Search Results (1,701)

Lipid nanoparticle (LNP)-based delivery systems are promising tools for advancing RNA-based therapies. However, there are underlying challenges for the oral delivery of LNPs. In this study, we optimized an LNP formulation, which we encapsulated in a pH-sensitive Eudragit^® S 100 (Eu) coating. LNPs were prepared using the DLin-MC3-DMA ionizable lipid, cholesterol, DMG-PEG, and DSPC at a molar ratio of 50:38.5:10:1.5. LNPs were coated with 1% Eu solution via nanoprecipitation using 0.25% acetic acid to get Eu-coated LNPs (Eu-LNPs). Particle characteristics of LNPs were determined by using dynamic light scattering (DLS). Ribogreen and agarose gel retardation assays were used to evaluate nucleic acid entrapment and stability. LNPs and Eu-LNPs were ~120 nm and 4.5 μm in size, respectively. Eu-LNPs decrease to an average size of ~191 ± 22.9 nm at a pH of 8. Phosphate buffer (PB)-treated and untreated Eu-LNPs and uncoated LNPs were transfected in HEK-293 cells. PB-treated Eu-LNPs showed significant transfection capability compared to their non-PB-treated counterparts. Eu-LNPs protected their nucleic acid payloads in the presence of a simulated gastric fluid (SGF) with pepsin and maintained transfection capacity following SGF or simulated intestinal fluid. Hence, Eu coating is a potentially promising approach for the oral administration of LNPs. Full article

Background: Metastatic melanoma is a highly aggressive malignancy with poor prognoses and frequent resistance to conventional chemotherapy. Approximately 40% of melanoma cases carry the BRAF^V600E mutation, for which vemurafenib, a selective BRAF^V600E inhibitor, is approved. Despite initial clinical benefits, vemurafenib often leads to drug resistance and relapse, highlighting the need for improved therapeutic strategies. Objectives, methods: In this study, we designed, synthesized, and characterized five novel vemurafenib analogs—RF-86A, RF-87A, RF-94A, RF-94B, and RF-96B—with the aim of enhancing anti-proliferative and anti-metastatic effects against human melanoma cells. Results: All compounds induced apoptosis in BRAF^V600E-mutated A375 cells, with RF-86A displaying the lowest IC₅₀ value among the series, comparable to that of vemurafenib. Moreover, RF-86A exhibited the highest selectivity index, as determined using HEK293T cells as a non-tumorigenic control. Additionally, migration assays and gelatin zymography demonstrated that the analogs, unlike vemurafenib, significantly inhibited matrix metalloproteinases MMP-2 and MMP-9, key enzymes involved in tumor invasion and metastasis. Conclusions: These findings suggest that structural modifications to the vemurafenib scaffold may improve therapeutic efficacy and offer a promising strategy to overcome acquired resistance. Full article

Bifora testiculata (L.) Spreng. (Apiaceae), an understudied species endemic to the Mediterranean and the only representative species of the genus Bifora in Sicily, was investigated for the first time for its essential oil (EO) chemical composition and cytotoxic properties. The EO was obtained via hydrodistillation and analyzed using GC-MS, revealing an aldehyde-rich profile (86.10%), dominated by trans-2-dodecenal (67.49%). Comparative analysis with previous studies on B. testiculata from Greece confirmed a similar aldehyde-rich profile, although minor compositional differences suggest potential chemotype variation. Given the biological relevance of trans-2-dodecenal and related aldehydes, further investigations into the cytotoxic properties of the EO of B. testiculata (Bt) and its main constituent against cancer cell lines were undertaken. Three human tumor cell lines (MDA-MB 231, A375, and CaCo2) and a human non-tumor cell line (HEK293) were subjected to viability tests using the MTT assay. The EO and trans-2-dodecenal exhibited remarkable cytotoxic activity against all cell lines, with IC₅₀ values ranging between 7.93 and 14.41 µg/mL for Bt and between 1.88 and 5.29 µg/mL for trans-2-dodecenal. AO/BE fluorescent staining and Hoechst nuclear staining showed the presence of apoptotic bodies in the treated cells. N-acetyl-L-cysteine was able to invert the effects of Bt and trans-2-dodecenal on cell lines, suggesting ROS involvement in cytotoxic activity. The results demonstrated that the Bt cytotoxic activity was mainly due to the presence of trans-2-dodecenal. Full article

The rise in multidrug-resistant bacterial strains and persistent infections such as Lyme disease caused by Borrelia burgdorferi highlights the need for novel antimicrobial agents. The present study explores the antioxidant, antibacterial, and cytotoxic properties of extracts from submerged mycelial biomass of Fomitopsis pinicola, cultivated in synthetic and lignocellulosic media. Four extracts were obtained using hot water and 80% ethanol. The provided analysis of extracts confirmed the presence of various bioactive compounds, including flavonoids, alkaloids, and polyphenols. All extracts showed dose-dependent antioxidant activity (IC₅₀: 1.9–6.7 mg/mL). Antibacterial tests revealed that Klebsiella pneumoniae was most sensitive, with the L2 extract producing the largest inhibition zone (15.33 ± 0.47 mm), while the strongest bactericidal effect was observed against Acinetobacter baumannii (MBC as low as 0.5 mg/mL for L1). Notably, all extracts significantly reduced the viability of stationary-phase B. burgdorferi cells, with L2 reducing viability to 42 ± 2% at 5 mg/mL, and decreased biofilm mass, especially with S2. Cytotoxicity assays showed minimal effects on NIH 3T3 cells, with slight toxicity in HEK 293 cells for S2 and L1. These results suggest that F. pinicola extracts, particularly ethanolic L2 and S2, may offer promising natural antimicrobial and antioxidant agents for managing resistant infections. Full article

Background/Objectives: Cancer suffers from unresolved therapeutic challenges owing to the lack of targeted therapies and heightened recurrence risk. This study aimed to investigate the new series of hydroxamate by structurally modifying the pharmacophore of vorinostat. Methods: The present work involves the synthesis of 15 differently substituted 2H-1,2,3-triazole-based hydroxamide analogs by employing triazole ring as a cap with varied linker fragments. The compounds were evaluated for their anticancer effect, especially their anti-breast cancer response. Molecular docking and molecular dynamics simulations were conducted to examine binding interactions. Results: Results indicated that among all synthesized hybrids, the molecule VI(i) inhibits the growth of MCF-7 and A-549 cells (GI₅₀ < 10 μg/mL) in an antiproliferative assay. Compound VI(i) was also tested for cytotoxic activity by employing an MTT assay against A549, MCF-7, and MDA-MB-231 cell lines, and the findings indicate its potent anticancer response, especially against MCF-7 cells with IC₅₀ of 60 µg/mL. However, it experiences minimal toxicity towards the normal cell line (HEK-293). Mechanistic studies revealed a dual-pathway activation: first, apoptosis (17.18% of early and 10.22% of late apoptotic cells by annexin V/PI analysis); second, cell cycle arrest at the S and G2/M phases. It also promotes ROS generation in a concentration-dependent manner. The HDAC–inhibitory assay, extended in silico molecular docking, and MD simulation experiments further validated its significant binding affinity towards HDAC 1 and 6 isoforms. DFT and ADMET screening further support the biological proclivity of the title compounds. The notable biological contribution of VI(i) highlights it as a potential candidate, especially against breast cancer cells. Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Plant flowers are recognized as a rich source of bioactive phenolic compounds. In this study, for the first time, the recovery of antioxidant phenolic compounds from Cytisus striatus flowers (CF) was optimized using microwave-assisted extraction (MAE). The variables (% of ethanol, temperature, and time) were studied using a response surface methodology (RSM). Extraction efficiency was assessed by total phenol content, total flavonoid content, and the antioxidant capacity through DPPH, ABTS, FRAP, and CUPRAC assays. Additionally, cytotoxicity and anti-inflammatory properties were evaluated in different cell lines. The optimal extraction conditions (87.6% ethanol, 160.8 °C and 8.76 min) yielded extracts rich in phenolics (85.9 mg GAE/g CF) and flavonoids (120.3 mg RE/g CF), with strong antioxidant capacity. LC-MS/MS analysis identified 27 phenolic compounds, including chrysin, apigenin, and quercetin derivatives. Cytotoxicity tests showed that CF extract maintained high viability (>80%) in human embryonic kidney (HEK293T) and human lung adenocarcinoma (A549) cells up to 2000 µg/mL, indicating low cytotoxicity. The anti-inflammatory potential was evidenced by a decrease in IL-1β levels and an increase in IL-10 cytokine production in LPS-stimulated macrophages. These results highlight the great potential of CF as a promising bioresource to obtain value-added compounds for the development of functional foods, nutraceuticals, and cosmetic products. Full article

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed, and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites, here, we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells, PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs, and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible, concentration-dependent, and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid, propionic acid, itaconic acid, and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids, including arachidonic acid. Although lactic acid inhibited PAC, it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude, weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids. Full article

Background: Modern viral vector production needs to consider process intensification for higher yields from smaller production volumes. However, innate antiviral immunity triggered in the producer cell may limit virus replication. While commonly used cell lines (e.g., Vero or E1A-immortalised cells) are already compromised in antiviral pathways, the redundancy of innate signaling complicates host cell optimization by genetic engineering. Small molecules that are hypothesized to target antiviral pathways (Viral Sensitizers, VSEs) added to the culture media offer a versatile alternative to genetic modifications to increase permissiveness and, thus, viral yields across multiple cell lines. Methods: To explore how the yield for a chimeric Zika vaccine candidate (YF-ZIK) could be further be increased in an intensified bioprocess, we used spin tubes or an Ambr15 high-throughput microbioreactor system as scale-down models to optimize the dosing for eight VSEs in three host cell lines (AGE1.CR.pIX, BHK-21, and HEK293-F) based on their tolerability. Results: Addition of VSEs to an already optimized infection process significantly increased infectious titers by up to sevenfold for all three cell lines tested. The development of multi-component VSE formulations using a design of experiments approach allowed further synergistic titer increases in AGE1.CR.pIX cells. Scale-up to 1 L stirred-tank bioreactors and 3D-printed mimics of 200 or 2000 L reactors resulted in up to threefold and eightfold increases, respectively. Conclusions: Addition of single VSEs or combinations thereof allowed a further increase in YF-ZIK titers beyond the yield of an already optimized, highly intensified process. The described approach validates the use of VSEs and can be instructive for optimizing other virus production processes. Full article

Angiotensin-converting enzyme 2 (ACE2) is a cell-surface receptor that helps the body regulate blood pressure and endocrine secretions. Transmembrane serine protease 2 (TMPRSS2) is a cell surface protein expressed mainly by endothelial cells of the respiratory and digestive tract, which participates in the cleavage of protein peptide bonds with serine as the active site. These two proteins have been studied to be highly associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Soybean trypsin inhibitor (SBTI) has special bioactivities such as anticarcinogenic and anti-inflammatory functions, which can be widely used in functional foods or drugs. Our study involved in vitro and in vivo experiments to elucidate the effect of SBTI on SARS-CoV-2 host invasion. First, it was confirmed that being under 250 μg/mL of SBTI was not toxic to HepG2, HEK293T, and Calu-3 cells. The animal study administered SBTI to mice once daily for 14 days. In the lungs, liver, and kidneys, the histopathologic findings of the SBTI group were not different from those of the control group, but the expression of ACE2, TMPRSS2, and CD147 was reduced. Thus, our findings suggest that the inhibition of ACE2, TMPRSS,2 and CD147 proteins by SBTI shows promise in potentially inhibiting SARS-CoV-2 infection. Full article

Three previously synthesized ketene dithioacetals were used as intermediates to obtain four nucleophiles to synthesize ten tetra-substituted pyrazoles (11–20). This was achieved through microwave irradiation in ethanol as the solvent, yielding superb results ranging from 68.4% to 90.1%, in agreement with some of the principles of green chemistry. The proposed structures were determined using various spectroscopic techniques, including infrared spectroscopy and hydrogen and carbon-13 nuclear magnetic resonance. Furthermore, the compounds underwent in-silico evaluations using CLC-Pred and AdmetSAR software to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. This was combined with molecular docking calculations for four main cancer-related targets for pyrazole core, to facilitate screening for subsequent biological assessments. Based on the data generated from these analyses, it was identified two pyrazoles (11 and 18) likely to exhibit anti-tumor activity, while also demonstrating low toxicity levels. Upon selection, these two pyrazoles were subjected to toxicity assessments using the Artemia salina method and evaluated for their effects on the viability of Jurkat cancer cells with a potency of 45.05 and 14.85 µM to 11 and 18, respectively, and with a potency of above 100 µM for the non-carcinogenic cells HEK 293. Overall, the findings from these studies indicate pyrazole derivatives as potential anti-tumor candidates. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article

Virus-like particles (VLPs) have the potential to become a versatile carrier platform for vaccination against multiple diseases. In the light of short process development timelines and the demand for reliable and robust processes, metabolic modeling of cell culture processes offers great advantages when coupled with a Quality-by-Design (QbD) development approach. A previous work was able to demonstrate the accurate prediction of HEK293F PiggyBac cell concentration as well as VLP titer and metabolite production with a reduced metabolic model. This work presents the reduced metabolic model for a more productive cell line Sleeping Beauty and emphasizes the need for model re-parameterization when the producer cell line changes. The goal of precise prediction for a fed-batch and continuous HEK293 cultivation can, therefore, be achieved. In terms of decision-making for downstream unit operations, a soft sensor for the prediction of main impurities like proteins and DNA was introduced for the first time for the production of lentiviral vectors with several terms describing the release of impurities like DNA and proteins, growth-related protein production, and enzymatic degradation activity associated with cell dissociation in an accurate manner. The additional information can contribute to a more efficient design phase by reducing experimental effort as well as during cultivation with data-based decision-making. With the aid of real-time process data acquisition through process analytical technology (PAT), its predictive power can be enhanced and lead to more reliable processes. Full article

Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9–VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9–VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA. Full article