Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5

Abstract

The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.

Figure 1. (A) Human CCR5 genomic DNA and transcribed mRNA transcript (GenBank accession number AF011539) (B) Chemical structure of unmodified PNA, ^MPγPNA, ^MPγGPNA units (C) Designed oligomer sequence of unmodified and gamma-modified PNA oligomers to target mRNA transcript. Bold letters indicate ^MPγPNA units, Bold underline indicates ^MPγGPNA units, and bold underlined and italics indicates the mismatch sites. All the PNAs positions are from 677 to 693 as shown above.

Figure 2. UV-melting profiles of PNA-RNA hybrid duplexes at 5 μM strand concentration each in 10 mM sodium phosphate buffer at pH 7.4. Both the heating and cooling runs were performed; they both had nearly identical profiles (only the heating runs are shown).

Figure 3. Nanoparticles show uniform size and morphology. Scanning electron microscope (SEM) images of PNA and γPNA nanoparticle batches. Average particle diameter and SD given under each batch.

Figure 4. PNA and γPNA nanoparticle release profile data of nucleic acid after indicated time points in a graph and incubation at 37°C.

Figure 5. Fluorescent confocal live-cell images of HeLa cells following 24 h incubation with nanoparticles containing ^MPγGPNA3 and naked ^MPγGPNA3.

Figure 6. CCR5 mRNA expression in THP1 cells after treatment with PLGA nanoparticles containing PNA as indicated in X-axis. CCR5 expression relative to average blank control (all normalized to GAPDH, n = 3, p < 0.05).

Figure 7. CCR5 mRNA expression in THP1 cells after treatment with ^MPγGPNA3 and MM−^ΜPγPNA4 containing PLGA nanoparticles. CCR5 expression relative to average blank control (all normalized to GAPDH, n = 3, * p < 0.05).

Figure 8. Dose-dependent effect in CCR5 mRNA expression level in THP1 cell lines after treatment with different doses of ^MP γGPNA3-containing PLGA nanoparticles. CCR5 expression relative to average blank control (all normalized to GAPDH, n = 3, * p < 0.05).

Figure 9. Western blot showing the corresponding changes of CCR5 protein levels in THP1 cells after treatment with blank, PNA1 and ^MP γGPNA3-containing nanoparticles for 24 h.

Figure 10. Cell survival graph data for THP1 cell lines after 10 h and 25 h treatment with nanoparticles containing PNA/γPNA oligomers.