Ligand exchange on noble metal nanocrystals assisted by coating and etching of cuprous oxide

Chunyu Zhouab, Yaocai Baib, Fan Yangb, Tao Suna, Liang Zhanga, Yuanqing Caia, Tao Gua, Yun Liua, Mingfu Gong*a, Dong Zhang*a and Yadong Yin*b
aDepartment of Radiology, Xinqiao Hospital, Army Medical University, Chongqing 400037, P. R. China. E-mail: hummer198625@163.com; hszhangd@tmmu.edu.cn
bDepartment of Chemistry, University of California, Riverside, CA 92521, USA. E-mail: yadong.yin@ucr.edu

Received 19th February 2020 , Accepted 1st April 2020

First published on 7th April 2020


We report an oxide-assisted coating-etching process to remove bio-incompatible capping ligands from the surface of noble metal nanocrystals. The method involves the growth of a layer of cuprous oxide (Cu2O) on the nanocrystal surface to compete with the existing ligands, followed by selective etching of the Cu2O layer in the presence of a new ligand. Such a ligand exchange process has its significance in the biomedical applications of noble metal nanocrystals as many of them are not biocompatible due to the cytotoxicity of the original capping ligands. We demonstrate the efficacy of this strategy by focusing on cetyltrimethylammonium bromide-capped gold (CTAB-Au) nanorods, a class of very useful plasmonic nanomaterials with well-known bio-incompatibility due to the cytotoxicity of CTAB. After coating and etching of Cu2O on AuNRs, the CTAB ligands on the nanocrystal surface can be readily replaced by a poloxamer ligand F127, and the resulting AuNRs can be used in computed tomography and optical coherence tomography imaging with higher contrast enhancement than those capped with CTAB ligands. This strategy is scalable, general, and extendable to other types of nanocrystals, and it is expected to open up many new opportunities that have been not possible previously due to the bio-incompatibility of the nanomaterial.


Introduction

Colloidal noble metal nanocrystals have received considerable interest owing to their unique properties and promising applications in biosensing,1 electronics,2,3 medical diagnostics,4–6 therapy,7–10 catalysis,11,12 and so forth. Thanks to the efforts from many research groups, now we can easily access noble-metal nanocrystals with different sizes, morphologies, compositions, and structures by a series of well-established synthesis protocols.13–16 Capping ligands are indispensable in wet chemical synthesis of colloidal nanocrystals. One of its important roles is to be adsorbed on the surface of noble metal nanocrystals, providing interparticle repulsion forces responsible for colloidal stabilization. The other one is to help direct particle growth, controlling the morphology of nanocrystals by selectively adsorbing on specific facets and modulating their relative growth rates.17,18 Various types of capping ligands have been reported, including organic ions (e.g. citrate),19,20 molecules (e.g. alkyltrimethylammonium,13,21,22 mercaptoacetic acid (MAA),23,24 cysteamine,25,26 oleylamine (OAm)),27,28 or polymers (polyvinylpyrrolidone (PVP)).29,30 While some of the capping ligands are biocompatible, such as citrate and cysteamine, many others are detrimental to cells.

Cetyltrimethylammonium bromide (CTAB), a cationic surfactant, is often used as a morphology guiding agent, playing a crucial role in shape controlling and stabilizing of noble metal nanocrystals.1,22,31 However, CTAB-capped nanocrystals cannot be directly used for biological and medical applications,2,32 especially for in vivo applications, because the CTAB molecules have been found to be highly cytotoxic owing to their reaction with the phospholipids in cytolemma or mitochondrial membrane.6,33 Various strategies have been developed to reduce the toxicity of the CTAB-capped nanocrystals. Harsh physical and chemical treatments (cleaning by diluted hydrochloric acid34 or thermal annealing35), coating the surface with an oxide or polymer layer (silica shell36 and polymer shell,37,38 biologic layer39–42), exchange with diethylamine molecules43 and direct surface exchange were the most studied methods to remove CTAB. However, these treatments usually cause significant interparticle sintering or reconstruction of the surface structure, which causes problems for many applications, especially those require a specific size or surface structure.12 The coating process could partially reduce the cytotoxicity, however the cellular uptake decreased accordingly.44,45 Direct surface exchange is straightforward to conduct, though its effectiveness is often questionable.31,46 Unless the second ligand binds more strongly than the initial one, it will be difficult to achieve complete replacement. To this end, Xia et al. reported an indirect ligand exchange method to remove CTAB from the surface of Au nanocrystals through the deposition and etching of Ag,47 which points out some new opportunities for changing unwanted surface ligands with the desired ones.

In this synthesis, we report an indirect ligand exchange method for effectively replacing ligands on different noble metal nanocrystals (Au nanorods, Ag nanodisks, and Pd nanocubes) by coating and etching of cuprous oxide (Cu2O). Compared with Ag, Cu2O has a number of advantages, including simplicity, generality, lower cost, less tendency of forming an alloy with the metal so less contamination to the nanocrystal surface. More importantly, the chemical reactions involved in the coating and etching of Cu2O are considerably mild so this strategy can be used for treating nanocrystals of metals with a higher tendency of oxidation (e.g. Ag).

We started with Au nanorods (AuNRs), because CTAB has a strong binding affinity toward Au, and it is essential in determining the rod shape of AuNRs. To remove the CTAB bilayer bound on the surface of AuNRs, a two-step method was carried out. The indirect ligand exchange involved the coating of a layer of Cu2O in the presence of a poloxamer capping ligand, F127, which is biocompatible and approved by U.S. Food and Drug Administration (FDA) for biomedical uses,48,49 followed by selectively etching the Cu2O layer in the presence of F127 capping ligand (Scheme 1). This strategy is based on our hypothesis that the deposition of cuprous oxide is quasi-epitaxial which excludes the attachment of organic ligand molecules on the nanocrystal surface.50 Once detached, the probability of the reabsorption of CTAB molecules back to the nanocrystal surface is very low due to their extremely low concentration in the solution.


image file: d0qm00092b-s1.tif
Scheme 1 Schematic illustration of capping ligands removal route for AuNRs.

Experimental

Chemicals

Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O) was purchased from Acros Organics. Silver nitrate (AgNO3, 99.9%), sodium hydroxide (NaOH, 98%), ammonium hydroxide (NH3·H2O, 28% in H2O), sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) were purchased from Fisher Scientific. L-Ascorbic acid (AA, BioXtra, ≥99.0%), cetyltrimethylammonium bromide (CTAB) (CTAB, ≥99%), sodium borohydride (NaBH4, 99%), hydroxylamine hydrochloride (NH2OH·HCl, ≥99.0%) and Pluronic F-127 (PEO106-PPO70-PEO106) were purchased from Sigma-Aldrich. Copper(II) chloride dihydrate (CuCl2·2H2O, ACS, ≥99.0%) was obtained from Alfa Aesar. Palladium(II) nitrate dehydrate (Pd(NO3)2·2H2O) and sodium ascorbate were obtained from Aldrich. All chemicals were used as received without further treatment. Milli-Q water with a resistivity of 18.2 MΩ was used in all preparations.

Synthesis of CTAB-capped Au nanorods (AuNRs)

CTAB-capped AuNRs were fabricated using a process similar to a previous report.21 The seed solution for AuNRs was prepared as follows: 5 mL of 0.5 mM HAuCl4 was injected into 5 mL of 0.2 M CTAB solution, followed by injection of 0.6 mL fresh 0.01 M NaBH4 under vigorous stirring, the color of the solution changed from yellow to brownish-yellow immediately. The stirring of seed solution was continued for 2 min and the seed solution was aged for 30 min at 30 °C before use.

To prepare the growth solution, 40 mL of 0.2 M CTAB was added 0.64 mL of 0.01 M AgNO3, followed by addition of 40 mL of 1 mM HAuCl4. Then 0.52 mL of 0.0788 M ascorbic acid was added after the gentle mixing of the solution. The color of growth solution changed from dark yellow to colorless as ascorbic acid is a mild reducing agent. Finally, 96 μL of seed solution was injected into the growth solution. The resultant mixture was stirred for 2 min and left undisturbed at 30 °C for 45 min for AuNRs growth. The final products were isolated by centrifugation at 7000 rpm for 20 min and washed one more time to remove the surfactant, then dispersed in 20 mL Milli-Q water.

Synthesis of CTAB-capped Ag nanodisks

Ag nanodisks capped with CTAB were synthesized based on seed-mediated growth51 with some modification. The seed solution for Ag nanodisks was prepared as follows: 0.3 mL of 10 mM NaBH4 was rapidly injected into the stirring mixture containing 0.25 mL of 10 mM AgNO3 and 10 mL of 1.25 mM sodium citrate. The reaction was processed using continuous vigorous magnetic stirring for 2 min and aged for 2 h at room temperature before use. The final solution displayed a deep yellow color. A particle growth solution was prepared: 2.5 mL of 10 mM AgNO3 and 5 mL of 100 mM L-ascorbic acid were added into 73 mL of 0.1 M CTAB. Then, 2.5 mL of the seed solution was introduced to the reactive system. Finally, 1.0 mL of 1 M NaOH was rapidly added. With gently shaking, the solution immediately showed a color change, varying from light yellow to brown, to red, and finally to green within 5 min. After aging of the above solution at 90 °C for 2 h, the Ag nanodisks were obtained. This solution was centrifuged at 3000g for 10 min to remove micelle CTAB, and this operation was repeated twice. The final sedimentation was stably re-dispersed in 3.5 mL Milli-Q water and later used for Cu2O growth.

Synthesis of CTAB-capped Pd nanocubes

CTAB-capped Pd nanocubes with an average edge length of ∼17 nm were synthesized according to a procedure published elsewhere.22 0.1820 g CTAB and 0.01 g of sodium ascorbate were dissolved in 15 mL Milli-Q water (18.2 MΩ). The solution was heated 50 °C while stirring in order to dissolve the CTAB. Then, 0.0108 g Pd(NO3)2·2H2O in 5 mL H2O was added quickly and the solution was stirred at 50 °C for 30 min. Then, the particles were centrifuged twice (6000 rpm, 10 min) and redispersed in 5 mL Milli-Q water for Cu2O coating.

Synthesis of Metal–Cu2O (Metal = AuNRs, Ag nanodisks, Pd nanocubes) core–shell nanostructures

The core–shell nanostructures were synthesized through the reduction of copper chloride (CuCl2) by hydroxylamine hydrochloride (NH2OH·HCl) under alkaline solution. In a typical synthesis, 1.0 mL of metal nanoparticles from the stock solution was mixed with 8.0 mL of 1 wt% F127 solution containing 50 μL of 0.1 M CuCl2 aqueous solution. After stirring for about 5 min, 125 μL of 1.0 M NaOH was injected into the mixture. Then 350 μL of 0.1 M NH2OH·HCl solution was added and gently shaken for 20 s. The color of the solution turned from purplish red to blue, then gradually became green, and ultimately stabilized in yellow-green after undisturbed The solution was aged at room temperature for 1 h to form the hybrid nanostructures.

Etching of Cu2O shell

The Cu2O shell was removed by dilute sulfuric acid. Typically, the metal@Cu2O nanocrystals were dispersed in a water solution containing 0.5 wt% F127, 1.84 mM H2SO4, in order to thoroughly etching the Cu2O shell, the solution was kept at room temperature for 0.5 h. The F127-capped nanocrystals were obtained by centrifugation and washing with 1.0 wt% F127.

Cells culture

Human non-small cell lung cancer cell line (A549) and human hepatocellular liver carcinoma cell line (HepG2) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). They were both routinely cultured with Dulbecco's high glucose modified eagle medium (DMEM/High glucose, Hyclone, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA) and 1% of penicillin/streptomycin in a humidified incubator (Thermo Scientific, USA) with 5% CO2 at 37 °C. The medium was replaced every other day until a ca. 90% confluency has been reached.

Cytotoxicity analysis

Both A549 and HepG2 cells were seeded in cell culture flask with 25 cm2 effective area at a density of 4 × 104 cells per cm2, and cultured until a ca. 90% confluency was reached. Afterward, the culture medium was discarded, and 1 mL fresh DMEM medium (no serum) containing either CTAB- or F127-capped AuNRs with a series of concentrations of 0, 10, 50, 100, 200 μg mL−1 was added into each well. The serum-free condition was used in our study to minimize the impact of the protein corona. Then, the biotoxicity of the CTAB-capped AuNRs and F127-capped AuNRs toward A549 and HepG2 cells were quantitatively studied by flow cytometry (FCM). According to the manufacturer's instructions (Annexin V: FITC Apoptosis Detection Kit, BD Pharmingen, CA, USA), the A549 and HepG2 cells incubated with serial Au concentration of 0, 10, 50, 100, 200 μg mL−1 for 24 h were firstly washed twice with warm phosphate buffer saline (PBS) solution and resuspended in 1X binding buffer at a concentration of 106 cells per mL. Then the cells were stained with 5 μL Annexin V-FITC and 10 μL PI for 15 min at room temperature in dark. After adding 400 μL of binding buffer, the cells were counted within 60 min by FCM. Live (quadrant I), early apoptotic (quadrant II), late apoptotic (quadrant III), and necrotic (quadrant IV) cells were measured before subsequent analysis by Cell Quest software (Becton-Dickinson). Additionally, the morphology changes of the incubated A549 cells were imaged by using an inverted fluorescence microscope (DMIRB, Leica, Germany).

Characterization

UV-Vis-NIR spectra of the composites were carried on an Ocean Optics HR2000CGUV-NIR Spectrophotometer. Zeta potential measurements were tested on a BECKMAN COULTER Delsa Nano Zeta-potential & particle size analyzer. The morphology of the nanorods was characterized by using a Tecnai T12 transmission electron microscope (TEM) system. The structure of the samples was studied by high-resolution transmission electron microscopy (HR-TEM, FEI Tecnai G2 F30 S-TWIN). The FTIR spectra were collected by a Nicolet 6700 spectrometer with 16 scans at 4 cm−1 solution in the 4000–400 cm−1 region. The concentration of Br was quantified using a Dionex ICS-2000 Ion chromatography. The concentration of Au and Cu in the equilibrated aqueous phase were determined by inductively-coupled plasma atomic emission spectrometry (Thermo Fisher Scientific, iCAP 6300 Duo). To prepare samples for the ICP-MS measurement, an aliquot of nanoparticle suspension (50 μL) was mixed with 450 μL aqua regia for 60 min. Then, the mixture was diluted with 1% HNO3 to reach the level of ppb for each element (Au or Cu) before the ICP-MS measurement.

CT imaging

The CTAB-capped AuNRs and F127-capped AuNRs solutions in the cuvette were imaged in a clinical CT system (Light Speed VCT CT99, GE Healthcare, USA). The suspensions in cuvettes were scanned using a shaped Styrofoam assembly to hold the samples in place. A simultaneous dual-energy scan was acquired at tube voltages of 80 kVp and current of 155 mA. Images were reconstructed in 0.625 mm-thick slices with a field of view of 96 mm, pitch of 0.984[thin space (1/6-em)]:[thin space (1/6-em)]1, gantry rotation time of 0.4 s with table speed of 40 mm per rotation. CT imaging of water filled in the cuvette provides negative control.

OCT imaging

The CTAB-capped AuNRs and F127-capped AuNRs solutions in the cuvette were imaged using an OCT imaging system (Cirrus HD-OCT, Model 4000, ZEISS, America) to determine their OCT enhancement. As a control, water in the cuvette was also imaged with OCT instrumentation in the same condition.

Results and discussion

The CTAB-capped AuNRs were synthesized by the AgI-assisted seed-mediated method.21 They had an average length of 48 ± 3 nm and a diameter of 14 ± 2 nm and exhibited two typical localized surface plasmon resonance (LSPR) bands at 512 and 710 nm, corresponding to the transverse and longitudinal surface plasmon resonance, respectively (Fig. 1a). In the presence of F127, a Cu2O layer was coated on the surface of AuNRs through the reduction of CuCl2 by hydroxylamine hydrochloride (NH2OH·HCl) at room temperature, leading to red-shift in both the transverse and longitudinal plasmon bands owing to the increase in the dielectric constant of the surrounding medium.52 Meanwhile, an intensity increase of the transverse band and decrease of the longitudinal one were also observed, which was resulted from the decrease of the overall aspect ratio of the Au@Cu2O nanorods (Au@Cu2O NRs). As shown in Fig. 1c, the Cu2O coating shell had an average thickness of 11 nm along the length and 16 nm along the width based on the TEM image.
image file: d0qm00092b-f1.tif
Fig. 1 (a) UV-Vis-NIR spectra of CTAB-capped AuNRs, Au@Cu2O NRs, and F127-capped AuNRs. TEM images of CTAB-capped AuNRs, (b) Au@Cu2O NRs, (c) and F127-capped AuNRs (d). (e) HRTEM image of Au@Cu2O NRs. (f and g) HRTEM images and FFT patterns of the sample in (e), with panels (f) and (g) corresponding to regions 1 and 2, respectively. The red dash lines highlight the boundary between Au and Cu2O. The right panels show the FFT patterns of the Au core and Cu2O shell regions. (h) Zeta potential of CTAB-capped AuNRs, Au@Cu2O NRs, and F127-capped AuNRs.

After Cu2O coating, the Au@Cu2O NRs were collected and then re-dispersed in a dilute H2SO4 solution in the presence of Pluronic F127 to selectively remove the Cu2O layer. The solution was left undisturbed for 1 h, and then the nanoparticles were collected and re-dispersed in aqueous F127 solution. The UV-Vis-NIR spectrum and TEM image of the resultant F127-capped AuNRs (Fig. 1a and d) showed no difference from the original AuNRs (Fig. 1b), confirming that the optical property and morphology of AuNRs could be well preserved during the coating-etching process. Our ICP-MS data further confirmed that only a trace amount of Cu (<0.01 wt%) remained in the final products (Table S1, ESI).

HRTEM was used to observe the microstructure. The boundary between the Au nanorod core and Cu2O shell could be clearly distinguished by the brightness contrast (Fig. 1e). Fig. 1f exhibits an HRTEM image of the end region of the Au@Cu2O NRs (region 1 in Fig. 1e) and fast Fourier transform (FFT) patterns of Au and Cu2O, confirming the epitaxial growth of the (111) plane of Cu2O over the facet of Au nanorod.53 Accordingly, HRTEM image and FFT patterns taken over the side view of the Au@Cu2O NRs (region 2 in Fig. 1e) are displayed in Fig. 1g, which indicates the epitaxial growth of the (200) plane of Cu2O along the facet of Au nanorod.

The surface chemistry of AuNRs was further studied to confirm the completeness of the ligand exchange. Since the ligands before and after exchange carry different charges, zeta potential measurement was adopted to qualitatively analyze the surface charges on nanoparticles during the ligand exchange process (Fig. 1h). The ζ-potential of CTAB-capped AuNRs was measured to be +42.1 ± 2 mV, which was caused by the exposure of cationic trimethylammonium head group to the solvent. After being covered with a layer of Cu2O, the NRs showed a zeta potential of −28.4 ± 1.2 mV, which might originate from the adsorption of NH2OH·HCl. After etching the Cu2O shell, the AuNRs showed a zeta potential of −13.8 ± 0.8 mV, which confirmed the binding of F127. Prior studies have shown that F127 could exhibit a slightly negative charge in water due to the presence of negative partial charges on the oxygen sites along its backbone, despite that F127 is typically considered a neutral molecule.54,55

To further prove the effectiveness of this strategy, the nanorods were further examined by Fourier-transform infrared spectroscopy (FTIR) and ion chromatography (Fig. 2). The structural differences between the stabilizers CTAB and F127 offered an opportunity to use FTIR to accurately analyze the surface species (Fig. 2a). Pure CTAB and F127 were also measured as references. Two strong peaks from the stretching modes of the –CH2– group in the region of 2850–2950 cm−1 were observed for the ligand CTAB due to the hydrocarbon chain.56,57 Differ from CTAB, F127 only shows one characteristic peak of –CH2– in the region of 2850–2950 cm−1. Subsequently, FTIR spectra from all the nanoparticles were collected during the ligand exchange process. The CTAB-capped AuNRs showed strong doublet peaks from the stretching modes of the CH2 group in 2850–2950 cm−1, confirming the presence of CTAB on the surface of AuNRs. After the coating and etching process, the doublet peaks changed to be one around 2900 cm−1 in the Au@Cu2O NRs and F127-capped AuNRs, demonstrating successful ligand exchange from CTAB to F127.


image file: d0qm00092b-f2.tif
Fig. 2 (a) FTIR spectra of the AuNRs capped with different ligands and the reference spectra of the pure ligands. (b) Br ion chromatography analysis for remained CTAB in CTAB-capped AuNRs, Au@Cu2O NRs, and F127-capped AuNRs. The stretching modes of the CH2 group in CTAB and F127 are highlighted by the boxes.

As bromide is the characteristic ion in CTAB, the concentration of the CTAB molecules could be monitored through the detection of Br by ion chromatography, quantitatively confirming the effectiveness of this strategy. The concentration of CTAB in CTAB-capped AuNRs was regarded as a reference and set as 100%. The Br ion chromatography analysis showed that after the epitaxial coating process, the CTAB remained in Au@Cu2O NRs significantly decreased (Fig. 2b). After etching the Cu2O shell, the content of CTAB remained in the F127-capped AuNRs continue to reduce to below 0.2%. The trace CTAB in F127-capped AuNRs may come from the residual solution during the washing process. The above results confirmed the effectiveness of the coating and etching strategy in removing CTAB molecules from the surface of AuNRs.

We further demonstrated that this method was effective in achieving complete ligand exchange on other noble metal nanoparticles such as CTAB-capped Ag nanodisks51 and Pd nanocubes.22 The growth of Cu2O shell over the surface of Ag nanodisks and Pd nanocubes was also carried out through the reduction of CuCl2 by using NH2OH·HCl as a reducing agent. TEM images in Fig. 3a–f revealed that the morphology of Ag nanodisks and Pd nanocubes capped with F127 showed no difference from the original CTAB-capped nanocrystals, confirming that the morphology of Ag nanodisks and Pd nanocubes can be well preserved during the coating-etching process. The UV-Vis spectra of Ag nanodisks before and after the ligand exchange process shown in Fig. 3g were almost identical, further prove that the optical property could also be well retained. Zeta potential and FTIR spectroscopy were further employed to prove the effectiveness of this strategy (Fig. S1 and S2, ESI). The zeta potentials of Ag nanodisks during the coating and etching process show a similar trend with the case of AuNRs, as well as the Pd nanocubes. After coating a layer of Cu2O shell, the positive charged CTAB-capped nanocrystals turned into negative and the nanocrystals stay negative after etching the Cu2O shell, indicating the successful ligand exchange. The structural difference between CTAB and F127 was identified by FTIR. Fig. S1b and S2b (ESI) show that the two strong peaks in the region of 2850–2950 cm−1 from CTAB capped Ag nanodisks and Pd nanocubes were replaced by one single peak from F127, indicating the successful ligand exchange from CTAB to F127. The success of extending the ligand exchange process to Pd nanostructures suggests that strict epitaxy may not be required since it has different lattice parameters than Au and Ag.


image file: d0qm00092b-f3.tif
Fig. 3 TEM images of the CTAB-capped Ag nanodisks (a), Ag@Cu2O nanodisks (b), F127-capped Ag nanodisks (c), CTAB-capped Pd nanocubes (d), Pd@Cu2O nanocubes (e), and F127-capped Pd nanocubes (f). (g) UV-Vis spectra of CTAB-capped Ag nanodisks, Ag@Cu2O nanodisks, and F127-capped Ag nanodisks. The scale bar is 50 nm for all the TEM images.

AuNRs are currently used in a range of biological applications including sensing, drug delivery, photothermal therapy, and imaging applications.36,58–61 The ligand exchanged F127-capped AuNRs are expected to show significantly enhanced biocompatibility. To verify it, the cytotoxicity of F127-capped AuNRs was evaluated through flow-cytometry analysis toward human non-small cell lung cancer cell line (A549) and human hepatocellular liver carcinoma cell line (HepG2). Meanwhile, CTAB-capped AuNRs were also investigated for comparison (Fig. 4). The results showed that the toxicity of CTAB-capped AuNRs was in a concentration-dependent manner regardless of the cell line, which can be attributed to the ability of quaternary amines to disrupt the plasma membrane integrity. When the concentration of CTAB-capped AuNRs increased from 10 to 200 μg mL−1, the viability of A549 cells decreased from 93.3% to 5.45%, and the viability of HepG2 cells showed a similar trend from 90.4% to 18.3%. In contrast, when the capping agent was changed to F127, the toxicity of F127-capped AuNRs was largely inhibited, the cells exhibited much higher cell viability. The viability of A549 and HepG2 cells only slightly decreased to 92.5% and 86.8%, respectively, even being cocultured with 200 μg mL−1 F127-capped AuNRs for 24 h. The morphology changes of the incubated A549 and HepG2 cells were also recorded by using an inverted fluorescence microscope (Fig. S3 and S4, ESI). The inverted fluorescence microscope results are consistent with the flow-cytometry analysis. The cells exhibited shrinkage and became round and loosely arranged after even cocultured with 50 μg mL−1 CTAB-capped AuNRs. Differ from the CTAB-capped AuNRs, cells cocultured with different concentrations of F127-capped AuNRs barely showed any morphology changes, indicating negligible toxicity of the F127-capped AuNRs to the cells. Based on these results, it can be concluded that the biocompatibility of F127-capped AuNRs has been greatly improved by the ligand exchange process, further demonstrating the effectiveness of the coating-etching process in removing CTAB molecules from AuNRs.


image file: d0qm00092b-f4.tif
Fig. 4 Flow cytometric analysis of apoptosis induced by the CTAB-capped AuNRs and F127-capped AuNRs in A549 (a) and HepG2 cells (b) for 24 h. The cells were incubated with the CTAB-capped and F127-capped AuNRs respectively, at different concentrations (10, 50, 100, 200 μg mL−1). The apoptosis assay was co-stained by PI and Annexin V-FITC to label live (quadrant I), early apoptotic (quadrant II), late apoptotic (quadrant III), and necrotic (quadrant IV) cells. Viability of A549 (c) and HepG2 cells (d) after co-incubation with 10, 50, 100 and 200 μg mL−1 CTAB-capped AuNRs and F127-capped AuNRs for 24 h.

Computed tomography (CT) and optical coherence tomography (OCT) are two important noninvasive biomedical examination techniques, which produce cross-sectional images based on the absorption of X-ray and scattering of light by bodily structures, respectively. AuNRs as a class of anisotropic nanostructures, exhibit a relatively high X-ray attenuation coefficient, unique localized surface plasmon resonance, and high stability, and therefore represent an ideal contrast agent for both CT and OCT. However, the high toxicity of the surface ligand CTAB molecules limits the use of AuNRs for these applications. Since the cell viability was greatly enhanced upon changing the ligands from CTAB to F127, as shown in the above cytotoxicity test, we further demonstrate that the ligand-exchanged AuNRs can serve as suitable contrast agents in CT and OCT measurements. Fig. 5a and b are the CT images obtained with AuNRs before and after ligand exchange at variable concentrations (0.3, 0.6, 1.2, 1.8, and 2.4 mg mL−1). Interestingly, a moderate signal enhancement (∼25%) was observed when F127-capped AuNRs were used instead of CTAB-capped AuNRs. Fig. 5d and e show the OCT B-scan images for the cases of AuNRs with different ligands in Milli-Q water. The image of water without particles was employed as a negative control, which, as expected, showing little to no back-reflected signal (Fig. 5c). Compared with the case of CTAB-capped AuNRs, the B-scan images of water with F127-capped AuNRs also show considerable enhancement in intensity. The log of the OCT signal as a function of depth is plotted in Fig. 5f, which shows more clearly the improvement in contrast efficiency upon ligand exchange from CTAB to F127. The enhancement effect can be ascribed to the reduced dampening of surface plasmon resonance upon switching the strong-binding CTAB ligand to the weak-binding F127.62


image file: d0qm00092b-f5.tif
Fig. 5 (a) In vitro CT images of CTAB-capped AuNRs and F127-capped AuNRs with different concentrations. (b) The linear fitting of CT value at various concentrations of CTAB-capped AuNRs and F127-capped AuNRs. OCT B-scan images of pure water (c) and aqueous dispersions containing CTAB-capped AuNRs (d) and F127-capped AuNRs (e). (f) A-scan profile of these AuNRs with different ligands in water. The scale bar is 200 μm and applies to all the images.

Conclusions

In summary, we have demonstrated a convenient ligand exchange method for replacing undesired ligands on noble metal nanocrystals. The method involves the coating of a layer of cuprous oxide onto the nanocrystal surface to deport the original strongly bonded CTAB molecules. Upon selective etching of the Cu2O layer, a more biocompatible capping ligand such as poloxamer F127 can be introduced onto the nanocrystal surface. The ligand exchange process greatly enhances not only the biocompatibility of AuNRs but also their performance as contrast agents in CT and OCT imaging thanks to the reduced dampening of surface plasmon resonance. This novel ligand exchange strategy is scalable, expandable to noble metals of different reactivity, and involves relatively mild chemical processes that do not significantly alter the morphology and optical property of the nanocrystals. It is therefore expected to bring many new opportunities for the application of colloidal noble metal nanocrystals, especially in biomedicine where surface ligands play a critical role in determining not only their cytotoxicity but also their surface derivatization.

Author contributions

Y. Y. proposed this work. C. Z. and Y. B. carried out the experiments. C. Z., Y. L., and M. G. analyzed the data and wrote the manuscript. T. S., L. Z., Y. C., T. G., and D. Z. contributed to the imaging experiments. Y. B., F. Y., and Y. Y. revised the manuscript. All authors contributed to the discussion of the results.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

C. Z. thanks the fellowship support by the China Scholarship Council (CSC), and the financial support from Army Medical University Foundation (2018XLC2020) and the National Natural Science Foundation of China (81901815). M. G. acknowledges the support from the National Natural Science Foundation of China (81501521). Y. Y. thanks the UCR Academic Senate for providing the Committee on Research (CoR) Grant. We also thank M. Ye at Zhejiang University for the ion chromatography analysis and J. Han at Yangzhou University for help with HRTEM analysis.

Notes and references

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/d0qm00092b

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