Nano-anticoagulant based on carrier-free low molecular weight heparin and octadecylamine with an albumin shuttling effect.

Low-molecular-weight heparin (LMWH), derived from unfractionated heparin (UFH), has enhanced anticoagulant efficacy, long duration of action, and extended half-life. Patients receiving LMWH for preventive therapies would strongly benefit from its long-term effects, however, achieving this is challenging. Here, we design and evaluate a nanoengineered LMWH and octadecylamine conjugate (LMHO) that can act for a long time while maintaining close to 97 ± 3% of LMWH activity via end-specific conjugation of the reducing end of LMWH. LMHO can self-assemble into nanoparticles with an average size of 105 ± 1.7 nm in water without any nanocarrier and can be combined with serum albumin, resulting in a lipid-based albumin shuttling effect. Such molecules can circulate in the bloodstream for 4-5 days. We corroborate the self-assembly capability of LMHO and its interaction with albumin through molecular dynamics (MD) simulations and transmission electron microscopy (TEM) analysis. This innovative approach to carrier-free polysaccharide delivery, enhanced by nanoengineered albumin shuttling, represents a promising platform to address limitations in conventional therapies.

The mechanism of interaction between tri-para-cresyl phosphate and human serum protein: A multispectroscopic and in-silico study.

Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet-visible (UV-vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease in the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was -24.452 kJ/mol, -25.907 kJ/mol, -27.363 kJ/mol, and - 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 60.83 kJ/mol and 291.08 J/(mol·>k), respectively, indicating that hydrophobic force was the major driving force in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OPFRs in humans.

Exploring glycated sites in human serum albumin: impact of sample processing techniques on detection and analysis.

Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both in vitro glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.

Interaction of Cephalosporins with Human Serum Albumin: A Structural Study.

Modification of the R1 and R2 side chain structures has been used as the main strategy to expand the spectrum of cephalosporins and impart resistance to hydrolysis by ß-lactamases. These structural modifications also result in a wide range of plasma protein binding, especially with human serum albumin (HSA). Here, we determined the crystal structures of the HSA complexes with two clinically important cephalosporins, ceftriaxone and cefazolin, and evaluated the binding of cephalosporin to HSA by susceptibility testing and competitive binding assay. Ceftriaxone and cefazolin bind to subdomain IB of HSA, and their cephem core structures are recognized by Arg117 of HSA. Tyr161 of HSA changes its rotamer depending on the cephalosporin, resulting in alterations of the cavity shape occupied by the R2 side chain of cephalosporins. These findings provide structural insight into the mechanisms underlying the HSA binding of cephalosporins.

Glycation and drug binding by serum albumin.

Accumulation of glycation products in patients with hyperglycaemic conditions can lead to their reaction with the proteins in the human system such as serum albumin, haemoglobin, insulin, plasma lipoproteins, lens proteins and collagen among others which have important biological functions. Therefore, it is important to understand if glycation of these proteins affects their normal action not only qualitatively, but also importantly quantitatively. Glycation of human serum albumin can easily be carried out over period of weeks and its drug transportability may be examined, in addition to characterisation of the amadori products. A combination of ultrasensitive isothermal titration calorimetry, differential scanning calorimetry, spectroscopy and chromatography provides structure-property-energetics correlations which are important to obtain mechanistic aspects of drug recognition, conformation of the protein, and role of amadori products under conditions of glycation. The role of advance glycation end products is important in recognition of antidiabetic drugs. Further, the extent of glycation of the protein and its implication on drug transportability investigated by direct calorimetric methods enables unravelling mechanistic insights into role of functionality on drug molecules in the binding process, and hinderance in the recognition process, if any, as a result of glycation. It is possible that the drug binding ability of the protein under glycation conditions may not be adversely affected, or may even lead to strengthened ability. Rigorous studies on such systems with diverse functionality on the drug molecules is required which is essential in deriving guidelines for improvements in the existing drugs or in the synthesis of new molecular entities directed towards addressing diabetic conditions.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each ß-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.

Plumbagin accelerates serum albumin's amyloid aggregation kinetics and generates fibril polymorphism by inducing non-native ß-sheet structures.

The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka âˆ¼ 10-4 M-1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of ß-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native ß-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.

Aggregation of Apo/Glycated Human Serum Albumins and Aptamer-Saturated Graphene Quantum Dot: A Simulation Study.

Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer-graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer-GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin-GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.

Serum albumin-embedding copper nanoclusters inhibit Alzheimer's ß-amyloid fibrillogenesis and neuroinflammation.

Increasing evidence suggests that the accumulations of reactive oxygen species (ROS), ß-amyloid (Aß), and neuroinflammation are crucial pathological hallmarks for the onset of Alzheimer's disease (AD), yet there are few effective treatment strategies. Therefore, design of nanomaterials capable of simultaneously elimination of ROS and inhibition of Aß aggregation and neuroinflammation is urgently needed for AD treatment. Herein, we designed human serum albumin (HSA)-embedded ultrasmall copper nanoclusters (CuNCs@HSA) via an HSA-mediated fabrication strategy. The as-prepared CuNCs@HSA exhibited outstanding multiple enzyme-like properties, including superoxide dismutase (>5000 U/mg), catalase, and glutathione peroxidase activities as well as hydroxyl radicals scavenging ability. Besides, CuNCs@HSA prominently inhibited Aß fibrillization, and its inhibitory potency was 2.5-fold higher than native HSA. Moreover, CuNCs@HSA could significantly increase the viability of Aß-treated cells from 60 % to over 96 % at 40 µg/mL and mitigate Aß-induced oxidative stresses. The secretion of neuroinflammatory cytokines by lipopolysaccharide-induced BV-2 cells, including tumor necrosis factor-α and interleukin-6, was alleviated by CuNCs@HSA. In vivo studies manifested that CuNCs@HSA effectively suppressed the formation of plaques in transgenic C. elegans, reduced ROS levels, and extended C. elegans lifespan by 5 d. This work, using HSA as a template to mediate the fabrication of copper nanoclusters with robust ROS scavenging capability, exhibited promising potentials in inhibiting Aß aggregation and neuroinflammation for AD treatment.

Predicting the Stability of Formulations Containing Lyophilized Human Serum Albumin and Sucrose/Trehalose Using Solid-State NMR Spectroscopy.

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the ß-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.

Kinetics of Human Serum Albumin Adsorption on Polycation Functionalized Silica.

The adsorption kinetics of human serum albumin (HSA) on bare and poly-L-arginine (PARG)-modified silica substrates were investigated using reflectometry and atomic force microscopy (AFM). Measurements were carried out at various pHs, flow rates and albumin concentrations in the 10 and 150 mM NaCl solutions. The mass transfer rate constants and the maximum protein coverages were determined for the bare silica at pH 4.0 and theoretically interpreted in terms of the hybrid random sequential adsorption model. These results were used as reference data for the analysis of adsorption kinetics at larger pHs. It was shown that the adsorption on bare silica rapidly decreased with pH and became negligible at pH 7.4. The albumin adsorption on PARG-functionalized silica showed an opposite trend, i.e., it was negligible at pH 4 and attained maximum values at pH 7.4 and 150 mM NaCl, the conditions corresponding to the blood serum environment. These results were interpreted as the evidence of a significant role of electrostatic interactions in the albumin adsorption on the bare and PARG-modified silica. It was also argued that our results can serve as useful reference data enabling a proper interpretation of protein adsorption on substrates functionalized by polyelectrolytes.

Exploring high hydrostatic pressure effects on anthocyanin binding to serum albumin and food-derived transferrins.

This study investigated the effects of high hydrostatic pressure (HHP) on the binding interactions of cyanindin-3-O-glucoside (C3G) to bovine serum albumin, human serum albumin (HSA), bovine lactoferrin, and ovotransferrin. Fluorescence quenching revealed that HHP reduced C3G-binding affinity to HSA, while having a largely unaffected role for the other proteins. Notably, pretreating HSA at 500 MPa significantly increased its dissociation constant with C3G from 24.7 to 34.3 µM. Spectroscopic techniques suggested that HSA underwent relatively pronounced tertiary structural alterations after HHP treatments. The C3G-HSA binding mechanisms under pressure were further analyzed through molecular dynamics simulation. The localized structural changes in HSA under pressure might weaken its interaction with C3G, particularly polar interactions such as hydrogen bonds and electrostatic forces, consequently leading to a decreased binding affinity. Overall, the importance of pressure-induced structural alterations in proteins influencing their binding with anthocyanins was highlighted, contributing to optimizing HHP processing for anthocyanin-based products.

Optimizing the pharmacokinetics of an 211At-labeled RGD peptide with an albumin-binding moiety via the administration of an albumin-binding inhibitor.

PURPOSE: A probe for targeted alpha therapy (TAT) using the RGD peptide (Ga-DOTA-K([211At]APBA)-c(RGDfK) ([211At]1)) with albumin-binding moiety (ABM) was recently developed. [211At]1 highly accumulated in tumors and significantly inhibited tumor growth in U-87 MG tumor-bearing mice. However, high [211At]1 retention in blood may cause critical adverse events, such as hematotoxicity. Therefore, we attempted to accelerate the blood clearance of [211At]1 by competitively inhibiting the binding of [211At]1 to albumin to modulate the pharmacokinetics of the former. METHODS: To evaluate the effects of albumin-binding inhibitors in normal mice, sodium 4-(4-iodophenyl)butanoate at 2, 5, or 10 molar equivalents of blood albumin was administered at 1-h postinjection of [211At]1. The biodistribution of [211At]1, SPECT/CT imaging of [67Ga]Ga-DOTA-K(IPBA)-c(RGDfK) ([67Ga]2), and the therapeutic effects of [211At]1 were compared with or without IPBA administration in U-87 MG tumor-bearing mice. RESULTS: Blood radioactivity of [211At]1 was decreased in a dose-dependent manner with IPBA in normal mice. In U-87 MG tumor-bearing mice, the blood radioactivity and accumulation in nontarget tissues of [211At]1 were decreased by IPBA. Meanwhile, tumor [211At]1 accumulation was not changed at 3-h postinjection of IPBA. In SPECT/CT imaging of [67Ga]2, IPBA administration dramatically decreased radioactivity in nontarget tissues, and only tumor tissue was visualized. In therapeutic experiments, [211At]1 with IPBA injected-group significantly inhibited tumor growth compared to the control group. CONCLUSION: IPBA administration (as an albumin-binding inhibitor) could modulate the pharmacokinetics and enhance the therapeutic effects of [211At]1.

Fundamental evaluation regarding the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands.

OBJECTIVE: The marked success of prostate-specific membrane antigen (PSMA)-targeting radioligands with albumin binder (ALB) is attributed to the improvement of blood retention and tumor accumulation. [111In]In-PNT-DA1, our PSMA-targeting radioligand with ALB, also achieved improved tumor accumulation due to its prolonged blood retention. Although the advantage of ALBs is related to their reversible binding to albumin, the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands remains unclear because of the lack of information about radioligands with stronger albumin-binding than ALBs. In this study, we designed and synthesized [111In]In-PNT-DM-HSA, a new radioligand that consists of a PSMA-targeting radioligand covalently bound to albumin. The pharmacokinetics of [111In]In-PNT-DM-HSA was compared with those of [111In]In-PNT-DA1 and [111In]In-PSMA-617, a non-ALB-conjugated radioligand, to evaluate the relationship between albumin-binding and tumor accumulation. METHOD: The [111In]In-PNT-DM-HSA was prepared by incubation of [111In]In-PNT-DM, a PSMA-targeting radioligand including a maleimide group, and human serum albumin (HSA). The ability of [111In]In-PNT-DM-HSA was evaluated by in vitro assays. A biodistribution study using LNCaP tumor-bearing mice was conducted to compare the pharmacokinetics of [111In]In-PNT-DM-HSA, [111In]In-PNT-DA1, and [111In]In-PSMA-617. RESULTS: The [111In]In-PNT-DM-HSA was obtained at a favorable radiochemical yield and high radiochemical purity. In vitro assays revealed that [111In]In-PNT-DM-HSA had fundamental characteristics as a PSMA-targeting radioligand interacting with albumin covalently. In a biodistribution study, [111In]In-PNT-DM-HSA and [111In]In-PNT-DA1 showed higher blood retention than [111In]In-PSMA-617. On the other hand, the tumor accumulation of [111In]In-PNT-DA1 was much higher than [111In]In-PNT-DM-HSA and [111In]In-PSMA-617. CONCLUSIONS: These results indicate that the moderate reversible binding of ALB with albumin, not covalent binding, may play a critical role in enhancing the tumor accumulation of PSMA-targeting radioligands.

Investigating the Serum Albumin Binding Behavior of Naphthalimide Based Fluorophore Conjugates: Spectroscopic and Molecular Docking Approach.

In the present study, naphthalimide-pyrazole-benzothiazole based fluorescent analogs were synthesized by substituting different primary and secondary amines on the naphthalimide nucleus and were evaluated for their sensitivity and selectivity towards serum albumin. Among various synthesized analogues compound 25 showed the most significant change with serum albumin and was further studied for selective detection and mode of interaction with serum albumin. Here, we compared the binding interaction of fluorescent probe 25 for variation/detection of two 76 % structurally resembling proteins HSA and BSA, by spectroscopic experiments. The compound shows more selectivity for HSA and BSA with a higher binding constant and evident visible change in the emission spectra of two serum albumins among different bioanalytes. The mode of interaction of 25 with human serum albumin and bovine serum albumin was investigated by FT-IR, circular dichroism, and DLS techniques to find out the change in the microenvironment and variation in the structure of serum albumin proteins. Higher binding affinity and specific selectivity of 25 with a limit of detection of 0.69 µM and 1.4 µM towards HSA and BSA compared to other bioanalytes make it a significant fluorescent probe for quantitatively detecting serum albumins at the very early stage of many fatal diseases.

The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures.

Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple "mix-and-match" method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non-phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be effectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy-aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics.

BODIPY(aryl)iodonium salts in the efficient synthesis of diversely functionalized BODIPYs and selective detection of serum albumin.

BODIPY(aryl)iodonium salts were readily accessible from the high-yielding reaction of BODIPY with iodoarenes or hydroxyl(tosyloxy)iodoarenes in the presence of m-CPBA. The prepared BODIPY(aryl)iodonium salts bearing substituents of varied electronic nature were utilized for the direct syntheses of thiocyanate, azide, amine and acrylate functionalized BODIPYs and ß,ß'-bis-BODIPYs. The regioselective syntheses of α-piperidinyl and ß-piperidinyl substituted BODIPYs were achieved through the reaction of BODIPY(aryl)iodonium salts with piperidine in the absence and presence of copper(I). Expeditious and high yielding (79-82%) synthesis of ß,ß'-bis-BODIPYs was also developed through the palladium-catalyzed reductive coupling of the easily accessible BODIPY(aryl)iodonium salts. Some of the indole-appended BODIPYs and bis-BODIPYs displayed strong absorption in the visible region (∼610 nm). The BODIPY(aryl)iodonium salts also showed significant binding with serum albumin and were observed to be selective serum protein sensors with estimated limits of detection as low as 7 µg mL-1 in some cases.

Construction of Biosensing System for Glycated Albumin Using an Electron Transfer Peptide-Modified Protein Probe.

Glycated albumin (GA) is one of the proteins that replaces several sugar moieties and can be used as an indicator of diabetes mellitus. We developed a sensing system that uses GA in the early detection of diabetes mellitus. In this study, H6Y4C acetylated (Ac-) at the N-terminals of the peptide was combined with wheat germ agglutinin (WGA) to recognize glucose moieties. The Ac-H6Y4C-WGA was constructed as a GA-sensing probe. The tyrosine residues of Y4C exhibited an oxidation peak, and His-tag moieties were introduced to separate Ac-H6Y4C-WGA in the synthesis of the probe. The Ac-H6Y4C-WGA probe binds with the 1-2 molecules of Ac-H6Y4C per WGA using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS. Next, the functions of Ac-H6Y4C-WGA were evaluated using voltammetry. The number of electron-transfers was calculated based on the relationship between the peak potential and logarithm of scan rate and was 3.03. In the electrochemical measurements with mannose and bovine serum albumin, the peak currents were similar to that of GA alone. By contrast, a decrease in the peak current was suppressed when glucose was added to the solution containing the probe. As a result, Ac-H6Y4C-WGA was selectively bound to the glucose moieties of GA. The calibration curve via differential pulse voltammetry was proportional to the concentrations of GA and ranged from 1.0 × 10-12 to 2.0 × 10-11 M with a detection limit of 3.3 × 10-13 M.

Developing a Copper(II) Isopropyl 2-Pyridyl Ketone Thiosemicarbazone Compound Based on the IB Subdomain of Human Serum Albumin-Indomethacin Complex: Inhibiting Tumor Growth by Remodeling the Tumor Microenvironment.

To develop a next-generation metal agent and dual-agent multitargeted combination therapy, we developed a copper (Cu) compound based on the properties of the human serum albumin (HSA)-indomethacin (IND) complex to remodel the tumor microenvironment (TME). We optimized a series of Cu(II) isopropyl 2-pyridyl ketone thiosemicarbazone compounds to obtain a Cu(II) compound (C4) with significant cytotoxicity and then constructed an HSA-IND-C4 complex (HSA-IND-C4) delivery system. IND and C4 bind to the hydrophobic cavities of the IB and IIA domains of HSA, respectively. In vivo, the HSA-IND-C4 not only showed enhanced antitumor efficacy relative to C4 and C4 + IND but also improved their targeting ability and decreased their side effects. The antitumor mechanism of C4 + IND involved acting on the different components of the TME. IND inhibited tumor-related inflammation, while C4 not only induced apoptosis and autophagy of cancer cells but also inhibited tumor angiogenesis.

Alleviation of albumin glycation-induced diabetic cardiomyopathy by L-Arginine: Insights into Nrf-2 signaling.

In hyperglycemia, accelerated glycation and oxidative stress give rise to many diabetic complications, such as diabetic cardiomyopathy (DCM). Glycated human serum albumin (GHSA) has disturbed structural integrity and hampered functional capabilities. When GHSA accumulates around cardiac cells, Nrf-2 is dysregulated, aiding oxidative stress. L-Arginine (L-Arg) is prescribed to patients with diabetes and cardiovascular diseases. This research contributes to the mechanistic insights on antiglycation and antioxidant potential of L-Arg in alleviating DCM. HSA was glycated with methylglyoxal in the presence of L-Arg (20-640 mM). Structural and functional modifications of HSA were studied. L-Arg and HSA, GHSA interactions, and thermodynamics were determined by steady-state fluorescence. H9c2 cardiomyocytes were given treatments of GHSA-L-Arg along with the inhibitor of the receptor of AGEs. Cellular antioxidant levels, detoxification enzyme activities were measured. Gene, protein expressions, and immunofluorescence data examined the activation and nuclear translocation of Nrf-2 during glycation and oxidative stress. L-Arg protected HSA from glycation-induced structural and functional modifications. The binding affinity of L-Arg was more towards HSA (104 M-1). L-Arg, specifically at lower concentration (20 mM), upregulated Nrf-2 gene, protein expressions and facilitated its nuclear translocation by activating Nrf-2 signaling. The study concluded that L-Arg can be of therapeutic advantage in glycation-induced DCM and associated oxidative stress.