Peptide Anchor for Folate-Targeted Liposomal Delivery

Abstract

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

“…We previously demonstrated that the hydrophobic NPD inserts deeply into the lipid bilayer and anchors folate onto liposomal surface. 9 Also others have used hydrophobic peptides derived from bacteriophages and genetically fused them to targeting peptides to functionalize liposomes. 30,31 Now we show that NDP anchors onto liposomes without the need of harmful cross-linkers not only small ligands but also Fabs with the antigen-binding site well exposed.…”

“…Previously, NDP was successfully used to anchor folate into the lipid bilayer of the liposomes without affecting liposomal integrity. 9 Further, bioinformatic analysis of the NDP-heavy chain constructs revealed that NDP is extended away from the Fab variable and constant domains ( Supplementary Figure 2), indicating that the Fab fragments should be efficiently inserted into the liposomal membrane via the C-terminal hydrophobic peptide, with the N-terminal antigen-binding sites well exposed. Indeed, introducing NDP into the Fab sequences had no detrimental effect onto the Fab folding ( Figure 1, A and B) or their functionality (Figure 2, A right, 2, B right).…”

“…([N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoylsn-glycero-3-phosphoethanolamine]) were prepared using a thin film hydration method as previously described. 9,10 Briefly, DOPE, CH and DSPE-MPEG were dissolved in chloroform. After its evaporation, the dried lipid film was dispersed in PBS containing Fab-NDP antibody fragments and Alexa Fluor 647 (AF647).…”

“…Liposomes were characterized by the dynamic light scattering using a Malvern zetasizer NS (Malvern Instruments, Malvern, UK) as previously described. 9 Further methods describing liposome specificity and toxicity tests are provided in Supplementary data.…”

“…8 Previously we showed that the SP-D neck domain peptide efficiently anchored folic acid into the liposomal membrane. 9 Now we show that the same strategy can be used to equip liposomes with targeting proteins. We report here a full process of converting a mouse mAb into a recombinant Fab antibody fragment with C-terminal neck domain peptide (NDP), its expression in the yeast Pichia pastoris and functionalization of liposomes with an isolated Fab-NDP construct.…”