Temperature-sensitive vesicles designed by inclusion of leucine zipper peptides within a lipid bilayer (Lp-Peptide hybrids) encapsulating Doxorubicin have been reported. Intravenous administration of these constructs prolonged blood circulation kinetics and increased tumour accumulation in vivo with local mild hyperthermia. In this study, the biological activity of the Doxorubicin-loaded Lp-Peptide hybrid vesicles was further investigated at the cellular level and in vivo compared to lysolipid-containing temperature-sensitive liposomes (LTSL) and traditional temperature-sensitive liposomes (TTSL). Lp-Peptide vesicles were not toxic to cell cultures at 37 ºC, while effective cancer cell toxicity was observed after 1 h of heating at 42 ºC. The activity of Lp-Peptide vesicles in vivo was studied using two different heating protocols to obtain tumour intravascular or interstitial drug release. Lp-Peptide vesicle treatment allowing intravascular Doxorubicin release showed equally effective tumour growth retardation and survival to that of LTSL treatment. The Lp-Peptide vesicles also offered therapeutic responses using the alternative heating protocol to maximise drug release within the tumour interstitium. Matching the drug release kinetics of temperature-sensitive vesicles with the heating protocol applied is considered the most critical factor to determine therapeutic efficacy in the clinical translation of such modalities.