OFF/ON galvanic replacement reaction for preparing divergent AuAg nano-hollows as a SERS-visualized drug delivery system in targeted photodynamic therapy

Abstract

A polymer-inhibition (off) and proton-triggered (on) galvanic replacement reaction was developed for preparing divergent growth of the core-free AuAg nanoparticles. We demonstrated an excellent surface plasmon resonance behavior and surface-enhanced Raman scattering (SERS) of 3-D AuAg nano-hollows for potential SERS sensor as well as Raman imaging-guided and folic acid-targeted photodynamic therapy of cancer cells.

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Interest in the development of plasmonic core-free metal materials has grown because of their tunable surface plasmonic resonance (SPR) features for prospective biomedical and industrial applications.^1–9 AuAg nano-hollow nanoparticles (NPs) with significant absorbance at near-infrared (NIR) wavelengths could be used for various photochemical and photophysical applications, such as SPR sensors, surface-enhanced Raman scattering (SERS) tagged probes for tracking, photothermal therapy, and photoacoustic imaging.^2,10–12 Recent synthesis methods for producing AuAg nano-hollows have also shown that galvanic replacement reactions affected by metal ion precursors,³ pH values,⁴ redox agents,^5,6 halides,^7,9 and dopamine/Ag⁺ co-additives⁸ are an effective approach for shifting the growth balance between the oxidation of Ag sacrificial templates and the reduction of Au ions.

Despite the fact that the highly successful modified galvanic replacement reactions^13–15 and examples discussed above^3–8 have been developed, the out-growth of the shell-like structure at the surface of AuAg NPs still remains a difficult challenge. The divergent growth of the three-dimensional (3-D) structure of plasmonic NPs can improve not only the extinction intensity¹⁶ to enhance the electromagnetic field but also the specific surface-to-volume ratio to carry a large dosage of a drug. However, the previous surface engineering with soft PVP coatings,¹⁰ silica scaffold nanoshells,^10,14 and polyphenol-based nanoshells¹⁵ did not achieve 3-D growth of Au nanostructures at the interface of Ag NPs.

Based on the chemistry of the Lewis acid and base interaction, we introduced poly(styrene-alt-maleic anhydride) (PSMA) with carboxylate anions as electron donors (Lewis bases) to bind Au³⁺ ions as electron acceptors. The Au-complex could be dissociated with protons to form carboxylic acid and instantly release the Au ion species. This induces the redox reaction at the Ag nanoparticle surface, leading to the growth of the 3-D structure of AuAg nano-hollows. Optical measurements of the 3-D AuAg nano-hollows presented largely show a shift toward red-NIR wavelengths with a strong and broadened absorption band that improved the photothermal conversion and increased the SERS activity to toxic, drug, and pigment analysts in comparison with “old” AuAg nano-hollows. When grafted with folic acid (FA), the 3-D AuAg nano-hollow has a high affinity for folate receptors (FRs)¹⁶ of cancer cells for targeted photodynamic therapy (PDT). This developed SERS nano-enhancer was able to perform site-specific recognition for cellular imaging of the drug delivery interaction between a non-fluorescent photosensitizer (PS) and living cells. A laser light source at 785 nm in micro-Raman is inactive for PDT. We found either long exposure PDT or combined photothermal-photodynamic therapy delivered by 3-D AuAg nano-hollows acquired with low laser fluence rate for great stimulating cell death.

Significantly, the introduction of irrelevant small thiolate ligands combined with a secondary surface protection layer and subsequent conjugation with targeting molecules at the outmost surface^17–19 were not needed for our integrated platform of PDT, PTT, and SERS.

Fig. S1† shows the preparation of a PVP-protected Ag colloid (62 ± 4.4 nm) in ethylene glycol according to previously reported methods.⁹ The mixture of Ag colloids (2.625 mL at 0.67 mM) with 0–4 mL of an HAuCl₄ solution (0.04 mM) resulted in the original color being mostly maintained (Fig. 1a) when the PSMA solution was added before the HAuCl₄ solution (see the experimental section for details). The remaining NPs had a solid core, and only a few had a single cave structure (Fig. 1b), providing strong evidence that the oxidation of the Ag NPs by Au(III) ions was suppressed. An enlarged view of the same TEM image clearly showed a thin polymer nanolayer (∼2 nm) coating the resulting NPs (Fig. S2†). Upon the addition of 200 μL of HNO₃ (0.22 M), the solution color changed immediately from yellow to blue. The TEM images showed that the blue colloids resulted in the formation of 3-D nano-hollows (73.5 ± 7.9 nm) with a nodular surface structure (Fig. 1c). The EDS and ICP measurements showed that the Au and Ag ratio was approximately 1 : 4 for these 3-D AuAg nano-hollows. In the TEM images, the amorphous PSMA polymer layer was visible on these 3-D AuAg hollow NPs (Fig. S3†). Indeed, the FT-IR spectra (Fig. S4†) showed an additional peak at 1596 cm⁻¹, produced by the C=C stretching of the styrene moiety of the PSMA polymer in the PVP/PSMA-protected Ag NPs and AuAg nano-hollows.

Fig. 1 (a) Images obtained the color change and (b) a TEM image of the PSMA/PVP-protected Ag nanoparticles after the addition of the HAuCl₄ solution. (c) A TEM image observation of 3-D AuAg nano-hollows from the H⁺-induced reaction of the sample in (b). The inset in (c) is a bright field image of the resulting colloidal solution. (d) HR-TEM images (200 kV) of a single 3-D AuAg nano-hollow. (e) STEM-HAADF image and (f–h) EDS elemental maps of the 3-D AuAg nano-hollows.

Fig. 1d shows HR-TEM images of 3-D AuAg nano-hollow composed of 4–12 nm polycrystalline particles in the wall structure. The generation of worm-like piled pores (over 5 nm) stacked with several small nanocrystals was observed. The d-spacing distance was determined to be 0.24 nm, which matched the lattice fringes of the (111) planes of the AuAg alloy. Accordingly, the dark-field imaging in Fig. 1e shows lower contrast in the core site; the bright contrast that homogeneously appeared in the surface structure specifically appeared in the outermost nano-isolates. Using element mapping (Fig. 1f–h), as shown in the HAADF STEM image on the right, one can see that the 3-D nano-hollow consists of Ag atoms together with Au atoms. Notably, the Au atoms present in high concentrations and distributed in these nanocrystals at the surfaces of the particles (Fig. S5†).

Furthermore, we performed additional SEM and TEM imaging (Fig. S6†) to study the concentration effect of HNO₃ on the formation of 3-D AuAg nano-hollows. The critical concentration in the formation of a rough surface on the resulting nanoparticle was found after the addition of 0.16 M HNO₃. In addition, we conducted UV-visible spectroscopy to monitor this acid-induced Ag structure hollowing process. A trend toward the red-shifting of the SPR bands was observed during the formation of 3-D AuAg nano-hollows with increasing HNO₃ concentrations (Fig. S7a and Table S1†). The maximum absorption intensity rarely decreased, and the peak became broader at all NIR wavelengths. When more than 0.22 M of HNO₃ was used, the optical curve of the resulting 3-D AuAg nano-hollow was rarely changed because of the consistent structure morphology (Fig. S8†).

Again, to directly validate that the galvanic replacement reaction relied on the H⁺ concentration, a thermal imaging camera was utilized for in situ monitoring of the conversion of NIR photons at 808 nm to thermal energy after the addition of the HNO₃ solution to Ag@PSMA/HAuCl₄ (Fig. S7b†). The results showed that this photothermal conversion was independent of the HAuCl₄ concentration.

Obviously, the carboxylate groups of the PSMA polymer that bound to the Au ions indeed played a vital role that not only violates the classical galvanic rule but also assists in the 3-D growth of AuAg nanograins at the surface of the nano-hollows. To validate the chemical state of the COO–Au complex and the subsequent metallic Au–Ag alloy of the solid particle, we used X-ray photoelectron spectroscopy (XPS) to provide evidence of the interface interaction between Ag NPs and HAuCl₄ (Fig. S9†).

Parallel experiments consisting of the replacement of the HNO₃ solution with a basic solution (NaOH), redox agent (FeCl₂), electrolyte (NaCl), or another acidic system (HCl) were conducted (Fig. S10†). Few Ag NPs are oxidized in reactions with NaOH, FeCl₂, and NaCl reagents. Consistently, similar to the use of HNO₃, a 3-D AuAg nano-hollow was produced using a 0.22 M HCl solution (0.2 mL), which did not exhibit the additional complex effect of Au ions with NO₃⁻ and Cl⁻ anions.

For the various agents (anionic PAA(COOH), natural PEG(–CH₂CH₂O–), and cationic PAH(NH₂) polymers), the colloidal solution remained yellow only during synthesis with PAA, indicating the same inhibition of the oxidation of Ag NPs as that which was produced by the HAuCl₄ solution. Once the solution reacted with HNO₃, the resulting sample became blue and consequently yielded AuAg nano-hollows with antler-like structures on the surface (Fig. S11†).

Despite the urchin-like nanostructure of AuAg nano-hollows with random size distribution was successfully produced from spherical Ag nanoparticles (50–75 nm), we demonstrated that the well-defined and 3-D/porous AuAg nano-hollows could also be fabricated by using homogeneous templates of Ag nanocubes, Ag nanoplates, and Cu₂O nanoparticles via the developed OFF/ON galvanic replacement reaction (Fig. S12–S15†).

Notably, traditional^1,2 and modified^3–10,14,15 synthesis of AuAg nano-hollows using PVP results in a HAuCl₄ concentration-dependent oxidation of Ag NPs. Concomitantly, the yellow color of the Ag solution gradually turned dark brown with the addition of the HAuCl₄ solution (Fig. S16a†). An ICP-AES measurement quantified the composite as Au_0.22Ag_0.78. A TEM image shows a core-free interior and a smooth surface structure (Fig. S16b†). At the same HAuCl₄ concentration, this “old” synthesis of smooth surface structured AuAg colloids approached the SPR band in red wavelength regions, which is different from our 3-D AuAg nano-hollows possessing strong NIR absorption (Fig. S7c†). Either at 808 nm and 1064 nm, 3-D AuAg nano-hollows exhibited greater photothermal conversion than smooth surface AuAg nano-hollows (Fig. S7d†).

To exploit the electromagnetic interaction between excitation laser light and plasmonic 3-D AuAg nano-hollow absorption, a 785 nm laser line was used as a light source. Specifically, the amphoteric PSMA on the surface of AuAg nano-hollow made the targeting molecules closely attach to the nanoparticle surface through van der Waal interactions (e.g., pi–pi and electrostatic absorption),^16,20 which may also give rise to multivariate applications of molecular sensing. We compared the SERS spectra (with 1 s of acquisition time) of malachite green solution, an antibacterial agent used in fisheries which acts as a toxin in the human liver, using equal amounts of 3-D and smooth surface AuAg nano-hollows. The SERS signal is independent of the sample volume and highly reproducible (Fig. 2a). Fig. 2b shows the increase in the intensity in Raman peaks with analyte concentrations ranging from 500 nM to 50 μM. However, smooth-surface AuAg nano-hollows did not significantly improve the Raman peaks of the MG molecule, even when the concentration was increased to 1 mM (Fig. S17†). Using 3-D AuAg nano-hollows, we analyzed the SERS activity of MG with a low limit of experimental detection (LOD) of 500 nM and a Raman enhancement factor (EF) of approximately 2 × 10⁵.

Fig. 2 Raman spectra and imaging records of (a) the addition of 1–16 μL of 3-D AuAg nano-hollows (250 ppm) including 5 × 10⁻³ mM-MG molecules. Raman spectra records of 10 μL of 3-D AuAg nano-hollows (250 ppm) including (b) MG molecules with concentrations ranging from 5 × 10⁻⁵ mM to 5 × 10⁻² mM, (c) MB molecules with concentrations ranging from 5 × 10⁻⁶ mM to 5 × 10⁻² mM, and (d) iron chlorophyll molecules with concentrations ranging from 5 × 10⁻⁷ mM to 5 × 10⁻³ mM.

In addition, the 3-D AuAg nano-hollows were able to greatly enhance the Raman signal of other components, such as a phenothiazine-based methylene blue (MB) photosensitizer (Fig. 2c) and a chlorophyll food pigment (Fig. 2d). For the MB analysis, a superior LOD of 50 nM with a ∼2 × 10⁶ EF value was detected. Again, we found a poor SERS sensitivity to MB by PVP-protected AuAg nano-hollows (Fig. S18†).

However, 3-D AuAg nano-hollows carrying MB might aid in the detection of non-fluorescent MB PS for targeting in biological systems. In a proof of concept experiment, we used HS–PEG–NH₂ as a thiol linker molecule to directly bind the surface of AuAg@MB nano-hollows and the subsequently conjugate the amine groups to the carboxylate groups of the folic acid with EDC/NHS coupling. The nanoparticle modified with folic acid was suggested to display high affinity for binding to a folate receptor protein on the HeLa cell surface.²¹ The appearance of Raman peaks of MB immobilized on the AuAg nano-hollows indicate the robust protection of MB against the oxidant NaNO₂ and HNO₃/H₂O₂ (Fig. S19†). These species generally appear in mammals.

Fig. 3a presents a bright field image of the incubation (0.25 h) of HeLa cancer cells with folic acid-conjugated 3-D AuAg@MB nano-hollows; the selection and an enlarged image of a single cell is shown in Fig. 3b. The micro-Raman spectra (Fig. 3c) show the MB-based peak signals at the cell surface, indicated by the red star, and the background signal (no peaks) indicated by the black star. Raman mapping imaging (Fig. 3d) was used to determine the signal distribution of the MB-based peak at 1619 cm⁻¹. Fig. 3e shows a merged image from Fig. 3b and d, with a high degree of overlap of the MB signal intensity in the vicinity of the plasma membrane with the location of black dots by 3-D AuAg nano-hollows. Fig. 3f and g show additional examinations after six hours of cell–particle interaction, which resulted in the promotion of the entry of numerous nano-hollows into the cell body. A Raman map facilitates tracking the movement of folic acid-conjugated 3-D AuAg nano-hollows that still carried MB photosensitizers within the HeLa cell.

Fig. 3 The delivery of 3-D AuAg@MB nano-hollows (25 ppm_metal) to HeLa cells after (a–e) 0.25 h and (f, g) 6 h. The imaging analysis: (a, b, f) bright field image, where (b) the selected zone corresponded to the red-square marked area in (a), and (d, e, g) mapping images constructed of the Raman peak at 1619 cm⁻¹ corresponding to the white-square marked area in (a), and (f), where (e, f) merge their bright field picture. (c) Raman spectra from the picture (b) labeled with the red star and the black star.

Finally, we utilized a 660 nm LED light to excite the FA-conjugated AuAg@MB nano-hollows (FA-AuAg@MB)-treated HeLa cells after 0.25 h and 2 h of incubation time. The PDT-treated cells were then kept in the incubator for 1 day, again followed by an MTT assay to quantitatively determine the cell viability. Fig. 4a shows cell death rate to ∼15% for the 0.25 h-sample and ∼77% for the 6 h-sample. The long-term exposure favored a conspicuous elimination of cancer cells, which was probably due to a high degree of MB-based particle accumulation in the cell organelles according to the SERS imaging (Fig. 3g). To prove the generation of reactive oxygen species in the cell (with green fluorescent DCF), the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)²⁰ staining method was employed. Fig. 4b shows a brighter green fluorescence was detectable in the FA-AuAg@MB-treated HeLa cell after 6 h in contrast to 0.25 h-treated sample group. However, enhanced loss of cell viability to inhibitory concentration 50% (IC50) doses was observed at 0.25 h-treated time when an additional illumination with 808 nm light was applied, as shown PDT → PTT in Fig. 4a. This therapeutic improvement could be explained by combining photodynamic and photothermal therapy to evoke a synergistic way of killing cancer cells. For the PTT first followed by PDT we did not found a difference in phototherapy resulted by PDT → PTT. Fig. S20 and S21† show that HeLa cells were not received significant phototoxicity at a short irradiation time and FA-free AuAg@MB nano-hollows, respectively. Our demonstration provided direct evidence that FA-AuAg@MB could exhibit FA targeted and energy density stimulated therapy of cervical cancer cells. Compared to the previous Raman integrated PTT-PDT system,^19,20,22,23 our current FA-AuAg@MB-based biophotonics platform has advantage of acquired with lower laser fluence rate to execute phototherapy of PTT by 0.38 W cm⁻² and PDT by 10 mW cm⁻² for PDT.

Fig. 4 (a) MTT assay of the AuAg nano-hollows@PS@FA-treated HeLa cells received LED light at 660 nm (51 mW), laser light at 808 nm for PTT (115 mW), 660 nm → 808 nm, and 808 nm → 660 nm. (b) Fluorescence images of DCF (green) in HeLa cells incubated with AuAg nano-hollows@PS@FA for 0.25 h and 2 h. Samples received 660 nm LED exposure for 4 min and 808 nm light for 3 min.

In conclusion, a new polymer-induced confined (off) and pH-assisted triggered (on) redox reaction of Ag NPs was demonstrated by carefully using different functional groups for the polymer side chain and electrolyte/redox/basic/acidic reagents. The complicated surface reaction processes were not necessary for approaching targeted PDT and tagged SERS by using AuAg@PSMA nano-hollows. Prior to stimulation of the PDT activity with excitation at 660 nm, the SERS imaging assisted in monitoring the local accumulation of PS in cancer cells. Efficient PDT–PTT resulted in cancer cell damage by the intracellular localization of AuAg nano-hollows@PS at the FA-receptor and after endocytosis.

Acknowledgements

This work was supported in part by grants from the Ministry of Science and Technology (grant no. MOST 103-2113-M-010-001-MY2 and MOST 102-2221-E-006-300-MY3) of Taiwan. We would like to thank Yao-Tzu Yang for help with cell culture experiments.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12971d

‡ These authors contributed equally.

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