2.1 Nucleoside phosphocholine derivatives

One of the biggest challenges for bio-inspired amphiphiles is finding easy ways to take advantages of their building blocks to construct complex supramolecular systems. To achieve this objective, a clear understanding of the chemical structural parameters that dictate self-assembly is of high importance. Our general approach was to modify the molecular structures of conventional lipids such as fatty acids via the incorporation of either a nucleoside or a nucleotide. A first family of zwitterionic uridine phosphocholine amphiphiles was initially synthesized as shown in Scheme 1 [10]. This four-step synthetic strategy was developed to afford the phosphocholine uridine amphiphiles 5a through 5e. As expected, the uridine derivatives synthesized provided an easy access to supramolecular assemblies in aqueous and non-aqueous media. Interestingly, by changing the temperature, the nucleoside amphiphiles form fibers or bilayers in aqueous solvent. This transformation between the two supramolecular structures, which depends on the melting temperature (T_m) of the amphiphiles, was found to be fully reversible (Fig. 2). The hydrogels stabilized by the nano-fibers can be prepared in the presence of plasmid DNA, suggesting that the system could be used as functional delivery matrix.

An adenosine phosphocholine derivative with fatty esters on the 2′ and 3′ positions was also prepared by using a convenient synthetic route involving unprotected intermediates [11]. Similarly as for uridine amphiphiles, the adenine moiety does not prohibit the formation of lamellar organizations above the T_m and stable small unilamellar vesicles were prepared by using extrusion techniques.

The behavior of the perfluorocarbon analogues was explored more recently. In the context of fluorinated molecules, it was straightforward to achieve the substitution of the 2′ and 3′ nucleoside ribose positions by fluorocarbon chains. Consequently, the 2H,2H,3H,3H-perfluoroundecanoic acid was DCC coupled to the 2′ and 3′ secondary hydroxyls of 5′-phosphocholine uridine 4 (Scheme 1) to afford the fluorinated nucleoside-based amphiphile (2′,3′-O-di-2H,2H,3H,3H-perfluoro-undecanoyl-uridine-5′-phosphocholine, DiF₁₇UPC) shown in Fig. 3 [12]. Surprisingly, despite the high hydrophobic character of the amphiphile, lamellar systems were observed (Fig. 3). Indeed, hydrocarbon analogues possessing saturated alkyl chains such as DSUPC (1,2-Distearoyluridinophosphocholine) self-assemble into helical structures stabilizing nano-fibers in the crystalline state below its T_m. The lamellar systems, observed in the case of the fluorinated derivative, were explained by the greater stiffness and volume of the fluorocarbon chains, which avoid the chain–chain packing requested for the formation of the nano-fibers.

2.2 Nucleotide-based lipids

There are several ways of designing novel bio-inspired amphiphiles featuring nucleobases. One of the simplest ways consists of using a nucleotide instead of a nucleoside. A variety of synthetic strategies have been explored to synthesize nucleotide-based amphiphiles [13], The large literature dedicated to this class of amphiphiles reports mainly hydrophobic modification of nucleoside-5′-monophosphate derivatives. Another strategy was to develop a synthetic route to 3′-O-alkyl nucleotide. The rationale behind this synthetic approach was to use the 3′-phosphoramidites 5′-protected nucleosides commercially available as starting materials for the preparation of the nucleotide amphiphiles. Accordingly, single chain nucleoside-3′-monophosphate amphiphiles were synthesized as shown in Fig. 4. Briefly stated, starting material 6 was reacted with different fatty alcohols in the presence of tetrazole, the resulting lipidic intermediates were oxidized with iodine in a THF/pyridine/water mixture at room temperature. Removal of the cyanoethyl and dimethoxytrityl protecting groups led to a series of nucleotide-based amphiphiles 8. It was discovered that the aqueous dispersion of these nucleotide amphiphiles could induce the formation of nano-objects. The collected information that emerged from numerous investigations (TEM, SEM, FT-IR and WAXS) of these nucleotide amphiphiles demonstrated that these nucleotide amphiphiles possess different aggregating properties compared to both their non-nucleotide analogues and simple nucleotides. Nano-structured assemblies such as ribbon-like structures for the thymidine derivatives (Fig. 5) and small aggregates for adenine amphiphiles were observed, indicating that the nature of the base has a direct impact on the aggregates morphology.

2.3 2D-supramolecular assemblies on graphite surface

Beside nano-structured assemblies in colloidal suspensions, the preparation of functional surfaces with nanometer dimensions is also currently attracting a substantial interest from both a fundamental and an applied point of view [14]. In many cases the functionalization of a surface is required to take advantage of the properties of a given material. For example, to make nanotubes more easily dispersible in liquids, it is necessary to physically or chemically attach certain molecules, or functional groups, to their smooth sidewalls. This process, which is called functionalization, has to be achieved for biological applications especially for sensing [15]. In this context, nucleoside amphiphiphiles are ideal candidates for surface nanopatterning and/or functionalization.

Since the assembly processes of amphiphilic molecules on surfaces and interfaces can be imaged at the nanometer scale by using scanning probe microscopies, The behavior of nucleotide amphiphiphiles at the liquid/solid interface was investigated in collaboration with Dr. Fichou's group (CEA Saclay, France). Interestingly, two-dimensional supramolecular organizations on graphite were successfully imaged by high resolution Scanning Tunnelling Microscopy (STM) by self-assembly of physisorbed amphiphile nucleotides [16]. Here again, it was found that the organization of the molecules was dictated by the nature of nucleobases. In the case of the thymidine amphiphiles a head-to-tail self-assembly was imaged (Fig. 6C), whereas a head-to-head organization was observed for adenosine derivatives (Fig. 6A). Surprisingly, the co-adsorption of complementary mixtures allowed the formation of a two-dimensional supramolecular structure likely stabilized via base pairing (Fig. 6E). Several examples of 2D structures observed on the graphite surface and their corresponding calculated models are shown in Fig. 6.

2.4 Hollow spheres

An important extension of the nucleoside-based lipids involves the formation of supramolecular assemblies composed of f-block elements. These objects, in particular hollow spheres, could be of interest for many applications in several fields including photonics, catalysis, energy storage, and medicine [17]. In order to construct the microspheres, we took advantages of the unique self-organization capabilities of nucleoside-based lipids. Accordingly, microspheres featuring a hybrid lipid-cation multilamellar shell were prepared simply by hydration of nucleoside-based lipids with aqueous solutions containing actinide or lanthanide salts. It is worthy to note that due to the lack of nucleobase natural phosphocholine lipids cannot induce the formation of microsphere. It was demonstrated that the microsphere is stabilized thanks to both hydrophobic and base–base interactions. In addition, phosphate/f-block element salt binding allows the formation of the hollow supramolecular structures shown in Fig. 7.

3.1 Cationic species

A feature of supramolecular systems is hierarchy, where primary building blocks associate into more complex secondary structures that are integrated into the next size level. These hierarchical constructions may provide useful systems for drug delivery applications. In particular, when bioactive molecules such as oligonucleotides are inserted in the hierarchical self-assemblies constructed with building blocks such as lipids or amphiphiles. Such combinations can lead to effective drug vehicles capable to transport and deliver polynucleotides. In the framework of biomedical research, the delivery of nucleic acids to cells and the resulting ability to correct a defective gene, introduce a new gene, or knock-down a gene provides a method to potentially treat many diseases. Cationic-based synthetic nonviral systems are routinely used in vitro as vehicles for nucleic acids.

Numerous cationic lipids have been developed and evaluated as cationic agents for plasmid DNA delivery [18]. Different structural features have been modified on the basic cationic lipid structures to enhance the transfection efficiency. Importantly, most of the synthetic vectors currently in use rely either on electrostatic forces or electrostatic and hydrophobic interactions for DNA binding and gene delivery. Our group has pioneered the use of nucleoside-based lipids with additional hydrogen bonding and ð stacking capabilities to favor the interactions between nucleic acids and the synthetic vector [19]. Several promising nucleic acid vectors based on a cationic amphiphilic uridine were synthetized (Fig. 8). The in vitro transfection assays realized with these derivatives were very encouraging. For example, O-Et-DOUPC at high nucleolipid:DNA (w:w) ratios (18:1–36:1) was an efficient transfecting reagent in CHO cells using a reporter β-gal gene assay. These results compare favorably with DOTAP and Transfast both used under optimal conditions (Fig. 9). In contrast to conventional cationic transfecting reagents, the cytotoxicity of nucleoside-based lipids such as O-Et-DOUPC or DOTAU (Fig. 8) was shown to be negligible. Hence, nucleoside-based lipids can be used in a wider range of concentration compared to Lipofectamine and for cell lines sensitive to the toxicity of conventional cationic lipids.

3.2 Nucleoside-based lipids–DNA complexes

The supramolecular systems involving calf thymus DNA (CT-DNA) and nucleoside-based lipids feature particular characteristics. Data collected from several experiments, including SAXS (small angle X ray scattering) show that CT-DNA/nucleoside-based lipid complexes arrange into L_α^C phases (lamellar complexes, with DNA intercalated between planar lipid membranes) [19,20]. Notably, In the case of DOTAU, it was found that the DOTAU/poly A lipoplexes were more compact systems than DOTAP/poly A lipoplexes due to tighter association between the uracil base of DOTAU and adenine of the poly A single strand.

While most of the lipoplexes rely only on electrostatic interaction between the polyanionic DNA and cationic lipids, DNA complexes were also observed in the presence of neutral nucleoside-based lipids. For example, the nucleic acid binding properties of the glycosyl nucleoside derivatives (Fig. 10) were investigated by different techniques including quasi-elastic light scattering (QELS), UV–vis, transmission electronic microscopy (TEM), NMR, IR, and circular dichroism (CD) [21]. It was found that lipid–nucleic acid complexes are stabilized thanks to the amphiphilic character of the glycosyl derivatives and phosphate–sugar interactions. Note that the uridine moiety of the lipid contributes to the stabilization of the lipid/polyA–polyU complex.

Interestingly, lipoplexes only stabilized via base pairing have been reported recently [22]. Baglioni and co-workers demonstrated the proof of the concept of “nucleolipoplexes” by exposing polynucleotides to anionic nucleolipids. It is worthy to note that these supramolecular systems were the results of complementary molecular recognition between micellar systems made of short chains nucleolipids and complementary polynucleotides. It will be interesting to watch the field of nucleolipoplexes in the years ahead to see if these systems are able to transfect polynucleotide.