The functional dissection of the plasma corona of SiO₂-NPs spots histidine rich glycoprotein as a major player able to hamper nanoparticle capture by macrophages

Abstract

A coat of strongly-bound host proteins, or hard corona, may influence the biological and pharmacological features of nanotheranostics by altering their cell-interaction selectivity and macrophage clearance. With the goal of identifying specific corona-effectors, we investigated how the capture of amorphous silica nanoparticles (SiO2-NPs; Ø = 26 nm; zeta potential = -18.3 mV) by human lymphocytes, monocytes and macrophages is modulated by the prominent proteins of their plasma corona. LC MS/MS analysis, western blotting and quantitative SDS-PAGE densitometry show that Histidine Rich Glycoprotein (HRG) is the most abundant component of the SiO2-NP hard corona in excess plasma from humans (HP) and mice (MP), together with minor amounts of the homologous Kininogen-1 (Kin-1), while it is remarkably absent in their Foetal Calf Serum (FCS)-derived corona. HRG binds with high affinity to SiO2-NPs (HRG Kd ∼2 nM) and competes with other plasma proteins for the NP surface, so forming a stable and quite homogeneous corona inhibiting nanoparticles binding to the macrophage membrane and their subsequent uptake. Conversely, in the case of lymphocytes and monocytes not only HRG but also several common plasma proteins can interchange in this inhibitory activity. The depletion of HRG and Kin-1 from HP or their plasma exhaustion by increasing NP concentration (>40 μg ml(-1) in 10% HP) lead to a heterogeneous hard corona, mostly formed by fibrinogen (Fibr), HDLs, LDLs, IgGs, Kallikrein and several minor components, allowing nanoparticle binding to macrophages. Consistently, the FCS-derived SiO2-NP hard corona, mainly formed by hemoglobin, α2 macroglobulin and HDLs but lacking HRG, permits nanoparticle uptake by macrophages. Moreover, purified HRG competes with FCS proteins for the NP surface, inhibiting their recruitment in the corona and blocking NP macrophage capture. HRG, the main component of the plasma-derived SiO2-NPs' hard corona, has antiopsonin characteristics and uniquely confers to these particles the ability to evade macrophage capture.

The nanoparticle protein corona formed in human blood or human blood fractions

“…We have previously pointed out the effect of different ratios between particle surface and protein concentrations on the amount of detectable pellet after centrifugation [15, 28]. Furthermore, gradually decreasing the available surface area showed that in human blood proteins the detected proteins in the corona on silica particles was dependent on the ratio of proteins and particle surface area [29] and in an excess of proteins, the corona was dominated by a single protein. A general problem with the centrifugation technique is that the pellet is analyzed.…”

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

“…Optimal fitting of free protein concentration according to the one-site equilibrium equation, being the NPs molar concentration known, allows for obtaining K D and N max values. This has been recently demonstrated for the interaction of human Histidine Rich Glycoprotein (HRG), human Kininogen 1 (Kin 1) and FIB with SiO 2 -NPs [35]. However such method, being based on the measurement of what is left free, is limited by the accuracy of the protein determination assays and by the affinity and binding capability of the NPs to be analyzed.…”

“…Low affinity (high K D ) and/or low capacity (low N max ) binding may introduce a too high experimental error to get reasonable quantitative information. For example, the affinity of HSA for SiO 2 -NPs could be just extrapolated (> 1 µM) and the Nmax was not defined [35]. Alternatively, the amount of NP-bound protein can be determined after nanoparticle washings with any suitable method, as above, and plotted versus free protein (deduced) by fitting data with the Hill model [50].…”

“…With all the caveats emerged, the available data indicate that zeta-potential of the NPs thus seems to be a very influential parameter. [24,35,50,51]. The yellow line acts merely as a guide to the eye to follow the trend of data, without analytical rational behind the shape of the curve.…”

Combined Action of Human Commensal Bacteria and Amorphous Silica Nanoparticles on the Viability and Immune Responses of Dendritic Cells

“…Since the reciprocal interactions between the microbiome and the immune system (22)(23)(24) influence a delicate balance of defensive/proinflammatory and tolerance/anti-inflammatory responses (25)(26)(27)(28)(29)(30), the presence of NPs may represent a factor perturbing host-microbe interactions and, in the end, tissue homeostasis. Such a possibility is indeed suggested by the little evidence that is available: endocytosed NPs and the lipopolysaccharide (LPS) of Gram-negative bacteria synergistically increase the levels of cytokine production by monocytes, macrophages, and DCs (17,(31)(32)(33)(34)(35)(36) and cytotoxicity in A549 epithelial cells (37). In addition, prokaryotic agonists of formyl peptide receptors (FPRs) synergize with different NP types [SiO 2 NPs, bare or pegylated organically modified silica NPs, poly(lactic-coglycolic acid) NPs, and liposomes] in determining specific inflammatory responses in monocytes and PMNs (38,39).…”