Cytotoxicity Evaluation Research Articles

Photothermal Hydrogel Composites Featuring G4-Carbon Nanomaterial Networks for Staphylococcus aureus Inhibition.

Microbial infections represent a significant health risk, often leading to severe complications and, in some cases, even fatalities. As a result, there is an urgent need to explore innovative drug delivery systems and alternative therapeutic techniques. The photothermal therapy has emerged as a promising antibacterial approach and is the focus of this study. Herein, we report the successful synthesis of two distinct supramolecular composite hydrogels by incorporating graphene oxide (GO) and single-walled carbon nanotubes (SWNTs) into guanosine quadruplex (G4) based hydrogels containing covalently bound β-cyclodextrin (β-CD). The G4 matrix was synthesized through a two-step process, establishing a robust network between G4 and β-CDs, followed by the encapsulation of either GO or SWNTs. Comprehensive characterization of these composite hydrogels were conducted using analytical techniques, including circular dichroism, Raman spectroscopy, rheological investigations, X-ray diffraction, and scanning electron microscopy. A notable discovery from the conducted research is the differential photothermal responses exhibited by the hydrogels when exposed to near-infrared laser irradiation. Specifically, SWNT-based hydrogels demonstrated superior photothermal performance, achieving a remarkable temperature increase of up to 52 °C, in contrast to GO-based hydrogels, which reached a maximum of 34 °C. These composite hydrogels showed good cytotoxicity evaluation results and displayed synergistic antibacterial activity against Staphylococcus aureus, positioning them as promising candidates for antibacterial photothermic platforms, particularly in the context of wound treatment. This study offers a valuable contribution to the development of advanced and combined therapeutic strategies for combating microbial infections and highlights the potential of carbon nanomaterial-enhanced supramolecular hydrogels in photothermal therapy applications.

Synthesis of N-oxide Poly(1-vinylimidazole) for pDNA delivery systems in vivo

Abstract N-oxide poly(1-vinylimidazole) (PVIm+–O−) has been synthesized as a unique biocompatible polyampholyte. Measurements of solution turbidity prove that PVIm+–O− had no significant effect on rapid aggregate formation with serum proteins. Agarose gel retardation assay, particle size/ζ-potential measurement, and cytotoxicity evaluation prove that the PVIm+–O− coated binary complexes between branched poly(ethyleneimine) (bPEI) and plasmid DNA (pDNA). The resulting PVIm+–O−/bPEI/pDNA ternary complex mediated higher gene expression than the bPEI/pDNA binary complex in vivo mouse skeletal muscle. These results suggest that PVIm+–O− is a polyampholyte for biocompatibility.

Synthesis, Cytotoxic Activity Evaluation and Molecular Docking Studies of Some Benzimidazole Derivatives

In this study, the synthesis of 2-(2-acetyl-1H-benzimidazol-1-yl)-1-arylethanone (3a-3d) and 1-methyl-3-phenyl-benzo[4,5]imidazo[1,2-a]pyrazine derivatives (4a-4d) and to investigate their cytotoxic activity were aimed. APCI, IR, 1HNMR, and 13CNMR spectra were utilized to determine the structure of the synthesized compounds. The cytotoxic activity of selected compounds were detected in A549 (human lung carcinoma) and NIH3T3 (mouse embryonic fibroblasts) cell lines. Compounds 4c and 4d were found to be selectively cytotoxic against A549 and NIH3T3 cell lines. Molecular docking studies were performed using the data retrieved from the Protein Data Bank server (PDBID: 4QTX).

Synthesis of novel ursolic acid-gallate hybrids via 1,2,3-triazole linkage and its anti-oxidant and anti-inflammatory activity study

Excessive oxidation can lead to inflammation and affect health. Studies have shown that ursolic acid (UA) and gallic acid are widely employed in medicine and cosmetics owing to their obvious antioxidant and anti-inflammatory properties. In this study, a series of 16 novel UA hybrids tethered via 1,2,3-triazole to the modified gallate moieties were synthesized via CuAAC 1,3-cycloaddition reactions. In vitro, all derivatives were proved to be effective in antioxidation and anti-inflammation activities. Interestingly, compound 20 significantly inhibited the expression of pro-inflammatory cytokines including mRNA of inducible nitric oxide synthase (iNOS) (p < 0.05) and cyclooxygenase-2 (COX-2) (p < 0.01) in lipopolysaccharide (LPS)-induced RAW264.7 cells in a dose-dependent manner. The inhibitory effect of compound 20 on pro-inflammatory cytokines was closely associated with suppression of the LPS-activated PI3K/Akt signal pathway. Compound 20 significantly alleviated inflammation in the gastrointestinal tract of zebrafish in vivo. In addition, compound 20 showed good biosafety in cytotoxicity evaluation. This study provides a novel reference for the development of treatments for health problems related to anti-oxidation and anti-inflammation properties.

Synthesis and cytotoxic evaluation of glycosyl derivatives of icaritin

A series of glycosyl alkyl/triazol-linked icaritin derivatives have been designed and synthesised. The target glycosyl derivatives were evaluated for their anticancer activity against three human cancer cell lines. The results of preliminary anticancer tests in vitro revealed that mannose derivatives 10a–10c (100 μM) with different aliphatic chain lengths exhibited increased cytotoxicity against HepG2 and SK-OV-3 cells compared with the parent compound icaritin. The data indicated that the kind of glycosyl groups and linkers affected the anticancer potency significantly. The ADME analysis of derivatives 10a–10c was also performed.

Abstract 92: Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune cells and cancer cells. Immune effector T-cells are a promising cancer therapy due to their innate cytotoxicity. In particular, CAR T-cell therapy uses genetically engineered T-cells that express a chimeric antigen receptor that binds to a specific antigen on tumor cells. Assessing the efficacy and potency of CAR T-cell therapies in vitro and at high throughputs is vital for the preclinical development of these promising therapies. Here, we describe an in vitro potency assay that uses an automated imaging platform to quantify immune cell-mediated cytotoxicity of cancer target cells by immune effector cells. A GFP fluorescent and HER-2 expressing lung cancer cell line, A549, was seeded into a 96-well cell culture plate. Then, HER-2 CAR T-cells were added at 24 hours post target cell seeding at various E:T ratios (1:10, 1:5, 1:2, 1:1, and 5:1). Fluorescent images of the target cells were captured every 6 hours by the Omni Pro 12, an automated, high-throughput, live-cell analysis platform designed for continuous multi-well imaging inside an incubator. Automated image analysis was performed using the Confluency Module in the Axion Portal to view attachment and proliferation and quantify cytolysis of fluorescent A549 target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent confluency of treated wells to no treatment control wells. A549-GFP cells showed a dose-dependent decrease in confluency over time that correlated with increasing amounts of CAR T-cells. At 92 hours post HER-2 CAR T-cell addition, the 5:1 ET group demonstrated approximately 91.8% +/- 2.0% cytolysis of A549-GFP cells, while the 1:10 group demonstrated approximately 69.2% +/- 3.0% cytolysis. Future work will evaluate differences in CAR T-cell potency when co-cultured with target cells that express different levels of HER-2. Overall, the Omni Pro 12 platform enables continuous quantification of the potency and kinetics of immune cell-mediated cytolysis. Citation Format: Denise Sullivan, Lieke Stemkens, Inge Thijssen, Svenja Meiler, Danielle Califano, Stacie Chvatal, Daniel Millard. Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 92.

Abstract 6787: Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids

Abstract Introduction: Over the last decade, chimeric antigen receptor T cell (CAR-T)-based immunotherapy has achieved unprecedented clinical benefits for the treatment of hematological malignancies. However, application of CAR-T therapy to solid tumors remains challenging, in part due to tumor antigen heterogeneity and microenvironmental complexity present in these tumors. Therefore, selecting physiologically-relevant models is critical for validation of the on-target efficacy and off-target effect of CAR-T therapy. Emerging 3D patient-derived tumoroid models better recapitulate the morphological organization, mutational status, and gene expression levels of patient tumors. Hereby, we propose a screening platform to evaluate mesothelin (MSLN)-targeted CAR-T therapy for solid tumors using patient-derived tumoroids. Methods: T cells were isolated from peripheral blood mononuclear cells of healthy human donors. Meso3 (region III of MSLN) or CD19 CARs (served as control) were knocked-in (KI) to activated T cells using electroporation-based CRISPR/Cas9 system followed by CAR Cassette containing adeno-associated virus (AAV) transduction. T-cell receptor (TCR) knockout (KO) and CAR-KI efficiency were validated by flow cytometry. Patient-derived tumoroids with high and low MSLN expression were co-cultured with various effector-to-target ratios of Meso3 CAR, CD19 CAR and Control (TCR KO without CAR KI) T cells. Cytotoxicity was evaluated by caspase-based live cell imaging. Key cytokine secretion was measured using a multiplexing platform 2-3 days post co-culture. Results: T-cell electroporation of CRISPR/Cas9 yielded a high KO efficiency (&amp;gt;90%) of TCR as well as high AAV-mediated CAR cassette-KI efficiency (&amp;gt;70%). The killing efficiency of Meso3 CAR-T cells with different effector to target ratios was successfully captured in a ratio-dependent manner via live cell imaging and analysis platform in the MSLN-high tumoroids, while minimum killing was observed in MSLN-low tumoroids. As expected, control and CD19 CAR-T cells showed no killing towards MSLN-high or -low tumoroids, which do not express CD19. Moreover, the observed killing ability correlated with secretion of cytokines associated with T cell-mediated cytotoxicity. Conclusion: These findings demonstrated the ability of 3D patient-derived models as a screening platform for validations of CAR-T cell therapy in vitro. This platform has potential advantages over commonly used impedance-based and flow cytometry-based killing assay since it better captures the 3D architecture and microenvironment of the tumor origin and allows real-time quantification. With the ability of accurately detecting on and off-target killing, quick turn-around time, and cost-effectiveness compared to in vivo systems, this platform allows for high-throughput screening workflows for CAR-T cell therapies. Citation Format: Xiaoyu Yang, Maryam Mafreshi, Garrett Wong, Vivek Chandra, Colin Paul, Chris Yankaskas, Brittany Balhouse, Jason Sharp, Matt Dallas, Erik Willems, David Kuninger. Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6787.

Abstract 478: Targeted combination nano-drug delivery system to enhance anti-cancer efficacy and reduce side effects

Abstract Background: Colorectal cancer (CRC) is a global health concern, with significant morbidity and mortality. Oxaliplatin (OXL) is a common treatment for CRC but its effectiveness is hampered by side effects, including gastrointestinal and neurotoxic effects. APX3330 exerts anti-angiogenic and neuroprotective effects. It is proposed that the combined use of OXL and APX3330-loaded nanoparticles (NPs) would improve drug delivery to cancer cells with enhanced therapeutic efficacy while reducing side effects of OXL. Methods: We developed dual-loaded NPs containing OXL and APX3330 linked to a cancer targeting monoclonal antibody (NPs+mAb). We characterized the physicochemical properties of the NPs and evaluated their cytotoxicity and drug release using CRC cell lines. In vivo, we assessed their therapeutic efficiency and side effects using an orthotopic CRC mouse model. Results: NPs exhibited an average size of 229±26 nm with a zeta potential of -24.1±2.1 mV. The drug loading and encapsulation efficiency were 3.3±0.2% and 71.9%, respectively. Morphological analysis via scanning electron microscopy confirmed NP structure. In vitro drug release studies showed a slower release profile from NPs compared to free OXL, with a cumulative release of 34% (w/w) after 48 hours. Notably, cytotoxicity evaluation on CT-26 cells revealed a significant reduction in the IC50 for NPs compared to free APX3330 and OXL. In vivo studies demonstrated a remarkable 31% reduction in tumor growth rate in animals treated with NPs+mAb compared to those receiving free forms of OXL and APX3330. Body weight for animals treated with NPs+mAb remained similar to the healthy group, indicating minimal systemic toxicity. Moreover, the length of the colon in healthy organs remained steady compared to animals treated with OXL, which experienced a reduction in colon length. Animals treated with the free form of OXL exhibited diarrhoea, while the stool water content for animals treated with NPs+mAb remained at a similar level as the healthy group, suggesting reduced gastrointestinal side effects. Conclusions: This study demonstrates the potential of NPs with improved retention and encapsulation efficiency for effective drug delivery. Our research validates our novel method for creating NPs with mAb conjugation, enhancing cytotoxicity against CRC cell lines. In conclusion, the designed NPs+mAb formulation improves OXL treatment efficacy, providing targeted delivery of anti-cancer drugs to the tumor site and reducing tumor size while minimizing side effects. Citation Format: Niloufar Rashidi, Majid Davidson, Vasso Apostolopoulos, Mark R. Kelley, Kulmira Nurgali. Targeted combination nano-drug delivery system to enhance anti-cancer efficacy and reduce side effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 478.

Abstract 4257: A novel 3D co-culture platform for patient-specific immune-oncology treatment

Abstract Lung cancer is the leading cause of global cancer-related mortality resulting in ~1.8 million deaths. annually. Over the past decade, the contribution of tumor microenvironment (TME) in lung cancer progression has been highly appreciated and provided significant improvements of survival outcomes for patients. That includes the complexity of the tumor immune microenvironment (TIME) playing a critical role in cancer progression. However, drug resistance usually arises and there is an urgent need for novel immune cell-based therapies. 3D tumor cell structures that recapitulate the architecture and functionality of the original tumor have revolutionized the understanding of disease mechanisms and testing potential therapeutics. However, there are certain drawbacks that need to be overcome. For example, these models mainly consist of only the epithelial compartment and lack signals from the microenvironment. Recent advances with engineering co-culture systems with immune cells at Champions Oncology help to emulate cell-cell and immune cell infiltration. We have successfully established a biobank of TumorGraft3D (CTG3Ds) derived from non-small cell lung cancer (NSCLC) patient-derived xenografts as well as their autologous and allogeneic immune cells (TILs, PBMCs) to establish a proprietary co-culture platform to investigate disease mechanisms and test potential therapeutics. In brief, surgical resections from patients were used to generate patient-derived xenografts organoids, and matched immune cells were expanded using standard rapid expansion protocol. The combination of a comprehensive staining system and high-content imaging as an endpoint allowed for 3D confocal analysis and deep evaluation of cancer-immune cellular interaction at the single cell level. Our pipeline of image analysis not only differentiates cancer-immune cell spatial distribution and cellular death but also allows for the evaluation of tumor infiltration and drug cytotoxicity. The characterization of the platform showed proliferation of the cancer cells and functional immune profiling by flow cytometry. The molecular profile of the organoids composing the platform showed a tight overlap with the original tumor highlighting the clinical relevance of the model. In addition, this novel co-culture platform has been tested with several NSCLC models to test the efficacy of various therapeutics such as immune-checkpoint inhibitors (ICIs) in reproducible, cost-effective, and patient-specific manner. In summary, Champion’s novel co-culture platform allows screening of novel immune targeting agents to determine impacts tumor cytotoxicity. Furthermore, our lung cancer tumorGraft3D platform serves as a powerful tool to facilitate the practice of precision medicine by investigating a variety of therapeutic modalities, alone or in combination, to identify patient-specific treatment strategies. Citation Format: Taylor Light, Samaneh Kamali, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. A novel 3D co-culture platform for patient-specific immune-oncology treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4257.

Abstract 4100: AMP-peptide vaccination against mutant BRAF epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background Mutations in BRAF cause ~8% of human cancers[1]. Nonetheless, therapies designed to promote immune responses against mutated BRAF (mBRAF) have led to limited clinical success[2], despite evidence confirming T cell recognition of these mutations[3,4]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by resident immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components including peptide antigens and molecular adjuvants results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety, and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT04853017). Application of this strategy to prevalent BRAF mutations with ELI-007 offers the potential for improved therapeutic activity in a setting of significant unmet need, as BRAF inhibitors benefit only a fraction of patients, with near universal progression. Methods Following multi-dose immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mBRAF V600E/K peptides and CpG adjuvant, immunological readouts were performed 7 days after dosing. Antigen-specific T cell responses were assessed after ex vivo re-stimulation of cells with mBRAF overlapping peptides. Assays included analysis of antigen-specific T cell frequencies, multiplexed proteomics determining polyfunctionality, and effector function and cytotoxic potential evaluation. Responses were determined in secondary lymphoid tissues (spleen), as well as lung, to which many BRAF driven cancers metastasize. Results AMP-immunization generated robust in vivo immune responses yielding strong T cell activation against mBRAF epitopes. Non-lymph node targeted vaccination using soluble comparators were inactive with no response above mock treated mice. Responses to AMP-vaccination were characterized by the generation of significantly increased frequency of Th1-skewed, polyfunctional T cells (IFNγ, TNFα, IL2) specific to mBRAF V600E and V600K. Induced T cells further exhibited mBRAF-specific cytotoxic function. Conclusions AMP-conjugation enhances efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These enhanced immune responses provide an encouraging therapeutic opportunity to treat &amp;gt;90% of BRAF-mutated cancers[5] with the AMP-mBRAF vaccine, ELI-007. Moreover, the AMP-platform technology is simple, rapidly executable, and scalable, promising broad clinical off-the-shelf application for BRAF. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against mutant BRAF epitopes promotes lymph node delivery to generate potent, functional T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4100.

Abstract 5807: Bicycle Toxin Conjugates®for the treatment of solid tumors

Abstract Bicycle Therapeutics is developing a unique class of chemically synthesized medicines. Based on its proprietary bicycle peptide (Bicycle®) phage display platform, Bicycles are a unique class of highly constrained peptides, which have demonstrated utility in the targeted delivery of different classes of payloads (for example cytotoxic agents, radioisotopes, immune modulators) into tumors. Bicycles are currently being explored in the clinic as Bicycle Toxin Conjugates® (BTCs) for targeted delivery of cytotoxic payloads into tumors. BTCs consist of a bicyclic peptide that is conjugated to a cytotoxic payload via a cleavable linker, which allows payload release in the tumor microenvironment or within the tumor cell. BTCs were developed to address the shortcomings of antibody drug conjugates (ADCs) in several ways. First, the small size of BTCs (∼4 kDa) compared to large biologic entities such as monoclonal antibody (mAb)-based conjugates (∼150 kDa) allows rapid distribution to tissues and extensive tumor penetration, which enables rapid delivery of payload into the tumor. Second, the peptidic nature of BTCs results in relatively short, yet tunable, duration of systemic exposure and liver-sparing renal elimination. These properties limit the body’s exposure to payload and should therefore minimize damage to normal tissue. In this body of work, we used in vitro cytotoxicity and cell uptake assays and mouse and rat cell line derived xenograft models for 1) cytotoxicity and anti-tumor activity evaluation, and 2) Bicycle and toxin uptake and biodistribution evaluation. Here, we show that BTCs targeting a number of different tumor antigens can deliver toxins to tumor tissue producing durable responses in a range of preclinical in vivo models, spanning several solid tumor indications. Citation Format: Stephen J. Walsh, Johanna Lahdenranta, Philip Huxley, Gemma Mudd, Gavin Bennett, Amy Brown, Katerine an Rietschoten, Liuhong Chen, Heather Scott, Gabriella Ivanova-Berndt, Katarzyna Dzionek, Mike Rigby, Olga Burenkova, Phil Jeffrey, Paul Beswick, Michael Skynner, Nicholas Keen. Bicycle Toxin Conjugates®for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5807.

Anti-cancer activity of crude Slrp protein conjugated mesoporous silica nanoparticles in HeLa Cell Lines: An in vitro approach

Microbial based therapeutics for cancer have gained a significant interest in recent decades. The present study relies on the synthesis, analysis, and conjugation of Salmonella Leucine-rich Proteins (SlrP) with mesoporous silica nanoparticles (MSNN) to evaluate their potential anticancer activity. The SlrP proteins were effectively produced and isolated from Salmonella enterica using Tryptic Soy Broth (TSB), and the subsequent SDS-PAGE analysis verified the presence of a band at around 72 KDa. The MSN synthesis yielded particles with an average diameter of 68.05±0.87 nm and a pore diameter of 7.1 nm. In addition, we synthesized MSNMPA and then conjugated them with SlrP. Characterization studies confirmed the effective conjugation. The cytotoxicity evaluation conducted on HeLa cells revealed no substantial modification in cell viability upon treatment with MSN alone. Nevertheless, when MSNMPA/SlrP was done, it demonstrated significant cytotoxic properties, as evidenced by an IC50 value of 10 µg/mL. The results indicate that SlrP-conjugated MSN (MSNMPA/SlrP) could be utilized as promising nanocarriers for delivering anticancer proteins.

Purification, Molecular Docking and Cytotoxicity Evaluation of Bioactive Pentacyclic Polyhydroxylated Triterpenoids from Salvia urmiensis.

Triterpenoids, as one of the largest classes of naturally occurring secondary metabolites in higher plants, are of interest due to their high structural diversity and wide range of biological activities. In addition to several promising pharmacological activities such as antimicrobial, antiviral, antioxidant, anti-inflammatory and hepatoprotective effects, a large number of triterpenoids have revealed high potential for cancer therapy through their strong cytotoxicity on cancer cell lines and, also, low toxicity in normal cells. So, this study was aimed at discovering novel and potentially bioactive triterpenoids from the Salvia urmiensis species. For this, an ethyl acetate fraction of the acetone extract of the aerial parts of the plant was chromatographed to yield five novel polyhydroxylated triterpenoids (1: -5: ). Their structure was elucidated by extensive spectroscopic methods including 1D (1H, 13C, DEPT-Q) and 2D NMR (COSY, HSQC, HMBC, NOESY) experiments, as well as HRESIMS analysis. Cytotoxic activity of the purified compounds was also investigated by MTT assay against the MCF-7 cancer cell line. Furthermore, a molecular docking analysis was applied to evaluate the inhibition potential of the ligands against the nuclear factor kappa B (NF-κB) protein, which promotes tumor metastasis or affects gene expression in cancer disease. The 1β,11β,22α-trihydroxy-olean-12-ene-3-one (compound 4: ) indicated the best activity in both in vitro and in silico assays, with an IC50 value of 32 µM and a docking score value of - 3.976 kcal/mol, respectively.

Wound microenvironment-responsive peptide hydrogel with multifunctionalities for accelerating wound healing.

The fabrication of wound microenvironment-responsive peptide hydrogels with hemostatic ability, antibacterial activity, and wound healing potential remains a challenge. Herein, we constructed a multifunctional dressing by inducing the self-assembly of a peptide (Pep-1) and water-soluble new methylene blue (NMB) through electrostatic interaction. The self-assembly mechanism was demonstrated using a combination of transmission electron microscopy, circular dichroism spectrum, fluorescence spectrum, Zeta potential, and rheological analysis. The Pep-1/NMB hydrogel also exhibited a faster drug release rate in wound acidic environment. Furthermore, when Pep-1/NMB was exposed to a 635 nm laser, its antibacterial ratios increased sharply to 95.3%, indicating remarkably improved antibacterial effects. The findings from the blood coagulation and hemostasis assay indicated that Pep-1/NMB effectively enhanced the speed of blood clotting in vitro and efficiently controlled hemorrhage in a mouse liver hemorrhage model. Meanwhile, hemolytic and cytotoxicity evaluation revealed that the hydrogel had excellent hemocompatibility and cytocompatibility. Finally, the findings from the wound healing studies and H&E staining indicated that the Pep-1/NMB hydrogel had a significant impact on cell migration and wound repair. The results indicated that wound microenvironment-responsive Pep-1/NMB hydrogel had significant potential as a highly effective wound dressing platform, offering rapid hemostasis, antibacterial, and wound healing acceleration properties.

A Facile Access to Green Fluorescent Albumin Derivatives.

A Morita-Baylis-Hillman Adduct (MBHA) derivative bearing a triphenylamine moiety was found to react with human serum albumin (HSA) shifting its emission from the blue to the green-yellow thus leading to green fluorescent albumin (GFA) derivatives and enlarging the platform of probes for aggregation-induced fluorescent-based detection techniques. A possible interaction of MBHA derivative 7 with a lipophilic pocket within the HSA structure was suggested by docking studies. DLS experiments showed that the reaction with HSA induce a conformational change of the protein contributing to the aggregation process of GFA derivatives. The results of investigations on the biological properties suggested that GFA retained the ability of binding drug molecules such as warfarin and diazepam. Finally, cytotoxicity evaluation studies suggested that, although the MBHA derivative 7 at 0.1 μg/mL affected the percentage of cell viability in comparison to the negative control, it cannot be considered cytotoxic, whereas at all the other concentrations≥0.5 μg/mL resulted cytotoxic at different extent.

Solvent-free strategy for facile synthesis and cytotoxicity evaluation of benzimidazole derivatives

This study reports a facile synthesis and the cytotoxic evaluation of N–H benzimidazoles. This synthetic method made use of the condensation reaction between o-phenylenediamine and benzylamine and was highlighted by its solvent-free reaction conditions and short reaction time with the use of Na2S2O5 as an inexpensive and environmentally friendly oxidizing agent. The cytotoxicity assays revealed that the benzimidazole derivatives 3l, 3h, and 3r displayed strong activity against the HeLa cancer cell line with the IC50 of 1.54 μM, 0.86 μM, and 1.03 μM, respectively, which were 5.6-fold, 10-fold, and 8-fold stronger than the positive control 5-fluorouracil (5-FU, IC50 = 8.61 μM). Furthermore, both compounds 3h and 3r exhibited similar cytotoxicities against MCF-7, which were equivalent to that of 5-FU and about 2.5-fold stronger than 5-FU towards the A549 cell line.

Trifluoroacetyl‐effect on amino‐single benzene‐based fluorophores: Synthesis, optical properties, and cytotoxicity

AbstractAmino‐single benzene‐based fluorophores (SBBFs) are representative small‐size fluorescent dyes that have an electron push‐pull structure within the benzene core. In this work, a new library of amino‐SBBFs, which have trifluoroacetyl moiety at the electron‐pushing amine group (named SBBF‐TFA), featuring a variety of methylamines, dimethylamines, as well as acetyl. To explore the SBBF‐TFA library, we conducted (i) library synthesis, (ii) assessment of various photophysical properties in nine different types of organic/aqueous solvents, and (iii) evaluation of cytotoxicity in the HeLa cell line. We believe that our findings provide valuable insights for advancing novel small‐molecule fluorescent probes and materials with diverse applications across pharmacochemical fields.

Synthesis, computational study, and antileishmanial and antifungal evaluation of phenoxyacetophenone hydrazones bearing a 4-arylthiazole moiety

A series of new phenoxyacetophenone hydrazones 6a-l bearing a 4-arylthiazole moiety were synthesized and tested as antileishmanial agents. After spectroscopic and computational characterization of the synthesized compounds, their antileishmanial and antifungal activities were investigated in vitro. All compounds exhibited good antileishmanial activity, demonstrating IC50 values = 3.63–17.4 μg/ml against promastigotes of Leishmania major. Certainly, the para-tolyl analog 6b showed the most potent activity against promastigotes. Compounds 6b, 6c, 6f, and 6i-6k with good activity against promastigotes (IC50 values ≤ 7.59 μg/ml) were subjected to further bioassay on the amastigotes of L. major, as well as cytotoxicity evaluation against host-cell macrophages. The representative compounds displayed substantial anti-amastigote activity (IC50 values ≤ 21.3 μg/ml) with good selectivity index (SI > 18). Additionally, the in vitro antifungal screening against yeasts, molds and dermatophytic fungi revealed that several compounds had respectable activity against dermatophytes. These results demonstrate that the thiazolyl-hydrazones 6 are proper leads for further design and development of potential antileishmanial agents.

Exploring Borate-Modified Calcium Phosphate Ceramics: Antimicrobial Potential and Cytocompatibility Assessment.

Addressing periprosthetic infections, which present significant healing challenges that often require revision surgeries, necessitates the development of novel antibacterial materials and implants. Current research focuses on creating materials that hinder bacterial adhesion, colonization, and proliferation in surrounding tissues. Boron (B)-containing compounds are known for their antibacterial properties and potential in bone metabolism for regenerative medicine. In this study, we synthesized B-containing tricalcium phosphate (0.3B-TCP) with 1.1 wt.% B content via precipitation from aqueous solutions and sintering at 1100 °C. X-ray diffraction confirmed the ceramic's primary crystalline phase as β-TCP, with B evenly distributed according to energy-dispersive spectroscopy data. Electron paramagnetic resonance (EPR) data verified stable paramagnetic borate anions, indicating successful BO33- substitution for phosphate groups. The microstructural properties of 0.3B-TCP ceramic were assessed before and after soaking in a saline solution. Its bending strength was approximately 30 MPa, and its porosity was about 33%. 0.3B-TCP ceramic demonstrated significant antimicrobial efficacy against various bacterial strains and a fungus. Cytotoxicity evaluation using equine adipose tissue-derived mesenchymal stem cells and osteogenic differentiation assessment were conducted. The combination of antibacterial efficacy and good cytocompatibility suggests 0.3B-TCP ceramic as a promising bone substitute material.

A high-throughput gut-on-chip platform to study the epithelial responses to enterotoxins

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins’ distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.