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.     

The preferential adsorption of hemoglobin to polyethylene.

Hemoglobin adsorption to foreign surfaces has not previously been considered in studies of blood-material interactions, despite the fact that hemoglobin is the most abundant protein present in blood. A hemoglobin-like protein was detected on a number of surfaces exposed to blood plasma, serum, and red cell suspensions. Hemoglobin adsorption to polyethylene from plasma was found to approximately equal the amount of adsorption of albumin and fibrinogen. The high relative affinity of hemoglobin for polyethylene was further confirmed by adsorption isotherm and direct competition experiments. The data from all four experimental methods support the following ranking of plasma protein affinity for polyethylene: Hemoglobin greater than fibrinogen greater than albumin congruent to gamma-globulin.     

Competitive adsorption of human fibrinogen on an amorphic quartz surface

The kinetics of adsorption of fibrinogen on the surface of amorphous quartz from binary solutions containing human serum albumin and gamma-globulin was studied by the method of fluorescence of total internal reflection. A model of energetically nonhomogeneous interactions in the surface/protein system was used to explain the mechanism of competitive adsorption of proteins.     

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.     

Protein Adsorption on Erythrocytic Membranes and Its Effect on Erythrocyte Rheology in Athletes during Competition Exercise

The adsorption of high-molecular-weight plasma proteins on erythrocyte membranes was studied in athletes after prolonged exercise under competition conditions. The adsorption of individual high-molecular-weight protein fractions depended on their concentration. The adsorption index changed biphasically at submaximum exercise. The adsorption of plasma high-molecular-weight protein fractions was associated with the fluidity of concentrated erythrocyte suspensions. The adsorbed high-molecular-weight globulins and fibrinogen had different effects on the parameters of erythrocyte rheology.     

Interaction of fibrinogen with magnetite nanoparticles

The interaction between fibrinogen and magnetite nanoparticles in solution has been studied by the methods of spin labeling, ferromagnetic resonance, dynamic and Rayleigh light scattering. It is shown that protein molecules adsorb on the surface of nanoparticles to form multilayer protein covers. The number of molecules adsorbed on one nanoparticle amounts to ∼65 and the thickness of the adsorption layer amounts to ∼27 nm. Separate nanoparticles with fibrinogen covers (clusters) form aggregates due to interactions of the end D domains of fibrinogen. Under the influence of direct magnetic field, nanoparticles with adsorbed proteins form linear aggregates parallel to the force lines. It is shown that the rate of protein coagulation during the formation of fibrin gel under the action of thrombin on fibrinogen decreases ∼2 times in the presence of magnetite nanoparticles, and the magnitude of the average fiber mass/length ratio grows.     

PM-IRRAS investigation of the interaction of serum albumin and fibrinogen with a biomedical-grade stainless steel 316LVM surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the interaction of bovine serum albumin (BSA) and fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics and adsorption-induced secondary structure changes of the proteins. Highly negative apparent Gibbs energy of adsorption values revealed a spontaneous adsorption of both proteins onto the surface, accompanied by significant changes in their secondary structure. It was determined that, at saturated surface coverages, lateral interactions between the adsorbed BSA molecules induced rather extensive secondary structure changes. Fibrinogen's two coiled coils appeared to undergo negligible secondary structure changes upon adsorption of the protein, while large structural rearrangements of the protein's globular domains occurred upon adsorption. The secondary structure of adsorbed fibrinogen was not influenced by lateral interactions between the adsorbed fibrinogen molecules. PM-IRRAS was deemed to be viable for investigating protein adsorption and for obtaining information on adsorption-induced changes in their secondary structures.     

Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

The solubility of fibrinogen in dilute salt solutions

Highly purified human fibrinogen was dialyzed versus eleven different sodium salts at ionic strengths of 0.005–0.3 and pH values of 4.5–8.0. After equilibration and centrifugation of the protein solutions, fibrinogen solubilities were determined spectrophotometrically and were analyzed as functions of pH, ionic strength, and specific anion. Bell-shaped curves are obtained when fibrinogen solubility is plotted as a function of pH. The solubility exhibits a minimum at a given pH and rises at acid and alkaline values. As the ionic strength is increased, the solubility curves shift toward more acid pH values. At constant pH values between 6 and 7, fibrinogen solubility increases with an increase in ionic strength. At constant pH values below pH 6, a decrease in solubility occurs as the ionic strength is increased. The isoionic pH of a saturated aqueous fibrinogen solution has been determined to be 6.25, meaning that fibrinogen in pure water behaves as a weak acid with a mean net charge of ?0.9. At pH values acid to 6.25, the anions solubilize fibrinogen in the following order of increasing efficacy: thiocyanate, perchlorate, sulfate, citrate, bromide, nitrate, phosphate, chloride, acetate, fluoride, and formate. This order is reversed at pH values alkaline to 6.25. Anion binding parameters calculated from the solubility data indicate that those anions which most effectively solubilize fibrinogen at alkaline pH and precipitate it at acid pH have the highest apparent binding affinities for the protein. Anions with less pronounced solubility effects have lower binding affinities.     

New hydrophobic charge-induction resin with 2-mercaptoimidazole as the ligand and its separation characteristics for porcine IgG

New hydrophobic charge-induction chromatography (HCIC) resin coupled with 2-mercaptoimidazole (MI) as the ligand was prepared and used to purify IgG from porcine plasma. The cellulose matrix was activated by divinylsulfone (DVS), and was then coupled with MI as the functional ligand. The reaction conditions were optimized as pH: 11, volume ratio of DVS to matrix: 1.0, reaction time: 4 h. The ligand density reached about 100 μmol/mL gel. The adsorption isotherms of porcine IgG was determined at different pH values, and high saturated adsorption capacities of 78.02 mg IgG/mL gel were found at pH 8. The adsorption of IgG showed a typical pHdependent property of HCIC, and the adsorption capacity decreased significantly in acidic conditions. The prepared resin was used to separate IgG from porcine plasma. After precipitating the fibrinogen by salting-out, the supernatant was loaded onto the column at pH 7 and the elution pH was optimized. The results indicated that acidic elution pH was necessary to recover the IgG. The purity of IgG in the elution fractions was in the range of 81 ～ 90%, which demonstrated that HCIC with the new ligand showed the excited separation performs and is a potential effective technique to purify IgG from the complex feedstock.     

Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G

Protein A immobilized polyhydroxylmethyacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma.     

Generation of functional coatings on hydrophobic surfaces through deposition of denatured proteins followed by grafting from polymerization

Hydrophilic coatings were produced on flat hydrophobic substrates featuring n-octadecyltrichlorosilane (ODTS) and synthetic polypropylene (PP) nonwoven surfaces through the adsorption of denatured proteins. Specifically, physisorption from aqueous solutions of α-lactalbumin, lysozyme, fibrinogen, and two soy globulin proteins (glycinin and β-conglycinin) after chemical (urea) and thermal denaturation endowed the hydrophobic surfaces with amino and hydroxyl functionalities, yielding enhanced wettability. Proteins adsorbed strongly onto ODTS and PP through nonspecific interactions. The thickness of adsorbed heat-denatured proteins was adjusted by varying the pH, protein concentration in solution, and adsorption time. In addition, the stability of the immobilized protein layer was improved significantly after interfacial cross-linking with glutaraldehyde in the presence of sodium borohydride. The amino and hydroxyl groups present on the protein-modified surfaces served as reactive sites for the attachment of polymerization initiators from which polymer brushes were grown by surface-initiated atom-transfer radical polymerization of 2-hydroxyethyl methacrylate. Protein denaturation and adsorption as well as the grafting of polymeric brushes were characterized by circular dichroism, ellipsometry, contact angle, and Fourier transform infrared spectroscopy in the attenuated total reflection mode.     

Effect of Acetylation of Ovalbumin on Its Adsorption Behavior at Solid/Liquid Interface

This paper reports the effect of modification of lysine residues on the adsorption of ovalbumin at alumina/water interface. It has been shown that the pH dependence of the adsorption changes on acetylation of lysine. Thus at pH 7.6 acetylated ovalbumin does not show any affinity for alumina surface although unmodified protein does. It seems that although electrostatic interactions are operative, surface unfolding of proteins and surface hydrophobicity of protein also control the adsorption of ovalbumin onto alumina.     

Adsorption of Bovine Serum Albumin from salt solution onto activated carbon

One of the wastewaters from tanning industry (soak liquor) contains 0.4 g/l of dissolved protein. During coagulation and flocculation 41 % of protein was removed. A suggestion has been made to remove the residual protein by adsorption technique. The optimum conditions for adsorption of Bovine Serum Albumin (BSA) on rice bran based activated carbon (RBAC) have been determined. Maximum adsorption of BSA took place at pH 7.O. Ionic strength was found to have influence on the adsorption behaviour. Adsorption capacity of BSA onto charcoal surface decreased with increase in temperature. Enthalpy of adsorption in all cases was found to be within –19 to –57 kJ/mole, indicating exothermic nature of the process. Applicability of adsorption technique to the removal of dissolved protein from soak water has been studied. The maximum removal of protein occurred at pH 7.0 and the ratio of protein removed to weight of adsorbent was 3.22×10^–3 g/g.     

Surface modification with poly(sulfobetaine methacrylate-co-acrylic acid) to reduce fibrinogen adsorption, platelet adhesion, and plasma coagulation

Zwitterionic sulfobetaine methacrylate (SBMA) polymers were known to possess excellent antifouling properties due to high hydration capacity and neutral charge surface. In this study, copolymers of SBMA and acrylic acid (AA) with a variety of compositions were synthesized and were immobilized onto polymeric substrates with layer-by-layer polyelectrolyte films via electrostatic interaction. The amounts of platelet adhesion and fibrinogen adsorption were determined to evaluate hemocompatibility of poly(SBMA-co-AA)-modified substrates. Among various deposition conditions by modulating SBMA ratio in the copolymers and pH of the deposition solution, poly(SBMA(56)-co-AA(44)) deposited at pH 3.0 possessed the best hemocompatibility. This work demonstrated that poly(SBMA-co-AA) copolymers adsorbed on polyelectrolyte-base films via electrostatic interaction improve hemocompatibility effectively and are applicable for various substrates including TCPS, PU, and PDMS. Furthermore, poly(SBMA-co-AA)-coated substrate possesses great durability under rigorous conditions. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices.     

Kinetic studies of protein-surface interactions: A two-stage model of surface-induced protein transitions in adsorbed biofilms

The irreversible adsorption of proteins on artificial surfaces plays an important role in a wide variety of practical problems. The simple analytical models based on definite concepts regarding the mechanisms of interfacial evolution can be used efficiently for characterization of protein-surface interactions by analyzing the intrinsic kinetics of the process. In this article, analytical expressions are derived for the adsorption kinetics that take into account the presence of more than one adsorbed state for proteins in biofilms. It is shown that the experimentally observed dependence of the adsorbed mass on the concentration of protein in solution can be reproduced with this model, and the approach provides a rapid method for obtaining quantitative parameters for the adsorption process. It is shown by analytical approximation of the kinetic curves for fibrinogen adsorption onto an unmodified gold surface studied by a surface plasmon resonance biosensor that this model is in good quantitative agreement with experiments. It is found that the rate of adsorption, controlled mainly by the mass flow from the solution, determines the contribution both to self-assembling and spreading, resulting in variations of adsorbed fibrinogen interfacial structures.     

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.     

Amino acid composition of human fibrinogen and anticoagulant derivatives

1. The amino acid compositions of human fibrinogen and three intermediate anticoagulant derivatives were determined by column chromatography. The derivatives were isolated by ammonium sulphate fractionation and column electrophoresis from solutions of fibrinogen undergoing spontaneous breakdown. One derivative, isolated as the large electrophoretic peak at the end of the clottable period (100% CP) of the parent fibrinogen solution, was labelled LP(100) and others obtained at twice this period (200% CP) were designated as LP(200) and SP(200) (LP, large peak; SP, small peak). 2. Maximal ;molecular' weights of approx. 294000 for LP(100), 137000 for LP(200) and 37000 for SP(200) were calculated for the protein moieties. At least 265 amino acid residues must have been lost from each fibrinogen molecule during the formation of LP(100), and 1362 during the formation of the other two derivatives. 3. Only one derivative (LP(200)) had a partial specific volume ([unk] 0.725ml./g.) different from that of fibrinogen ([unk] 0.721ml./g.). 4. No significant differences in refractive index at 589mmu were detected. 5. Calculation of the total number of ionizable groups/10(5)g. of each protein moiety showed a preponderance of the following numbers of negative charges: 22 in fibrinogen; 24 in LP(100); 26 in LP(200); 49 in SP(200). The isoionic points were estimated to be approx.+0.03pH unit (for fibrinogen), -0.06pH unit for (LP(100)) and +0.28pH unit (for LP(200)) from the pK of imidazole, and 0.78pH unit above the average pK of aspartyl and glutamyl ions (for SP(200)). These figures agree closely with experimentally determined values of the isoelectric point of fibrinogen and its derivatives.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.