pharmaceuticals Review Multifunctional Delivery Systems for Peptide Nucleic Acids Stefano Volpi , Umberto Cancelli, Martina Neri and Roberto Corradini * Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124 Parma, Italy; [email protected] (S.V.); [email protected] (U.C.); [email protected] (M.N.) * Correspondence: [email protected]; Tel.: +39-0521-905410 Abstract: The number of applications of peptide nucleic acids (PNAs)—oligonucleotide analogs with a polyamide backbone—is continuously increasing in both in vitro and cellular systems and, parallel to this, delivery systems able to bring PNAs to their targets have been developed. This review is intended to give to the readers an overview on the available carriers for these oligonucleotide mimics, with a particular emphasis on newly developed multi-component- and multifunctional vehicles which boosted PNA research in recent years. The following approaches will be discussed: (a) conjugation with carrier molecules and peptides; (b) liposome formulations; (c) polymer nanopar- ticles; (d) inorganic porous nanoparticles; (e) carbon based nanocarriers; and (f) self-assembled and supramolecular systems. New therapeutic strategies enabled by the combination of PNA and proper delivery systems are discussed. Keywords: peptide nucleic acids; delivery; nanoparticles; conjugates; multifunctional systems 1. Introduction 1.1. Peptide Nucleic Acids and Their Uses Peptide nucleic acids (PNAs, Figure1) are DNA analogues in which the sugar- Citation: Volpi, S.; Cancelli, U.; phosphate units connecting the nucleobases are replaced by N-(2-aminoethyl)glycine Neri, M.; Corradini, R. moieties [1]. These molecules are excellent binding partners for cognate DNA and RNA Multifunctional Delivery Systems for strands, being able to exploit both the canonical Watson–Crick (WC) base pairing to form Peptide Nucleic Acids. stable duplexes [1,2] and a combination of WC and Hoogsteen hydrogen bonding to be Pharmaceuticals 2021 14 , , 14. arranged with their counterpart in triple-helical adducts [3], eventually performing dsDNA https://dx.doi.org/10.3390/ strand invasion [4,5]. PNAs and their open-chain analogs (bearing modifications in the ph14010014 polyamide backbones as indicated in Figure1b) have been used in a series of important applications [6] encompassing drug development [7,8], RNA and DNA detection for di- Received: 2 December 2020 Accepted: 23 December 2020 agnostics [9–11], nanofabrication [12,13], and chemical encoded libraries [14] (Figure1c) . Published: 25 December 2020 PNAs are also particularly suited for the development of sensory systems (Figure1c), thanks to their high stability in biological media [15] and to their high fidelity in the Publisher’s Note: MDPI stays neu- recognition of relevant DNA and RNA sequences [16]. In fact, they are able to tightly tral with regard to jurisdictional claims bind complementary nucleic acids with high selectivity, presenting a particular ability in published maps and institutional to discriminate mismatched sequences even in the presence of a single mismatched base affiliations. (i.e., in point mutations or single nucleotide polymorphisms). In this review, however, we will focus only on the applications of diagnostics in which PNA probes are delivered into cells (in vitro or in vivo detection), leaving to the readers some general review for their use in the sensory sector [17,18]. Another application of PNAs which has recently attracted Copyright: © 2020 by the authors. Li- increasing attention is their use as “smart” materials for self-assembly, nanofabrication, censee MDPI, Basel, Switzerland. This and computing, as they are able to form PNA:PNA duplexes which can be easily modulated article is an open access article distributed in stability and helical handedness [18–20]. under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Pharmaceuticals 2021, 14, 14. https://dx.doi.org/10.3390/ph14010014 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 14 2 of 31 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 32 Figure 1.1. Schematic structures of (a)) PNAPNA andand ((b)) modifiedmodified PNAsPNAs withwith pendantpendant side chains,chains, and (cc)) typestypes ofof complexescomplexes formedformed withwith DNA,DNA, RNA,RNA, and and PNA PNA and and general general applications applications derived derived from from these these interactions. interactions. The The structures structures reported reported in (inc) are(c) are 3D 3D models models derived derived from from Protein Protein Data Data Bank Bank (entries (entries inr8, inr8, 176d, 176d, 1pnn, 1pnn, and and 1pup, 1pup, respectively). respectively). Being very efficient efficient tools for modulating gene gene expression expression both both inin vitro vitro andand inin vivo, vivo, PNAs and and their their analogues analogues have have been been proposed proposed as as antisense antisense molecules molecules capable capable of ofblocking block- mRNAing mRNA or correcting or correcting aberrant aberrant pre-mRNA pre-mRNA splicing splicing (Figure (Figure 2a) [21–23],2a) [ 21 a– field23], athat field has that re- centlyhas recently boosted boosted research research on novel on novelantimicrobial antimicrobial agents agents [24–26], [24 –as26 anti-gene], as anti-gene agents, agents, thus blockingthus blocking transcription transcription from DNA from DNAto mRNA to mRNA (Figure (Figure 2b) [27–29],2b) [ 27 as– 29anti-miR], as anti-miR agents, agents, block- ingblocking this important this important regulative regulative class classof non-coding of non-coding RNAs RNAs (Figure (Figure 2c) [30–33],2c) [ 30 –and33], as and “de- as “decoy”coy” molecules molecules capable capable of sequestering of sequestering transcription transcription factors factors (Figure (Figure 2d)2d) [34]. [ 34 It]. should It should be notedbe noted that that the the antisense antisense action action of of PNAs PNAs occu occurr by by steric steric blockage blockage of of the corresponding mRNA [35–37], [35–37], without the occurrence of enzymaticenzymatic digestion as instead observed in the RNAse-H- or RISC-mediated cl cleavageeavage of other antisense oligonucleotidesoligonucleotides and siRNA. Similarly, PNA-based anti-gene or anti-miR systemssystems rely only on their tight bindingbinding to their targets, and hence bothboth theirtheir bindingbinding abilityability andand intracellularintracellular concentrationconcentration shouldshould be maximized maximized to to obtain obtain a atherapeutic therapeutic effect. effect. Another Another important important and and relatively relatively recent recent use ofuse PNAs—especially of PNAs—especially those those able ableto form to form triplexes triplexes with with target target DNAs DNAs (i.e., (i.e.,bis-PNAs bis-PNAs and and tail-clamp PNAs)—is the induction of homologous recombination processes with an tail-clamp PNAs)—is the induction of homologous recombination processes with an ex- exogenous DNA segment, leading to genome editing or “repair” (Figure2e) [38]. ogenous DNA segment, leading to genome editing or “repair” (Figure 2e) [38]. One of the most important issues in the development of PNA technology for biological One of the most important issues in the development of PNA technology for biolog- applications, either for therapy or in vivo diagnosis, is their poor uptake by target cells. ical applications, either for therapy or in vivo diagnosis, is their poor uptake by target In order to solve this drawback, the various approaches that have been considered (Figure2) cells. In order to solve this drawback, the various approaches that have been considered can be differentiated into: (a) cellular delivery, (b) specific delivery in the subcellular (Figure 2) can be differentiated into: (a) cellular delivery, (b) specific delivery in the sub- compartment, and (c) tissue delivery for in vivo studies. In this review, we will focus on cellular compartment, and (c) tissue delivery for in vivo studies. In this review, we will the use of multifunctional and multicomponent delivery systems to enable the cellular focus on the use of multifunctional and multicomponent delivery systems to enable the uptake of PNAs, with the aim to give a general view on this subject that update what cellular uptake of PNAs, with the aim to give a general view on this subject that update reported in previous reviews [39] or extend more recent works describing only some whataspect reported of nanoparticle-based in previous reviews PNA [39] transport or extend [40, more41]. Additionally, recent works thedescribing description only ofsome the aspectapproaches of nanoparticle-based exploiting these types PNA of transport oligonucleotides [40,41]. asAdditionally, building blocks the fordescription multifunctional of the approaches exploiting these types of oligonucleotides as building blocks for multifunc- Pharmaceuticals 2021, 14, 14 3 of 31 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 32 materials will be discussed, with a particular emphasis on recent works and concepts unveilingtional materials the enormous will be possibilities discussed,opened with a byparticular the rational emphasis use of on PNAs recent as works components and con- forcepts “biomolecular unveiling engineering”the enormous [42 possibilities]. opened by the rational use of PNAs as compo- nents for “biomolecular engineering”[42]. FigureFigure 2. Uses 2. Uses and