To date, the knowledge gained by nanotechnology has offered a plenty variety of nanoparticles (NPs), which have unique properties, such as tunable surface functionality. Gold nanoparticles (AuNPs) are highly remarkable in biomedicine due to their physicochemical, electronic, and intrinsic optical properties (1–4).

AuNPs are widely used in biomedicine as antitumor drug delivery vehicle (5–7), theragnostic platforms (8, 9), thermotherapy (10, 11), gene therapy (12, 13), and diagnostic (14).

AuNPs can be synthesized tuning size and shape particles by different strategies; for instance, thermal, electrochemical, chemical methods, and biosynthesis (15–21). Particularly, chemical methods use a gold salt (Au³⁺) precursor and different reducing agents, such as sodium borohydride, sugars, polyols, gallic acid (GA), and citric acid (CA). Molar proportions of reagents, temperature and reaction time influence the shape and particle size (22).

In this sense, the formation of AuNPs occurs in various steps. Firstly, the dissociation of HAuCl₄ (strong acid) in water to H⁺ + AuCl4-, and CA to citrate (weak conjugated base) + H⁺. Later, citrate molecules provide electrons to the metallic ions, which form seed particles. Then, some AuCl₃OH⁻ molecules interact with the seed particles to growth into AuNPs (23). Finally, the excess of citrate molecules interacts with the AuNPs surface to increase steric and electrostatic repulsions between particles to increase colloidal stability (24). Similarly, the formation and stabilization of AuNPs occurs in presence if GA. However, this last synthesis is carried out at high pH, favoring the complexation of Au³⁺ by -OH ions and gallate base and consequently, controlling the nucleation process to obtain smaller nanoparticles with homogeneous particle size (25).

The different AuNPs synthesis methods can confer other biological effects. For instance, the spherical AuNPs shape, synthesized with citrate (AuNPsCA; 16–20 nm/150 mM), inhibits cell proliferation in human cholangiocarcinoma cells after 24 h (26). The use of other reduced agents such as GA in spherical AuNPsGA (15 nm/150 μM) reduced the ability to inhibit the growth of cervical cancer cells after 24 h. However, AuNPsGA at the same concentration did not affect normal Vero kidney cells (27).

Interestingly, [Freese et al. (28)] reported that spherical AuNPsCA (18 and 65 nm, 250 μg/mL) can be internalized by human dermal microvascular endothelial cells after 24 h exposure, showing no toxic effects and suggesting that target biology plays a decisive role in toxicity of AuNPsCA (28).

Various spherical sizes of AuNPsCA (3, 5, 7, 10, 30, 60 nm), in concentration of 0.24 to 15.6 μg/mL do not alter the endothelial permeability either promote the release of pro-inflammatory mediators, such as prostaglandins I₂ and E₂ in rat brain microvessel endothelial cells. However, the smallest AuNPsCA (3–7 nm) tend to accumulate into these cells. Moreover, AuNPsCA of 3 nm (7.8 μg/mL or higher) show a moderate decline of the viability cell but unmodified the morphology after 24 h exposure (29).

Pan et al. (30) showed that the cytotoxicity induced by spherical AuNPsCA (1–2 and 5 nm; 110 μM, 24 h of exposure) is size-dependent in evaluations performed in cancer cell cultures of SK-Mel-20 human melanoma, HeLa human cervix carcinoma, L929 mouse fibroblast and J774A1 cells after 24 h of exposure (30), meanwhile, Chi-Ming et al. (31), showed that AuNPs in the range of 3–5 nm after 30 min of exposure, suppressed the vascular endothelial growth factor (VEGF)-induced activation of Akt/eNOs signaling pathway in rhesus macaque choroid-retinal endothelial cell line RF/6A derived from the choroid-retina with no signs of cytotoxicity.

Moreover, 10 nm spherical AuNPsCA were exposed to the NO donor ruthenium complex Cis-[Ru(bpy)₂(NO)(4PySH)]. (PF₆)₃ in a range concentration of 0.3 nM to 10 μM, the combination induced a vasodilator effect from the concentration of 5 μM in precontracted isolated rat aortic rings (32). Recently, 20 nm AuNPsCA promoted a transient vasodilation in mouse 4T1 tumors after intragastric and intravenous administration of these NPs. This effect could be mediated at least in part by the NO production and did not accelerate the tumor growth (33).

On the other hand, a study with spherical AuNPs (14 nm, 1 μg/mL) synthesized with eggplant extract and coated with HA evaluated the incorporation of a small-interfering ribonucleic acid-specific (to silence the expression of IAP-2, an inhibitor of apoptosis). The results showed that the modified AuNPs decreased the cell proliferation and triggered pronounced cell apoptosis in A456 human lung carcinoma cells after 48 h of exposure (34). Also, spherical AuNPs of 30 nm synthesized with eggplant extract coated with HA and metformin 4 μg/mL, induced a reduction in G2/M phase and molecular level apoptosis in HePG2 human liver cancer cells after 48 h exposure, while for that free metformin ranged from 10 μg/mL (35).

The HA is a macromolecule that makes up the endothelial glycocalyx (among other glycosaminoglycans and proteoglycans), which is produced by endothelial cells (36, 37). HA has been implicated in NO production when endothelial cells are exposed to wall shear stress (37, 38). NO is an essential mediator in the regulation of vascular tone since it promotes muscle relaxation and is synthesized from L-arginine by activation of different NO synthase (NOS) isoforms, endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (39, 40).

In this context, the glycocalyx can potentially mediate mechanical transduction; since when the glycocalyx layer is removed, flow-dependent vasodilation and NO production is altered (36, 41). For example, in an experiment where 4–6 cm length of the rat's right superficial femoral artery was incubated with 14 μg/mL of hyaluronidase (HIA) for 20 min to remove HA, NO levels decreased as well as the vasodilation (42).

Our research group has shown that other metallic nanoparticles, such as silver nanoparticles can modulate smooth muscle contraction (43–45) and moreover the AuNPsGA promoted a transient smooth muscle contraction in precontracted rat isolated tracheal rings (46). However, studies on the actions of the AuNPs and their interaction with structures of blood vessels are poorly studied. Thereby, we aim to evaluate the participation of glycocalyx structures in vascular actions induced by AuNPs synthesized by two different methods.

It is undeniable that studies of AuNPs are supplying relevant information for their biomedical applications. However, the biological mechanisms involved are not fully described in the literature. Considering the leading site of AuNPs distribution is blood circulation, most of the toxicological evaluations of AuNPs in animals have been conducted by intravenous administration of AuNPs (4, 7). The vascular system is divided into (1) heart, as a central pump focuses on distributing blood to (2) major vessels, which delivers and returns blood from the heart to (3) minor vessels, which are finally distributed in the organs and tissues (49). Both major and minor vessels have an inner endothelial cell layer (endothelium). Together, the muscle cells and endothelial cells regulate the vascular tone through vasoactive factors (50, 51). One of these factors is nitric oxide (NO), which is synthesized by the nitric oxide synthase (NOS) (39, 40).

The vascular effects induced by NO are controversial, including the regulation of the vascular tone, which are in function of a list of factors such as: (a) the presence of O2.-, this kind of factor can modify the half time, bioavailability, and the concentration of NO, for instance the NO varies in function of the oxygen tension and the O2.-. The concentration range of 10-50 nM, NO has a half-life time around 3-5 s, in excessive concentration of 300 nM, the half-life time could be longer than 30 s; (b) the presence of scavengers like oxyhemoglobin to yield methemoglobin and inorganic nitrate; (c) the reaction of NO with thiols groups presents in the proteins, which formed the S-nitroso thiols; (d) The biological location of the NOS isoforms in conditions by the normal physiological situation, which can produce a balance of NO concentration in the order or picomolar/nanomolar (eNOS/nNOS), or in altered conditions produces NO in the order or micromolar (iNOS). For example, the high NO production that characterized iNOS isoform is expressed in the vascular smooth muscle cells following exposure to pro-inflammatory cytokines promote; hypotension, cardiodepression and vascular hyporeactivity in septic shock (40, 52, 60).

In the vascular smooth muscle cells of thoracic aorta from rat, 5 nm AuNPs (100 mM) synthesized with sodium ascorbate induced vasodilatation dependent on the NP concentration and endothelium-independent by the ability to activate sensitive potassium channels calcium (53). Our findings demonstrated induction of the vasodilation only with AuNPsGA, while AuNPsCA showed contractile effects in aortic rings. A preliminary analysis showed that AuNPsCA could be deposited onto the tissue and then induce contractile effects (Supplementary Figure 2).

An earlier report with AuNPsCA coupled to NO donors induced a dilatory effect on rat aortic rings (32). In our work, the role of NO on the AuNPs vasodilator and vasoconstriction effects were evaluated; AuNPsGA and AuNPsCA actions were related to NO production. The variation on NO levels suggests the activation of different isoforms of NOS, which leads to vasodilation and vasoconstriction, saw that AuNPsGA concentrations increased the basal NO production inducing vasodilation. Furthermore, 0.1 to 10 μg/mL of AuNPs-CA did not increase the basal NO production and caused vasoconstriction, but the AuNPsCA at 100 μg/mL induced NO production with no vasoconstriction effect may explain these results by activating various signaling pathways or the AuNPsCA interaction with the endothelial surface structures. For instance, [Santos et al. (54)] compared the vasorelaxation induced by AuNPs capped with either thioglycolic acid (AuNPTGA) or thioglycolic acid modified with berberine (AuNPTGA-BS). AuNPTGA did not induce vasorelaxation, but the incorporation of berberine onto the particle surface triggered vasorelaxation by cytosolic calcium ions concentration decreased (54). Mohamed et al. (55) reported a vasodilation effect in isolated aorta rings (male Wistar rats) when exposed to AuNPs with different chemical surfaces. The percentage of relaxation was associated with the chemical surface. At 0.030 M, AuNPsCA induced relaxation of ~70%, while AuNPs with polyvinylpyrrolidone (PVP) and mercaptopolyethylene glycol (mPEG) at the same concentration generated ~60 and ~50%, respectively. The authors reported that citrate capped particles did not alter endothelial-dependent vasodilation previously induced by ACh but attenuated endothelial-independent responses induced by sodium nitroprusside. The capping with PVP attenuated the ACh-induced relaxation, whereas mPEG did not (55).

On the other hand, the slight differences in particle size and the apparent value dispersion could trigger different cellular mechanisms. The synthesis of AuNPs with gallic acid allows a more controlled nucleation of Au due to both gallic acid and -OH ions (pH 10) complexing Au³⁺, resulting in smaller nanoparticles (25) with apparent better dispersion than AuNPsCA, as characterization presents (Table 1). Thus, AuNPsGA (11.6 could ± 2.82 nm) may be more compatible than AuNPsCA with higher diameter (15.8 ± 3.56 nm), and which induced contractions in smooth muscle due accumulation and aggregation of particles (Supplementary Figure 2).

However, the evidence found in this work regarding the glycocalyx's role, particularly from hyaluronic acid (HA), suggest that functional groups and/or chemical conformation of the capping agent exhibit variations in the affinity with HA.

With respect to glycocalyx role, a report by Kumagai et al. (42) in the superficial femoral artery of porcine showed that HIA 15 mU/mL (2 h 37°C) decreased about 50% the NO level (concerning a control with no enzyme) and about 15% vasodilation effect (concerning same control). Thus, alterations of glycocalyx may modify intracellular and cytoskeletal structures and activate NO synthase (eNOS), associated with a low NO production (56). It has been showed that citrate-coated iron oxide NP show strong interaction with proteoglycans and glycosaminoglycans in THP-1 monocytes (57); and particularly, AuNPsCA exhibit high affinity to HA in physiological-like solutions (58). The glycocalyx can entrap and accumulate AuNPs and take part in the receptor-mediated endocytosis (59). In the present work, when aortic rings were treated with HIA and AuNPs, the NO production depended on the concentration of the enzyme concentration being more evident, the physiological effects and NO production induced by AgNPsGA at the concentration of 100 μg/mL in the presence of HIA 50 U, which increased the aortic relaxation associated with NO production; however, 100 U of HIA instead promoted the vascular relaxation, did not change the NO levels at the concentrations of AuNPsGA 100 μg/mL, suggesting that other endogenous agents HA-dependent could modulate the relaxation.

In contrast, AgNPsCA has a different profile upon the physiological effect and NO levels with respect to those showed by AgNPsGA. Our results suggest that AuNPs effects are dependent on the capping agents, and endothelial glycocalyx plays an important role in these actions.

Further details are underway to investigate the interaction of AgNPsCA and AgNPsGA with glycocalyx and the physiological effects under normal and pathological conditions.

This work shows at the vascular level the effects of AuNPs with two different capping agents. Contractile effects induced by AuNPs may be due to an interaction between AuNPs and the endothelial glycocalyx. Notably, the removal of HA led to NO production modifications, which triggered an intracellular signal for either vasodilation or vasoconstriction effects. The data generated show the biological importance of the reduce/stabilizing agents (GA and CA) used in the chemical synthesis of AuNPs and turn settings applications of NMs in the biomedical area, bioaccumulation, and route of administration. Our findings contribute to understand the AuNPs effects, their mechanism of action in the vascular system and to benefit biosafety of AuNPs.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study protocols CEID2014033 and CEID202003 were reviewed and approved by CONBIOETICA-24-CEI-003-20190726.

DM-O: investigation, visualization, writing—original draft, and data curation. GN-T: writing—review and editing, investigation, visualization, and data curation. GM-C and GP: validation and data curation. CG: conceptualization, visualization, writing—original draft, supervision, funding acquisition, project administration, and investigation. All authors contributed to the article and approved the submitted version.

This work was supported by Consejo Nacional de Ciencia y Tecnologia through the fellowship of DM-O (633022), CI6-PIFI-09-08-08, Problemas Nacionales CONACyT PN-2017-01-4710.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.