Liposomal chemotherapy brings the advantage of minimizing systemic toxicity towards healthy organs and tissues, while has the drawbacks of limited nanoparticle accumulation and low drug bioavailability at targeted tumors. The aim of our study is to apply a clinically available mild hyperthermia (HT) treatment with thermosensitive liposomes (TSL) to tackle both issues. A two-step HT approach was combined with systemic administration of doxorubicin (Dox) TSL, in a first step to maximize nanoparticle accumulation in tumors and second step to actively trigger Dox release. The therapeutic activity of the two-step approach was compared to a one-step HT triggering intravascular Dox release from circulating TSL. Whereas the intravascular drug release approach requires fast releasing Dox-TSL (Dox-fTSL), the TSL formulation used in the two-step approach is fine-tuned to prolong Dox retention at physiological temperature in circulation, while releasing their drug content at mild HT at a slower rate (Dox-sTSL). Cytotoxicity assays show that a first-step HT at 41 degrees C for 1 h causes no drug resistance on murine BFS-1 sarcoma, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) towards subsequent exposure to Dox. However, HT sensitizes HUVEC towards Dox at higher concentrations (10-100 mu M). After 2 h of intratumoral Dox-TSL accumulation, HT at 42 degrees C for 1 h was applied to trigger Dox release fromDox-sTSL. Quantification of intratumoral Dox accumulation revealed that the two-step HT approach increased TSL accumulation and Dox bioavailability reaching levels comparable to the intravascular release approach. The two-step HT in combination with Dox-sTSL delayed tumor growth for 12 days compared to PBS group, however, was less effective compared to intravascular Dox release from Dox-fTSL using one-step HT. The two-step approach focuses on interstitial drug release upon mild HT, instead of intravascular drug release. This novel two-step approach represents an attractive alternative for the treatment of large and deep seated tumors, which are difficult to heat precisely and require loco-regional HT of the tumor area and accumulated Dox-sTSL therein to obtain a precise intratumoral drug delivery. (C) 2013 Elsevier B.V. All rights reserved.