Hydrogel containing minocycline and zinc oxide-loaded serum albumin nanopartical for periodontitis application: preparation, characterization and evaluation.

Periodontal disease is a complex problem which often interrelates with several serious systemic diseases. However, the satisfactory clinical therapy has yet to be achieved. Herein, serum albumin microspheres containing minocycline and zinc oxide nanoparticals (ZnO NPs) were prepared and incorporated in a Carbopol 940® hydrogel. Compared with 2% minocycline ointment (Perio®), the hydrogel has shown obvious therapy effects and the ability of gingival tissue self-repairing. The serum albumin microspheres containing 0.06% of minocycline and 0.025% of ZnO NPs presented an average size of 139 ± 0.42 nm using electrophoretic light scattering (n = 3). Photomicrographs obtained by TEM showed homogeneous and spherical-shaped particles. The encapsulation efficiency was 99.99% for minocycline and the slow-release time was more than 72 h with pH-sensitive property. The in vitro skin adhesion experiment showed that the largest bioadhesive force is 0.35 N. Moreover, the hydrogel showed broad-spectrum antimicrobial and effective antibacterial ability when concentration of the ZnO NPs was over 0.2 µg/mL. The cell survival rates were more than 85% below 0.8 mg/L of ZnO NPs, which proved its low toxicity and high security.

One-pot synthesis and biodistribution of fluorine-18 labeled serum albumin for vascular imaging.

INTRODUCTION: Equilibrium single-photon radionuclide imaging methods for assessing cardiac function and the integrity of the vascular system have long been in use for both clinical and research purposes. However, positron-emitting blood pool agents that could provide PET equivalents to these (and other) clinical procedures have not yet been adopted despite technical imaging advantages offered by PET. Our goal was to develop a PET blood pool tracer that not only meets necessary in vivo biological requirements but can be produced with an uncomplicated and rapid synthesis method which would facilitate clinical translation. Herein, albumin labeled with fluorine-18 was synthesized using a one-pot method and evaluated in vitro and in vivo in rats. METHODS: A ligand (NODA-Bz-TFPE), containing NODA attached to a tetrafluorophenylester (TFPE) via a phenyl linker (Bz), was labeled with aluminum fluoride (Al[18F]F). Conjugation of the serum albumin with the ligand (Al[18F]F-NODA-Bz-TFPE), followed by purification (size exclusion chromatography), yielded the final product (Al[18F]F-NODA-Bz-RSA/HSA). In vitro stability was evaluated in human serum albumin by HPLC. Rat biodistributions and whole-body PET imaging over a 4 h time course were used for the in vivo evaluation. RESULTS: This synthesis exhibited an overall radiochemical yield of 45 ±â€¯10% (n = 30), a 50-min radiolabeling time, a radiochemical purity >99% and apparent stability up to 4 h in human serum. Blood had the highest retention of Al[18F]F-NODA-Bz-RSA at all times with a blood half-life of 5.2 h in rats. Al[18F]F-NODA-Bz-RSA distribution in most rat tissues remained relatively constant for up to 1 h, indicating that the tissue radioactivity content represents the respective tissue plasma volume. Dynamic whole-body PET images were in agreement with these findings. CONCLUSIONS: A new ligand has been developed and radiolabeled with Al[18F]F that allows rapid (50-min) preparation of fluorine-18 serum albumin in one-pot. In addition to increased synthetic efficiency, the construct appears to be metabolically stable in rats. This method could encourage wider use of PET to quantify cardiac function and tissue vascular integrity in both research and clinical settings.

Combined local blood-brain barrier opening and systemic methotrexate for the treatment of brain tumors.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood-brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)-HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA-HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA-HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.

Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.

We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent.

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Site-selective labeling of a lysine residue in human serum albumin.

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo half-life of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK-242, a small molecule inhibitor of Toll-like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK-242-based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site-specific manner. Additionally, HSA-cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.

Evaluation of a rapid serological test for leprosy classification using human serum albumin as the antigen carrier.

The presence of anti-BSA antibodies may interfere in serological tests, as ELISA or immunochromatographic assays. BSA is frequently used as a blocking agent or as "inert" carrier of antigens, such as the NT-P-BSA, the semi-synthetic trisaccharide analogue of the PGL-I (phenolic glycolipid-I) antigen from the cell wall of the Mycobacterium leprae. PGL-I was prepared and linked to human serum albumin based in the hypothesis that replacing BSA by a human protein carrier would enhance the performance of leprosy serological tests. A total of 1162 serum samples were tested by ELISA and by the ML Flow rapid test using NT-P-BSA or NT-P-HSA antigens. When grouping leprosy patients as paucibacillary (PB) or multibacillary (MB) according to the Ridley & Jopling classification, ML Flow BSA and ML Flow HSA tests correctly allocated 70.9% and 68.6% of patients in the PB group, and 87% and 81% of patients in the MB group, respectively. Concordant results were found in 82.0% (953/1162) (kappa value=0.637; sd=0.023) of samples between ML Flow tests and 85.7% (996/1162) (kappa value=0.703; sd=0.021) between ELISA tests. ML Flow results were statistically similar and the same was true for ELISA tests using HSA or BSA. However, we noticed a tendency to decreased capacity to detect MB patients and an increased positivity among PB patients, HHC, TB patients and healthy controls by the HSA carrier in both ML Flow and ELISA. The PGL-I serology performed by the ML Flow test with BSA or HSA as antigen carriers can be a useful, friendly auxiliary tool to identify patients with higher bacterial load.

An Escherichia coli trap in human serum albumin microtubes.

We describe the template synthesis of human serum albumin microtubes (MTs) and highlight their Escherichia coli (E. coli) trapping capability with extremely high efficiency. The E. coli was loaded into the one-dimensional pore space interior of the tubule. Similar MTs including an Fe3O4 layer also captured E. coli and were manipulated by exposure to a magnetic field.

Hemodynamics and tissue oxygenation after hemodilution with ultrahigh molecular weight polymerized albumin.

BACKGROUND: Compared to blood transfusion, plasma expanders (PEs) are more cost effective, have a longer shelf-life, and elicit a milder immune response. High molecular weight (MW) dextrans preserve microvascular function during extreme hemodilution. Dextrans, however, evokes negative hemostatic effects, including red blood cell (RBC) aggregation and reduce platelet adhesion, that limit their clinical use. Therefore, polymerization of human serum albumin (HSA) presents a simple strategy to increase HSA's molecular size. METHODS: This study was designed to test the hypothesis that polymerized HSA (PolyHSA) biophysical properties improves systemic and microvascular hemodynamics when used as a PE under anemic conditions. The study was implemented using the hamster window chamber model. Animals were first hemodiluted to 18% hematocrit (Hct) using 6% dextran 70 kDa and then to 11% Hct using either 10% PolyHSA, 10% unpolymerized HSA, or 6% dextran 70 kDa. Systemic and microvascular hemodynamics, including cardiac output (CO), mean arterial blood pressure (MAP), functional capillary density (FCD), microvascular perfusion, and oxygen tension were measured. RESULTS: Posthemodilution, PolyHSA improved MAP, CO, and oxygen delivery compared to HSA and dextran. Additionally, PolyHSA improved microvascular function in terms of blood flow and FCD. Although oxygen carrying capacity is limited at 11% Hct, tissue pO2 and oxygen delivery were higher for PolyHSA compared to HSA and dextran. CONCLUSION: PolyHSA during extreme anemia supported systemic and microvascular hemodynamics by increasing plasma viscosity without increasing vascular resistance. These findings can aid to design of studies to understand the role of the PE biophysical properties in clinical scenarios.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Design and synthesis of glycoprotein-based multivalent glyco-ligands for influenza hemagglutinin and human galectin-3.

We report a facile synthesis of glycoprotein-based glyco-ligands and their binding with influenza hemagglutinin and human galectin-3. Human serum albumin (HSA) was used as the scaffold and an Asn-linked complex type N-glycan prepared from chicken eggs was used as the glycan building block. It was found that Cu(I)-catalyzed alkyne-azide cycloaddition reaction (click chemistry) between the alkyne-labeled glycan and the azide-tagged HSA led to an efficient formation of the glycoconjugates. The density of glycan ligands on the protein scaffold was readily varied by changing the molar ratios of the two reactants. Binding studies indicated that the sialylated and desialylated multivalent glycoligands could selectively bind to influenza hemagglutinin and human galectin-3, respectively, with high affinity. In the two glycan-lectin interactions, a clear multivalent effect was observed. Moreover, a cell-based assay showed that the synthetic multivalent glyco-ligands could efficiently inhibit the attachment of galectin-3 to human prostate cancer and lung cancer cell lines. This study suggests that the synthetic glycoprotein-based glyco-ligands can be useful for different applications, including blocking the function of galectin-3 in cancer metastasis.

Serum albumin targeted, pH-dependent magnetic resonance relaxation agents.

The objective of this work was the synthesis of serum albumin targeted, Gd(III)-based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH-dependent relaxivity. Two new complexes (Gd-glu and Gd-bbu) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH-dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r(1)). In the absence of human serum albumin (HSA), r(1) increases from 2.0 to 6.0 mM(-1) s(-1) for Gd-glu and from 2.4 to 9.0 mM(-1) s(-1) for Gd-bbu from pH 5 to 8.5 at 37 °C, 0.47 T, respectively. These complexes (0.2 mM) are bound (>98.9 %) to HSA (0.69 mM) over the pH range 5-8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r(1) increased 120 % (pH 5) and 550 % (pH 8.5) for Gd-glu and 42 % (pH 5) and 260 % (pH 8.5) for Gd-bbu. The increases in r(1) at pH 5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd-bbu system was investigated further. At pH 5, it binds in a stepwise fashion to HSA with dissociation constants K(d1)=0.65, K(d2)=18, K(d3)=1360 µM. The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu(III) analogue revealed that the inner-sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r(1) at pH 5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r(1) of the albumin-bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1-2 ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin-bound pH-dependent relaxivity.

S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects.

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.

Cellular uptake mechanisms and responses to NO transferred from mono- and poly-S-nitrosated human serum albumin.

Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects.

Development of 68Ga-labeled mannosylated human serum albumin (MSA) as a lymph node imaging agent for positron emission tomography.

INTRODUCTION: Although many sentinel lymph node (SLN) imaging agents labeled with (99m)Tc have been developed, no positron-emitting agent has been specifically designed for SLN imaging. Furthermore, the development of the beta probe and the requirement for better image resolution have increased the need for a positron-emitting SLN imaging agent. Here, we describe the development of a novel positron-emitting SLN imaging agent labeled with (68)Ga. METHODS: A mannosylated human serum albumin (MSA) was synthesized by conjugating α-d-mannopyranosylphenyl isothiocyanate to human serum albumin in sodium carbonate buffer (pH 9.5), and then 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid was conjugated to synthesize NOTA-MSA. Numbers of mannose and NOTA units conjugated in NOTA-MSA were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. NOTA-MSA was labeled with (68)Ga at room temperature. The stability of (68)Ga-NOTA-MSA was checked in labeling medium at room temperature and in human serum at 37°C. Biodistribution in normal ICR mice was investigated after tail vein injection, and micro-positron emission tomography (PET) images were obtained after injecting (68)Ga-NOTA-MSA into a tail vein or a footpad. RESULTS: The numbers of conjugated α-d-mannopyranosylphenyl isothiocyanate and 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid units in NOTA-MSA were 10.6 and 6.6, respectively. The labeling efficiency of (68)Ga-NOTA-MSA was greater than 99% at room temperature, and its stability was greater than 99% at 4 h. Biodistribution and micro-PET studies of (68)Ga-NOTA-MSA showed high liver and spleen uptakes after intravenous injection. (68)Ga-NOTA-MSA injected into a footpad rapidly migrated to the lymph node. CONCLUSIONS: (68)Ga-NOTA-MSA was successfully developed as a novel SLN imaging agent for PET. NOTA-MSA is easily labeled at high efficiency, and subcutaneously administered (68)Ga-NOTA-MSA was found to migrate rapidly to the lymph node.

Synthesis and application of lactosylated, 99mTc chelating albumin for measurement of liver function.

Neogalactosylated and neolactosylated albumins are currently used as radiopharmaceutical agents for imaging the liver asialoglycoprotein receptors, which allows the quantification of hepatic liver function in various diseases and also in healthy liver transplant donors. We developed an original process for synthesizing a chelating neolactosylated human albumin using maleimidopropyl-lactose and maleimidopropyl-diethylene triamine pentaacetic acid (DTPA) derivatives. The lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to nonlactosylated protein and a very high signal-to-noise ratio, based on functional assessment of biodistribution in mice using (99m)Tc-scintigraphy.

Bionanoprobes to study particle-cell interactions.

A variety of research reports have provided evidence that the interplay between nanoparticles and biological systems is strongly dependent on the composition as well as on the size of the particles. However, irrespective of that a fine tuning of the interaction characteristics can be attained by functionalisation of the particle surface. In order to be able to monitor such interactions, an analytically accessible model system for potentially target-specific therapeutically relevant nanoparticles (NP) was generated by encapsulation of the highly hydrophobic fluorophore BODIPY 493/503 into poly(D,L-lactide-co-glycolide) (PLGA) nanodroplets. Analyses of the mean particle size and zeta potential revealed that plain and human serum albumin (HSA) conjugated NP can be stored without agglomeration for at least 28 days at 4 degrees C as well as -80 degrees C. Although transfer of the particles into commonly used buffers and cell culture medium did not affect the system's stability, protein-containing dispersants should not be used for experiments demanding high sensitivity as distinct quenching effects were observed. Concerning liberation of the fluorescent marker, no release occurred when incubating the suspension for 7 days at 4 degrees C, while an onset of release was expectedly detected after 3 h at 37 degrees C. These experimental limitations were taken into account for the particle-cell interaction studies which, as a consequence of the more hydrophilic particle surface, showed an enhanced adhesion of HSA-conjugated colloids to Caco-2 cells by means of flow cytometry and fluorescence microscopy.

Docetaxel-albumin conjugates: preparation, in vitro evaluation and biodistribution studies.

Docetaxel (DTX) is one of the most active chemotherapeutic agents for treating metastatic breast cancer. Its aqueous solubility is very low, hence the available formulation of DTX for clinical use consists of high concentrations of tween80, which has been associated with several hypersensitivity reactions. To reduce the systemic toxicity of DTX as well as to avoid the use of tween80, in this study DTX was chemically conjugated with human serum albumin via a succinic spacer. A high-performance liquid chromatography method was developed for the determination of DTX-albumin conjugate. T47D and SKOV3 cells were used for the evaluation of the in vitro cytotoxicity of the conjugate by MTT assay. Studies were then done on balb/c mice to elucidate the tissue distribution of conjugates after intravenous administration. The albumin-conjugated formulation of DTX with the particle size of 90-110 nm showed enhanced solubility and in vivo characteristics and significantly higher cytotoxicity against tumor cells, for example, IC50 of 6.30 +/- 0.73 nM for T47D cell line compared to free DTX with IC50 of 39.4 +/- 1.75 nM. Conjugation also maintained DTX plasma level at 16.19% up to 2 h after injection compared with 2.51% for Taxotere, hence increasing the chance of nanoparticles uptake by tumor cells.

Effect of processing methods on colouration of human serum albumin preparations.

Human serum albumin is a well tolerated therapeutic for the treatment of hypovolemia. Despite all commercial human albumin preparations being derived from plasma, these products can have a highly variable colour. Albumin samples derived from ethanol precipitation and chromatographic fractionation procedures were evaluated for bilirubin and biliverdin levels and by spectrophotometry. It was shown that albumin derived from a chromatographic process, which had a bilirubin:albumin ratio similar to that observed in plasma, had a vibrant yellow appearance. The albumin derived from ethanol precipitation had undetectable levels of bilirubin, and the amber colour of this product was attributed mainly to residual haem. The presence of bilirubin during pasteurisation led to oxidation to biliverdin, with a resultant colour change from yellow to yellow/green. Given that the antioxidant properties of bilirubin are well established, it is possible that bilirubin helps protect albumin from oxidation during the pasteurisation step.

Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins.

Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. 99mTc-LSA and 99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of 99mTc-LSA and 99mTc-MSA metabolites were examined. Both 99mTc-LSA and 99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. 99mTc-MSA showed higher spleen and kidney uptakes than 99mTc-LSA. 99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of 99mTc-LSA injected, respectively, while 99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.