In order to obtain nanocarriers suitable for the delivery of drugs in the treatment of cancer, pH-responsive nanovesicles capable of facilitating fusion (fusogenic nanovesicles) were synthesized and then their physicochemical characteristics were modified. These nanovesicles were made by combining polysorbates having different physicochemical features with the aim of realizing multiple-targeting nanoformulations suitable for in vitro treatment of cancer cells. Tween21 and Tween80 were self-assembled at different molar concentrations resulting in pH-responsive fusogenic nanovesicles with an average size of less than 150 nm, and a narrow size distribution (polydispersity index) value of less than 0.2. Hydrophobic and hydrophilic fluorescent probes were loaded inside the nanovesicles in order to study their pH-responsiveness and fusogenic properties and it was noted that this process did not modify their physicochemical features. The pH-responsiveness and fusogenic assay demonstrated that the nanovesicles containing Tween21 at different molar ratios were pH-responsive and interacted with a synthetic model of a biological membrane supplemented with Ca2+ in the incubation medium. Fifty percent (molar ratio) of Tween21 was replaced with Tween80, since Tween80 can promote the adsorption of apolipoproteins (A–E) onto the surfaces of nanovesicles without altering their pH-responsiveness or fusogenic properties. In fact this equivalent molar concentration of Tween21 and Tween80 also maintained their degree of interaction with the apolipoproteins (A–E). Doxorubicin hydrochloride-loaded nanovesicles were synthesized and physicochemically characterized in order to obtain nanoformulations suitable for anticancer treatment. The therapeutic nanovesicles showed physicochemical properties similar to those of empty nanoformulations, and maintained pH-responsiveness, fusogenic properties and targeting versus the apolipoproteins (A–E). The doxorubicin hydrochloride was loaded into the nanovesicles using both passive and pH gradient remote loading procedures. The latter provided the nanovesicles with an entrapment efficiency percentage of over 30%, which was much higher than the 10% that was obtained using the passive loading procedure. The entrapment efficiency improved up to 60% for the nanovesicles made from the same molar concentration of Tween21 and Tween80. The anticancer activity of doxorubicin hydrochloride-loaded nanovesicles was further tested in vitro using human neuroblastoma (SH-SY5Y) cells which respond to treatment with this chemotherapeutic drug, but the nanovesicles carrying it must cross the BBB by means of specific receptors before the drug can provide a therapeutic effect in vivo. The anticancer activity of these doxorubicin hydrochloride-loaded nanovesicles was time- and dosage- dependent, and the surfactant components making up the nanoformulations was also a determining factor in the efficiency of their activity. These nanovesicles could provide innovative nanotherapeutics for potential in vitro multidrug targeting therapy.