Highlights

Figure 2. Top panels: 1D SDS-PAGE gel of human plasma proteins obtained from (a) DOTAP cationic liposome-protein complexes and (b)
DOTAP/DNA lipoplex-protein complexes following incubation at different plasma concentrations. Bottom panels: schematic presentation of the
evolution of the protein corona that forms around both CLs (c) and lipoplexes (d) upon exposure to plasma. Passing from low to high plasma
concentrations the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes (c)
while the protein corona of lipoplexes changes in abundance but not in composition (d). Adapted with permission from (Langmuir, 2011, 27 (24),
pp 15048–15053). Copyright (2011) American Chemical Society.

The effective unit of interest in cell-nanomaterial interactions is not the nanoparticle itself but the particle and its hard corona of associated proteins from plasma or other bodily fluids.
Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concentrations (2.5%-80%). The composition of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. As a consequence, the biological identity of lipid gene vectors whose surfaces are entirely lipidic may change dramatically as the amount of protein in the environment changes.        

Recently, it has been shown that the effective unit of interest in the cell-nanomaterial interaction is not the nanoparticle itself but the particle and its hard corona of associated proteins from plasma or other bodily fluids. This corona of proteins at the surface of the particle is sufficiently long-lived that actually the entity is “seen” and processed by living cells. This is a key issue that has broad implications for in vitro – in vivo extrapolations and will determine the future road map of nanomedicine and perhaps impact the overall field of nanoscience.
Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes as a function of increasing plasma concentration. According to recent findings, CLs are excellent model systems of lipid nanoparticles (LNP) in which a DNA/polycation core is coated with a lipid envelope. This study allowed us to elucidate more quantitatively the degree to which the protein corona of lipid gene vectors can change, depending on the biological environment. To better investigate the protein corona-nanoparticle complex, we performed a preliminary physicalchemical characterization of both DOTAP CLs and DOTAP/DNA lipoplexes. SAXS data collected at the SAXS beamline at Elettra are reported in Fig. 1: Fig. 1a shows the SAXS pattern of DOTAP CLs characteristic of uncorrelated bilayers (e.g., unilamellar vesicles) and Fig. 1b shows the SAXS pattern of DOTAP/DNA lipoplexes characteristic of a multilamellar structure. Combined sizing, ζ-potential, and SAXS data well support the accepted model of the DNA-induced restructuring of CLs upon lipoplex formation. Taken together, these results confirm the recent suggestion that lipoplexes are hybrid structures with the lipid surface partially decorated by negatively charged DNA chains.

To focus on the evolution of the protein corona that forms around both CLs and lipoplexes upon exposure to plasma, one-dimensional (1D) Sodium Dodecyl Sulphate/PolyAcrylamide Gel Electrophoresis (SDS/PAGE) experiments were performed. Fig. 2 shows 1D SDS/PAGE gel results in which DOTAP CLs and DOTAP/ DNA lipoplexes were incubated in plasma, over a wide range of plasma concentrations (2.5%-80%). With increasing plasma concentration, the protein pattern for DOTAP CLs changes considerably (Fig. 2a), whereas for lipoplexes the intensity of the protein bands seem to increase monotonously with increasing plasma concentration (Fig. 2b). The identities of the proteins were determined by mass spectrometry analysis of selected bands cut from the gels reported in Figure 2a,b. We observed that the protein corona of CLs is made of both low-affinity and competitivebinding proteins whose relative abundance changes with the plasma concentration (Fig. 2c). On the other side, passing from low to high plasma concentrations, the protein corona of lipoplexes changes in abundance but not in composition (Fig. 2d). Such effects may be so striking that the biological identity of lipid gene vectors with DNA cargo confined in the interior space (e.g., LNP) may change dramatically as the amount of protein in the environment changes. Therefore, the evolution of the protein corona passing from in vitro to in vivo conditions is severely affected by the presence of DNA. This aspect should be carefully considered for the rational design of lipid gene vectors. Synchrotron SAXS experiments have revealed the structure of DOTAP CLs and DOTAP/DNA lipoplexes on the nanoscale. Such a nanostructure would have a deep impact on the adsorption of plasma proteins, which is to a large extent an electrostatically driven phenomenon.  
 
Figure 1.  (a) SAXS pattern of DOTAP cationic liposomes. (b) SAXS pattern of DOTAP/DNA
lipoplexes. Bragg peaks arise from the multilayered lipid membrane/DNA structure. The black
arrow indicates the DNA peak arising from the 1D DNA-DNA in-plane correlation. Adapted
with permission from (Langmuir, 2011, 27 (24), pp 15048–15053). Copyright (2011) American
Chemical Society.

Retrieve article

Evolution of the Protein Corona of Lipid Gene Vectors as a Function of Plasma Concentration;
G. Caracciolo, D. Pozzi, A.L. Capriotti, C. Cavaliere, P. Foglia, H. Amenitsch and A. Laganà;
Langmuir 27, 15048 (2011).
10.1021/la202912f