Cancer-specific drug delivery represents an attractive approach to prevent undesirable side-effects and increase the accumulation of the drug in the tumor. Surface modification of nanoparticles such as liposomes with targeting moieties specific to the up-regulated receptors on the surface of tumor cells thus represents an effective strategy. Furthermore, since this receptor expression can be heterogeneous, using a dual-combination of targeting moieties may prove advantageous. With this in mind, the anti-cancer activity of PEGylated doxorubicin-loaded liposomes targeted with folic acid (F), transferrin (Tf) or both (F+Tf) was evaluated. The dual-targeted liposomes showed a 7-fold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cell monolayers. The increased penetration and cell association of the dual-targeted liposomes was also demonstrated using HeLa cell spheroids. The in vitro cytotoxicity of the doxorubicin liposomes (LD) was then evaluated using HeLa and A2780-ADR ovarian carcinoma cell monolayers. In both these cell lines, the (F+Tf) LD showed significantly higher cytotoxic effects than the untargeted, or single-ligand targeted liposomes. Ina HeLa xenograft model in nude mice, compared to the untreated group, though the untargeted LD showed 42% tumor growth inhibition, both the (F) LD as well as (F+Tf) LD showed 75% and 79% tumor growth inhibition respectively. These results thus highlight that though the dual-targeted liposomes represent an effective cytotoxic formulation in the in vitro setting, they were equally effective as the folic acid-targeted liposomes in reducing tumor burden in the more complex in vivo setting in this particular model.

The treatment of glioblastoma (GBM) is a challenge for the biomedical research since cures remain elusive. Its current therapy, consisted on surgery, radiotherapy, and concomitant chemotherapy with temozolomide (TMZ), is often uneffective. Here, we proposed the use of zoledronic acid (ZOL) as a potential agent for the treatment of GBM. Our group previously developed self-assembling nanoparticles, also named PLCaPZ NPs, to use ZOL in the treatment of prostate cancer. Here, we updated the previously developed nanoparticles (NPs) by designing transferrin (Tf)-targeted self-assembling NPs, also named Tf-PLCaPZ NPs, to use ZOL in the treatment of brain tumors, e.g., GBM. The efficacy of Tf-PLCaPZ NPs was evaluated in different GBM cell lines and in an animal model of GBM, in comparison with PLCaPZ NPs and free ZOL. Tf-PLCaPZ NPs were characterized by a narrow size distribution and a high incorporation efficiency of ZOL. Moreover, the presence of Tf significantly reduced the hemolytic activity of the formulation. In vitro, in LN229 cells, a significant uptake and cell growth inhibition after treatment with Tf-PLCaPZ NPs was achieved. Moreover, the sequential therapy of TMZ and Tf-PLCaPZ NPs lead to a superior therapeutic activity compared to their single administration. The results obtained in mice xenografted with U373MG, revealed a significant anticancer activity of Tf-PLCaPZ NPs, while the tumors remained unaffected with free TMZ. These promising results introduce a novel type of easy-to-obtain NPs for the delivery of ZOL in the treatment of GBM tumors.

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