Pierre Vierling

Extended in vivo blood circulation time of fluorinated liposomes

The clearance from blood circulation of fluorinated liposomes made with perfluoroalkylated phosphatidylcholines was investigated in mice using liposome‐entrapped 5(6)‐carboxyfluorescein. The presence of a fluorinated core inside the membrane strongly retards their blood clearance. The fluorinated vesicles showed circulation half‐lives of up to 8.6 h, which are 6–13 and 3–6 times larger than those of similarly sized conventional distearoylphosphatidylcholine and distearoylphosphatidylcholine/cholesterol liposomes, respectively. Their blood clearance was similar to that of some polyethylene glycol (PEG)‐labelled ‘stealth’ liposomes and was dose‐independent in a 3.3–330 body weight dose range.

Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression.

A molecular-diversity-oriented approach for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene-delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic-hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure-activity relationship studies. Gel electrophoresis revealed that paCDs self-assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70-150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL-CL2 and COS-7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen-bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space-oriented dendritic polycationic constructs.

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.

Highly fluorinated amphiphiles as drug and gene carrier and delivery systems

Abstract A short review on fluorinated liposomes made from highly fluorinated double-chain phospho- or glyco-lipids as well as fluorinated lipoplexes, e.g. complexes made from highly fluorinated polycationic lipospermines and a gene, is presented. These systems display a number of interesting physico-chemical and biological properties which make them very attractive alternatives as drug or gene carrying, targeting and delivering devices. The properties of the fluorinated liposomes that are discussed include liposome formation and shelf-stability, membrane structure, thermotropism and permeability, encapsulation, release and stability in various media (serum, detergents), bioavailability and biodistribution, anti-HIV activity. The properties of the fluorinated lipoplexes that are reported include lipoplex formation, stability, in vitro cell transfection in the presence of serum or of bile.

Fluorinated phosphatidylcholine-based liposomes: H+/Na+ permeability, active doxorubicin encapsulation and stability in human serum

The active encapsulation of doxorubicin (DOX) into fluorinated liposomes, the stability of these liposomes with respect to encapsulated DOX release in buffer and in human serum, and their H+/Na+ membrane permeability have been investigated and compared to those of their conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. We found that the presence of this fluorinated core is not a barrier for the active encapsulation of DOX. Efficient (> 90%) and stable loading could be achieved using a transmembrane ammonium sulfate or even, in the absence of Na+, a transmembrane pH gradient. The higher H+/Na+ permeability found for the fluorinated membranes, as compared to conventional ones, is responsible for the lower stability observed for the DOX-loaded fluorinated liposomes when incubated in a physiological buffer (PBS) or in human serum. It is also noticeable that the retention of DOX is increased in human serum and for the liposomes whose membranes are in a gel or in a semi-fluid semi-gel state at 37 degrees C.

On the Origin of Primitive Cells: From Nutrient Intake to Elongation of Encapsulated Nucleotides

Recent major discoveries in membrane biophysics hold the key to a modern understanding of the origin of life on Earth. Membrane bilayer vesicles have been shown to provide a multifaceted microenvironment in which protometabolic reactions could have developed. Cell-membrane-like aggregates of amphiphilic molecules capable of retaining encapsulated oligonucleotides have been successfully created in the laboratory. Sophisticated laboratory studies on the origin of life now show that elongation of the DNA primer takes place inside fatty acid vesicles when activated nucleotide nutrients are added to the external medium. These studies demonstrate that cell-like vesicles can be sufficiently permeable to allow for the intake of charged molecules such as activated nucleotides, which can then take part in copying templates in the protocell interior. In this Review we summarize recent experiments in this area and describe a possible scenario for the origin of primitive cells, with an emphasis on the elongation of encapsulated nucleotides.

Polymorphic phase behavior of perfluoroalkylated phosphatidylcholines

The polymorphic phase behavior of the F-alkyl modified phosphatidylcholines FnCmPC with Fn = CnF2n + 1 and Cm = -(CH2)m- and the physicochemical properties of their aqueous dispersions have been investigated. We show that the supramolecular assemblies formed by F4C4PC, F6C4PC, F8C4PC and F4C10PC dispersed in water consist of liposomes. F6C10PC forms, as does F8C10PC, a ribbon-like phase (two-dimensional centered rectangular lattice) at 25 degrees C, but on heating, it forms a lamellar phase. Upon cooling, the lamellar gel phase is metastable and converts slowly back into the ribbon-like phase. Analyses of the dispersions before and after heat sterilization and upon storage at 25 degrees C reveal an exceptional stability of the FnCmPC-based liposomes which contrasts strongly with that of DPPC vesicles. This enhanced stability most likely arises from the increased hydrophobic character resulting from the presence of the perfluoroalkyl tails. The gel to fluid phase transition temperature of the FnCmPCs is found to be related to the total length of the hydrophobic chain and more markedly to the length of the perfluoroalkyl tail. This phase transition is first induced by the melting of the fluorocarbon chain. Each portion of the Fn tail and of the hydrocarbon spacer experiences intrinsic changes of molecular motion with temperature. The partitioning of a lipophilic/hydrophilic paramagnetic probe between the aqueous and lipidic phases present in the FnCmPC dispersions shows that an increase in fluorophilic character results in a lower solubility of the probe in the membrane, thus reflecting a dramatic decrease of the membranes lipophilicity.

Tailoring β-Cyclodextrin for DNA Complexation and Delivery by Homogeneous Functionalization at the Secondary Face

An efficient general strategy for the incorporation of functional elements onto the secondary hydroxyl rim of beta-cyclodextrin has been developed and applied to the synthesis of a novel series of C7-symmetric homogeneous macromolecular polycations with improved DNA complexing and delivery properties.

Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid.

One of the main drawbacks of synthetic, non‐viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F‐PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE.

Synthesis and preliminary data on the biocompatibility and emulsifying properties of perfluoroalkylated phosphoramidates as injectable surfactants

Abstract Perfluoroalkylated dimorpholinophosphoramidates, 1–4 , were synthesized in 65–80% yield by phosphorylation of the appropriate perfluoroalkylated alcohol by phosphorus oxytrichloride, followed by reaction with morpholine. Preliminary biological tests (effect on cell cultures, hemolysis of red blood cells, and acute in vivo toxicity in mice) performed on compounds 1 to 5 show that the F-alkylated derivatives 1 to 4 are better tolerated than their hydrocarbon analog 5 in spite of their higher surface activity. Where the emulsification of fluorocarbons is concerned, the incorporation of the perfluoroalkylated compounds 1 to 4 in 20% w/v F-decalin/lecithin-based emulsions results in a slight stabilizing effect (not found for the hydrocarbon analog 5 ), detectable only after one month of storage at 25°C. However, when used as the sole surfactant, the F-alkylated dimorpholinophosphoramidates show no emulsifying properties; this may be assigned to insufficient hydrophilicity of the dimorpholinophosphoramidate head.