Gold Nanoparticles Modulate Testosterone Metabolism in Human Liver Microsomes: Size, Surface Chemistry, and Protein Corona Effects

Abstract

Gold nanoparticle (AuNP)–protein corona complexes can alter cytochrome P450 (CYP)-mediated testosterone (TST) metabolism by changing their physicochemical properties. We examined how NP size, surface chemistry, and human plasma protein corona (PC) influence TST metabolism in pooled human liver microsomes (pHLM). Twenty‑forty‑nanometer and eighty‑nanometer AuNPs functionalized with branched polyethylenimine (BPEI), lipoic acid (LA), or polyethylene glycol (PEG) were studied, both naked and with PC. In pHLM, 40‑nm naked AuNPs inhibited the formation of five hydroxylated TST metabolites, while 80‑nm particles had a weaker effect. PC largely mitigated these inhibitions. Surprisingly, naked AuNPs increased androstenedione production. In single‑donor HLM, interindividual variability was observed; most AuNPs suppressed TST metabolism at non‑inhibitory concentrations, whereas PC‑PEG‑AuNPs stimulated androstenedione production. These findings highlight AuNPs as potential endocrine disruptors by modulating TST metabolism and provide a framework for screening other nanomaterials.

Introduction

AuNPs are widely used in drug delivery, diagnostics, and consumer products due to their unique optical and physical properties (Refs. 1–3). Upon exposure to biological fluids, AuNPs rapidly adsorb proteins, forming a corona that modifies surface chemistry, protein conformation, and downstream biological responses, including cytotoxicity, cellular uptake, and CYP enzyme activity (Refs. 4–7). In vitro studies have shown that AuNPs can be toxic to human hepatocytes, the C3A hepatoma line, and sperm cells (Refs. 6–8), but the presence of a PC can attenuate or potentiate these effects depending on surface chemistry (Refs. 6–7). PC formation also interferes with cellular uptake in various cell types regardless of NP size or charge (Refs. 6–12).

CYP enzymes in the liver metabolize endogenous and exogenous compounds. Several agents—including drugs, pesticides, and nanoparticles—can disrupt steroid hormone synthesis and metabolism, leading to altered physiological functions (Refs. 13–17). Testosterone (TST) is a primary androgen and a substrate for CYP3A4, which hydroxylates it to 6β‑OH‑TST. Additional hydroxylations by CYP3A4 yield 2β‑OH‑TST, 15β‑OH‑TST, 16α‑OH‑TST, and 16β‑OH‑TST, while CYP2D6 dealkylates TST to androstenedione (AD) (Refs. 17, 19, 27). Prior work indicates that naked and PC‑coated AuNPs modulate a broad spectrum of CYP enzymes, including CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 (Refs. 6, 7, 20–21). AgNPs also suppress CYP3A4 activity in HLM (Ref. 22). BPEI- and LA‑functionalized AuNPs decrease CYP3A4 activity in C3A cells, but PC attenuates this effect (Ref. 7). In vivo, small AuNPs accumulate in liver and spleen and alter hepatic Cyp1a1 and Cyp2b expression (Ref. 23). Given these findings, the current study investigates how PC influences AuNP size, charge, and surface chemistry on CYP‑mediated TST metabolism in pHLM and single‑donor HLM.

Methods

Chemicals

Testosterone, its metabolites, and ¹³C₃‑labeled TST were sourced from MilliporeSigma (St. Louis, MO). Additional reagents were purchased from Steraloids or MilliporeSigma. LC‑MS grade solvents were from Fisher Scientific; ultrapure water was generated by a Merck Synergy® UV‑R system.

Human Liver Microsomes

Pooled HLM (pHLM) from 200 donors (100 male, 100 female) and single‑donor HLM were obtained from Corning Inc. (Charlotte, NC). Donor‑specific CYP activities are detailed in Supplementary Table S1.

Gold Nanoparticle Synthesis

Biopure™ 40‑ and 80‑nm AuNPs functionalized with BPEI, LA, or PEG were purchased from nanoComposix. Core synthesis involved reducing HAuCl₄·3H₂O in K₂CO₃, followed by aging and tangential flow filtration (TFF). Surface functionalization: LA (0.2:1 w/w) or PEG (0.5:1 w/w) was added, followed by TFF and sterile filtration. BPEI surfaces were prepared via EDC/NHS chemistry linking LA carboxyl to BPEI amines; unbound BPEI was removed by TFF and centrifugation.

Protein Corona Preparation

Pooled human plasma (n=5) was incubated with 40‑ or 80‑nm AuNPs at 55 % plasma volume in a 37 °C shaker (250 rpm, 1 h). PC‑AuNPs were collected by centrifugation (20 000×g, 20 °C, 20 min), washed thrice with PBS, and resuspended in PBS for characterization.

Physical Characterization

DLS and TEM measured particle size, polydispersity, and zeta potential. Measurements were taken in DI water, PBS, and microsomal incubation buffer (pH 7.4) at 0 min and 45 min (37 °C). TEM grids were examined on a Tecnai G2 Spirit BioTWIN (120 kV). UV‑Vis spectra were recorded with a Spectra Max i3 reader.

In Vitro Testosterone Metabolism

Linear TST metabolism (10 µM) was confirmed over 1.3–9.3 mg mL⁻¹ microsomal protein for up to 60 min. pHLM were incubated with TST and varying concentrations of naked or PC AuNPs (0–571 µg mL⁻¹). Reactions were initiated with or without NADPH (0.25 mM NADP, 2.5 mM glucose‑6‑phosphate, 2 U mL⁻¹ dehydrogenase) and stopped after 45 min at 37 °C with 4 % phosphoric acid. Supernatants were centrifuged, stored at –20 °C, and processed for LC‑MS/MS. Single‑donor HLM were treated similarly at 10 µg mL⁻¹ AuNPs.

Standards and Sample Preparation

Primary standards (1 mM) and ISTD (¹³C₃‑TST) were prepared in methanol. Working standards ranged from 0.01 to 200 µM. QC samples were 0.01, 0.05, and 0.1 µM. Samples were spiked with ISTD, extracted via Oasis PRIME HLB plates, washed, eluted, diluted, and analyzed by LC‑MS/MS.

Liquid Chromatography‑Mass Spectrometry

Separation used a Waters UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a 600 µL min⁻¹ flow. Gradient: 30 % B to 98 % B over 8.4 min. ESI⁺ source operated at 4000 V, 150 °C; desolvation at 450 °C. MRM transitions are in Supplementary Table S2. Calibration curves spanned 0.001–20 µM. LOD and LOQ were 0.001 µM and 0.005 µM, respectively.

Statistical Analysis

Student’s t‑test assessed D_H and PDI differences. IC₅₀ and EC₅₀ were calculated via Hill equation in GraphPad Prism®. One‑way ANOVA followed by Tukey’s HSD (p < 0.05) evaluated AuNP effects. Pearson r correlated CYP activity with metabolite production.

Results and Discussion

Physicochemical Properties of AuNPs

TEM confirmed monodispersity of all naked and PC AuNPs except 40‑ and 80‑nm PC‑BPEI‑AuNPs, which displayed aggregation in PBS. D_H values increased for 40‑nm naked BPEI‑ and LA‑AuNPs and PC‑PEG‑AuNPs over 45 min, while 80‑nm PC‑LA‑AuNPs decreased. PDI increased for 40‑nm naked and PC‑PEG‑AuNPs, and zeta potential decreased over time for naked BPEI‑AuNPs. These changes mirror previous findings where PC alters NP size, absorbance, and morphology (Refs. 6, 7, 10, 26).

TST Metabolism in Pooled HLM

Six TST metabolites (five hydroxylated and AD) were detected in pHLM. Naked 40‑ and 80‑nm AuNPs inhibited 2β‑OH‑TST, 6β‑OH‑TST, and 15β‑OH‑TST production in a dose‑dependent manner (IC₅₀: 416–1113 µg mL⁻¹). They did not inhibit 16α‑ or 16β‑OH‑TST, except 40‑nm BPEI‑AuNP slightly suppressed 16β‑OH‑TST at the highest dose. Interestingly, naked AuNPs increased AD production at high concentrations (4.3 pmol mg⁻¹ min⁻¹ for 40‑nm BPEI‑AuNP). PC‑coated AuNPs largely abolished these inhibitions; PC also mitigated the stimulatory effect on AD for 80‑nm LA‑AuNP. Overall, naked AuNPs reduced hydroxylation while sometimes enhancing dealkylation, an effect attenuated by PC.

Single‑Donor HLM Variability

Donor‑specific CYP activity correlated strongly with metabolite formation: 6β‑OH‑TST correlated with CYP2C19 (r = 0.99, p = 0.01) and CYP3A4 (r = 0.99, p = 0.03); AD correlated inversely with CYP4A11 (r = –0.98, p = 0.04). AuNPs at non‑inhibitory concentrations modulated metabolite production differently across donors, reflecting CYP polymorphisms and phenotypes (Refs. 33). Size, surface coating, and PC formation all significantly affected metabolite levels (p < 0.0001). PC‑PEG‑AuNPs, for example, activated AD production in donor HDC3 regardless of size.

Conclusions

AuNPs, particularly naked 40‑ and 80‑nm particles, inhibit TST hydroxylation and can stimulate dealkylation in pooled HLM. The presence of a biologically relevant PC mitigates these effects, especially for cationic BPEI‑AuNPs. In single‑donor HLM, AuNP effects are surface‑chemistry dependent and vary with individual CYP activity, underscoring the potential of AuNPs as endocrine disruptors. These results provide a framework for assessing other nanomaterials’ impact on steroid hormone metabolism and identifying vulnerable subpopulations.

Availability of Data and Materials

All data generated or analyzed during this study are included in this article and its supplementary information file.

Abbreviations

11β‑OH TST

11β‑hydroxytestosterone

15β‑OH TST

5β‑hydroxytestosterone

16α‑OH TST

16α‑hydroxytestosterone

16β‑OH TST

16β‑hydroxytestosterone

2α‑OH TST

2α‑hydroxytestosterone

2β‑OH TST

2β‑hydroxytestosterone

6α‑OH TST

6α‑hydroxytestosterone

6β‑OH TST

6β‑hydroxytestosterone

AD

Androstenedione

AgNP

Silver nanoparticles

ANOVA

One‑way analysis of variance

AuNP

Gold nanoparticles

BPEI

Branched polyethylenimine

CFS

Chlorpyrifos

CYP

Cytochrome P450

DEET

Diethyltoluamide

D_H

Hydrodynamic diameter

DI

Deionized water

DLS

Dynamic light scattering

EC₅₀

Half maximal activation concentration

EDC/NHS

1‑Ethyl‑3‑(3‑dimethylaminopropyl) carbodiimide/N‑hydroxysuccinimide

ESI⁺

Electrospray positive

HLM

Human liver microsomes

HSD

Tukey’s honest significant difference

IC₅₀

Half maximal inhibitory concentration

ISTD

Internal standard

LA

Lipoic acid

LC‑MS/MS

Liquid chromatography‑mass spectrometry

LOD

Limit of detection

LOQ

Limit of quantitation

MRM

Multiple reaction monitoring

NADP

Reduced nicotinamide adenine dinucleotide phosphate

NADPH

Reduced NADP

naked

No PC

NP

Nanoparticle

PBS

Phosphate‑buffered saline

PC

Human plasma protein corona

PDI

Polydispersity index

PEG

Polyethylene glycol

pHLM

Pooled human liver microsomes

QC

Quality control

SWCNT

Single‑walled carbon nanotube

TEM

Transmission electron microscopy

TFF

Tangential flow filtration

TiO₂

Titanium dioxide

TST

Testosterone

UPLC TQD

Ultra performance liquid chromatography system with Triple quadrupole Detector