Enhanced In Vitro Cancer Cell Cytotoxicity of Drug-Loaded Dual Ligand-Targeted Liposomes Shravan Sriraman and Vladimir Torchilin
ABSTRACT Cancer, one of the leading causes of human deaths worldwide, originates from the uncontrolled cell growth of normal cells. Many of the current anticancer drug therapies are fraught with toxicity issues resulting from non-specific drug accumulation in healthy cells. A broad variety of these cancer cells over-express different types of cell-surface receptors like the transferrin (TfR) and folic acid receptors (FR). This receptor expression increases as the tumor progresses. With this in mind, polyethylene glycol (PEG)-coated long-circulating liposomes targeted with both, transferrin (TL) and folic acid (FL) were loaded with doxorubicin (Dox) and evaluated on human cervical cancer cells (HeLa). Flow cytometry demonstrated a 7-fold increase in cell association of the dual ligand-targeted liposomes (F+T L) compared to any single ligand-targeted liposomes (SL). The association and subsequent internalization of the (F+T) L was further confirmed using confocal microscopy. The (F+T) L also significantly increased cytotoxicity of Dox when compared to SL and non-targeted Dox-liposomes in vitro. In conclusion, the simultaneous targeting of TfR and FR has been shown to be an effective strategy to deliver drugs selectively and enhance their cytotoxic effects on cancer cells in vitro over SL. This selectivity allows us to work with lower drug concentrations thereby reducing the potential for off-target drug toxicity.
INTRODUCTION INTRODUCTION
RESULTS
Traditional chemotherapeutic agents kill cancer cells as well as a majority of rapidly dividing healthy cells in the body leading to disastrous side-effects. Ligand-targeted liposomes allow for the increased accumulation of the liposomal cargo at the target site thus resulting in improved therapeutic outcomes and reduced drug-associated toxicity.1 It has been found that cancer cells over-express different types of cell-surface receptors like TfR and FR when compared to normal cells in order to fuel their aggressive growth.2, 3 This led us to hypothesize that the simultaneous targeting of these receptors using an optimized combination of ligands could enhance therapeutic outcomes in a variety of regular and drug-resistant cancer cells.
FL
TL
F+T L
METHODS METHODS Liposomes were prepared by the thin film hydration method followed by extrusion
Figure 3. TEM image of plain liposome shows a uniform size distribution
Mean Fluorescent Intensity (MFI)
Figure 1. Extravasation of Liposome and subsequent binding to cellular receptors The liposomes extravasate out of systemic circulation and accumulate at the pathological site by the EPR effect. Here the dual-ligand targeted liposome is able to bind to the complimentary receptors on the cell surface.
Figure 2. Cellular internalization of dual-targeted liposome (1)Liposome specifically binds to membrane-bound receptors (2) Receptor binding initiates the formation of clathrincoated pits (3) Clathrin-coated pits pinch off of the membrane (4) Liposome is internalized via formation of an endosome and trafficked into the cell
Figure 4. Confocal microscopy image showing the enhanced cell association of the dual-targeted liposomes (F+T L) Red: Rhodamine-labeled liposomes Blue: Hoechst cell nucleus stain
Transferrin and folic acid were conjugated to the distal end of the PEG chain and post-inserted into the liposomes along with DSPE-PEG2000
100 90 80 70
Figure 5. Mean fluorescent Intensity of rhodaminelabeled liposomes targeted with folic acid (F),transferrin (T) or both (F+T) analyzed by flow cytometry
60 50 40 30 20 10 0
Control
PL
FL
FL + 2mM free folate
TL
TL + 0.5mM free transferrin
F+T L
140%
For cell association studies, liposomes fluorescently labeled with rhodamine were incubated with the cell for 4 hours and subsequently analyzed by confocal microscopy and flow cytometry
120%
Table 1. IC50 values of dox-loaded formulations
For cytotoxicity studies, the liposomes were loaded with doxorubicin by the pH-gradient method Cells were incubated with the dox-loaded formulations for 4 hours and then washed with media Cell viability was measured after 48 hours using the Promega® cytotoxicity assay
Cell Viability
100% 80% 60% 40% 20%
N=3 for all experiments
Formulation
Doxorubicin IC50 Values (µM)
LD
> 60
F LD
14.59
T LD
50.89
F+T LD
2.52
Free Dox
0.58
0%
51.7
16.30
5.13
1.62
0.51
0.16
0.05
Doxorubicin Concentration (µM)
REFERENCES REFERENCES 1) Abu Lila, Amr S., Tatsuhiro Ishida, and Hiroshi Kiwada. "Recent advances in tumor vasculature targeting using liposomal drug delivery systems." Expert Opinion on Drug Delivery 6.12 (2009): 1297-1309 2) Daniels, Tracy R., et al. "The transferrin receptor and the targeted delivery of therapeutic agents against cancer." Biochimica et Biophysica Acta (BBA)-General Subjects 1820.3 (2012): 291-317. 3) Xia, Wei, and Philip S. Low. "Folate-targeted therapies for cancer." Journal of medicinal chemistry 53.19 (2010): 6811-6824.
ACKNOWLEDGEMENTS This work was supported by the NIH grant U54CA151881 to VT.
LD
F LD
T LD
F+T LD
Free Dox
Figure 6. Cytotoxicity of dox-loaded liposomes (LD) targeted with folic acid (F), transferrin (T) or both (F+T)
CONCLUSIONS The simultaneous use of two targeting ligands, such as folate and transferrin, has been shown to be an effective strategy to deliver liposomal drugs specifically and enhance their cytotoxic effects on cancer cells in vitro over SL This selectivity allows to use lower drug concentrations thereby reducing the off-target drug toxicity