Alberto Rodríguez-Pulido

Light-Triggered Sequence-Specific Cargo Release from DNA Block Copolymer–Lipid Vesicles†

Nanocontainers have gained much importance because of their versatile properties and broad application potential in the fields of chemistry,1 biophysics,2 and nanomedicine.2b, 3 Lipid vesicles have proven to be a particularly effective class of nanocontainers, able to encapsulate and protect diverse small molecules, such as ions and drugs,4 as well as larger biomacromolecules, such as proteins or DNA.2a, 5 Moreover, the engineering of lipid vesicles has sufficiently advanced to a level which enables functionalization and manipulation of their surfaces with specific ligands to improve their poor chemical and physical specificity. For example, proteins (including antibodies),5a, 6 carbohydrates,7 and vitamins2a, 8 have all been used as targeting units anchored to the liposome surfaces to direct these nanocontainers to the site of action. More recently, single-stranded DNA covalently attached to cholesterol or lipid moieties has been incorporated into vesicle bilayers in order to exploit the specific recognition ability of oligonucleotides (ODNs) by hybridization with their complementary strands. These DNA hybrids have been shown to be critical building blocks in the construction of novel self-assembled supravesicular structures in which vesicles were linked by double-stranded ODNs,9 or utilized to induce programmed fusion.10 Moreover, DNA–lipids have been used to construct hybridization-sensitive nanocontainers,11 to improve liposome marking,12 to mimic cellular systems,13 and for multiplexed DNA detection.14 As demonstrated by the numerous examples above, the decoration of vesicles with DNA amphiphiles has resulted in significant advances in the functionality of these containers; the bilayer barrier itself remains a significant hindrance to the release of cargo, however. There have been several successful attempts to liberate cargo molecules from vesicles. One possibility is the generation of pores in the lipid bilayer through the incorporation of natural or synthetic ion channels.15 Another approach, which entails enzymes, makes use of selective lipases for cargo release.16 A promising alternative is the design of “smart” liposomes that are able to release cargo through physicochemical responses to external stimuli (such as nanoparticle incorporation into the membrane, or changes in pH or temperature).17 Furthermore, photosensitizers that generate singlet oxygen (1O2) upon light irradiation have been incorporated into the bilayer or the vesicle interior to mediate cargo release.18 Nevertheless, the liberation of cargo molecules from such functionalized nanocontainers is unfortunately not selective for mixed populations of vesicles and further work is needed to increase the specificity of these container systems. Herein, we report a powerful new approach for selective cargo release from lipid vesicles that is based on amphiphilic DNA block copolymers (DBCs) and the hybridization of photosensitizer units (Scheme 1). It was demonstrated that this new class of nucleic acid amphiphiles, DBCs, can be stably anchored in the phospholipid membrane of liposomes (step 1). The protruding ODN was functionalized with ODN-photosensitizer conjugates through Watson–Crick base pairing (step 2) and after light irradiation (step 3) selective cargo release was achieved (step 4) depending on the DNA code on the surface of the vesicles. DBCs, as used here for cargo release, consist of a single-stranded ODN covalently bound to an organic polymer block. The combination of highly specific DNA interactions with the hydrophobic properties of the polymer block make DBCs ideally suited to diverse nanoscience applications, for example, as gene and drug delivery systems, or as building blocks in nanoelectronic devices.19 Herein, we introduce a new application for DBCs: as a functionalization and release reagent for liposomes. DNA-b-polypropyleneoxide (DNA-b-PPO) was selected because of its amphiphilic nature, which leads the hydrophobic polymer segments to interact with the internal region of the lipid bilayer while the hydrophilic nucleotides remain on the liposome surface free to bind with the complementary DNA sequences. Additional features of DNA-b-PPO include its fully automated synthesis,20 the known ability of PPO to insert into the hydrophobic part of phospholipid bilayers,15d, 21 and its susceptibility to oxidation.22

A Theoretical and Experimental Approach to the Compaction Process of DNA by Dioctadecyldimethylammonium Bromide/Zwitterionic Mixed Liposomes

The compaction of DNA by cationic liposomes constituted by a mixture of a cationic lipid, dioctadecyldimethylammonium bromide (DODAB), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), has been evaluated by means of experimental studies (electrophoretic mobility, conductometry, cryogenic electron transmission microscopy or cryo-TEM, and fluorescence spectroscopy) as well as theoretical calculations. This information reveals that DODAB/DOPE and DODAB/DLPC liposomes are mostly spherical and unilamellar, with a mean diameter of around 70 and 61 nm, respectively, a bilayer thickness of 4.5 nm, and gel-to-fluid transition temperatures, T(m), of around 19 and 28 degrees C, respectively. Their positively charged surfaces efficiently compact the negatively charged DNA by means of a strong entropically driven surface interaction that yields DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes as confirmed by zeta potential and ethidium bromide fluorescence intercalation assays. These experiments have permitted as well the evaluation of the different microenvironments of varying polarity of the DNA helix, liposomes, and/or lipoplexes. DODAB/DOPE-DNA and DODAB/DLPC-DNA lipoplexes have been characterized by isoneutrality ratios (L/D)(phi) of around 4.7 and 4.8, respectively, a more fluid membrane than that of the parent liposomes, and T(m) around 24 and 28 degrees C, respectively, as revealed by fluorescence anisotropy. Cryo-TEM micrographs reveal a rich scenario of nanostructures and morphologies, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures. Phase diagrams (aggregation and re-entrant condensation phenomena), calculated by means of a phenomenological theory, have confirmed the experimental concentration domains and the isoneutrality conditions. The influence of helper lipid in the compaction process, as well as the optimum choice among those herein chosen, has been analyzed.

Compaction process of calf thymus DNA by mixed cationic-zwitterionic liposomes: a physicochemical study.

The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH = 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 +/- 20 nm and a positively charged surface with a charge density of sigmazeta = (21 +/- 1) x 10(-3) C m(-2). When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)phi = 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.

Experimental and Theoretical Approach to the Sodium Decanoate−Dodecanoate Mixed Surfactant System in Aqueous Solution

The mixed system consisting of two anionic surfactants of identical headgroups but with 10 and 12 carbon atoms on the hydrophobic tail, sodium decanoate (C(10)Na) and sodium dodecanoate (C(12)Na), has been studied in aqueous solution at 298.15 K by means of conductivity and fluorescence spectroscopy experiments and from a theoretical point of view. The monomeric and micellar phases of the mixed aggregates were analyzed through the experimental determination of the total critical micelle concentration, cmc*, the degree of ionization of the mixed micelle, beta, and the total aggregation number, N*. Results indicate that, compared to the ideal behavior, the mixed system with two anionic surfactants differing only in two methylenes in the hydrophobic tail shows a negative deviation in the cmc* and a positive one in N*. Pure surfactants (C(10)Na and C(12)Na) form spherical micelles, but mixed micelles must aggregate with a rodlike shape to allow more surfactant molecules than expected. In addition, rodlike micelles result in more compacted aggregation (i.e., less area per polar head). From the experimental data in this work, several theoretical models for mixed surfactant systems have been checked: Rubinghs model predicts lower deviations from ideality than Motomuras model. The stability of the micelles has been analyzed by computing the standard Gibbs energy of micelle formation, Delta G(mic,0), of pure and mixed micelles. Results of this work reinforce the feature that mixed systems formed by alkylsurfactants with the same polar head that differ in the hydrocarbon length, usually admitted as roughly ideal systems, may show nonideal behavior. This deviation, being mostly related to the difference in the chain length, Delta n(c), between surfactants can be analyzed only when very accurate experimental techniques as well as adequate theoretical models are used.

Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study

A double approach, experimental and theoretical, has been followed to characterize from a physicochemical standpoint the compaction process of DNA by means of cationic colloidal aggregates. The colloidal vectors are cationic liposomes constituted by a mixture of a novel cationic lipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC) and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). A wide variety of high precision experimental techniques have been used to carry out the analysis: electrophoretic mobility, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy (ethidium bromide intercalation assays). On the other hand, a theoretical model that considers the renormalization of charges of both the polyelectrolyte and the colloidal aggregates sheds light as well on the characteristics of the compaction process. This global information reveals that the compaction of DNA by the cationic liposomes is mostly driven by the strong electrostatic interaction among the positively charged surfaces of the colloidal aggregates and the negatively charged DNA, with a potent entropic component. DOEPC/DOPE liposomes are mostly spherical, with a mean diameter of around 100 nm and a bilayer thickness of 4.4 nm. From a morphological viewpoint, an appreciable amount of multilamellar structures has been found not only on the lipoplexes but also on the parent liposomes. The isoneutrality of the lipoplexes is found at liposome/DNA mass ratios that decrease with the molar fraction of cationic lipid in the mixed liposome (α). This liposome composition has a clear effect as well on the lipoplex structure, which goes from an inverted hexagonal phase (HII), usually related to improved cell transfection efficiency, at low cationic lipid molar fraction (α ≈ 0.2), to a lamellar structure (Lα) when the cationic lipid content in the mixed liposomes increases (α ≥ 0.4), irrespective of the lipoplex charge ratio. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work, by using renormalized charges. The model allows us to confirm and predict the experimental isoneutrality conditions as well as to determine the maximum magnitude of this charge as a function of the composition of the resulting lipoplexes.

Electrochemical and spectroscopic study of octadecyltrimethylammonium bromide/DNA surfoplexes.

The use of cationic micelles consisting of octadecyltrimethylammonium bromide (C18TAB) to compact calf thymus DNA has been investigated in aqueous buffered solution at 310.15 K by means of conductometry, electrophoretic mobility, and several fluorescence spectroscopy methods. The results indicate that C18TAB micelles, consisting of 44 monomers on average, may compact DNA molecule by an electrostatic interaction that takes place at the cationic spherical micelle surface. The surfoplexes thus formed show a surface density charge that goes from negative to positive values at a Lmic/D mass ratio of around 1.0 (where Lmic and D are the masses of micellized cationic surfactant and DNA), called the isoneutrality ratio (Lmic/D)phi. Values of this characteristic parameter, determined in this work not only from the electrochemical experimental data but also from spectroscopic measurements, are in very good agreement with those ones calculated from molecular parameters and some other properties also obtained in this work. The electrostatic character of the DNA-micelle interaction has been confirmed by analyzing the decrease in fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the surfoplexes are formed. Fluorescence anisotropy experiments have revealed that micelle packing becomes more rigid in the presence of DNA, but once the surfoplex is formed, the fluidity increases with the Lmic/D mass ratio, attaining its maximum when the isoneutrality ratio is exceeded. This fact, together with the net positive charge of the surfoplexes with the Lmic/D mass ratio over the isoneutrality ratio, makes this regimen of lipid and DNA content the optimum for efficiency in the transfection process.

Probing the shielding properties of aptameric protective groups

Site-specific derivatization of chemically equivalent functional groups has recently been facilitated by the introduction of high-affinity aptamers as non-covalent protective groups. More specifically, a series of RNA aptamers have proven to be highly efficient in enhancing the regioselectivity of reactions with the aminoglycoside antibiotic neomycin B, which carries several chemically indistinguishable amino and hydroxy groups. Since small-molecule targets tend to exhibit multiple modes of binding with a single aptamer, the impact of secondary binding sites on the regioselectivity should be considered. To address this issue, we investigated a series of well-characterized RNA aptamers that bind neomycin B and propose a mechanism that accounts for the regioselective outcome of these transformations. We further demonstrate that the regioselectivity induced by non-covalent aptamer protective groups is determined by the number of binding sites, their affinity, and the mode of interaction with the guest molecule.