Gold Nanoparticles Functionalized with Angiogenin for Wound Care Application

Abstract

In this work, we aimed to develop a hybrid theranostic nano-formulation based on gold nanoparticles (AuNP)-having a known anti-angiogenic character-and the angiogenin (ANG), in order to tune the angiogenesis-related phases involved in the multifaceted process of the wound healing. To this purpose, spherical were surface "decorated" with three variants of the protein, namely, the recombinant (rANG), the wild-type, physiologically present in the human plasma (wtANG) and a new mutant with a cysteine substitution of the serine at the residue 28 (S28CANG). The hybrid biointerface between AuNP and ANG was scrutinized by a multi-technique approach based on dynamic light scattering, spectroscopic (UV-visible, circular dichroism) and microscopic (atomic force and laser scanning confocal) techniques. The analyses of optical features of plasmonic gold nanoparticles allowed for discrimination of different adsorption modes-i.e.; predominant physisorption and/or chemisorption-triggered by the ANG primary sequence. Biophysical experiments with supported lipid bilayers (SLB), an artificial model of cell membrane, were performed by means of quartz crystal microbalance with dissipation monitoring acoustic sensing technique. Cellular experiments on human umbilical vein endothelial cells (HUVEC), in the absence or presence of copper-another co-player of angiogenesis-were carried out to assay the nanotoxicity of the hybrid protein-gold nanoassemblies as well as their effect on cell migration and tubulogenesis. Results pointed to the promising potential of these nanoplatforms, especially the new hybrid Au-S28CANG obtained with the covalent grafting of the mutant on the gold surface, for the modulation of angiogenesis processes in wound care.

Keywords: AFM; QCM-D; angiogenesis; confocal microscopy; copper; endothelial cells; mutant protein; nanomaterial; nanomedicine; plasmonics.

Figure 1

Ribonucleolytic activity of wtANG and S28CANG. Assay was performed at increasing protein concentration (0.2, 0.5, 1, 1.5 μg/mL), in 33 mM MOPS buffer, at 37 °C for 2 h, with the addition of 6 × 10² μg/mL tRNA. Averaged values and standard deviation from three experiments.

Figure 2

(a) UV-visible spectra of (a) 8.24 × 10⁸ NP/mL AuNPs in 1 mM MOPS (pH = 7.4) before (wine) and after the addition of 5 × 10⁻⁸ M of ANG (red = wtANG; green = rANG; cyan = S28CANG). In the inset a magnification of the plasmon peak is shown. (b) UV-visible spectra of the pellets collected after the washing procedure and re-suspended in MOPS buffer (dashed lines) for bare AuNP and hybrids AuNP-ANG.

Figure 3

Hydrodynamic size of the bare AuNP (4.3 × 10⁸ NP/mL) and the functionalized nanosystems Au-wtANG (2.9 × 10⁷ NP/mL), Au-rANG (1.4 × 10⁷ NP/mL), Au-S28CANG (7.3 × 10⁶ NP/mL), measured by DLS. (***) = p < 0.001 vs. AuNP; (###) = p < 0.001 vs. Au-wtANG (one-way ANOVA).

Figure 4

Atomic force microscopy micrographs of (a) bare AuNP, (b) Au-wtANG, (c) Au-rANG and (d) Au-S28CANG pellets; scale bar = 200 nm. The graphs on the right of each image show the histograms calculated for Z max.

Figure 5

QCM-D curves of frequency (a,b) and dissipation (c,d) shifts corresponding to the fifth overtones (n = 5) for POPC SUVs adsorption followed by the addition of 5 × 10⁻⁸ M free proteins (open symbols; wtANG = red; rANG = green; S28CANG = dark cyan) or AuNP-based systems (solid symbols; 2.3 × 10⁷ NP/mL citrate-capped bare nanoparticles = wine; 2.5 × 10⁶ NP/mL Au-wtANG = red; 1.6 × 10⁶ NP/mL Au-rANG = green; 1.6 × 10⁶ NP/mL Au-S28CANG = dark cyan. Experiments were performed in 1 mM MOPS buffer (pH = 7.4).

Figure 6

Cell viability assay (MTT) on HUVECs untreated (negative control) or treated for 24 h with: 20 nM free ANG proteins; 100 nM CuSO₄; ANG—copper complexes ([Cu(II)]/[ANG] = 5); bare AuNP and AuNP-ANG hybrids, in the absence or presence of 100 nM CuSO₄. The following nanoparticle concentrations were used: 0.5 nM AuNP (8.45 × 10⁶ NP/mL); 0.2 nM Au-wtANG (2.77 × 10⁶ NP/mL); 0.3 nM Au-rANG (2.32 × 10⁷ NP/mL); 0.2 nM Au-S28CANG (1.96 × 10⁶ NP/mL). The bars represent means ± S.D. of three independent experiments performed in triplicate (S.D. = standard deviation).

Figure 7

Representative micrographs of HUVECs, untreated (a): negative control) or treated with the different samples at time 0, 5 h and 24 h after scratch: (b) 20 nM free ANG proteins; (c) AuNP-ANG hybrids in the absence of copper; (d) 100 nM CuSO₄; (e) AuNP-ANG hybrids in the presence of 100 nM CuSO₄. The following nanoparticle concentrations were used: 0.2 nM Au-wtANG (2.77 × 10⁶ NP/mL); 0.3 nM Au-rANG (2.32 × 10⁷ NP/mL); 0.2 nM Au-S28CANG (1.96 × 10⁶ NP/mL). In (f): quantitative analysis of migration assay (wound edge advancement in percent vs. time). Values (means ± SEM) are from three independent experiments. Statistical analysis was performed by pairwise Student’s t-test. (*) p < 0.05, (**) p < 0.01 vs. CTRL; (#) p < 0.05, (##) p < 0.01 vs. the corresponding free ANG protein. Results are expressed as percentage of wound closure with respect to time 0.

Figure 8

Tube formation assay. (a) Representative bright-field optical images of HUVEC cells cultured on Matrigel matrix, untreated and treated for 5 h in M200 supplemented with 1% v/v FBS with: 20 nM free ANG proteins; 100 nM CuSO₄; ANG—copper complexes ([Cu(II)]/[ANG] = 5); bare AuNP and AuNP-ANG hybrids, either in the absence or presence of 100 nM CuSO₄. The following nanoparticle concentrations were used: 0.5 nM AuNP (8.45 × 10⁶ NP/mL); 0.2 nM Au-wtANG (2.77 × 10⁶ NP/mL); 0.3 nM Au-rANG (2.32 × 10⁷ NP/mL); 0.2 nM Au-S28CANG (1.96 × 10⁶ NP/mL). (b) The number of closed networks of vessel-like tubes was counted from three experiments. Data are mean ± S.D. (*) p < 0.05, (**) p < 0.01 vs. CTRL (Student’s t-test).

Figure 9

LSM micrographs of HUVECs (in blue, nuclear staining, λex/em = 405/425–450 nm; in green, CS1-copper probe, λex/em = 543/550–600 nm, in red mitochondrial staining, MitoTracker d.r λex/em = 633/650–655 nm) after 2 h of treatment with: (a) CuSO₄ (1 × 10⁻⁷ M); (b–d) AuNP-ANG hybrids in the presence of CuSO₄ (1 × 10⁻⁷ M) (Au-wtANG: 2.77 × 10⁶ NP/mL corresponding to [AuNP] = 2.1 × 10⁻¹⁰ M; Au-rANG: 2.32 × 10⁷ NP/mL corresponding to [AuNP] = 3.1 × 10⁻¹⁰ M; Au-S28CANG: 1.96 × 10⁶ NP/mL, corresponding to [AuNP] = 2.1 × 10⁻¹⁰ M). Scale bar 10 μm. Intensity value of fluorescence corresponding to the (e) CS1 copper probe and (f) Mitotracker deep red staining. Bars represent means ± SEM of at least 3 experiments; (*) = p < 0.05, (**) = p < 0.01, (****) = p < 0.0001 vs. Cu; (###) = p < 0.01, (####) = p < 0.0001 vs. Au-wtANG (Student’s t-test).