Sunfish cationic amphiphiles: toward an adaptative lipoplex morphology

A detailed physicochemical study is presented on a new class of cationic amphiphiles, Sunfish amphiphiles, recently designed, synthesized, and tested for gene delivery. These materials have two hydrophobic tails, connected to the cationic pyridinium headgroup at the 1- and 4-positions. Two extreme morphologies can be visualized, i.e. one by back-folding involving association of both tails at one side of the pyridinium ring and one by independent unfolding of the tails, the two molecular geometries leading to considerable differences in the aggregate morphology. The behavior of six members of the Sunfish family in mixtures with DOPE, applying different conditions relevant for transfection, has been studied by a combination of techniques (DLS, DSC, NMR, SAXS, Cryo-TEM, fluorescence, etc.). The effects of structural parameters such as the presence of unsaturation in the tails and length of the alkyl chains on the properties of the aggregates have been assessed. A correlation of these structural data with cellular transfection efficiencies reveals that the highest transfection efficiency is obtained with those amphiphiles that are easily hydrated, form fluid aggregates, and undergo a transition to the inverted hexagonal phase in the presence of plasmid DNA (p-DNA) at physiological ionic strength.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Structural evolution of environmentally responsive cationic liposome-DNA complexes with a reducible lipid linker

Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2-5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL-DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL-DNA complexes form the lamellar (L(α)(C)) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L(β') phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L(βF), L(βL), and L(βI)) constituting the L(β') phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.     

Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery.     

Dynamic octopus amphiphiles as powerful activators of DNA transporters: differential fragrance sensing and beyond

We report the design, synthesis and evaluation of dynamic "octopus" amphiphiles with emphasis on their efficiency as activators in synthetic membrane-based sensing systems. Previously, we found that the in situ treatment of charged hydrazides with hydrophobic aldehydes or ketones gives amphiphilic counterion activators of polyion transporters in lipid bilayers, and that their efficiency increases with the number of their hydrophobic tails. Herein, we expand this series to amphiphiles with one cationic head (guanidinium or ammonium) and four exchangeable hydrophobic tails. These results, with the highest number of tails reported to date, confirm that dynamic octopus amphiphiles provide access to maximal activity and selectivity. Odorants, such as muscone, carvone, or anisaldehyde are used to outline their usefulness in differential sensing systems that operate based on counterion-activated DNA transporters in fluorogenic vesicles. The enhanced ability of octopus amphiphiles to enable the discrimination of enantiomers as well as that of otherwise intractable ortho, meta, and para isomers and short cyclo-/alkyl tails is demonstrated. These findings identify dynamic octopus amphiphiles as being promising for application to differential sensing, "fragrant" cellular uptake, and slow release.     

Comprehensive screening of octopus amphiphiles as DNA activators in lipid bilayers: implications on transport, sensing and cellular uptake

Dynamic octopus amphiphiles contain one charged "head," here a guanidinium cation, together several hydrophobic "tails" (or "tentacles") that can be attached and exchanged in situ by reversible hydrazone formation. Quite surprisingly, their ability to activate DNA as transporters in lipid bilayer membranes was found to increase with the number of tails (up to four) as well as with their length (up to eight carbons). Both encouraged and puzzled by these results, we decided that a comprehensive screening of octopus amphiphiles with regard to number (from one to six) and length (from three to eighteen carbons) of their tails would be appropriate at this point. For this purpose, we here report the synthesis of cationic hexahydrazide peptide dendrons together with that of aldehydes with long, saturated, unsaturated and branched hydrophobic tails. Comprehensive screening of the completed collection of tails and heads reveals that the ability of octopus amphiphiles to activate DNA transporters shifts with increasing number of tails to decreasing length of the tails. Moreover, cis-alkenyl and branched alkyl tails are more active than their linear analogs, branched aromatic tails are best. These overall very meaningful trends for octopus amphiphiles will be of importance for sensing applications and fragrant cellular uptake.     

On the gene delivery efficacies of pH-sensitive cationic lipids via endosomal protonation: a chemical biology investigation

In an effort to probe the importance of endosomal protonation in pH-sensitive, cationic, lipid-mediated, non-viral gene delivery, we have designed and synthesized a novel cholesterol-based, endosomal pH-sensitive, histidylated, cationic amphiphile (lipid 1), its less pH-sensitive counterpart with an electron-deficient, tosylated histidine head group (lipid 2) as well as a third new cholesterol-based, cationic lipid containing no histidine head group (lipid 3). For all the novel liposomes and lipoplexes, we evaluated hysicochemical characteristics, including lipid:DNA interactions, global surface charge, and sizes. As anticipated, lipid 2 showed lower efficacies than lipid 1 for the transfection of 293T7 cells with the cytoplasmic gene expression vector pT7Luc at lipid:DNA mole ratios of 3.6:1 and 1.8:1; both lipids were greatly inhibited in the presence of Bafilomycin A1. This demonstrates the involvement of imidazole ring protonation in the endosomal escape of DNA. Conversely, endosome escape of DNA with lipid 3 seemed to be independent of endosome acidification. However, with nuclear gene expression systems in 293T7, HepG2, and HeLa cells, the transfection efficacies of lipid 2 at a lipid:DNA mole ratio of 3.6:1 were found to be either equal to or somewhat lower than those of lipids 1 and 3. Interestingly, at a lipid:DNA mole ratio of 1.8:1, lipids 2 and 3 were remarkably more transfection efficient than lipid 1 in both HepG2 and HeLa cells. Mechanistic implications of such contrasting relative transfection profiles are delineated.     

Enhanced intravenous transgene expression in mouse lung using cyclic-head cationic lipids

Herein, we report enhanced intravenous mouse lung transfection using novel cyclic-head-group analogs of usually open-head cationic transfection lipids. Design and synthesis of the new cyclic-head lipid N,N-di-n-tetradecyl-3,4-dihydroxy-pyrrolidinium chloride (lipid 1) and its higher alkyl-chain analogs (lipids 2-4) and relative in vitro and in vivo gene transfer efficacies of cyclic-head lipids 1-4 to their corresponding open-head analogs [lipid 5, namely N,N-di-n-tetradecyl-N,N-(2-hydroxyethyl)ammonium chloride and its higher alkyl-chain analogs, lipids 6-8] have been described. In stark contrast to comparable in vitro transfection efficacies of both the cyclic- and open-head lipids, lipids 1-4 with cyclic heads were found to be significantly more efficient (by 5- to 11-fold) in transfecting mouse lung than their corresponding open-head analogs (5-8) upon intravenous administration. The cyclic-head lipid 3 with di-stearyl hydrophobic tail was found to be the most promising for future applications.     

High transfection efficiency of cationic lipids with asymmetric acyl-cholesteryl hydrophobic tails

The ability of a nonviral gene delivery system to overcome extra- and intracellular barriers is a critical issue for the future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common nonviral gene delivery system. One hundred and eighty novel cationic lipids with asymmetric acyl-cholesteryl hydrophobic tails were synthesized by parallel solid-phase chemistry. The liposomes were prepared and gel retardation assays were used to study the binding efficiency between the prepared liposome and the DNA. Transfection efficiencies of the lipids were evaluated against various mammalian cells, such as human embryonic kidney (HEK293), human cervical adenocarcinoma (HeLa), canine osteosarcoma (D17), colorectal adenocarcinoma (COLO 205), and human prostate adenocarcinoma (PC3) cells. The lipids with an acyl portion at the terminal part of the polyamine backbone exhibited higher transfection efficiency than those with the acyl portion as an internal part of the backbone. These compounds also showed higher transfection efficiency and lower cytotoxicity than the commercially available agents, Effectene, DOTAP, and DC-Chol.     

Phosphonodithioester–Amine Coupling as a Key Reaction Step for the Design of Cationic Amphiphiles Used for Gene Delivery

A convergent synthesis of cationic amphiphilic compounds is reported here with the use of the phosphonodithioester–amine coupling (PAC) reaction. This versatile reaction occurs at room temperature without any catalyst, allowing binding of the lipid moiety to a polar head group. This strategy is illustrated with the use of two lipid units featuring either two oleyl chains or two-branched saturated lipid chains. The final cationic amphiphiles were evaluated as carriers for plasmid DNA delivery in four cell lines (A549, Calu3, CFBE and 16HBE) and were compared to standards (BSV36 and KLN47). These new amphiphilic derivatives, which were formulated with DOPE or DOPE-cholesterol as helper lipids, feature high transfection efficacies when associated with DOPE. The highest transfection efficacies were observed in the four cell lines at low charge ratios (CR = 0.7, 1 or 2). At these CRs, no toxic effects were detected. Altogether, this new synthesis scheme using the PAC reaction opens up new possibilities for investigating the effects of lipid or polar head groups on transfection efficacies.     

Inverse-phosphocholine lipids: a remix of a common phospholipid

Zwitterionic inverse-phosphocholine (iPC) lipids contain headgroups with an inverted charge orientation relative to phosphocholine (PC) lipids. The iPC lipid headgroup has a quaternary amine adjacent to the bilayer interface and a phosphate that extends into the aqueous phase. Neutral iPC lipids with ethylated phosphate groups (CPe) and anionic iPC lipids nonethylated phosphate groups (CP) were synthesized. The surface potential of CPe liposomes remains negative across a broad pH range and in the presence of up to 10 mM Ca(2+). CP liposomes aggregate in the presence of Ca(2+), but at a slower rate than other anionic lipids. Hydrolysis of CP lipids by alkaline phosphatases generates a cationic lipid. CPe liposomes release encapsulated anionic carboxyfluorescein (CF) 20 times faster than PC liposomes and release uncharged glucose twice as fast as PC liposomes. As such, iPC lipids afford a unique opportunity to investigate the biophysical and bioactivity-related ramifications of a charge inversion at the bilayer surface.     

Charge-reversal amphiphiles for gene delivery

Delivering a missing gene or a functional substitute of a defective gene has the potential to revolutionize current medical care. Of the two gene delivery approaches, viral and synthetic vectors, synthetic cationic vectors possess several practical advantages but suffer from poor transfection efficiency. A new approach to gene delivery using charge-reversal amphiphiles is described. This synthetic vector transforms from a cationic to an anionic amphiphile intracellularly. This amphiphile performs two roles: first, it binds and then releases DNA, and second, as an anionic multicharged amphiphile, it destabilizes lipid bilayers. A charge-reversal amphiphile was synthesized and fully characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using these vectors compared to current cationic amphiphiles.     

Molecular packing of functionalized fluorinated lipids in Langmuir monolayers

Fluorinated amphipaths are a fascinating class of compounds, which, despite significant challenges associated with their syntheses, have found use across a number of areas of biotechnology. Applications range from the in vitro stabilization of membrane proteins to the development of enhanced stability intravenous drug and gene delivery systems. More recently, monolayer-forming fluorinated lipids have found use in the 2D crystallization of detergent-solubilized hydrophobic or partially hydrophobic proteins at the air-water interface. In this study, we investigate the surface properties of a novel suite of monolayer forming, partially fluorinated lipids. These modular lipid structures contain a densely fluorinated insertion in the hydrocarbon tail and a synthetically modifiable headgroup. Analyses of surface-pressure area isotherms and X-ray reflectometry profiles reveal that the lipids spread into fluid monolayers and are more compressible than their non-fluorinated counterparts. Furthermore, the data support a model whereby the partially fluorinated chains of the lipid tails form a film which is fundamentally incompatible with detergents and other destabilizing amphipaths.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Amphiphilic cationic [dendritic poly(L-lysine)]-block-poly(L-lactide)-block-[dendritic poly(L-lysine)]s in aqueous solution: self-aggregation and interaction with DNA as gene delivery carriers

A new series of triblock [dendritic poly(L-lysine)]-block-PLLA-block-[dendritic poly(L-lysine)]s (DL(2) -PLLA-DL(2) ) with PLLA block lengths of 11.5-26.5 and double 2-generation PLL dendrons DL(2) as model cationic amphiphiles were synthesized and characterized. Their CAC, self-aggregation and plasmid DNA binding affinities in pure water and PBS were studied. The PLLA block length dependence of particle size, morphology and ξ potential for organized pDNA/amphiphile polyplex aggregates were examined. Finally, toxicities of these DL(2) -PLLA-DL(2) amphiphiles and their polyplexes were assayed by MTT with HeLa, SMMC-7721 and COS-7 cells, and COS-7 cell luciferase and eGFP gene transfection efficacies with these amphiphiles as the delivery carriers were investigated.     

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.     

Natural surfactants

The ever-increasing environmental concern about surfactants triggers an interest in natural surfactant. This review, which has an emphasis on work published since 1998, covers three categories of natural surfactants: amphiphiles produced by yeast or bacteria, amphiphiles containing a natural polar headgroup and amphiphiles containing a natural hydrophobic tail. Microorganisms produce both high molecular weight and low molecular weight surfactants. Only the low molecular weight compounds are included in the review. Sugars and amino acids are the two most important examples of surfactant polar headgroups of natural origin. The research is particularly intense in the area of sugar surfactants and the review covers three types: alkylglucosides, alkylglucamides and sugar esters. Surfactants based on two types of natural hydrophobic tails are included: fatty acid monoethanolamides and sterol ethoxylates. Routes of preparation as well as physico-chemical properties are discussed for the surfactants prepared by organic synthesis.     

Lipothiophosphoramidates for gene delivery: critical role of the cationic polar headgroup

When considering a family of cationic lipids designed for gene delivery, the nature of the cationic polar head probably has a great influence on both the transfection efficacy and toxicity. Starting from a cationic lipothiophosphoramidate bearing a trimethylammonium headgroup, we report herein the impact on gene transfection activity of the replacement of the trimethylammonium moiety by a trimethylphosphonium or a trimethylarsonium group. A series of three different human epithelial cell lines were used for the experimental transfection studies (HeLa, A549 and 16HBE14o(-)). The results basically showed that such structural modifications of the cationic headgroup can lead to a high transfection efficacy at low lipid/DNA charge ratios together with a low cytotoxicity. It thus appears that the use of a trimethylarsonium cationic headgroup for the design of efficient gene carriers, which was initially proposed in the lipophosphoramidate series, can be extended to other series of cationic lipids and might therefore have great potential for the development of novel non-viral vectors in general.     

Synthesis of an enlarged library of dynamic DNA activators with oxime, disulfide and hydrazone bridges

Dynamic amphiphiles have a "bridge" between their charged head and their hydrophobic tails. The presence of dynamic covalent bonds is of interest for differential and biosensing applications as well as for rapid access to the libraries needed to screen for gene delivery or cellular uptake of siRNA. However, efforts to develop libraries have so far concentrated on hydrazone bridges to monocationic heads. Here, we report synthesis efforts to enlarge this focused library with oxime and disulfide bridges and dynamic amphiphiles with more than one positive charge. Evaluation in fluorogenic vesicles reveals best activation of DNA as ion transporters by dynamic amphiphiles with dendritic scaffolds, doubly charged heads and four tails. Moreover, oximes, contrary to hydrazones, remain active under acidic conditions. Linear elongation of dendritic head-groups seems to cause increasing detergent effects and should therefore be avoided.     

Electrostatic model for mixed cationic-zwitterionic lipid bilayers

The current interest in mixed cationic-zwitterionic lipid membranes derives from their potential use as transfer vectors in nonviral gene therapy. Mixed cationic-zwitterionic lipid membranes have a number of structural properties that are distinct from the corresponding anionic-zwitterionic lipid membranes. As known from experiment and reproduced by computer simulations, the average cross-sectional area per lipid changes nonmonotonically with the mole fraction of the charged lipid, passing through a minimum at a roughly equimolar mixture. At the same time, the average orientation of the zwitterionic headgroup dipoles changes from more parallel to the membrane plane to more perpendicular. We suggest a simple mean-field model that reveals the physical mechanisms underlying the observed structural properties. To backup the mean-field calculations, we have also performed Monte Carlo simulations. Our model extends Poisson-Boltzmann theory to include (besides the cationic headgroup charges) the individual charges of the zwitterionic lipid headgroups. We model these charges to be arranged as dipoles of fixed length with rotational degrees of freedom. Our model includes, in a phenomenological manner, the changes in steric headgroup interactions upon reorientation of the zwitterionic headgroups. Our numerical results suggest that two different mechanisms contribute to the observed structural properties: one involves the lateral electrostatic pressure and the other the zwitterionic headgroup orientation, the latter modifying steric headgroup interactions. The two mechanisms operate in parallel as they both originate in the electrostatic properties of the involved lipids. We have also applied our model to a mixed anionic-zwitterionic lipid membrane for which neither a significant headgroup reorientation nor a nonmonotonic change in the average lateral cross-sectional area is predicted.