Liposomes engineering for tumor-selective delivery of radionuclides

Radiopharmaceuticals, central tools in nuclear medicine, are composed of a carrier and an unstable nuclide. The ISOLPHARM project, involving Legnaro National Laboratories (LNL) of the Nuclear Physics National Institute and the University of Padua, aims to obtain highly pure radionuclides, such as 111Ag and 64/67Cu. However, radionuclides require ad hoc delivery systems to reach the tumor selectively. Thus, ISOLPHARM aims at developing PEGylated liposomes bearing a targeting agent to preferentially associate with cancer cells and a chelator to stably entrap the desired nuclide. The selected targeting agent is Nastorazepide (Z-360), a small molecule with high affinity for Cholecystokinin type 2 receptor (CCK2-R), overexpressed in several cancers. Hence, this thesis focused on generating PEGylated liposomes formulated with model tetraazacyclododecane derivative (DOTA) as chelator for Cu2+. The studies were performed with cold metal as proof of concept. In particular, we explored the most efficient Cu2+ loading conditions on liposomes produced with thin film hydration method. Two techniques were used: (1) surface chelation and (2) remote loading. To this aim, liposomes were formulated using Hydrogenated soy L-α-phosphatidylcholine (HSPC) and cholesterol at 2:1 molar ratio. For method 1, we synthesized DSPE-PEG2KDa-DOTA by conjugating DSPE-PEG2KDa-NH2 and DOTA with a quantitative conjugation yield. The conjugate was directly added at 2.5 or 5 mol/mol% (with respect to the lipid content) in the film. In method 2, 10 mM free DOTA solution was directly employed for liposomes re-hydration. To compare the influence of formulation conditions (i.e. pH and salt), different buffers were used to re-hydrate: phosphate buffered saline (PBS) pH 7.4, or 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) pH 7.4 (methods 1 and 2), or 200 mM ammonium acetate (NH4+CH3COO−) pH 5.5 (method 1). Liposomes thus formulated were loaded with Cu2+ by co-incubation with Cu(NO3)2 standard solution at 50°C, 300 rpm for 1h. To evaluate the buffer effect on Cu2+ loading, liposomes underwent a buffer exchange with either PBS, MOPS or NH4+CH3COO− before incubation with Cu2+. Liposomes size and polydispersity index (PDI) were characterized by Dynamic Light Scattering (DLS) while surface charge was measured by z-potential analysis. All the formulations showed low polydispersity (<0.22), z-potential in the neutrality range (-12.2 mV and 0 mV), and average size of around 150 nm before and after Cu2+ loading, with no relevant differences highlighted neither comparing the buffers used. The unloaded Cu2+ was removed purificating with PD-10 desalting colums containing Sephadex G-25 fine resin and the loading was defined by graphite furnace-atomic absorption spectroscopy (GF-AAS). Data were normalized for the lipidic concentration calculated by the Stewart assay. Results indicated higher Cu2+/lipid ratio using NH4+CH3COO− in Cu2+ loading. In particular, the two best formulations were RLα and SL5: remote chelation at pH 5.5 (RLα) gave the best results (64 ng Cu/μg lipid), but also the highest variability among the tests. On the contrary, surface chelation with PBS (SL5) entrapped limited amount of Cu2+ (13 ng Cu/μg lipid), but with more reliable and reproducible results. On SL5 and RLα were performed further release and stability studies in simulated physiological conditions. Preliminary liposomes association tests were performed on human epidermoid carcinoma cell line A431, transfected to stably express CCK2-R on the cell membrane (A431-CCK2R+). The internalization ability was evaluated by incubating the cells with the liposomes for 4 hrs. GF-AAS analysis showed a better uptake for RLα than SL5, with 5x and 2x higher Cu2+ intracellular levels as compared to untreated control cells, respectively. Future studies will be devoted to the addition of Z-360 and dedicated in vitro studies to evaluate the formulation specificity for A431-CC2R+ cells.