Impact of Lipid Composition on Vesicle Protein Adsorption: A BSA Case Study

Abstract

Investigating the interaction between liposomes and proteins is of paramount importance in the development of liposomal formulations with real potential for bench-to-bedside transfer. Upon entering the body, proteins are immediately adsorbed on the liposomal surface, changing the nanovehicles' biological identity, which has a significant impact on their biodistribution and pharmacokinetics and ultimately on their therapeutic effect. Albumin is the most abundant plasma protein and thus usually adsorbs immediately on the liposomal surface. We herein report a comprehensive investigation on the adsorption of model protein bovine serum albumin (BSA) onto liposomal vesicles containing the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in combination with either cholesterol (CHOL) or the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). While many studies regarding protein adsorption on the surface of liposomes with different compositions have been performed, to the best of our knowledge, the differential responses of CHOL and DOTAP upon albumin adsorption on vesicles have not yet been investigated. UV-vis spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong influence of the phospholipid membrane composition on protein adsorption. Hence, it was found that DOTAP-containing vesicles adsorb proteins more robustly but also aggregate in the presence of BSA, as confirmed by DLS and TEM. Separation of liposome-protein complexes from unadsorbed proteins performed by means of centrifugation and size exclusion chromatography (SEC) was also investigated. Our results show that neither method can be regarded as a golden experimental setup to study the protein corona of liposomes. Yet, SEC proved to be more successful in the separation of unbound proteins, although the amount of lipid loss upon liposome elution was higher than expected. In addition, coarse-grained molecular dynamics simulations were employed to ascertain key membrane parameters, such as the membrane thickness and area per lipid. Overall, this study highlights the importance of surface charge and membrane fluidity in influencing the extent of protein adsorption. We hope that our investigation will be a valuable contribution to better understanding protein-vesicle interactions for improved nanocarrier design.

Figure 1

Chemical structures of the lipids used in this work.

Figure 2

ζ-Potential for generated liposomes. Data are presented as mean ± SEM (n = 4–6).

Figure 3

Representative TEM images of aqueous suspensions of liposomes with different concentrations of CHOL in the bilayer and after extrusion. (A) CHOL-containing liposomes, (B) DOTAP-containing liposomes, and (C) (CHOL)DOTAP-containing liposomes after BSA adsorption.

Figure 4

Lipid loss upon centrifugation, as determined by phospholipid content determination, relative to the expected content in the measured volume of sample. Data are presented as mean ± SEM (n = 3).

Figure 5

Liposome size and PDI before centrifugation (CFG), after centrifugation, and after incubation with BSA and centrifugation for (A) CHOL-containing liposomes in PBS, (B) CHOL-containing liposomes in PB, and (C) DOTAP-containing liposomes in PBS. Data are presented as mean ± SEM (n = 3). A one-way ANOVA followed by pairwise comparison using the Benjamini–Hochberg method for multiple testing correction was used for assessing statistical significance; “Incubated CFG” refers to the liposomes after BSA adsorption; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns—not significant.

Figure 6

Size exclusion chromatography elution profiles for BSA-incubated (A) DPPC:CHOL 70:30 in PB and (B) DPPC:DOTAP 60:40 in PBS.

Figure 7

ζ-Potential before centrifugation, after centrifugation, and after incubation with BSA and centrifugation for (A) CHOL-containing liposomes in PBS, (B) CHOL-containing liposomes in PB; and (C) DOTAP-containing liposomes in PBS, “Incubated CFG” refers to the liposomes after BSA adsorption; Data are presented as mean ± SEM (n = 3).

Figure 8

Adsorbed BSA in g/mol lipid following SDS-PAGE from pelleted liposomes after centrifugation and washing and UV–vis determinations from the supernatant. Data are presented as mean ± SEM (n = 3). A one-way ANOVA followed by pairwise comparison using the Benjamini–Hochberg method for multiple testing correction was used for assessing statistical significance; *p < 0.05, **p < 0.01, ns–not significant.

Figure 9

Membrane transverse snapshots in the final frame of production molecular dynamics simulations (30 ns). (A) DPPC:CHOL 100:0, (B) DPPC:CHOL 90:10, (C) DPPC:CHOL 80:20, (D) DPPC:CHOL 70:30, (E) DPPC:CHOL 60:40, (F) DPPC:CHOL 50:50, (G) DPPC:DOTAP 90:10, (H) DPPC:DOTAP 80:20, (I) DPPC:DOTAP 70:30, (J) DPPC:DOTAP 60:40. DPPC are depicted as cyan, CHOL are yellow and DOTAP are magenta. Na⁺ and Cl^– are shown as blue and cyan spheres, respectively.

Figure 10

Number of Na⁺: membrane contacts within 5 Å throughout the salt-containing simulations. Data are presented as mean ± SD.