Search Results (65)

(1) Background: Our study investigates the green synthesis of Ag₂O/Ag nanoparticles using the isoflavone Puerarin as a bioreductor. (2) Methods: The PUE@Ag₂O/Ag nanoparticles were characterized using various techniques, including X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), electronic microscopy (TEM, SEM), energy dispersive X-ray spectroscopy (EDX), and dynamic light scattering (DLS). Biological activities were assessed through antimicrobial testing, cytotoxicity assays on human keratinocytes and melanoma cells, and an in ovo screening using the HET-CAM assay. (3) Results: The formation of crystalline Ag₂O/Ag nanoparticles with sizes below 100 nm was accomplished with Puerarin. Despite their high cytotoxicity at all tested concentrations, the nanoparticles showed antioxidant activity with IC₅₀ 981.5 ± 94.2 μg/mL, antibacterial activity against several clinically relevant nosocomial strains (Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa), and no local irritant effects or inhibition of angiogenesis in the HET-CAM assay. (4) Conclusions: This study provides insights into the synthesis, characterization, and biological profile of PUE@Ag₂O/Ag nanoparticles for potential biomedical applications. Full article

Background/Objectives: Angiogenesis is the multistep process of the formation of new blood vessels. It is beneficial in scenarios that require tissue repair and regeneration, such as wound healing, bone fracture repair, and recovery from ischemic injuries like stroke, where new blood vessel formation restores oxygen and nutrient supply to damaged areas. Extremely low-frequency electromagnetic stimulation (ELF-EMS), which involves electromagnetic fields in the frequency range of 0–300 Hz, have been shown to reduce ischemic stroke volume by improving cerebral blood flow and recovery effects that are dependent on eNOS. Based on previous results, we herein explore the effects of ELF-EMS treatment (13.5 mT/10 and 60 Hz) on the activation of angiogenic processes in vitro in homeostatic conditions. Methods: Using human microvascular endothelial cells (HMEC-1), we studied cell proliferation, migration, and tube formation in vitro, as well as nitric oxide production and the effect of calcium and nitric oxide (NO) on these processes. Moreover, blood vessel formation was studied using a chicken chorioallantoic membrane (CAM) assay. Results: Our results showed that ELF-EMS increases proliferation, tube formation, and both the migration and transmigration of these cells, the latter of which was mediated via NO. In turn, calcium inhibition decreased ELF-EMF-induced NO production. Furthermore, ELF-EMS significantly increased blood vessel formation in the CAM assay. Conclusions: Our results indicated that ELF-EMS exposure (13.5 mT/10 and 60 Hz) significantly induces angiogenesis in vitro and in ovo, underscoring its potential application in the treatment of conditions characterized by insufficient blood supply. Full article

Fibrosarcomas represent a rare but highly aggressive tumor entity among soft tissue tumors. Due to its rarity, questions regarding its development and pathophysiology remain unclear. The chorioallantoic membrane (CAM) assay represents an easily available method to investigate tumors on a growth membrane, live and in ovo. The following study was established to test whether the growth of fibrosarcoma spheroids on the CAM was possible and to critically review their applicability for downstream investigations. The shells of fertilized chicken eggs were opened and the previously prepared HT1080 cell spheroids (50,000, 75,000, and 100,000 cells per spheroid) were applied to the CAM. After 7 days, tumors were examined for size, weight, and vascularization. After 7 days, 80 of 163 chicken eggs showed sufficient tumor growth. Of these 80 eggs with confirmed tumor growth, 32 (40%) were from the 50,000 spheroid group, 18 (22.5%) were from the 75,000 spheroid group, and 30 (37.5%) were from the 100,000 spheroid group. The 100,000-cell spheroid group exhibited the highest weights, with a mean of 110.7 mg, as well as tumor size expansion. This cell number also showed the highest vascularization rates. Tumor growth of fibrosarcoma spheroids could successfully be initiated on the CAM. Consequently, the CAM assay presents a good base for future studies involving human fibrosarcoma cell spheroids. Full article

A comparative evaluation of Moringa oleifera Lam. ethanolic leaf extracts was performed using different extraction methods (maceration or ultrasound-assisted) and the qualitative and quantitative profile of the bioactive compounds contained were further assessed. The antioxidant potential and antimicrobial activity were evaluated, as well as the antiangiogenic effects through in ovo studies. Six ethanolic extracts were tested in this study. Moringa MAC 70% and Moringa US 70% extracts displayed the highest concentration of phenolic compounds and also showed a significant AOA at the highest tested dose. Regarding the antimicrobial effect, the extracts obtained with 70% ethanol (maceration or ultrasound-assisted) had antimicrobial activity against S. aureus, S. pyogenes and E. coli, followed by Candida parapsilosis. On the Pseudomonas aeruginosa strain, the extracts showed no effect. The HET-CAM assay showed that the extracts did not cause any irritation compared to the used positive control. Furthermore, the extracts Moringa MAC 70% and Moringa US 70% did not affect the normal process of blood vessel formation. The data obtained highlights that, from the six tested extracts, the ones obtained with 70% ethanol using maceration and ultrasound-assisted methods (Moringa MAC 70% and Moringa US 70%) showed the highest phenolic content and exhibited the strongest antioxidant activity. The same two extracts did not show signs of irritation in the HET-CAM model. Full article

Background and Objectives: Cutaneous melanoma (CM) poses a continuous challenge in oncology due to the developing resistance to available treatments. Doxorubicin (DOX) is noted as one of the most effective chemotherapeutics, although associated toxicity and resistance limit its use in CM treatment. Consequently, DOX has become a promising candidate for combination therapies targeting this neoplasm. Genistein (GEN) gathered significant attention due to its anti-neoplastic properties and ability to enhance the effects of DOX against several cancers, yet this association remains underexplored in CM. Therefore, this study investigated the combination therapy regimen comprising GEN and DOX in terms of anti-melanoma activity and safety profile. Materials and Methods: The in vitro experiments were performed on SK-MEL-28 and HaCaT cells. Cell viability was determined using MTT assay. Cell morphology and confluence were inspected microscopically. Nuclear and cytoskeletal aspects were assessed via immunofluorescence. Apoptosis and oxidative stress were quantified through caspase activity and intracellular reactive oxygen species (ROS) production, respectively. The irritant effect was evaluated on the chorioallantoic membrane. Results: The results revealed that the combination of GEN 10 µM with DOX (0.5 and 1 µM) provided augmented cytotoxic events (e.g., reduced cell viability, altered cell morphology and confluence, apoptotic-like impairments in nuclear shape and cytoskeletal network, increased caspases-3/7 and -9 activity, and elevated ROS) in SK-MEL-28 cells, compared to individual treatments, and exerted a strong synergistic interaction. Simultaneously, GEN 10 µM efficiently surpassed the toxic effects (e.g., viability and confluence loss, hypertrophy, and cytoskeletal condensation) of DOX (0.5 and 1 µM) in HaCaT cells. In ovo, GEN 10 µM + DOX 1 µM treatment was classified as non-irritant. Conclusions: These findings stand as one of the first contributions revealing the beneficial therapeutic interplay between GEN and DOX at physiologically achievable concentrations that resulted in elevated anti-tumor properties in CM cells and alleviated toxicity in keratinocytes. Full article

Tendon ruptures and tendinopathies represent a major part of musculoskeletal injuries. Due to the hypovascular and hypocellular nature of tendons, the natural healing capacity is slow and limited. Cell-free approaches for tendon injuries are being investigated as the next generation of therapeutic treatments. The aim of this study was to compare the proteomic profiles and biological activities of two different secretomes, obtained from New Zealand white rabbit adipose-tissue-derived mesenchymal stem cells (ADSCs) or a 3:1 mixed culture of ADSCs and rabbit tenocytes. The secretomes were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and their functional properties, such as gene expression, migration and angiogenesis, were investigated in vitro in rabbit tenocytes and in ovo using the chicken chorioallantoic membrane (CAM) assay after stimulation with secretomes or medium control. Both secretomes had a positive effect on angiogenesis and showed similar changes in relative gene expression levels associated with extracellular matrix (ECM) remodeling. Proteomic data showed that the two secretomes were clearly distinguishable, with 182 proteins significantly differentially expressed. The ADSC secretome was more effective in enhancing tenocyte migration under both healthy and inflammatory conditions. In the upregulated protein fraction of the mixed secretome, the tendon-related protein biglycan (BGN) and tenascin C (TNC) were increased. Based on our results, the mixed secretome shows great potential for promoting tendon healing as its composition is more effective in enhancing ECM-related processes and tendon development than the secretome of ADSCs. Full article

Background/Objectives: Angiogenesis plays a crucial role in tumor development and is a driving force for the aggressiveness of several types of cancer. Our team developed a novel series of thiosemicarbazone-containing quinazoline derivatives, TSC1-TSC10, as potential VEGFR2 inhibitors with proven anti-angiogenic and antiproliferative potential. Methods: The TSC1-TSC10 series was synthesized and characterized by spectral data. Extensive methodology was applied both in vitro (Alamar Blue assay, Scratch assay, CAM assay, and VEGFR2 kinase assay) and in silico (docking studies, MDs, and MM-PBSA) for the confirmation of the biological potential. Results: TSC10 emerged as the most promising compound, with a favorable cytotoxic potential across the cell panel (Ea.Hy296, HaCaT, and A375) in agreement with the in vitro VEGFR2 kinase assay (IC₅₀ = 119 nM). A comparable motility reduction in the vascular endothelial cells to that of the reference drug sorafenib was provided by TSC10, with a similar anti-angiogenic potential in the more complex in ovo model of the CAM assay. The in silico experiments confirmed the successful accommodation of the active site of the kinase domain similar to sorafenib for the entire TSC1-TSC10 series, providing valuable key insight into the complex stability driving force for the evaluated compounds. Conclusions: The in vitro evaluations of the biological potential correlated with the in silico predictions by computer-aided complex simulations provided a solid confirmation of the initial hypothesis for the TSC1-TSC10 series. Full article

Cardiovascular diseases such as myocardial infarction or limb ischemia are characterized by regression of blood vessels. Local delivery of growth factors (GFs) involved in angiogenesis such as fibroblast blast growth factor-2 (FGF-2) has been shown to trigger collateral neovasculature and might lead to a therapeutic strategy. In vivo, heparin, a sulfated polysaccharide present in abundance in the extracellular matrix (ECM), has been shown to function as a local reservoir for FGF-2 by binding FGF-2 and other morphogens and it plays a role in the evolution of GF gradients. To access injectable biomaterials that can mimic such natural electrostatic interactions between soluble signals and macromolecules and mechanically tunable environments, the backbone of agarose, a thermogelling marine–algae-derived polysaccharide, was modified with sulfate, phosphate, and carboxylic moieties and the interaction and release of FGF-2 from these functionalized hydrogels was assessed by ELISA in vitro and CAM assay in ovo. Our findings show that FGF-2 remains active after release, and FGF-2 release profiles can be influenced by sulfated and phosphorylated agarose, and in turn, promote varied blood vessel formation kinetics. These modified agaroses offer a simple approach to mimicking electrostatic interactions experienced by GFs in the extracellular environment and provide a platform to probe the role of these interactions in the modulation of growth factor activity and may find utility as an injectable gel for promoting angiogenesis and as bioinks in 3D bioprinting. Full article

Cutaneous melanoma (CM) represents a severe skin cancer with a rising incidence at present and limited treatment options. 5-Fluorouracil (5-FU) is widely used, including for CM; however, the innate resistance of this cancer to conventional therapy remains problematic. Quercetin (QUE) is a flavonoid that can sensitize cancer cells to antitumor agents such as 5-FU. However, the potential sensitization capability of CM cells to 5-FU has scarcely been determined, and is investigated herein. Therefore, A375 CM cells were tested in terms of their cell viability, cell confluence, and morphological changes. Their nuclear and cytoskeletal aspects, clonogenic potential, and in ovo properties were also followed. The results showed that the 50% inhibitory concentrations (IC50s) of 5-FU and QUE determined by a cell proliferation assay were 11.56 and 11.08 µM, respectively. The addition of QUE (10 µM) to 5-FU (5–50 µM) increased the cytotoxic potential. A significant decline in cell viability (up to 43.51%), the loss of cell confluence, chromatin condensation and nuclear dysmorphology, tubulin and F-actin constriction, and a suppressed clonogenic ability were noted. The QUE + 5-FU association was non-irritating to the chorioallantoic membrane and showed an antiangiogenic effect in ovo. Thus, our results highlight that combining QUE with 5-FU can enhance the cytotoxic effect of 5-FU in A375 melanoma cells and present a safe profile in ovo. Full article

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico—an ESE-15-one and an ESE-16 one—with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class. Full article

Determining the safety of a newly developed experimental product is a crucial condition for its medical use, especially for clinical trials. In this regard, four hydrogel-type formulations were manufactured, all of which were based on carbomer (Blank-CP940) and encapsulated with caffeine (CAF-CP940), phosphorus derivatives (phenyl phosphinic (CAF-S1-CP940) and 2-carboxyethyl phenyl phosphinic acids (CAF-S2-CP940)). The main aim of this research was to provide a comprehensive outline of the biosafety profile of the above-mentioned hydrogels. The complex in vitro screening (cell viability, cytotoxicity, morphological changes in response to exposure, and changes in nuclei morphology) on two types of healthy skin cell lines (HaCaT—human keratinocytes and JB6 Cl 41-5a—murine epidermal cells) exhibited a good biosafety profile when both cell lines were treated for 24 h with 150 μg/mL of each hydrogel. A comprehensive analysis of the hydrogel’s impact on the genetic profile of HaCaT cells sustains the in vitro experiments. The biosafety profile was completed with the in vivo and in ovo assays. The outcome revealed that the developed hydrogels exerted good biocompatibility after topical application on BALB/c nude mice’s skin. It also revealed a lack of toxicity after exposure to the hen’s chicken embryo. Further investigations are needed, regarding the in vitro and in vivo therapeutic efficacy and safety for long-term use and potential clinical translatability. Full article

Diabetes mellitus is a spreading global pandemic. Type 2 diabetes mellitus (T2DM) is the predominant form of diabetes, in which a reduction in blood glucose uptake is caused by impaired glucose transporter 4 (GLUT4) translocation to the plasma membrane in adipose and muscle cells. Antihyperglycemic drugs play a pivotal role in ameliorating diabetes symptoms but often are associated with side effects. Hence, novel antidiabetic compounds and nutraceutical candidates are urgently needed. Phytogenic therapy can support the prevention and amelioration of impaired glucose homeostasis. Using total internal reflection fluorescence microscopy (TIRFM), 772 plant extracts of an open-access plant extract library were screened for their GLUT4 translocation activation potential, resulting in 9% positive hits. Based on commercial interest and TIRFM assay-based GLUT4 translocation activation, some of these extracts were selected, and their blood glucose-reducing effects in ovo were investigated using a modified hen’s egg test (Gluc-HET). To identify the active plant part, some of the available candidate plants were prepared in-house from blossoms, leaves, stems, or roots and tested. Acacia catechu (catechu), Pulmonaria officinalis (lungwort), Mentha spicata (spearmint), and Saponaria officinalis (common soapwort) revealed their potentials as antidiabetic nutraceuticals, with common soapwort containing GLUT4 translocation-activating saponarin. Full article

Silver nanoparticles (AgNPs) were successfully synthesized via the biological route using a 1 M silver nitrate (AgNO₃) aqueous solution and an ethanolic peel extract of Punica granatum (Pg), at 60 °C. The physicochemical analysis revealed the formation of green synthesized Pg-AgNPs with a semi-spherical shape, non-uniformly distributed, and a particle size distribution between 5 and 100 nm. As regards the preliminary in vitro toxicological screening, the green synthesized Pg-AgNPs did not significantly affect the neonatal BALB/c epidermal cells’ viability (JB6 Cl 41-5a) at lower concentrations and did not produce visible changes in the morphology of the JB6 Cl 41-5a cells. In contrast, at higher concentrations (>50 μg/mL), the green Pg-AgNPs exhibited an important decrease in cell viability and confluency. In addition, the impact of Pg-AgNPs on cell membrane integrity suggests a potential cytotoxic effect. Contrary to the in vitro assays, after the evaluation of the anti-irritant effect in ovo, the lower concentration of Pg-AgNPs (10 μg/mL) produced hemorrhage and lysis when applied to the chorioallantoic membrane, while at 50 μg/mL, only slight coagulation was observed. Therefore, regarding the in ovo toxicological screening, the higher concentration of the Pg-AgNPs exhibited a better safety profile compared to the lower concentration, as indicated by the irritation score. Full article

Colorectal cancer (CRC) is a heterogenous pathology with high incidence and mortality rates globally, but it is also preventable so finding the most promising candidates (natural compounds or repurposed drugs) to be chemopreventive alternatives has become a topic of interest in recent years. The present work aims to elucidate the potential effects of a combination between genistein (GEN), an isoflavone of natural origin, and aspirin (ASA) in CRC prevention/treatment by performing an in vitro evaluation in human colorectal cancer cells (HCT-116) and an in ovo analysis using the chick embryo chorioallantoic membrane (CAM) model. Cell viability was verified by an MTT (migratory potential by scratch) assay, and the expressions of MMP-2 and MMP-9 were analyzed using RT-qPCR. Our results indicated a dose-dependent cytotoxic effect of ASA (2.5 mM) + GEN (10–75 µM) combination characterized by reduced cell viability and morphological changes (actin skeleton reorganization and nuclei deterioration), an inhibition of HCT-116 cells’ migratory potential by down-regulating MMP-2 and MMP-9 mRNA expressions, and an antiangiogenic effect by modifying the vascular network. These promising results raise the possibility of future in-depth investigations regarding the chemopreventive/therapeutical potential of ASA+GEN combination. Full article

The antimicrobial effect of eight essential oils’ vapors against pathogens and spoilage bacteria was assayed. Oreganum vulgare L. essential oil (OVO) showed a broad antibacterial effect, with Minimum Inhibitory Concentration (MIC) values ranging from 94 to 754 µg cm⁻³ air, depending on the bacterial species. Then, gaseous OVO was used for the treatment of stainless steel, polypropylene, and glass surfaces contaminated with four bacterial pathogens at 6–7 log cfu coupon⁻¹. No viable cells were found after OVO treatment on all food-contact surfaces contaminated with all pathogens, with the exception of Sta. aureus DSM 799 on the glass surface. The antimicrobial activity of OVO after the addition of beef extract as a soiling agent reduced the Sta. aureus DSM 799 viable cell count by more than 5 log cfu coupon⁻¹ on polypropylene and glass, while no viable cells were found in the case of stainless steel. HS-GC-MS analysis of the headspace of the boxes used for the antibacterial assay revealed 14 different volatile compounds with α-Pinene (62–63%), and p-Cymene (21%) as the main terpenes. In conclusion, gaseous OVO could be used for the microbial decontamination of food-contact surfaces, although its efficacy needs to be evaluated since it depends on several parameters such as target microorganisms, food-contact material, temperature, time of contact, and relative humidity. Full article