Carbohydrate- and related polyol-derived fluorosurfactants: an update.

A short review of recent literature is presented on the synthesis, biological properties, colloid and surface chemistry, and applications of carbohydrate- and related polyol-derived amphiphiles with perfluoroalkyl hydrophobes.

Development of an ex vivo model of pig kidney perfused with human lymphocytes. Analysis of xenogeneic cellular reactions.

BACKGROUND: Because of the explosive nature and the extremely rapid process of hyperacute rejection (HAR), significant infiltration of the xenograft by immunocompetent cells is not observed, and the role and the mechanism of action of cell-mediated rejection in discordant xenografts are therefore still under discussion. METHOD: We developed an experimental approach using pig kidneys perfused with human peripheral blood lymphocytes (PBL) in which the immunologic barrier of hyperacute rejection was excluded and which mimics the in vivo situation. RESULTS: PBL retention in the kidney was evaluated at 20-minute intervals for 3 hours. Retention increased from 30% to 80% with the time of perfusion and was specific because significantly fewer syngeneic lymphocytes were retained. Phenotype analysis of recovered PBL showed a significant decrease in natural killer (NK) cells. Immunohistochemical studies revealed the presence of NK cells and T lymphocytes in the glomerular and interstitial tubular structures of the kidney. Functional studies showed a progressive cessation of diuresis and augmentation of renal vascular resistance when the kidney was perfused with PBL. Electron microscopy examinations of kidney sections perfused with PBL showed swollen endothelial zones, suggesting alterations to and damage of the endothelium. CONCLUSIONS: This system provides a valuable model for the study of early discordant xenogeneic cellular rejection and demonstrates the predominance of xenograft infiltration by NK cells.

Highly fluorinated amphiphiles and colloidal systems, and their applications in the biomedical field. A contribution.

Fluorocarbons and fluorocarbon moieties are uniquely characterized by very strong intramolecular bonds and very weak intermolecular interactions. This results in a combination of exceptional thermal, chemical and biological inertness, low surface tension, high fluidity, excellent spreading characteristics, low solubility in water, and high gas dissolving capacities, which are the basis for innovative applications in the biomedical field. Perfluoroalkyl chains are larger and more rigid than their hydrogenated counterparts. They are considerably more hydrophobic, and are lipophobic as well. A large variety of well-defined, modular fluorinated surfactants whose polar head groups consist of polyols, sugars, sugar phosphates, amino acids, amine oxides, phosphocholine, phosphatidylcholine, etc, has recently been synthesized. Fluorinated surfactants are significantly more surface active than their hydrocarbon counterparts, both in terms of effectiveness and of efficiency. Despite this, they are less hemolytic and less detergent. Fluorosurfactants appear unable to extract membrane proteins. Fluorinated chains confer to surfactants a powerful driving force for collecting and organizing at interfaces. As compared to non-fluorinated analogs, fluorosurfactants have also a much stronger capacity to self-aggregate into discrete molecular assemblies when dispersed in water and other solvents. Even very short, single-chain fluorinated amphiphiles can form highly stable, heat-sterilizable vesicles, without the need for supplementary associative interactions. Sturdy microtubules were obtained from non-chiral, non-hydrogen bonding single-chain fluorosurfactants. Fluorinated amphiphiles can be used to engineer a variety of colloidal systems and manipulate their morphology, structure and properties. Stable fluorinated films, membranes and vesicles can also be prepared from combinations of standard surfactants with fluorocarbon/hydrocarbon diblock molecules. In bilayer membranes made from fluorinated amphiphiles the fluorinated tails segregate to form an internal teflon-like hydrophobic and lipophobic film that increases the stability of the membrane and reduces its permeability. This fluorinated film can also influence the behavior of fluorinated vesicles in a biological milieu. For example, it can affect the in vivo recognition and fate of particles, or the enzymatic hydrolysis of phospholipid components. Major applications of fluorocarbons currently in advanced clinical trials include injectable emulsions for delivering oxygen to tissues at risk of hypoxia; a neat fluorocarbon for treatment of acute respiratory failure by liquid ventilation; and gaseous fluorocarbon-stabilized microbubbles for use as contrast agents for ultrasound imaging. Fluorosurfactants also allow the preparation of a range of stable direct and reverse emulsions, microemulsions, multiple emulsions, and gels, some of which may include fluorocarbon and hydrocarbon and aqueous phases simultaneously. Highly fluorinated systems have potential for the delivery of drugs, prodrugs, vaccines, genes, markers, contrast agents and other materials.

Fluorinated materials for in vivo oxygen transport (blood substitutes), diagnosis and drug delivery.

Fluorocarbons are characterized by exceptional chemical and biological inertness, extreme hydrophobicity, lipophobicity, high gas-dissolving capacities, low surface tensions, high fluidity and spreading coefficients, high density, absence of protons, and magnetic susceptibilities comparable to that of water. These unique properties are the foundation for a range of biomedical applications. An injectable fluorocarbon-in-water emulsion is in advanced clinical trials as a temporary oxygen carrier (blood substitute) to prevent tissue hypoxia or ischemia in the surgical and critical care patient. A liquid fluorocarbon is in Phase II/III clinical trials for treatment of acute respiratory failure through liquid ventilation. Several fluorocarbon-based contrast agents for ultra-sound imaging are in various stages of clinical investigation. Multiple families of well-defined pure fluorinated surfactants have recently been synthesized. These surfactants have a modular structure which allows stepwise adjustment of their physicochemical characteristics. Their polar head group derives from polyols, sugars, aminoacids, amides, amine oxides, phosphocholine, phosphatidylcholine, etc. Fluorinated surfactants are significantly more surface-active than their hydrocarbon analogs and they display a greater tendency to self-assemble, thus forming well-ordered, stable supramolecular assemblies such as vesicles, tubules, fibers, ribbons, etc. Fluorinated amphiphiles also allowed the obtaining of a variety of stable reverse and multiple emulsions and gels. These systems are being investigated as drug delivery devices.

Effects of perfluorocarbon emulsions on cultured human endothelial cells.

Perfluorocarbons (PFCs) and their emulsions (PFCEs) were used in organ preservation before transplantation, but not in organ perfusion. Our purpose was to achieve organ perfusion with a PFCE at room temperature or at 37 degrees C, i. e. with oxygenation, to prevent damages related to reoxygenation after hypoxia. Therefore, we first investigated the effect of such emulsions on endothelial cells, the first cells to be in contact with the emulsion. A stem emulsion was prepared from perfluorooctyl bromide (90% w/v), emulsified with egg yolk phospholipids (2% w/v) and stabilized with a mixed fluorocarbon-hydrocarbon "molecular dowel" (1.4% w/v) (droplets of ca 0.2 micron in diameter). This emulsion was found to be stable when diluted with cell culture media or organ preservation fluids. Endothelial cells from human umbilical vein (HUVECs) were cultured in multiwell plates in M199 medium (with growth factors, 10% foetal calf serum and 5% human serum). Confluent cells were incubated overnight with 51Cr, washed and overlayed with M199 (control) or the above PFCE diluted 2x or 4x with M199 (test). After incubation, the cytotoxicity of the PFCEs was estimated by measuring 51Cr release and observing cell morphology by electron and light microscopy. The percentages of released 51Cr were identical to those of the control cells for the 2x, 3x or 4x diluted PFCEs at 4, 25 or 37 degrees C. After return to the M199 medium, the cells grew and multiplied normally. We conclude that the diluted PFCEs were devoid of cytotoxicity. The 2x diluted PFCE was however partially taken up by the cells: by microscopy, we observed intracellular PFC droplets and by density gradient analysis we found a slight increase in cellular density. The diluted PFCEs were compared to classical organ preservation solutions : HUVECs were incubated with UW (University of Wisconsin) or EC (EuroCollins) solutions at +4 and 37 degrees C (3, 17 or 24 h of incubation). The solutions were observed to be toxic to the cells under these conditions, with cell mortality after return to the M199 medium. This cytotoxicity may be attributed to the high K+ concentration of UW and EC, since similar assays performed on HUVECs with Hank's solution adjusted to 100 mM K+ showed a similar % of 51Cr release. UW and EC are therefore not acceptable as dilution media for PFCEs.

Advanced fluorocarbon-based systems for oxygen and drug delivery, and diagnosis.

Fluorocarbons and fluorocarbon-derived materials constitute a vast family of synthetic components that have a range of remarkable properties including exceptional chemical and biological inertness, gas-dissolving capacity, low surface tension, high fluidity, excellent spreading characteristics, unique hydro- and lipophobicity, high density, absence of protons, and magnetic susceptibility close to that of water. These properties lead to a diversity of products and applications as illustrated by those products that are already in advanced clinical trials, which comprise: 1) an injectable oxygen carrier, i.e. blood substitute, consisting of a fluorocarbon-in-water emulsion for use in surgery to alleviate the problems raised by the transfusion of homologous blood; the same emulsion is also being evaluated with cardiopulmonary bypass patients; 2) a neat fluorocarbon for treatment of acute respiratory failure by liquid ventilation; and 3) fluorocarbon-based or stabilized gas bubbles to be used as contrast agents for the assessment of heart function and detection of perfusion defects by ultrasound imaging. Proper selection of the fluorocarbon best suited for the intended application, formulation optimization, and advanced stabilization and processing procedures led to effective, ready-for-use products with minimal side-effects. Further highly fluorinated materials, including amphiphiles and various fluorocarbon-based colloidal systems that have potential as pulmonary, topical and ophthalmological drug delivery agents, and as skin protection barriers, are now being investigated. Such systems include drug-in-fluorocarbon suspensions, reverse water-in-fluorocarbon emulsions, oil-in-fluorocarbon emulsions, multiple emulsions, microemulsions, fluorocarbon gels, fluorinated liposomes, fluorinated tubules and other novel supramolecular systems.

Vesicles made of glycophospholipids with homogeneous (two fluorocarbon or two hydrocarbon) or heterogeneous (one fluorocarbon and one hydrocarbon) hydrophobic double chains.

The vesicle-forming ability of the new anionic double chain glycophospholipids 1-4, with either two hydrocarbon or two perfluorocarbon chains, or a mixed double chain (one fluorinated, one hydrogenated), was investigated. When dispersed in water, 1a-c,e, 2b,c and 4b,c readily gave heat-sterilizable vesicles, 30-70 nm in diameter. The galactose and mannose-based fluorinated vesicles were also highly stable on aging. The 6-substituted glucose derivatives 3 formed tubules that reversibly interconverted into vesicles, depending on temperature. The leakage rate in buffer of carboxyfluorescein or calcein from vesicles made from 1a-c,e 2b,c and 4b,c depended on the sugar (t1/2 galactose > mannose > glucose). It decreased significantly with increasing fluorination and length of the hydrophobic tails. The mixed perfluorocarbon/hydrocarbon-tailed amphiphiles were found to be miscible with both the two fluorocarbon chains and the two hydrocarbon chains derivatives. Such admixing tended, however, to increase the small unilamellar vesicles' permeability. In buffered serum, all the vesicles investigated were highly permeable, but incorporation of cholesterol or DSPC in vesicles made of 1e significantly reduced their permeability in serum. The new vesicle and membrane components have i.v. maximum tolerated doses as high as 500 mg/kg body weight in mice; hemolytic activity sharply decreases with increasing degree of fluorination.

Hydrolysis of DMPC or DPPC by pancreatic phospholipase A2 is slowed down when (perfluoroalkyl) alkanes are incorporated into the liposomal membrane.

The effect of the incorporation of linear (perfluoroalkyl)alkanes (CmF2m + 1CnH2n + 1, FmHn) into liposomes made of DMPC or DPPC on the activity of porcine pancreatic phospholipase A2 was investigated. A large decrease in enzyme activity and modifications of the kinetic profile, especially at and above the phospholipid's phase transition temperature, were observed; both depend on the relative lengths of the phospholipid's fatty acid chains and of the Hn segment of the FmHn molecule. With DMPC Hn must have a minimum of 10 carbon atoms to be effective, as in F6H10, F8H10 and F4H12; F8H8 had no significant hydrolysis-rate-reducing effect. With DPPC Hn must have a minimum of 12 carbon atoms, as in F4H12, while F8H8, F6H10 and F8H10 were ineffective. The absence of effect when C10H22 or C16H34 was incorporated establishes that the fluorinated segment, although its length (from C4 to C8) is not crucial, is required to hinder hydrolysis by PLA2, indicating that this segment plays an important role in structuring the liposomal membrane.

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.

Vesicles and other supramolecular systems from biocompatible synthetic glycolipids with hydrocarbon and/or fluorocarbon chains.

A series of double-tailed hydrocarbon and/or fluorocarbon glycolipids derived from galactose and glucose have been prepared. These compounds were obtained upon opening a lactono- and maltonolactone moiety by the amino group of either a glycine, glycylglycine or lysine residue. The carboxyl terminus of the glycyl and glycylglycine conjugates was further reacted with the appropriate double-tailed amine. In the case of lysine, the lactonamide conjugate was functionalized with a hydrocarbon and/or fluorocarbon fatty amine and acid, respectively. The ability of such glycolipids to disperse in water, the morphology of self-assemblies formed and the stability of the supramolecular structure obtained were shown to depend on the presence or absence and on the nature of the aminoacid spacer. Most of the compounds described were shown by conventional techniques (TEM, Cryo-TEM, LLS, etc.) to produce stable vesicular systems.

Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes.

The stability (with respect to encapsulated carboxyfluorescein release) of fluorinated liposomes and their membrane permeability have been investigated in buffer and in human serum as compared to conventional hydrogenated analogues. These fluorinated liposomes are made from highly fluorinated phosphatidylcholines and contain a fluorinated core within their membrane. In buffer and in their fluid state, the fluorinated liposomes retain much more efficiently their entrapped content and display lower membrane permeability coefficients than any of their hydrogenated counterparts. This indicates that the fluorinated core acts as a very efficient barrier to permeation. In terms of molecular structure/permeability relationships, the thicker the fluorinated lipophobic core, the more efficient the barrier to permeation. In their gel state, the fluorinated core has, however, almost no effect on permeation. Interestingly, some of the 'fluid' fluorinated liposomes were even less permeable than 'gel' or 'gel-like' ones, including egg phosphatidylcholines/cholesterol liposomes. Human serum destabilizes the 'fluid' fluorinated liposomes but to a lesser extent than the 'fluid' hydrogenated ones, indicating that the fluorinated lipophobic core inside the liposomal membrane protects the vesicles, possibly by reducing their interactions with serum components. 'Gel' or 'gel-like' fluorinated liposomes are significantly more stable in serum than in buffer. They are also more stable than conventional 'gel' or 'gel-like' liposomes.

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 membrane's lipophilicity.

Improved control over particle sizes and stability of concentrated fluorocarbon emulsions by using mixed fluorocarbon/hydrocarbon molecular dowels.

The use of a surfactant system consisting in equimolar amounts of egg yolk phospholipids and of a mixed fluorocarbon/hydrocarbon amphiphile (C8H17CH = CHC8F17) allows the preparation of concentrated (90% w/v, i.e. 47% v/v) emulsions of perfluorooctyl bromide (perflubron), with average particle sizes ranging from 0.12 to 16 microns post-sterilization, depending on the surfactant/fluorocarbon ratio. Emulsion droplet diameters varied linearly as a function of the emulsifier's concentration, thus allowing easy pre-determination of the emulsion's average particle size. Excellent stability was observed for the dowel-containing emulsions for at least 6 months at 40 degrees C over the whole domain of particle sizes investigated.

Biodistribution and excretion of a mixed fluorocarbon-hydrocarbon "dowel" emulsion as determined by 19F NMR.

To investigate the biodistribution, possible metabolism and excretion of mixed fluorocarbon-hydrocarbon "dowel" molecules used as stabilizers of fluorocarbon emulsions, we have prepared a 25% w/v emulsion of such a molecule, and quantitatively evaluated, by means of 19F NMR, its behavior in the blood and reticuloendothelial system (RES) of rats. C6F13CH = CHC10H21 (F6H10E) was emulsified using egg yolk phospholipids (EYP). The emulsion (F6H10E/EYP: 25/6% w/v) was injected intravenously into 33 Sprague Dawley female rats at a 3.6 g/kg body weight dose of F6H10E. The animals were sacrificed at regular intervals of time. 24 hours after the injection, 70% of the injected dose was located in the liver, 17% in the spleen, 4% in the lungs, 2% in the kidneys and 2% in the blood. The half-time retention of the dowel molecule in the liver was estimated to be 25 +/- 5 days. None of the 33 treated animals died prior to the planned sacrifice date. The dowel molecule F6H10E proved to be well tolerated, and excreted reasonably fast, without metabolism. This appears to warrant the use of such molecules as stabilizers in injectable fluorocarbon emulsions destined to serve as oxygen carriers, contrast agents or drug delivery systems.

Mixed fluorocarbon/hydrocarbon molecular dowels help protect concentrated fluorocarbon emulsions with large size droplets against coalescence.

Mixed fluorocarbon/hydrocarbon amphiphiles, the so-called molecular dowels, have previously been reported to strongly stabilize concentrated (90% w/v, i.e. 47% w/v) submicronic size perfluorooctyl bromide emulsions emulsified by egg yolk phospholipids. The dowel molecules, used in equimolar amounts with phospholipids, enable the preparation of emulsions with large-sized particles which are impossible to obtain with phospholipids alone. We report here that molecular dowels (C6F13C10H21, F6H10) also hinder droplet coalescence induced by mechanical stress in sterilized emulsions with average particle sizes ranging from ca 1 to 16 microns. In contrast, the addition of equimolar amounts of perfluorodecyl bromide was found to have little influence on these emulsions' resistance to mechanical stress. This is consistent with the view that mixed fluorocarbon/hydrocarbon dowels are held in the interfacial film and reinforce its cohesion with the fluorocarbon phase.

Amphiphilic sugar phosphates with single or double perfluoroalkylated hydrophobic chains for use in oxygen and drug delivery systems.

New anionic amphiphiles with a phosphate ester junction between the fluorophilic-lipophilic tail and the sugar-based hydrophilic head were synthesized and evaluated. The single hydrophobic chain surfactants 1 a, b and 2a allowed the preparation of stable and fine highly concentrated emulsions of perfluorodecalin or perfluoroocytl bromide, either when used alone or in conjunction with egg yolk phospholipids (EYP). Surfactants 3d, 5d, 6d and 6e, with two hydrophobic chains, one fluorinated the other not, gave liposomal structures, and displayed encapsulation properties for carboxyfluorescein. The phosphodiesters tested cause no significant inhibition of the growth and viability of Namalva cell cultures (0.1-1 g/L range). Single chain phosphodiesters manifest no detectable hemolytic activity (at 100 g/L for 1a) whereas double chain compounds do moderately (ca 20% hemolysis at 20 g/L). The maximum tolerated dose compatible with the survival of all of a series of 10 intravenously injected mice is in 130 mg/kg body weight range.

Fluorinated phospholipid-based vesicles as potential drug carriers: encapsulation/sustaining of drugs and stability in human serum.

The release of entrapped 5(6)-carboxyfluorescein from fluorinated vesicles in a buffer or in human serum is considerably less than that from hydrogenated liposomes. The presence of a fluorinated core inside the liposomal membrane definitely reduces its permeability. In some cases, the stability of liposomes made from fluorinated phospholipids alone is better than that of egg phosphatidylcholine cholesterol 1/1 vesicles, which are among the least permeable and most stable hydrogenated liposomes.