Feasibility study of click-modified liposomes targeting folate receptors in mouse tumor model

Objectives: The aim of this study is to demonstrate utility of folate receptor targeting liposomes prepared by click reactions in mice with folate receptor positive tumors. Methods: Clickable liposomes (Click-LIP) were prepared by mixing a predetermined ratio of distearoylphosphatidylcholine (DSPC), cholesterol, and distearoylphosphatidylethanolamine (DSPE) with 2.5 mol% DBCO-PEG2000-DSPE, and then the crude mixture was extruded. To conjugate folate on the liposomal surface, 100 nmole of Folate-GGCEK-azide (Fol-N3) was added to 5 mg/mL of Click-LIP solution, purified afterwards by PD-10 desalting column (Fol-LIP). Lastly, radiolabeled 64Cu-NOTA-N3 was conjugated with Fol-LIP by click reaction (64Cu-LIP-Fol). The size distribution of liposomes was measured by dynamic light scattering and nanoparticle tracking analysis. Six weeks old BALB/c nu/nu mice received folate-free diet for three weeks, and one million folate receptor positive KB cells cultured in folate-free media were subcutaneously inoculated in each mouse (3 or 4 mice in each group). For biodistribution study, 0.15 to 0.2 MBq of 64Cu-LIP-Fol was injected through the tail vein of each mouse. Twenty-four hours later, the mice were sacrificed, and weight and radioactivity of each dissected organ were measured. For in-vivo imaging study, 1.85 MBq of 64Cu-LIP-Fol was injected through the tail vein of each mouse, and PET images were taken after 0.5, 4, and 24 hours later. For comparison, excessive free folate by about 100 times was pre-injected 30 minutes before the tail vein injection of 64Cu-LIP-Fol. Results: The size distribution of 64Cu-LIP-Fol was as follows: mean diameter of 48.4±1.4 nm and standard deviation of 21.5±15.0 nm. In biodistribution study, tumoral uptakes of 64Cu-LIP-Fol were significantly higher in mice without excessive free folate pre-injection (2.66±0.46 ID%/g) than those with excessive free folate pre-injection (1.91±0.10 ID%/g, p=0.029). Liver, spleen, and kidneys showed high uptake of 64Cu-LIP-Fol, but there was no significant difference between the groups with and without excessive free folate pre-injection. In in-vivo imaging study, persistently high tumoral uptake was shown at 4 and 24 hours after the tail vein injection, and liver uptake decreased as time passed. There was minimal tumoral uptake shown in mice with excessive free folate pre-injection. Conclusion: Folate receptor positive tumor cells in mice were successfully accumulated by 64Cu-LIP-Fol and blocked by pre-inje