Development of Folic Acid-Conjugated and Methylene Blue-Adsorbed Au@TNA Nanoparticles for Enhanced Photodynamic Therapy of Bladder Cancer Cells

Abstract

Photodynamic therapy (PDT) is a promising treatment for malignancy. However, the low molecular solubility of photosensitizers (PSs) with a low accumulation at borderline malignant potential lesions results in the tardy and ineffective management of recurrent urothelial carcinoma. Herein, we used tannic acid (TNA), a green precursor, to reduce HAuCl₄ in order to generate Au@TNA core-shell nanoparticles. The photosensitizer methylene blue (MB) was subsequently adsorbed onto the surface of the Au@TNA nanoparticles, leading to the incorporation of a PS within the organic shell of the Au nanoparticle nanosupport, denoted as Au@TNA@MB nanoparticles (NPs). By modifying the surface of the Au@TNA@MB NPs with the ligand folate acid (FA) using NH₂-PEG-NH₂ as a linker, we demonstrated that the targeted delivery strategy achieved a high accumulation of PSs in cancer cells. The cell viability of T24 cells decreased to 87.1%, 57.1%, and 26.6% upon treatment with 10 ppm_[Au] Au@TNA/MB NPs after 45 min, 2 h, and 4 h of incubation, respectively. We also applied the same targeted PDT treatment to normal urothelial SV-HUC-1 cells and observed minor phototoxicity, indicating that this safe photomedicine shows promise for applications aiming to achieve the local depletion of cancer sites without side effects.

Keywords: Au@TNA nanoparticles; bladder cancer; photodynamic therapy; photomedicine; photosensitizers; phototoxicity.

Figure 1

(a) Transmission electron microscopy (TEM) image and (b,c) High-resolution TEM (HR-TEM) images of Au@TNA nanoparticles (NPs). (d) TEM image of Au@TNA@MB NPs. (e) X-ray diffraction (XRD) pattern of Au@TNA@MB NPs. (f) X-ray photoelectron spectroscopy (XPS) gold (Au) 4f orbitals of Au@TNA (red) and Au@TNA@MB (black) NPs.

Figure 2

(a) UV-visible and (b) fluorescence spectra of Au@TNA NPs, Au@TNA@MB NPs, and methylene blue (MB) molecules. (b) SERS measurement of Au@TNA@MB NPs at 633 and 785 nm.

Figure 3

UV-visible spectra (a) and relative intensity at 440 nm (b) of the N,N-dimethyl-4-nitrosoaniline (RNO)/imidazole indicator for measuring single oxygen generation from Au@TNA@MB NPs as a function of the irradiation time at 650 nm. Pure MB and Au@TNA are presented as control groups. (c) Thermographic images mapping the temperature increase of 25 ppm_[Au] Au@TNA@MB NPs at 650 nm (125 mW/cm²).

Figure 4

(a) Thiazolyl blue tetrazolium bromide (MTT) assay of HeLa cells cotreated with particles for 24 h: Au@TNA, Au@TNA@MB, Au@TNA plus 650 nm light, and Au@TNA@MB plus 650 nm light. (b) 2’,7’-dichlorofluorescin diacetate (DCFHDA)-stained HeLa cells, (c) bright field image of HeLa cells, and (d) trypan blue-stained HeLa cells. The cells were stained after 4 h of treatment with Au@TNA@MB and then exposed to 650 nm light. (e) Bright field images of HeLa cell apoptosis after pretreatment with 10 ppm_[Au] Au@TNA@MB (4 h) plus 650 nm light. The 650 nm laser power density was 125 mW/cm².

Figure 5

MTT assays of (a) T24 cells cotreated with particles for 24 h: Au@TNA, Au@TNA@MB, Au@TNA plus 650 nm light, and Au@TNA@MB plus 650 nm light. T24 cells cotreated with (b) Au@TNA@MB NPs and (c) folic acid (FA)-conjugated Au@TNA@MB NPs for 0.75–4 h upon excitation at 650 nm. Photodynamic therapy (PDT) treatment of T24 cells stained with DCFH-DA dye by using (d) Au@TNA@MB NPs and (e) FA-conjugated Au@TNA@MB NPs. Top: bright field image, middle: fluorescent image, and bottom: merged image. The 650 nm laser power density was 125 mW/cm².

Figure 6

MTT assays of SV-HUC-1 cells cotreated with (a) Au@TNA@MB and (b) FA-conjugated Au@TNA@MB NPs for 0.75–4 h plus 650 nm light (125 mW/cm²), followed by another 24 h of incubation in fresh medium.