Abstract
Peptide nucleic acids (PNAs) are a class of artificial oligonucleotide mimics that have garnered much attention as precision biotherapeutics for their efficient hybridization properties and their exceptional biological and chemical stability. However, the poor cellular uptake of PNA is a limiting factor to its more extensive use in biomedicine; encapsulation in nanoparticle carriers has therefore emerged as a strategy for internalization and delivery of PNA in cells. In this study, we demonstrate that PNA can be readily loaded into porous silicon nanoparticles (pSiNPs) following a simple salt-based trapping procedure thus far employed only for negatively charged synthetic oligonucleotides. We show that the ease and versatility of PNA chemistry also allows for producing PNAs with different net charge, from positive to negative, and that the use of differently charged PNAs enables optimization of loading into pSiNPs. Differently charged PNA payloads determine different release kinetics and allow modulation of the temporal profile of the delivery process. In vitro silencing of a set of specific microRNAs using a pSiNP-PNA delivery platform demonstrates the potential for biomedical applications.
Highlights
Peptide nucleic acids (PNAs) are a class of artificial oligonucleotide mimics that have garnered much attention as precision biotherapeutics for their efficient hybridization properties and their exceptional biological and chemical stability
The presence of this unnatural backbone is why PNAs have exceptional biological stability: in contrast to natural nucleic acids and peptides, they are not recognized and degraded by nucleases and proteases. Another crucial feature of PNAs is that by using standard solid-phase manual or automated synthesis[4] typical of peptide synthesis, PNA monomers can be assembled and conjugated with a range of diverse functional groups, such as fluorophores, amino acids, or small molecules. These properties have all been harnessed to exploit the use of PNAs in a wide range of biomedical applications, from precision medicine therapeutics to chemical biology to molecular diagnostics.[5,6]
Some of the most promising approaches involve conjugation with cell-penetrating peptides,[8−12] or formulation with liposomes,[13−15] polymer nanoparticles,[16−21] and carbon-based nanocarriers.[22−25] Porous inorganic nanocarriers have been demonstrated to be suited for the loading and the delivery of PNA.[26−28] Among these, porous silicon nanoparticles have emerged as a class of nanomaterials with a suite of properties that makes them especially amenable to accommodating and releasing biomolecular payloads
Summary
PNA 1- R8 PNA 1- E8 PNA 1 PNA 2- R8 PNA 2- E8 PNA 2 PNA 3- R8 PNA 3- E8 PNA 3 loading (nmol/mg). The encapsulation of the eight arginine PNA (PNA-R8) into Ca-pSiNPs suggests that other positively charged payloads can be incorporated into pSiNPs, which is ideal for minimizing the typical cytotoxicity associated with cationic drugs.[48−50] In addition, using a calcium silicate-based approach for encapsulation of negatively charged payloads, such as siRNA or other therapeutic oligonucleotides, allows for bypassing the need for cationic polymers that are often proposed for loading anionic payloads in nanoparticle carriers through electrostatic interaction and that raise concern for their potential cytotoxicity.[35,40,51] PSiNPs loaded with neutral PNA payloads successfully delivered their cargo in a model cell line relevant to cystic fibrosis and enabled silencing of three specific target microRNAs. Delivery of CFTR-upregulating anti-miRNA PNAs can support new therapeutic strategies for the treatment of cystic fibrosis.[52,53] These results all demonstrate a strategy for formulating PNA into pSiNPs for therapeutic applications in precision medicine.
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