Nucleic Acid Based Fluorinated Derivatives: New Tools for Biomedical Applications

Nucleic acid-based fluorinated derivatives, e.g., nucleosides or oligonucleotides connected to highly fluorinated chains or labeled with one or more fluorine atoms, have been investigated recently due to their high potential for biomedical applications. This review deals with recent works on nucleoside and oligonucleotide fluorocarbon amphiphiles as well as with properties and applications of fluorine-labeled oligonucleotide analogues.


Introduction
The nucleic acid architectures stabilized by base-pairing and aryl π−π stacking interactions provides to scientists one of the most sophisticated supramolecular models.Since the determination of the structure of double-stranded DNA (dsDNA) by Watson, Crick, Wilkins and Franklin [1][2][3], artificial bio-inspired molecules featuring nucleic acid units have become a very powerful strategy to construct supramolecular systems [4][5][6].Of particular interest is the design of hybrid amphiphilic nucleolipids featuring a bi-functionality based on the combination of nucleic acids and lipids [7][8][9][10][11][12][13][14][15].Interestingly, different bottom-up approaches involving nucleolipids have been used for the construction of nanostructures for different applications, including materials [16][17][18] and biomedical devices [19][20][21].In parallel to nucleolipid derivatives, the combination of nucleobase units with fluorocarbon OPEN ACCESS hydrophobic and lipophobic moieties to create novel materials, for diagnostics or in medical applications, is still in its infancy.It has been extensively reported that highly fluorinated derivatives possess unique properties, including chemical and biological inertness, strong hydro-and lipophobia.Interestingly, fluorocarbon colloids used for biomedical applications feature high gas solubility, low lipid and water solubility, and outstanding surface characteristics [22].
Hence, material and biomedical sciences could take advantage of the new properties coming from the combination of nucleic acids and fluorocarbon synthetic units for new applications.The availability of chemically modified nucleotides and fluorocarbon synthons and/or reagents, means that Nucleic Acids Fluorocarbon Amphiphiles (NAFAs) could be used in numerous fields of research including engineering, biology and medicine (Figure 1).Recently, the use of nucleoside-based fluorocarbon derivatives has been mainly motivated by three fundamental objectives: (i) construct novel supramolecular materials and/or devices; (ii) develop new drug delivery systems; and (iii) find new therapeutic molecules.This short, non-exhaustive review intends to illustrate the role that highly fluorinated amphiphiles may play in chemistry, biology and material science.It consists primarily of an account of the authors' own efforts and experience in the field, hence will be limited to some specific aspects of this chemistry.It also intends to outline the potential applications of fluorinated nucleolipids, ranging from chemistry to biomedical research.
First, we will focus on the recent progress achieved in the fields of fluorocarbon and nucleoside amphiphiles.This first section includes hybrid molecules bearing both a nucleic acid and fluorocarbon chains: namely the Fluorocarbon Nucleoside amphiphiles (FNs).Second, we will present several Fluorocarbon OligoNucleotide amphiphiles (FONs) and fluorinated oligonucleotide analogues.

Fluorocarbon Amphiphiles
Due to their unique properties, fluorocarbon chains represent a useful building block for the construction of supramolecular systems.Perfluoroalkyl chains have a larger cross section and are more rigid than their hydrogenated counterparts.They are considerably more hydrophobic and are lipophobic as well.As a result, when dispersed in water, fluorinated amphiphiles exhibit an enhanced capacity to self-assemble into highly stable and well-defined assemblies such as films, membranes, micelles, vesicles and other stable supramolecular systems [23,24].The self-assembling behavior of a variety of synthesized single and double chain neutral, zwitterionicor anionic fluorinated phospholipids [25][26][27][28], glycolipid [29,30], glycophospholipids, glycopeptides [31,32] was already explored (Figure 2) [33], to evaluate the impact of the fluorinated chains on the properties of the resulting aggregates.It enhances stability, low permeability to both hydrophilic and lipophilic material and reduces interaction with biological media.These fluorinated amphiphiles possess a considerable potential for the delivery of materials such as drugs, prodrugs, markers, contrast agents and nucleic acids.Recently, new perfluorinated dimerizable polycationic amphiphiles were designed by Vierling et al. to evaluate the ability of the monomer to condense DNA and of the dimer to form stable nanoparticles capable of efficient cell transfection [34].Although formulation of small-sized cationic monomolecular DNA nanoparticles (<40 nm) with the dimerizable perfluorinated succeeded, spermine-based amphiphiles proved to be poor non-specific transfection agents in vitro.Improvements are still in progress [35,36].[32]; and (c) perfluorinated dimerizable polycationic amphiphile [34].

Nucleoside Amphiphiles
Nucleoside-based lipids commonly named nucleolipids [7] are hybrid molecules composed of a nucleobase, a nucleoside, a nucleotide or an oligonucleotide (either DNA or RNA), and a lipophilic moiety, which might be either simply a single-or double-chained alkyl moiety.Several improved nucleolipids have been reported hereafter (Figure 3) [37][38][39][40][41][42][43][44][45][46][47].Most nucleolipids have a polar head group at the 5' position and hydrophobic groups at both the 2' and 3' positions, patterned after natural glycerol phospholipids, which are key components of the cellular membrane.Nucleolipids were designed to interact with nucleic acids through hydrogen bonding, π-π stacking, and nucleobase recognition, as well as electrostatic interactions and the hydrophobic effect.Several cationic [12,[48][49][50][51] and anionic [52,53] nucleolipids were first investigated as potential agents for the delivery of nucleic acids into cells.Indeed their self-assembled structures into lipoplexes mimic cellular membranes and their cationic properties attract both anionic oligonucleotides and negatively charged cellular membranes.New versions comprising neutral, zwitterionic, and anionic nucleolipids are currently evaluated.[39]; and (c) a zwitterionic nucleolipid.

Hybrid Fluorocarbon Nucleoside Amphiphiles
The combination of the self-assembly potential of nucleoside amphiphiles with the hydrophobic character of highly fluorinated chains is of high relevance to prepare new self-assembled structures with tunable physico-chemical properties and functions.To assess whether fluorinated chains can enhance the scope of supramolecular assemblies formed by hybrid fluorocarbon nucleoside amphiphiles, different molecules were synthesized (Figure 4).
The fluorocarbon amphiphile 1 is based on a uridine backbone modified by a phosphocholine moiety at the 5' position, and two 2H,2H,3H,3H-perfluoroundecanoyl side chains at the 2' and 3' positions.One of the goals of the study was to characterize the properties of such new compounds and to compare those results with that observed for the analogous hydrocarbon nucleoamphiphiles.In aqueous solution, fluorocarbon nucleoamphiphiles were surprisingly able to form large vesicular assemblies and unexpected lamellar phases below their T m , contrary to hydrocarbon analogs that self-assemble into helical structures below their T m and bilayers above it [54].These highly hydrophobic fluorocarbon amphiphiles exhibited interesting hybrid microspherical supramolecular assemblies in the presence of thorium that were characterized by Scanning Electron Microscopy (SEM) [55].Recently, two glycosyl-nucleoside fluorinated amphiphiles (GNFs) were designed to construct new supramolecular hydrogels.Molecules 2 and 3 feature both sugar moieties attached to a thymidine base and a 2H,2H,3H,3H-perfluoroundecanoyl side chain at the 5' position, linked via triazole bridges resulting from "click" chemistry.The self-assembly and gelation properties of such molecules were investigated.Similarly to their hydrocarbon analogs [56,57] GNFs stabilize hydrogels at low concentrations (0.1% w/w for compound 2).GNFs gelators displayed supramolecular networks of varying morphologies, such as nanofibers and helical springs, which are correlated to the presence of fluorocarbon chains.Moreover, fluorocarbon amphiphile GNF 2 proved to be not toxic for human cells (Huh7) whereas hydrocarbon analogue becomes toxic above 100 µM, demonstrating the greater potential of such fluorocarbon nucleoside amphiphiles for biomedical applications [58].

Fluorocarbon OligoNucleotide Amphiphiles (FONs) and Fluorinated Oligonucleotide Analogues
In the previous section, several examples of nucleoside-based fluorinated amphiphiles have been discussed for gel stabilization.In this section we will discuss on Fluorocarbon OligoNucleotide amphiphiles (FONs).The fluorine atom is likely a mimic of either a hydroxyl group (in terms of electronegativity) [59][60][61][62] or a hydrogen atom (in terms of steric factor).In this context, we will also provide an overview on properties and applications of oligonucleotides carrying fluorine modifications at different positions.

Fluorocarbon OligoNucleotide Amphiphiles (FONs)
Recently our group has reported on the synthesis and biological properties of FONs [63].In previous studies, our group already reported on the synthesis and cellular uptake studies of lipid-oligonucleotide conjugates (LONs) [64,65].The main purpose of attaching a fluorocarbon chain to a polyanionic oligonucleotide (ONs) was to study how cellular uptake or internalization of ONs are influenced by the nature of the hydrophobic moieties.To address this issue, Godeau et al. have synthesized two FONs featuring C 8 F 17 and C 6 F 13 chains using 1,3-dipolar Huisgen's reaction [66] via a "post synthetic" modification approach.Three different human cell lines were incubated with either FONs or LONs or unconjugated ONs for comparative study of cellular internalization (Figure 5).Moreover, internalization kinetics of FONs, LONs and unconjugated ONs with Huh7 cells were also evaluated.Keeping in mind the hydrophobic and lipophobic properties of fluorocarbon chains, it can be hypothesized that FONs self-assemble outside the cell to form nano aggregates.It is likely that these nanoobjects would be uptaken by the cell via an endocytosis pathway.It is worth mentioning here that to the best of our knowledge, there is no earlier report on oligonucleotide amphiphiles featuring perfluorinated chains.

Conclusions
The nucleic acids fluorocarbon amphiphiles belong to a new class of hybrid bioinspired molecules, which is clearly connected to soft materials, nanosciences and biomedical technology.It is a promising and exciting time for NAFAs and the emerging devices developed so far clearly open up interesting routes for the design and development of nanodevices, therapeutic strategies and biotechnological tools.The potential innovations using these fluorocarbon molecules are numerous in terms of chemical architectures, physicochemical and biological properties.This short survey highlights the interest to combine nucleic acids with fluorocarbons for developing soft and complex matters.The history of this field is still at the beginning and we can imagine that remarkable achievements will be realized in the future.

Figure 5 .
Figure 5. Conjugation of fluorocarbon chain to the oligonucleotide (FONs) allows the delivery of highly polyanionic oligonucleotides into human cells.

Figure 8 .
Figure 8. Monomer building blocks for the incorporation of fluorine derivative into RNA oligonucleotides by solid-phase synthesis.