The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax.

Background/Objectives: The development of polymer–lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core–shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs’ preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery.

The aim of the article. The aim was to test the method for determining certain protein markers in rat urine by electrophoretic separation followed by mass spectrometric identification for early diagnostic of nephrotoxicity. The objectives of the study included assessing the reproducibility of the protein separation method, comparing rats urine protein profiles normal and after gentamicin sulfate administration, as well as a biochemical and morphological study of kidney biopsy specimens. Materials and methods. Gentamicin sulfate (GS) was administered intramuscularly to rats of both sexes at a dose of 60 mg/kg b.w. for consequence 5 days. Daily urine was collected in the background, on the 3rd, 7th and 14th day from the start of the administration of GS in the metabolic cages. We measured the concentrations of total protein and creatinine in urine samples, the level of lipids in the homogenates of the kidneys. Electrophoretic separation of urine proteins was performed in the Mini-PROTEAN Tetra Vertical Electrophoresis. The gels were stained, and the stained zones were excised and enzymatic hydrolysis of the proteins was performed. Spectra of tryptic peptides were recorded on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany). Proteins were identified in the Biotools program by accessing the Mascot database (matrixscience.com). Results. Assessment of reproducibility of the results of electrophoretic separation of rat urine proteins showed an acceptable value of the coefficient of variation (on average 8.03%) and the error of analysis (analysis = 14.53%). It that normally in rats of males the alpha-2-uroglobulin precursor dominates in urine and in females immunoglobulin G light chains dominate was show. Against the background of the administration of GS, leukocytes in pathological concentrations appear in the urine in rats of both sexes; in males, the proteomic profile has a strong intraspecific dispersion, including due to sperm contamination; in females, well identified zones of albumin, alpha-1-antitrypsin, aminopeptidase M, beta-2-microglobulin, and transferrin was appear. A biochemical study of kidney homogenates an increase in total cholesterol (males) and triacylglycerides (females) revealed. Pathomorphological changes were similar in male and female rats in the form of fatty degeneration of the proximal tubule nephrothelium and leukocyte infiltration of interstitium and confirmed changes in the spectrum of urine proteins. Conclusion. Based on the analysis of the experimental results it was found that the use of the technology of electrophoretic separation of proteins in PAGE under denaturing conditions followed by MALDI mass spectrometric identification (PAGE-MALDI-TOF/TOF) and densitometric determination of the percentage of protein fractions is applicable for the detection of nephrotoxicity. Due to gender differences in the protein spectrum it is preferable to examine the urine of female rats. Pathomorphological changes did not have gender differences.

BACKGROUND: The delivery of nucleic acids to mesenchymal stem cells, which are used as model objects in in vitro experiments or as therapeutic agents in regenerative medicine and oncology, is an actively developing area of research. Existing non-viral delivery systems either have low effectiveness or highly toxic to mesenchymal stem cells. Therefore, the development of new carriers has become an urgent priority. AIM: To demonstrate the feasibility of delivering model messenger RNA and small interfering RNA to rat heart mesenchymal stem cells (MSCs) in vitro using original cationic liposomes 2X3-DOPE (1:3 molar ratio) and to evaluate the influence of exosomes incorporated into hybrid nanoparticles with 2X3-DOPE on the efficiency of RNA delivery. METHODS: Exosomes were isolated using a standard ultracentrifugation technique followed by characterization of the obtained vesicles through Western blotting, transmission electron microscopy, atomic force microscopy, and hydrodynamic diameter measurement using dynamic light scattering. Small interfering RNA was chemically synthesized; whereas messenger RNA was obtained by in vitro transcription. Complexes of liposomes or hybrid nanoparticles with RNA were prepared by mixing; the properties of the resulting particles were assessed using dynamic light scattering and atomic force microscopy. To evaluate the efficiency of RNA delivery to rat heart mesenchymal stem cells derived from both healthy and ischemic myocardium, we used fluorescence microscopy, laser scanning confocal microscopy, and flow cytometry. RESULTS: Complexes of cationic liposomes 2X3-DOPE (1:3 molar ratio) with messenger RNA and 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) complexed with small interfering RNA were successfully prepared and characterized. It was demonstrated that 2X3-DOPE is ineffective for messenger RNA delivery to rat cardiac mesenchymal stem cells; whereas hybrid nanoparticles incorporating exosomes based on these liposomes exhibited up to 40% transfection efficiency. In addition, 2X3-DOPE modified with DSPE-PEG2000 (0.62 mol%) was effective for small interfering RNA delivery to rat cardiac mesenchymal stem cells, achieving up to 90% transfection efficiency; whereas the use of hybrid nanoparticles based on this formulation resulted in 100% transfected cells with more than a twofold increase in small interfering RNA in the cells as indicated by the average fluorescence intensity. CONCLUSION: Cationic liposomes 2X3-DOPE (1:3 molar ratio) modified with DSPE-PEG2000 (0.62 mol%) are promising vehicles for small interfering RNA delivery to mesenchymal stem cells, both independently and in combination with exosomes. Exosomes integrated in hybrid nanoparticles based on 2X3-DOPE improve the transfection efficiency of both messenger RNA and small interfering RNA in rat cardiac mesenchymal stem cells in vitro.

Viral, bacterial, and mixed acute respiratory infections are a global health challenge, claiming more than four million lives annually. This condition is particularly prevalent among infants, children, and the elderly, with the highest rates reporting in low- and middle-income countries. Acute respiratory infections are primarily caused by bacterial and viral pathogens. Therefore, for effective treatment, the pathogen should be accurately identified. Currently, viral and bacterial respiratory infections are mainly differentiated by clinical symptoms, blood leukocyte count with differential, erythrocyte sedimentation rate, and various laboratory parameters. However, these diagnostic tools are inadequate for accurate diagnosis because of their limited sensitivity and frequent lack of specificity. Viral infections significantly increase the likelihood of secondary bacterial infections, which tend to present with a more severe clinical profile and higher rates of complications. The underlying mechanisms of bacterial superinfections are multifactorial processes that involve interactions between viruses, bacteria, and the host immune system. Differentiation between viral and bacterial infections in patients with respiratory diseases is a significant diagnostic challenge that often leads to overuse of antibiotics. Although current biomarkers can evaluate the likelihood of bacterial infections, reliable biomarkers for viral infections have yet to be discovered. This diagnostic gap highlights the need for new methods, especially given the strict antibiotic stewardship regulations in many countries, which require precise differential diagnoses to prevent antibiotic misuse.

Вирус гриппа вызывает высококонтагиозное заболевание людей, птиц и млекопитающих. Подсчитано, что ежегодно от эпидемий гриппа погибает от 250 000 до 500 000 человек и эта цифра может увеличиваться до 1 млн в период возможных пандемий вируса гриппа. Самым эффективным путем профилактики болезни или ее тяжелых последствий является вакцинация. На данный момент существует несколько подходов для создания противогрипозных вакцин, но они имеют ряд недостатков, которые снижают общую эффективность вакцинных препаратов. Соответственно, создание универсальной вакцины, способной обеспечить надежную защиту от существующих и возможных реассортантных штаммов вируса гриппа, на данный момент является приоритетной задачей. Использование вакцин на основе рекомбинантных белков позволяет избежать рисков, связанных с введением вируса в организм, пусть и инактивированного. Данная работа посвящена получению высокоочищенного гибридного рекомбинантного белка, включающего фрагменты белков гемагглютинина вирусов гриппа А и В, а также компоненты флагеллина в качестве адъюванта. Указанные фрагменты белков представляют собой консервативные части гемагглютининов H1, H3, H5 и В, к которым в процессе естественной инфекции образуются специфичные антитела, перекрестно реагирующие с гомологичными эпитопами среди различных штаммов вирусов гриппа А и В. Выбранные участки белков наиболее иммуногенны. Их присутствие обеспечивает как В-клеточный, так и Т-клеточный иммунный ответ. Использование эпитопов нескольких белков позволяет увеличить эффективность вакцины, а использование гибких мостиков между эпитопами позволяет сохранить правильную пространственную укладку белка и, соответственно, обеспечивает полноценное функционирование каждого эпитопа. Гибридный белок, содержащий В- и Т-клеточные эпитопы разных субтипов вирусов гриппа А и В, а также флагеллин, позволят обеспечить сильный иммунный ответ при меньших затратах на производство. Нами был смоделирован гибридный рекомбинантный белок Flu-Chim, содержащий иммуногенные эпитопы вирусов гриппа А/H1N1, A/H3N2, A/ H5N1 и В, слитые с фрагментами флагеллина. Был создан высокопродуктивный штамм-продуцент, на основе клеток E. coli BL21(DE3). Была изучена экспрессия гибридного гена flu-chim при различных условиях индукции. Также был получен высокоочищенный гибридный рекомбинантный белок Flu-Chim с использованием металлоаффинной хроматографии. Чистота белка после финальной стадии очистки составила 98%. В дальнейшем планируется изучение иммунологических свойств полученного рекомбинантного белка.