The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake

Abstract

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation-a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.

Keywords: cancer cell targeting; chimeric carbohydrate; drug delivery; giant unilamellar vesicles; hypersialylation; live-cell imaging; protein engineering.

Figure 1

Hypothetical model of FS-Janus lectin-mediated crosslinking of glyco-decorated liposomes and human cancer cells leading to the cellular uptake of complete liposomes and liposomal content. (a) The peptide sequence of FS-Janus lectin with two blades of RSL (green) connected to CBM40_NanI (yellow) through a peptide linker. (b) Schematic representation of FS-Janus lectin presenting six fucose-binding sites at the upper face and three sialic acid-binding sites at the bottom face. (c) FS-Janus lectin binds simultaneously to glyco-decorated liposomes and the cell surface (1) and crosslinks them (2). These tight interactions lead to the cellular uptake of complete liposomes and liposomal content (3). Intracellular liposome transport and burst (4).

Figure 2

FS-Janus lectin triggers crosslinking and lipid exchange between GM3- and blood group A trisaccharide- (FSL-A) functionalized liposomes. GM3 GUVs (red color; labeled with the fluorescent lipid DOPE-Atto 647N) and FSL-A GUVs (green color; labeled with the fluorescent lipid DOPE-Atto 488) were incubated with 200 nM FS-Janus lectin (unlabeled) at room temperature and were monitored for 120 min using confocal microscopy. The crosslinking between GUVs starts immediately after the addition of FS-Janus lectin, and lipid exchange becomes visible after 55 min of incubation. Some examples are highlighted by yellow arrows. Without the addition of FS-Janus lectin, the liposomes do not crosslink and maintain their round shape over the entire incubation time. The scale bars represent 10 μm.

Figure 3

Dose-dependent binding of FS-Janus lectin and di-CBM40 to H1299 lung epithelial cells. Flow cytometry analysis of gated living H1299 cells incubated for 30 min on ice with FS-Janus lectin (a), di-CBM40 (b), and FS-Janus lectin in the presence of soluble l-fucose (c). (a) Histogram of fluorescence intensity of H1299 cells stimulated with different concentrations of FS-Janus lectin AF488 (red: negative control, green: 16 nM, blue: 32 nM, orange: 64 nM). (b) Histogram of fluorescence intensity of H1299 cells stimulated with different concentrations of di-CBM40 AF488 (red: negative control, green: 15 nM, blue: 21 nM, orange: 42 nM, grey: 107 nM, light blue: 214 nM). (c) Histogram of fluorescence intensity of H1299 cells stimulated with FS-Janus lectin AF488 (32 nM) pre-incubated with different concentrations of soluble l-fucose for 30 min at room temperature (RT) to saturate fucose-binding sites (red: negative control, light blue: FS-Janus lectin 32 nM, dark green: FS-Janus lectin 32 nM + 25 mM L-fucose, green: FS-Janus lectin 32 nM + 50 mM L-fucose, orange: FS-Janus lectin 32 nM + 100 mM L-fucose).

Figure 4

Fluorescence imaging revealed the internalization of FS-Janus lectin and di-CBM40 into H1299 cells. (a) Confocal imaging of fluorescently labeled FS-Janus lectin (green color) incubated with H1299 cells at different time points. FS-Janus lectin is internalized within 60 min and accumulates in the perinuclear region of H1299 cells (white arrows). Nuclei were counterstained by DAPI. (b) Confocal imaging of di-CBM40 (green color) binding and uptake into H1299 cells at different time points. di-CBM40 was observed intracellularly in H1299 cells after 60 min (as indicated by white arrows). (c) Confocal imaging of fluorescently labeled FS-Janus lectin (green color) incubated with H1299 cells at different time points. In the presence of soluble l-fucose, FS-Janus lectin was internalized within 60 min and accumulated in the perinuclear region of H1299 driven by its CBM40 domains. Nuclei were counterstained by DAPI. Scale bars represent 10 μm.

Figure 5

Crosslinking of blood group A trisaccharide-decorated GUVs and H1299 cells is mediated by 200 nM FS-Janus lectin. FS-Janus lectin is enriched at the interfaces (white arrows). The GUVs (red color; labeled with the fluorescent lipid DOPE-Atto 647N) were incubated with the H1299 cells (blue color; partially stained with CellTrace™ Violet) and FS-Janus lectin (green color; labeled with AF488). The yellow arrow indicates perinuclear accumulations of liposomal lipids and FS-Janus lectin. Live-cell imaging experiments were performed at 37 °C by using confocal microscopy and were recorded for 120 min. Scale bars represent 10 μm.

Figure 6

Kinetics of the cellular uptake and perinuclear accumulation of liposomal lipids and FS-Janus lectin. H1299 cells (blue color; partially stained with CellTrace™ Violet) were incubated with 200 nM FS-Janus lectin (red color, labeled with Atto 647) and blood group A trisaccharide-decorated GUVs (green color; labeled with the fluorescent lipid DOPE-Atto 488). Yellow arrows indicate perinuclear accumulation of fluorescent liposomal lipids and FS-Janus lectin. Live-cell imaging experiments were performed at 37 °C by using confocal microscopy, and the kinetics of cellular uptake were recorded for 180 min. Scale bars represent 10 μm.

Figure 7

Complete uptake of blood group A-decorated GUVs into H1299 cells. FS-Janus lectin (200 nM, red color; labeled with Atto 647) triggers the internalization of complete liposomes (green color; labeled with fluorescent lipid DOPE-Atto 488) into H1299 cells (blue color; partially stained with CellTrace™ Violet). Yellow arrows point to one GUV that is taken up between 25 and 30 min. Additionally, inside the cell, the liposome remains covered by FS-Janus lectin. Live-cell imaging experiments were performed at 37 °C by using confocal microscopy, and the kinetics of uptake were recorded for 120 min. Scale bars represent 10 μm.

Figure 8

An internalized blood group A decorated GUV undergoes deformations, shrinkage, and bursts inside H1299 cells. FS-Janus lectin (500 nM, red color; labeled with Atto 647) triggered the internalization of complete liposomes (green color; labeled with fluorescent lipid DOPE-Atto 488) into H1299 cells (blue color; partially stained with CellTrace™ Violet). Once internalized, the selected GUV became deformed, reduced its size, and burst. The yellow arrows point to these events. Live-cell imaging experiments were performed at 37 °C by using confocal microscopy and were recorded for 120 min. Scale bars represent 10 μm.