Impact of the spacer on the condensing effect of fluorinated chains investigated by grazing incidence X-ray diffraction on ultrathin Langmuir monolayers.

Langmuir monolayers of double perfluoroalkyl(alkyl) chain amphiphiles fitted with a monomorpholinophosphate polar head, [C(n)F(2n+1)(CH(2))(m)O](2)P(O)[N(CH(2)CH(2))(2)O] (di(FnHm)MP with n = 6, 8, or 9; and m = 1 or 2), were investigated by surface pressure (π)/molecular area (A(0)) compression isotherms for temperatures ranging from 15 to 50 °C, and by grazing-incidence X-ray diffraction (GIXD) at 25 °C. Ultrathin monolayers were obtained for these short surfactants. Though the hydrocarbon spacer is short, it has a remarkable impact on the monolayer's organization. At 25 °C, whereas di(F8H2)MP monolayer presents a liquid expanded (LE)/liquid condensed (LC) transition, simply replacing one CH(2) by a CF(2) in the latter compound's structure at constant chain length, i.e. shortening the spacer from 2 to 1 CH(2) (as in di(F9H1)MP), suppresses the LE phase. At 25°, GIXD established that for both di(F8H2)MP and di(F9H1)MP, the chains form an hexagonal lattice in the LC phase. The collective tilt of the two compounds is close to zero. The lattice of the dense phase can be compressed, as assessed by the continuous linear decrease of the d spacing with increasing pressure. This indicates that the azimuthal distribution of the molecular tilts is progressively reduced upon compression. The d value for di(F9H1)MP is significantly lower than that of di(F8H2)MP, providing evidence for strong condensing effect of the fluorinated chains. Molecular areas were determined directly from the compression curves and also from the X-ray data, the latter allowing reconstruction of the compression isotherms. The calculated lattice compressibilities are ~30% and 50% of the macroscopic compressibilities for di(F9H1)MP and di(F8H2)MP, respectively. Comparison with the experimentally determined isotherms shows that the monolayer of di(F9H1)MP is more stable than that of di(F8H2)MP. The enthalpies and entropies determined for di(F9H1)MP and di(F8H2)MP, derived from the Clausius-Clapeyron equation, confirm that the observed transitions are both of the LE/LC type, although the triple point temperatures are strikingly different (27 °C vs -18 °C); this large difference further illustrates the stabilizing effect of the fluorinated chains. Disorder is hindered by the fluorinated chains and facilitated by a hydrocarbon spacer when larger than 1 CH(2).

Perfluorocarbon emulsions prevent hypoxia of pancreatic ß-cells.

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on ß-cell lines and rat pancreatic islets. RINm5F ß-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). ß-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 µg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 µg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 µg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 µg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Improved in vivo two-photon imaging after blood replacement by perfluorocarbon.

Two-photon microscopy is a powerful method in biomedical research that allows functional and anatomical imaging at a subcellular resolution in vivo. The technique is seriously hampered by absorption and scattering of light by blood, which prevents imaging through large vessels. Here, we demonstrate in the rat cerebral cortex that blood replacement by perfluorocarbon emulsion, a compound also used in human critical care medicine, yields superior image quality, while preserving neuronal integrity. Shadows of large superficial vessels disappear completely and cells can be imaged underneath them. For the first time, it is possible to image complete populations of neurons and astrocytes in the upper layers of neocortex in vivo.

Perfluorocarbons: new tool for islets preservation in vitro.

Pancreatic islet transplantations to treat type 1 diabetes often fail to function because of hypoxia. Perfluorocarbons (PFCs) exhibit a high oxygen solubility coefficient and maintain high oxygen partial pressure for extended times. They also serve as oxygen "reservoirs" for harvested organs in pancreas organ transplantation. Previous studies have shown the PFCs display antiadhesive effects on beta cells. The aim of this study was to evaluate the effects of PFC on islet viability and functionality and on extracellular matrix (ECM) disruption of islets via inhibition of adhesion. Primary cultures of rat islets were incubated for 24 hours in the presence or absence of 3.5% (weight/volume) PFCs in culture media. We studied viability (FDA/PI), stimulation index linked to insulin secretion (ELISA), and expression of insulin and laminin messenger RNAs (mRNAs). Immunostaining was performed on insulin and laminin. Islet viability was similar in the presence or absence of PFCs (about 80%). Stimulation index showed preservation of islet functionality in the presence of PFC (4.9 +/- 0.7) as compared with controls (2.8 +/- 0.5). Moreover, laminin mRNA expression was lower compared with controls (55% of PFC incubated vs control islets). Immunohistochemistry studies showed preservation of ECM inside the islets in the presence of PFCs versus controls at 24 hours after islet isolation. In conclusion, PFCs preserved islet viability and functionality and prevented ECM disruption. PFCs may represent a new tool for islet preservation in vitro.

In vivo evaluation of a reverse water-in-fluorocarbon emulsion stabilized with a semifluorinated amphiphile as a drug delivery system through the pulmonary route.

The potential of a reverse water-in-fluorocarbon (w-in-FC) emulsion stabilized with a semifluorinated amphiphile, namely C8F17(CH2)11OP(O)[N(CH2CH2)2O]2 (F8H11DMP) for drug delivery through intrapulmonary administration was investigated in the mouse. This study involved assessment of the effect of single or repeated intranasal instillations of a plain emulsion on lung tissue integrity, and evaluation of blood glucose levels in mice treated with an insulin-loaded emulsion. When instilled intranasally to mice, the plain emulsion did not alter lung tissue integrity, as demonstrated by histological staining, and did not induce any airway inflammatory reaction. Treated mice exhibited decreased body weight within the 3-4 days that followed the first emulsion administration, but this decrease was reversible within few days. Mice instilled intranasally with the insulin-loaded emulsion displayed decreased blood glucose levels within the 20 min that followed the administration, thus demonstrating the potential of the reverse w-in-FC emulsion stabilized with F8H11DMP to systemically deliver drugs, including peptides, upon lung administration.

Evaluation of cytotoxicity of new semi-fluorinated amphiphiles derived from dimorpholinophosphate.

Water-in-fluorocarbon reverse emulsions and microemulsions stabilized by semi-fluorinated amphiphiles derived from the dimorpholinophosphate polar head group, C(n)F(2n+1)(CH(2))(m)OP(O)[N(CH(2)CH(2))(2)O](2) (FnHmDMP), are being investigated as new delivery systems for drugs or genetic materials into the lung. Since information related to the toxicity of fluorinated surfactants is still very limited, we evaluated herein the cytotoxicity of a series of FnHmDMP (n=4, 6, 8 and 10 and m=2, 5, and 11). Both solutions of FnHmDMP in fluorocarbons, and reverse water-in-fluorocarbon emulsions stabilized by FnHmDMP were assessed in order to determine the relation between surfactant structure and cell toxicity, and select the most innocuous emulsifier. A first short-term evaluation on mouse fibroblasts using a viability/cytotoxicity assay indicated that amphiphiles (in solution) with a chain length longer than C12 exhibit less toxicity than amphiphiles with shorter chain. Moreover cytotoxicity decreased also with length of the fluorinated segment. The protective effect of the fluorinated chain was strongly supported by the fact that the hydrogenated analog, C(15)H(31)OP(O)[N(CH(2)CH(2))(2)O](2) (H15DMP), was highly toxic. Qualitative evaluation on human lung epithelial cells (HLEC) using a colorimetric method (Mayer's hematoxylin) confirmed that amphiphiles (in solution) with longer chain were the least cytotoxic. The protective effect of the fluorinated chain appeared, however, to be significant only at low amphiphile concentrations (0.1% w/v). In contrast, at higher concentrations (1% and 5% w/v), the total chain length was the determining factor. Quantitative evaluation of the least cytotoxic amphiphiles using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method then showed that F10H11DMP (in solution) was harmless until its solubility limit (1% w/v); cell growth was even enhanced due to improved oxygenation provided by the fluorocarbon phase. F8H11DMP exhibited some cytotoxicity at both 1% and 5% w/v, but the toxicity appeared to level off with concentration. Reverse water-in-perfluorooctyl bromide (PFOB) emulsions stabilized by either F10H11DMP or F8H11DMP were found to be non-cytotoxic. In conclusion, the present evaluation indicates that the cytotoxicity of FnHmDMP depends on both total and fluorinated amphiphile chain length, and leads us to select F8H11DMP and F10H11DMP as the less cytotoxic amphiphiles among a series of FnHmDMP compounds. Furthermore, water-in-fluorocarbon emulsions stabilized with F8H11DMP and F10H11DMP appeared to be non-cytotoxic towards HLEC in culture.

Reverse water-in-fluorocarbon emulsions for use in pressurized metered-dose inhalers containing hydrofluoroalkane propellants.

Pulmonary administration of drugs has demonstrated numerous advantages in the treatment of pulmonary diseases due to direct targeting to the respiratory tract. It enables avoiding the first pass effect, reduces the amount of drugs administered, targets drugs to specific sites and reduces their side effects. Reverse water-in-fluorocarbon (FC) emulsions are potential drug delivery systems for pulmonary administration using pressurized metered-dose inhalers (pMDI). The external phase of these emulsions consists of perfluorooctyl bromide (PFOB, perflubron), whereas their internal phase contains the drugs solubilized or dispersed in water. These emulsions are stabilized by a perfluoroalkylated dimorpholinophosphate (F8H11DMP), i.e. a fluorinated surfactant. This study demonstrates the possibility of delivering a reverse fluorocarbon emulsion via the pulmonary route using a CFC-free pMDI. Two hydrofluoroalkanes (HFAs) (Solkane(R) 134a and Solkane(R) 227) were used as propellants, and various solution (or emulsion)/propellant ratios (1/3, 1/2, 2/3, 1/1, 3/2, 3/1 v/v) were investigated. The insolubility of water (with or without the fluorinated surfactant F8H11DMP) in both HFA 227 and HFA 134a was demonstrated. PFOB and the reverse emulsion were totally soluble or dispersible in all proportions in both propellants. This study demonstrated also that the reverse FC emulsion can be successfully used to deliver caffeine in a homogeneous and reproducible way. The mean diameter of the emulsion water droplets in the pressured canister was investigated immediately after packaging and after 1 week of storage at room temperature. Best results were obtained with emulsion/propellant ratios comprised between 2/3 and 3/2, and with HFA 227 as propellant.

Detection of perfluorocarbons in blood by headspace solid-phase microextraction combined with gas chromatography/mass spectrometry.

A new method of detection of perfluorocarbon molecules (PFCs) in blood sample has been established. After an extraction and pre-concentration step performed by headspace solid-phase microextraction (HS-SPME), the PFCs are detected by gas chromatography-mass spectrometry (GC/MS) with an ion trap mass spectrometer in MS and MS/MS modes. The influence of different parameters on the SPME process is discussed. The limit of detection and the linearity of the procedure have been determined for two PFCs.

Seventy-two hours hypothermic intestinal preservation study using a new perfluorocarbon emulsion.

We investigated the effect of a perfluorocarbon emulsion (FC) added to the University of Wisconsin (UW) solution on hypothermic (4 degrees C, 12-72h) preservation of rat small bowel grafts. The FC was 90%w/v perfluorooctylbromide, 2%w/v egg yolk phospholipids and 1.4%w/v mixed fluorocarbon-hydrocarbon molecular dowels. Four groups were defined: [1] UW flush and UW storage; [2] UW flush and FC storage; [3] flush with FC diluted 2 times with UW (FU) and FU storage; [4] FU flush and storage in oxygenated FU. Preservation was estimated with a histological score based on villus epithelium adhesion, on villus sloughing and on crypt cell adhesion to the basal membrane. Antioxidant potential was estimated by measurement of total thiol functions (SH) and activities of glutathione-peroxidase (GSH-P), superoxide dismutase (SOD) and catalase. FC in flush improved preservation during the first 24h (p<0.01). Storage in FC appeared superior to UW for the first 24h (p<0.01). Oxygenation (100% O2) of the storage medium yielded superior results at 12h and 24h (p<0.01 and p<0.001 versus group [1] respectively). After 72h, SOD and catalase activities increased in groups [3] and [4], and SOD decreased in group [1] (p<0.05). SH progressively decreased in group [1] (p<0.05) and GSH-P increased at 24 and 48h in groups [3] and [4] (p<0.01). The increase of O2 in the perfusion flush or storage medium ameliorated the preservation status and protected the antioxidant potential of the small bowel.

Fluorocarbons and fluorinated amphiphiles in drug delivery and biomedical research.

The specific properties of fluorocarbons, exceptional chemical and biological inertness, high gas-dissolving capacity, low surface tension, excellent spreading characteristics and high fluidity, have triggered numerous applications of these compounds in oxygen delivery. An injectable emulsion of fluorocarbon-in-water destined to deliver oxygen to tissues at risk of hypoxia has now completed Phase III clinical trials in Europe. A neat fluorocarbon is currently investigated in Phase II for treatment of acute respiratory failure by liquid ventilation. Fluorinated lipids and fluorinated surfactants can be used to elaborate and stabilize various colloidal systems, including different types of emulsions, vesicles and tubules, that also show promise for controlled release drug delivery.

Determination of perfluorodecalin and perfluoro-N-methylcyclohexylpiperidine in rat blood by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry method (SIM mode) was developed for the determination of perfluorodecalin (cis and trans isomers, 50% each) (FDC), and perfluoromethylcyclohexylpiperidine (3 isomers) (FMCP) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Analysis was performed by electronic impact ionization. The ions m/z 293 and m/z 181 were selected to quantify FDC and FMCP due to their abundance and to their specificity, respectively. The ion m/z 295 was selected to monitor internal standard. Before extraction, blood samples were stored at -30 degrees C for at least 24 h in order to break the emulsion. The sample preparation procedure involved sample clean-up by liquid-liquid extraction. The bis(F-butyl)ethene was used as the internal standard. For each perfluorochemical compound multiple peaks were observed. The observed retention times were 1.78 and 1.87 min for FDC, and 2.28, 2.34, 2.48 and 2.56 min for FMCP. For each compound, two calibration curves were used; assays showed good linearity in the range 0.0195-0.78 and 0.78-7.8 mg/ml for FDC, and 0.00975-0.39 and 0.39-3.9 mg/ml for FMCP. Recoveries were 90 and 82% for the two compounds, respectively with a coefficient of variation <8%. Precision ranged from 0.07 to 15.6%, and accuracy was between 89.5 and 111.4%. The limits of quantification were 13 and 9 microg/ml for FDC and FMCP, respectively. This method has been used to determine the pharmacokinetic profile of these two perfluorochemical compounds in blood following administration of 1.3 g of FDC and 0.65 g of FMCP per kg body weight, in emulsion form, in rat.

Assay method for the perfluorooctyl bromide (perflubron) in rat blood by gas chromatography-mass spectrometry.

This paper describes a GC-MS method (SIM mode) for the analysis of perfluorooctyl bromide (perflubron, I) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Following destruction of the emulsion by addition of ethanol, the analytical procedure involves a liquid-liquid extraction with 1,1,2-trichlorotrifluoroethane. The bis(F-butyl)ethene (II) was used as internal standard. Observed retention times were 3.22 min for I and 2.32 min for II. Two calibration curves were used; linear detection responses were obtained for concentrations ranging from 0.009 to 0.9 mg/ml and from 0.9 to 13.5 mg/ml. The extraction efficiency averaged 50% for I and 93% for II. Precision ranged from 0.7 to 14%, and accuracy was between 91 and 109%. The limit of quantification was 9 microg/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay method for use in pharmacokinetic studies.

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.

Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes.

We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This could be due to a delipidating, denaturing effect of these detergents (octyl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F17(CH2)2O-P(O2-)-O(CH2)2N+(CH3)3(F8C2PC), extracted about as much protein from neutrophil plasma membranes into a 100,000 g supernatant as octyl glucoside. The second compound, C8F17(CH2)11O-P(O2-)-O(CH2)2N+(CH3)3(F8C11PC), was less efficient. We found that flavin was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained without addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside extracts. These experiments appeared to make F8C2PC an interesting membrane-solubilizing agent. Nevertheless, no protein in the F8C2PC extract could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octyl glucoside, some of the proteins, such as myeloperoxidase, could be adsorbed (and eluted), but not cytochrome b558. Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicles and aggregates of small shapeless particles. Higher centrifugal forces sedimented most proteins of the 100,000 g supernatant. As a check, the effect of F8C2PC was tested on sarcoplasmic reticulum vesicles, the behavior of which with respect to the usual non-denaturating detergents has been well studied. There was little, if any, solubilization. We conclude that, although supernatants of F8C2PC extracts of neutrophil membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes.

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.

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.

Longitudinal studies on the interaction of perfluorochemicals with liver cytochromes P-450 by means of testing the rate of detoxification of pentobarbital.

Four perfluorochemicals, Bis-[F-butyl]ethene, perfluorocyclohexylmorpholine, perfluorodecalin and perfluorooctylbromide were compared by their influence on the liver cytochrome P-450 system, measuring the pentobarbital sleeping time as defined by the time of loss of the righting reflex in rats. In all experiments first a prolongation of barbital detoxification was observed, which lasted at least 2-4 days. Thereafter a very long extended period of abbreviated sleeping time followed which was only missed after perfluoroctylbromide. Thus substrate competition, uncoupling of monooxygenation and enzyme induction determine the detoxifying processes in the liver that follow the administration of perfluorochemicals.