Salt- and pH-resistant gold nanoparticles decorated with mixed-charge zwitterionic ligands, and their pH-induced concentration behavior

Abstract

In this work, salt- and pH-resistant gold nanoparticles were obtained by decorating their surfaces with mixed-charge zwitterionic ligands, and their concentration was changed by exploiting their pH-induced reversible aggregation properties.

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Nanoparticles (NPs) have received increasing interest because of their unique physicochemical properties, which give them potential for applications in a variety of fields.^1,2 Decorating the surfaces of NPs with functional ligands may amplify existing properties, or generate new properties, making the modified NPs suitable for applications that undecorated NPs could not be used for.^2–4 For example, decorating Au NPs with external stimuli-responsive ligands can endow Au NPs with pH-,^5–7 photo-,⁸ temperature-⁹ or bimolecular-responsive¹⁰ properties. Because of their smart response to external stimuli, decorated Au NPs can be used as nanosensors,⁵ recycling catalysts,⁶ photothermal therapeutic cancer agents,⁷ and bioanalytical nanosystems.¹⁰

Among all of the ligands used to date, zwitterionic ligands have emerged as an excellent alternative to PEGylation in the field of biomaterials.¹¹ First, zwitterionic surfaces can effectively resist nonspecific protein adsorption, avoiding adverse complications due to protein accumulation, thus extending the use of certain biomaterials.^12–15 Second, decorating the NPs with small zwitterionic ligands minimizes their total size, facilitating access to confined biological compartments.^11,16 Third, zwitterionic ligands show good stability, allowing NPs to withstand elevated salt concentrations and a wide pH range, which facilitates their application under complex biological conditions.^17–19 Finally, the control of their chemical composition, synthesis, purification, and functionalization is relatively straightforward, because of their small molecular size. The zwitterionic ligand-decorated NPs thus show high stability, minimized size, biocompatibility, and improved resistance to protein adsorption, properties that greatly favor their downstream applications (for example, in vivo applications).²⁰

Inspired by the pioneering work of Whitesides's¹² and Cooper's²¹ groups, many groups have used zwitterionic ligands to stabilize/functionalize Au (or Ag) NPs,^{17–19,22,23} copolymer micelles,²⁴ quantum dots,¹⁶ antifouling silica hydrogels,²⁵ superparamagnetic iron oxide nanoparticles,²⁰ silica nanoparticles,²⁶ and nanogels²⁷ for biomedical applications. We previously reported that zwitterionic ligand-decorated Au NPs showed a size-dependent critical coagulation concentration and reversible aggregation properties, which allowed for the size-selective separation, concentration, and long-term preservation of Au NPs.²⁸ The zwitterionic ligands typically used in these studies are laboratory-synthesized, single-component zwitterionic ligands terminating in groups combining negatively and positively charged moieties in the same molecule. Alternatively, zwitterionic ligands can also be constructed from 1 : 1 mixtures of negatively and positively charged ligands.¹² Following this concept, Ji's group reported a facile method for stabilizing large gold NPs (16–100 nm), using a mixed-charge zwitterionic self-assembled monolayer with relatively long chains,¹⁹ while Lin's group improved the surface biocompatibility.²⁹ In this work, we used commercially available, neutral and negatively charged thiol ligands to construct the mixed-charge zwitterionic ligands, taking into account the following considerations: To avoid the steric stabilization introduced by long chains,³⁰ short-chain ligands were deliberately chosen. The zwitterionic structure could easily be constructed/deconstructed by protonating/deprotonating the terminal group via variation of the pH. The objectives of this work were to study the stability of Au NPs under pH and salt stimuli, and to elucidate the stability mechanism originating from the zwitterionic structure itself. The results of this work may help in the design and synthesis of functional zwitterionic ligands with excellent pH and salt resistant properties for biomaterials.

Au NPs with a diameter of 4 nm were synthesized according to a previously reported method.³¹ The Au NPs were then decorated (through a ligand exchange reaction) with a single-component ligand, 3-mercapto-1-propanesulfonic acid sodium (MPS) or 2-(dimethylamino) ethanethiol (DAET) and with a 1 : 1 molar ratio mixture of MPS and DAET (the decorating procedures are detailed in the Electronic Supporting Information, ESI†). The 4 nm Au NPs showed a surface plasmon (SP) absorption band at 510 nm,³² which was used to investigate the stability of the Au NPs. Before focusing on the mixed MPS/DAET ligands, we first studied the stability behavior of single ligand-decorated Au NPs. Fig. 1a shows that as ∼2–10 μL of a 1 × 10⁻² M solution of DAET was added, the SP band of the 4 nm Au NPs broadened, red-shifted, and decreased in absorption intensity. These spectral features were caused by the formation of particle aggregates, and their sedimentation from the solutions, indicating that the DAET alone had no stabilization abilities. A possible reason for this is that the DAET ligand caused the aggregation of the NPs via bridging; its two terminal –SH and –N(CH₃)₂ groups (or –NH(CH₃)₂⁺ in the protonated form) both show great affinity to the Au surface. Fig. 1b–d shows that the addition of MPS allowed the 4 nm Au colloids to withstand salt concentrations of up to 0.8 M, and a pH range of 4–12. As the pH was further decreased below 4, the MPS-decorated Au colloids began to aggregate, indicating that there was a critical aggregation pH value, below which the Au colloids lost their stability (Fig. 1c). These stability behaviors can be reasonably well understood in terms of the classical Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, which explains the colloidal stability in terms of van der Waals attraction and electrostatic repulsion (DLVO interactions).³⁰ When the Au NP surfaces were decorated with MPS, the electrostatic repulsion between the Au NPs was further enhanced, and prevailed over the van der Waals attraction; this provided stability to the Au colloids, and allowed them to withstand NaCl concentrations of up to 0.8 M. At higher salt concentrations, the thickness of the double layer was significantly decreased, and the electrostatic repulsion therefore no longer made a significant contribution to the stability. Under strongly acidic conditions, the protonation of the –SO₃⁻ groups weakened the electrostatic repulsion, and created hydrogen bonds between the –SO₃H groups on different NPs; under strongly alkaline conditions, the excess NaOH electrolyte shielded the electrostatic repulsion. The electrostatic repulsion therefore no longer stabilized the MPS-decorated Au NPs outside of the pH range of 4–12.

Fig. 1 UV-vis spectra for citrate- and DAET-decorated 4 nm Au NPs (a), and UV-vis spectra for MPS-decorated 4 nm Au NPs in NaCl (b), acidic (c), and alkaline solutions (d), upon the addition of one monolayer amount of MPS in the solution.

We then studied the stability behavior of the mixed MPS/DAET-decorated 4 nm Au NPs in NaCl solutions, over a wide pH range. Fig. 2 shows the UV-vis spectra for the mixed MPS/DAET-decorated 4 nm Au NPs, at various NaCl concentrations. As revealed by Rouhana et al.¹⁷ and us,²⁸ monolayer-type coverage with single-component zwitterionic ligands can stabilize 4 nm Au NPs in a 3.0 M NaCl solution. Therefore, the amount of the 1 : 1 molar-ratio mixture of MPS/DAET needed to provide nearly one monolayer of adsorption was used to decorate the 4 nm Au NPs. Fig. S1 shows that the MPS/DAET-decorated 4 nm NPs aggregated in 1.0 M NaCl, indicating the amount of the MPS/DAET was not sufficient to provide stability (ESI†). When the amount of MPS/DAET was increased to provide two-monolayer adsorption, the Au colloids were stable even in a saturated (6.02 M at 20 °C) NaCl solution (Fig. 2), far beyond the highest concentration (0.8 M) that the MPS-decorated 4 nm Au NPs could withstand (Fig. 1b). Thus, to ensure a saturated monolayer-type adsorption on the Au NP surfaces, the amount of MPS/DAET in the solution had to be in excess of that needed to provide one-monolayer coverage. In this case, the mixed MPS/DAET ligands provided stability comparable with that observed for 4 nm Au NPs with single-component zwitterionic ligands of similar chain length.^17,28

Fig. 2 UV-vis spectra for the 4 nm Au colloids in different-concentration NaCl solutions, upon the addition of a two monolayer amount of 1 : 1 mixed MPS/DAET in the solution.

Fig. 3a and b shows the UV-vis spectra for the MPS/DAET-decorated 4 nm Au colloids in the pH range 3–13.5. The MPS/DAET-decorated 4 nm Au colloids remained stable at pH > 5.85, and had a critical aggregation pH located at ∼5.85, slightly higher than that (∼4) of the MPS-decorated 4 nm Au colloids. To reveal whether their pH-induced aggregation was reversible or not, three Au colloid samples were induced to aggregate by adjusting their pH to 5, 4, and 3, respectively, and then returning the pH value to 7. As can be seen from Fig. 4, the SP band of the Au NPs recovered to its original position and shape; in addition to these spectral changes, the blue color associated with the aggregated colloids changed back to the orange color produced by the unaggregated colloids. These results showed that the pH-induced aggregation was reversible. This aggregation and redispersion cycle could be repeated further without the need to dialyze the NaCl (via the repeated addition of HCl and NaOH to adjust the pH), because the MPS/DAET-decorated 4 nm Au NPs remained stable in 3 M NaCl solutions with pH values of 7, 9, and 13 (Fig. S2, ESI†). Fig. S3 shows TEM images of the MPS/DAET-decorated Au NP samples prepared from the colloids with pH = 7 and pH = 5, respectively, which further confirmed the dispersed and aggregated states at these two pH values (ESI†).

Fig. 3 UV-vis spectra for the 1 : 1 MPS/DAET-decorated 4 nm Au NPs under acidic (a) and alkaline (b) conditions.

Fig. 4 UV-vis spectra for the MPS/DAET-decorated 4 nm Au NPs at pH values of 3, 4, and 5, their corresponding spectra upon adjusting the pH back to 7, and digital photographs of the MPS/DAET-modified 4 nm Au colloids at pH values of 7 and 5.

The mixed MPS/DAET ligands showed stabilizing abilities that were distinctly different from those of each of the single ligands; this was due to the unique zwitterionic structure. Being a strongly polarized dipole, the zwitterionic structure induces the organization and orientation of water. This creates not only a hydration layer—which confers antifouling, water-soluble, and biocompatibility properties on the zwitterionic surface^21,33,34—but also a repulsive hydration force between the Au NPs.^35,36 Due to its origin, the hydration force is a salt-insensitive, non-charge-based repulsion, which accounts for the high stability of the 4 nm Au NPs, even in a saturated (6.02 M at 20 °C) NaCl solution. Another example of this behavior was provided by silica particles that were much more stable at high ionic strengths than predicted by DLVO theory.³⁷ We previously found that short chain-length zwitterionic ligands provided much better stabilization for smaller NPs than for larger NPs,²⁸ because larger NPs have a higher van der Waals attraction and are therefore more unstable.³⁰ Ji's group found that relatively long chain-length zwitterionic ligands could stabilize large Au NPs in the range 16–100 nm.¹⁹ We therefore concluded that the hydration force alone was not sufficient to stabilize large Au NPs; to achieve high stability in this case requires the synergistic stabilization effects of both the hydration force and the long chain-related steric stabilization.

To understand the pH-dependent stability, Fig. 5 schematically illustrates the construction/deconstruction of the zwitterionic structure and hydrogen bonds at varying pH values. At pH < 5.85, the –N(CH₃)₂ and –SO₃⁻ terminal groups might both be protonated, creating hydrogen bonds between the –SO₃H groups and the neighboring–NH(CH₃)₂⁺ groups on the same and other particles, at the cost of the zwitterionic structure, i.e., the hydration repulsion. As a result, the hydrogen bonds mediated the self-assembly of the NPs, and destabilized the Au colloids (Fig. 3a). With increases in the pH (>5.85), the –NH(CH₃)₂⁺ groups were partially deprotonated, and the –SO₃H groups were completely deprotonated, deconstructing the hydrogen bonds and constructing zwitterionic structures between the residual –NH(CH₃)₂⁺ groups and the neighboring –SO₃⁻ groups. The salt-insensitive hydration force created therefore allowed the 4 nm Au NPs to withstand the conditions in elevated-pH solutions, in the absence (Fig. 3b) or presence of 3 M NaCl (Fig. S2, ESI†). Upon further increases in the pH, the –NH(CH₃)₂⁺ and –SO₃H were both completely deprotonated, generating neutral –N(CH₃)₂ and negatively charged –SO₃⁻ groups at the NP surfaces. In this case, it seems likely that the Au NPs were stabilized solely by the electrostatic repulsion, and should not have been stable at high ionic strengths (3 M, NaCl); this, however, was contradicted in the experimental observations (see Fig. S2, ESI†). We therefore speculate that the neighbors of the –N(CH₃)₂ and –SO₃⁻ groups still induced the organization and orientation of water, and created a hydration force to stabilize the Au NPs at high ionic strengths. These different interactions between the particles account for the fact that the MPS/DAET-decorated Au NPs showed a pH-resistant range that was different from that observed for the MPS-decorated Au NPs; the pH-resistant range was determined by the properties of the charged groups that formed the zwitterionic structure. As discussed before, under strong acidic conditions, the construction of the hydrogen bonds mediated the self-assembly of the NPs at the cost of the hydration repulsion, while at pH values above the critical coagulation pH the hydration force recovered, and disassembled the NP aggregates. The repeated construction and deconstruction of the zwitterionic structures and hydrogen bonds thus imparted pH-dependent reversible aggregation properties to the mixed MPS/DAET-decorated Au NPs.

Fig. 5 A schematic illustration of the construction/deconstruction of zwitterionic structures and hydrogen bonds at varying pH values.

These salt- and pH-resistant Au NPs may find wide applications in a variety of fields. To facilitate their storage, transport, and downstream applications, a pH-induced concentration strategy based on the pH-dependent reversible aggregation properties was demonstrated. The right panel of Fig. 6 shows photographs for each concentration step. The concentration process was begun in 7 mL of an MPS/DAET-decorated 4 nm Au colloidal dispersion (photograph 1). The particles were induced to aggregate by adjusting the pH to below 5 (photograph 2), and the sample was then left undisturbed, to allow the formed aggregates to completely settle (photograph 3). After removing the supernatant and neutralizing the residual acid using a few drops of NaOH solution, the aggregates were redispersed in water (here 1.5 mL), and formed concentrated colloids with a desired concentration (photograph 4). The left panel of Fig. 6 shows the UV-vis spectra for the original and concentrated colloids, and indicates that the 4 nm Au NPs were well dispersed in the concentrated colloids.

Fig. 6 UV-vis spectra for MPS/DAET-decorated 4 nm Au NPs before (1), and after concentration (from 7 to 1.5 mL) (4), and digital photographs showing the concentration evolution processes: (1) original, (2) aggregated (upon the addition of HCl), (3) settled (stood undisturbed), and (4) concentrated colloids.

In conclusion, 4 nm Au NPs decorated with 1 : 1 mixed-charge MPS/DAET showed enhanced stability, with the ability to withstand elevated salt concentrations and a wide pH range. They also showed pH-dependent reversible aggregation properties, which allowed the pH-induced concentration of dilute colloids. These properties were attributed to the unique zwitterionic structure constructed at the NP surface. The zwitterionic structure and appropriate-length ligand chains provided synergetic stability to differently sized NPs, while minimizing their total size. The synergetic properties (i.e., pK values) of the charged groups forming the zwitterionic structure not only gave the NPs antifouling, water-soluble, and biocompatibility properties, but also determined their pH-resistant range.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (Grant Nos 20603008, 20873037, and 91027037).

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Footnote

† Electronic Supplementary Information (ESI) available: Experimental details and supporting figures. See DOI: 10.1039/c2ra22165a

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