Structure-Dependent Stability of Lipid-Based Polymer Amphiphiles Inserted on Erythrocytes

Abstract

Cell-based therapies have the potential to transform the treatment of many diseases. One of the key challenges relating to cell therapies is to modify the cell surface with molecules to modulate cell functions such as targeting, adhesion, migration, and cell-cell interactions, or to deliver drug cargos. Noncovalent insertion of lipid-based amphiphilic molecules on the cell surface is a rapid and nontoxic approach for modifying cells with a variety of bioactive molecules without affecting the cellular functions and viability. A wide variety of lipid amphiphiles, including proteins/peptides, carbohydrates, oligonucleotides, drugs, and synthetic polymers have been designed to spontaneously anchor on the plasma membranes. These molecules typically contain a functional component, a spacer, and a long chain diacyl lipid. Though these molecular constructs appeared to be stably tethered on cell surfaces both in vitro and in vivo under static situations, their stability under mechanical stress (e.g., in the blood flow) remains unclear. Using diacyl lipid-polyethylene glycol (lipo-PEG) conjugates as model amphiphiles, here we report the effect of molecular structures on the amphiphile stability on cell surface under mechanical stress. We analyzed the retention kinetics of lipo-PEGs on erythrocytes in vitro and in vivo and found that under mechanical stress, both the molecular structures of lipid and the PEG spacer have a profound effect on the membrane retention of membrane-anchored amphiphiles. Our findings highlight the importance of molecular design on the dynamic stability of membrane-anchored amphiphiles.

Keywords: amphiphiles; erythrocytes; lipid; membranes.

Figure 1

Kinetic stability of lipo-PEGs with varying length of lipid and PEG on RBC surface. (A–C), mouse RBCs were loaded with different Fam-labeled lipo-PEGs and cocultured with fleshly isolated mouse blood. The decay of fluorescence on RBC surface was quantified by flow cytometry. (A) Dot plot of RBC at different time points. (B,C) Time dependent Fam-positive frequencies (B) and relative mean fluorescence intensities (C) of RBCs. Each sample was assayed with three replicates. Error bars represent the SEM. Data show the mean values ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Figure 2

Shear stress-induced release of lipo-PEGs on RBC surface. C18-PEGs-loaded RBCs were mixed with whole blood, or unloaded RBCs, or serum, and shear stress was applied for 15 min. Cells were assayed by flow cytometer. Flow dot plot (A) and percentage of release (B,C) of lipo-PEGs from RBCs w/wo shear stress. Plot (B) shows the percentage of fluorescence release of lipo-PEGs in blood with or without shear stress. Plot (C) shows the fluorescence release after 15 min shearing at 10 Pa from lipo-PEGs loaded RBCs in each of the suspension media. Each sample was assayed with three replicates. Error bars represent the SEM. Data show the mean values ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Figure 3

In vivo release kinetics of C18EG6, C18EG48, and DSPE-PEG2K loaded RBCs. (A) Representative flow cytometry dot plots. (B,C) The relative frequencies of Fam-positive RBCs (B) and mean fluorescence intensities (C) at different time points after injection. Each time point was assayed with three replicates. Error bars represent the SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. Statistical analyses were made by comparing C18EG6 and C18EG48 groups.

Figure 4

Cationic lipid prolongs the retention time of long PEG amphiphiles in vitro and in vivo. (A), Percentages of release cationic C18PEG2K after in vitro culture with whole blood. (B–D) Representative flow cytometry dot plots (B) and relative frequencies of Fam-positive RBCs (C) and mean fluorescence intensities (D) at different time points after injection. Each time point was assayed with three replicates. Error bars represent the SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.