Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia
Graphical abstract
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 Tm, 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 Tm [26]. Therefore, all TSL formulations in this study were extruded above their Tm 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.
Section snippets
Chemicals
The phospholipids 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) were provided by Lipoid (Ludwigshafen, Germany), and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) was purchased from Avanti Polar Lipid Inc. Carboxyfluorescein (CF) purified by recrystallization [28]. Other chemicals were obtained from Sigma Aldrich (Netherlands) unless otherwise
Characterization of the TSL
In vitro characteristics of the TSL are summed up in Table 1. TSL encapsulation ratio of CF/lipid (mole/mole) of the different formulations was comparable except for TSL with 10 mol% DSPE-PEG2000, which was significantly lower than any other formulation (p value ≤ 0.05). TSL with 1–5 mol% DSPE-PEG2000 in 90% serum retained over 96% CF and TSL with 6 mol% DSPE-PEG2000 retained 91% CF, while TSL with 10 mol% DSPE-PEG2000 were the least stable to retain 83% CF after 1 h incubation at 37 °C. Size of the
Discussion
Stealth TSL with 5 mol% DSPE-PEG2000 were optimal, considering stability at 4, 25, 37 °C and release kinetics at mild HT. Stealth TSL with 1–5 mol% DSPE-PEG2000 were stable (< 4% CF leakage) at 37 °C in 90% serum, while TSL with 6 and 10 mol% DSPE-PEG2000 leaked out 9 and 17% CF, after 1 h incubation. High DSPE-PEG2000 density, especially 10 mol%, collapsed the membrane integrity, causing content leakage at relatively low temperatures. Based on in vitro stability study of stealth TSL (Table 1), 1–5 mol%
Acknowledgements
The authors thank Dr. Kristina Djanashvili, Dr. Daniel Schuhle, Dr. Aurelie M. A. Brizard at ChemTech, Delft University of Technology, Delft, for DSC measurements; Dr. Martin Hossann at Medical Clinic and Pre-clinic III, Ludwig-Maximilians University, Munich, and Dr. Gert van Cappellen at Erasmus MC, Rotterdam, for technical support; Stichting Vanderes, Stichting Fondsen, SEHK, Dr. Mildred Scheel Stiftung and Erasmus MC grants (Mrace) for financial support.
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