Biological Media Research Articles

Fullerene C60-chitosan conjugate applied in the photoinactivation of Staphylococcus aureus

A novel fullerene/chitosan (C60-CS) conjugate was synthesized. The addition reaction of the amine groups of chitosan (CS) to fullerene (C60) was carried out in N,N-dimethylformamide (DMF), and triethylamine at room temperature. The conjugate C60-CS was purified by dialysis in water. C60-CS showed an absorption spectrum characteristic of modified fullerenes. The ability to produce O2(1Δg) was determined by the decomposition of 9,10-dimethylanthracene (DMA) in DMF, finding a quantum yield value of 0.07. Besides, to obtain information about the contributions of the photoreaction mechanisms, the L-tryptophan photodecomposition was studied in the presence of reactive oxygen species (ROS) scavengers, such as D-mannitol and sodium azide. The results indicated a significant contribution of the type II mechanism to the photooxidation of the amino acid. This effect was confirmed by studies of the kinetics of nitrotetrazolium blue photoreduction in the presence of nicotinamide adenine dinucleotide. This conjugate's photodynamic inactivation (PDI) capacity was investigated in Staphylococcus aureus. The photodynamic effect induced a 4 log decrease in cell viability when the cells were treated with 10 μM C60-CS and 30 min of irradiation with white light. The photoreaction mechanisms involved in the photosensitized inactivation of S. aureus by C60-CS were studied with the addition of azide ions, deuterated saline water, D-mannitol, and potassium iodide. It was confirmed that the photodynamic action of C60-CS was mainly mediated by type II photoprocess. Photooxidation of DMA induced by C60-CS in S. aureus cells indicated the production of O2(1Δg) in this biological medium. Finally, PDI studies were performed on agar surfaces, treating the cells with C60-CS before deposition on the surface. The subsequent inactivation of S. aureus sensitized by C60-CS after 30 min of irradiation with white light produced a marked elimination of bacteria as a function of increasing concentration. Therefore, this conjugate is a promissory photosensitizing agent to inactivate S. aureus.

Validation studies of the spiral plating method to determine the potency of lactobacillus-containing probiotics

Scientific relevance. Viable cell counting is an important microbiological test for the quality assessment of medicinal products containing live microorganisms. To optimise labour and material costs and enhance testing precision and reproducibility, it is practical to use partially automated instrumental methods, such as the spiral plating method.Aim. The aim was to conduct a validation study of the spiral plating method for assessing the potency of biologicals containing live bacterial cells, using a lactobacillus-containing probiotic medicinal product as a case study.Materials and methods. This study used a culture of Lactiplantibacillus plantarum isolated from a probiotic medicinal product. Spiral plating on agar-based biological culture media was performed using an automatic Eddy Jet 2 plating system. The study used an IUL Flash & Go colony counter for automatic reporting of the results. The validation study was conducted according to the general chapter on the validation of microbiological testing methods (OFS.1.1.0021.18) of the State Pharmacopoeia of the Russian Federation.Results. According to the validation results for the main parameters, the spiral plating method had the range of 104–105 CFU/mL; the limit of quantification of 102 CFU/mL; and the coefficient of linear determination, R2, of 0.99. The accuracy of the spiral plating method in determining the potency of the test sample was 93%; the repeatability was 4.9%.Conclusions. The study results confirm the similarity of the spiral plating method with an automatic colony counter to Koch’s plating method, which is currently used in accordance with the State Pharmacopoeia of the Russian Federation, in terms of the validated parameters. Therefore, spiral plating can be used to evaluate the potency of lactobacillus-containing probiotics. Spiral plating can help improve the cost-effectiveness and accuracy of testing.

Gold Nanoparticle-Loaded Porous Poly(ethylene glycol) Nanosheets for Electrochemical Detection of H2O2.

The effective detection of hydrogen peroxide (H2O2) in different environments and, above all, in biological media, is an important practical issue. To this end, we designed a novel electrochemical sensor for H2O2 detection by introducing gold nanoparticles (AuNPs) into the porous poly(ethylene glycol) (PEG) matrix formed by the thermally activated crosslinking of amino- and epoxy-decorated STAR-PEG precursors. The respective composite PEG-AuNP films could be readily prepared on oxidized Si substrates, separated from them as free-standing nanosheets, and transferred as H2O2 sensing elements onto the working electrode of the electrochemical cell, with the performance of the sensing element relied on the established catalytic activity of AuNPs with respect to H2O2 decomposition. The sensitivity, detection limit, and the operation range of the composite PEG-AuNP sensors were estimated at ~3.4 × 102 μA mM-1 cm-2, 0.17 μM of H2O2, and 20 μM-3.5 mM of H2O2, respectively, which are well comparable with the best values for other types of H2O2 sensors reported recently in literature. The particular advantages of the composite PEG-AuNP sensors are commercial source materials, a simple fabrication procedure, the bioinert character of the PEG matrix, the 3D character of the AuNP assembly, and the possibility of transferring the nanosheet sensing element to any secondary substrate, including the glassy carbon electrode of the electrochemical cell. In particular, the bioinert character of the PEG matrix can be of importance for potential biological and biomedical applications of the designed sensing platform.

Resveratrol and Resveratrol-Loaded Galactosylated Liposomes: Anti-Adherence and Cell Wall Damage Effects on Staphylococcus aureus and MRSA.

Antibiotic resistance due to bacterial biofilm formation is a major global health concern that makes the search for new therapeutic approaches an urgent need. In this context,, trans-resveratrol (RSV), a polyphenolic natural substance, seems to be a good candidate for preventing and eradicating biofilm-associated infections but its mechanism of action is poorly understood. In addition, RSV suffers from low bioavailability and chemical instability in the biological media that make its encapsulation in delivery systems necessary. In this work, the anti-biofilm activity of free RSV was investigated on Staphylococcus aureus and, to highlight the possible mechanism of action, we studied the anti-adherence activity and also the cell wall damage on a MRSA strain. Free RSV activity was compared to that of RSV loaded in liposomes, specifically neutral liposomes (L = DOPC/Cholesterol) and cationic liposomes (LG = DOPC/Chol/GLT1) characterized by a galactosylated amphiphile (GLT1) that promotes the interaction with bacteria. The results indicate that RSV loaded in LG has anti-adherence and anti-biofilm activity higher than free RSV. On the other side, free RSV has a higher bacterial-growth-inhibiting effect than encapsulated RSV and it can damage cell walls by creating pores; however, this effect can not prevent bacteria from growing again. This RSV ability may underlie its bacteriostatic activity.

Design, synthesis, chemical and biological evaluation of 2,5,5-trisubstituted-1,2-thiazepan-6-one 1,1-dioxides

In this article, we designed and synthesized a series of novel 2,5,5-trisubstituted (including spirocyclic) 1,2-thiazepan-6-one 1,1-dioxides (put simply, γ,γ-disubstituted β-keto ε-sultams), prepared a number of derivatives and evaluated their cytotoxic activity against MDA-MB-231 breast cancer cell line. In particular, alkylation of N-monosubstituted methanesulfonamides with α,α-disubstituted β-halogenated esters (including cyclic representatives) afforded the corresponding N-mesylated β-amino acid esters. The latters were involved in CSIC (Carbanion-mediated Sulfonamide Intramolecular Cyclization) reaction to give the target γ,γ-disubstituted β-keto ε-sultams (including spirocyclic representatives) in synthetically useful yields. This class of compounds can be considered as valuable building blocks since they possess carbonyl functionality and an EWG-activated methylene group capable of further functionalization. For instance, the condensation with DMFDMA afforded the corresponding α-dimethylaminomethylidene derivatives – the direct precursors for the heterocyclization reactions. Their treatment with hydrazine hydrate or guanidine hydrochloride provided the corresponding pyrazolo- and pyrimidofused ε-sultams. Despite the prepared β-keto ε-sultams showing weak cytotoxicity against the MDA-MB-231 breast cancer cell line, their pyrazolofused derivatives appeared perspective pharmacological templates with stable cytotoxic effects. Moreover, β-keto ε-sultams and their heterofused derivatives form water-soluble conjugates with branched polymers based on dextran-polyacrylamide (D-PAA) that can be used as the transport module for targeted drug delivery in biological media.

Ginger extract loaded Fe2O3/MgO-doped hydroxyapatite: Evaluation of biological properties for bone-tissue engineering.

Since antiquity, the medicinal properties of naturally sourced biomolecules such as ginger (Zingiber officinale) extract are documented in the traditional Indian and Chinese medical systems. However, limited work is performed to assess the potential of ginger extracts for bone-tissue engineering. Our work demonstrates the direct incorporation of ginger extract on iron oxide-magnesium oxide (Fe2O3 and MgO) co-doped hydroxyapatite (HA) for enhancement in the biological properties. The addition of Fe2O3 and MgO co-doping system and ginger extract with HA increases the osteoblast viability up to ~ 1.4 times at day 11. The presence of ginger extract leads to up to ~ 9 times MG-63 cell viability reduction. The co-doping does not adversely affect the release of ginger extract from the graft surface in the biological medium at pH 7.4 for up to 28 days. Assessment of antibacterial efficacy according to the modified ISO 22196: 2011 standard method indicates that the combined effects of Fe2O3, MgO, and ginger extract lead to ~ 82 % more bacterial cell reduction, compared to the control HA against S. aureus. These ginger extract-loaded artificial bone grafts with enhanced biological properties may be utilized as a localized site-specific delivery vehicle for various bone tissue engineering applications.

Fluorescent pH‐sensing bandage for point‐of‐care wound diagnostics

AbstractDiabetic foot ulcers (DFUs) are a serious and prevalent complication of diabetes. Current diagnostic options are limited to macroscopic wound analysis such as wound size, depth, and infection. Molecular diagnostics promise to improve DFU diagnosis, staging, and assessment of treatment response. Here, we developed a rapid and easy‐to‐use fluorescent pH‐sensing bandage for wound diagnostics. In a fluorescent dye screen, we identified pyranine as the lead compound due to its suitable pH‐sensing properties in the clinically relevant pH range of 6–9. To minimize the release of this dye into the wound bed, we screened a library of ionic microparticles and found a strong adhesion of the anionic dye to a cationic polymeric microparticle. These dye‐loaded microparticles showed a strong fluorescence response in the clinically relevant pH range of 6–9 and a dye release below 1% after 1 day in biological media. The dye‐loaded microparticles were subsequently encapsulated in a calcium alginate hydrogel to minimize the interaction of the microparticles with the wound tissue. This pH‐sensing diagnostic wound dressing was tested on full thickness dorsal wounds of mice, and a linear fluorescence response (R2 = 0.9909) to clinically relevant pH values was observed. These findings encourage further development of this pH‐sensing system for molecular diagnostics in DFUs.

Nanogels with covalently bound and releasable trehalose for autophagy stimulation in atherosclerosis

Atherosclerosis, cholesterol-driven plaque formation in arteries, is a complex multicellular disease which is a leading cause of vascular diseases. During the progression of atherosclerosis, the autophagic function is impaired, resulting in lipid accumulation-mediated foam cell formation. The stimulation of autophagy is crucial for the recovery of cellular recycling process. One of the potential autophagy inducers is trehalose, a naturally occurring non-reducing disaccharide. However, trehalose has poor bioavailability due to its hydrophilic nature which results in poor penetration through cell membranes. To enhance its bioavailability, we developed trehalose-releasing nanogels (TNG) for the treatment of atherosclerosis. The nanogels were fabricated through copolymerization of 6-O-acryloyl-trehalose with the selected acrylamide-type monomers affording a high trehalose conjugation (~ 58%, w/w). TNG showed a relatively small hydrodynamic diameter (dH, 67 nm) and a uniform spherical shape and were characterized by negative ζ potential (-18 mV). Thanks to the trehalose-rich content, TNG demonstrated excellent colloidal stability in biological media containing serum and were non-hemolytic to red blood cells. In vitro study confirmed that TNG could stimulate autophagy in foam cells and enhance lipid efflux and in vivo study in ApoE−/− mice indicated a significant reduction in atherosclerotic plaques, while increasing autophagic markers. In conclusion, TNG hold great promise as a trehalose delivery system to restore impaired autophagy-mediated lipid efflux in atherosclerosis and subsequently reduce atherosclerotic plaques.Graphical abstract

Fixation and Visualization of Full Protein Corona on Lipid Surface of Composite Nanoconstruction.

Spontaneous sorption of proteins on the nanoparticles' surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins-the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

Effects of friction stir processing and nano-hydroxyapatite on the microstructure, hardness, degradation rate and in-vitro bioactivity of WE43 alloy for biomedical applications

Nowadays, magnesium alloys are emerging in biomedical implants for their similar properties to natural bones. However, the rapid degradation of magnesium alloys in biological media hinders successful implantation. Refinement of microstructure, as well as reinforcement particles can significantly improve the degradation rate. In this work, multi-pass friction stir processing (FSP) was proposed to synthesize WE43/nano-hydroxyapatite (nHA) surface composite, the microstructure, reinforced particle distribution, micro-hardness, corrosion behavior and in-vitro bioactivity were studied. The subsequent FSP passes of WE43 alloy and WE43/nHA composite refined the grain size which was reduced by 94.29% and 95.92% (2.63 and 1.88 µm, respectively) compared to base metal after three passes. This resulted in increasing the microhardness by 120% (90.86 HV0.1) and 135% (105.59 HV0.1) for the WE43 and WE43-nHA, respectively. It is found that increasing FSP passes improved the uniform distribution of nHA particles within the composite matrix which led to improved corrosion resistance and less degradation rate. The corrosion rate of the FSPed WE43/nHA composite after three passes was reduced by 38.2% (4.13 mm/year) and the degradation rate was reduced by 69.7% (2.87 mm/y). This is attributed to secondary phase (Mg24Y5 and Mg41Nd5) particle fragmentation and redistribution, as well as a homogeneous distribution of nHA. Additionally, the growing Ca-P and Mg(OH)2 layer formed on the surface represented a protective layer that reduced the degradation rate. The wettability test revealed a relatively hydrophilic surface with water contact angle of 49.1 ± 2.2° compared to 71.2 ± 2.1° for base metal. Also, biomineralization test showed that apatite layer grew after immersion 7d in simulated body fluid with atomic ratio of Ca/P 1.60 approaching the stoichiometric ratio (1.67) indicating superior bioactivity of FSPed WE43/nHA composite after three passes. These results raise that the grain refinement by FSP and introduction of nHA particles significantly improved the degradation rate and in-vitro bioactivity of WE43 alloy for biomedical applications.

Designed Polyhydroxyproline Helical Peptide with Ultrarobust Antifouling Capability for Electrochemical Sensing in Diverse Complex Biological Fluids.

Developing a generalized strategy for the nonfouling detection of biomarkers in diverse biological fluids presents a significant challenge. Herein, a polyhydroxyproline helical peptide (PHHP) was designed and adopted to fabricate electrochemical microsensors capable of detecting targets in various biological media. The PHHP possessed unique properties such as strong hydrophilicity, rigid structure, and lack of ionizable side-chain groups. Compared with common zwitterionic peptides (ZIPs), the PHHP exhibited similar antifouling capability but exceptional stability, allowing its antifouling performance to be unaffected by environmental alteration. The PHHP can prevent biofouling even in fluctuating pH conditions, high ionic strength environments, and the presence of high-valence ions and resist the protease hydrolysis. The PHHP-modified carbon fiber microelectrode was further immobilized with an aptamer to construct an antifouling microsensor for cortisol detection across diverse biofluids, and the microsensor exhibited acceptable accuracy and higher sensitivity than the ELISA method. In addition, different biological samples of mice were collected in situ using a microsensing device, and cortisol levels were analyzed in each specifically tailored region. This nonfouling sensing strategy based on PHHP allows a comprehensive assessment of biomarkers in both spatial and temporal dimensions in diverse biological environments, holding promising potential for early disease diagnosis and real-time health monitoring.

Using SRIM code for calculating Bragg curve, projected range, straggling of 12C ion in biological materials

‘The Stopping and Range of Ions in Matter’ SRIM program, utilizing the built-in compound directory has been used to compute Bragg curve, projected range, longitudinal, lateral straggle of 12C ion with energies of 1 − 150 MeV in biological matter: liquid water (benchmark) human blood, skin, thyroid, and spleen are assessed. Results show that in the studied biological media for higher energy beam i): noticeable modification are seen in Bragg peak, shift in peak-position; ii) maximum projected range: 595.2 μm suggests effective use of 12C ions for cancer tumor therapy, especially for superficial cancer tumors, without much damage to healthier cells; iii) longitudinal straggling ~ 22.6 μm, low lateral straggling ~ 4.7 μm suggests potential application of 12C ions in treating cancer tumor nearby critical organs, causing minimal damage in nearby normal tissues.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Biocompatible Ferrofluid-Based Millirobot for Tumor Photothermal Therapy in Near-Infrared-II Window.

Ferrofluidic robots with excellent deformability and controllability have been intensively studied recently. However, most of these studies are in vitro and the use of ferrofluids for in vivo medicinal applications remains a big challenge. The application of ferrofluidic robots to the body requires the solution of many key problems. In this study, biocompatibility, controllability, and tumor-killing efficacy are considered when creating a ferrofluid-based millirobot for in vivo tumor-targeted therapy. For biocompatibility problems, corn oil is used specifically for the ferrofluid robot. In addition, a control system is built that enables a 3D magnetic drive to be implemented in complex biological media. Using the photothermal conversion property of 1064nm, the ferrofluid robot can kill tumor cells in vitro; inhibit tumor volume, destroy the tumor interstitium, increase tumor cell apoptosis, and inhibit tumor cell proliferation in vivo. This study provides a reference for ferrofluid-based millirobots to achieve targeted therapies in vivo.

A mathematical model for simulating photoacoustic signal generation and propagation in biological tissues

Photoacoustic (PA) medical imaging is a crossbred technique relying on light-induced ultrasonic waves due to the PA effect phenomenon recorded primarily in 1880 by A. G. Bell. Numerical simulation, also known as in-silico, studies assist scientists in minimizing incorrect PA experiments in both in-vitro and in-vivo. Numerical modeling techniques help to achieve a fast simulation process in contrast to pure mathematics alone. However, if a suitable facilitated mathematical model can be established prior to applying numerical modeling, it will be of great interest to the whole numeric model. Numerous mathematical equations, theorems, and propositions have been proposed to model the whole PA signal generation and propagation process in biological media. However, most of them are complicated and difficult to be understood by researchers, especially beginners. That’s why this paper was introduced. Our paper aims to simplify the understanding of the generation and propagation process of biomedical PA waves. We have developed a facilitated mathematical model for the entire process. The introduced developed mathematical model is based on three steps: (1) pulsed laser stimulation, (2) light diffusion, and (3) PA stress wave generation and propagation. The developed mathematical model has been implemented utilizing COMSOL Multiphysics, which relies on the finite element method (FEM) numerical modeling principle. The in-silico time-dependent study's results confirmed that the proposed mathematical model is a simple, efficient, accurate, and quick starting point for researchers to simulate biomedical PA signals' generation and propagation process utilizing any suitable software such as COMSOL multiphysics.

Acetylacetonate Ruthenium Nitrosyls: A Gateway to Nitric Oxide Release in Water under Near-Infrared Excitation by Two-Photon Absorption.

A fundamental challenge for phototriggered therapies is to obtain robust molecular frameworks that can withstand biological media. Photoactivatable nitric oxide (NO) releasing molecules (photoNORMs) based on ruthenium nitrosyl (RuNO) complexes are among the most studied systems due to several appealing features that make them attractive for therapeutic applications. Nevertheless, the propensity of the NO ligand to be attacked by nucleophiles frequently manifests as significant instability in water for this class of photoNORMs. Our approach to overcome this limitation involved enhancing the Ru-NO π-backbonding to lower the electrophilicity at the NO by replacing the commonly employed 2,2'-bipyridine (bpy) ligand by an anionic, electron-rich, acetylacetonate (acac). A versatile and convenient synthetic route is developed and applied for the preparation of a large library of RuNO photoNORMs with the general formula [RuNO(tpy)(acac)]2+ (tpy = 2,2':6',2″-terpyridine). A combined theoretical and experimental analysis of the Ru-NO bonding in these complexes is presented, supported by extensive single-crystal X-ray diffraction experiments and by topological analyses of the electron charge density by DFT. The enhanced π-back-bonding, systematically evidenced by several techniques, resulted in a remarkable stability in water for these complexes, where significant NO release efficiencies were recorded. We finally demonstrate the possibility of obtaining sophisticated water-stable multipolar NO-delivery platforms that can be activated in the near-IR region by two-photon absorption (TPA), as demonstrated for an octupolar complex with a TPA cross section of 1530 GM at λ = 800 nm and for which NO photorelease was demonstrated under TPA irradiation in aqueous media.

Pharmacokinetics of a New Dimethylamineethanol Derivative Administered Orally to Laboratory Animals

A new derivative of dimethylaminoethanol, butanedioic and trans-butenedioic acids (laboratory code ADK-17) was synthesized at the Organic Chemistry Department of the Saint Petersburg State Chemical and Pharmaceutical University (SPSPU). This promising nootropic and anti-asthmatic agent is planned for use in an oral dosage form. The aim was to calculate the pharmacokinetic parameters of the synthesized compound and to determine its possible biotransformation pathways. Rabbits of the Soviet Chinchilla breed were used as a test system in the preclinical study of the compound pharmacokinetics. In accordance with the designed schedule, blood was taken from the marginal vein of the ear, and urine samples were taken. Quantitative determination in biological media was carried out using an Ultimate 3000 liquid chromatograph with a Q-Exactive mass-selective detector with electrospray ionization in accordance with OFS 1.2.1.100015 (Chromatography) of the State Pharmacopoeia by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS). To study the pharmacokinetics of the ADK-17 preparation, a measurement procedure was developed with a detection limit in biological media ranging from 10-6 to 10-3 mg/ml. The determined harmacokinetic parameters show that the absorption of ADK-17 into the systemic circulation from the gastrointestinal tract occurs at an average rate, with its maximum concentration in blood plasma during the oral route of administration being observed between 60–90 min. The elimination half-life from the systemic circulation comprises 4.3 h. Urinary excretion in the first 4 h proceeds at a high rate; however, the compound is detected in the urine within 3 days and in the blood within 2 days from the moment of administration.

A DNA micro-complex containing polyaptamer for exosome separation and wound healing

Exosomes (EXOs) have showed great potential in regenerative medicine. The separation of EXOs from complex biological media is essential for the down-stream applications. Herein, we report a deoxyribonucleic acid (DNA)-based micro-complex (DMC) containing polyaptamers, which realized the specific separation of EXOs from cell culture media and the significant promotion of wound healing. The synthesis of DMCs was based on a biomineralization process via rolling circle amplification (RCA) under the catalysis of phi29 DNA polymerase. To endow DMCs with the ability to capture EXOs, the DNA template of RCA was integrated with complementary sequence of aptamer that specifically recognized the CD63 proteins on EXOs. The obtained DMCs contained polyaptamers that can specifically capture the EXOs in cell culture media. The EXOs-capturing DMCs were collected by centrifugation, achieving the separation of EXOs. Mesenchymal stem cell (MSC)-derived EXOs (MSC-EXOs) were separated by this DMC-based strategy, and the separated MSC-EXOs significantly enhanced the migration ability of cells. In particular, the significant therapeutic efficacy of the DMCs with MSC-EXOs was verified in full-thickness wound excision mouse models, in which the wounds completely healed in 10 days. We envision that this DMC-based separation strategy can be a promising route to promote the development of EXOs in biomedicine.

Biomarkers of stress in children with asthenic syndrome associated with aluminum contamination in biological media and A/G polymorphism of the HTR2A (rs7997012) serotonin receptor gene

Introduction. Ambient air pollution with heavy metals, included aluminum, creates their elevated levels in children and leads to rather early decompensation of weak genetically heritable systems of the body including the nervous system.
 Materials and methods. We examined eighty six 4–7 years children with asthenic syndrome. Fifty two of them (the test group) lived and attended preschool facilities in a zone influenced by emissions from aluminum production (a dose created by a non-ferrous metallurgic plant is 5.65•10–5 mg/(kg•day)). The reference group was made of 34 children who lived on a conditionally clean territory and were exposed to airborne aluminum in a dose of 3.0•10–6 mg/(kg•day). Within this study, there were examined several children’s biological media including urine (aluminum), blood (serotonin and cortisol), and buccal epithelium (HTR2A rs7997012 gene). Differences were considered to be significant at a p<0.05.
 Results. We established statistically significantly higher aluminum levels in urine (1.3 times higher) in the test group against the reference one (0.012±0.0001 and 0.0092±0.0012 respectively); authentically higher prevalence of the minor allele G of the HTR2A (rs7997012) serotonin receptor gene (authentically 1.6 times higher in the test group against the reference one). Both these factors create an elevated risk of asthenic syndrome, 1.86 times higher in the test group (OR=1.86; 95%CI (1.02–3.67)) together with lower serotonin levels (130.28±32.42 ng/ml and 183.35±34.57 ng/ml respectively) and higher cortisol levels (278.08±25.90 nmol/cm3 and 191.28±21.62 nmol/cm3 respectively).
 Limitations. The limitation is application of the identified stress biomarkers for diagnostics of nervous system disorders that manifest themselves in children through asthenic syndrome associated with exposure to aluminum.
 Conclusion. A/G replacement of the HTR2A (rs7997012) gene creates a 1.86 times higher risk of asthenic syndrome (OR=1.86; 95%CI (1.02–3.67)) for children with elevated aluminum levels in urine under inhalation exposure to it in a dose equal to 5.65•10–5 mg/kg•day). The disease would have such a peculiarity as longer recurrence and its biomarkers include an imbalance between indicators of a developing stress, cortisol, and serotonin.

The origins of nonideality exhibited by monoclonal antibodies and Fab fragments in human serum.

The development of therapeutic antibodies remains challenging, time-consuming, and expensive. A key contributing factor is a lack of understanding of how proteins are affected by complex biological environments such as serum and plasma. Nonideality due to attractive or repulsive interactions with cosolutes can alter the stability, aggregation propensity, and binding interactions of proteins in solution. Fluorescence correlation spectroscopy (FCS) can be used to measure apparent second virial coefficient (B2,app ) values for therapeutic and model monoclonal antibodies (mAbs) that capture the nature and strength of interactions with cosolutes directly in undiluted serum and similar complex biological media. Here, we use FCS-derived B2,app measurements to identify the components of human serum responsible for nonideal interactions with mAbs and Fab fragments. Most mAbs exhibit neutral or slightly attractive interactions with intact serum. Generally, mAbs display repulsive interactions with albumin and mildly attractive interactions with IgGs in the context of whole serum. Crucially, however, these attractive interactions are much stronger with pooled IgGs isolated from other serum components, indicating that the effects of serum nonideality can only be understood by studying the intact medium (rather than isolated components). Moreover, Fab fragments universally exhibited more attractive interactions than their parental mAbs, potentially rendering them more susceptible to nonideality-driven perturbations. FCS-based B2,app measurements have the potential to advance our understanding of how physiological environments impact protein-based therapeutics in general. Furthermore, incorporating such assays into preclinical biologics development may help de-risk molecules and make for a faster and more efficient development process.