Folate receptor-targeted liposomes as vectors for therapeutic agents.

The folate receptor is a cell surface protein that has recently been identified as a tumor marker, due to its differential overexpression in several malignancies. Current research indicates that folate can be covalently attached to the surface of liposomes to mediate their selective internalization by tumor cells through the folate receptor-mediated endocytic pathway. Optimized liposome formulations, characterized by improvements in drug loading, extended residence times in the circulation and improved drug release, have been developed to improve the biodistribution of therapeutic molecules. Theoretically, folate receptor-targeting can be combined with liposome encapsulation to synergistically affect disease outcome by enhancing the delivery of chemotherapeutic agents to neoplastic cells, while reducing systemic toxicities to normal tissues. The purpose of this chapter is to characterize the components of folate receptor-targeted liposomes, and summarize their applications in gene and drug delivery.

Characterization of a novel diolein-based LPDII vector for gene delivery.

LPDII vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA (polyplexes) complexed with anionic pH-sensitive liposomes. Here, we describe a novel LPDII formulation containing polyethylenimine (PEI) polyplexes complexed with anionic pH-sensitive liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol). The pH-sensitivity of diolein/CHEMS liposomes was evaluated through quantitative fluorescence measurements of calcein release and particle size analysis. The results indicated that diolein/CHEMS liposomes are stable at physiological pH, but undergo rapid aggregation and fluorescence dequenching at pH values < or =5.0. Using a luciferase reporter gene, in vitro transfection of KB oral cancer cells showed that the transfection efficiency of LPDII vectors was superior to other well-characterized polyplexes and lipoplexes. Results further showed that gene delivery using diolein-containing LPDII vectors was dependent on the PEI nitrogen/DNA phosphate (N/P) ratio, the lipid/DNA weight ratio and the cell line being transfected. Replacing PEI with poly-L-lysine as the DNA condensing agent resulted in only a moderate reduction in transfection activity. Moreover, in contrast to LPDII formulations incorporating dioleoylphosphatidylethanolamine (DOPE), the transfection efficiency of diolein-based LPDII vectors was sustained in media containing up to 50% fetal bovine serum. Since diolein-based LPDII vectors mediate efficient gene transfer and retain their transfection activity in the presence of serum, diolein may be a promising alternative to DOPE for the construction of non-viral vectors for in vivo gene delivery.

Incorporation of reversibly cross-linked polyplexes into LPDII vectors for gene delivery.

LPDII vectors are synthetic vehicles for gene delivery composed of polycation-condensed DNA complexed with anionic liposomes. In this study, we evaluated the stability and transfection properties of polyethylenimine (PEI, 25 kDa)/DNA polyplexes before and after covalent cross-linking with dithiobis(succinimidylpropionate) (DSP) or dimethyl x 3,3'-dithiobispropionimidate x 2HCl (DTBP), either alone or as a component of LPDII vectors. We found that cross-linking PEI/DNA polyplexes at molar ratios > or =10:1 (DSP or DTBP:PEI) stabilized these complexes against polyanion disruption, and that this effect was reversible by reduction with 20 mM dithioerythritol (DTE). Transfection studies with polyplexes cross-linked at molar ratios of 10:1-100:1 in KB cells, a folate receptor-positive oral carcinoma cell line, showed decreasing luciferase gene expression with increasing cross-linking ratio. Subsequently, polyplexes, cross-linked with DSP at a molar ratio of 10:1, were combined with anionic liposomes composed of diolein/cholesteryl hemisuccinate (CHEMS) (6:4 mol/mol), diolein/CHEMS/poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) (6:4:0.05 mol/mol), or diolein/CHEMS/folate-PEG-cholesterol (folate-PEG-Chol) (6:4:0.05 mol/mol) for LPDII formation. Transfection studies in KB cells showed that LPDII vectors containing cross-linked polyplexes mediated approximately 2-15-fold lower gene expression than LPDII prepared with un-cross-linked polyplexes, depending on the lipid:DNA ratio. Inclusion of PEG-DSPE at 0.5 mol % appeared to further decrease transfection levels approximately 2-5-fold. Compared with LPDII formulated with PEG-DSPE, LPDII incorporating 0.5 mol % folate-PEG-Chol exhibited higher luciferase activities at all lipid:DNA ratios tested, achieving an approximately 10-fold increase at a lipid:DNA ratio of 5. Compared with cross-linked LPDII vectors without PEG-DSPE, inclusion of folate-PEG-Chol increased luciferase activities 3-4-fold between lipid:DNA ratios of 1 and 5. Interestingly, inclusion of 1 mM free folate in the growth media during transfection increased transfection activity approximately 3-4-fold for cross-linked LPDII vectors and LPDII containing folate-PEG-Chol, but had no effect on the transfection activity of LPDII formulated with PEG-DSPE. However, in the presence of 5 mM free folate, the luciferase activity mediated by LPDII vectors containing folate-PEG-Chol was reduced approximately 6-fold. Transmission electron micrographs were also obtained to provide evidence of LPDII complex formation. Results showed that cross-linked LPDII vectors appear as roughly spherical aggregated complexes with a rather broad size distribution ranging between 300 and 800 nm.