(743j) Cytosolic Delivery of Doxorubicin to Overcome Multidrug Resistance

Cytosolic Delivery of Doxorubicin to
Overcome Multidrug Resistance

Jacob B. Williams¹, William G. Pitt¹

¹ Chemical Engineering Department,
Brigham Young University

           
We have developed a method to deliver a payload directly to the cytosol of
multidrug resistant cancer cells. The payload is encapsulated inside of a
liposome along with a liquid nanodroplet of immiscible perfluorocarbon having a
high vapor pressure (eLiposome) [1].
Since folate receptors are overexpressed on over 1/3 of cancer cells [2], folate is attached to the
surface of the liposome as a targeting ligand to induce folate-mediated endocytosis.
Insonation causes the nanodroplet to expand to gas and rupture the liposome,
thereby rapidly releasing the payload [3].
If the payload is released after the liposome has been endocytosed, then there
is a sudden high concentration of drug released directly to the cytosol of the
cell.

The multidrug
resistance of cancer cells is often a result of an increased the number of
permeability glycoprotein (P-gp) pumps on the surface of the cell that export
undesired compounds out of the cytosol [4].
Normally the resistant cancer cells are able to keep the cytosol at sub-lethal
levels of anti-cancer drugs, but the sudden high concentration of drug released
directly to the cytosol allows the drug to have a cytotoxic response before all
of it can be pumped out of the cell. Furthermore, the combination of
folate-induced endocytsosis and triggered release (via ultrasound) localizes
the drug to the inside of the cell and will therefore minimize negative
side-effects of cancer drugs commonly observed when delivered systemically.

           
Our liposomes were synthesized with DPPC (dipalmitoylphosphatidylcholine) and
cholesterol and phospholipid-folate ligand was inserted in the surface of the
liposome to bind the liposome to the cell and induce endocytosis. The liposomes
were loaded with a liquid nanodroplet of perfluorohexane and the anti-cancer
drug doxorubicin (Dox). Resistant breast cancer cells (MCF-7/ADR) were grown in
a 24-well plate and kept in folate-free media (RPMI) for 48 hours prior to drug
delivery. Cells were then exposed to Dox at 10 µM as 1) free Dox; 2) Dox in
eLiposomes (eLipoDox); 3) Dox in eLiposomes with folate ligands incorporated in
the surface (folated eLipoDox). The media was aspirated 2 hours after the drug
was administered and replaced with fresh media. Ultrasound (1W/cm²)
was applied to some of the cells. Cell viability was measured with a trypan
blue exclusion after 24 hours.

           
As shown in Figure 1, ultrasound applied to folated eLipoDox (red solid bar) is
more effective at killing MCF-7/ADR cells than the other conditions tested. If
ultrasound is not applied to folated eLipoDox (red hatched bar) or folate is
not incorporated into the eLipoDox (yellow bars) there is no significant
killing of MCF-7/ADR cells. This suggests that both ultrasound and folate
ligands are necessary to achieve significant killing of resistant breast cancer
cells.

Figure 1. Viability of MCF-7 resistant
cells to treatment with Dox, eLipodox, and folated eLipodox. Each drug formulation
had a 10 µM Dox concentration. Ultrasound was applied at 20-kHZ, 1 W/cm².
Error bars indicate the standard deviation (n = 3). Folated eLipoDox with US
cell viability is statistically different from all other conditions except for
No Drug without US.

References

[1] M. Javadi, W.G. Pitt, D.M. Belnap, N.H. Tsosie, J.M.
Hartley, Encapsulating Nanoemulsions Inside eLiposomes for Ultrasonic Drug
Delivery, Langmuir, 28 (2012) 14720-14729.

[2] L.A. Bareford, P.W. Swaan, Endocytic mechanisms for targeted
drug delivery, Adv Drug Deliver Rev, 59 (2007) 748-758.

[3] M. Javadi, W.G. Pitt, C.M. Tracy, J.R. Barrow, B.M.
Willardson, J.M. Hartley, N.H. Tsosie, Ultrasonic gene and drug delivery using
eLiposomes, J Control Release, 167 (2013) 92-100.

[4] M.M. Gottesman, T. Fojo,
S.E. Bates, Multidrug resistance in cancer: role of ATP-dependent transporters,
Nature reviews. Cancer, 2 (2002) 48-58.