Dopant concentration-dependent size-controlled spherical Cu²⁺-doped Zn Au nanocluster assemblies for efficient cancer theragnostic

Abstract

Assembled metal nanoclusters are a promising material for imaging and optical applications, but face challenges in anticancer therapy due to poor cellular uptake arising from their large size. To address this, a Cu²⁺ ion-doping strategy was employed in a zinc ion-induced gold nanocluster (Zn Au NC) assembly to enhance cellular uptake efficiency. This was achieved by varying doping levels, which enabled control over the size and crystallinity of the doped assembly, thereby enhancing cellular uptake and therapeutic performance. The smallest Cu²⁺ ion-doped Zn Au NC assembly (3.02 nm) exhibited the best anticancer activity with an IC₅₀ value of 107 μg mL⁻¹ (equivalent to 1.6 μg mL⁻¹ of Cu) against HeLa cells. It also exhibited the highest efficacy against MCF-7 cells, with an IC₅₀ value of 132 μg mL⁻¹. Moreover, the dopant-induced red luminescence at 590 nm facilitated cellular imaging, demonstrating combined therapeutic and diagnostic functionality. These findings establish metal ion doping as an effective route to tune the structural and biomedical properties of nanocluster assemblies.

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Introduction

Nanoclusters that form part of an assembly exhibit unique properties, owing to their collective behaviour, which significantly differs from the characteristic properties of individual nanoclusters or bulk materials. These distinct attributes arise from quantum confinement, surface effects, and inter-cluster interactions, giving rise to novel electronic, optical, and catalytic properties. One effective approach to further enhance or introduce new functionalities in such systems involves the integration of trace amounts of chemical species into nanoscale particles. In particular, chemical doping – the intentional introduction of foreign atoms or ions – has emerged as a powerful strategy to generate additional states within materials, thereby enabling emergent properties that are absent in the pristine systems.

Over the past decade, doping has been widely explored in nanomaterials such as semiconductor quantum dots (QDs), carbon nanotubes, and carbon dots.^1–9 Semiconductor QDs are of particular interest due to their size-dependent strong luminescence, enabling applications in solid-state lighting and biological labelling.^10–12 Their electronic and optical properties can be tuned by altering their size, shape, or composition, particularly via metal ion doping.^13–15 Transition metal ions like Cu²⁺ and Mn²⁺ are commonly employed for this purpose. For example, Mn²⁺-doped ZnS and ZnO QDs emit from yellow to red depending on the doping site, while Cu⁺-doped ZnSe QDs shift fluorescence from blue to green due to new acceptor levels.^16–18

Beyond tuning emission properties, doping can significantly impact the structure of QDs. Dopant concentration can modulate the size and crystallinity, as seen with Cu²⁺-doped ZnS QDs forming hexagonal crystals with altered crystallite size.¹⁹ Similarly, Ag⁺ doping changes the shape and size of CdTe QDs, while the size of SnO₂ QDs can be tailored by dopant concentration.^20,21 These examples highlight metal ion doping as a versatile approach to control physicochemical properties through structural modification of QDs. Typically, dopant ions interact with the host lattice, modifying charge distribution, electronic states, stoichiometry, and crystallinity. Additionally, dopants can influence the nucleation and growth process during synthesis via interactions with surface ligands, affecting the crystallinity and morphology of the resulting QDs.²²

While doped QDs offer considerable tunability, doped ultra-small nanoclusters (NCs) extend functionality by combining atomic precision with nanoscale tunability. Unlike QDs, NCs exhibit molecule-like chemical stability, precise compositions, and distinctive reactivity, making them highly attractive for advanced applications.^23–28 For instance, systematic doping of silver atoms into gold NCs has been shown to alter their nonlinear optical properties.²⁹ Importantly, when nanoclusters are organised into assemblies, their collective behaviour can enhance key features including the luminescence quantum yield, luminescence lifetime, temporal stability, and chemical reactivity.^30–38 These attributes position nanocluster assemblies not only as effective platforms for chemical doping but also as superior alternatives to isolated NCs, owing to the greater diversity of potential dopant sites and improved control over property modulation.

In line with these developments, our earlier work demonstrated that introducing Mn²⁺ ions into a Zn²⁺-complexed assembly of Au NCs not only modulated their optical characteristics but also introduced magnetic properties.^39,40 Building on this strategy, in this work, we have incorporated copper ions into zinc ion-induced gold nanocluster (Zn Au NC) assemblies, during synthesis, to impart anticancer activity and modulate the optical properties. Our study indicates that Cu²⁺ doping levels modulated the crystallinity, size, and optical properties, with smaller amorphous assemblies exhibiting enhanced anticancer efficacy. This represents a significant advancement, given that the size-dependent therapeutic potential of NC assemblies remains largely underexplored in comparison with semiconductor QDs.^41,42 Notably, Cu²⁺ doped semiconductor QDs have shown little anticancer activity and have mainly been used for cell imaging.^43,44 In contrast, our copper-doped assemblies combine therapeutic activity with modified optical properties that enable cell imaging capability. This work demonstrates, for the first time, that copper-ion doping can tune the size of a metal nanocluster assembly, in direct correlation with anticancer efficacy, while also imparting optical properties for dual therapeutic and imaging applications.

Results and discussion

Gold nanoclusters (Au NCs) were synthesised by reacting tetrachloroauric acid with mercapto-propanoic acid and L-histidine following a modified previous protocol.³⁰ The as-synthesised Au NCs exhibited weak luminescence at 577 nm under 300 nm excitation (Fig. S1A) and a broad UV-vis absorbance between 230 and 400 nm (Fig. S1B), consistent with the formation of Au NCs over gold nanoparticles (Au NPs). Transmission electron microscopy (TEM) revealed the formation of ∼1.09 nm sized Au NC particles (Fig. S1C). Electrospray ionization mass spectrometry (ESI-MS) indicated an Au₉ unit with isotopic distributions m/z = 3175.84 (Fig. S2A). The species formulated is [Au₉(HIS)₃(MPA)₉ + H]⁺, consistent with the simulation results generated by the mMass software (Fig. S2B). Mass analysis suggests that both ligands (MPA and HIS) act as stabilizers and reducing agents for the Au NCs.

Addition of zinc acetate dihydrate to Au NCs produced a red emission at 642 nm under 300 nm excitation, which is red-shifted by 65 nm from the original NC emission peak (Fig. S3A). The Zn²⁺ ion-induced Au NC assemblies exhibited a delayed fluorescence lifetime of 182 μs and a normal component of 9.45 ns (Fig. S3B, C and Tables S1, S2). TEM revealed formation of 354.9 nm spherical assemblies (Fig. S3D) as the reason for the delayed emission, consistent with earlier reports of Zn-mediated delayed fluorescence in Au and Cu NCs.^30,45,46 SAED confirmed a crystalline hexagonal structure with a 2.59 nm lattice parameter (Fig. S3E). The absence of a surface plasmon resonance band (Fig. S3F) excluded Au NP formation, supporting the Zn²⁺-mediated assembly of Au NCs.

Lastly, zinc acetate dihydrate and varying amounts of copper acetate dihydrate were added to Au NCs and stirred for 2 h. Notably, Cu addition induced a new emission at 590 nm under 300 nm excitation, while the 642 nm peak from Zn Au NC assemblies disappeared (Fig. 1A). The luminescence colour of the reaction mixture shifted from red to orange under UV light (Fig. 1A1 and A2). When the Zn : Cu ratio decreased, the 590 nm emission intensity first increased and then decreased, with the optimum ratio (98.8 : 1.2) giving maximum emission (Fig. 1B), indicating the dopant role of Cu within the Zn Au NC assemblies. The quantum yield at 590 nm was 2.95%, compared to 1.26% for undoped Zn Au NCs at 642 nm. According to literature reports, Cu⁺ complexes exhibit much higher quantum yields (20–90%) than Cu²⁺ complexes due to better quenching efficiency of the latter.^47–50 Thus, Cu in doped Zn Au NCs is likely in the +2 oxidation state. Studies show that increasing the Cu²⁺ dopant concentration in CdZnS QDs shifts cyan emission to yellow, orange, and red.⁴⁹ Likewise, Cu²⁺ complexation with tetraphenyl ethylene-PAR microgels (TPE-PAR MG) yields new peaks at 465 and 580 nm.⁵¹ Considering these reports along with the observed quantum yield and emission maxima, the 590 nm emission is attributed to Cu²⁺ incorporation within the Zn Au NC assemblies.

Fig. 1 (A) Emission spectra of (a) Zn Au NCs and (b) Cu doped Zn Au NCs under 300 nm excitation. Digital images of (A1) Zn Au NCs and (A2) Cu doped Zn Au NCs illuminated under 300 nm excitation. (B) Emission spectrum recorded at 300 nm excitation, (C) excitation spectrum, and (D) UV-visible spectrum of Cu doped Zn Au NC assemblies at different Zn : Cu weight ratios (w : w, %).

The excitation spectra for the emission at 590 nm (doped assembly) and 642 nm (undoped assembly) showed clear changes with doping. At Zn : Cu = 100 : 0, three features were observed: a strong peak at 376 nm, a weaker broad band at 283 nm, and a weak peak at 334 nm (Fig. 1C). At Zn : Cu = 99.5 : 0.5, the 375 nm peak got quenched and blue-shifted, the 334 nm peak disappeared, and a stronger peak emerged at 289 nm. With a further decrease in the Zn : Cu ratio, the 375 nm peak vanished completely, while an intense peak appeared at 293 nm along with a shoulder peak at 328 nm.

In the absorption spectra, the peak at 356 nm shifted to 334 nm as the Zn : Cu ratio decreased, with a strong new peak appearing around 330 nm (Fig. 1D). These changes in absorption and excitation spectra suggest modifications in the electronic states of the assembly, likely due to the incorporation of dopant states within Zn Au NCs. Furthermore, Tauc plot analysis from the UV-vis spectra showed that the band gap energy of the Zn Au NC assembly increased from 2.18 eV to 2.35 eV upon copper ion incorporation (Fig. S4). This increase indicates changes in the optical properties, as evidenced by the blue shift observed in the absorption, emission, and excitation maxima, in clear correlation with the UV-vis and photoluminescence spectra.

Luminescence lifetime measurements (Table S3 and Fig. S5) revealed that lifetimes varied with the Zn : Cu (w/w, %) ratio added to the reaction mixture containing Au NCs. For undoped Zn Au NCs (Zn : Cu = 100 : 0), the 642 nm peak exhibited a lifetime of 9.45 ns. In doped assemblies, the 590 nm peak's lifetime increased from 6.43 ns to 39.67 ns, as the Zn : Cu ratio decreased from 99.5 : 0.5 to 98.8 : 1.2 and then dropped to 19.37 ns at 98 : 2. This decrease suggests enhanced non-radiative relaxation at a higher Cu content, correlating with quenching of the 590 nm emission.

The cause of enhanced non-radiative relaxation at high copper levels in the doped assembly, as well as the stability of the assembly after copper ion doping, was investigated by TEM. TEM analysis confirmed structural changes, showing a reduction in assembly size from 354.9 nm to 3.02 nm as the Zn : Cu ratio decreased from 100 : 0 to 98 : 2 (Fig. 2A–F). The most dramatic size change (113.4 → 13.5 nm) occurred between 99.2 : 0.8 and 98.8 : 1.2, coinciding with rapid spectroscopic changes (Fig. 2A–F and Table S3, Fig. S6). Thus, it appears that small Zn : Cu ratios drastically reduce the assembly size, enhancing non-radiative relaxation, explaining the quenching and shortened lifetime of the 590 nm emission.

Fig. 2 TEM images of Cu doped Zn Au NC assemblies prepared with different Zn : Cu weight ratios (w/w in %): (A) 100 : 0.0, (B) 99.5 : 0.5, (C) 99.2 : 0.8, (D) 98.8 : 1.2, (E) 98.4 : 1.6, and (F) 98 : 2.0. The inset displays the corresponding size distribution for each TEM image.

Several control experiments were carried out to identify the origin of the 590 nm emission in the doped assembly. When copper acetate was added to Au NCs without zinc acetate, the 590 nm emission peak did not appear (Fig. S6A). Adding extra zinc acetate to this mixture of copper acetate and Au NCs also failed to produce the 590 nm emission (Fig. S7A). These results show that the Zn Au NC assembly is essential for the 590 nm emission peak to appear after copper ion doping.

To rule out ion exchange between Zn²⁺ in the Zn Au NC assembly and added Cu²⁺ ions, further control experiments were performed. When copper acetate was added to a pre-formed Zn Au NC assembly, the 642 nm emission of the assembly was quenched, but no new 590 nm peak emerged (Fig. S7B), consistent with Cu²⁺ being a known quencher. Likewise, post-treatment of the copper-doped Zn Au NC assembly with zinc acetate caused no significant change in its emission features (Fig. S7C), and the 642 nm peak of the undoped assembly did not reappear. Together, these experiments confirm that the 590 nm emission develops only when zinc acetate and copper acetate are introduced together during the synthesis of the doped Zn Au NCs assembly.

Control experiments using TEM showed that when extra zinc acetate was added to the copper-doped Zn Au NC assembly (Zn : Cu = 98.8 : 1.2), the particles disaggregated from 13.47 nm (Fig. 2D) into smaller ones of 1.81 nm (Fig. S7D). This structural change may explain the partial quenching of the 590 nm fluorescence (Fig. S7C).

Elemental mapping, using TEM, confirmed the co-presence of elements Au, Cu, Zn, O, N, and S in the doped assembly, substantiating the homogeneous distribution of all the elements within the copper-doped assembly of Zn Au NCs (Fig. S8A–H).

The uptake of copper ions vis-à-viś zinc ions within the doped assembly was monitored using inductively coupled mass spectrometric analysis (ICP-MS) of dispersed doped assembly samples. ICP-MS studies revealed that initially, the doping percentage of Cu increased from 8.68% to 8.92% and thereafter sequentially decreased to 3.63% while the percentage uptake of Zn was relatively stable as the Zn : Cu ratio was decreased from 99.5 : 0.5 to 99.2 : 0.8 and finally to 98 : 2 (Table S4). This trend in the uptake was further confirmed in solid doped samples using a field emission scanning electron microscope fitted with an energy dispersive X-ray spectroscopy (FESEM-EDX) system, i.e., the doping percentage of Cu increased and then decreased (Table S5) as the ratio of Zn : Cu added to the synthesis mixture was decreased. It should be noted that ICP-MS provides bulk elemental quantification after complete dissolution of the assemblies, whereas FESEM-EDX measures the surface or near-surface composition in the solid state. Despite these methodological differences, both techniques consistently revealed that the relative uptake of copper increased and then decreased as the Zn : Cu ratio decreased from 99.5 : 0.5 to 98 : 2. This factor could have contributed to the reduced fluorescence intensity at 590 nm when the Zn : Cu ratio was changed from 98.8 : 1.2 to 98 : 2 (Fig. 1B).

X-ray photoelectron spectroscopic (XPS) analysis was undertaken to confirm the presence of various elements and shed light on their oxidation/chemical states within the doped Zn Au NC assembly. The survey spectrum revealed the presence of electrons with binding energies corresponding to the elements, C, Au, Cu, Zn, O, S and N in the sample (Fig. 3A). High-resolution XPS of Au 4f indicated the presence of Au in the zero-oxidation state (Fig. 3B).³⁹ Similarly, the high-resolution spectrum of Zn confirmed the presence of Zn in the +2 oxidation state (Fig. 3C).³⁹ Interestingly, the high-resolution XPS of Cu 2p confirmed the presence of majority Cu²⁺ species (78.9%) and to a lesser extent Cu¹⁺ species (21.1%) (Fig. 3D), but no Cu(0) species.^52,53 The high-resolution spectrum of N 1s showed the presence of the sp³ and sp² C–N bond (Fig. 3E), while that of C 2p revealed the presence of –C–C, –C=C, –C–N, C=N, –C–S and –C–O groups (Fig. 3F).^54,55 Furthermore, high-resolution spectra of O 1s and S 2p are consistent with the presence of carboxylate groups and thiol groups within the assembly and rule out the formation of oxides and sulphides of Cu and Zn (Fig. 3G and H).^56–59 Thus, XPS results are consistent with the doping of copper within the assembly of Zn Au NCs. These findings were further corroborated by FTIR studies.

Fig. 3 (A) XPS survey spectrum and high-resolution spectra of (B) Au, (C) Zn, (D) Cu, (E) N, (F) C, (G) O and (H) S for the Cu²⁺ doped Zn Au NC assembly.

FTIR studies were carried out to investigate interactions of copper and zinc ions with the ligands MPA and HIS stabilizing the Au NCs within the assembly. The FTIR spectrum of Zn Au NC showed peaks at 1541 cm⁻¹, 1424 cm⁻¹ and 690 cm⁻¹, corresponding to the asymmetric stretch, symmetric stretch, and bending modes of COO⁻, respectively.^31,60 Upon copper ion doping, these peaks shifted: the asymmetric stretching moved to 1536 cm⁻¹, the symmetric stretch broadened, and the bending mode shifted to 675 cm⁻¹ (Fig. S9 and Table S6). These changes indicate that some Zn²⁺ ions coordinated with carboxylates in MPA and HIS were replaced by Cu²⁺. The stronger polarizing interaction of Cu²⁺ with the oxygen of the carboxylate groups altered the COO⁻ frequencies compared to those observed with Zn²⁺ coordination.

Additional peaks at 1444, 1314, 1273, 1145, 1080, and 1057 cm⁻¹ in Zn Au NCs corresponded to the N–H in-plane bending, C=N and C–N stretching (Nτ), =C–H bending (Nπ), =C–N stretching and N–H bending combination, C–C–N stretching, and C–H in-plane bending, respectively. After copper doping, these peaks also shifted: the N–H bending red-shifted to 1437 cm⁻¹, the =C–N stretching and N–H bending modes broadened, the =C–H bending shifted to 1264 cm⁻¹, the C–C–N stretch shifted to 1073 cm⁻¹, and the C–H bending mode broadened completely.^61,62 These changes indicate that Cu²⁺ ions were also incorporated into the assembly by complexation with the N-end of histidine ligands. In the undoped Zn Au NC assembly, zinc ions primarily bonded via carboxylate groups, leaving N–H groups of histidine unbound. Upon Cu²⁺ incorporation, Cu–N bonds readily formed due to the stronger polarizing power, favourable d⁹ ligand field stabilization, and better hard-soft-acid–base (HSAB) compatibility of Cu²⁺ with N-donors, leading to the observed changes in N–H stretching frequencies.

As seen in Fig. S3E, the original Zu Au NC assembly was crystalline. Interestingly, upon gradual Cu²⁺ introduction, SAED pattern analysis revealed that the crystalline hexagonal Zn Au NC assembly gradually transformed into an amorphous state (Fig. 4). At low Cu²⁺ doping levels (Zn : Cu = 99.2 : 0.8), the diffraction spots become more diffused, likely due to partial disintegration of the assembly into smaller particles. With further doping (Zn : Cu = 98.8 : 1.2), the SAED pattern showed a complete transformation to an amorphous state (Fig. 4). These structural changes are also observed in XRD studies. The host (undoped) Zn Au NC lattice shows reflections at 2θ values of 14.12° and 21.62° (Fig. S10 and Table S7). When small amounts of Cu²⁺ were introduced, the assembly retained the original crystalline structure. However, with decreasing Zn : Cu ratio, the diffraction peaks shifted, consistent with lattice parameter changes caused by increasing dopant incorporation into the host lattice.⁶³ The peaks also broadened considerably, correlating with the reduction in assembly size observed by TEM. Together, SAED and XRD results indicate that Cu²⁺ doping induced progressive lattice distortion, disintegration into smaller assemblies, and ultimately transformation from the crystalline to the amorphous state. However, due to the broad nature of the XRD peaks, detailed crystallographic analysis could not be carried out, and the specific doping sites of Cu²⁺ ions could not be identified from these studies.

Fig. 4 SAED pattern of Cu²⁺ doped Zn Au NC assemblies at different Zn : Cu (w/w, %) ratios: (A) 100 : 0.0, (B) 99.5 : 0.5, (C) 99.2 : 0.8, (D) 98.8 : 1.2, (E) 98.4 : 1.6, and (F) 98 : 2.0 as obtained from TEM analysis.

To identify the doping site of Cu²⁺ in the doped assembly of Zn Au NCs, a room temperature electron paramagnetic resonance (EPR) spectroscopy study was undertaken, following repeated washing of the reaction product. The EPR spectrum revealed four distinct lines arising from the hyperfine interaction between the unpaired electron of Cu²⁺ ions and the magnetic moment of its nucleus^63,65 (I = 3/2) (Fig. 5). These four lines are clearly resolved in all Zn : Cu ratios of Cu²⁺ doped Zn Au NCs. The X-band EPR spectrum of the Cu²⁺ doped assembly of Zn Au NCs recorded at room temperature exhibited anisotropic behaviour with g_‖ = 2.297, g_⊥ = 2.057 and A_II = 91 × 10⁻⁴ cm⁻¹. The spectrum is axial in nature with g_‖ > g_⊥ > 2.0023, indicating that the unpaired electron is localized in the d_x²−y² orbital of Cu²⁺. This is characteristic of a square planar geometry about the Cu²⁺ ion, as it is reported in the literature that g_‖ = 2.42 indicates a distorted octahedral geometry while g_‖ = 2.279 indicates a square planar geometry.^64,65 Therefore, we can conclude that these EPR data show a square planar coordination around the Cu²⁺ ion in the doped Zn Au NC assembly. Additionally, the consistent observation of the hyperfine structure throughout all the different Zn : Cu ratios suggested that there are no electron–electron or spin–spin interactions between Cu²⁺ ions, indicating that the doped Cu²⁺ ions are spaced significantly apart within the assembly of Zn Au NCs at all studied compositions. Thus, FTIR analysis revealed how Cu²⁺ ions bind within the Zn Au NC assemblies, while XPS confirmed their oxidation state. Complementary EPR studies clarified the structural environment around Cu²⁺, together providing a comprehensive view of copper incorporation into the copper doped Zn Au NC assemblies.

Fig. 5 EPR spectra of Cu²⁺ doped Zn Au NC assemblies at varying Zn : Cu ratios (w/w%).

To summarize, the introduction of Cu²⁺ ions into the Zn Au NC assembly resulted in the quenching of the original 645 nm emission and emergence of a new emission peak at 590 nm, attributed to dopant-induced states. In the doped assemblies, Cu²⁺ ions coordinate with a square planar geometry by interacting with the functional groups of MPA and HIS ligands that stabilize the Au NCs. The differing ionic radii, electronic structures, and bonding geometries of Zn²⁺ and Cu²⁺ ions likely contributed to a structural rearrangement, wherein the initially crystalline Zn Au NC assembly particles disintegrated into smaller polycrystalline/amorphous particles upon Cu²⁺ doping. This size transformation, along with the incorporation of Cu²⁺ ions, significantly altered the optical properties of the Cu²⁺ doped Zn Au NC assemblies. Fig. S11 illustrates how the percentage of Cu²⁺ dopant affects the size of the Zn Au NC assemblies. Thus, the incorporation of Cu²⁺ ions led to a decrease in the size of the doped Zn Au NC assemblies. This size reduction is likely due to the difference in ionic radii and electronic structures between Cu²⁺ and Zn²⁺, inducing lattice strain. Furthermore, the binding of Cu²⁺ to stabilising ligands (MPA and HIS) could have restricted the assembly growth of the Au NCs. Such effects are well established in doped QD systems.⁶⁶

The intense emission observed at 642 nm in the Zn induced Au NC assembly is attributed to restricted intramolecular rotations, which effectively suppress non-radiative relaxation pathways in the larger assemblies. Upon doping with Cu²⁺ ions, the formation of a smaller-sized doped assembly induces strong aurophilic interactions along with LMCT/LMMCT interactions. This structural change further restricts non-radiative relaxation processes, thereby increasing both the luminescence lifetime and intensity. Notably, the doped assembly exhibits a new emission at 590 nm, arising from enhanced aurophilic interactions owing to reduced assembly size.⁶⁷ However, the intensity of this new emission diminishes with increasing Cu²⁺ concentration. This decline is attributed to the quenching effect of Cu²⁺ ions, which act as luminescence quenchers at higher concentrations.⁶⁸

The potential of the Cu²⁺ doped Zn Au NC assemblies for cancer theragnostic was explored by evaluating both their therapeutic efficacy and photoluminescence-based imaging capability. While the anticancer activity of zinc and copper ions in various forms is well-documented, reports addressing the size-dependent anticancer efficacy of nanoparticles remain limited.^69–71 Notably, to date, no prior studies have examined the size dependent anticancer efficacy of metal NC assemblies. This study is the first to investigate the impact of doped assembly size on anticancer efficacy, offering new insights into the role of Cu²⁺ doping in enhancing their therapeutic potential. For in vitro cytotoxicity studies, HeLa and MCF-7 were utilized as representative cancer cell lines, while HEK cells served as a normal cell model. MTT assay results revealed no significant cytotoxicity of Cu²⁺ doped Zn Au NCs (Zn : Cu = 98 : 2, IC₅₀ = 320 μg mL⁻¹ and 98.8 : 1.2, IC₅₀ = 348 μg mL⁻¹) in the HEK cell line (Fig. S12A and B), while cytotoxicity was significant in both the cancer cell lines, as discussed below. It is noteworthy that without copper doping, the assembly of Zn Au NCs reveals no significant anticancer efficacy in HeLa and MCF-7 cell lines (Fig. 6A and Fig. S13A).

Fig. 6 Cell viability assay on HeLa cell line using MTT dye in the presence of Cu²⁺ doped Zn Au NCs prepared at different Zn : Cu (w/w, %) ratios: (A) 100 : 0, (B) 99.5 : 0.5, (C) 99.2 : 0.8, (D) 98.8 : 1.2, (E) 98.4 : 1.6, and (F) 98 : 2. The results are expressed as the mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined in comparison with untreated HeLa cells, with the significance level set at p < 0.05(*), p < 0.01(**), p < 0.001(***), and p < 0.0001(****).

As discussed above, the size of the doped assembly was found to vary based on the Zn : Cu ratio used during synthesis (Fig. 2), allowing for systematic evaluation of size-dependent anticancer activity. The in vitro anticancer efficacy of differently sized Cu²⁺ doped Zn Au NC assemblies was assessed against HeLa cells (Fig. 6B–F). The results of this investigation reveal an inverse relationship between the assembly size and anticancer potency: as the size of the copper doped Zn Au NC assembly decreased (from 354.9 nm, 273.4 nm, 113.4 nm, 13.47 nm, and 6.54 nm to 3.02 nm), the IC₅₀ value also decreased (from 282 μg mL⁻¹, 259 μg mL⁻¹, 220 μg mL⁻¹, 199 μg mL⁻¹, and 151 μg mL⁻¹ to 107 μg mL⁻¹) (Fig. 6A–F). A plot of IC₅₀ values versus assembly size (Fig. 7) reveals a marked increase in anticancer activity when the size drops below 14 nm. Next, we wanted to check this size-dependent anticancer activity of the copper-doped assembly of Zn Au NCs in a different cancer cell line. For this, we have used MCF-7 cancer cells to check the size-dependent efficacy of copper-doped assembly of Zn Au NCs. Interestingly, like HeLa cells, a similar trend was observed for MCF-7 cancer cells – the IC₅₀ value decreased (from 603 μg mL⁻¹, 316 μg mL⁻¹, 229 μg mL⁻¹, 212 μg mL⁻¹, and 197 μg mL⁻¹ to 132 μg mL⁻¹) with decreasing size of the copper-doped Zn Au NC assembly (from 354.9 nm, 273.4 nm, 113.4 nm, 13.47 nm, and 6.54 nm to 3.02 nm, respectively) (Fig. S13A–F). Furthermore, the plot of IC₅₀ value versus size indicates that the anticancer efficacy is enhanced effectively when the size drops below 14 nm (Fig. S14). Thus, the size effect on cancer cell lines of the doped assembly appears to be more general. These findings establish that size modulation via Cu²⁺ doping not only alters the physicochemical characteristics of Zn Au NC assemblies but also dramatically enhances their therapeutic performance, highlighting a promising strategy for size optimized nanocluster-based cancer therapeutics.

Fig. 7 Plot of IC₅₀ value of HeLa cell versus the size of Cu²⁺ doped Zn Au NC assemblies at different Zn : Cu ratios.

Control experiments were conducted in which HeLa cells were either treated with only Au NCs, with Zn Au NCs post-synthetically treated with 2 mg of copper acetate, or with copper acetate in Au NCs. In these cases, no significant cancer cell death was observed (Fig. S15A–C). Furthermore, the MCF-7 cancer cell line was treated with only Au NCs, with Zn Au NCs post-synthetically treated with 2 mg of copper acetate, and with copper acetate in Au NCs. In these cases also, no significant cancer cell death was observed (Fig. S15D–F).

Smaller-sized nanomaterials have been reported to more easily penetrate bacterial and mammalian cells.^69–71 In our studies too, the smaller sized doped Zn Au NC assemblies (≤10 nm) probably exhibited greater penetration ability into the HeLa cells, leading to more efficient cancer cell destruction. For larger-sized assemblies, despite having a higher concentration of doped Cu ions (Table S8), their limited cellular entry leads to reduced anticancer efficacy. In contrast, smaller-sized assemblies, which can more effectively penetrate cancer cells, demonstrate enhanced anticancer efficacy even with a lower concentration of doped Cu ions. Therefore, in the presence of Cu ions, the size of the doped assembly plays a crucial role in efficient killing of cancer cells.

Interestingly, as the assembly size decreases, other factors such as zeta potential, the relative amount of surface exposed copper ions, and the total number of assembly particles are also affected (Table S8). These parameters may further influence the anticancer efficacy of the doped assemblies. For instance, an increase in zeta potential (with reduced doped assembly size) could enhance particle attachment to cancer cell membranes (Fig. S16).⁷⁰ Hence, the improved anticancer effect observed with decreasing Cu doped Zn Au NC assembly size may be due to multiple contributing factors. Ultimately, by modulating the Zn Au NC assembly size through Cu²⁺ doping, its anticancer efficacy against HeLa cells was effectively tuned.

Metal ions are known to induce apoptotic cell death by generating reactive oxidative stress (ROS), primarily through Fenton-type reactions.⁷² For example, Zn²⁺ is known to disrupt the mitochondrial function, leading to an increase in ROS levels.⁷³ The Cu-doped Zn Au NC assemblies, in this study, have the potential to induce ROS due to the presence of Cu²⁺ ions and Zn²⁺ ions within the assembly. Additionally, functional groups such as –COOH, present in the ligands of the doped assembly, along with the redox properties of the transition metals, may further enhance ROS production. Furthermore, with increasing Cu²⁺ incorporation, the Zn Au NC assemblies become smaller, leading to higher surface-to-volume ratios and enhanced ROS activities. Cu²⁺ doping also alters the crystallinity of the assemblies, shifting from crystalline to amorphous. This transition increases the number of reactive surface sites, which likely contributes to higher ROS generation. Similar effects have been reported for nanocrystal systems.⁷⁴

Notably, our data revealed a clear correlation between reduced assembly size and increased ROS generation in HeLa cells (Fig. S17 and Table S9), which likely contributes to the enhanced anticancer efficacy observed with smaller-sized doped assemblies. Among the various compositions tested, the Zn : Cu ratio of 98 : 2, when evaluated at its IC₅₀ value (107 μg mL⁻¹)), exhibited the highest anticancer activity (Table S10 and Fig. S18). ICP-MS analysis confirmed that the corresponding Cu concentration at this IC₅₀ value was 1.6 μg mL⁻¹, which falls well within the safe, non-toxic range recommended by the World Health Organization (0.005–30 mg L⁻¹ of Cu²⁺ in water). These findings underscore that the smaller Cu²⁺-doped Zn Au NC assemblies not only possess potential anticancer properties but also maintain biocompatibility, as evidenced by their minimal toxicity towards normal cells. Interestingly, Md Palashuddin et al. reported Cu²⁺ doped carbon nanoparticles as anticancer agents, where the concentration of Cu²⁺ was 2.55 μg mL⁻¹.⁷⁵ In addition, Priya et al. reported copper nanocluster assembly for cancer theragnostic, where the Cu concentration was 10 μM.⁷⁶ Thus, in our case, the therapeutic copper concentration is much lower at 1.6 μg mL⁻¹.

Copper is an essential trace element, and the human body has a built-in mechanism for its absorption, transport, and excretion, with excess copper primarily eliminated through bile into the intestine.⁷⁷ Reports indicate that copper nanoclusters mainly accumulate in the liver and kidneys after administration.⁷⁸ Ultra-small nanoclusters, however, show efficient renal and bowel clearance, limited off-target distribution, and low non-specific tumour uptake, making them attractive candidates for translational cancer theranostics.^79–82 For example, biocompatible Au NPs have been applied for the treatment of triple-negative breast cancer.⁸³ Cu NPs have also been applied for cancer cell imaging and therapy.⁸⁴ These findings highlight that smaller Cu²⁺-doped Zn Au NC assemblies combine anticancer potential with good biocompatibility, as shown by our minimal toxicity towards normal HEK cells. Although in vitro IC₅₀ values cannot fully predict long-term safety due to the complexity of biological systems, they provide useful preliminary evidence of biocompatibility. Future studies are needed to evaluate the pharmacokinetics, biodistribution, and toxicology of copper-doped nanocluster assemblies under physiologically relevant conditions, to better understand their long-term safety and behaviour.

Furthermore, the potential of fluorescent Cu²⁺ doped Zn Au NC assemblies as cell imaging agents was evaluated in HeLa cells. To this end, HeLa cells were incubated for 6 hours with the doped assemblies of two compositions, Zn : Cu = 98.8 : 1.2 (IC₅₀ = 199 μg mL⁻¹) and Zn : Cu = 98 : 2 (IC₅₀ = 107 μg mL⁻¹), and subsequently analysed using fluorescence microscopy. As shown in Fig. 8, the intracellular fluorescence intensities of the assemblies closely mirrored their solution-phase fluorescence profiles (Fig. 1B). Notably, consistent with their emission behaviour in solution, the Zn : Cu = 98.8 : 1.2 assembly exhibited stronger intracellular fluorescence than the Zn : Cu = 98 : 2 assembly.

Fig. 8 Fluorescence imaging of HeLa cells treated with Cu²⁺ doped Zn Au NC assemblies at varying Zn : Cu ratios. The scale bar represents 50 μm.

In addition to fluorescence visualization, the imaging data in Fig. 8 reveal significant morphological changes and reduced cell count with decreasing assembly size, corroborating the size-dependent anticancer efficacy trends observed in Fig. 7. These findings further substantiate that smaller Cu²⁺ doped Zn Au NC assemblies are more effective in inducing cytotoxicity in HeLa cells. Thus, Cu²⁺ doping not only enables modulation of the assembly size and optical characteristics, but also affects therapeutic efficiency, as demonstrated through combined imaging and cytotoxicity outcomes in vitro.

Conclusions

In conclusion, this study demonstrates the effective incorporation of Cu²⁺ ions into zinc ion-induced gold nanocluster (Zn Au NC) assemblies through a controlled doping strategy using copper acetate dihydrate. The introduction of Cu²⁺ ions not only induced a blue shifted emission from 642 nm to 590 nm but also significantly altered the crystallinity and reduced the assembly size. These structural changes had a strong influence on biological activity, with smaller doped assemblies exhibiting markedly enhanced anticancer efficacy against HeLa cells and MCF-7 cancer cells, while remaining non-toxic to normal HEK cells. In addition, the luminescence properties of the doped assemblies enabled effective fluorescence imaging of cancer cells, establishing their dual functionality as therapeutic and diagnostic agents. Overall, this work highlights Cu²⁺ doping as a powerful strategy for modulating the physicochemical and biomedical properties of nanocluster assemblies, opening new avenues for precision design of size-controlled theragnostic platforms.

Materials and methods

Chemicals

Tetrachloroauric acid (Sigma-Aldrich, ≥99.99%), mercapto propionic acid (Sigma-Aldrich, ≥99%), L-histidine (Sigma-Aldrich, ≥99%), zinc acetate dihydrate (Sigma-Aldrich, ≥98%), copper acetate monohydrate (Sigma-Aldrich, ≥98%) and Milli-Q grade water were used to perform experiments.

Synthesis of gold nanoclusters (Au NCs)

Au NCs were synthesized by modifying the following established protocol. 1 mL of 10 mM HAuCl₄ solution was added to 10 mL of Milli-Q water, followed by 0.35 mL of 0.11 M mercapto-propionic acid added under stirring conditions, and then 100 mg of L-histidine was added to the solution and stirred for 15 minutes at 40 °C which gave weak luminescent, ultra-small nanoclusters, as viewed under TEM.

Assembly of zinc-induced Au NCs (Zn Au NCs)

100 mg of zinc acetate dihydrate was added to the previously synthesized Au NCs and stirred for 2 h. Then centrifugation was performed at 12 °C for 15 min at 12 000 rpm and the pellet was collected. Then it was dispersed in water and again centrifugation was performed. Then the final pellet was dispersed in 3 mL of water and utilized for further experiments.

Cu²⁺ doped assembly of Zn Au NCs

In this step, zinc acetate and copper acetate were added at different weight ratios to the synthesized Au NCs and stirred for 2 h (Table S11). Then centrifugation was performed at 12 °C for 15 min at 12 000 rpm and the pellet was collected. Then it was dispersed in water and again centrifugation was performed. Then the final pellet was dispersed in 3 mL of water and utilized for further experiments.

Optical measurements

All fluorescence-related measurements for Au NCs, Zn Au NCs and the Cu²⁺ doped assembly of Zn Au NCs were performed using a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. UV measurements for all the samples were performed using a PerkinElmer Lambda 750 UV-visible spectrophotometer.

Fourier transform infrared spectroscopy

FTIR analysis for Zn Au NCs and the Cu²⁺ doped assembly of Zn Au NCs was performed using a PerkinElmer Spectrum Two spectrophotometer.

Field emission transmission electron microscopy (FETEM) and selected area electron diffraction (SAED) analysis

TEM and SAED of Au NCs, Zn Au NCs and the Cu²⁺ doped assembly of Zn Au NCs were performed using a JEOL JEM 2100F and a FETEM instrument at a maximum acceleration voltage of 200 kV. Samples were prepared by drop casting an aqueous dispersion of samples on a nickel grid and samples were allowed to dry under vacuum.

Field emission scanning electron microscopy (FESEM)

FESEM analysis was performed for the Cu²⁺ doped assembly of Zn Au NCs using the instrument Gemini 300 to find the elemental composition.

Electron spin resonance (ESR) spectroscopy

ESR measurements were performed on all samples using a JEOL JES-FA200.

Inductively coupled mass spectrometry (ICP-MS)

ICP-MS measurements of all samples were performed using an Agilent-7850 ICP-MS system.

Powder X-ray diffraction (XRD) measurements

XRD measurements of all samples were performed using a Rigaku TTRAX III diffractometer instrument.

X-ray photoelectron spectroscopy (XPS) measurement

XPS measurements on all samples were performed using a PHI 5000 Versaprobe III XPS instrument.

MTT assay for cell viability

To determine the IC₅₀ value and cell viability, MTT assay was performed using a HeLa cancer cell line, an MCF-7 cancer cell line and a HEK normal cell line. In a 96-well plate, 10 000 cells per well were seeded, followed by treatment for 48 h. MTT dye (0.5 μg mL⁻¹) was added, followed by incubation for 4 h and the crystals thus formed were dissolved using DMSO. The absorbance reading was taken using a GloMax plate reader at 560 nm wavelength. The data were further processed using GraphPad Prism software.

Uptake study

80 000 cells were seeded in a 35 mm plate and incubated for 24 h in a CO₂ chamber. For bio-imaging, cells were treated with respective obtained IC₅₀ concentrations of the samples. After 3 h of treatment, the medium was discarded and the cells were washed twice using phosphate buffered saline (PBS) and fixed with 4% formaldehyde. Further imaging was carried out using a ZOE microscope.

ROS study

To assess the ROS fold change, 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dye was used. 10 000 cells were seeded in a 96 well-plate for 24 h in a CO₂ incubator and cells were treated with respective IC₅₀ concentrations of the samples at different time points. After treatment, 10 μm of DCFDA dye was added to each well, followed by incubation for 30 minutes. To evaluate the generated ROS in the GloMax plate, fluorescence reading was measured using an excitation wavelength of 475 nm and an emission wavelength of 500–550 nm. Further data were processed using GraphPad Prism software.

Cell imaging

In a 35 mm plate, 75 000 cells were seeded and incubated for 24 h in a CO₂ incubator. Cells were treated with the obtained IC₅₀ concentration of Cu doped assembly of Zn Au NCs for 6 h. Furthermore, cells were washed using PBS and fixed with 4% formaldehyde. Imaging of the cellular uptake of the compound was carried out using a ZOE fluorescence microscope.

Author contributions

The idea was conceived by SD and AP. Experiments were conducted by SD and SR. Data were analysed by all the authors. All the authors have contributed to the preparation and editing of the manuscript. All authors have given consent to the final version of the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The code for the mMass software used in this work is freely available to download at https://mmass.findmysoft.com/. The version of the mMass used was 5.5.0.

Additional data supporting this article have been included as part of the supplementary information (SI). Supplementary information: Additional fluorescence, mass spectra, XRD, XPS, EDX, TEM, MTT assay results, and ROS data. See DOI: https://doi.org/10.1039/d5tb01418b.

Acknowledgements

We thank the Department of Electronics and Information Technology, Government of India [no. 5(9)/2012-NANO (Vol. II)] for financial assistance. We also acknowledge the instrument support from the Centre for Nanotechnology. The Central Instruments Facility, Indian Institute of Technology Guwahati is acknowledged for providing instrumentation facilities. The authors thank Prof. Arun Chattopadhyay and Prof. Shidhartha Sankar Ghosh for helpful discussions. The authors thank Mr. Souradeep Dey for his kind assistance in preparing the cover art figures using BioRender.com.

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