSPE-PEG₂₀₀₀ is commonly grafted on lipid membranes for prolonged circulation time, and 5 mol% DSPE-PEG₂₀₀₀ is the sufficient and optimal concentration [17]. In addition, DSPE-PEG₂₀₀₀ also prevents liposome aggregation in plasma [23]. Formulations containing up to 10 mol% DSPE-PEG₂₀₀₀ have been described [20], [22], [24], though PEG-lipids tend to form micellar structures at higher concentrations [25], and disk-like micelle formation can be enhanced by extrusion repeatedly crossing T_m [26]. Therefore, all TSL formulations in this study were extruded above their T_m to avoid PEG-micelle formation.

Stealth TSL typically adopt conventional DSPE-PEG₂₀₀₀ concentrations between 1 and 5 mol% in stealth non-thermosensitive liposomes to prolong half-lives in plasma for drug delivery [17], [18], [27]. However, the ideal DSPE-PEG₂₀₀₀ concentration for TSL content release has not yet been determined. This study provides a valuable guide on the use of DSPE-PEG₂₀₀₀ in TSL by identifying the optimum DSPE-PEG₂₀₀₀ concentration with regard to intrinsic stability, in vitro and in vivo content release properties, to improve content release of stealth TSL triggered by mild HT.