A side by side comparison of PLGA and SLN particles for the delivery of diethyldithiocarbamate-copper complex in the treatment of breast cancer.

Diethyldithiocarbamate-copper complex ((DDC)2-Cu) is a new and promising drug that, thanks to several mechanisms of action, can efficiently inhibit the growth of both cancer and cancer stem cells. Importantly, (DDC)2-Cu was found to be active against triple-negative breast cancer (TNBC) cells. The main limitation to (DDC)2-Cu application is its insolubility in water. In this thesis project, we carried out a side-by-side comparison of two delivery systems, based either on polymeric or lipid nanoparticles, suitable for the systemic administration of (DDC)2-Cu with the aim to increase its water solubility and, in turn, its therapeutic effect. Polymeric nanoparticles (NPs) were made of poly (lactic-co-glycolic acid) (PLGA, MW of 24-38 kDa) partially modified by conjugation with polyethylene glycol (MeO-PEG2kDa-NH2), to endow the system with stealth properties. PLGA-PEG2kDa conjugation was performed through a coupling procedure, and the conjugation yield was assessed by 1H NMR after purification. NPs were formulated by nanoprecipitation technique, using DMSO as organic phase containing 50 mg/mL PLGA-PEG2kDa:PLGA 1:1 weight ratio and 0.5 mg/mL of (DDC)2-Cu, and as aqueous phase a 20 mg/mL polyvinyl alcohol solution in milliQ water. The organic phase was added dropwise to the aqueous one at a 1:9 ratio. After the purification process, which was optimized to completely remove the unloaded drug, NPs were characterized by Dynamic Light Scattering (DLS), showing a mean diameter of 171.1 ± 9.3 nm and an almost neutral Z-potential (-2.25  0.86 mV). NPs showed a Loading Capacity (LC%) and an Encapsulation Efficiency (EE%) of 0.72  0.14% and 72.2  14.5% respectively. Moreover, they displayed excellent stability in physiological medium (PBS, pH 7.4) over 3 weeks and for 48 hrs in PBS+5% FBS. Lipidic-based nanoparticles (SLNs) were produced with Hydrogenated Soybean Phosphatidyl Choline (HSPC):cholesterol at 1:3 molar ratio and a total lipid concentration of 35 mg/mL. Two different techniques were used to produce the SLNs, nanoprecipitation and microfluidics. Different parameters were varied during the assembling process, including the organic phase composition and the drug-containing phase. The best formulation in terms of reproducibility, LC% and stability was identified as the SLNs produced by microfluidics at a total flow rate of 4 mL/min and a milliQ water:organic phase flow rate ratio of 3:1. The organic phase was a mixture of ethanol:acetone 1:1 containing 0.5 mg/mL of (DDC)2-Cu. After purification, nanoparticles were characterized by DLS, showing a mean size of 105.7  3.7 nm and an almost neutral Z-potential (-4.797  2.662 mV). UV-visible spectrophotometric analysis resulted in a LC% and an EE% of 0.085  0.023% and 4.35  1.33%, respectively. Like PLGA-PEG2kDa NPs, SLNs showed a good stability for 48 hrs in presence of FBS, but they demonstrated a limited stability in presence of salt concentration (PBS) or in aqueous environment. The anticancer activity of polymer- and lipid-based selected formulations was preliminarily tested via MTS assay on MDA-MB-231 cell line as model for TNBC and compared to the free drug (0.05-1 M (DDC)2-Cu concentration range). Treated cells showed a decreased viability in a drug concentration-dependent manner, and nanoparticles activity was comparable with that of the free drug (IC50 values of 0.252  0.065 M, 0.203  0.041 M, and 0.291  0.021 M, for free (DDC)2-Cu, (DDC)2-Cu-loaded PLGA-PEG2kDa NPs and SLNs, respectively). Future studies will be devoted to confirm the anticancer activity in vitro and to find the correct lyophilization conditions for long-term storage. Finally, release and trafficking studies will complete the particles characterization. The most promising nanocarrier will be co-loaded with a second anticancer drug for combination therapy.