Prostate-specific membrane antigen targeted gold nanoparticles for prostate cancer radiotherapy: does size matter for targeted particles?

Abstract

Since the introduction of PSA testing, significantly more men have been diagnosed and treated for prostate cancer. Localized prostate cancer typically is treated with prostatectomy, however there is still a high risk of recurrence after surgery, and adjuvant radiation has been shown to mitigate disease progression. X-ray therapy is frequently used as an adjuvant to treat prostate cancer, but is an imperfect tool. In this report we describe the development of a targeted-radiosensitizing nanoparticle that significantly improves X-ray therapy. Taking advantage of the demonstrated radiosensitizing activity of gold nanoparticles (AuNPs) we developed targeted AuNPs and varied both surface ligand density and AuNP size to develop an optimized AuNP for X-ray radiotherapy. We conjugated a prostate-specific membrane antigen (PSMA) targeting ligand, PSMA-1, to AuNPs and found that the targeting ligand dramatically improved gold uptake by PSMA-expressing PC3pip cells compared with PC3flu cells lacking the PSMA receptors. Further, enhancement of radiotherapy was significantly more pronounced by internalization of smaller PSMA targeted-AuNPs. Our studies provide a foundation for design of size-selected AuNPs for targeted radiotherapy and, for the first time, systematically investigate both the effect of ligand and AuNP size on the cell uptake, tumor targeting and radiotherapy efficacy.

Scheme 1. Schematic illustration of targeted radiotherapy of prostate cancer using PSMA-targeted AuNPs of different sizes.

Fig. 1. Synthesis of AuNPs with PSMA-1 targeting. (a) Molecular structure of PSMA-1 conjugated PEG_2k (PSMA-1 in pink and PEG_2k in blue), and schematic of AuNPs modified with both PEG_2k-PSMA-1 and PEG_2k; (b) ESI-MS spectrum of PSMA-1 with m/z at 1087; (c) MOLDI-TOF mass spectrum of PEG_2k-PSMA-1 conjugations; (d) Agarose gel electrophoresis demonstrates the successful binding of PEG_2k-PSMA-1 to AuNPs and mobility of AuNPs with different sizes (T, PSMA-targeted AuNPs; NT, nontargeted AuNPs); (e–g) TEM images AuNPs with average core sizes of (a) 2 nm, (b) 5 nm and (c) 19 nm, with PEG_2k shell stained with phosphotungstic acid.

Fig. 2. PSMA-1 ligand mediated cell uptake of 5 nm AuNPs. AuNPs were conjugated with PEG_2k-PSMA-1 and PEG_2k at ratios of 0, 1 : 8, 1 : 4, 1 : 1 and 2 : 1. The Au content in each cell was verified as a function of PSMA-1 conjugation ratio as determined by ICP-MS. Data are presented as mean ± SD (n = 3).

Fig. 3. Effect of particle size on cell uptake of the PSMA-targeted AuNPs. (a) PSMA-targeted AuNPs with core sizes of 2 nm, 5 nm and 19 nm were incubated with PC3pip and PC3flu cells for 1 h, 6 h and 24 h and then stained by silver staining kits. The silver nucleates around AuNPs and thus reveals AuNP uptake by the cells. (b) Quantitative Au content in cells incubated with 2 nm, 5 nm and 19 nm AuNPs as determined by ICP-MS divided by total cell number. Data are presented as mean ± SD (n = 3), and differences between groups are compared with two-tailed t-tests, *p ≤ 0.05 **p ≤ 0.01.

Fig. 4. In vitro radiation enhancement by AuNPs with different sizes. PC3pip and PC3flu cells were incubated with both PSMA-targeted (T) and non-targeted (NT) AuNPs with sizes of (a) 2 nm, (b) 5 nm and (c) 19 nm for 24 h and irradiated with X-ray at 2 Gy, 4 Gy and 6 Gy. (d) Radiation enhancing factor (REF, ratio of eradicated cells with and without AuNPs) for PC3pip cells incubated with PSMA-targeted AuNPs at radiation doses of 2 Gy, 4 Gy and 6 Gy. Data are presented as mean ± SD (n = 3), and differences between groups are compared with two-tailed t-tests, *p ≤ 0.05 **p ≤ 0.01.

Fig. 5. Tumor uptake and biodistribution of AuNPs with different sizes. (a) In vivo 3D CT images of PC3pip (blue circle) and Pc3flu (green circle) tumor-bearing mice at 4 h post-injection of both PSMA-targeted and untargeted AuNPs. (b) Quantitative analysis of the CT signals at PC3pip and PC3flu tumor regions after injection of PSMA-targeted (T) and non-targeted (NT) AuNPs at 0.5 h, 1 h, 2 h, 4 h, 6 h and 24 h, (c) biodistributions of AuNPs after 24 h as determined by ICP-MS. Data are presented as mean ± SD (n = 3), and differences of Au content in PC3pip and PC3flu tumors are compared with two tailed t-tests, *p ≤ 0.05 **p ≤ 0.01.

Fig. 6. In vivo AuNP enhanced radiotherapy. (a) Schematic PC3pip and PC3flu tumor bearing mouse, and X-ray radiation of tumors and timeline. (b and c) PC3pip and PC3flu tumor growth curves after injection of (b) PSMA-targeted and (c) untargeted AuNPs with different sizes. Data are presented as mean ± SD (n = 3), and growth inhibition of PC3pip and PC3flu tumors are compared with two-tailed t-test, **p ≤ 0.01.