Galactosylated silver nanoparticles as a biocompatible intrinsic SERS probe for bladder cancer imaging and ex vivo tumor detection

Abstract

The biological application of silver nanoparticles (Ag NPs), which are commonly used as SERS substrates, is often limited by issues related to uncontrolled Ag ion release, resulting in instability and cytotoxicity. In this study, we developed galactosylated Ag@PGlyco-PSMA NPs, a novel biocompatible and bio-SERS platform for sensing small molecules at nanomolar concentration levels, achieving an analytical enhancement factor of 1.71 × 10⁴ alongside intrinsic imaging and labeling capabilities for bladder cancer cells. These Ag NPs were co-synthesized during the polymerization of o-nitrophenyl-β-D-galactopyranoside to form an Ag@polyaniline-based glycopolymer (PGlyco) nanostructure, which was subsequently reacted with poly(styrene-alt-maleic acid) (PSMA). This process stabilized the particle dispersion while generating robust SERS signals due to PGlyco immobilization. By controlling the formation kinetics through the addition of the PSMA polymer at 30 seconds after the reaction of Ag@PGlyco NPs, we observed the formation of aggregate-induced hot spots to evolve PGlyco-related SERS signals arising from interparticle interactions. Our results demonstrated that Ag@PGlyco-PSMA NPs exhibit minimal Ag ion release, resulting in over 80% cell viability across T24, MB49, VERO, and SV-HUC-1 cell lines. Among these cells, Ag@PGlyco-PSMA nanoparticles demonstrated remarkable capability for enhancing cellular uptake, effectively distinguishing bladder cancer cells from normal cells with over 2.6 folds of the signal difference in SERS imaging. The galactose moieties in the PGlyco coating around the Ag@PGlyco-PSMA nanoparticles served as a SERS probe for multivalent binding to bladder cancer cells, enabling cancer imaging diagnosis and tumor-specific detection in accordance with tumor volume growth. Our findings indicated that Ag@PGlyco-PSMA nanoparticles offered intrinsic SERS capability for galactose-mediated bio-interaction and minimal Ag ion release, showing an ideal diagnostic optical platform for in vitro cancer cell imaging and ex vivo tumor progression tracking through bladder SERS detection.

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1. Introduction

Surface-enhanced Raman spectroscopy (SERS) has garnered significant attention due to its ability to provide rapid, highly sensitive, and non-invasive molecular fingerprinting analysis,^1–8 making it an invaluable tool for a wide range of applications, including cancer diagnosis,^1,4 bacterial detection,⁹ and metabolite analysis.¹⁰ Among the noble metals commonly used in SERS, silver nanoparticles (Ag NPs) exhibit superior SERS enhancement due to their excellent optical properties and high molar extinction coefficients, surpassing gold and copper.¹¹ However, the inherent instability of Ag NPs,¹² particularly their tendency to release toxic Ag ions,^12,13 poses significant challenges for their application in biological systems. This toxicity, combined with the short-wavelength absorption of Ag NPs,⁵ limits their use in high-biocompatibility SERS-based imaging and tracking experiments, especially when near-infrared (NIR) light excitation is required for deeper tissue penetration.^14,15

Ag NPs are ideal materials for high-sensitivity biosensors due to their excellent surface plasmon resonance (SPR) effect,⁵ which can enhance Raman signals from target molecules, such as in the detection of bacteria and the analysis of cancer biomarkers.^16,17 However, the application of Ag nanostructures in live cell imaging remains limited because of their propensity to easily degrade and release Ag ions.¹² Several research groups have attempted to address this issue through structural engineering, as well as surface modifications^18,19 to enhance stability and biocompatibility. Furthermore, the development of Ag@Au²⁰ or AuAg alloys²¹ has shown promise in further amplifying optical signal intensity, despite the challenges posed by the cost of gold and the decrease in SERS efficiency that occurs with higher concentrations of gold. The bio-immobilization of Ag-based SERS particles is essential for effective biomolecular recognition, ensuring specificity and reliable analytical signals, while also necessitating methods to label cancer cell membranes and improve cellular uptake of synthesized Ag NPs.²² Current approaches are yet to address economic constraints and the time-consuming processes involved in producing Ag SERS structures without excessive surface modifications that could reduce SERS efficiency.

To address these challenges, this study focused on the development of Ag@PGlyco-PSMA NPs, which feature a galactose-related polyaniline-based glycopolymer (PGlyco) coating that not only enhances biocompatibility but also generates intrinsic SERS signals without the need for additional Raman probes.^23–25 Our experimental design was based on the co-synthesis of glycopolymer polymerization and Ag nucleation growth, enabling the PANI segments of the glycopolymer to be positioned extremely close to the Ag nanoparticle surface. After a reaction at 30 seconds, poly(styrene-alt-maleic acid) (PSMA) was introduced to self-assemble onto the Ag@PGlyco NPs, improving the stability of the Ag NPs and forming a novel class of galactosylated Ag NPs, as shown in Scheme 1. In contrast to conventional Ag NPs, which often release Ag ions unpredictably¹² and can cause inflammatory diseases,²⁶ harm organisms,^27–31 and descend their optical properties,³² the use of a glycopolymer combined with a PSMA polymer coating effectively restricted Ag ion release. This approach significantly reduced cytotoxicity while preserving strong SERS activity. A key innovation in this work lied in designing Ag NPs decorated with the PANI-based glycopolymer that did not require external SERS probes. During synthesis, the addition of o-nitrophenyl-β-D-galactopyranoside (ONPG) led to spontaneous polymerization on the Ag surface, generating intrinsic bio-SERS signals. Moreover, the exposed galactose side chains on the glycopolymer allowed for diagnostic selectivity and nanoparticle–cell interaction studies by binding to galactose-related lectins³³ and glucose transporters (GLUT)³⁴ that are overexpressed in cancer cells. The PSMA coating^23,35–38 not only stabilized the Ag NPs but also improved their optical properties, facilitating the generation of strong SERS signals even under biologically relevant conditions. Recently, Liao and co-workers^36–38 have demonstrated that PSMA layer protection can increase metallic Cu NPs’ absorption and improve their SERS signals’ stability. Furthermore, the timing of PSMA polymer addition during synthesis allowed for controlled NP aggregation, resulting in a tail absorption effect that enhances SERS intensity. This controlled aggregation enhanced plasmonic coupling, further amplifying the SERS signal for sensitive detection and imaging applications. This design enabled the use of galactosylated and biocompatible Ag@PGlyco-PSMA NPs in cancer cell imaging and tracking, capitalizing on their high biocompatibility, robust SERS signals, and selective recognition of solid tumors (Scheme 1). This advancement overcame the limitations of traditional Ag NPs, providing a novel platform for non-invasive cancer detection and imaging.

Scheme 1 A schematic illustration for the developed redox reaction involving Ag, ONPG, and NaBH₄, facilitated by the PSMA polymer. This process yields galactosylated biocompatible and SERS-active Ag@PGlyco-PSMA NPs, which enhance imaging capabilities for cellular and tumor detection.

2. Experimental

2.1. Chemicals and materials

Silver trifluoroacetate (CF₃COOAg) and 4-nitrothiophenol (4-NTP, 97%) were purchased from Alfa Aesar. 2-Nitrophenyl-β-galactoside (ONPG, >99%) was purchased from Biosynth Carbosynth. Sodium tetrahydridoborate (NaBH₄, 95%) was purchased from Riedel-de Haën. Poly(styrene-alt-maleic acid) sodium salt solution (PSMA, 13 wt% in H₂O), polyvinylpyrrolidone (PVP, M_w ∼ 55  000), polyethylenimine (PEI, M_w ∼ 25  000), malachite green oxalate salt (MG), rhodamine 6G (R6G) and hydrogen peroxide solution (H₂O₂, 34.5–36.5%) were purchased from Sigma-Aldrich. Ethanol was purchased from J.T. Baker. Phosphate buffered saline (PBS, pH 7.4) and LIVE/DEAD® BacLight™ Bacterial Viability Kit were purchased from Thermo Fisher Scientific.

2.2. Preparation of Ag@PGlyco-PSMA NPs

Ag@PGlyco-PSMA NPs were synthesized by adding 1 mL of ONPG (75 mM) and 2 mL of CF₃COOAg (5 mM) solution to 8 mL of D.I. water. The mixture was heated with magnetic stirring in an oil bath at 80 °C for 10 minutes. After an additional 10 minutes of heating, a reducing agent, 0.2 mL of NaBH₄ (40 mM), was injected into the solution. The reaction was allowed to proceed for 30 seconds, after which PSMA (1.2 g in 50 mL) was added as a protective polymer. The mixture was then cooled to room temperature while stirring for 30 minutes. The samples were purified by centrifugation at 4500 rpm for 10 minutes, repeated three times. The purified samples dispersed in D. I. water were stored at room temperature for subsequent experiments. Different concentrations of NaBH₄ (20, 30, and 50 mM) and polymer species (PSMA, PEI, and PVP) were employed to assess the formation of the Ag-based NPs under varying parameters.

2.3. SERS measurement of analyte molecules

The analytes (MG, R6G, and 4-NTP) were prepared at five different concentrations and combined with equal volumes of Ag@PGlyco-PSMA, Ag nanospheres, and Ag nanocubes at an optical density (OD) of 2. The SERS spectrum was subsequently detected using a 671 nm Raman system (LiveStrong Optoelectronics, LS-PT 671) with a power of 100 mW and a low noise spectrometer module (<13 cm⁻¹ resolution). The spectrum was employed with an integration time of 1 s with three accumulations.

2.4. Bacterial viability

The viability of E. coli was assessed by cultivating NPs with the bacteria and calculating their survival rate. A volume of 100 μL of bacterial suspension (10⁶ CFU mL⁻¹) treated in Mueller–Hinton Broth was mixed with 100 μL of either Ag@PGlyco-PSMA, Ag nanospheres, Ag nanocubes or Ag ions (at concentrations of 1.5, 3, 6.25, 12.5, 25, and 50 ppm_[Ag]), and incubated overnight at 37 °C. After another co-incubation time of 24 h, bacterial viability was determined by measuring absorbance at 600 nm using UV-Vis spectrometry.

2.5. Release of Ag ions in solution

Ag@PGlyco-PSMA, Ag nanospheres, and Ag nanocubes at a concentration of 100 ppm were dispersed in 1 mL of deionized water and a cellular medium (RPMI) at 37 °C. After 24 h incubation, it was subjected to high-speed centrifugation to remove the NPs. The concentration of Ag ions released into the resulting supernatant was measured using atomic absorption spectroscopy (AAS).

2.6. Cell culture

T24 cells (human bladder cancer cells) were cultured in McCoy's 5A medium, MB49 cells (murine bladder cancer cells) were cultured in RPMI-1640 medium, VERO cells (African green monkey kidney epithelial cells) were cultured in DMEM medium, and SV-HUC-1 cells (human urinary tract epithelium cells) were cultured in F-12K medium. All mediums were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). All cell lines were maintained at 37 °C in a 5% CO₂ incubator.

2.7. In vitro cell viability

T24, MB49, VERO and SV-HUC-1 cells were seeded in a 96-well cell culture plate at a density of 8000 cells per well. Afterward, the cells were cultured with Ag@PGlyco-PSMA NPs, Ag nanospheres, Ag nanocubes and Ag ions with the different concentrations of 0.1, 1, 5, 10, 25, 50, and 100 ppm, respectively. After another 24, 48 and 72 h culture, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) solution (1.2 mM) was added into each well for 1 h. Subsequently, DMSO was added into each well to dissolve formazan crystals, and then the absorbance at 565 nm was measured by using a microplate reader (Synergy H1, BioTek).

2.8. In vitro ROS level detection

SV-HUC-1 cells were seeded in a 3.5 cm dish at a density of 10⁵ cells per mL overnight. Afterward, the cells were cultured with Ag@PGlyco-PSMA NPs, Ag nanospheres, and Ag nanocubeswith the different concentrations of 25, 50, and 100 ppm, respectively. After another 24 h culture, dichlorodihydrofluorescein diacetate (DCFH-DA) solution (10 μM) was added to the dish. After 30 minutes of incubation, the cells were washed with PBS before fluorescence microscope observation (Olympus BX53 microscope).

2.9. LDH test

SV-HUC-1 cells were seeded in a 96-well cell culture plate at a density of 10⁵ cells per mL overnight. Afterward, the cells were cultured with Ag@PGlyco-PSMA NPs, Ag nanospheres, and Ag nanocubes with the different concentrations of 0, 1, 10, 25, 50, and 100 ppm, respectively. After another 24 h culture, the 50 μL supernatant was transferred from each well to a new clear 96-well plate. Then, add 50 μL of the LDH assay working solution to each well and incubate it at room temperature for 30 minutes. Add 25 μL of the stop solution to each well. Finally, measure the absorbance at 490 nm by using a microplate reader (Synergy H1, BioTek).

2.10. Cell uptake quantification

T24 and SV-HUC-1 cells were seeded in a 6-well cell culture plate at a density of 10⁵ cells per mL overnight. Afterward, the T24 and SV-HUC-1 cells were treated with 25 ppm Ag@PGlyco-PSMA NPs for 0, 3, 6, 12, and 24 h, respectively. After treatment, the cells were washed with PBS three times to remove the residual NPs and subsequently dissolved in 12 M HCl. The dissolved solutions were subjected to the NP uptake measurement by atomic absorption spectroscopy (AAS) for Ag concentration determination.

2.11. In vitro SERS mapping

T24 and SV-HUC-1 cells were seeded in a 3.5 cm culture dish (glass bottom) at a density of 10⁵ cells per mL overnight. Afterward, the T24 and SV-HUC-1 cells were treated with 25 ppm Ag@PGlyco-PSMA NPs for 24 h. After treatment, the cells were washed with PBS three times to remove the residual NPs and subjected to SERS measurement by using a BX53 microscope (Olympus) equipped with spectrometers (MRS-iHR 320). The Raman signals were acquired using a 671 nm laser with 3 mW for an integration time of 1 s. For SERS mapping of cells, the SERS signals at 600 cm⁻¹ were acquired with 40 × 40 pixels for a mapping space of 200 × 200 μm².

2.12. Characterization

The morphology of the NPs was examined using transmission electron microscopy (TEM, H-7500, Hitachi) at 80 keV, and elemental mapping was performed with high-resolution transmission electron microscopy (JEM-2100F, JEOL). The crystalline structure of the NPs was analyzed using an X-ray thin-film diffractometer (Rigaku). The chemical states of the NPs were determined by X-ray photoelectron spectroscopy (Thermo Fisher Scientific ESCALAB Xi+). Surface-enhanced Raman scattering (SERS) spectra were recorded using a portable 671 nm Raman system (LiveStrong Optoelectronics, LS-PT 671). The hydrodynamic diameter of the NPs was measured with a dynamic light scattering spectrometer (Zetasizer Nano ZS90, Malvern, U.K.), and the PDI value was calculated by the equation below:
where σ is the standard deviation of the particle size distribution and d is the mean particle size.

Absorption spectra were recorded with an ultraviolet-visible spectrophotometer (V-730, JASCO), while Fourier-transform infrared (FT-IR) spectra of dried samples were recorded using Fourier-transform infrared spectroscopy (JASCO-4700). The concentrations of Ag in the Ag-related materials were quantified through atomic absorption spectroscopy (SensAA GBC).

2.13. In vivo animal model

C57BL/6 female mice, aged 12 weeks, were sourced from the Laboratory Animal Center at National Cheng Kung University (NCKU). Animal care was conducted in accordance with the Laboratory Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee (IACUC) of National Cheng Kung University (NCKU), Tainan, Taiwan, Republic of China. All experimental protocols received approval from the Institutional Animal Care and Use Committee (IACUC) at the Animal Experiment Center of NCKU (IACUC No. 111341). To create the orthotopic bladder tumor model, MB49 cancer cells (1 × 10⁷ cells suspended in 100 μL of PBS) were orthotopically injected into the bladder wall and allowed to settle for 90 minutes.

2.14. Ex vivo bladder tumor SERS detection

Bladder tumors were induced at intervals of 7, 14, and 21 days to obtain tumors of varying sizes. The size of the bladder was evaluated using ultrasound imaging. Following this, Au@PGlyco-PSMA nanoparticles (250 ppm_[Ag]) were instilled into the bladder for 1 hour, after which the bladder was washed three times with PBS. For SERS detection, ex vivo tumor samples were obtained through surgical removal of the bladder. Raman signals were collected using a 671 nm laser Raman system with 90 mW power, and the integration time was set at 10 seconds.

2.15. Histological examination

Histological analysis of mice tissues was performed after 1 h of instillation of Ag@PGlyco-PSMA NPs. The bladder tissue (with/without tumor) of C57BL/6 female mice was stained with hematoxylin and eosin stain (H&E stain). The H&E stained sections were then visualized by using a microscope (at 20× objective, Olympus BX53 microscope).

3. Results and discussion

3.1. Preparation and characterization of Ag@PGlyco-PSMA NPs

The Ag@PGlyco-PSMA NPs were synthesized by reacting CF₃COOAg with NaBH₄ in the presence of ONPG molecules, followed by the addition of the PSMA polymer at 30 seconds after the reaction of Ag@PGlyco NPs. Fig. 1a shows a selective zoom-in transmission electron microscopy (TEM) image for the detection of the structure wherein most particles exhibit an elliptical structure due to self-aggregation, while some others were dispersed separately with non-spherical structures. The average diameter of each nanodomain is 43.9 ± 8.2 nm (more than 100 particles). Notably, a distinct organic layer (3.7 ± 0.4 nm) surrounded each particle. Dynamic light scattering (DLS) analysis shown in Fig. S1 (ESI†) was additionally performed for characterizing 83.9 ± 8.9 nm in the resulting particle size and distribution, suggesting a microstructure of coagulated colloid in agreement with the TEM image observation (Fig. 1a and b). Moreover, the polydispersity index (PDI) of Ag@PGlyco-PSMA NPs was calculated to be 0.011, demonstrating their well-dispersed property.³⁹ TEM-EDS mapping results (Fig. 1b) indicated that the particle cores were primarily composed of Ag atoms, which were co-hybridized with O and N elements originating from the organic structures of the ONPG and PSMA molecules. High-resolution TEM (HR-TEM) imaging revealed a well-defined crystal structure with distinct periodic patterns at 2.28 Å, 2.33 Å, and 2.02 Å, which correspond to the (111)_Ag and (200)_Ag planes (Fig. 1c) and consisted of the fcc crystal structure of Ag material in the X-ray diffraction (XRD) pattern (Fig. 1d).⁴⁰

Fig. 1 (a) TEM image, (b) TEM-EDS mapping of Ag, N and O elements, (c) high-resolution TEM image, (d) XRD pattern, (e) FT-IR spectrum, (f) zeta potential, and (g) high-resolution XPS spectra of Ag 3d, C 1s and O 1s for characterizing Ag@PGlyco-PSMA NPs. (h) SERS spectrum of Ag@PSMA and Ag@PGlyco-PSMA NPs. The inset in (a) corresponds to the particle size distribution by TEM image measurement.

Fourier-transform infrared (FT-IR) spectroscopy was performed to investigate the surface molecular structure of the particles. Fig. 1e shows that the signals at 1024 cm⁻¹ and 1078 cm⁻¹ were attributed to C–O stretching and C–H bending vibrations, respectively, originating from the galactose structure in PGlyco.²³ In contrast, the signals observed at 2854 cm⁻¹ and 2927 cm⁻¹ corresponded to the stretching vibrations of –CH₃ and –CH₂ groups in methylene, which were associated with the PSMA organic backbone. Additionally, the signals detected at 1450 cm⁻¹, 1680 cm⁻¹, and 1741 cm⁻¹ were linked to other organic structures on PSMA, specifically the aromatic C–C stretching of styrene, the carbonyl group attached to the phenyl group, and ester bonds, respectively.⁴¹ At lower pH levels, the increased concentration of H⁺ ions in the solution neutralized the R-OH groups on the PSMA layer and generated protonation of polyaniline frameworks,⁴² leading to the display of a positive charge (Fig. 1f). The Ag@PSMA NPs could not perform significant positive charge feature because of the lack of polyaniline in the surface structure.

The chemical state of the nanoparticle's surface structure was analyzed by using X-ray photoelectron spectroscopy (XPS). Fig. 1g shows the XPS spectra of Ag 3d, C 1s, and O 1s, respectively. The Ag XPS spectra exhibited four peaks located at 367.2/367.8 (Ag 3d_5/2) and 373.2/373.8 eV (Ag 3d_3/2), which were attributed to the presence of metallic⁴³/ionic⁴⁴ Ag species. The presence of ionized Ag at higher binding energy was believed to be the result of Ag ions in the outermost surface area undergoing complexation with the carboxylate groups of PSMA and chemical adsorption of the condensed ONPG (forming polyaniline-containing galactosylated polymer) organic layer and a small amount of oxidation of Ag on the surface of the particles.⁴⁵ Consistently, O1s spectra show a peak at 532.4 eV attributed to the C–OH and C–O–C bonds,⁴⁶ and another peak at 533.3 eV, corresponding to the O–C–O group.⁴⁷ The C 1s spectra showed the multiple binding energies at 284.4 eV of the C–C and C–H bonds,⁴⁸ 285.0 and 285.7 eV of the polyaniline-based backbone (i.e., C–N, C=N, C–N⁺ and C=N⁺ bonds),⁴⁸ and 286.5 eV of galactose-related C–O–C and C–OH bonds.⁴⁹ The binding energy at 288.7 eV was assigned to the PSMA-protected layer of C–O=C bonds.⁵⁰ Furthermore, the SERS spectra (Fig. 1h) determined the vibrational peaks at 876 cm⁻¹ and 1145–1620 cm⁻¹ by using a 671 nm laser, being related to the benzenoid and semiquinoid ring structures of polyaniline (PANI),^23–25 as shown in (Table S1, ESI†). Such SERS signal enhancement would be due to the close adsorption of the condensed polyaniline-containing galactosylated layer around the surface of Ag NPs. Note that the peak profile of Ag@PGlyco-PSMA NPs was different from that of Ag@PSMA NPs (Fig. 1h), indicating its minimal contribution to SERS peaks. Such lack of PSMA signals would be due to the existence of the PANI spacer for the deposition of the PSMA protection layer to Ag NPs.

Therefore, based on the aforementioned analytic results and reaction steps, we presumed that the reaction mechanism of the Ag@PGlyco-PSMA NPs involved at least three stages (see Scheme 1): (1) the reductant (NaBH₄) assists in the reduction of the nitro group of ONPG to ortho-aminophenyl-β-D-galactopyranoside (OAPG) upon Ag nuclei generation, acting as a catalyst. (2) OAPG molecules could donate electrons to reduce Ag ions in the solution and initiate an aniline-related polymerization process on the surface of the Ag seeds, followed by the continuous growth of Ag@PGlyco NPs. (3) Subsequently, the addition of the PSMA polymer led to the formation of an organic protective layer on the surface of Ag@PGlyco NPs, effectively stopping further particle size from growing.

3.2. Reactions of Ag@PGlyco NPs with different polymers

The optimum reaction time for producing a strong SERS signal was 30–120 seconds after the addition of the PSMA polymer (Fig. S2, ESI†). Fig. S2a (ESI†) shows TEM images indicating that delaying the addition of the PSMA polymer improved the particle dispersion (>150 seconds, Fig. S2a-iii, ESI†). Still, increased delay led to a significant reduction in SERS intensity (Fig. S2b, ESI†) because of a few hot-spot enhancements from the inter-particle junction sites.⁴ This kinetic formation, arising from the clustering of several primary particles, resulted in the generation of clearly separated particles. This phenomenon could be attributed to the destabilization of these nanoaggregates through Coulomb repulsion and/or steric interactions, akin to the Turkevich method for synthesizing Au NPs.^51–53 The formation of dispersed Ag nanoaggregates with high SERS intensity of PGlyco was advised to occur after a reaction time of 30 seconds (Fig. 1a). The other parameter was 40 mM of NaBH₄ (Fig. S3, ESI†), which offered a sufficient electron number to reduce both Ag and ONPG and contribute to the strong SERS performance by great PANI–Ag interaction.²⁵ When PSMA was replaced with polyethyleneimine (PEI) or polyvinylpyrrolidone (PVP), TEM images revealed that the polymer-coated Ag@PGlyco NPs primarily adopt an elliptical shape, similar to Ag@PGlyco-PSMA, with varying degrees of aggregation depending on the polymer used (Fig. 2a–c). Without a protective agent, particles tended to generate Ag@PGlyco nanoaggregates significantly (Fig. 2d and e). The absorption spectrum of Ag@PGlyco-PSMA featured a peak around 390.5 nm (Fig. 2f), indicating its nano-size effect,⁵ with tail absorption extending to the NIR wavelength region,⁵⁴ suggesting a remarkable evolution of inter-particle aggregation. Ag@PGlyco samples prepared with PSMA, PEI, and PVP show similar PANI-related SERS peaks, but the intensity varies. While PSMA- and PVP-coated Ag@PGlyco NPs provided the relevant strong SERS signal intensity, the amine groups of the PEI polymer might also bind to the surface of Ag NPs to partly remove the PGlyco molecules, thereby weakening the SERS signal (Fig. 2g). In contrast, Ag@PGlyco NPs (polymer-free group) showed the lowest SERS intensity due to the lack of nano-sized SPR-related SERS properties.^1–3,5–8

Fig. 2 TEM images of (a) Ag@PSMA, (b) Ag@PGlyco-PVP, (c) Ag@PGlyco-PEI and (d) Ag@PGlyco NPs. (e) Picture of Ag@PGlyco NPs aggregation. The (f) UV-visible and (g) SERS spectra of the Ag@PSMA, Ag@PGlyco-PVP, Ag@ PGlyco-PEI, Ag@PGlyco-PSMA and Ag@PGlyco NPs.

3.3. Stability of Ag@PGlyco-PSMA NPs

We used UV-visible spectra to determine the SPR stability of Ag@PGlyco-PSMA NPs in D.I. water (Fig. S4a, ESI†) and 1/10× phosphate-buffered saline (PBS) over 0 to 5 days (Fig. S4b, ESI†). A decrease of ∼11.5% in the optical density (O.D.) value was observed in D.I. water. Moreover, under 14 mM chloride ion in the 1/10× PBS solution, the O.D. value change rate was similar to that observed in the water dispersion (Fig. S4c, ESI†). This result indicated that Ag@PGlyco-PSMA NPs resisted oxidation by Cl⁻ ions.

3.4. SERS performance of Ag@PGlyco-PSMA NPs

To validate the metallic property of Ag, we utilized a 671 nm laser to investigate the SERS performance of Ag@PGlyco-PSMA NPs by reacting with malachite green (MG), 4-nitrothiophenol (4-NTP), and rhodamine 6G (R6G) at different concentrations. A signal subtraction between Ag@PGlyco-PSMA NPs and Ag@PGlyco-PSMA NPs plus analytes was performed for all samples (Fig. 3a) to identify the analyte molecular signature cleaner; an example for this is shown in Fig. 3b. The assignments of the three samples are listed in Tables S2–S4 (ESI†) according to the literature.^6–8 It could be observed that the LOD of SERS was 8 × 10⁻⁸ M for MG (Fig. 3c and f), which was superior to 2 × 10⁻⁶ M for R6G (Fig. 3d and g) because the negatively charged surface of the Ag@PGlyco-PSMA NPs facilitates the adsorption of the cationic MG analyte at pH = 7. For 4-NTP, the detection limit of 8 × 10⁻⁸ M was achieved with Ag@PGlyco-PSMA NPs (Fig. 3e and h) by evolving binding interaction between the thiol group of 4-NTP and the Ag atoms of the Ag@PGlyco-PSMA NPs, which evoked a chemical enhancement mechanism.^1–5,8 The SERS intensity diagrams obtained for different analytes exhibit non-linear variations with respect to the analyte concentration. This non-linear response was likely due to the complicated PGlyco-PSMA coating layer (Fig. 1a), impeding the inward diffusion of the analytes to the Ag NP.

Fig. 3 (a) Schematic illustration of the particle structure of Ag@PGlyco-PSMA NPs. (b) SERS spectra of 4-NTP with Ag@PGlyco-PSMA NPs after mutual subtraction. (c)–(e) SERS spectra and (f)–(h) SERS intensity records (n = 5) of (c) and (f) MG, (d) and (g) R6G, and (e) and (h) 4-NTP in different concentrations.

The analytical enhancement factor (AEF) of Ag@PGlyco-PSMA NPs at a concentration of 15 ppm_[Ag], calculated from the SERS signal of the physisorption MG molecule, was 1.71 × 10⁴ (the calculation formula and details can be found in the ESI†) (Fig. S5). To further evaluate the SERS performance, we synthesized Ag nanospheres⁵⁵ (69.3 ± 14.3 nm, Fig. S6a, ESI†) and Ag nanocubes⁵⁶ (47.9 ± 6.3 nm, Fig. S6b, ESI†) synthesized according to the literature and compared their SERS intensity enhancement for MG molecules (Fig. S6c–e, ESI†). A 5.80 fold and 1.23 fold increase in SERS intensity was obtained by utilizing Ag@PGlyco-PSMA NPs (with inter-particle aggregates contributing to hotspots) in contrast to the use of free-standing Ag nanospheres and Ag nanocubes, respectively. Additionally, for 4-NTP, the signal intensity measured using Ag@PGlyco-PSMA NPs increased by 25.47 folds and 1.19 folds compared to those measured using Ag nanospheres and Ag nanocubes, respectively (Fig. S6f–h, ESI†).

Previous studies in our laboratory indicated that substituting reaction precursors with Au ions resulted in the formation of Au@PGlyco NPs.²³ However, the SERS signal of Au@PGlyco-PSMA NPs was obtained using the same preparation method as that of Ag@PGlyco-PSMA NPs. It could see a significant decrease after the addition of PSMA (Fig. S7a, ESI†). Using the peak at 1599 cm⁻¹ as a reference for comparison, it was found that the SERS intensity of Ag@PGlyco-PSMA was approximately 34 times higher than that of Au@PGlyco-PSMA (Fig. S7a and b, ESI†). The various differences in peak intensity and shifting were observed due to the different coordination environments of PANI with metal ions.^23–25 In addition, a comparative analysis of the enhancement in SERS intensity for MG was conducted using the two types of metal-based particles mentioned (Fig. S7c and d, ESI†), providing direct and clear evidence of the superior SERS enhancement capability of Ag@PGlyco-PSMA NPs.

3.5. Cytotoxicity of Ag@PGlyco-PSMA NPs

Given the harmful effects of Ag ions released from Ag NPs on living cells, further investigation into the toxicity of Ag-sensitive bacteria was conducted. The survival rate of E. coli was observed to be as high as 90 ± 3.14% at a concentration of 50 ppm_[Ag] (Fig. 4a). Fluorescence imaging of the co-culture of E. coli with Ag@PGlyco-PSMA NPs confirmed that the living cells displayed green fluorescence, with no indications of dead cells (red emission), in contrast to the results seen with free Ag ions at 50 ppm_[Ag] (Fig. 4b). After 24 hours, approximately 2.1 ± 0.3 ppm of Ag ions was detected in the PBS solution (Fig. 4c). This gradual and limited release of Ag ions from Ag@PGlyco-PSMA NPs into the medium helped to ensure the viability of E. coli. In stark contrast, significantly higher concentrations of Ag ions released from Ag nanospheres and Ag nanocubes (Fig. 4c) resulted in notable toxic effects on E. coli. The PSMA coating might effectively restrict the release of Ag ions from the Ag nanocore, thereby aiding in the preservation of bacterial survival (Fig. 4a).

Fig. 4 (a) Bacteria viability of E. coli after 1 day of culture time with Ag@PGlyco-PSMA NPs, Ag nanospheres, Ag nanocubes and Ag ions in different concentrations of 0–50 ppm_[Ag] (n = 4). (b) Live/death staining fluorescence images for co-culture of E. coli with 50 ppm of Ag@PGlyco-PSMA NPs and Ag ions for 24 h. (c) Ag release assessment of 50 ppm Ag@PGlyco-PSMA NPs, Ag nanospheres, and Ag nanocubes in D.I. water and medium at 37 °C for 24 h (n = 5). (d) MTT assays were conducted on VERO, SV-HUC-1, T24, and MB49 cells cultured with Ag@PGlyco-PSMA NPs at various concentrations ranging from 0 to 100 ppm_[Ag] for 24 h (n = 4). (e) SERS images of SV-HUC-1 and T24 cells cultured with 25 ppm_[Ag] Ag@PGlyco-PSMA NPs for 24 h. (f) AAS for the cellular uptake examination of SV-HUC-1 and T24 cells cultured with 25 ppm_[Ag] Ag@PGlyco-PSMA NPs for 0, 3, 6, 12, and 24 h (n = 4). The flow chart, depicted in panel (g), illustrates the timeline for tumor induction and the administration of instilled samples at 7, 14, and 21 days. The treatment for tumor induction at various time points for detection with (h) ultrasound images of orthotopic bladder tumors (scale bar: 2 mm) and their corresponding (i) SERS spectra obtained from post-treatment with Ag@PGlyco-PSMA NPs, and (j) records of bladder tumor volume (black line, quantified from Fig. 4h) and measurements of SERS intensity (blue line, quantified from Fig. 4i, n = 5).

We subsequently examined the cell viability of four mammalian cell lines following treatment with Ag@PGlyco-PSMA NPs. These include normal cell lines, such as SV-HUC-1 cells (human urinary tract epithelium cells) and Vero cells (African green monkey kidney epithelial cells), alongside cancer cell lines T24 (human bladder carcinoma cells) and MB49 (mouse bladder carcinoma cells). Fig. 4d demonstrates a robust cell viability rate exceeding 80% for all cell types at a concentration of 25 ppm (24 h), surpassing the biosafety standards for most Ag-related nanomaterials reported in the literature (Fig. S8a and d, ESI†).^27,31 A moderate tolerance level exceeding 60% was observed for all cells at 100 ppm_[Ag]. Ag@PGlyco-PSMA NPs showed enhanced biocompatibility of T24 cells and SV-HUC-1 cells in comparison to Ag nanospheres and Ag nanocubes during the 24–72 hours culture period (Fig. S8, ESI†). Significantly, the 25–100 ppm of Ag@PGlyco-PSMA NPs exhibited at least 84% of cell viability after 72-hour culture time (Fig. S8c and f, ESI†). We also examined the intracellular reactive oxygen species (ROS) levels in SV-HUC-1 cells induced by various Ag-related nanomaterials (Fig. S9, ESI†). The fluorescence images revealed a low intensity of green fluorescence in Ag@PGlyco-PSMA-treated cells (24 h) stained with DCFHDA. The substantial release of Ag ions (Fig. 4c) from both Ag nanospheres and Ag nanocubes resulted in an increase in ROS within the cell body. Similarly, the LDH assay indicated the low lactate dehydrogenase (LDH) release from the damaged cells caused by Ag@PGlyco-PSMA NPs (Fig. S10, ESI†). These results suggested that the PSMA/ONPG-coated surface layer plays a crucial role in preventing the excessive release of Ag ions from the Ag@PGlyco-PSMA NPs at 25 ppm_[Ag], preventing Ag-induced ROS generation by interacting with intracellular biomolecules, including DNA,^28,31 proteins,^29,31 and mitochondria.^30,31

3.6. In vitro bladder cancer cell tracking ability of Ag@PGlyco-PSMA NPs

Bladder cancer is one of the most common cancer types worldwide,⁵⁷ which might cause urinary frequency and dysuria in patients when the tumors progress to obstruct the bladder outlet or reduce bladder capacity.^58,59 Developing the rapid diagnostic method would aid in detecting the growth behavior of bladder tumors. Due to the galactose-related glycopolymer structure of Ag@PGlyco-PSMA NPs, which confer molecular binding capabilities at galactose-based receptors, high biocompatibility, and intrinsic PGlyco-related delivery, we conducted SERS studies to determine whether 25 ppm of Ag@PGlyco-PSMA NPs could specifically recognize T24 cells in co-culture experiments. Fig. 4e demonstrates that SERS imaging revealed a significant accumulation of Ag@PGlyco-PSMA NPs in T24 cells, with signal strength increasing over the co-culture duration. These signals consistently surpassed those observed for Ag@PGlyco-PSMA NPs in SV-HUC-1 cells at all examined time points. The optical intensity analysis of the accumulation regions correlated well with destructive atomic absorption spectroscopy (AAS) analysis (Fig. 4f), confirming the superior accumulation capability of Ag@PGlyco-PSMA NPs in cancer cells compared to normal cells. This phenomenon might be attributed to the heightened nutrient demands of T24 cells, which led to the overexpression of sugar-related receptors³³ or transport proteins³⁴ on the cell membrane. Such overexpression may facilitate enhanced recognition and subsequent endocytosis³⁵ of Ag@PGlyco-PSMA NPs to T24 cancer cells.

3.7. Ex vivo bladder tumor detection of Ag@PGlyco-PSMA NPs

To demonstrate tissue-level SERS detection using Ag@PGlyco-PSMA NPs, we utilized a surgical resection bladder tumor model with MB49-bearing mice for SERS evaluation and ultrasound (US) imaging monitoring of tumor growth (Fig. 4g). To confirm successful tumor induction, we assessed the bladder cavity volume through US imaging (Fig. 4h). The yellow dotted circle denotes the initial size of the bladder cavity, while the red dotted circle highlights the differences in residual volume following the introduction of cancer cells. US imaging results indicated a significant increase in tumor proliferation within the bladder wall at the 0, 7, 14, and 21-day intervals (Fig. 4h). Subsequently, we administered Ag@PGlyco-PSMA NPs (100 μL and 250 ppm_[Ag]) into the bladders of the mice for one hour, after which the bladders were surgically excised for further SERS analysis. The SERS signal at 1599 cm⁻¹ from the intrinsic signal of Au@PGlyco-PSMA NPs was used for comparison. The differences in SERS intensity reflected an increase in SERS signals corresponding to the days of tumor induction at 7, 14, and 21 (Fig. 4i). These tumor-associated SERS signals were notably higher than those obtained from the same labeling process in normal bladder tissue. Fig. 4j illustrates that the SERS intensity correlates with the trend in tumor volume growth, confirming that this SERS detection platform effectively monitors tumor volume ex vivo. The H&E staining results (Fig. S11, ESI†) indicated that there was no significant tissue damage following the instillation of Ag@PGlyco-PSMA NPs, confirming its in vivo safety attributed to the nanoparticle's surface stability and minimal Ag ion release (Fig. 4c) upon uptake by tumor cells. As a result, our developed Ag@PGlyco-PSMA NPs served as an intrinsic Raman imaging tool for bladder cancer cells, presenting promising prospects for cancer diagnosis and imaging in future medicine.

4. Conclusion

In summary, we successfully developed Ag@PGlyco-PSMA NPs, a novel class of Ag NPs with intrinsic SERS capabilities, high biocompatibility, and selective cancer cell recognition properties. By introducing a galactose-related glycopolymer coating, these NPs addressed the limitations of conventional Ag NPs, such as cytotoxicity caused by Ag ion release and insufficient biocompatibility for biological applications. The PSMA coating not only restricted the release of Ag ions, thereby minimizing toxicity, but also enhanced the stability and optical properties of the NPs, facilitating the generation of strong and reliable SERS signals. Additionally, the spontaneous polymerization of ONPG during synthesis eliminated the need for external Raman probes, simplifying the preparation process while maintaining robust SERS activity. The exposed galactose side chains on the glycopolymer enabled the selective recognition of cancer cells in vitro and ex vivo, which overexpressed sugar-related receptors and transport proteins, leading to higher NP accumulation in cancer cells than in normal cells. This selective recognition, coupled with the improved SERS signals, promoted cancer diagnostics and noninvasive optical imaging. Overall, the Ag@PGlyco-PSMA NPs developed in this study provided a promising platform for cancer diagnosis and imaging. Our future work with Ag@PGlyco-PSMA NPs will concentrate on integrating in vivo Raman detection systems that are optimized for minimal photodamage to monitor bladder tumors and to access in vivo imaging and pharmacokinetics in real time, advancing toward the translation of clinical applications.

Author contributions

TYC, KHC, and YJC conducted experiments, analyzed data, and TYC contributed to manuscript drafting. TYC, KHC, YCC, and CCH participated in data analysis and performed validation. TYC and YCC provided methodological and software support. YCC and CCH acquired funding. CCH conceptualized ideas, provided supervision, oversaw the research, and finalized the manuscript.

Conflicts of interest

The authors have reported no potential competing interests.

Data availability

The authors confirm that the data of this study are available within the article and ESI.†

Acknowledgements

This study was supported by the National Science and Technology Council, Taiwan, R.O.C. (ID: NSTC 112-2113-M-006-006-MY3, NSTC 114-2622-M-006-003-, and NSTC 113-2218-E-006 024-) and by a grant from the Taipei City Hospital and the Department of Health, Taipei City Government (TCH No. 11301-62-035 and 11401-62-044). This work was also financially supported by the Center of Applied Nanomedicine, National Cheng Kung University, the Featured Areas Research Center Program and the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. We are also grateful for the support from the Laboratory Animal Center, College of Medicine, and the Core Facility Center (EM000800, ESCA000200, EM02620, and XRD005100), National Cheng Kung University, and the Core Facility of Taiwan Mouse Clinic and Animal Consortium.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00546a

‡ These authors contributed equally.

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