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1.  Copper Selenide Nanosnakes: Bovine Serum Albumin-Assisted Room Temperature Controllable Synthesis and Characterization 
Nanoscale Research Letters  2010;5(6):949-956.
Herein we firstly reported a simple, environment-friendly, controllable synthetic method of CuSe nanosnakes at room temperature using copper salts and sodium selenosulfate as the reactants, and bovine serum albumin (BSA) as foaming agent. As the amounts of selenide ions (Se2−) released from Na2SeSO3 in the solution increased, the cubic and snake-like CuSe nanostructures were formed gradually, the cubic nanostructures were captured by the CuSe nanosnakes, the CuSe nanosnakes grew wider and longer as the reaction time increased. Finally, the cubic CuSe nanostructures were completely replaced by BSA–CuSe nanosnakes. The prepared BSA–CuSe nanosnakes exhibited enhanced biocompatibility than the CuSe nanocrystals, which highly suggest that as-prepared BSA–CuSe nanosnakes have great potentials in applications such as biomedical engineering.
PMCID: PMC2894339  PMID: 20672034
Copper selenide; Nanosnakes; Bovine serum albumin; Synthesis; Characterization; Mechanism; Biocompatibility
2.  Prepare dispersed CIS nano-scale particles and spray coating CIS absorber layers using nano-scale precursors 
In this study, the Mo-electrode thin films were deposited by a two-stepped process, and the high-purity copper indium selenide-based powder (CuInSe2, CIS) was fabricated by hydrothermal process by Nanowin Technology Co. Ltd. From the X-ray pattern of the CIS precursor, the mainly crystalline phase was CIS, and the almost undetectable CuSe phase was observed. Because the CIS powder was aggregated into micro-scale particles and the average particle sizes were approximately 3 to 8 μm, the CIS power was ground into nano-scale particles, then the 6 wt.% CIS particles were dispersed into isopropyl alcohol to get the solution for spray coating method. Then, 0.1 ml CIS solution was sprayed on the 20 mm × 10 mm Mo/glass substrates, and the heat treatment for the nano-scale CIS solution under various parameters was carried out in a selenization furnace. The annealing temperature was set at 550°C, and the annealing time was changed from 5 to 30 min, without extra Se content was added in the furnace. The influences of annealing time on the densification, crystallization, resistivity (ρ), hall mobility (μ), and carrier concentration of the CIS absorber layers were well investigated in this study.
PMCID: PMC3895740  PMID: 24380376
Nano-scale particle; Spray coating method; CIS absorber layer; Annealing
3.  Evidences For Charge Transfer-Induced Conformational Changes In Carbon Nanostructure-Protein Corona 
The binding of proteins to a nanostructure often alters protein secondary and tertiary structures. However, the main physical mechanisms that elicit protein conformational changes in the presence of the nanostructure have not yet been fully established. Here we performed a comprehensive spectroscopic study to probe the interactions between bovine serum albumin (BSA) and carbon-based nanostructures of graphene and single-walled carbon nanotubes (SWNTs). Our results showed that the BSA “corona” acted as a weak acceptor to facilitate charge transfer from the carbon nanostructures. Notably, we observed that charge transfer occurred only in the case of SWNTs but not in graphene, resulting from the sharp and discrete electronic density of states of the former. Furthermore, the relaxation of external α–helices in BSA secondary structure increased concomitantly with the charge transfer. These results may help guide controlled nanostructure-biomolecular interactions and prove beneficial for developing novel drug delivery systems, biomedical devices and engineering of safe nanomaterials.
PMCID: PMC3519440  PMID: 23243478
Protein corona; charge transfer; graphene; carbon nanotubes; Raman spectroscopy; FTIR spectroscopy
4.  Thallium(I) copper(I) thorium(IV) tris­elenide, TlCuThSe3  
Thallium(I) copper(I) thorium(IV) tris­elenide, TlCuThSe3, crystallizes with four formula units in the space group Cmcm in the KCuZrS3 structure type. There is one crystallographic­ally independent Th, Tl, and Cu atom at a site of symmetry 2/m.., m2m, and m2m, respectively. There are two crystallographically independent Se atoms at sites of symmetry m.. and m2m. The structure consists of sheets of edge-sharing ThSe6 octa­hedra and CuSe4 tetra­hedra stacked parallel to the (010) face, separated by layers filled with chains of Tl running parallel to [100]. Each Tl is coordinated by a trigonal prism of Se atoms.
PMCID: PMC3393138  PMID: 22807695
5.  Enzyme-Linked Immunosorbent Assay (ELISA) and Blocking with Bovine Serum Albumin (BSA) - Not all BSAs are alike 
Journal of immunological methods  2012;384(1-2):148-151.
The enzyme-linked immunosorbent assays (ELISA) is an extremely common and powerful laboratory technique for detecting proteins by antibodies. Researchers frequently use bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding of antigens and antibodies to the microtiter well. While studying the interactions of the vaccinia virus complement control protein (VCP) with complement, we found nonspecific binding of VCP to BSA and identify a BSA preparation that did not result in non-specific binding. This work draws attention to the fact that not all BSA preparations are alike. It also highlights the need to perform critical controls to ensure that ELISA reactants do not inappropriately bind to the blocking agent.
PMCID: PMC3432671  PMID: 22732194
Enzyme-Linked Immunosorbent Assay/methods*; False Positive Reactions; Serum Albumin, Bovine/immunology*; Protein Binding; Vaccinia virus complement control protein
6.  Electrically Polarized Biphasic Calcium Phosphates: Adsorption and Release of Bovine Serum Albumin 
In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled, as well as unpoled surfaces of the composites, was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as drug or growth factor delivery vehicle.
PMCID: PMC2979312  PMID: 20939493
7.  Psychosocial interventions and medication adherence in bipolar disorder 
Recent research has indicated that psychosocial interventions can have a valuable role in reducing the substantial psychosocial disability associated with bipolar disorder. Randomized controlled trials of these interventions indicate that improvements are seen in symptoms, psychosocial functioning, and treatment adherence. These interventions systematically presented in the form of standardized treatment manuals, vary in format, duration, and theoretical basis. All are meant to augment pharmacotherapy, which represents the standard of treatment in the field. Modalities that have gathered the most empirical support include cognitive-behavioral therapy, familyfo-cused therapy, interpersonal and social rhythms therapy, and psychoeducation. The enhancement of adherence to pharmacotherapy is a common therapeutic target, due to the association of nonadherence with higher relapse rates, hospitalization, and health care costs among people with bipolar disorder. Given the complexity of nonadherence behavior, multicomponent interventions are often required. In this review, we provide an overview of the rationale, evidence base, and major psychotherapeutic approaches in bipolar disorder, focusing on the assessment and enhancement of medication adherence.
PMCID: PMC3181867  PMID: 18689293
bipolar disorder; psychotherapy; psychoeducation; medication adherence; medication compliance
8.  Enhanced Fluorescence of Cy5-Labeled Oligonucleotides Near Silver Island Films: A Distance Effect Study Using Single Molecule Spectroscopy 
The journal of physical chemistry. B  2006;110(45):22557-22562.
We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.
PMCID: PMC2737407  PMID: 17092001
9.  Double-perovskite magnetic La2NiMnO6 nanoparticles for adsorption of bovine serum albumin applications 
Nanoscale Research Letters  2013;8(1):207.
Double-perovskite La2NiMnO6 (LNMO) nanoparticles were synthesized by co-precipitation process, and the adsorption of bovine serum albumin (BSA) protein on these nanoparticles was carried out. The powder samples were annealed at 750, 850, 950, and 1,050°C, respectively. X-ray diffraction (XRD) results reveal that there are double perovskites and exhibit mixed orientations, without any impurity phases. Transmission electron microscopy results as well as the XRD estimate results show that the crystalline size is about 34 to 40 nm. The adsorption of BSA on the magnetic nanoparticles was analyzed using a UV spectrophotometer at room temperature. The results show that the as-prepared LNMO nanoparticles display a good adsorbing ability for BSA, and the nanoparticle sintered at 850°C has the highest value of 219.6 mg/g, which is much higher than others.
PMCID: PMC3655024  PMID: 23639007
Chemical co-precipitation; LNMO; Magnetic property; Nanoparticles; Adsorption; Bovine serum albumin protein
10.  Effect of deposition temperature on the structural and optical properties of chemically prepared nanocrystalline lead selenide thin films 
Nanocrystalline lead selenide (PbSe) thin films were prepared on glass substrates by a chemical bath deposition method, using sodium selenosulfate (Na2SeSO3) as a source of Se2− ions, and lead acetate as a source of Pb2+ ions. Trisodium citrate (TSC) was used as a complexing agent. PbSe films were prepared at various deposition temperatures while the pH value was kept fixed at 11, and the effect on the resulting film properties was studied by X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM) and optical absorption studies. The structural parameters, such as the lattice constant (a), crystallite size (D), dislocation density (ρ) and microstrain (ε) were evaluated from the XRD spectra. It was found that average crystallite size, as calculated from Scherrer’s formula, increased from 23 to 33 nm as the deposition temperature was varied from 303 to 343 K. The dislocation density and microstrain were found to vary inversely with the crystallite size, whereas the lattice constant was found to increase with an increase in crystallite size. The optical absorption spectra of the nanocrystalline PbSe films showed a blue shift, and the optical band gap (E g ) was found to increase from 1.96 to 2.10 eV with the decrease in crystallite size.
PMCID: PMC3388368  PMID: 23016148
chemical bath deposition; lattice parameter; lead selenide; Nelson–Riley plot; optical absorption
11.  Dynamic Adsorption of Albumin on Nanostructured TiO2Thin Films 
Spectroscopic ellipsometry was used to characterize the optical properties of thin (<5 nm) films of nanostructured titanium dioxide (TiO2). These films were then used to investigate the dynamic adsorption of bovine serum albumin (BSA, a model protein), as a function of protein concentration, pH, and ionic strength. Experimental results were analyzed by an optical model and revealed that hydrophobic interactions were the main driving force behind the adsorption process, resulting in up to 3.5 mg/m2 of albumin adsorbed to nanostructured TiO2. The measured thickness of the adsorbed BSA layer (less than 4 nm) supports the possibility that spreading of the protein molecules on the material surface occurred. Conformational changes of adsorbed proteins are important because they may subsequently lead to either accessibility or inaccessibility of bioactive sites which are ligands for cell interaction and function relevant to physiology and pathology.
PMCID: PMC3065784  PMID: 21461339
Nanomaterials; protein adsorption; spectroscopic ellipsometry; titanium dioxide; bovine serum albumin
12.  Polycaprolactone coated porous tricalcium phosphate scaffolds for controlled release of protein for tissue engineering 
Polycaprolactone (PCL) was coated on porous tricalcium phosphate (TCP) scaffolds to achieve controlled protein delivery. Porous TCP scaffolds were fabricated using reticulated polyurethane foam as sacrificial scaffold with a porosity of 70–90 vol %. PCL was coated on sintered porous TCP scaffolds by dipping-drying process. The compressive strength of TCP scaffolds increased significantly after PCL coating. The highest strength of 2.41 MPa at a porosity of 70% was obtained for the TCP scaffold coated with 5% PCL solution. Model protein bovine serum albumin (BSA) was encapsulated efficiently within the PCL coating. The amount of BSA encapsulation was controlled by varying proteins’ composition in the PCL coating. The FTIR analysis confirmed that BSA retained its structural conformation and did not show significant denaturization during PCL coating. The release kinetics in phosphate buffer solution indicated that the protein release was controlled and sustained, and primarily dependant on protein concentration encapsulated in the PCL coating.
PMCID: PMC3058418  PMID: 19572301
porous tricalcium phosphate; polycaprolactone; coating; protein delivery
13.  Interaction of Avelox with Bovine Serum Albumin and Effect of the Coexistent Drugs on the Reaction 
The interaction between Avelox and bovine serum albumin (BSA) was investigated at different temperatures by fluorescence spectroscopy. Results showed that Avelox could quench the intrinsic fluorescence of BSA strongly, and the quenching mechanism was a static quenching process with Förester spectroscopy energy transfer. The electrostatic force played an important role on the conjugation reaction between BSA and Avelox. The order of magnitude of binding constants (Ka) was 104, and the number of binding site (n) in the binary system was approximately equal to 1. The binding distance (r) was less than 3 nm and the primary binding site for Avelox was located in subdomain IIA of BSA. Synchronous fluorescence spectra clearly revealed that the microenvironment of amino acid residues and the conformation of BSA were changed during the binding reaction. In addition, the effect of some antibiotics on the binding constant of Avelox with BSA was also studied.
PMCID: PMC3296219  PMID: 22505918
14.  Hollow hydroxyapatite microspheres as a device for controlled delivery of proteins 
Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li2O–CaO–B2O3 glass (106–150 μm) in K2HPO4 solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K2HPO4 solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m2/g), and a mesoporous shell wall (pore size ≈13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24–48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1–24 h at 600–900°C). Sustained release of BSA was achieved over 7–14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs.
PMCID: PMC3160344  PMID: 21290170
15.  Investigation of the Interaction of Naringin Palmitate with Bovine Serum Albumin: Spectroscopic Analysis and Molecular Docking 
PLoS ONE  2013;8(3):e59106.
Bovine serum albumin (BSA) contains high affinity binding sites for several endogenous and exogenous compounds and has been used to replace human serum albumin (HSA), as these two compounds share a similar structure. Naringin palmitate is a modified product of naringin that is produced by an acylation reaction with palmitic acid, which is considered to be an effective substance for enhancing naringin lipophilicity. In this study, the interaction of naringin palmitate with BSA was characterised by spectroscopic and molecular docking techniques.
Methodology/Principal Findings
The goal of this study was to investigate the interactions between naringin palmitate and BSA under physiological conditions, and differences in naringin and naringin palmitate affinities for BSA were further compared and analysed. The formation of naringin palmitate-BSA was revealed by fluorescence quenching, and the Stern-Volmer quenching constant (KSV) was found to decrease with increasing temperature, suggesting that a static quenching mechanism was involved. The changes in enthalpy (ΔH) and entropy (ΔS) for the interaction were detected at −4.11±0.18 kJ·mol−1 and −76.59±0.32 J·mol−1·K−1, respectively, which indicated that the naringin palmitate-BSA interaction occurred mainly through van der Waals forces and hydrogen bond formation. The negative free energy change (ΔG) values of naringin palmitate at different temperatures suggested a spontaneous interaction. Circular dichroism studies revealed that the α-helical content of BSA decreased after interacting with naringin palmitate. Displacement studies suggested that naringin palmitate was partially bound to site I (subdomain IIA) of the BSA, which was also substantiated by the molecular docking studies.
In conclusion, naringin palmitate was transported by BSA and was easily removed afterwards. As a consequence, an extension of naringin applications for use in food, cosmetic and medicinal preparations may be clinically and practically significant, especially in the design of new naringin palmitate-inspired drugs.
PMCID: PMC3604151  PMID: 23527100
16.  Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation: The response to glycated and nonglycated BSA is lost in metabolic syndrome 
Glycobiology  2008;18(7):517-525.
The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37°C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.
PMCID: PMC2430009  PMID: 18458031
AGE-BSA; BSA; metabolic syndrome; vascular relaxation
17.  Evaluation of the Validity of the Condom Use Self-Efficacy Scale (CUSES) in Young Men Using Two Behavioral Simulations 
Assessment of behavioral skills remains critical to the evaluation of HIV prevention interventions; however, investigators often rely upon participant reports of self-efficacy to estimate such skills. We evaluated the relationship between self-efficacy beliefs for condom use and behavioral performance. Forty-three men completed the Condom Use Self-Efficacy Scale (CUSES) and participated in two behavioral assessments. Regression analyses indicated that the CUSES subscales relevant to negotiation of condom use did not account for a significant amount of variability in interpersonal skills; similarly, the CUSES subscale relevant to technical condom use skill did not account for variability in the condom application scores. We caution investigators against the assumption that higher self-efficacy reflects behavioral competence for HIV-risk reduction.
PMCID: PMC2440312  PMID: 9269889
Self-efficacy; HIV; AIDS; assessment
18.  Spontaneous in vivo isomerization of bovine serum albumin as a determinant of its normal catabolism. 
Journal of Clinical Investigation  1976;57(2):398-407.
By combination of isoelectric focusing and immunoelectrophoresis of fresh bovine plasma it is shown that 10% of the albumin in plasma has isoionic points equal to the intramolecular SS-interchanged isomers of bovine serum albumin (BSA). It is also shown that (a) albumin with the isoionic point of SS-interchanged BSA is produced in the cow from radioiodinated BSA depleted from SS-interchanged albumin before injection and (b) purified radiolabeled SS-interchanged BSA can be converted in vivo to albumin with the native isoionic point. On this basis, it is proposed that SS-interchanged albumin in vivo is in postsynthetic equilibrium with the "native" albumin conformation. The SS-interchanged isomers purified either from commerical BSA or from BSA submitted to SH-SS interchange was, after radioiodination with 125I, compared metabolically with "native" albumin labeled with 131I in the same calf. Both species of SS-interchanged albumins have fast initial turnover rates but obtain a normal rate of degradation after the reversion to native albumin. If the isomers formed in vivo have the same properties as the ones present in commercial BSA, at least 50% of the physiological degradation of albumin can be accounted for by the 6-7 times faster catabolic rates of these isomers.
PMCID: PMC436664  PMID: 1254725
19.  Polymer microchip capillary electrophoresis of proteins either off- or on-chip labeled with chameleon dye for simplified analysis 
Electrophoresis  2009;30(24):4230-4236.
Microchip capillary electrophoresis of proteins labeled either off- or on-chip with the “chameleon” CE dye 503 using poly(methyl methacrylate) microchips is presented. A simple dynamic coating using the cationic surfactant cetyltrimethyl ammonium bromide prevented nonspecific adsorption of protein and dye to the channel walls. The labeling reactions for both off- and on-chip labeling proceeded at room temperature without requiring heating steps. In off-chip labeling, a 9 ng/mL concentration detection limit for bovine serum albumin (BSA), corresponding to a ~7 fg (100 zmol) mass detection limit, was obtained. In on-chip tagging, the free dye and protein were placed in different reservoirs of the microchip, and an extra incubation step was not needed. A 1 μg/mL concentration detection limit for BSA, corresponding to a ~700 fg (10 amol) mass detection limit, was obtained from this protocol. The earlier elution time of the BSA peak in on-chip labeling resulted from fewer total labels on each protein molecule. Our on-chip labeling method is an important part of automation in miniaturized devices.
PMCID: PMC2805013  PMID: 19924700
20.  Acid Cleavable Surface enhanced Raman Tagging for Protein Detection 
The Analyst  2010;136(3):520-526.
Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.
PMCID: PMC3023828  PMID: 21109888
21.  Effect of Albumin on the Biliary Clearance of Compounds in Sandwich-Cultured Rat Hepatocytes 
The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction (fu), biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Cl’biliary) were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Cl’biliary of these compounds. Surprisingly, BSA-FAF decreased both BEI and intrinsic Cl’biliary for digoxin and pravastatin, which represent low and moderately-bound compounds, respectively. The BEI and intrinsic Cl’biliary of taurocholate, a highly-bound compound, were not altered significantly by BSA-FAF. Neither BSA nor BSA-FAF had a discernable effect on the bile canalicular networks, based on carboxydichlorofluorescein (CDF) retention. The addition of physiological concentrations of calcium, or the addition of fatty acids to BSA-FAF, was unable to restore the BEI or intrinsic Cl’biliary of the model compounds to similar values in the absence or presence of BSA. Careful consideration is warranted when selecting the type of BSA for addition to in vitro systems such as sandwich-cultured rat hepatocytes.
PMCID: PMC2574866  PMID: 18653747
22.  Release and Activity of Anti-TNFα Therapeutics from Injectable Chitosan Preparations for Local Drug Delivery 
Tumor necrosis factor alpha (TNFα) is a cytokine that regulates immune and inflammatory overactivation in various pathological states. Protein therapeutics may antagonize this cytokine, but may also have systemic toxicities. Small molecule natural products are also efficacious, but can suffer from poor oral bioavailability. A drug delivery vehicle is needed to sustain release of active therapeutics and address localized inflammation.
Chitosan is a biocompatible aminopolysaccharide that undergoes thermally-initiated gelation in cosolutions with glycerophosphate (GP), and may entrap and sustain release of additive therapeutics. Gelation time and temperature of chitosan/GP were evaluated by turbidity (OD350), as was the kinetic effect of bovine serum albumin (BSA) entrapment. We investigated in vitro release of BSA and various anti-TNF agents (curcumin, sTNFRII, anti-TNF antibody) and confirmed in vitro activity of the released drugs using an established bioassay.
Turbidity results show that chitosan/GP thermogel achieves gelation at 37°C within 10 minutes, even with significant protein loading. Sustained BSA release occurred with 50% retained at 7 days. All anti-TNF therapeutics exhibited sustained release, with 10% of sTNFRII and anti-TNF antibody remaining after 7 days and 10% of curcumin remaining after 20 days. After release, each compound antagonized TNFα-cytotoxicity in murine fibrosarcoma cells.
This study demonstrates that thermogelling chitosan/GP entraps and sustains release of a broad range of anti-TNF agents. Such delivery of disease-modifying therapy could establish a drug depot to treat local inflammation. The breadth of molecular sizes demonstrates significant versatility, and slow release could protect against toxicities of systemic delivery.
PMCID: PMC2775925  PMID: 19072988
chitosan; drug delivery; drug depot; tumor necrosis factor alpha; inflammation; curcumin
23.  Protection Effect of Endomorphins on Advanced Glycation End Products Induced Injury in Endothelial Cells 
Journal of Diabetes Research  2013;2013:105780.
Endomorphins (EMs) have a very important bridge-function in cardiovascular, endocrinological, and neurological systems. This study is to investigate the effects of EMs on the synthesis and secretion of vasoactive substances induced by advanced glycation end products in primary cultured human umbilical vein endothelial cells (HUVECs). Firstly, HUVECs were stimulated with AGEs-bovine serum albumin (AGEs-BSA), bovine serum albumin (BSA), or both AGEs-BSA and EMs together, respectively. Then, HUVEC survival rate was calculated by MTT assay, the levels of NO, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were detected by colorimetric analysis, and the contents of endothelin-1 (ET-1) were detected by ELISA. The mRNA levels of eNOS and ET-1 were measured by RT-PCR. The expression of p38 mitogen-activated protein kinase (p38 MAPK) was detected by immunofluorescence assay. The results showed that the mRNA expression and secretion of eNOS were significantly enhanced after incubation with EMs compared to those with AGEs-BSA, while the secretion of NO and iNOS, mRNA expression, and secretion of ET-1 had opposite changes. The fluorescence intensity of p38MAPK in nuclear was decreased after pretreatment with EMs compared to incubation with AGEs-BSA. Conclusion. The present study suggests that EMs have certain protection effect on AGEs-BSA-induced injury in HUVEC.
PMCID: PMC3647545  PMID: 23671848
Journal of Clinical Investigation  1983;71(3):487-499.
Intravenous cationic bovine serum albumin (BSA, pI > 9.5) induces membranous nephropathy in immunized rabbits. In this study, unimmunized rabbits received intravenous injections of cationic (n = 3) or native (n = 3) or native (n = 3) BSA, followed by ex vivo isolated left renal perfusions with sheep anti-BSA antibody. Capillary wall deposits of IgG and C3 were seen exclusively in the group receiving cationic BSA, confirming an in situ pathogenesis for cationic, BSA-induced membranous nephropathy, and demonstrating the importance of a cationic antigen for its production. We then explored whether membranous nephropathy in this model is prevented by the concomitant injection of protamine sulfate, a filterable, relatively non-immunogenic polycation. An in vitro study demonstrated that protamine sulfate incubated with glomerular basement membrane (GBM) decreased the subsequent binding of radiolabeled cationic BSA (P < 0.05). In vivo, protamine sulfate was shown to bind to anionic sites in the glomerular capillary wall after intravenous injection.
Groups of rabbits received 3 wk of daily intravenous injections of cationic BSA alone (n = 15) or cationic BSA and protamine (n = 18). After 2 wk of injection of cationic BSA alone, typical membranous nephropathy developed. Granular deposits of IgG and C3 were present along the GBM associated with subepithelial dense deposits, foot process effacement, and marked albuminuria. Protamine significantly reduced or prevented the formation of deposits (P < 0.001) and in6 of 18 protamine-treated animals, existing deposits decreased or disappeared between 2 and 3 wk of injection. Albuminuria was significantly reduced in protamine-treated animals with a mean of 124±55 mg/24 h compared to 632±150 mg/24 h in the control group receiving cationic BSA alone. No significant differences between the groups were noted in serum lev9lsof IgG, C3, anti-BSA antibody, or circulating immune complex size. Studies in additional animals (n = 5) given radiolabeled cationic BSA showed that protamine did not alter the clearance of cationic BSA from serum.
Control experiments showed that protamine's beneficial effects were not related to its weak anticoagulant property or toits theoretical ability to deplete tissue histamine. The administration of heparin (n = 6) or diphenhydramine (n = 6) had no effect on the development of the epimembranous lesion compared to the group receiving cationic BSA alone. In addition, homogenized whole kidney histamine content was not significantly different in the group receiving cationic BSA alone compared to the group receiving cationic BSA and protamine.
This work shows that a cationic BSA-induced glomerular lesion can be produced by a renal perfusion technique involving in situ complex formation and that this process requires a cationic antigen for its development. We believe that the demonstrated beneficial effects of protamine are due to its ability to bind to glomerular anionic sites, and that this electrostatic interaction results in inhibition for the further binding of the cationic antigen, thereby limiting the severity of glomerulonephritis in this model.
PMCID: PMC436896  PMID: 6826719
25.  Increased Yield and Improved Transplantation Outcome of Mouse Islets with Bovine Serum Albumin 
Journal of Transplantation  2012;2012:856386.
Isolation and transplantation of rodent islets are frequently used as a tool for predicting the behavior of new protocols for islet allotransplants in type 1 diabetes patients. Bovine serum albumin (BSA) is recognized as a protease inhibitor possibly protecting function and viability in islets. For this study, the addition of 0.2% BSA to the isolation protocol resulted in a 30% increase in islet yields while other parameters, such as viability and function, retained high islet quality. In vivo, a minimal mass of 70 BSA treated islets showed their ability to control glycemia levels in diabetic mice by bringing the average blood glucose to 153 ± 13.2 mg/dL compared to 288 ± 22.6 mg/dL without BSA. Our results show that the simple addition of BSA to the isolation protocol constitutes a reliable and reproducible method for increasing islet yield. Also adding BSA to the transplantation medium improves islet function in vivo. The method outlined here can reduce the overall number of animals needed per experiment and also reduce the time and resources needed for islet preparation.
PMCID: PMC3523609  PMID: 23304445

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