High-Curvature Features on Branched Nanoconstructs Circumvent Protein Corona Interference

Abstract

This paper reports how the local nanoscale curvature on nanoparticle constructs determines the protein corona distribution in biological conditions. Using transmission electron microscopy, we found that DNA-gold nanostar nanoconstructs (DNA-AuNS) having positive-curvature tips <5 nm in radius showed less dense and less uniform protein corona layers compared to 50 nm gold nanospheres (DNA-50NPs). Statistical analysis based on type of curvature on AuNS revealed that the protein layer thickness on the tips was lower than that on the neutral and negative curvature regions. Since protein coronas screen ligands on nanoparticles, we used DNA hybridization to evaluate whether local ligand functionality was preserved after adsorption of proteins. DNA-AuNS nanoconstructs with less dense protein coronas hybridized more 5 nm gold nanosphere probes (5NPs) compared to DNA-50NPs. Without the protein corona layer, the two classes of nanoconstructs hybridized higher numbers of 5NPs, and differences due to NP shape were minimal. Notably, we found that the tips of DNA-AuNS nanoconstructs exhibited higher percentages of hybridization compared to neutral and negative curvature regions; this trend was independent of DNA sequence. Our work demonstrates the importance of nanoconstruct curvature in mitigating local protein adsorption and preserving ligand functionality at the single-particle level.

Keywords: DNA hybridization; gold nanostars; ligand functionality; nanoparticle shape; protein corona; surface curvature; targeting.

Figure 1.. Impact of nanoparticle shapes on protein corona distribution.

Negative-stained TEM images of HApt-functionalized 50NPs and AuNS (a) before and (b) after protein corona formation and binary images corresponding to the latter. d: Protein corona thickness measured along normal lines drawn on the contour of AuNP. Average values and relative standard deviation (S.D.) of protein corona thickness on (c) HApt nanoconstructs (50NP, N = 17; AuNS, N = 15) and (d) CpG nanoconstructs (50NP, N = 17; AuNS, N = 20). Statistical significance: ****p < 0.0001, **p < 0.01, Welch’s t-test (equal variance not assumed).

Figure 2.. Effect of curvature on protein corona distribution.

(a-b) Negative-stained TEM images of HApt-AuNS nanoconstructs showing normal lines used for the calculation of protein corona thickness at the tips (high positive curvatures) and flat/negative curvatures. (c) Average protein corona thickness on the tips and flat/negative curvatures of HApt-AuNS (tip regions, N = 77; flat/negative curvature regions, N = 79) and CpG-AuNS (tip regions, N = 123; flat/negative curvature regions, N = 121). Statistical significance: ****p < 0.0001, Welch’s t-test (equal variance not assumed).

Figure 3.. Influence of nanoparticle shapes on the hybridization of DNA nanoconstructs.

(a) Scheme of HApt and cDNA hybridization between nanoconstructs and 5NPs. PEG: Polyethylene glycol (b) TEM images of 5NPs hybridized to HApt-AuNP and PC@HApt-AuNP (AuNP = AuNS or 50NP). (c) Number of 5NPs hybridized to HApt nanoconstructs (PC@HApt-AuNS, N = 183; PC@HApt-50NP, N = 114; HApt-AuNS, N = 224; HApt-50NP, N = 100) and CpG nanoconstructs (PC@CpG-AuNS, N = 327; PC@CpG-50NP, N = 181; CpG-AuNS, N = 374; CpG-50NP, N = 160). Statistical significance: ****p < 0.0001, ns = non-significant. Welch’s t-test (equal variance not assumed).

Figure 4.. Influence of AuNS curvatures on nanoconstruct hybridization.

(a) TEM image analysis to identify 5NPs hybridized to tips of PC@HApt-AuNS. Shaded purple: tip region. Only 5NPs located within the length of HApt-cDNA duplex (15 nm) were considered hybridized to a nanoconstruct. (b) Number of 5NPs hybridized to the tips and flat/negative curvatures per nanoconstruct (PC@HApt-AuNS, N = 183; HApt-AuNS, N = 224; PC@CpG-AuNS, N = 327; CpG-AuNS, N = 374). Statistical significance: ****p < 0.0001. Welch’s t-test (equal variance not assumed).

Scheme 1.. Functionality of DNA Grafted on AuNP Nanoconstructs.

DNA hybridization on 50NP and AuNS nanoconstructs (left) without protein corona and (right) with protein corona.