Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer

Abstract

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

Keywords: PSMA; gold nanoparticles; photodynamic therapy; prostate cancer; theranostics.

Figure 1.

(a) Conjugation of PSMA-1 to OPSS-5kPEG-NHS followed by deprotection of thiol and (b) MALDI-TOF mass spectra of PSMA-1.

Figure 2.

(a) Synthesis of AuNP-5kPEG-PSMA-1 and loading of Pc4. (b) Agarose gel electrophoresis demonstrates the successful binding of 5kPEG-PSMA-1 to AuNP (c) UV−vis absorbance spectroscopy shows that Pc4 is incorporated into the nanoparticle system.

Figure 3.

Transmission electron microscopy of (a) unstained and (b) stained AuNP conjugates evidenced the formation of fairly monodispersed nanoparticles. (c) TEM image and (d) UV−vis spectra of the same nanoparticle after one year of storage at −20 °C.

Figure 4.

(a) ¹O₂ generation was confirmed by monitoring the oxidation of a singlet oxygen trap, diphenylisobenzofuran (DPBF). The inset shows the chemical transformation of DPBF in the presence of ¹O₂. (b) Evaluation of ¹O₂ generation of Pc4 in gold nanoparticles in EtOH solution. The data suggest that Pc4 molecules are indeed trapped in the polymer shell of the AuNP.

Figure 5.

(a) Competition binding study for different PSMA ligands: parent ZJ24, PSMA-1, and AuNP5kPEGPSMA-1. (b) Efficacy of AuNP-5kPEG-PSMA-1-Pc4 for killing prostate cancer cell lines. (c) Results of a DCFDA assay showing fluorescence images of PC3pip cells postirradiation (scale bar = 200 μm). Error bars ± SD.

Figure 6.

Cellular uptake study of targeted and nontargeted AuNP. Pc4 fluorescence was visualized using a fluorescence microscope, and Au nanoparticles were visualized using white light after enhancement with silver staining (dimension = 853 × 638 μm). Images were taken at 400×.

Figure 7.

(a, b) Maestro fluorescence images showing selective accumulation of AuNP-5kPEG-PSMA-1-Pc4 on PC3pip tumor. (c) Gold content of excised PC3pip and PC3flu tumors 3 h postinjection of AuNP-5kPEG-PSMA-1-Pc4 and (inset) sections of PC3pip and PC3flu stained with silver (scale bar = 1 cm).

Figure 8.

(a) Representative images of the same mouse monitored for GFP fluorescence after receiving 0.07 mg/kg AuNP-5kPEG-PSMA-1-Pc4 followed by PDT (300 J/cm²). The efficacy of the treatment was determined by monitoring (b) tumor volume, (c) total GFP signal, and (d) body weight (n = 5).

Scheme 1.

Design of PSMA-1-targeted gold nanoparticles containing Pc4 as imaging and PDT agent.