HEK293 Cell Lines Research Articles

Metabolic activation of WHO-congeners PCB28, 52, and 101 by human CYP2A6: evidence from in vitro and in vivo experiments.

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4'-trichlorobiphenyl (PCB28), 2,2',5,5'-tetrachlorobiphenyl (PCB52), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61µg/L (5,89nM) for PCB28, 316.98µg/L (1,03µM) for PCB52, and 151.1µg/L (441nM) for PCB101 from a 20µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners.

Eco-conscious synthesis of novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives as potent Anti-microbial agent and comparative study of cell viability and cytotoxicity in HEK-293 cell line utilizing Indian gooseberry (Phyllanthus emblica) fruit extract

Antimicrobial resistance (AMR) is a pressing global health challenge that necessitates the search for novel antimicrobial agents and synthetic methodologies. This study investigates the synthesis and antimicrobial efficacy of 25 novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives, catalyzed by Amla (Phyllanthus emblica) fruit juice, which is rich in organic acids and polyphenolic compounds, thus serving as an environmentally sustainable catalyst, in adherence to green chemistry principles. The synthesis is achieved through a one-pot, solvent-free process that yields high quantities of the desired compounds in significantly less time compared to traditional methods. Comprehensive antimicrobial evaluation was conducted against a range of clinically relevant microorganisms, including Chromobacterium violaceum, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Cryptococcus neoformans and Aspergillus niger. Concurrently, cytotoxic assays were performed on HEK-293 cells, to assess safety profiles, revealing that compounds B-1, B-6, B-7, B-14 and B-15 exhibited potent antimicrobial activity while maintaining low cytotoxicity and high cell viability. These findings underscore the therapeutic potential of the synthesized compounds in combatting infectious diseases and addressing the challenges posed by AMR, highlighting the critical importance of dose optimization in therapeutic applications. This study combats contagious diseases, mitigates AMR challenges and contributes significantly to the field of antimicrobial drug discovery, emphasizing the need for sustainable synthetic strategies that align with future pharmaceutical endeavors. Our research not only advances the understanding of these novel compounds but also supports ongoing efforts to develop safe and effective therapies against resistant pathogens.

Chemotherapeutic Drug Delivery Nanoplatform Development: From Physicochemical to Preclinical Evaluation

Through this study, the synergistic behavior of small-molecular-weight, amphiphilic surfactant molecules and the triblock copolymer Pluronic 188 was extensively evaluated based on their ability to formulate nanocarriers with novel properties for the delivery of class II and IV (biopharmaceutical classification system) chemotherapeutic compounds. The combination of four different surfactants at multiple weight ratios and twelve initially formulated nanosystems resulted in four hybrid delivery platforms, which were further studied in terms of multiple physicochemical characteristics, as well as their stability in protein-rich media (fetal bovine serum/phosphate-buffer saline). Finally, we obtained a single final nanoformulation that exhibited a high loading capacity (%EE ≥ 75%) and a sustained drug release profile under physiological conditions (model drug methotrexate), without altering the original physicochemical characteristics of the carrier. With a mean hydrodynamic radius (Rh) of less than 70 nm, a polydispersity index of 0.219, and no protein complexation, the system is a suitable candidate for in vivo, intravenous, and/or intramuscular administration. The cytotoxicity and genotoxicity of both loaded and unloaded carriers were evaluated through the examination of the upregulation or downregulation of apoptosis-related pathways. Multiple conventional 2D and 3D spheroidal conformations were used for these assessments, including HEK293, HCT-116, and MCF-7 cell lines, the results of which stressed the safety and biocompatibility of the empty nanocarrier. Additionally, experiments on Caenorhabditis elegans were conducted to evaluate the system’s in vivo toxicity, focusing on developmental stages, egg-laying behavior, and locomotion. Nanosystems studied in terms of chemotherapeutic encapsulation have mostly focused on the physiochemical aspect of the development of such novel delivery platforms, with only few exceptions proceeding step-by-step from cellular 2D to 3D to in vivo experimentation. The present study offers a holistic view of the behavior of such a novel system, advancing our understanding of the capabilities of polymeric/surfactant-based nanodelivery platforms.

Anti-cancer Activity Evaluation of Naphthalenonic and Chromanonic Spiroisoxazoline Derivatives as Selective COX-2 Inhibitors on HT29 Cell Lines

Background: Cyclooxygenase-2 (COX-2) is induced in response to proinflammatory conditions, and it is not only a key enzyme in the inflammatory process, but also seems to be highly expressed in various types of cancer cells. On the other hand, it is well documented that chemical compounds with spiro scaffolds in their structure could be effective chemical agents against cancer types. Objective: In this study, the cytotoxicity effects of spiroisoxazoline derivatives containing naphthalinone and chromanone spiro-bridge were investigated. Methods: The cytotoxicity effects of compounds 7a-7h were evaluated by performing the MTT assay on the HT-29 (colorectal cancer), MCF-7 (breast cancer), and HEK-293 (normal kidney) cell lines. After that, a compound with high yield and remarkable cytotoxic activity was selected to analyze the cell cycle and apoptosis mechanism. Results: The most effective cytotoxic activity was observed on HT-29 and MCF-7 cell lines of compounds 7b (IC50 value: 1.07±0.28 µM) and 7f (IC50 value: 11.92±1.07 µM). None of the compounds had a toxic effect on normal HEK-293 cells, except for compound 7g with an IC50 value of 21.30±16.14 µM, whose effect was much lower than that of cisplatin and doxorubicin, known as anti-cancer agents. Subsequently, compound 7e with significant yield and cytotoxic activity was investigated to evaluate cell cycle and apoptosis. The result showed that compound 7e induced significant G0/G1 cell cycle arrest and apoptosis in HT-29 cells Conclusion: The selective COX-2 inhibitor compounds with spiroisoxazoline core structure could be suitable scaffolds for cytotoxic effects.

Discovery of a novel benzimidazole conjugated quinazolinone derivative as a promising SARS-CoV-2 3CL protease inhibitor.

This report presents the design and synthesis of quinazolinone-based derivatives as promising SARS-CoV-2 3CL protease inhibitors. Two novel series, namely, febrifugine analogues 4a-i and quinazolinone conjugated benzimidazoles 9a-c, were successfully synthesized starting from isatoic anhydride. The synthesized quinazolinone derivatives were evaluated for their cytotoxicity against cancer cell lines and SARS-CoV-2 3CL inhibitory activity. The results showed that the synthesized compounds did not have significant toxicity for the non-cancer HEK293 cell line and MCF-7, MDA-MB-231, HEPG2 and HEPG2.2.15 cancer cell lines. Notably, compound 9b exhibited anti-3CL enzymatic activity in a dose-dependent manner, with the calculated IC50 value of 10.73 ± 1.17 μM. Docking results highlighted the interaction between 9b and 3CL protease through hydrogen bonding with key amino acids, including His41, Met49, Cys145, Met165, Arg188, His164, and Glu166, at the active site of the protease. Pharmacokinetic studies and ADME analyses provide valuable insights into the potential of compound 9b as a drug candidate. These findings support the new scaffold as a candidate for 3CLpro inhibition and advanced anti-coronavirus drug research.

Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.

Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.

GATA1 transcription factor targets the gene expression of B19 virus in HEK293 cell line

Backgrond/Aim: B19 virus (B19V) is a single strand DNA virus that has specific tropism to erythroid progenitor cells (EPCs). The virus enters to the cells via P antigen and coreseptors and induces infection and cell apoptosis. GATA1 has a high expression in EPC and is a critical transcription factor for the cells development and differentiation. As human EPCs are the main target of the virus infection that have high expression of GATA-1 as the critical transcription factor, the aim of this study was to investigate the effect of GATA1 cotransfection with B19V genome on expression of the viral mRNAs in HEK293 as non-permissive cell line to the virus that had no mRNA expression of GATA-1. Methods: HEK293 cells were transfected with pHI0 plasmid containing B19V genome and the plasmid of GATA1 genome. The quantity of B19V mRNAs (NS1, 7.5 kDa, 11 kDa) expression was evaluated after 24h of transfection. Results: The results showed a statistically significant increase in fold change expression of (NS1 ∽ 12.3, VP1 ∽ 27.6, 11kb protein ∽ 38) in cotransfected cells with GATA1 and B19 plasmids compare to control group (P<0.05). Conclusion: This research showed transfected cells with GATA1had elevation in expression of the B19V genes mRNAs in a non-permissive cell. This result may show a role of GATA1 as a critical transcription factor in support of the virus infection in EPCs. This suggests that GATA1 may potentially sport B19V replication or gene expression.

Physico-chemical properties and biological evaluation of graphene quantum dots for anticancer drug susceptibility

Graphene quantum dots (GQDs) possess unique optical and biocompatible properties, making them suitable candidates for biomedical and pharmaceutical applications. This study reports the hydrothermal synthesis of pristine-GQD and doped variants: Nitrogen-GQD and Sulfur-GQD. The materials underwent thorough characterization techniques such as UV–vis, fluorescence, XRD, FE-TEM/SEM, EDX, and Raman spectroscopy. The particle sizes of these GQDs range from 2 to 5 nm. We conducted a comprehensive study through MTT assays to evaluate the potential cytotoxic effect of GQD and the doped variants. This study demonstrated their synergistic interactions with an anti-cancer drug, methotrexate (MTX), and also improvement of cytocompatibility in the presence of folic acid (FA). Systematic MD simulations revealed a compacting effect on the dynamic behavior of GQD and its variants in the presence of drugs. Fluorescence spectroscopy and computational modeling suggest non-intercalative surface interactions between GQDs and the drugs. The cytotoxic activity of pristine GQD on HeLa cervical cancer cells is higher than that of N-GQD and S-GQD. When treated with GQD-IC50–MTX-IC50, only 5.6 % of HeLa cells remained viable. The doped variants exhibited bio-compatibility when tested on normal HEK cell lines. Overall, this study emphasizes the potential of GQDs for targeted cancer therapy through an interdisciplinary approach involving material characterization, computational modeling, and biological assays.

Study of TRAIL and SAHA Co-Treatment on Leukemia K562 Cell Line.

TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) is an attractive agent being considered a potential cancer treatment. It attaches to its death receptors, leading many cancer cells to apoptosis. However, some malignancies indicate substantial resistance to TRAIL, challenging anticancer scientists. Herein, combination therapy with TRAIL plus SAHA (Suberoyl Anilide Hydroxamic Acid) was conducted to evaluate the capability of SAHA to overcome TRAIL resistance in the leukemia K562 cell line. First, the IC50 for SAHA was calculated (2 µM) at 12, 24, 48, and 72 h of treatment using MTT assay. Second, the K562 cells were treated with concentrations of 50 and 100 nM of TRAIL and 2 μM of SAHA separately and together for 24, 48, and 72 h and the survival of these cells was evaluated by Flowcytometry following the annexin-V and PI staining. To demonstrate the non-toxicity of the combined treatment for normal cells, the HEK-293 cell line was treated with the TRAIL 100 nM and SAHA 2 μM combined and separated at the same periods. In the end, by performing real-time PCR, the amount of candidate genes' expression implicated in TRAIL resistance, and the levels of BCR-ABL expression was measured. The drug dosages were not toxic to normal cells. SAHA plus TRAIL strongly triggered apoptosis in K562 cells after 24, 48, and 72 h of exposure. Furthermore, it was shown that DR4, DR5, and CHOP expressions were enhanced, and PI3K, Akt, ERK, STAT3, c-FLIPL, NF-κB, and BCR-ABL expressions were decreased by SAHA in K562 cells. Our study indicated that SAHA combined with TRAIL can increase the sensitivity of K562 leukemic cells to TRAIL by suppressing intracellular anti-apoptotic molecules and augmenting the expressions of DR4/DR5 and CHOP.

Investigating the p21 Ubiquitin-Independent Degron Reveals a Dual Degron Module Regulating p21 Degradation and Function.

A group of intrinsically disordered proteins (IDPs) are subject to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDPs/IDRs are targeted to the 20S proteasome via interaction with the C-terminus of the PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using a split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interactions with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

Dimerization and lysine substitution of melittin have differing effects on bacteria.

Melittin is a potent antimicrobial peptide from bee venom that is effective against both Gram-positive and Gram-negative bacteria. However, it is extremely toxic to mammalian cells and, as yet, has no clinical use. Modifications to its amino acid sequence, cyclization, truncation, and dimerization have been attempted in order to reduce its toxicity whilst maintaining its antimicrobial activity. In this study, we targeted the three lysine residues present in melittin and substituted them with lysine homologs containing shorter side chains (ornithine, Orn, diaminobutyric acid, Dab, and diaminopropanoic acid, Dap) and made both parallel and antiparallel melittin dimers to observe how lysine substitution and dimerization affects its activity and toxicity. The antibacterial activity of melittin and its analogs was tested against S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria), and cytotoxicity was tested against the mammalian cell lines HEK293 and H4IIE. Overall, dimerization and lysine substitution exhibited improved antimicrobial activity toward E. coli and limited improvement toward S. aureus. However, mammalian cell toxicity was only marginally reduced compared to native melittin. Interestingly, the parallel dimer was found to be marginally more active than the antiparallel dimer, indicating orientation maybe important for activity, although both dimers were less effective than the native and Lys-analog peptides toward S. aureus. Of the Lys substitutions, Dab and Dap improved melittin's activity toward E. coli. Dimerization and Lys substitution of melittin improved the antimicrobial activity toward Gram-negative bacteria but did not significantly improve its activity toward Gram-positive bacteria. Some analogs also displayed reduced toxicity toward HEK293 and H4IIE cells but overall remained toxic at bactericidal concentrations. Our data indicates that although highly antibacterial, melittin's toxicity is the major drawback in its potential use.

Complement biosensors identify a classical pathway stimulus in complement-mediated thrombotic microangiopathy

AbstractComplement-mediated thrombotic microangiopathy (CM-TMA) or hemolytic uremic syndrome, previously identified as atypical hemolytic uremic syndrome, is a TMA characterized by germ line variants or acquired antibodies to complement proteins and regulators. Building upon our prior experience with the modified Ham (mHam) assay for ex vivo diagnosis of complementopathies, we have developed an array of cell-based complement “biosensors” by selective removal of complement regulatory proteins (CD55 and CD59, CD46, or a combination thereof) in an autonomously bioluminescent HEK293 cell line. These biosensors can be used as a sensitive method for diagnosing CM-TMA and monitoring therapeutic complement blockade. Using specific complement pathway inhibitors, this model identifies immunoglobulin M (IgM)–driven classical pathway stimulus during both acute disease and in many patients during clinical remission. This provides a potential explanation for ∼50% of patients with CM-TMA who lack an alternative pathway “driving” variant and suggests at least a subset of CM-TMA is characterized by a breakdown of IgM immunologic tolerance.

Pinonic Acid Derivatives Containing Thiourea Motif: Promising Antifungal Lead Compound Targeting Cellular Barrier of Colletotrichum fructicola.

In order to explore novel antifungal lead compounds from plant essential oil, thirty-two pinonic acid derivatives containing thiourea groups were designed and synthesized using α-pinene as a raw material. One of these pinonic acid derivatives compound 3a exhibited noteworthy in vitro antifungal activity against Colletotrichum fructicola (EC50 = 9.22 mg/L), which was comparable to that of the positive control kresoxim-methyl (EC50 = 9.69 mg/L). Structure-activity relationship (SAR) studies demonstrated that the introduction of thiourea groups, F atoms, and Cl atoms into the structure of pinonic acid derivatives significantly improved their antifungal activity. The in vivo antifungal test revealed that compound 3a could effectively control pear anthracnose. It also proved that compound 3a showed low acute oral toxicity to honeybees (LD50 > 100 μg/bee) and low or no cytotoxicity to LO2 and HEK293 cell lines. The preliminary mechanism of action studies revealed that compound 3a caused mycelium deformity, increased cell membrane permeability, blocked the normal process of phospholipase C on the cell membrane, and reduced mycelium protein content. The results of molecular docking studies demonstrated the stable binding of compound 3a to phospholipase C and chitin synthetase. This study suggested that compound 3a could be used as a promising lead compound for the development of novel antifungal agents targeting the cellular barrier of C. fructicola.

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

Evaluation of thiopyrano[2,3-d]thiazole derivatives as potential anticonvulsant agents.

Anticonvulsant drug discovery has achieved significant progress; however, pharmacotherapy of epilepsy continues to be a challenge for modern medicine and pharmacy. To expand the chemical space of heterocycles as potential antiepileptic agents, herein we report on the synthesis and evaluation of anticonvulsant properties of a series of thiopyrano[2,3-d]thiazoles. The studied heterocycles are characterized by satisfactory drug-likeness and pharmacokinetics properties, calculated in silico using SwissADME. The anticonvulsant activity of thiopyrano[2,3-d]thiazole derivatives was evaluated in vivo using the subcutaneous pentylenetetrazole test. Three hits, that is, compounds 12, 14, and 16, that caused a pronounced anticonvulsant effect were identified. Derivatives 12, 14, and 16 positively affected the latent period of onset of clonic seizures, number of seizures, mortality rate, and duration of the seizure period of animals under experimental conditions. The anticonvulsant properties of compound 14 were equivalent to the effect of the reference drug, sodium valproate. All hit compounds are characterized by satisfying toxicity properties in the human lymphocytes and HEK293 cell line. The most active hit 14 possesses a potential affinity with the GABAA receptor in the molecular docking study and forms a stable complex in the molecular dynamics experiments equal to diazepam. Preliminary SAR results were obtained and discussed based on screening data.

Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines.

Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to a continued need for diagnostic and therapeutic tools to monitor and respond to new variants. Glycosylation of the spike protein is critical for the protein's functions in viral attachment and host cell entry. For scalable and cost-effective production of the spike protein, expression system-driven divergence in glycosylation patterns on recombinant spike proteins needs to be fully understood. This study assessed the N-glycosylation profiles of a full-length trimeric spike protein expressed in either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using a tandem mass spectrometry workflow and BioPharma incorporating HEK and CHO glycan databases for protein characterisation. The results outline important differences in the variety and types of N-glycan generated by the two cell lines across the 22 known N-glycosylation sites of the spike protein. A notable increase in terminal sialylation, as well as the presence of the potentially immunogenic N-glycolylneuraminic acid at a functionally key N-glycosylation site, was observed in the CHO-S derived spike protein. With the potential for the relatively vast and more complex CHO glycan repertoire (182 glycans relative to 39 human glycans) to produce functional implications with CHO-S expressed spike protein, this study adds valuable knowledge to aid Quality by Design approaches and enable Multi Attribute Monitoring of specific N-glycosylation sites for proteoform analyses. This can further inform antigen development with future variants in order to devise updated diagnostic tests and therapeutic vaccine designs.

Comparison of the effects of photodynamic exposure with the use of chlorine E6 on glioblastoma cells of the U251 line and human embryonic kidney cells of the HEK293 line in vitro

Malignant gliomas of the brain are a global medical and social problem with a trend toward a steady increase in morbidity and mortality rates. A method that enables the visual identification of tumor tissue and simultaneously selectively destroys it is photodynamic therapy, which involves the introduction of a photosensitizer (PS) followed by its activation at a certain wavelength of light. The selectivity of the accumulation of PS in the tumor tissue of the malignant gliomas is one of the key issues in the problem of increasing the effectiveness of photodynamic therapy. Objective: to compare the effects of photodynamic exposure using PS chlorin E6 on human glioblastoma (GB) cells of the U251 line and non-malignant human embryonic kidney cells of the HEK293 line. Material and methods. Groups of cell cultures were formed depending on the conditions of cultivation and exogenous influence: 1) control - cultivated in a standard nutrient medium (Modified Eagle's Medium (MEM)) with L-glutamine, 1 mmol of sodium pyruvate, 10% fetal bovine serum) and experimental: 2) cultivated under the conditions of adding chlorin E6 (concentrations 1.0 and 2.0 μg/ml); 3) cultivated on a nutrient medium without the addition of PS and exposed to laser irradiation (LI) (λ=660 nm, power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode); 4) cultured under conditions of chlorin E6 addition and subsequent exposure to LI (power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode). After exposure to the specified experimental factors, dynamic observation with microphotographic registration was performed for 24 h, followed by microscopic and micrometric studies (number of viable cells, total number of cells, mitotic index (MI,%)). Results. PS chlorin E6 is incorporated into the cytoplasm of cells of U251 and HEK293 cell lines, the intensity of fluorescence is comparable. Upon exposure to chlorin E6 (1.0 and 2.0 μg/ml), cytodestructive and antimitotic effects are increased in a dose-dependent manner in the culture of human GB cells of the U251 line. The cytodestructive effect of chlorin E6 on cell cultures of the HEK293 line is less pronounced, but the antimitotic effect is comparable in both types of cell cultures. Under the influence of LI, cytodestructive and antimitotic effects increase in a dose-dependent manner in the culture of human GB cells of the U251 line. The level of cytodestructive and antimitotic effects is significantly lower in the cultures of non-neoplastic HEK293 cells. The most significant drop in the mitotic activity of GB U251 cells (~100%) was recorded at the lowest LI dose of 25 J/cm2, power of 0.6 W in pulse mode. For HEK293 cells, the most significant decrease in mitotic activity (~80%) was recorded at LI with a power of 0.6 W and dose of 75 J/cm2 in continuous mode. Under the combined effect of chlorin E6 (1 and 2 μg/ml, pre-incubation of 4 h) and LI in different modes, the viability of tumor cells in U251 culture decreases in a dose-dependent manner; the smallest dose of LI to achieve the maximum cytotoxic effect is 25 J/cm2, with a power of 0.6 W in pulse mode when using chlorin E6 at a concentration of 2 μg/ml. The specified characteristics of photodynamic exposure do not cause irreversible effects in HEK293 cultures (reference cells). Conclusions. An effective mode of photodynamic exposure to achieve a cytodestructive and antimitotic effect in the culture of human GB cells of the U251 line, which is relatively safe for non-malignant cells, has been established: the combined application of a laser irradiation dose of 25 J/cm2, with a power of 0.6 W in pulse mode during the preliminary incubation of the cell culture with chlorin E6 at a concentration of 2 μg/ml for 4 h.

Biosynthesis of Silver Nanoparticles Using Barleria albostellata C.B. Clarke Leaves and Stems: Antibacterial and Cytotoxic Activity

Silver nanoparticles (AgNPs) have increasingly gained attention owing to their distinctive physicochemical and biological properties. The objective of the investigation was to biologically synthesize AgNPs using plant extracts from Barleria albostellata. The synthesized AgNPs, obtained from B. albostellata (leaves and stems), were characterized through various techniques including UV-visible spectroscopy, scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray analysis, Fourier transform infrared (FTIR) spectral analysis, and nanoparticle tracking analysis (NTA). The antibacterial efficacy of the synthesized AgNPs was evaluated utilizing the disk diffusion method. The cytotoxicity effects of the synthesized AgNPs were determined using the MTT assay (3-[(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) in cervical cancer (HeLa), embryonic kidney (HEK293), and breast adenocarcinoma (MCF-7) cell lines. The results indicate that B. albostellata extracts could serve as eco-friendly biofactories for the synthesis of AgNPs. UV-vis spectroscopy of the leaf and stem extracts revealed absorption peaks within the range of 400–450 nm, thereby confirming the synthesis of AgNPs. Elemental Ag was highest in the methanol leaf extracts (16.87 ± 0.89%) and lowest in the powdered stem extracts (7.13 ± 1.44%). Synthesized AgNPs were predominantly spherical in shape. HRTEM revealed that synthesized AgNPs from the methanolic stem extracts (34.32 ± 16.99 nm) were larger in size, while those from the powdered stem extracts were smaller (16.57 ± 5.55 nm). AgNPs synthesized from both the leaf and stem extracts exhibited zeta potential values between −8.8 and −32.1 mV, with hydrodynamics diameters ranging from 34.3 to 111.3 nm. FTIR spectroscopy confirmed the presence of various functional groups on the AgNPs. AgNPs synthesized from the leaf and stem extracts displayed significant antibacterial activity and were sensitive to Gram-negative and Gram-positive bacteria. AgNPs exhibited significant antibacterial activity (diameter of the zone of inhibition) against Pseudomonas aeruginosa (21.67 ± 2.87 mm) in the leaf methanolic extract. Synthesized AgNPs exhibited selective in vitro cytotoxicity against HEK293, HeLa, and MCF-7 cell lines. The IC50 values of the AgNPs synthesized from the various extracts were all above 9 µg/mL. Significant cytotoxic levels (IC50 16.11 and 27.23 µg/mL) were observed for the MCF-7 cell line upon exposure to the methanolic leaf and stem AgNPs. This study recommends the use of medicinal plant extracts in producing economically effective AgNPs, due to their efficient capping. Overall, AgNPs synthesized from B. albostellata extracts comprised novel antibacterial and anticancer agents, and warrant further investigation. Bio-synthesized AgNPs show great potential in the area of nanotechnology and may be used as an affordable, eco-friendly alternative for the delivery of conventional therapeutics.

Phytochemical characterization and biological evaluation of lemongrass (Cymbopogon citratus) extracts: A systematic experimental study

Lemongrass () is widely used in traditional medicine, with growing interest in its pharmacological properties. This study aims to systematically investigate the phytochemical composition and biological activities of lemongrass extracts, focusing on optimizing extraction methods and evaluating their therapeutic potential.Various extraction techniques were employed, including solvent-based methods (ethanol, methanol, and water) and green extraction approaches (ultrasound-assisted and microwave-assisted extraction). Phytochemical profiling was conducted using qualitative and quantitative techniques, including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The biological activities of the extracts were assessed through in vitro assays for antioxidant (DPPH, ABTS), antimicrobial (agar diffusion), anti-inflammatory (COX inhibition), and cytotoxicity activities (on HeLa, MCF-7, and HEK-293 cell lines). Ethanol extracts showed the highest yield of bioactive compounds, including flavonoids, terpenoids, and phenolic acids. These extracts also exhibited significant antioxidant activity with DPPH and ABTS assays. The antimicrobial tests revealed strong activity against Staphylococcus aureus and Candida albicans, while the COX inhibition assay indicated notable anti-inflammatory effects. Cytotoxicity studies demonstrated selective toxicity toward cancer cell lines (HeLa, MCF-7) with minimal effects on normal cells (HEK-293). Lemongrass extracts, particularly those obtained via ethanol extraction, exhibit promising antioxidant, antimicrobial, anti-inflammatory, and anticancer properties. These findings highlight its potential for pharmaceutical and therapeutic applications, warranting further investigation in vivo and in clinical trials.

Green composite synthesis based on aluminum fumarate metal-organic framework (MOF) for doxorubicin delivery: An in vitro study

The development of an eco-friendly and cost-effective drug delivery system (DDS) remains a challenge in cancer treatment. This study suggests a novel, eco-friendly, and cost-effective method to synthesize aluminum fumarate (A520) MOF using tangerine peel extract (TPE) as a pH-sensitive coating agent on a reduced graphene oxide (rGO) substrate. The system also incorporates the 2,2′-bipyridine dimethyltin diisothiocyanate complex, (Sn(bpy), as an organotin complex) to enhance therapeutic efficacy while minimizing off-target effects. In vitro cellular and molecular cytotoxicity examinations were conducted using HEK-293 and MCF-7 cell lines to evaluate cytotoxicity after 24 and 72 h. The findings of the MTT assay indicated that the final nanocarriers, without and with doxorubicin (DOX), exhibited relative cell viability of 95 % and 55 %, respectively, highlighting the system's potential for efficient and safe drug delivery. The TPE coating created a pH-sensitive DDS with high drug loading efficiency (DLE) (59.1 %) and low cytotoxicity. Drug release was studied over 200 h at pH 5.5 and 7.2. The augmented drug release profile (47 %) demonstrated a targeted release and successful inhibition of premature release in acidic environments due to pH-sensitive TPE molecules. The efficacy of targeted drug delivery to the MCF-7 cell was evaluated using 2D fluorescence microscopy, which demonstrated successful delivery of drugs to the cells. This innovative approach addresses the limitations of existing studies while promoting biocompatibility, stability, and functionality in DDSs.