Impact of particle size and pH on protein corona formation of solid lipid nanoparticles: A proof-of-concept study

Abstract

When nanoparticles were introduced into the biological media, the protein corona would be formed, which endowed the nanoparticles with new bio-identities. Thus, controlling protein corona formation is critical to in vivo therapeutic effect. Controlling the particle size is the most feasible method during design, and the influence of media pH which varies with disease condition is quite important. The impact of particle size and pH on bovine serum albumin (BSA) corona formation of solid lipid nanoparticles (SLNs) was studied here. The BSA corona formation of SLNs with increasing particle size (120-480 nm) in pH 6.0 and 7.4 was investigated. Multiple techniques were employed for visualization study, conformational structure study and mechanism study, etc. "BSA corona-caused aggregation" of SLN2‒3 was revealed in pH 6.0 while the dispersed state of SLNs was maintained in pH 7.4, which significantly affected the secondary structure of BSA and cell uptake of SLNs. The main interaction was driven by van der Waals force plus hydrogen bonding in pH 7.4, while by electrostatic attraction in pH 6.0, and size-dependent adsorption was confirmed. This study provides a systematic insight to the understanding of protein corona formation of SLNs.

Keywords: BSA corona-Caused aggregation; Cell uptake; Conformational structure; Medium pH; Nanoparticle-protein interaction; Protein corona; Size effect; Solid lipid nanoparticles.

Graphical abstract

Scheme 1

The experimental framework of the current study.

Figure 1

The particle size distribution of prepared SLNS in pH 6.0 (A) and 7.4 (B) (C) The TEM images of SLNS. (D) The Zeta-potential of prepared SLNS in different pH. (Data are expressed as mean ± SD, n = 3.

Figure 2

The particle size of BSA incubated SLNS in 0, 24 and 48 h in pH 7.4 (A) and 6.0 (B). The zeta-potential of BSA incubated SLNS in 0, 24 and 48 h in pH 7.4 (C) and 6.0 (D). The zeta-potential of BSA incubated SLNS in different pH (E).∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001, Data are expressed as mean ± SD, n = 3, 0 h vs. other groups.

Figure 3

The colloidal stability of SLNS in pH 6.0: The heat map of D_H–BSA concentration (A) and zeta-potential of BSA incubated SLNS in different BSA concentrations (B). The normalized ACF curves of BSA incubated SLNS in different BSA concentrations (C) and the half decay time-BSA concentration plots of SLNS–BSA (D). The images of SLNS with or without BSA in tubes (E) and the 280 nm Abs values of subnatant of BSA incubated SLNS after centrifugal and its ratio (with BSA vs. without BSA) (F). The relative BSA adsorption amount of SLNS determined by BCA assay (SLNS1 vs. other groups) (G). The TEM images of BSA incubated SLNS (H). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001, Data are expressed as mean ± SD, n = 3.

Figure 4

The TEM (A) and CLSM (B) images of BSA incubated SLNS in pH 7.4. Scale bar = 20 μm.

Figure 5

The protein corona formation of SLNS in pH 7.4. The particle size (A) and zeta-potential (B) curves of BSA incubated SLNS in different BSA concentrations. The relative BSA adsorption amount of SLNS determined by BCA assay (SLNS1 vs. other groups) (C). The fluorescence quenching of BSA after incubation with different concentrations of SLNS in 48 h and the red stars represent the isosbestic point (D). The K_SV values of SLNS-BSA in 0, 24 and 48 h (E). The K_a values and the K_ass values of SLNS-BSA in 48 h (F). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001, Data are expressed as mean ± SD, n = 3.

Figure 6

The conformational change of BSA upon interaction with SLNS. The UV–Vis spectra of BSA with or without SLNS2 in pH 6.0 (A) and pH 7.4 (B). The FTIR spectra of BSA with or without SLNS2 in different pH of SLNS2 (C). The CD spectra of BSA with or without SLNS2 in pH 6.0 (D) and pH 7.4 (E).

Figure 7

The cell uptake of SLNS before and after incubation with BSA in different pH: The CLSM images (A) and normalized uptake efficiency (B) of B16 uptake of SLNS before and after incubation with BSA in pH 6.0. The CLSM images (C) and normalized uptake efficiency (D) of RAW 264.7 uptake of SLNS before and after incubation with BSA in pH 7.4. ∗∗∗∗P < 0.0001, Data are expressed as mean ± SD, n = 3.

Figure 8

The ITC power-time data (upper panel) and the subsequent binding isotherm of BSA to SLNS (lower panel).

Scheme 2

The mechanism of BSA corona formation and the impact of particle size and pH. The main driving force was marked by the red box.