Biocompatibility of electrospun human albumin: a pilot study.

Albumin is rarely used for electrospinning because it does not form fibres in its native globular form. This paper presents a novel method for electrospinning human albumin from a solution containing pharmaceutical grade protein and 25% polyethylene oxide (PEO) used as the fibre-forming agent. After spontaneous cross-linking at body temperature, with no further chemicals added, the fibres become insoluble and the excess PEO can be washed out. Albumin deposited along the fibres retains its native characteristics, such as its non-adhesiveness to cells and its susceptibility for degradation by macrophages. To demonstrate this we evaluated the mechanical properties, biocompatibility and biodegradability of this novel product. After subcutaneous implantation in mice, albumin mats were completely resorbable within six days and elicited only a limited local inflammatory response. In vitro, the mats suppressed cell attachment and migration. As this product is inexpensive, produced from human pharmaceutical grade albumin without chemical modifications, retains its native protein properties and fulfils the specific requirements for anti-adhesive dressings, its clinical use can be expedited. We believe that it could specifically be used when treating paediatric patients with epidermolysis bullosa, in whom non-healing wounds occur after minor hand injuries which lead to rapid adhesions and devastating contractures.

Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines.

Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation.

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

Controversies in nephrology: renal albumin handling, facts, and artifacts!

In this article, we discuss and contradict a recent publication by Russo et al., which suggests that the filtration of large amounts of albumin followed by transtubular transport of intact albumin is a physiological phenomenon.

Controversies in nephrology: response to 'renal albumin handling, facts, and artifacts'.

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.

Interactions of adsorbed albumin with underpotentially deposited copper on gold piezoelectrodes.

The adsorption of a model protein, bovine serum albumin (BSA), on Au electrodes was investigated using the Cu adatom probe method and Electrochemical Quartz Crystal Nanobalance (EQCN) technique. The adsorption of BSA was confirmed by AFM imaging and has been found to be controlled by kinetics. Using the Cu adatom probe method, we were able to reconstruct the entire BSA adsorption transient Theta(BSA) vs. t. The adsorption rate constant k(1), determined from this transient is k(1)=2.45x10(5) L mol(-1) s(-1). We have found that the bulk Cu(0) deposition process is blocked by BSA adsorption and it decays exponentially with time during BSA adsorption. It ceases completely when a full monolayer of BSA is formed. In contrast to that, the mass associated with Cu-u.p.d. decreases only to ca. 50% of that in the absence of BSA, indicating that Cu adatoms can penetrate (wedge) into the space between the surface Au atoms and the adsorbed BSA molecules. In addition to that, we have found that the degree of penetration of Cu adatoms can be controlled by the applied deposition potential. By selecting a sufficiently cathodic potential, we were able to deposit a full Cu-u.p.d. monolayer, independent of the BSA surface coverage extending from Theta(BSA)=0 to Theta(BSA) approximately 1. The positive shift of Cu(ad) desorption peak potential E(p), observed in the presence of adsorbed BSA, has been interpreted in terms of Frumkin exchange interaction forces between Cu(ad) and BSA(ad), on the basis of our earlier theoretical model, expanded here to include adsorbed species in two monolayers. This expansion is possible owing to the fast rate of Cu adatom penetration in the interfacial region. From the plots of E(p) vs. Theta(BSA), the presence of strong attractive interactions between Cu(ad) and BSA(ad) was deduced. These interactions result in a super-shift of the Cu-u.p.d. desorption peak potential, corresponding to the exchange interaction coefficient g(M,X)<-4, indicating on a possibility of the formation of a stable interface complex.

Enhancing efficacy of HIV gag DNA vaccine by local delivery of GM-CSF in murine and macaque models.

Controlled release of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific cytotoxic T lymphocyte (CTL) and T helper (Th) responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific antibody (Ab), Th, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and Th, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part III. Hydrogels as carriers for immobilization of proteins.

Four series of macroporous hydrogels based on crosslinked copolymers of 2-hydroxyethyl methacrylate (HEMA)-sodium methacrylate (MANa), copolymer HEMA-[2-(methacryloyloxy)ethyl]trimethylammonium chloride (MOETACl), terpolymer HEMA-MANa-MOETACl and on a polyelectrolyte complex were used as carriers for immobilization of proteins, chicken egg white albumin and avidin. The adsorption capacity of the hydrogels for the two proteins, kinetics and pH dependence of albumin adsorption and desorption were studied. The morphology of the hydrogels with and without immobilized albumin was studied by low-vacuum scanning electron microscopy.

Selective protein adsorption on micro-textured P-type and N-type silicon wafers.

There has recently been a great deal of effort put towards the development of bioMEMS-based electrochemical biosensors for use in implantable devices. Currently, the primary issue limiting the lifespan of implantable sensors is protein and cell adhesion (biofouling) to the sensor surface, which impedes the sensor's access to analyte. To better understand this problem, it would be useful to have an understanding of how silicon-based microdevices interact with proteins in a physiological environment. To help answer this question, we investigated the interactions of proteins with microtextured silicon wafers. Bulk micromachining techniques were used to create micro-textures that varied between 5 and 80 microns in size nd spacing. We used n-type and p-type silicon wafers with a <100> crystal orientation. Shapes such as rectangles, circles, and triangles were fabricated that were recessed into the silicon substrate. The features were estimated to be between 3 and 8 microns in depth. After the features were created, the wafers were coated with a layer of silicon dioxide. Once fabrication was complete, the wafers were incubated in vitro ith fluorescently tagged Albumin (500 microg/ml in Phosphate-Buffered Saline, PBS) for 5 minutes. The wafers were then rinsed with PBS solution and viewed using an epifluorescence microscope. Albumin adsorbed selectively onto the micropatterned wafers. Depending on the type of wafer we found that albumin adsorbed selectively onto either the bulk surface, the sidewalls, or the bottom of the etched feature.

Durability of the binding inhibition of albumin coating on tympanostomy tubes.

OBJECTIVE: Occlusion and prolonged otorrhea are typical problems associated with the use of middle-ear ventilation tubes. Albumin coating of ventilation tubes has been introduced to prevent tube occlusions by granulation tissue, blood clot, or pus. In this study, the durability of the binding inhibition (BI) of fibronectin was examined on the tube surface in albumin-coated tubes in different environments during an 8-month trial. METHODS: Human serum albumin (HSA) was used to coat silicone tympanostomy tubes. Fibronectin, a typical adhesive protein in serum and exudates, was used as a model representative of exudates of the ear. The durability of BI of this glue protein on the tube surface was tested in different time periods with radiolabelled fibronectin. Scanning electron microscopy (SEM) was performed on the tubes. RESULTS: The BI of fibronectin, achieved with the albumin coating, was still strong after 8 months of storage at +4 degrees C. A slight decline in BI was noted between the first and third months of storage at +37 degrees C. A significant difference between HSA-coated and uncoated tympanostomy tubes was noted in SEM. The uncoated surface generally appeared to be rougher than that of HSA-coated tubes when either titanium or silicone tubes were tested. CONCLUSIONS: Albumin coating markedly inhibits the binding of fibronectin on tube surfaces in vitro. A clear BI achieved by albumin coating on tube surfaces was shown to persist throughout an 8-month trial, although some reduction of the BI was seen over time. The result emphasizes the role of albumin coating in preventing the adherence of foreign material on tympanostomy tubes. No advantage was achieved by using a cross-linking chemical in the albumin coating.

Cell type-specific modulation of fibronectin adhesion functions on chemically-derivatized self-assembled monolayers.

Cell type-specific responses (microfilament stress fibers for fibroblasts or neurites for neuroblastoma cells) were evaluated in culture on inert and chemically-derivatized silane substrata adsorbed with fibronectin (Fn). Substrata of self-assembled monolayers contain a 14-17 carbon aliphatic chain terminating with different chemical endgroups -- [CH3], [C=C], [Br], [CN], [Diol], [COOH], [NH2], [SH], [SCOCH3], or [SO3H]. Fn adsorbed effectively to all derivatized surfaces. 3T3 fibroblasts or neuroblastoma cells attached equivalently to all surfaces preadsorbed with Fn, indicating availability of receptor binding sites on Fns. However, transmembrane signaling from Fn(adsorbed): receptor(cell) surface complexes yielded a range of abilities for generating F-actin stress fibers in fibroblasts or neurites in neuroblastoma cells. Efficiency for stress fiber formation was very different from that of neurite extension. The same chemical endgroups on glass, titanium, or germanium yielded the same patterns of cellular physiological responses, indicating that inert substrata do not act at a distance and that only chemical endgroups regulate Fn signaling functions. When adhesion-inert albumin is co-adsorbed with Fn, efficiency of neurite extension is improved on some surfaces or diminished on others. These results indicate that the conformation of Fn(adsorbed) changes in specific ways on derivatized substrata. Change in Fn conformation was confirmed by FTIR/ATR spectroscopy experiments of Fn(adsorbed). Overall, these studies indicate changes in Fn conformations on chemically-derivatized self-assembled monolayers leading to up- or down-regulation of cell type-specific physiological responses from receptors via their signaling pathways. They also offer predictability for regulating responses of specific cell types when these cells interact with biomaterial implants in vivo.

Efecto protector de la albúmina sobre las membranas del espermatozoide humano en la capacitación espermática in vitro / Albumin protective effect on human spermatozoa membranes during in vitro sperm capacitation

Se estudió el efecto de la preparación in vitro de semen sobre la estructura y sobrevida del espermatozoide humano, con el objeto de valuar si la albúmina protege las membranas durante la centrifugación. Se analizaron de manera cegada y aleatoria 12 muestras seminales de hombres fértiles a las cuales se les trató por tres métodos diferentes (swim-up, gradientes de albúmina, gradientes de percoll), para separar la fracción móvil de espermatozoides. Las membranas plasmática y acrosomal externa y la sustancia acrosomal de los espermatozoides fueron observadas al microscopio electrónico de transmisión en 50 espermatozoides por muestra y la viabilidad y movilidad progresiva se analizó después de incubación durante 24 horas a 37ºC. Se presenta evidencia que sugiere que la albúmina estabiliza la membrana acrosomal externa del espermatozoide humano e incrementa la viabilidad de esta célula

An adenosine agonist and prostaglandin E1 cause breakdown of the blood-retinal barrier by opening tight junctions between vascular endothelial cells.

Macular edema occurs in several disease processes, but little is known about the mechanisms by which it occurs in any disease process. Previously, the authors showed that intravitreous injection of adenosine agonists, prostaglandin E1 (PGE1), or epinephrine in rabbits, causes breakdown of the blood-retinal barrier (BRB) measured by vitreous fluorophotometry. N-ethylcarboxamidoadenosine (NECA), a nonspecific adenosine agonist, and PGE1, cause much greater breakdown of the BRB than the other agents tested. In this study, rabbit eyes were examined ultrastructurally and electron immunocytochemically for extravascular albumin as an indicator of BRB failure after intravitreous injection of these agents or vehicle alone to investigate potential mechanisms involved in BRB compromise. Six hours after injection, there were significantly more open tight junctions between retinal vascular endothelial cells in NECA-, PGE1-, and adenosine-injected eyes than in vehicle-injected eyes. Immunocytochemical staining for serum albumin showed that many of the junctions that appeared open were functionally open. Forty-eight hours after injection of PGE1 (10(-4) mol/l), the percentage of open vascular endothelial cell tight junctions had returned to that of the control specimens, but the opening of tight junctions by NECA (10(-3) mol/l) did not appear to be reversed after 48 hr. Pinocytotic vesicular transport was prominent in all eyes, and no difference was found between vehicle- and drug-injected eyes. These data suggest that NECA and PGE1 cause breakdown of the BRB, at least in part, by opening tight junctions between retinal vascular endothelial cells.

Self-complementary regions in human albumin mRNA encode important structural regions within the human albumin protein.

An analysis of the human albumin mRNA structure revealed a nonrandom distribution of self-complementary regions within the mRNA. The majority of these self-complementary mRNA stretches encode important structural regions of the human albumin protein. The amino acids contained within these regions of the protein exhibit a high degree of hydrophobic complementarity which could influence local protein conformation and contribute to the biological importance of the protein structures.

The spatial resolution of protein adsorption on surfaces of heterogeneous metallic biomaterials.

This study was designed to examine the heterogeneity of the adsorption of proteins onto metallic materials. The materials studied included pure Ag, Au, and Ti and sintered Ag 10% Ti and Ag 10% Ta. The distribution of the protein adsorption was studied using I-125 labeled albumin detected by microautoradiography. The surface morphology of the specimens was examined in the scanning electron microscope prior to exposure to the protein solution. A heterogeneous distribution in albumin adsorption was observed over the Ag surface. Similar regions were observed over parts of the mixed metal specimens, but superimposed on this pattern were distinct regions of very low protein adsorption which appeared to correlate closely with the regions of Ti or Ta observed in the scanning electron microscope. A uniform distribution of adsorbed albumin was observed on the Au and Ti, with Au giving a much denser microautoradiograph than Ti. This work demonstrates that variations in the protein adsorption to heterogeneous materials can be observed on a microscopic scale.