PSMA-specific theranostic nanoplex for combination of TRAIL gene and 5-FC prodrug therapy of prostate cancer

Abstract

Metastatic prostate cancer causes significant morbidity and mortality and there is a critical unmet need for effective treatments. We have developed a theranostic nanoplex platform for combined imaging and therapy of prostate cancer. Our prostate-specific membrane antigen (PSMA) targeted nanoplex is designed to deliver plasmid DNA encoding tumor necrosis factor related apoptosis-inducing ligand (TRAIL), together with bacterial cytosine deaminase (bCD) as a prodrug enzyme. Nanoplex specificity was tested using two variants of human PC3 prostate cancer cells in culture and in tumor xenografts, one with high PSMA expression and the other with negligible expression levels. The expression of EGFP-TRAIL was demonstrated by fluorescence optical imaging and real-time PCR. Noninvasive (19)F MR spectroscopy detected the conversion of the nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by bCD. The combination strategy of TRAIL gene and 5-FC/bCD therapy showed significant inhibition of the growth of prostate cancer cells and tumors. These data demonstrate that the PSMA-specific theranostic nanoplex can deliver gene therapy and prodrug enzyme therapy concurrently for precision medicine in metastatic prostate cancer.

Keywords: Gene therapy; PSMA; Prodrug enzyme therapy; Prostate cancer; TRAIL; Theranostic imaging.

Figure 1

Structure of PSMA-targeted nanoplex 1

Figure 2

Synthetic procedure of generating PSMA-targeted nanoplex 1.

Figure 3

A-E. Distribution of the hydrodynamic radius of PSMA-targeted nanoplex 1 at different N/P ratios. F. Average zeta potential of bCD, PSMA-targeted nanoplex 1 without pEGFP-TRAIL DNA (compound 8) and PSMA-targeted nanoplex 1 (N/P = 50). Values represent mean ± SEM of three measurements.

Figure 4

A-D. Laser confocal fluorescence microscopy of PC3-PIP (PSMA+ve) and PC3-Flu (PSMA−ve) cells. A. PC3-PIP cells treated with PSMA-targeted nanoplex 1; B. PC3-PIP cells treated with non-PSMA-targeted nanoplex; C. PC3-Flu cells treated with PSMA-targeted nanoplex 1; D. PC3-Flu cells treated with non-PSMA-targeted nanoplex. (DIC: differential interference contrast imaging; treatment time: 8 hours; concentration of pDNA: 2 μg/mL; N/P ratio: 50; scale bar: 50 μm).

Figure 5

A. Quantitative reverse transcription PCR mRNA expression of PC3-PIP and PC3-Flu cells following treatment with PSMA-targeted nanoplex 1 for eight hours (concentration of pDNA: 2 μg/mL; N/P ratio: 50; without 5-FC/bCD treatment; ** P < 0.01; bars are SD). B. and C. Cell viability of PC3-PIP and PC3-Flu cells with different treatments. Cells were treated for 24 and 48 hours (concentration of pDNA: 2 μg/ml; N/P ratio: 50; concentration of 5-FC: 3mM; values are generated from two separate experiments with three wells per experiment per condition; ** P < 0.01, * P < 0.05, bars are SD).

Figure 6

A. Representative longitudinal in vivo Cy5.5 NIR fluorescence optical images of SCID mice bearing PC3-PIP and PC3-Flu tumors. Mouse was injected IV. with nanoplex (150 mg/kg in 0.2 mL saline). B. Ex vivo Cy5.5 NIR fluorescence optical imaging of PC3-PIP and PC3-Flu tumors. Mouse was injected IV. with nanoplex (150 mg/kg in 0.2 mL saline). C. Quantification of Cy 5.5 fluorescence intensity in ex vivo imaging study. (** P < 0.01, * P < 0.05, bars are SEM, n = 3). D. In vivo ¹⁹F MR spectra acquired from a PC3-PIP tumor (~400 mm³) at 24 hours after IV. injection of the PSMA-targeted nanoplex (100 mg/kg) carrying bCD. Spectra were acquired after a combined IV. and IP. injection of 5-FC (450 mg/kg), on a Bruker Biospec 9.4 T spectrometer using a 1 cm solenoid coil tunable to ¹H and ¹⁹F frequency. Following shimming on the water proton signal, serial nonselective ¹⁹F MR spectra were acquired starting 20 min after the 5-FC injection and continued every 30 min for 110 min with a repetition time of 0.8 s, a number of scans of 2,000, and a spectral width of 10 KHz.

Figure 7

A. Real time PCR mRNA expression studies of PC3-PIP and PC3-Flu tumors with different treatments (control: non-treatment as the negative control; pEGFP-U6: PSMA-targeted pEGFP-U6 pDNA treatment as the positive control; pEFGP-TRAIL: PSMA-targeted pEFGP-TRAIL pDNA treatment). Tumor tissue was collected at 24 hours after mice were injected with PSMA-targeted nanoplex carrying pEGFP-U6 or pEGFP-TRAIL (dosage of DNA: 3.2 mg/kg; N/P ratio: 50; ** P < 0.01, bars represent SD, n = 3 or 4). B. Ex vivo EGFP fluorescence images of PC3-PIP and PC3-Flu tumor slices at 72 hours after treatment.

Figure 8

A. Inhibition of PC3-PIP tumor growth with different treatment (tumor volume was normalized to the pretreatment volume, values represent median ± SD (n =3 or 4), following a combined IV. and IP. injection of 5-FC (450 mg/kg) at 24 hours after IV. injection of the PSMA-targeted nanoplex (100 mg/kg). B. Representative high-resolution scanned images of H&E stained histological sections obtained at 10 days post treatment with NaCl solution, PSMA-targeted pEGFP-TRAIL pDNA only, PSMA-targeted 5-FC only, and combination of PSMA-targeted pEGFP-TRAIL pDNA and 5-FC. Purple hematoxiphilic regions indicate viable tumor tissues, and eosinophilic areas indicated tumor necrosis. C. Representative high-resolution scanned images of Ki-67 immunostained adjacent sections corresponding to the sections in B. Scale bar: 3.0 mm.