Targeted delivery of Quantum Dots-loaded liposomes.

VALERIA SIGOT; THOMAS JOVIN

Rosario

Taller; 1º Taller de Órganos Artificiales, Biomateriales e Ingeniería de Tejidos (BIOOMAT); 2009

Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR

The design of liposomal carriers for gene or drug delivery is an active field in current biomedical research. An important issue is the targeting of lipid-based carriers to specific cell types for which a therapeutic approach is intended. The combination of liposomes and imaging probes such as Quantum Dots (QDs) in a single nanoparticle offers an excellent tool for understanding the mechanisms of liposome uptake, required to improve the efficiency of gene or drug delivery.In the present work, biotinylated lipid particles (BLPs) were loaded with a red emitting QD655 and surface coated with a green emitting QD525 tagged with the Epidermal Growth Factor (EGF) ligand. This dual-color approach was employed to monitor the specificity of BLPs binding and uptake in A431 cells from human epidermoid carcinoma over-expressing the EGF receptor (EGFR) by confocal fluorescence microscopy. BLPs were formulated with 1.4% and 2.7 % of PEG-lipids composed by either a fusogenic or a pH-sensitive lipid to favor endosomal escape of the encapsulated QDs. Particle analysis by transmission electron microscopy showed that BLPs exhibited a spherical shape with a mean diameter of 120 nm and 2 to 5 encapsulated QDs per lipid particle for all the lipid formulations tested. BLPs loaded with QD655 were then conjugated to preformed complexes of biotin-EGF-streptavidin-QD525 (EGF-QD525). With this approach, colocalized red-green dots were expected to indicate specific uptake, whereas the sole detection of red fluorescence would reveal unspecific binding of non-targeted BLP-QD655. After 1 h incubation at 37°C of A431 cells with the BLPs, both QDs extensively colocalized and were distributed in clusters in the perinuclear region, indicating that targeted EGF-QD525-BLPQD655 particles were internalized. Negligible internalization as evidenced by a faint red fluorescence was detected for nontargeted BLP-QD655. Furthermore, no targeting effect was evident in CHO (Chinese Hamster Ovary) cells and in WM983A melanoma cells, both devoid of EGF receptor or in the presence of free competing ligand, indicating that enhanced celluptake is primarily mediated by EGF-EGFR interaction. Regardless of the lipid formulation tested, the clusters of colocalized QDs persisted over days in A431 cells, indicating the difficulty in releasing the encapsulated nanoparticles into the cytoplasm.We concluded that the dual-color QD labeling strategy allows the immediate detection of targeted BLPs from the untargeted counterparts, as well as the presence of free EGF-QD525 complexes during live cell imaging. However, the lipid formulations tested are unable to facilitate the endosomal release of QD655. From a therapeutic point of view, the specificity displayed bythe targeted BLPs may provide a platform for testing the specific delivery of anti-tumorigenic drugs into tumor cells overexpressing the EGFR. This approach was also intended to elucidate the fate of lipid particles in real time, taking advantages of the photostability and bright fluorescence of QDs and to provide refined information about the still poorly understood trafficking processes and the subcellular barriers to gene or drug delivery.