Water-soluble N-heterocyclic carbene stabilized gold nanoparticles by top-down synthesis: performance in catalysis and photoacoustic imaging

Abstract

N-heterocyclic carbenes (NHCs) have emerged as effective capping ligands for gold nanoparticles (AuNPs), offering improved stability and flexibility compared to sulfur-based alternatives. Two synthetic approaches, ‘bottom-up’ (BU) and ‘top-down’ (TD), have been described; however, comparative studies remain scarce. Herein, we report the TD-synthesized water-soluble NHC-stabilized AuNPs (NHC@AuNPs) for catalysis in water and biomedical applications, exhibiting exceptional stability in physiologically relevant conditions. Further, the catalytic ability of TD-NPs in model reactions was compared to their BU counterparts featuring the same NHC ligands. In detail, the catalysts were tested for the reduction of 4-nitrophenol and resazurin, as well as for the biomimetic oxidation of 3,3,5,5-tetramethylbenzidine (TMB) in aqueous conditions. The TD-NPs also showed distinct advantages over the BU NPs as contrast agents in photoacoustic imaging (PAI), further highlighting the differences between the two families of colloids.

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Introduction

Gold nanoparticles (AuNPs) are amongst the most prominent metal colloids and have become increasingly popular over the last few decades due to their array of applications. Their distinct surface plasmon resonance (SPR), high surface-area-to-volume ratio, and biocompatibility make them benchmark systems in catalysis, materials science, and medicine.^1–4 Over the years, the synthesis of AuNPs has been optimized to precisely control their size and shape, both of which are important for their applications.⁵ Another important feature essential for AuNP applicability is colloidal stability; thus, capping ligands are required as stabilizers in solution. Organosulfur-based ligands such as alkyl thiols are the most popular choice due to their high affinity to bind to the ‘soft’ gold surface.⁶ However, the long-term stability of the S–Au bond, especially in physiological conditions, is limited due to the reactivity of thiols to oxidize or be displaced from the NP's surface by competing nucleophiles.

Recently, N-heterocyclic carbenes (NHCs) have emerged as superior AuNP capping ligands.⁷ Not only do NHCs offer increased stability due to the strong σ donation of the NHC at the nanoparticle surface, but they are also less prone to oxidative cleavage compared to Au–S. Furthermore, the steric bulk of the NHC ligand is very different from thiol and phosphine ligands and can influence the association/dissociation behavior and general accessibility of the ‘buried’ AuNP surface. Overall, NHC ligands provide a versatile scaffold for surface functionalization at the heterocycle backbone and wingtip positions (Fig. 1A), opening new horizons in the applications of these materials in targeted medicine and hierarchical hybrid materials.^8–10 Of note, the carbene platform can also include less classical mesoionic carbenes^11–13 and nitrogen acyclic carbenes.¹⁴

Fig. 1 A. NHC@AuNP structure with possible sites for functionalization highlighted (backbone and wingtips). B. General synthesis scheme to produce NHC@AuNPs via either the ‘top-down’ or ‘bottom-up’ approach (NaBH₄ = sodium borohydride). C. Synthesis of water-soluble NHC@AuNPs by the ‘bottom-up’ approach (previous work) and by the ‘top-down’ approach (this work). Exert shows the selected hydrophilic sulfonate imidazolium salts as NHC precursors.

The two main synthetic routes to form NHC-stabilized AuNPs (NHC@AuNPs) are the ‘top-down’ (TD)¹⁵ and ‘bottom-up’ (BU)¹⁶ approaches (Fig. 1B). While the BU strategy involves the direct reduction of preformed Au(I)/(III) NHC complexes, the TD approach is based upon ligand exchange on preformed AuNPs typically capped with alkyl thiol ligands, which are exchanged for the NHC ligands.

The versatility of functionalizing NHCs allows the synthesis of systems bearing water-soluble groups, generating NPs with enhanced colloidal stability in aqueous media. The first examples of water-soluble NHC@AuNPs appeared in 2015, demonstrating the stability of these systems.^17,18 Although there have been a few reports of NHC@AuNP-mediated catalysis in water,^19–22 only one example includes water-soluble NHC@AuNPs.¹⁹

Furthermore, despite a number of water-soluble NHC@AuNPs being reported over the years,^{12,18,23–28} their use in biomedical applications, such as therapy and imaging, or for catalysis in living cells, is still limited.⁷ In 2017, Crudden and co-workers reported the ability of small (∼3 nm) spherical NHC@AuNPs, bearing carboxylic acid groups on the benzimidazole scaffold, to generate a photoacoustic signal upon irradiation, suggesting these colloids are suitable for photoacoustic imaging (PAI).²³

In 2019, Johnson and co-workers demonstrated the ability of a bidentate thiolate–NHC–Au(I) complex to be grafted onto the surface of cetyltrimethylammonium bromide (CTAB)-stabilized gold nanorods (NRs) via a TD approach.²⁹ The water-soluble gold nanorods, featuring triazole-conjugated polyethylene glycol, exhibited high stability in biologically relevant conditions and showed promise as photothermal therapeutic (PTT) agents in vitro.²⁹

Within this framework, Crudden and coworkers reported seminal results highlighting the superiority of ‘top-down’ synthesized NHC@AuNPs bearing long alkyl chain wingtips.³⁰ The stability of monodentate and bidentate NHC@AuNPs synthesized by the ‘top-down’ method was evaluated in tetrahydrofuran (THF) in the presence of dodecanethiol (DDT) and thiophenol, showing superior stability of the bidentate NHC@AuNPs under these conditions.³⁰

Furthermore, Reithofer and coworkers recently highlighted the superior stability of water-soluble azide-PEG-NHC@AuNPs formed by the TD vs. BU approach in various biomedically relevant conditions.²⁵

Despite the rising applications of NHC@AuNPs, and the known influence of the synthesis methodologies on the nanomaterial's properties, there is a lack of systematic studies characterizing the different outcomes of TD vs. BU synthetic approaches for the same NHC system in different applications, i.e., catalytic activity.

In 2022, our group studied water-soluble AuNPs with mono- or bidentate NHC capping ligands bearing sulfonate groups on the wingtips, obtained by the BU approach (Fig. 1C).¹⁹ Therefore, in this study, we report the first direct comparison of the catalytic and photoacoustic imaging performance between TD and BU NHC@AuNPs. Thus, first, we report the TD synthesis of AuNPs featuring the same water-soluble sulfonate-bearing NHC ligands of the published BU strategy (Fig. 1C). Then, the obtained NHC@AuNPs were fully characterized by various analytical and spectroscopic measurements, including UV-visible (UV-vis) absorption spectroscopy, NMR spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), inductively coupled plasma mass spectrometry (ICP-MS), and X-ray photoelectron spectroscopy (XPS). Furthermore, the ability of the TD NPs to catalyze three model reactions in aqueous solution was investigated, and the obtained results were compared to the corresponding BU-made NHC@AuNPs.¹⁹ Firstly, the catalytic reduction of 4-nitrophenol in the presence of sodium borohydride (NaBH₄) was used as a model reaction for wastewater remediation and to serve as a benchmark for the catalytic ability of AuNPs. Next, the reduction of resazurin forming fluorescent resorufin in the presence of hydroxylamine (NH₂OH) was considered, providing a model reaction for a NP-catalyzed bioorthogonal transformation in aqueous solution.³¹ Additionally, the NPs were tested as peroxidase mimics using the colorimetric 3,3,5,5-tetramethylbenzidine (TMB) oxidation reaction in the presence of hydrogen peroxide (H₂O₂) as an alternative to the enzyme horseradish peroxidase (HRP), which is commonly used in biosensing applications.³² Due to the elevated cost and low environmental stability of HRP, enzyme-mimicking NPs for biosensor development have become more popular over the years.^32,33 Finally, we studied the preliminary PAI performance of the NHC@AuNPs as contrast agents produced by either the TD or the BU synthesis using multispectral optoacoustic tomography (MSOT).³⁴ Altogether, our results demonstrate that the physicochemical, catalytic, and photoacoustic properties of NHC@AuNPs are significantly influenced by the selected synthetic strategy, highlighting the need for a mindful choice of approach when designing NHC@AuNPs for different applications.

Results and discussion

The soluble sulfonated N-heterocyclic imidazolium salts, NHC1 and NHC2 (Fig. 1C), and the corresponding BU nanoparticles (AuNP1-BU and AuNP2-BU, respectively) were synthesized according to previously reported methods.^19,35,36 To obtain the TD NPs, dodecyl sulfide (DDS)-stabilized gold nanoparticles (DDS@AuNPs) were prepared following the Brust-Schiffrin method.³⁷ Afterwards, the water-soluble TD-NHC@AuNPs were synthesized by the in situ deprotonation of the imidazolium salts (NHC1 and NHC2) with potassium tert-butoxide before the addition of DDS@AuNPs, yielding the NHC-stabilized AuNP1-TD and AuNP2-TD, respectively (Fig. 1C). The resulting NPs were then isolated by centrifugation and purified by extensive dialysis and lyophilization before characterization (see Experimental for details).

After ligand exchange, the synthesized TD-NHC@AuNPs’ SPR band was slightly blue-shifted compared to the precursor DDS@AuNPs (DDS@AuNPs = 520 nm, AuNP1-TD = 512 nm, AuNP2-TD = 514 nm, Fig. S1). Further, the displacement of DDS by the NHC ligand on the surface of the NPs after dialysis was confirmed by ¹H NMR spectroscopy (Fig. S2 and S3) with no evidence of DDS present in the purified material when compared to the ¹H NMR of DDS@AuNPs in the literature.¹⁷ The disappearance of the H_a imidazolium proton resonance, a slight shielding of the imidazole protons, and general broadening of the signals^38,39 for both AuNP1-TD and AuNP2-TD confirms the formation of the carbene species and completion of the metalation reaction via coordination to Au(0), meaning no free NHC ligand was available on the NP surface. Both ligands, NHC1 and NHC2, show diastereotopic peaks upon coordination to the AuNPs due to the restricted bond rotation.

By TEM, the TD NHC@AuNPs appear quasi-spherical, with a narrow size distribution and a similar size to that of the precursor DDS@AuNPs (5.7 ± 0.9 nm, 4.9 ± 0.9 nm, and 4.7 ± 1.6 nm for DDS@AuNPs, AuNP1-TD, and AuNP2-TD, respectively, Fig. S4 and Fig. 2A, B; Table 1). Additionally, no significant aggregation or etching of the NPs is observed after ligand exchange.

Fig. 2 TEM representative image of the A. AuNP1-TD and B. AuNP2-TD with size histogram and average particle size. Average DLS traces of C. AuNP1-TD and D. AuNP2-TD in MilliQ water over three measurements. E. Average zeta potentials (mV) of the two families of NHC@AuNPs with standard deviation over three measurements. F. TG graphs of AuNP1-TD (red line), AuNP2-TD (blue line), and DDS@AuNPs (grey line) under a controlled atmosphere. G. XPS spectra of the Au 4f region in AuNP1-TD and AuNP2-TD (Au(I) in orange and Au(0) in blue). Au(0) area was used for spectra normalization, while the normalized spectra are shown after Shirley background subtraction to provide a comparison.

Table 1 Properties of the investigated TD NHC@AuNPs in this work and previously investigated BU NHC@AuNPs

NHC@AuNPs	Au particle size^a [nm]	Hydrodynamic size^b [nm]	Au wt^c [%]	Au(0) content^d [%]	Zeta potential^e (mV)
n.d. = not determined.a Average diameter of the AuNPs and standard deviation values as determined by TEM analysis.b Average hydrodynamic size of the AuNPs in MilliQ H2O and standard deviation values over three measurements determined by DLS analysis.c Au weight percentage determined by ICP-MS.d Au(0) content percentage estimated by XPS analysis.e Zeta potential measurement obtained as an average over three experiments with standard deviation.f From ref. 19.
DDS@AuNPs	5.7 ± 0.9	n.d.	n.d.	n.d.	n.d.
AuNP1-TD	4.9 ± 0.9	13 ± 3	10.9	98	−20.1 ± 1.4
AuNP2-TD	4.7 ± 1.6	18 ± 5	38.3	96	−19.3 ± 0.5
AuNP1-BU	2.3 ± 1.1^f	n.d.	12.6^f	72^f	−26.4 ± 1.9
AuNP2-BU	2.6 ± 1.2^f	n.d.	29.0^f	38^f	−34.5 ± 3.3

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Furthermore, DLS in MilliQ H₂O shows hydrodynamic sizes of 13 ± 3 nm for AuNP1-TD (Fig. 2C and Table 1) and 18 ± 5 nm for AuNP2-TD (Fig. 2D and Table 1); the difference in size can be explained by the highly charged hydrophilic surface of the NPs creating a solvation/counterion shell of ∼8 nm for AuNP1-TD and ∼13 nm for the more densely charged AuNP2-TD. The low PDI values highlight the homogeneity of the samples.

Indeed, ζ-potential measurements were also performed to understand the electrostatic interactions between the particles, which can indicate charge-induced stability of the colloidal dispersions in aqueous solution.⁴⁰

The TD NHC@AuNPs exhibited ζ-potentials around −20 mV, while the BU NHC@AuNPs showed values ranging from −25 mV to −38 mV, all consistent with the presence of negatively charged sulfonated groups (Fig. 2E). This suggests that the layer of electrostatic stabilization is slightly higher for BU-AuNPs compared to the TD-AuNPs, with values above 30 mV or below −30 mV considered the most stabilizing.⁴¹

The thermal stability of the NHC@AuNPs was evaluated by TGA (Fig. 2F), with markedly lower desorption temperatures of ligand loss observed for the precursor DDS@AuNPs (∼210 °C, Fig. 2F) compared to the NHC@AuNPs (AuNP1-TD = ∼400 °C and AuNP2-TD = ∼420 °C, Fig. 2F) supporting the successful occurrence of complete ligand exchange based on the increased stability of the NHC ligands on the AuNP compared to thiol alternatives. The BU NPs also showed similar desorption temperatures for ligand loss.¹⁹ Furthermore, ICP-MS studies (Table 1) were conducted to assess the overall gold content, which is relevant for further catalysis studies.

Additionally, XPS was used to confirm the surface composition of the NPs, including the Au(0)/Au(I) ratio. The TD approach yielded NPs with Au(0) content >96% (Fig. 2G), which is significantly higher compared to the BU NHC@AuNPs, whereby a substantial percentage of Au(I) was retained.¹⁹ The presence of characteristic C 1s peaks corresponding to C–C and C–N could also be observed using XPS in both AuNP1-TD and AuNP2-TD at ca. 285 and 286 eV, respectively (Fig. S5A and B, Table S1), as well as the N 1s peak ca. 401 eV (Fig. S6A and B, Table S1). Furthermore, the ligand : Au ratio on the NP surface can be estimated by XPS due to the presence of sulfur on both of the NHC ligands, assuming quantitative replacement of the DDS ligand with the NHC ligand. This assumption is valid since no traces of DDS ligand are present in the corresponding ¹H NMR spectra of the purified AuNPs (Fig. S2 and S3). Using the S 2p peak, we can calculate for AuNP1-TD, the L : Au ratio to be 0.27, and for AuNP2-TD, the L : Au ratio to be 1.16 (taking into account the presence of one and two sulfoxide groups in NHC1 and NHC2, respectively, using relative sensitivity factors of 6.25 for Au 4f and 0.668 for S 2p),^42,43 highlighting the higher ligand coverage of NHC2 on the AuNP surface.

The colloidal stability of the TD NHC@AuNPs was further demonstrated over time (16 h) in MilliQ water at 37 °C by UV-visible absorption spectroscopy (Fig. S7A and B). Both TD-NPs exhibited high stability with no significant changes to the SPR bands over time. Similar results were observed in PBS 1× (phosphate buffered solution, pH 7.4) in the presence of serum albumin (bovine serum albumin, BSA 17 mM) (Fig. S8A and B), the most abundant protein in the mammalian bloodstream.⁴⁴ In this more complex solution, the high stability of the TD NHC@AuNPs remained, with negligible variations in the SPR band over 16 h, which was consistent with the results obtained for the BU NHC@AuNPs.¹⁹ In contrast, thiol-stabilized AuNPs, such as DDS@AuNPs, suffer low stability in these conditions (data not shown).⁴⁵

It should also be noted that the TD NHC@AuNPs showed no cytotoxicity in HGT-1 cells over 24 h (IC₅₀ = <100 µg mL⁻¹, data not shown), which is similar to the results observed for the BU NHC@AuNPs in PC-3 cells over 24 h.¹⁹

Overall, the TD and BU AuNPs present important differences in chemical and structural factors that typically play a role in the catalytic efficiency of gold nanoparticles. For example, a higher concentration of Au(0) may provide more active sites for catalysis, potentially leading to enhanced reaction rates. However, this varies depending on the reaction studied. Similarly, the size of NPs is another crucial factor. Smaller NPs generally possess a higher surface-area-to-volume ratio, offering more catalytically active sites per unit mass than larger NPs.⁴⁶ Additionally, the amount of ligand coverage must be carefully balanced: without stabilizing ligands, the NPs will suffer from aggregation. However, the ligand shell lowers the activity since there is reduced surface accessibility.⁴⁷ Therefore, since the TD and BU NHC@AuNPs display differences in the features described above, we evaluated the catalytic ability of the different NPs in three benchmark reactions, namely the reductions of 4-nitrophenol and resazurin and the peroxidase-mimicking oxidation of 3,3,5,5-tetramethylbenzidine (TMB), all of which are performed in an aqueous environment.

The TD NHC@AuNPs were first tested against the reduction of 4-nitrophenol with NaBH₄ in water, a model reaction used for wastewater remediation.¹⁹ In detail, the formation of 4-aminophenol (λ_max = 300 nm) was monitored by UV-vis absorption spectroscopy over time, following the decrease in the concentration of 4-nitrophenolate at 400 nm (see exemplary spectrum in Fig. S9A). The catalytic reduction of 4-nitrophenol was performed with 0.2 mg of AuNP1-TD (Au = 10.9 μg mL⁻¹) and 0.3 mg of AuNP2-TD (Au = 57.45 μg mL⁻¹), respectively, under the same conditions as the previously reported BU-formed NPs.¹⁹ To correct for the difference in NP mass, the rate constants (k) were normalized (k_nor = k/mass), as previously described by Ribot and coworkers.¹¹ In this reaction, AuNP1-TD displays a higher k of 0.74 ± 0.11 min⁻¹ (k_nor = 3.7 min⁻¹ mg⁻¹), with respect to AuNP2-TD (k = 0.39 ± 0.010 min⁻¹, k_nor = 1.4 min⁻¹ mg⁻¹) (Fig. S9B). This can be explained by the nature of the bidentate ligand NHC2, which could result in more hindered access of the substrate to the binding sites on the NP surface due to a thicker ligand shell, despite the higher Au(0) content on the surface of these AuNPs. This trend was also observed for the BU NPs¹⁹ with k = 1.4 min⁻¹ (k_nor = 7 min⁻¹ mg⁻¹) and 0.21 min⁻¹ (k_nor = 0.7 min⁻¹ mg⁻¹), for AuNP1-BU and AuNP2-BU, respectively.

Moreover, when comparing the AuNP1-TD to the corresponding AuNP1-BU, the rate constant of the latter appears nearly 2-fold higher than that of the TD NPs (k_nor = 3.7 min⁻¹ mg⁻¹vs. 7 min⁻¹ mg⁻¹ for AuNP1-TD and AuNP1-BU, respectively).¹⁹ Taking into account the similar gold content of both systems ([Au] = 10.9 μg mL⁻¹ and 12.6 μg mL⁻¹, respectively), the difference in catalytic activity can be explained by the favorable smaller size of the BU NPs (Table 1), which increases their surface-area-to-volume ratio, and the number of catalytically active sites. On the contrary, in the case of larger AuNP2-TD, the rate constant is almost double that of AuNP2-BU (k_nor = 1.4 min⁻¹ mg⁻¹vs. 0.7 min⁻¹ mg⁻¹, respectively). This can be due to the significantly higher percentage of Au(0) on the surface of the TD NPs (Table 1) and the slightly higher Au content (Au = 57.45 μg mL⁻¹ and 43.5 μg mL⁻¹ for AuNP2-TD and AuNP2-BU, respectively), which together outweighs their larger size.¹⁹

Additionally, the catalytic activity of the NPs was also assessed for the reduction of resazurin with NH₂OH in water, a biocompatible model reaction that is used to assess cell viability.⁴⁸ This reaction can be monitored by fluorescence spectroscopy due to the formation of the fluorescent product resorufin at 584 nm over time. Since an excess of NH₂OH is used, the rate constant (k) of the reaction can be determined by assuming a pseudo-first-order reaction.⁴⁹ To assess the rate constant for resazurin reduction, kinetic fluorescent studies were carried out in a plate reader with low concentrations of the NPs (1.8 µg mL⁻¹) and resazurin (4.5 µM) to avoid inner filter effects. Since all NP solutions have the same concentration in each well (1.8 µg mL⁻¹), the rate constants can be compared directly. In the case of AuNP2-BU, no catalytic activity was observed at these concentrations, which could be related to the lower Au(0) surface content. Instead, AuNP2-TD showed the highest rate constant (k = 3.7 × 10⁻³ ± 6 × 10⁻⁵ min⁻¹, k_nor = 0.02 min⁻¹ µg⁻¹) compared to AuNP1-TD (k = 7.0 × 10⁻⁴ ± 1 × 10⁻⁵ min⁻¹, k_nor = 0.004 min⁻¹ µg⁻¹) and AuNP1-BU (k = 6.0 × 10⁻⁴ ± 1 × 10⁻⁵ min⁻¹, k_nor = 0.003 min⁻¹ µg⁻¹) (Fig. S10).

Metal NPs are among the more promising artificial peroxidases, with AuNPs already proposed as effective peroxidase-mimicking catalysts;^50–53 however, to date, no NHC@AuNPs have been used for this purpose, and only one example uses a NHC-stabilized heterometallic system (NHC@AuPdNPs).⁵⁴ The generation of peroxidase-like active artificial systems, able to operate in conditions other than those of biological relevance, has big potential in chemical synthesis, wastewater remediation, biotechnology, and sensing, among other applications.⁵⁵ Peroxidases are a group of enzymes that allow hydrogen peroxide-mediated oxidations into water and free radicals.⁵⁵ To assess the catalytic activity of our NPs, we followed the oxidation of TMB in the presence of H₂O₂, profiting from the production of a charge transfer complex with distinctive absorptions at 370 and 650 nm,⁵⁶ which were monitored via UV-vis absorption spectroscopy (Fig. 3). The reaction was performed at pH 4 and 37 °C, over 25 min for the different NHC@AuNPs obtained via TD or BU approaches. To compare the different NPs, the turnover frequency (TOF) values for the oxidation of TMB were calculated using the initial rates approximation (see SI); the TOF values referred to the total amount of gold as measured by ICP-MS (Table 1).

Fig. 3 A. UV-vis absorption kinetics (658 nm) for the oxidation of TMB with NHC@AuNPs in the presence of H₂O₂ over 25 min at 37 °C in NaOAc buffer (pH 4.0). Kinetics of reaction shown without catalyst (blank, green line, two experiments), with the addition of AuNP1-TD (red line, Au = 0.05 μg mL⁻¹), AuNP1-BU (blue line, Au = 0.12 μg mL⁻¹), AuNP2-TD (orange line, Au = 0.05 μg mL⁻¹) and AuNP2-BU (teal line, Au = 0.12 μg mL⁻¹) with lines corresponding to an average of three experiments with standard deviation represented by splice. B. TOF values for NHC@AuNPs based on µg of Au (purple bars) in the sample. Statistical significance by nonparametric t-test (*P ≤ 0.05, ****P = 0.00006).

The results obtained show that both TD and BU NPs are able to catalyze this reaction. Interestingly, AuNP2 exhibits markedly faster conversion compared to AuNP1 for both synthetic methods (Fig. 3A). This can be due to an increased negative charge density when the NHC2 ligand is used, which will electrostatically interact with the positively charged TMB molecule, protonated at the reaction conditions.^57–59 However, while this explanation is corroborated by the more negative ζ-potential of AuNP2-BU compared with AuNP1-BU, the TD NPs display very similar ζ-potentials, suggesting different effects also play a role in this reaction.

When comparing the different synthetic approaches, the BU NHC@AuNPs show consistently lower TOF per mol of gold (mol_TMB mol_Au⁻¹ s⁻¹) regardless of the ligand used when compared to the TD counterparts (Fig. 3B, purple bars). Accordingly, the molecular Au(I) NHC compounds AuNHC1 and AuNHC2 did not show any catalytic activity (Fig. S11).

Finally, we carried out phantom studies to investigate the potential of these water-soluble NHC@AuNPs as contrast agents for photoacoustic imaging (PAI), which is a noninvasive imaging technique often used for the diagnosis of disease.⁶⁰ Thus, we tested the TD and BU NHC@AuNPs obtained with the NHC2 ligand (AuNP2-TD and AuNP2-BU) in a tissue phantom model using wavelengths within the ‘biological window’, where less blood and tissue attenuation are expected.⁶¹ We compared the performance of AuNP2 synthesized by BU and TD approaches to evaluate the influence of the Au(0) surface content and NP size on their performance (Table 1). Three different concentrations of the NPs were measured using the multispectral optoacoustic tomography (MSOT, Fig. S12) device within a tissue phantom. MSOT is an advanced optoacoustic approach whereby tissues can be imaged at multiple wavelengths rather than a single excitation wavelength and then processed by unmixing algorithms. This allows the intrinsic chromophores, such as hemoglobin and melanin, to be distinguished, as well as any exogenous agents used,⁶² such as AuNPs.

First, the photoacoustic spectra of the samples were measured between 660–980 nm by calculating the average photoacoustic response in a cross-sectional area of 5 mm² located inside the sample straw. Both synthesis approaches exhibit a consistent linear decline in response to the wavelength increases, with a pronounced dip occurring at 950 nm (Fig. S13A and B). To assess the relative photoacoustic amplitude in relation to NP concentration, we focused on three distinct wavelengths: 675 nm, commonly utilized for melanin sensing; 805 nm, representing the oxy-deoxy isosbestic point; and 930 nm, chosen for its relevance to lipids.⁶⁰ Specifically, AuNP2-TD demonstrated a notably stronger PA response and more linear relationship (R² > 0.99) with increasing concentrations, as illustrated in Fig. S14A. Conversely, the AuNP2-BU showed a non-linear effect for the higher concentrations (Fig. S14B). The concentration and 3D distribution of nanoparticles throughout the entire measured volume were then visualized using MSOT. The results revealed that the TD NPs (Fig. 4A) exhibit exceptionally higher photoacoustic contrast compared to the BU NPs (Fig. 4B), with a uniform response even within the ‘biological window’ despite the location of the SPR band at ca. 500 nm, far from 660–980 nm. The difference between the two samples is particularly evident at the lower concentrations. Furthermore, since the NPs cannot resonate with the NIR irradiation directly, this effect can be due to two-photon absorption, which we previously observed for the photothermal effect of the BU NHC@AuNPs.¹⁹

Fig. 4 Cartoon representation of the photoacoustic imaging (PAI) effect. Optoacoustic images showing the orthogonal views of the measured phantoms when irradiated with 65 different wavelengths ranging from 660 to 980 nm. The color intensity represents an unmixed spectrum for the respective NHC@AuNP concentrations. The grayscale background corresponds to the total PA response at 915 nm. A. AuNP2-TD and B. AuNP2-BU.

Due to the photoacoustic amplitude being an arbitrary value that is dependent on many factors within the setup, it is difficult to directly compare the response to existing data. Usually, larger AuNPs and gold nanorods are used for these studies due to their NIR SPR bands.^63–65 For example, AuNRs at a concentration of 0.08 mg mL⁻¹ showed a PA signal of ca. 250 a.u. in a similar phantom setup.⁶⁶ Furthermore, biodegradable gold vesicles formed from an assembly of AuNPs exhibited a significantly higher photoacoustic response (approximately 10-fold) compared to AuNRs, both with an optical density of 1.0.⁶⁷ However, studies with small nanoparticles are scarce, and no data has been previously reported on applying NHC-stabilized AuNPs within this setup. Overall, the results obtained in this tissue-mimicking phantom are encouraging and suggest further applicability that should be studied in vivo to assess clinical translation.

Conclusions

Here, we have successfully synthesized two different water-soluble NHC@AuNPs by the ‘top-down’ (TD) approach and compared their properties to ‘bottom-up’ (BU) NHC@AuNPs, featuring the same hydrophilic NHC ligands.¹⁹ Various methods were used to fully characterize the NPs, demonstrating the successful ligand exchange between the DDS and NHC ligands on the NP surface. In addition, the NPs formed by the TD approach were slightly larger (∼5 nm) than the BU NPs (∼2 nm) due to retention of the precursor DDS@AuNPs size, with a significantly higher percentage of Au(0) (>96%). Importantly, the high stability in physiologically relevant solutions was retained, as observed for the BU NPs.¹⁹

Moreover, the catalytic ability of the TD NPs was demonstrated for the reduction of 4-nitrophenol and compared to the corresponding BU NPs. Within the general trend, it is worth noting that the larger size of the AuNP1-TD resulted in a lower catalytic activity compared to the smaller AuNP1-BU, despite the markedly increased Au(0) content. In the case of the resazurin reduction, AuNP2-TD showed the highest rate constant, with AuNP1-TD and -BU performing similarly. AuNP2-BU showed no activity at these concentrations, following a similar trend as observed for the 4-nitrophenol reduction.

Additionally, the NPs were identified as peroxidase mimics by catalyzing the decomposition of hydrogen peroxide in the presence of TMB, which can result in more stable and cheaper alternatives to horseradish peroxidase⁵⁵ for applications such as DNA sensing. Interestingly, AuNP2 colloids synthesized by both methods outperformed the respective AuNP1, which we postulate may be due to the increased negative charge localization on the NHC ligands, offering electrostatic attraction to the TMB molecule.

Finally, in preliminary studies, the NPs were investigated as photoacoustic imaging (PAI) probes using multispectral optoacoustic tomography (MSOT) within a tissue-mimicking phantom. Despite their small size compared to classical nanorod systems, the investigated AuNP2-TD showed a high photoacoustic signal that increased linearly with concentration despite the use of wavelengths within the biological window. Overall, our work highlights that besides the generally desirable properties of NHC@AuNPs for catalysis and medicinal applications, significant differences emerge when different synthetic routes are applied to generate otherwise closely related materials, calling for attention to tailoring the synthetic method to the desired application. These cautions must be applied in future work involving the bioconjugation of targeting molecules to the NHC ligand, such as peptides, which could be used for targeted PAI and PTT. This could also be combined with catalysis in cells for the activation of a prodrug or uncaging of a fluorophore, providing a novel multimodal platform for disease diagnosis and management.

Author contributions

S. R. T. and A. C. were responsible for the project conceptualization and supervision. S. R. T. and A. L. A. Q. wrote the first draft of the manuscript, and all co-authors contributed to the results and discussion sections. S. R. T., A. L. A. Q., G. M.-A., and A. C. designed the graphics. S. R. T., A. L. A. Q., and T. T. performed the synthesis and characterization of the AuNPs. G. M.-A. performed the catalysis reactions and evaluated the data. T. R.-P performed the ICP-MS experiments with expertise and resources from V. S. S. Q. carried out XPS experiments and data analysis. D. D. G., P. A., and V. N. contributed to and supervised the photoacoustic measurements. H. S. provided expertise on synthesis. R. A. F. provided expertise on material characterization.

Conflicts of interest

V. N. is a founder and equity owner of Maurus OY, sThesis GmbH, Spear UG, Biosense Innovations P.C. and I3 Inc. All other authors declare that they have no competing interests.

Data availability

The authors confirm that the data supporting this article is available within the article and the supplementary information (SI). Supplementary figures, materials, and methods are included in the SI. See DOI: https://doi.org/10.1039/d5nr03709c.

Acknowledgements

S. R. T. acknowledges the TUM Global Postdoc Fellowship Scheme for funding and the FWF (Austrian Science Fund) for the ESPRIT fellowship (10.55776/ESP708). A. L. A. Q. acknowledges the funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES, Finance Code 001). T. R. P. acknowledges Margarita Salas research grant from the Ministry of Universities of Spain for funding. Dr Carsten Peters (TUM) is kindly acknowledged for his assistance recording TEM data. S. Q. gratefully acknowledges funding by the Deutsche Forschungs-Gemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy-EXC 2089/1-390776260. H. S. thanks CNPq (314399/2023-2) for research fellow.

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