Gold nanorods/siRNA complex administration for knockdown of PARP-1: a potential treatment for perinatal asphyxia

Abstract

Background: Perinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated with systemic and neurological diseases. Despite the important role of poly (ADP-ribose) polymerase 1 (PARP-1) in the regulation of gene expression and DNA repair, overactivation of PARP-1 in asphyxia-exposed animals worsens the ATP-dependent energetic crisis. Inhibition of PARP-1 offers a therapeutic strategy for diminishing the effects of perinatal asphyxia.

Methods: We designed a nanosystem that incorporates a specific siRNA for PARP-1 knockdown. The siRNA was complexed with gold nanorods (AuNR) conjugated to the peptide CLPFFD for brain targeting.

Results: The siRNA was efficiently delivered into PC12 cells, resulting in gene silencing. The complex was administered intraperitoneally in vivo to asphyxia-exposed rat pups, and the ability of the AuNR-CLPFFD/siRNA complex to reach the brain was demonstrated.

Conclusion: The combination of a nanosystem for delivery and a specific siRNA for gene silencing resulted in effective inhibition of PARP-1 in vivo.

Keywords: PARP-1 knockdown; PC12; gold nanorods; in vivo administration; neonatal hypoxia; rats; siRNA delivery.

Figure 1

UV-visible and TEM characterization. Notes: (A) UV-visible spectra characterization. AuNR (blue line) produces a longitudinal 512 nm peak and a transverse 762 nm peak (blue arrow). After conjugation with the CLPFFD peptide, the AuNR-CLPFFD (red line) presented a shift to a longitudinal 514 nm peak and a transverse 771 nm peak (red arrow). The graphic representation shows the peak displacement, indicating the conjugation of the CLPFFD peptide to the surface of the AuNR. (B) Characterization of the shape and aspect ratio of AuNR-CLPFFD. A representative TEM image of AuNR-CLPFFD showing the cylindrical shape of the nanoparticles is presented. The inset shows the aspect ratio histogram of the observed nanoparticles. The average aspect ratio, which was 5±0.9 (length/width), was determined by measuring 150 nanoparticles in different images. Abbreviations: TEM, transmission electron microscopy; AuNR, gold nanorod.

Figure 2

Hydrodynamic diameter and zeta potential characterization. Notes: (A) Hydrodynamic diameter of AuNR-CLPFFD and the AuNR-CLPFFD/siRNA complex. The average hydrodynamic diameter of AuNR-CLPFFD was 90 nm in length and 6 nm in width with a polydispersity index (PDI) of 0.5. The hydrodynamic diameter of the AuNR-CLPFFD/siRNA complex increased to 206 nm in length and 9 nm in width with a PDI of 0.7 (column graphic), suggesting the association of multiple AuNR-CLPFFD with siRNA molecules and their molecular rearrangement. (B) Zeta potential changes from 47.8 mV in AuNR-CLPFFD (red line) to 25 mV in AuNR-CLPFFD/siRNA complex (green line) (quantified in the column graphic) were observed, suggesting that electrostatic interactions occur between positively charged AuNR-CLPFFD and negatively charged siRNA. Abbreviation: AuNR, gold nanorod.

Figure 3

Effect of AuNR-CLPFFD and AuNR-CLPFFD/siRNA complexes on PC12 cell viability. Notes: PC12 cells were treated with increasing concentrations of (A) AuNR-CLPFFD and (B) AuNR-CLPFFD/siRNA complexes for 24 hours. The MTS assay shows decreased mitochondrial activity only in the cells that were treated with the highest concentrations (0.5–1 nM) of the compounds. Comparisons were analyzed using Student’s t-test (*P<0.05 compared to the control; n=6 for the respective experimental groups). Abbreviations: AuNR, gold nanorod; MTS, 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Figure 4

Cellular uptake of the AuNR-CLPFFD/siRNA complex and knockdown of PARP-1 protein. Notes: (A) PC12 cells treated with 1) nuclease-free H₂O (CS), 2) naked PARP-1 siRNA (siRNA, 0.1 nM), 3) FuGENE^® loaded with siRNA (3:1) (FuGene), or 4) AuNR-CLPFFD/siRNA complex (Complex, 0.1 nM) 1 hour after the treatment. Images were obtained by confocal microscopy: Hoechst 33342-stained nuclei (blue), early endosomes (green), fluorescent siRNA label dye 647 (red). The images shown represent the merging of three pictures taken at 1 µm intervals. Scale bar: 50 µm; magnification, 60×. The image suggests the colocalization of siRNA with the dotted pattern of early endosomes. (B) Relative quantification of siRNA fluorescence. (C) Quantification of PARP-1 protein levels in PC12 cells. The results show that the complex was able to transport the siRNA into the cells at 1 hour post-treatment. The siRNA inhibited the expression of PARP-1 protein 24 hours post-treatment (*P<0.03 compared to the saline control; F-ANOVA followed by Dunnett’s post hoc test [n=4 for each group]). Abbreviations: AuNR, gold nanorod; CS, cesarean-delivered control rats.

Figure 5

Gold content of neonatal rat brain. Notes: Distribution of gold (ng/g dry tissue) in brain samples from AuNR-CLPFFD/siRNA complex treated neonatal rats after a single dose (2.2 nM, ip). Samples were obtained at 30 minutes, 1 hour, and 2 hours after treatment. The result shows that gold accumulates in the brains of the neonatal rats after passing through the BBB. No differences between CS and AS animals were observed. ANOVA followed by Dunnett’s post hoc test (n=6 for each group). Abbreviations: AuNR, gold nanorod; ip, intraperitoneal; CS, cesarean-delivered control rats; AS, asphyxia-exposed rats; BBB, blood–brain barrier.

Figure 6

In vivo knockdown of PARP-1 in AS-exposed animals. Notes: Administration of a single dose of AuNR-CLPFFD/siRNA complex (ip) to AS-exposed animals. The animals were treated with 1) saline solution (Saline), 1) naked siRNA (siRNA), or 3) AuNR-CLPFFD/siRNA complex (Complex) 1 hour after birth. Total protein extracts were obtained 24 hours after treatment. PARP-1 protein levels in the mesencephalon, hippocampus, and telencephalon were quantified by Western blotting. The quantification shows that treatment with the AuNR-CLPFFD/siRNA complex decreased PARP-1 levels in the mesencephalon and hippocampus (^aP<0.05 compared to the saline treatment, Student’s t-test; n=6 for each condition). Abbreviations: AuNR, gold nanorod; ip, intraperitoneal; AS, asphyxia-exposed rats.