Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.     

Mechanisms of human plasma protein adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion stabilized with proxanol 268 is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to an increase in the total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and fibrinogen is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been set that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicate considerable conformational rearrangements of this protein during adsorption. It was shown that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction is completely or partially quenched.     

Sorption of plasma proteins by fluorocarbon emulsions stabilized by various surfactants

It has been found in in vivo and in vitro experiments that, as a perfluorocarbon emulsion stabilized by Proxanol 268 comes in contact with blood plasma proteins, plasma proteins with molecular masses from 25 to 170 kDa and above are adsorbed on the surface of emulsion particles. Among the adsorbed proteins, fibronectin and fibrinogen were identified by immunoblotting. In in vivo experiments, during circulation in the blood flow, considerable amounts of plasma proteins are adsorbed on Proxanol-stabilized emulsion particles; the amount of adsorbed proteins increases with the time the particles are in the blood flow. Considerably lesser amounts of proteins are adsorbed during circulation in the blood flow on emulsion particles stabilized by egg yolk phospholipids, and their qualitative composition differs from the composition of proteins adsorbed on Proxanol-stabilized emulsion particles. A preliminary incubation of the Proxanol-stabilized emulsion with heparin decreases the amount of the adsorbed proteins and changes their qualitative composition.     

Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.     

Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo Studies)

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 mL of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 h or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorbed new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5–5 mg of proteins per 1 mL of the emulsion) after 24 h.     

Application of perfluorocarbon emulsions for the administration of photosensitizing preparations into bone marrow stem cells

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5–7 days.     

Use of perfluorocarbon emulsions for administration of photosensitizing preparations into bone marrow stem cells

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5-7 days.     

Effect of perfluorocarbon emulsions on function of enzyme systems responsible for generating active forms of oxygen in neutrophils

The ability of the emulsion of perfluoroorganic compounds stabilized with proxanol 268 to affect the functions of peritoneal neutrophils was evaluated. The functional activity of neutrophils was estimated from the intensity of generation of reactive oxygen species using the method of chemiluminescent analysis. The emulsion was shown to suppress the neutrophil responses to phorbol-12-myristate-13-acetate in a dose-dependent manner. No inhibition of the activity of neutrophils in the presence of the emulsion was observed in N-formylmethionylleucylphenylalanine stimulated cells. The data obtained indirectly confirm the suggestion that the perfluoride emulsion inhibits neutrophil NADPH oxidase activity. In the presence of the perfluoride emulsion, myeloperoxidase plays a more important role in the generation of luminescent responses in both N-formylmethionylleucylphenylalanine- and phorbol-12-myristate-13-acetate-stimulated neutrophils. The effect of perfluoride emulsion results in the preferential myeloperoxidase-produced generation of reactive oxygen species in the neutrophil respiratory burst.     

A study of the nature of plasma protein adsorption on the surface of perfluorocarbon emulsions stabilized by different triblock copolymers

The composition of plasma proteins adsorbed on the surface of perfluorocarbon emulsions stabilized by different triblock copolymers and their quantitative ratio were analyzed. The results allowed us to describe three types of protein adsorption on the surface of emulsion droplets. Opsonin proteins prevailed during the first type of adsorption. Their adsorption occurred on a dense and inactive layer of triblock copolymers. The second type of adsorption occurred due to the hydrophobic effect on a dense and mobile layer, with low-molecular-weight proteins being predominantly adsorbed (monoadsorption). The third type of adsorption occurred on a loose layer of triblock copolymers. In this case, the adsorption of a large amount of proteins with a molecular weight of 10–500 kDa was observed, while the total molecular weight was distributed over a large number of proteins.     

Effects of different doses of perfluorocarbon emulsion on hemodynamics and contractility of ischemic heart]

The effect of different doses of perfluorocarbon emulsion (5, 10, and 15 ml/kg) on the hemodynamics and contractility of heart was studied on anesthetized dogs. The emulsion was introduced intravenously by the 60th minute of acute myocardium ischemia caused by partial coronary occlusion. When pO2 = 120 mm Hg, the emulsion was efficient only at doses of 10 and 15 ml/kg (an increase in cardiac ejection, in the rate of contraction and relaxation of the myocardium, reduction of vascular resistance). However, the efficiency of the emulsion at a dose of 15 ml/kg was lower, possibly, due to hypervolemia and cardiodepressive effect of introduction of excess quantity of the surface-active substance proxanol, a component of the emulsion.     

Effect of perfluorocarbon emulsions on rheological parameters of blood

The effect of a perfluorocarbon emulsion on the rheological parameters of blood in a living organism at the hematocrit twice lower than the norm was studied. It was shown that an increase in emulsion concentration leads to an increase in blood asymptotic viscosity and the aggregation of erythrocytes. It was concluded that only a small volume of the fluorocarbon phase of particles, as compared with the total amount of erythrocytes, can improve the blood fluidity upon disturbed circulation.     

Formation,Stability and In Vitro Digestion of β-carotene in Oil-in-Water Milk Fat Globule Membrane Protein Emulsions

The formation, stability and in vitro digestion of milk fat globule membrane (MFGM) proteins stabilized emulsions with 0.2 wt% β-carotene were investigated. The average particle size of β-carotene emulsions stabilized with various MFGM proteins levels (1%, 2%, 3%, 4%, 5% wt%) decreased with the increase of MFGM proteins levels. When MFGM proteins concentration in emulsions is above 2%, the average particle size of β-carotene emulsions is below 1.0 μm. A quite stable emulsion was formed at pH 6.0 and 7.0, but particle size increased with decrease in acidity of the β-carotene emulsion. β-carotene emulsions stabilized with MFGM proteins were stable with a certain salt concentrations (0–500 mMNaCl). β-carotene emulsions were quite stable to aggregation of the particles at elevated temperature and time (85 °C for 90 min). At the same time, β-carotene emulsions were stable against degradation under heat treatment conditions. In vitro digestion of β-carotene emulsion showed the mean particle size of β-carotene emulsions stabilized with MFGM proteins in the simulated stomach conditions and intestinal conditions is larger than that of initial emulsions and simulated mouth conditions. Confocal laser scanning microscopy of β-carotene MFGM proteins emulsions also showed the corresponding results to different vitro digestion model. There was a rapid release of free fatty acid (FFA) during the first 10 min and after this period, an almost constant 70% digestion extent was reached. Approximately 80% of β-carotene was released within 2 h of incubation under the simulated intestinal fluid. These results showed that MFGM protein can be used as a good emulsifier in emulsion stabilization, β-carotene rapid release as well as lipophilic bioactive compounds delivery.     

Apolipoprotein A-V-heparin interactions: implications for plasma lipoprotein metabolism

Transgenic and gene disruption experiments in mice have revealed that apolipoprotein (apo) A-V is a potent regulator of plasma triglyceride (TG) levels. To investigate the molecular basis of apoA-V function, the ability of isolated recombinant apoA-V to modulate lipoprotein lipase (LPL) activity was examined in vitro. With three distinct lipid substrate particles, including very low-density lipoprotein (VLDL), a TG/phospholipid emulsion, or dimyristoylphosphatidylcholine liposomes, apoA-V had little effect on LPL activity. In the absence or presence apolipoprotein C-II, apoA-V marginally inhibited LPL activity. On the other hand, apoA-V-dimyristoylphosphatidylcholine disc particles bound to heparin-Sepharose and were specifically eluted upon application of a linear gradient of NaCl. The interaction of apoA-V with sulfated glycosaminoglycans was further studied by surface plasmon resonance spectroscopy. ApoA-V showed strong binding to heparin-coated chips, and binding was competed by free heparin. ApoA-V enrichment enhanced binding of apoC-II-deficient chylomicrons and VLDL to heparin-coated chips. When LPL was first bound to the heparin-coated chip, apoA-V-enriched chylomicrons showed binding. Finally, human pre- and post-heparin plasma samples were subjected to immunoblot analysis with anti-apoA-V IgG. No differences in the amount of apoA-V present were detected. Taken together, the results show that apoA-V lipid complexes bind heparin and, when present on TG-rich lipoprotein particles, may promote their association with cell surface heparan sulfate proteoglycans. Through such interactions, apoA-V may indirectly affect LPL activity, possibly explaining its inverse correlation with plasma TG levels.     

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.     

A Relationship between Papaincatalyzed Aminolysis of Ethyl Hippurate by Amino Acid Esters and Their Incorporation into Ovalbumin Hydrolysate during the Plastein Reaction

The effects of the colloidal properties of emulsion particles and the conformation of tryptic digests of soybean glycinin, the emulsifiers, on emulsion properties were investigated. The digests were separated into some fractions, and the properties of intact glycinin, two kinds of the best were examined. The diameter of the emulsion particles measured by spectroturbidimetry was not very different among the best emulsifiers and intact glycinin in spite of the difference in emulsifying ability; however, a new parameter, flocculation strength, which is the rigidity of the flocculated structure and is defined as the minimum detergent concentration for the dissociation of flocculation, closely and negatively related to the short term emulsion stability. The amount of adsorbed protein on the surface of the emulsion particles was also related to the long term emulsion stability. The two best emulsifiers were analyzed by gel filtration and circular dichroism. The emulsifiers contained large molecular components whose molecular weight and secondary structures were similar to intact glycinin. The conformational stability of the emulsifiers was evaluated by the change in emission maxima of the intrinsic fluorescence of the proteins against changing urea concentration, and the surface hydrophobicity of the proteins was estimated by the binding of l-anilino-8-naphthalene sulfonate (ANS). The emulsion stability increased with decreasing conformational stability and increasing surface hydrophobicity of the emulsifier proteins.     

Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.     

The anti-ischemic action of a perfluorocarbon emulsion (PFCE) on the canine myocardium

On the 60th minute after inducing the acute ischemia in the canine myocardium by the occlusion of the left anterior descending coronary artery, the animals were intravenously infused with perfluorochemical emulsion (PFCE), its salt composition (SC) or 4% surface-active substance (SAS), proxanol, at a dose of 10 ml/kg. Two hours after infusion, the occlusion was removed (reperfusion). The arterial pO2 was maintained at 120 mm Hg. Analysis of the blood flow, oxygen supply, acid-alkali balance, ECG, as well as creatine phosphokinase activity, measured in the ischemic area, has shown that PFC emulsion is capable of reducing ischemic damage and preventing reperfusion-induced myocardium injury. Thus, the presence of perfluorochemicals in the PFC emulsion is an essential factor in its ischemia-protective effect.     

Effects of a perfluorocarbon emulsion, Fluosol-DA, on rat lymphoid tissue and immunological competence

The effects of administration of low doses (5 or 10 ml/kg body wt) of the proprietary perfluorocarbon emulsion blood substitute, Fluosol-DA 20%, on rat lymphoid tissue and antibody production against sheep red blood cells (SRBC) have been studied. Spleen and mesenteric lymph nodes (MLN) were significantly heavier at eight days after injection of Fluosol, whereas liver and thymus weights were unchanged. The mean plasma antibody titre to sheep red blood cells was significantly (P less than 0.05) increased in animals injected intraperitoneally with both doses of Fluosol-DA before immunization. These results show that there are differences in the uptake of perfluorochemicals by certain rat lymphoid tissues and that immunological competence can be altered following administration of such materials.     

皮克林乳液酶反应体系的构建与应用研究进展

皮克林乳液是依靠固体颗粒稳定的乳液,具有抗聚结性、稳定性好,颗粒易于分离、成本低、毒性小等优点.研究表明将其应用于多相酶催化反应中,可以大大提高体系中的界面反应面积,加快传质速率.综述了皮克林乳液常用的稳定剂颗粒以及颗粒表面改性对乳液稳定的影响.在此基础上,进一步详细论述了皮克林乳液在酶反应体系中应用以及具有"开关"调...     

Blood levels of immunoglobulins G,A,M, circulating immune complexes, complement and proteins in patients on intermittent peritoneal dialysis

The study involved 17 patients on IPD. Blood serum levels of IgG, IgA, IgM, circulating immune complexes, complement and proteins were determinated at the beginning of therapy, after 3, 6, 12 months on IPD and after 1 year on hemodialysis. The frequency of peritonitis was noted during this time. Peritonitis was the most frequent during first 3 months on IPD. No differences in blood serum levels of IgG, IgA, IgM, in the specific periods of IPD were noted. A significant increase in blood serum circulating immune complexes in patients on IPD and hemodialysis compared to the control group was found. A significant decrease in blood serum of C3 complement in patients on IPD and hemodialysis in comparison with the controls were found. A significant decrease in blood serum proteins at the beginning of IPD and after 3 months IPD in comparison with proteins concentration in patients on hemodialysis was observed.