Unraveling the In Vivo Protein Corona

Abstract

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed "ex vivo" as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

Keywords: biodistribution; in vivo; nanoparticle; plasma; protein corona; serum.

Figure 1

Monitoring the biodistribution and blood circulation of magnetic HES nanoparticles in vivo. (A) Magnetic HES nanoparticles (mgHES) were injected into mice and the blood was isolated after distinct time points (1 min–2 h). Nanoparticles were recovered from the blood via magnetic separation and further the protein corona surrounding nanoparticles after in vivo circulation was analysed. (B) A representative IVIS image of the different organs after treatment with PBS and clodronate-liposomes as control or mgHES nanoparticles. (C) The nanoparticle concentration in blood after distinct time points was measured via a Tecan Plate Reader and normalized based on the initial injected amount of nanoparticles (n = 2–6).

Figure 2

The in vivo corona pattern is not comparable with the in vitro situation. (A) mgHES nanoparticles were incubated with serum or plasma (heparin, EDTA or citrate) for in vitro protein corona analysis for 1 min. (B) Nanoparticles were recovered from the blood stream after 1 min of circulation and purified for protein corona analysis. As visualized by SDS PAGE (A,B) the pattern for the in vivo and in vitro situation highly differs. (C) LC-MS analysis indicates that the total number of proteins identified for the in vivo corona is significantly higher compared to the in vitro situation. Both corona types share a minor number of proteins. (D) The five most abundant proteins for the in vitro coronas after 1 min of incubation and their amount in the in vivo corona after 1 min of blood circulation. The average amount of each protein in % is shown and calculated from technical triplicates.

Figure 3

In vivo protein corona formation occurs rapidly and does not change significantly over time. (A) The protein corona composition in vivo was compared for three different time points (1 min, 10 min or 1 h). The average amount of each protein in % is shown (n = 3–5). The 10 most abundant proteins contribute to ~50 % and indicate no significant difference in their relative abundance over time. (B) To mimic in vivo corona formation, nanoparticles were incubated directly in blood (ex vivo) for 1 min. The heat map of the 20 most abundant corona proteins highlights the similarity between the ex vivo and in vivo corona after 1 min of blood circulation. The average amount of each protein in % is shown and calculated from technical triplicates and biological replicates for ex vivo (n = 2) and in vivo (n = 5). For comparison, nanoparticles were incubated with serum or plasma for 1 min. All identified proteins are summarized in a separate Excel sheet.

Figure 4

Cellular interaction of protein corona coated nanoparticles depends on the anticoagulant and differs for nanoparticles recovered from the blood stream (in vivo) (A) Nanoparticles were incubated with serum, plasma, blood or isolated after in vivo administration. Protein corona coated nanoparticles were subsequently added to macrophages (RAW264.7) for 2 h at a concentration of 75 µg/mL. Cellular association was analysed via flow cytometry. The amount of nanoparticle (NP) positive cells in % is shown. (B) Representative images illustrate the intracellular distribution of the nanoparticles. RAW264.7 cells were treated with serum or citrate plasma coated nanoparticles for 2 h at a concentration of 75 µg/mL. Scale bar: 10 µm. For statistical analysis, a student’s t-test (two-tailed, unpaired) was performed comparing EDTA versus serum, serum versus ex vivo and ex vivo versus in vivo incubated mgHES nanoparticle. ns = not significant, ** p < 0.01 (n = 3).

Figure 5

The biodistribution of the mgHES nanoparticles into different organs over time. (A–D) Animals were treated with PBS, clodronate-liposomes or mgHES nanoparticles (1 mg). Organs were isolated and imaged with IVIS^®. The fluorescent intensity of all organs was analysed over time 1 min–2 h. (A) = Liver, (B) = Spleen, (C) = Lung, (D) = Kidney. For statistical analysis, a two-way ANOVA test was performed comparing PBS versus mgHES nanoparticle treated animals * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 3–5).