Evaluation of New Folate Receptor-mediated Mitoxantrone Targeting
Liposomes In Vitro
Authors: Wen Tianjiao , Gao Yuan , Zheng Ying , Shan Bin , Song Cong , An Yahui , Cui Jingxia 
Published: November 2023

Background::
Ligand-mediated liposomes targeting folate receptors (FRs) that are
overexpressed on the surface of tumor cells may improve drug delivery. However, the properties
of liposomes also affect cellular uptake and drug release.

Objective::
Mitoxantrone folate targeted liposomes were prepared to increase the enrichment of
drugs in tumor cells and improve the therapeutic index of drugs by changing the route of drug
administration.

Methods::
Liposomes were prepared with optimized formulation, including mitoxantrone folatetargeted
small unilamellar liposome (MIT-FSL), mitoxantrone folate-free small unilamellar liposome
(MIT-SL), mitoxantrone folate-targeted large unilamellar liposome (MIT-FLL), mitoxantrone
folate-free large unilamellar liposomes (MIT-LL). Cells with different levels of folate alpha
receptor (FRα) expression were used to study the differences in the enrichment of liposomes,
the killing effect on tumor cells, and their ability to overcome multidrug resistance.

Results::
The results of the drug release experiment showed that the particle size of liposomes
affected their release behavior. Large single-compartment liposomes could hardly be effectively
released, while small single-compartment liposomes could be effectively released, MIT-FSL vs
MIT-FLL and MIT-SL vs MIT-LL had significant differences in the drug release rate
(P<0.0005). Cell uptake experiments results indicated that the ability of liposomes to enter folic
acid receptor-expressing tumor cells could be improved after modification of folic acid ligands
on the surface of liposomes and it was related to the expression of folate receptors on the cell
surface. There were significant differences in cell uptake rates (p<0.0005) for cells with high
FRα expression (SPC-A-1 cells), when MIT-FSL vs MIT-SL and MIT-FLL vs MIT-LL. For
cells with low FRα expression (MCF-7 cells), their cell uptake rates were still different (p<0.05),
but less pronounced than in SPC-A-1 cells. The results of the cell inhibition experiment suggest
that MIT-FLL and MIT-LL had no inhibitory effect on cells, MIT-FSL had a significant inhibitory
effect on cells and its IC50 value was calculated to be 4502.4 ng/mL, MIT-SL also had an
inhibitory effect, and its IC50 value was 25092.1 ng/mL, there was a statistical difference
(p<0.05), MIT-FSL had a higher inhibitory rate than MIT-SL at the same drug concentration.
Afterward, we did an inhibitory experiment of different MIT-loaded nanoparticles on MCF-7
cells compared to the drug-resistant cells (ADR), Observing the cell growth inhibition curve,
both MIT-FSL and MIT-SL can inhibit the growth of MCF-7 and MCF-7/ADR cells. For MCF-
7 cells, at the same concentration, there is little difference between the inhibition rate of MITFSL
and MIT-SL, but for MCF-7/ADR, the inhibition rate of MIT-FSL was significantly higher
than that of MIT-SL at the same concentration (P<0.05).

Conclusion::
By modifying folic acid on the surface of liposomes, tumor cells with high expression
of folic acid receptors can be effectively targeted, thereby increasing the enrichment of intracellular
drugs and improving efficacy. It can also change the delivery pathway, increase the
amount of drug entering resistant tumor cells, and overcome resistance.