Human Serum Albumin Solution Research Articles

Albumin adjuvant therapy for acute ischemic stroke with large vessel occlusion (AMASS-LVO): rationale, design, and protocol for a phase 1, open-label, clinical trial.

Acute ischemic stroke (AIS) is an acute brain injury caused by sudden occlusion of a blood vessel. Endovascular therapy is the most effective way to restore blood flow. However, despite the restoration of blood flow in some patients, their clinical prognosis often remains unsatisfactory. Albumin has shown neuroprotective effects in animal models of AIS. Therefore, this study aims to evaluate the safety, feasibility, and efficacy of local arterial infusions of 20% human serum albumin solution as an adjuvant therapy after endovascular therapy in patients with AIS. This study is a prospective, therapeutic exploratory, non-randomized, open-label, phase 1 clinical trial testing the use of 20% human serum albumin solution injected via the artery immediately after successful reperfusion in patients with AIS. The study is divided into two stages. In the first stage, a single-dose-finding will explore the maximum safe dose according to the 3 + 3 dose escalation principle;, with the maximum dose being 0.60 g/kg. After recanalizing the occluded blood vessel, human serum albumin solution will be injected into the internal carotid artery region through a guiding catheter for 30 min. The second stage involves an albumin adjuvant therapy cohort (AT) and an endovascular treatment lonely cohort (ET). The AT cohort will encompass at least 15 additional participants to complete safety trials at the maximum safe dose determined in the first stage. The ET cohort will include well-matched patients receiving endovascular therapy alone, derived from a contemporaneous prospective registry, who will be excluded from having cardiopulmonary disorders and from receiving any neuroprotective therapy. The primary outcome of this study will be symptomatic intracranial hemorrhage. Efficacy outcomes will include the proportion of patients with the progression of cerebral infarction volume, a modified Rankin Scale of 0-2 on day 90 after randomization. An exploratory secondary outcome will be the analysis of thromboinflammatory and neuroprotective molecule profiles. This pilot trial aims to explore the safety and efficacy of arterial infusion of an albumin solution after occlusive vessel opening in AIS. The results will provide data parameters for subsequent tests on the arterial infusion of albumin solutions. ClinicalTrials.gov, NCT05953623.

Aggregation of Albumins under Reductive Radical Stress.

The reactions of radicals with human serum albumin (HSA) under reductive stress conditions were studied using pulse radiolysis and photochemical methods. It was proved that irradiation of HSA solutions under reductive stress conditions results in the formation of stable protein aggregates. HSA aggregates induced by ionizing radiation are characterized by unique emission, different from the UV emission of non-irradiated solutions. The comparison of transient absorption spectra and the reactivity of hydrated electrons (eaq-) with amino acids or HSA suggests that electron attachment to disulfide bonds is responsible for the transient spectrum recorded in the case of albumin solutions. The reactions of eaq- and CO2•- with HSA lead to the formation of the same products. Recombination of sulfur-centered radicals plays a crucial role in the generation of HSA nanoparticles, which are stabilized by intermolecular disulfide bonds. The process of creating disulfide bridges under the influence of ionizing radiation is a promising method for the synthesis of biocompatible protein nanostructures for medical applications. Our Raman spectroscopy studies indicate strong modification of disulfide bonds and confirm the aggregation of albumins as well. Low-temperature measurements indicate the possibility of electron tunneling through the HSA protein structure to specific CyS-SCy bridges. The current study showed that the efficiency of HSA aggregation depends on two main factors: dose rate (number of pulses per unit time in the case of pulse radiolysis) and the temperature of the irradiated solution.

Human Serum Albumin Protein Corona in Prussian Blue Nanoparticles.

Prussian Blue nanoparticles (PBnps) are now popular in nanomedicine thanks to the FDA approval of PB. Despite the numerous papers suggesting or describing the in vivo use of PBnps, no studies have been carried out on the formation of a protein corona on the PBnp surface and its stabilizing role. In this paper, we studied qualitatively and quantitatively the corona formed by the most abundant protein of blood, human serum albumin (HSA). Cubic PBnps (41 nm side), prepared in citric acid solution at PB concentration 5 × 10-4 M, readily form a protein corona by redissolving ultracentrifuged PBnp pellets in HSA solutions, with CHSA ranging from 0.025 to 7.0 mg/mL. The basic decomposition of PBnp@HSA was studied in phosphate buffer at the physiological pH value of 7.4. Increased stability with respect to uncoated PBnps was observed at all concentrations, but a minimum CHSA value of 3.0 mg/mL was determined to obtain stability identical to that observed at serum-like HSA concentrations (35-50 mg/mL). Using a modified Lowry protocol, the quantity of firmly bound HSA in the protein corona (hard corona) was determined for all the CHSA used in the PBnp@HSA synthesis, finding increasing quantities with increasing CHSA. In particular, an HSA/PBnp number in the 1500-2300 range was found for CHSA 3.0-7.0 mg/mL, largely exceeding the 180 HSA/PBnp value calculated for an HSA monolayer on a PBnp. Finally, the stabilization brought by the HSA corona allowed us to carry out pH-spectrophotometric titrations on PBnp@HSA in the 3.5-9-0 pH range, revealing a pKa value of 6.68 for the water molecules bound to the Fe3+ centers on the PBnp surface, whose deprotonation is responsible for the blue-shift of the PBnp band from 706 nm (acidic solution) to 685 nm (basic solution).

Human serum albumin and curcumin heteroassociate in aqueous solutions

Complex formation in aqueous solutions of human serum albumin (HSA) was studied with the medical drug curcumin, using the methods of UV absorption, emission, and excitation of fluorescence, the Stern equation (Vollmer, Hill, Foerster, and Van-Hoff), and computer modeling. It was established that the heteroassociation in the HSA-Cur system is realized due to the formation of stable complexes in the ground state, mainly due hydrophobic interaction in binding sites I and II.

A comparative study of cross-linked human serum albumin nanoparticles using ultrasonication or glutaraldehyde; The impact on the corona protein adsorption using SDS-PAGE and MALDI-TOF

Protein-based nanoparticles (NPs) are promising delivery systems designed for many drugs, including anticancer drugs. In this study, human serum albumin (HSA) in solution and once nanoparticulated by the desolvation method were crosslinked using ultrasonication, and their characteristics were compared to glutaraldehyde (GA) crosslinked HSA solution and nanoparticles (NPs). The results revealed that ultrasonic irradiation caused the development of new intermolecular disulfide bonds between albumin molecules, perhaps via the production of free radical species. The sonicated NPs were then compared with the GA cross-linked NPs in terms of size, shape, surface charge, and functional groups. Furthermore, the corona protein of the produced HSA NPs was examined using SDS-PAGE and MALDI-TOF since protein adsorption plays a significant role in the biological fate of NPs. The findings showed that while the morphologies of the sonicated and GA-NPs were comparable, the particle sizes (224 vs. 174 nm) and zeta potentials (+27.5 vs. −6.4 mV) were different. SDS-PAGE results showed that the protein bands adsorbed onto the surface of the sonicated nanoparticles were more intense. This could be connected to the distinct surface characteristics and surface charge of the ultrasonically irradiated NPs. In summary, the preparation of cross-linked HSA NPs using disulfide bound rearrangement, which led to the positively charged albumin NPs is reported. Moreover, ultrasonic irradiation can be introduced as an alternative method to avoid the toxic residual of cross-linking agents in HSA NPs production.

Desalting Plasma Protein Solutions by Membrane Capacitive Deionization.

Plasma protein therapies are used by millions of people across the globe to treat a litany of diseases and serious medical conditions. One challenge in the manufacture of plasma protein therapies is the removal of salt ions (e.g., sodium, phosphate, and chloride) from the protein solution. The conventional approach to remove salt ions is the use of diafiltration membranes (e.g., tangential flow filtration) and ion-exchange chromatography. However, the ion-exchange resins within the chromatographic column as well as filtration membranes are subject to fouling by the plasma protein. In this work, we investigate the membrane capacitive deionization (MCDI) as an alternative separation platform for removing ions from plasma protein solutions with negligible protein loss. MCDI has been previously deployed for brackish water desalination, nutrient recovery, mineral recovery, and removal of pollutants from water. However, this is the first time this technique has been applied for removing 28% of ions (sodium, chloride, and phosphate) from human serum albumin solutions with less than 3% protein loss from the process stream. Furthermore, the MCDI experiments utilized highly conductive poly(phenylene alkylene)-based ion exchange membranes (IEMs). These IEMs combined with ionomer-coated nylon meshes in the spacer channel ameliorate Ohmic resistances in MCDI improving the energy efficiency. Overall, we envision MCDI as an effective separation platform in biopharmaceutical manufacturing for deionizing plasma protein solutions and other pharmaceutical formulations without a loss of active pharmaceutical ingredients.

Evaluation of organophosphorus pesticide tyrosine adducts for postmortem change by human serum albumin with liquid chromatography quadrupole Orbitrap mass spectrometry.

Organophosphorus pesticides (OPPs) having a phosphate ester moiety, such as malathion (MA) and methidathion (DMTP), are widely used and have been detected in many fatal cases of accidental exposure or suicide in Japan. In forensic toxicology, the accurate determination of blood OPP concentration is mandatory to prove death by OPP poisoning. However, fatal pesticide concentration in blood at autopsy varies depending on the circumstances surrounding the dead body. In this study, we found that 16 OPPs were degraded by human serum albumin (HSA) in a temperature-dependent fashion. The mechanism underlying MA, DMTP, azinphos-methyl, etrimfos, fenthion (MPP), pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, edifenphos (EDDP), fosthiazate, and pyraclofos degradation involves the formation of adducts with tyrosine residues in HSA. The mass spectra obtained by liquid chromatography quadrupole Orbitrap mass spectrometry revealed that phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5 were formed in HSA solution incubated with OPPs. These results indicate that the 16 OPPs are postmortem changed by HSA. The detection of phosphate ester amino acid adducts such as Y-adduct1, Y-adduct2, Y-adduct3, Y-adduct4, and Y-adduct5, instead of MA, DMTP, azinphos-methyl, etrimfos, MPP, pirimiphos-methyl, (E)-dimethylvinphos, (Z)-dimethylvinphos, vamidothion, EDDP, fosthiazate, and pyraclofos per se, may be used to determine death by these OPPs poisoning.

Small molecule interactions with biomacromolecules: selective sensing of human serum albumin by a hexanuclear manganese complex - photophysical and biological studies.

A covalently bonded hexanuclear neutral complex, [Mn6(μ3-O)2(3-MeO-salox)6(OAc)2(H2O)4] (1), has been synthesized and characterized by single crystal X-ray diffraction analysis along with IR and HRMS studies. Complex 1 has been found to selectively interact with human serum albumin (HSA), a model transport protein. The interaction of 1 with HSA was investigated by monitoring the change in the absorbance value of HSA at λ = 280 nm with increasing concentration of 1. Likewise, fluorescence titrations were carried out under two conditions: (i) titration of a 5 μM solution of complex 1 with the gradual addition of HSA, showing a ∼9-fold fluorescence intensity enhancement at 424 nm, upon excitation at 300 nm; and (ii) upon excitation at 295 nm, titration of 5 μM HSA solution with the incremental addition of complex 1, showing a quenching of fluorescence intensity at 334 nm, with simultaneous development of a new emission band at 424 nm. A linear form of the Stern-Volmer equation gives KSV = 9.77 × 104 M-1 and the Benesi-Hildebrand plot yields the binding constant as KBH = 1.98 × 105 M-1 at 298 K. The thermodynamic parameters, ΔS°, ΔH°, and ΔG°, were estimated by using the van't Hoff relationship which infer the major contribution of hydrophobic interactions between HSA and 1. It was observed that quenching of HSA emission arises mainly through a dynamic quenching mechanism as indicated by the dependence of average lifetime 〈τ〉 on the concentration of 1. The changes in the CD (circular dichroism) spectral pattern of HSA in the presence of 1 clearly establish the variation of HSA secondary structure on interaction with 1. The most probable interaction region in HSA for 1 was determined from molecular docking studies which establish the preferential trapping of 1 in the subdomain IIA of site I in HSA and substantiated by the results of site-specific marker studies. Complex 1 was further evaluated for its antiproliferative effects in lung cancer A549 cells, which strictly inhibits the growth of the cells in both 2D and 3D mammospheres, indicating its potential application as an anticancer drug.

Рeculiarities of static light scattering by aqueous solutions of human serum albumin

The paper presents the results of the study of molecular light scattering (MLS) by aqueous solutions of human serum albumin.Using static light scattering methods, it was found that the concentration dependence of molecular scattering intensity differs from similar dependences for solutions of polymers such as polyethylene glycol or polyvinyl alcohol.In contrast, the concentration dependences of MLS intensity have a scattering peak at concentrations of 6%, which can be considered as anomalous scattering similar to the scattering of light in aqueous solutions of some substances, more often - alcohols.The position of the light scattering peak obtained in the work coincides with the characteristic point of aqueous solutions of albumins of various origins, which is determined by the concentration of the protein component in the blood plasma of mammals.A detailed analysis of the obtained results requires new studies on the temperature dependence of the MLS intensity peaks and the results of laser correlation spectroscopy of the intensity of scattered light.

Comparison of Different Ultrafiltration Devices for the Study of Plasma Protein Binding of Carvedilol

Abstract The aim of the present study was to assess the suitability of different Amicon Ultra and Centrifree ultrafiltration devices for the study of the plasma protein binding process in the case of carvedilol, a highly protein-bound and lipophilic beta-blocking agent. Samples at different levels of concentration were prepared in both proteic and non-proteic matrices (human plasma, 5% human serum albumin solution and saline solution) and subjected to the classical ultrafiltration method using the different devices considered. Furthermore, an attempt to apply a previously described modified ultrafiltration method was also made. The analysis and quantification was achieved using a validated LC-MS/MS method. For the Centrifree devices, the determined unbound fractions of carvedilol and the corresponding binding degree were in accordance to literature data, while for the Amicon Ultra devices a great degree of carvedilol adsorbtion to the sample reservoir was observed, the analyte not being detected in the ultrafiltrate samples. Thus, it was further demonstrated that the type of ultrafiltration device used has a significant influence on the outcome of a plasma protein binding study. In the case of carvedilol, the evaluation of the protein binding interaction could be achieved using the Centrifree ultrafiltration devices, but not the Amicon Ultra devices.

A comparison of surface properties for bovine serum albumin and human serum albumin – Dynamic/equilibrium surface tension and dilational modulus

The biological functionalities of Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) have been extensively studied in the literature. However, limited information is available on their surface properties [dynamic/equilibrium surface tension (ST) and dilational modulus (E)]. This study compared the dynamic/equilibrium ST of HSA and BSA solutions and the E of the adsorbed films. A pendant bubble tensiometer was employed for measuring the relaxations of ST of HSA/BSA solutions at C = 0.01 – 90 (10−10 mol/cm3). The data revealed that while the dynamic ST of HSA(aq) and BSA(aq) exhibited a similar relaxation in general, the ST of HSA(aq) relaxed notably faster at the post-induction regime than BSA(aq). The equilibrium ST of both HSA and BSA was constant at 52.6 and 52.3 mN/m over a wide concentration range, C = 0.01 – 90 (10−10 mol/cm3). The E of HSA and BSA films exhibited a near-identical concentration dependency to each other: E was reasonably constant at ∼ 21 mN/m from C = 0.05 to 0.2 (10−10 mol/cm3), and at further concentration elevations, E rose notably, and eventually leveled off at ∼ 52 and ∼ 45 mN/m for HSA and BSA films, respectively.

Serum albumin acted as an effective carrier to improve the stability of bioactive flavonoid.

The health-improving functions of bioactive flavonoids in vitro and in vivo are often limited by their low stability, which could be counteracted by the application of proteins as carriers of flavonoids. Clarification of the mechanism of protein-ligand interaction is crucial for the encapsulation of bioactive components. Herein, common plasma proteins [i.e., bovine serum albumin (BSA), human serum albumin (HSA), human immunoglobulin G (IgG) and fibrinogen (FG)] were compared for their binding characteristics to quercetin, the main component of flavonoids in human diet, in the absence and presence of free Cu2+ (anaccelerator for flavonoids' instability) using multi-spectroscopic and computational methods. As a flexible open structure of proteins, both BSA and HSA were found to be the most promising carriers for quercetin and Cu2+ with an affinity on the order of 104M-1. HSA-diligand complex (i.e., HSA-quercetin-Cu2+) was successfully generated when both quercetin and Cu2+ were added to the HSA solution. The stability and free radical scavenging activity of bioactive quercetin during incubation was promoted in the HSA-diligand complex relative to quercetin-Cu2+ complex. Quercetin/Cu2+ system could induce the formation of reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxide radical (·OH), which were significantly suppressed upon HSA binding. Consistently, the cytotoxicity of the quercetin/Cu2+ system to endothelial cells was reduced in the HSA-diligand complex. These results demonstrate the possibility of developing serum albumin-based carriers for the protection of bioactive flavonoids in their nutritional application.

In Vitro Characterization of the Interactions of Human Serum Albumin with Rosuvastatin and Atorvastatin Using Fluorescence Spectroscopy and Molecular Modeling

Human serum albumin (HSA) is one of the most important transporters for drugs in the systemic circulation. In this study, we investigated the interaction of rosuvastatin (ROS) and atorvastatin (ATO) with HSA. Binding of a drug molecule to HSA significantly affects the pharmacokinetics of the drug as it increases drug solubility in plasma, decreases toxicity and protects molecules from oxidation. This study was made using fluorescence spectroscopy and molecular modeling approach. Fluorescence spectra were recorded for two different statins brands at seven different concentrations. The results revealed that both statins (ROS and ATO) cause the fluorescence quenching of the HSA solution. ROS and ATO binds strongly to HSA with the binding constant (Kb) of 1.0246×106 and 0,9018×106, respectively. In addition, it was observed that high concentrations of ATO cause a shift of the emission maximum towards longer wavelengths (red-shift), which may be due to the unfolding of protein chains or denaturation. Furthermore, it was calculated that HSA possesses one binding site for ROS and ATO. Results from molecular docking showed that ROS has a higher affinity for Sudlow site I compared to Sudlow site II and the main binding forces are hydrogen bonds. ATO has nearly equal affinity for both binding sites on HSA, and the main binding forces are hydrophobic interactions.

N-Acetyl cysteine amide and cerium oxide nanoparticles as a drug delivery for ischemic stroke treatment: Inflammation and oxidative stress crosstalk

BackgroundInflammation and oxidative stress crosstalk is involved in the ischemic stroke(IS) pathogenesis and the new therapeutic options should be offered based on the targets that are critical in the golden hour of IS. YKL-40 and total antioxidant capacity(TAC), the inflammation and oxidative stress biomarkers, provide us with clues for proper intervention targets. N-acetyl cysteine amide (NACA), a lipophilic antioxidant, with a nanoparticle-based drug delivery system is permeable enough to penetrate blood-brain barrier (BBB) and was proposed as a new treatment option for IS. In this study, we evaluated the YKL-40 and TAC levels in the sera of IS patients to elucidate the best intervention target. A rat tissue model is used to assess the NACA efficiency. The microbiology tests performed to figure out the potential NACA and antibiotics interactions. Material and methodsThe YKL-40 and TAC were measured in the serum of IS patients by ELISA and FRAP methods, respectively. The serum samples were obtained 12 h after the patient's admission and meantime other laboratory findings and NIHSS-based prognosis were recorded. In the animal study, the brain cortex, liver, kidney, adipose, and the heart of healthy rats were dissected and then incubated in DMEM cell culture media containing 50 micrograms/milliliter of nanoparticles; the nanoparticles were titanium dioxide nanoparticles (TiO2 NPs), copper oxide nanoparticles (CuO NPs) and cerium dioxide nanoparticles (CeO2 NPs). Olive oil and human serum albumin solution were exposed to the nanoparticles with and without NACA. TAC was measured in the supernatant culture media. With similar concentrations and settings, we evaluated the NACA, nanoparticle, and antibiotics interactions on pseudomonas aeruginosa. ResultsThere was a nonparametric correlation between YKL-40 levels and post stroke serum TAC levels. Nonsmokers had higher YKL-40 and TAC levels than smokers. A new calculated variable, urea*lymphocyte/age, predicts a poor prognosis with an acceptable AUC (0.708). Exposing to the nanoparticles, the liver, kidney, and brain had a significantly higher TAC than adipose and cardiac tissue. The NACA had an ameliorative effect against TiO2 NPs in the brain. This effectiveness of NACA was also observed against CuO NPs treatment. However, the CeO2 NPs exert a strong antioxidant property by reducing the TAC in the brain tissue but not the others. Albumin showed antioxidant properties by itself, but olive oil had an inert behavior. NACA had no interaction with the action of routine antibiotics. ConclusionOxidative stress but not inflammation is the best point for intervention in IS patients because YKL-40 has not a relationship with NIHSS score. The CeO2 NPs and NACA combination are eligible option to develop antioxidant-based drug for the treatment of IS. As a complementary finding, the urea*lymphocyte/age is proposed as a NIHSS-based prognosis biomarker.

Detection and analysis of moving reaction boundary-based electrophoresis distance using smartphone images

Electrophoresis titration (ET) based on the moving reaction boundary (MRB) theory can detect the analyte contents in different samples by converting content signals into distance signals. However, this technique is only suitable for on-site qualitative testing, and accurate quantification relies on complex optical equipment and computers. Hence, applying this method to real-time point-of-care testing (POCT) is challenging. In this study, we developed a smartphone-based ET system based on a visual technique to achieve real-time quantitative detection. First, we developed a portable quantitative ET device that can connect to a smartphone; this device consisted of five components, namely, an ET chip, a power module, a microcontroller, a liquid crystal display screen, and a Bluetooth module. The device measured 10 cm×15 cm×2.5 cm, weighed 300 g, and was easy to hold. Thus, it is suitable for on-site testing with a run time of only 2-4 min. An assistant mobile software program was also developed to control the device and perform ET. The colored electrophoresis boundary can be captured using the smartphone camera, and quantitative detection results can be obtained in real time. Second, we proposed a quantitative algorithm based on ET channels. The software was used to recognize the boundary migration distance of three channels, a standard curve based on two given contents of the standards was established using the two-point method, and the content of the test sample was calculated. Human serum albumin (HSA) and uric acid (UA) were used as a model protein and biosample, respectively, to test the performance of the detection system. For HSA detection, different HSA solutions were mixed with a polyacrylamide gel (PAG) stock solution, phenolphthalein was added as an indicator, and sodium persulfate and tetramethyl ethylenediamine (TEMED) were used to promote polymerization to form a gel. For UA detection, agarose gel was filled into the ET channel, the UA sample, urate oxidase, and leucomalachite green were added into the anode cell and incubated for 20 min. ET was then performed. The fitting goodness (R2) values of HSA and UA were 0.9959 and 0.9935, respectively, with a linear range of 0.5-35.0 g/L and a log-linear range of 100-4000 μmol/L. The limits of detection for HSA and UA were 0.05 g/L and 50 μmol/L, respectively, and the corresponding relative standard deviations (RSDs) were not greater than 2.87% and 3.21%, respectively. These results demonstrate that the detection system has good accuracy and sensitivity. Clinical samples collected from healthy volunteers were used as target blood samples, and the developed system was used to measure serum total protein and UA levels. Serum samples from five volunteers were selected, standard curves of total serum protein and UA were established, and the test results were compared with hospital standard testing results. The relative errors for serum total protein and UA were less than 6.03% and 6.21%, respectively, and the corresponding RSDs were less than 3.72% and 5.84%, respectively. These findings verify the accuracy and reliability of the proposed detection system. The smartphone-based ET detection system introduced in this paper presents several advantages. First, it enables the portable real-time detection of total serum protein and UA. Second, compared with traditional ET strategies based on colored boundaries, it does not rely on optical detection equipment or computers to obtain quantitative detection results; as such, it can reduce the complexity of the operation and provide portability and real-time metrics. Third, the detection of two biomarkers, serum total protein and UA, is achieved on the same device, thereby improving the multitarget detection potential of the ET method. These advantages render the developed method a promising detection platform for clinical applications and real-time POCT.

Semi-synthetic human albumin isoforms: Production, structure, binding capacities and influence on a routine laboratory test

Human Serum Albumin (HSA) undergoes Post-Translational-Modifications (PTMs) leading to isoforms affecting its oncotic and non-oncotic properties. HSA is comprised of several isoforms whose abundance may vary with pathologies such as diabetes, kidney and liver diseases. Studying their impact separately may help to understand their sources and potential pathogenicity and further their evaluation as biomarkers.The present study examined semi-synthetic HSA isoforms to investigate independently their structure by means of advanced mass spectrometry techniques (LC-TOF-MS and ICP-MS), influence on the HSA binding/antioxidant activities using a binding capacity test, and potential impact on albumin quantification by a routine immunoturbidimetric assay.Applying different chemical reactions to a commercial HSA solution, we obtained different solutions enriched up to 53 % of native HSA, 78 % of acetylated HSA, 71 % of cysteinylated HSA, 94 % of oxidized HSA, 58 % of nitrosylated HSA and 96 % of glycated HSA, respectively. Moreover, the semi-synthetic isoforms showed differently altered binding capacities for a panel of ligands (Cu, Cd, Au, Ds and L-T4). Furthermore, immunoturbidimetry was found to be insensitive to the presence and abundance of the different isoforms.The fully characterized semi synthetic HSA isoforms obtained should be useful to further investigate their pathogenicity and potential roles as biomarkers.

RENAL4: Indoxyl Sulfate Removal by NORIT RO Carbon and the Adsorba 300 Hemoperfusion Column

Purpose: Indoxyl sulfate is a protein-bound uremic toxin typically cleared by the kidneys. In end-stage renal disease (ESRD) it accumulates, contributing to vascular disease and possible early mortality. A number of novel charcoal-based adsorbents remove indoxyl sulfate, but the FDA-approved Adsorba 300 column has never been tested for this. Indoxyl sulfate removal using a currently approved medical device would be of great practical value. Methods: We studied indoxyl sulfate removal from albumin solutions. We first used a charcoal column designed in our lab and a commercially available Norit RO 0.8 based charcoal. Norit RO 0.8 resembles the charcoal in the Adsorba 300. We tested bovine and human serum albumin solutions (BSA and HSA respectively). Indoxyl sulfate was measured by mass spectrometry. After this proof of concept, we used Adsorba 300 columns. Results: We found that adsorbent columns made with Norit RO 0.8 can remove indoxyl sulfate from BSA and HSA solutions. With the Adsorba 300 column indoxyl sulfate concentration fell below the limit of detection within 15 minutes from a BSA solution. Summary: The clinically available Adsorba 300 hemoperfusion column can remove the albumin bound uremic toxin indoxyl sulfate in vitro. This toxin is not dialyzable. This detoxification option may improve treatment of ESRD. In vivo testing should be considered.

Theoretical investigation on the effect of volume fraction in the optical trapping of gold nanoparticles

Stable optical trapping of gold nanoparticles is essential and desirable because of its wide applications in nanotechnology. While several factors have been proposed to affect optical trapping stability, the sample’s volume fraction during optical trapping has often been neglected. To address this, by utilizing the effective medium theory, we analyze the stability of optical trapping of a gold nanoparticle in human serum albumin solutions, HIV-1,virus solutions, and gold nanoparticle solutions in this article. Our comparative analysis of the optical force and potential on a single gold nanoparticle in solutions of varying volume fractions reveals that both parameters decrease with increasing volume fraction. This finding can aid in more effective control of gold nanoparticles in various applications.

PH-Responsible Doxorubicin-Loaded Fe3O4@CaCO3 Nanocomposites for Cancer Treatment

A magnetic nanocomposite (MNC) is an integrated nanoplatform that combines a set of functions of two types of materials. A successful combination can give rise to a completely new material with unique physical, chemical, and biological properties. The magnetic core of MNC provides the possibility of magnetic resonance or magnetic particle imaging, magnetic field-influenced targeted delivery, hyperthermia, and other outstanding applications. Recently, MNC gained attention for external magnetic field-guided specific delivery to cancer tissue. Further, drug loading enhancement, construction stability, and biocompatibility improvement may lead to high progress in the area. Herein, the novel method for nanoscale Fe3O4@CaCO3 composites synthesis was proposed. For the procedure, oleic acid-modified Fe3O4 nanoparticles were coated with porous CaCO3 using an ion coprecipitation technique. PEG-2000, Tween 20, and DMEM cell media was successfully used as a stabilization agent and template for Fe3O4@CaCO3 synthesis. Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) data were used for the Fe3O4@CaCO3 MNC's characterization. To improve the nanocomposite properties, the concentration of the magnetic core was varied, yielding optimal size, polydispersity, and aggregation ability. The resulting Fe3O4@CaCO3 had a size of 135 nm with narrow size distributions, which is suitable for biomedical applications. The stability experiment in various pH, cell media, and fetal bovine serum was also evaluated. The material showed low cytotoxicity and high biocompatibility. An excellent anticancer drug doxorubicin (DOX) loading of up to 1900 µg/mg (DOX/MNC) was demonstrated. The Fe3O4@CaCO3/DOX displayed high stability at neutral pH and efficient acid-responsive drug release. The series of DOX-loaded Fe3O4@CaCO3 MNCs indicated effective inhibition of Hela and MCF-7 cell lines, and the IC 50 values were calculated. Moreover, 1.5 μg of the DOX-loaded Fe3O4@CaCO3 nanocomposite is sufficient to inhibit 50% of Hela cells, which shows a high prospect for cancer treatment. The stability experiments for DOX-loaded Fe3O4@CaCO3 in human serum albumin solution indicated the drug release due to the formation of a protein corona. The presented experiment showed the "pitfalls" of DOX-loaded nanocomposites and provided step-by-step guidance on efficient, smart, anticancer nanoconstruction fabrication. Thus, the Fe3O4@CaCO3 nanoplatform exhibits good performance in the cancer treatment area.

Deep ensemble learning enables highly accurate classification of stored red blood cell morphology

Changes in red blood cell (RBC) morphology distribution have emerged as a quantitative biomarker for the degradation of RBC functional properties during hypothermic storage. Previously published automated methods for classifying the morphology of stored RBCs often had insufficient accuracy and relied on proprietary code and datasets, making them difficult to use in many research and clinical applications. Here we describe the development and validation of a highly accurate open-source RBC morphology classification pipeline based on ensemble deep learning (DL). The DL-enabled pipeline utilized adaptive thresholding or semantic segmentation for RBC identification, a deep ensemble of four convolutional neural networks (CNNs) to classify RBC morphology, and Kalman filtering with Hungarian assignment for tracking changes in the morphology of individual RBCs over time. The ensembled CNNs were trained and evaluated on thousands of individual RBCs from two open-access datasets previously collected to quantify the morphological heterogeneity and washing-induced shape recovery of stored RBCs. Confusion matrices and reliability diagrams demonstrated under-confidence of the constituent models and an accuracy of about 98% for the deep ensemble. Such a high accuracy allowed the CNN ensemble to uncover new insights over our previously published studies. Re-analysis of the datasets yielded much more accurate distributions of the effective diameters of stored RBCs at each stage of morphological degradation (discocyte: 7.821 ± 0.429 µm, echinocyte 1: 7.800 ± 0.581 µm, echinocyte 2: 7.304 ± 0.567 µm, echinocyte 3: 6.433 ± 0.490 µm, sphero-echinocyte: 5.963 ± 0.348 µm, spherocyte: 5.904 ± 0.292 µm, stomatocyte: 7.080 ± 0.522 µm). The effective diameter distributions were significantly different across all morphologies, with considerable effect sizes for non-neighboring classes. A combination of morphology classification with cell tracking enabled the discovery of a relatively rare and previously overlooked shape recovery of some sphero-echinocytes to early-stage echinocytes after washing with 1% human serum albumin solution. Finally, the datasets and code have been made freely available online to enable replication, further improvement, and adaptation of our work for other applications.