Design and comparative evaluation of nanoformulations for the delivery of a novel colchicine derivative

Colchicine, an alkaloid compound belonging to Microtubule-Targeting Agents (MTAs), has gained interest for its anti-cancer activity. By interacting with tubulin, colchicine can block the formation of the mitotic spindle during mitosis, thereby arresting cell proliferation. The potential of this drug to block the growth of cancer cells is significant. However, colchicine has been associated with gastrointestinal side effects after oral administration, while intravenously, it has been shown to cause necrosis and even death. For this reason, new derivatives have been developed to improve the pharmacological performances of Colchicine. CCI-001 is a Colchicine derivative, which has demonstrated efficacy in vitro against different cancer cells at extremely low doses due to its high affinity to β-III tubulin. Since β-III tubulin is overexpressed in many tumors and shows limited expression in healthy tissues, CCI-001 has the potential to act more selectively on cancer cells, with reduced toxicity to normal cells. Despite these advantages, CCI-001 shows low absorption rate, due to its high hydrophobicity which results in difficult systemic administration. Therefore, new tools to improve the transport and tumor delivery of this drug are urgently needed. Nano-scaled delivery systems, such as nanoparticles (NPs), have the potential to improve cancer targeting and to reduce side effects of traditional chemotherapy, circumventing unspecific transport and off-target accumulation. In this work, we investigated the use of polymer NPs for the delivery of CCI-001. CCI-001 NPs were obtained through a previously optimized nanoprecipitation protocol (nNPs), as well as through a nano-emulsion method (eNPs), which was optimized in this thesis work. A comparative evaluation of eNPs and nNPs was then performed in terms of size distribution, toxicity, drug encapsulation and release, as well as in vitro efficacy against different tumor cells. For both nNPs and eNPs, small size were obtained, ranging from ~170 nm for nNPs to ~240nm for eNPs. This range is deemed acceptable for passive tumor targeting. Additionally, both NPs showed negative zeta potential (ZP) values around -35 mV, compatible with cellular endocytosis. Drug entrapment efficiency (EE%) was 6 % for nNPs and 25% for eNPs. Release tests yielded satisfactory results, highlighting a burst release within the first 24 hours and achieving total drug release after 7 days. In vitro tests were performed on glioblastoma multiforme (U87-MG), pancreatic adenocarcinoma (MIA PaCa-2) and ovarian adenocarcinoma (OVCAR-3) cell lines. Empty nNPs and eNPs did not elicit signs of toxicity, up to a concentration of 1 mg/ml, while CCI-001-loaded-NPs exerted anticancer activity resulting in 20%-28% residual viability after 72 h treatments with nNPs. eNPs exhibited similar results, with 56%, 43% and 33% residual viabilities for U-87, Mia-PaCa-2, and OVCAR-3, respectively (at the same equivalent drug concentration). Overall, CCI-001-NPs, with satisfactory loading efficacy and good toxicity profiles were obtained. NPs displayed high cells internalization and were able to control the release of CCI-001, without altering its anti-cancer effect, warranting their further investigation.