New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Ultralow fouling polyacrylamide on gold surfaces via surface-initiated atom transfer radical polymerization

In this work, polyacrylamide is investigated as an ultralow fouling surface coating to highly resist protein adsorption, cell adhesion, and bacterial attachment. Polyacrylamide was grafted on gold surfaces via surface-initiated atom transfer radical polymerization (ATRP). Protein adsorption from a wide range of biological media, including single protein solutions of fibrinogen, bovine serum albumin, and lysozyme, dilute and undiluted human blood serum, and dilute and undiluted human blood plasma, was studied by surface plasmon resonance (SPR). Dependence of the protein resistance on polyacrylamide film thickness was examined. With the optimal film thickness, the adsorption amount of all three single proteins on polyacrylamide-grafted surfaces was <3 pg/mm(2), close to the detection limit of SPR. The average nonspecific adsorptions from 10% plasma, 10% serum, 100% plasma, and 100% serum onto the polyacrylamide-grafted surfaces were 5, 6.5, 17, and 28 pg/mm(2), respectively, comparable (if not better) than the adsorption levels on poly(ethylene glycol) (PEG) and zwitterionic poly(sulfobetaine methacrylate) surfaces, the best antifouling materials known to date. The polyacrylamide-grafted surfaces were also shown strongly resistant to adhesion from bovine aortic endothelial cells and two bacterial species, Gram-positive Staphylococcus epidermidis ( S. epidermidis ) and Gram-negative Pseudomonas aeruginosa ( P. aeruginosa ). Strong hydrogen bond with water is considered the key attribute for the ultralow fouling properties of polyacrylamide. This is the first work to graft gold surfaces with polyacrylamide brushes via ATRP to achieve ultralow fouling surfaces, demonstrating that polyacrylamide is a promising alternative to traditional PEG-based antifouling materials.     

Antibody immobilization onto glow discharge treated polymers.

Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2–2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1–2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

Plasma fibrinogen levels in type II diabetics

Plasma fibrinogen levels measured by an immunoassay method on 170 type II diabetic patients exhibited a bimodal distribution with one small population demonstrating levels greater than those of the normal reference range. The mean plasma level of fibrinogen in the type II diabetics was higher than that of the normal population. Spearman's correlations demonstrated statistically significant positive relationships in type II diabetic patients between fibrinogen levels and fasting glucose levels, serum cholesterol, glycosylated hemoglobin and urinary albumin excretion rate. These relationships suggest that increased plasma fibrinogen may be another marker for coronary heart disease complications encountered by diabetics.     

A biophysical mechanism by which plasma proteins inhibit lung surfactant activity

These in vitro experiments study a potential mechanism by which plasma proteins, found in the alveoli during pulmonary edema and hemorrhage, may act to inhibit the surface activity of pulmonary surfactant. The results indicate that the inhibition of the adsorption facility and surface tension lowering ability of a calf lung surfactant extract (CLSE) by albumin, hemoglobin, or fibrinogen may be completely abolished by centrifugation of the protein-surfactant mixture at 12,500 x g. Furthermore, albumin, hemoglobin and fibrinogen (1.25 mg/ml) were shown to inhibit the adsorption of high concentrations of CLSE (0.32 mg/ml), normally unaffected by the addition of exogenous proteins, when the CLSE was injected into the subphase under a preformed protein surface film. Similarly, injection of large amounts of these proteins (2.5 mg/ml) into the subphase beneath a preformed CLSE surface film was without effect, even though the CLSE concentration was only 0.06 mg/ml, a surfactant concentration which is normally inhibited by even small amounts of exogenous protein. Taken together, the data suggest that some proteins may inhibit surfactant function by preventing the surfactant phospholipids from adsorbing to the air-liquid interface, possibly by a competition between the proteins and CLSE phospholipids for space at the air-liquid interface rather than direct molecular interactions between proteins and surfactant.     

An in vivo method for measuring the adsorption of plasma proteins to titanium in humans

A novel method of collecting in vivo plasma proteins of humans from osteotomies prepared during insertion of an oral implant is described. A rod containing a collecting portion with a predetermined surface is introduced into the osteomy, removed, and transferred for enzyme-linked immunosorbent assay analysis. Two experiments were used to examine the feasibility of the method. In the first, titanium (Ti) rods with different roughness were exposed for 10 min to the blood. Blasted and acid-etched surfaces adsorbed four times more and acid-etched surfaces adosorbed two times more plasma proteins as compared to machined surfaces. In the second experiment, blasted and acid-etched rods were wetted for 10 s prior to the insertion. The adsorption for fibronectin, albumin, fibrinogen, and IgG was enhanced significantly compared with nonwetted rods. These results are discussed in the light of previous methods used in studies on adsorption. Thus, use of the collecting instrument enables aspects of human plasma–implant interface to be studied in a more realistic manner.     

Effect of exercise and thermal stress on plasma volume.

Six male subjects exercised for 50 min at 25% (light exercise) and 55% (moderate exercise) of their estimated aerobic capacities in environments of 42 degrees C db, 35 degrees C wb and 30 degrees C db, 24 degrees C wb, respectively. Alterations in the hematocrit, hemoglobin, and plasma protein concentrations, and in the activity of an injected aliquot of isotopically labeled albumin were each used to calculate the percentage change in plasma volume occurring during exercise and recovery. Changes in each measure were consistent with a reduction in plasma volume during exercise and a return to preexercise levels during recovery. There was no significant difference between the measures when exercising in the heat, but during the more severe exercise in the cooler environment disproportional changes in protein, hematocrit, and hemoglobin were observed. Disproportional changes were also seen during the recovery phase, when the hematocrit and hemoglobin concentration indicated a more rapid return of the plasma volume to preexercise levels than did either the plasma protein concentration or albumin activity. During moderate exercise and recovery there was a 1% decrease in red cell volume. It is concluded that exercise accelerates the rate of protein movement from extravascular compartments to the intravascular compartment, leading to elevated plasma protein levels during recovery which favor the return of water to the intravascular space. Hemoglobin concentration is considered to be the most reliable measure of plasma volume change during exercise.     

Interaction of fluorescent molecular rotors with blood plasma proteins

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.     

Selenite metabolism in rat and human blood

The binding of selenite, the form of selenium used in the treatment of Keshan disease, to plasma proteins and the role of erythrocytes in this process have been studied. The experiments were carried out by incubating⁷⁵Se as selenite with plasma and whole bloodin vitro (human and rat) andin vivo (rat) and subsequent fractionation by Sephadex G-150 gel filtration. Human and rat plasma proteins were unable to incorporate selenium from selenite, as shown by the negligible amount present in proteins after incubation of plasma with selenite. The incorporation can be carried out after internalization of selenite by the erythrocytes in a fast, temperature dependent process. Hemoglobin, being the major binding protein for the newly reduced selenium in the erythrocyte, might have a role in the uptake of selenite by erythrocytes. The greater affinity of plasma proteins for the final selenium compound resulting from reduction could be the cause of the efflux. However, this is minimal in the absence of plasma, as is evident from the results obtained from blood reconstituted with saline solution instead of plasma. At least two proteins, one albumin like, probably albumin itself and the other of molecular weight close to or greater than 200,000 in the plasma, are involved in the binding and efflux processes.     

A microfluidic biosensor based on competitive protein adsorption for thyroglobulin detection

We report a microfluidic sensing platform for the detection of thyroglobulin (Tg) using competitive protein adsorption. Serum Tg is a highly specific biomarker for residual thyroid tissue, recurrence and metastases after treatment for differentiated thyroid cancer (DTC). Conventional Tg detection techniques require complicated immobilization of antibodies and need to form a sandwich assay using additional secondary antibodies to enhance the sensitivity. We present a fundamentally different sensing technique without using antibody immobilization on a microfluidic platform. We engineer two surfaces covered by two known proteins, immunoglobulin G (IgG) and fibrinogen, with different affinities onto the surfaces. The microfluidic device offers a selective protein sensing by being displaced by a target protein, Tg, on only one of the surfaces. By utilizing the competitive protein adsorption, Tg displaces a weakly bound protein, IgG; however, a strongly bound protein, fibrinogen, is not displaced by Tg. The surface plasmon resonance (SPR) sensorgrams show that five human serum proteins, albumin, haptoglobin, IgG, fibrinogen and Tg, have different adsorption strengths to the surface and the competitive adsorption of individuals controls the exchange sequence. The adsorption and exchange are evaluated by fluorescent labeling of these proteins. Tg in a protein mixture of albumin, haptoglobin, and Tg is selectively detected based on the exchange reaction. By using the technique, we obviate the need to rely on antibodies as a capture probe and their attachment to transducers.     

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes

The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin--an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.     

Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml(-1) for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes

Abstract

The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin – an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.     

Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry

The observation that protein-A conjugated gold sols bound to fibronectin-collagen (FNC) fibres in human fibroblast cultures prompted a series of studies on the binding of gold particles stabilized in various ways (Staphylococcal protein A, bovine serum albumin, avidin, streptavidin, gelatin, hemoglobin, polyethylene glycol (MW 20 000), methylcellulose and the nonionic detergent Tween 20) to cell and tissue components, to protein dot blots and SDS-PAGE blots on nitrocellulose paper. We found that binding of gold particles to certain cell and tissue components and to various immobilized proteins did occur irrespective of the stabilizing agent. We argue that, albeit gold sols are stabilized against salt coagulation by adsorption of proteins and other stabilizing agents, "naked areas" are (constantly or intermittently) present on particle surfaces, available for interaction with cell and tissue components that have a high electrostatic affinity for the charged gold surface under prevailing experimental conditions. Non-specific binding may be reduced or abolished by competing proteins (i.e. proteins with a higher affinity for gold than any component in the object studied) provided the proteins and the gold conjugate are present concomitantly during incubation. We found gelatin (Bloom number 60-100) to be an effective competitive protein probably due to its high affinity for gold over a wide pH range. Further, gelatin did not appreciably inhibit the specific interaction in dot blots between SpA and IgG except at very low IgG concentrations. A protocol for the use of gold-protein conjugates to circumvent the hazards of unspecific gold binding is suggested.     

Fixation of aldehydic dextrans onto human deoxyhemoglobin.

A procedure commonly used to transform native adult human hemoglobin (Hb) into a physiological oxygen carrier consists of a pyridoxylation of the protein to lower its oxygen affinity, followed by its polymerization in the presence of glutaraldehyde, with or without further reduction, to increase its circulating half-life. This series of reactions yields derivatives presenting a great molecular heterogeneity that have to be fractionated for use in vivo. Hemoglobin derivatives with low oxygen affinity and a narrow distribution of molecular weights were obtained by linking a dextran polyaldehydic derivative to deoxyhemoglobin at pH 8. From oxygen-binding measurements carried out in the presence of inositolhexaphosphate, a strong effector of hemoglobin, it appeared that the allosteric site of hemoglobin was blocked, probably by crosslinking bonds, which stabilizes its deoxy structure. On the other hand, when the reaction was performed in the presence of inositolhexaphosphate, the resulting conjugates exhibited an oxygen affinity identical to that of unmodified hemoglobin. After treatment with NaBH4, the polymer-hemoglobin derivatives were stable and possessed a reversible oxygen-carrying capacity similar to that of blood. The conjugates prepared from oxyhemoglobin all possessed a lower P50 than native hemoglobin whatever the reaction conditions.     

Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml^?1 for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

Environmentally-related changes in red cell levels of ionic modulators of hemoglobin-O2 affinity in rainbow trout, Salmo gairdneri

Hemoglobin content, plasma and red cell levels of chloride and magnesium and molar ion:hemoglobin ratios were examined in trout acclimatized to eight combinations of two treatment levels of temperature (5, 20 degrees C), O2 availability (less than or equal to 30%, greater than or equal to 75% saturation) and photoperiod (16L:8D, 8L:16D). Increases in hemoglobin content were associated with exposure to higher temperature, abbreviated daylength and hypoxia, with hypoxia greater than photoperiod greater than temperature. Under nominal "summer" conditions (20 degrees C, hypoxia, 16L:8D) photoperiodic influence was apparently masked by hypoxic and thermal effects. Temperature was the principal determinant of plasma and cellular chloride levels as well as [Cl:Hb]. O2 availability and photoperiod had little effect. Temperature was also the primary factor influencing magnesium, with hypoxia exerting a lesser influence. Photoperiod effects were negligible. With increased temperature and reduced O2 availability, plasma magnesium increased white cell magnesium levels and [Mg:Hb] declined. These observations suggest that with normal seasonal changes in environmental conditions, temperature-induced increases in the O2 requirements of summer trout are probably accompanied by increases in blood O2-carrying capacity and reductions in hemoglobin-O2 affinity with consequent increases in O2 delivery to tissues.     

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).