Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy

Abstract

Background: miR-15a and miR-16-1 have been identified as tumor suppressor genes in prostate cancer, but their safe and effective delivery to target cells is key to the successful use of this therapeutic strategy. RNA aptamer A10 has been used as a ligand, targeting prostate cancer cells that express prostate-specific membrane antigen (PSMA). Compared with A10, the binding of the second-generation RNA aptamer, A10-3.2, to PSMA is more efficient.

Methods: A10-3.2 was investigated as a PSMA-targeting ligand in the design of a polyamidoamine (PAMAM)-based microRNA (miR-15a and miR-16-1) vector to prostate cancer cells. Using polyethyleneglycol (PEG) as a spacer, PAMAM was conjugated to aptamer (PAMAM-PEG-APT) and used as a vehicle for miRNA target delivery.

Results: Luciferase assays of pGL-3 expression against PC3 (PSMA(-)) and LNCaP (PSMA(+)) cells demonstrated that the transfection efficiency of the synthesized DNA/PAMAM-PEG-APT complex was higher than that of the DNA/PAMAM-PEG complex. In addition, cell viability assays of LNCaP (PSMA(+)) cells showed that, with a N/P ratio of 15:1, the IC(50) value of miRNA/PAMAM-PEG-APT was approximately 4.7-fold lower than that of miRNA/PAMAM-PEG.

Conclusion: This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of prostate cancer cells.

Keywords: aptamer; miRNA; polyamidoamine; prostate cancer; prostate-specific membrane antigen; targeted delivery.

Figure 1

NMR spectra of (A) PAMAM, (B) PAMAM-PEG, and (C) PAMAM-PEG-APT in D₂O at 300 mHz. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Figure 2

(A) Fluorescence microscopy images (scale bar 50 μm) and (B) fluorescent-activated cell sorting analysis (n = 3, error bars represent a standard deviation) after a 60-minute incubation of DyLight-633-labeled PAMAM-PEG-APT as a function of concentration range against PC3 and LNCaP cells, respectively. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Figure 3

Sizes and zeta potentials of pEGFP/PAMAM-PEG-APT complexes at various N/P ratios. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Figure 4

(A) Fluorescence microscopy images (scale bar 50 μm) and (B) luciferase activity analysis (n = 4, error bars represent the standard deviation) after transfection of pGL-3/PAMAM-PEG or pGL-3/PAMAM-PEG-APT as a function of N/P ratios against PC3 and LNCaP cells, respectively. *P < 0.05. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Figure 5

Bcl-2, Cyclin D1, and Wnt3a knock down following miRNA/PAMAM-PEG-APT complexes delivery to LNCaP cells. The cells were treated with miRNA/PAMAM-PEG-APT (miRNA) and NC-miRNA/PAMAM-PEG-APT (NC-miRNA) complexes and processed for immunoblotting with anti-Bcl-2, anti-Cyclin D1, and anti-Wnt3a antibodies at 24, 48, and 72 hours post-transfection. β-actin was a loading control. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Figure 6

Cell viabilities of LNCaP cells treated with miRNA/PAMAM-PEG-APT, NC-miRNA/PAMAM-PEG-APT, miRNA/PAMAM-PEG complexes, respectively, (n = 3, error bars represent the standard deviation). Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.