Cell Cytotoxicity Assay Research Articles

Differential effects of SARS-CoV-2 amyloidogenic segments on the aggregation and toxicity of human islet amyloid polypeptide within membrane environments

Human islet amyloid polypeptide (hIAPP), an intrinsically disordered protein (IDP), plays a significant role in the pathogenesis of type 2 diabetes through its aggregation. Recent studies have suggested that certain viral protein segments exhibit amyloidogenic potential and may influence its amyloid aggregations associated with pathogenesis. However, the potential link between recurrent SARS-CoV-2 infections and the exacerbation of type 2 diabetes remains poorly understood. In this study, we explore how the amyloidogenic segments of SARS-CoV-2, specifically SK9 and FI10, influence the aggregation of hIAPP and the toxicity of the resulting conformers in a membrane environment. To investigate this, we utilized a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance, atomic force microscopy, dynamic light scattering, fluorescence assays, and cell cytotoxicity assays, complemented by molecular dynamics simulations. Our results indicate that SK9 and FI10 promote hIAPP aggregation in a membrane-mimicking environment, forming distinct aggregate structures. Specifically, SK9 accelerates rapid fibril formation due to inter-chain interactions, while FI10 stabilizes oligomeric aggregates primarily through intra-chain contacts. These results reveal the differential effects of viral protein segments on amyloid formation pathways and aggregate characteristics, providing new insights into the mechanisms of amyloid aggregation for developing better therapeutic strategies against amyloid-associated diseases, particularly diabetes.

Impact of 5-Flurouracil loaded chitosan nanoparticle on A375 and A431 cell line for the therapy of skin cancer

Skin cancer is an uncontrolled proliferation of abnormal skin cells that is usually caused by damaging UV radiation. Skin cancer is classified into two types: non-melanoma skin cancer and melanoma skin cancer. The entire studies focus on cytotoxic effect of prepared 5-Flurouracil loaded chitosan nanoparticle using A375 and A431 skin cancer cell line. In-vitro cell cytotoxicity assay, DAPI test of prepared formulation was conducted to observe the cytotoxic effect. The cell cytotoxicity investigation revealed that nanoparticles had higher cytotoxicity, inducing greater apoptosis within 72 h. The experimental results show that the produced nanoparticle is a good candidate for use as a 5-FU carrier in the treatment of skin cancer.

Anti-IL-1RAP scFv-mSA-S19-TAT fusion carrier as a multifunctional platform for versatile delivery of biotinylated payloads to myeloid leukemia cells

Acute myeloid leukemia (AML) is an aggressive blood cancer with frequently poor clinical outcomes. This heterogeneous malignancy encompasses genetically, molecularly, and even clinically different subgroups. This makes it difficult to develop therapeutic agents that are effective for all subtypes of the disease. Therefore, a selective, universal, and adaptable delivery platform capable of carrying various types of anti-neoplastic agents is an unmet requirement in this area. Two multifunctional fusion proteins were designed for the delivery of biotinylated cargoes to human myeloid leukemia cells by fusing an anti-IL-1RAP single-chain antibody with streptavidin (tetramer or monomer), a cell-penetrating peptide (CPP), and an endosomolytic peptide in a single biomacromolecule. The designed fusions were analyzed primarily in silico, and the biofunctionality of the selected fusion was fully characterized via several binding assays, hemolysis assay, confocal microscopy and cell cytotoxicity assay after production via the Escherichia coli (E. coli) system. The refolded protein exhibited desirable binding activity to leukemic cells, pure antigen and biotinylated BSA. Further analyses revealed efficient cellular uptake, endosomolytic activity, and nuclear penetration without any detectable cytotoxicity toward normal epithelial cells. The described platform seems to have great potential for targeted delivery of different therapeutics to malignant myeloid cells.

Transcriptomic analysis reveals Streptococcus agalactiae activation of oncogenic pathways in cervical adenocarcinoma.

Cervical adenocarcinoma (AC), a subtype of uterine cervical cancer (CC), poses a challenge due to its resistance to therapy and poor prognosis compared with squamous cervical carcinoma. Streptococcus agalactiae [group B Streptococcus (GBS)], a Gram-positive coccus, has been associated with cervical intraepithelial neoplasia in CC. However, the underlying mechanism interaction between GBS and CC, particularly AC, remains elusive. Leveraging The Cancer Genome Atlas public data and time-series transcriptomic data, the present study investigated the interaction between GBS and AC, revealing activation of two pivotal pathways: 'MAPK signaling pathway' and 'mTORC1 signaling'. Western blotting, reverse transcription-quantitative PCR and cell viability assays were performed to validate the activation of these pathways and their role in promoting cancer cell proliferation. Subsequently, the present study evaluated the efficacy of two anticancer drugs targeting these pathways (binimetinib and ridaforolimus) in AC cell treatment. Binimetinib demonstrated a cytostatic effect, while ridaforolimus had a modest impact on HeLa cells after 48 h of treatment, as observed in both cell viability and cytotoxicity assays. The combination of binimetinib and ridaforolimus resulted in a significantly greater cytotoxic effect compared to binimetinib or ridaforolimus monotherapy, although the synergy score indicated an additive effect. In general, the MAPK and mTORC1 signaling pathways were identified as the main pathways associated with GBS and AC cells. The combination of binimetinib and ridaforolimus could be a potential AC treatment.

Evaluation of the bioactivity of Berberis microphylla G. Forst (Calafate) leaves infusion

Berberis microphylla G Forst (Calafate) have been used in traditional medicine from prehispanic times in Patagonia. In the last decade the consumption of the fruit has been increased due to their antioxidant capacity, and because several studies demonstrated health benefits associated with the protection against atherosclerosis and other metabolic diseases. Nevertheless, the bioactivity properties of the leaves, a by-product of agronomic management, have been poorly studied. Recently, 108 compounds mainly hydroxycinnamic acids, flavonols, and berberine were identified in a methanolic extract of the leaves, demonstrating great potential for the development of new functional beverages. Based on these, for first time a comprehensive chemical characterization and bioactivity was evaluated for a Calafate leaves infusion prepared in hot water. For this, chemical characterization of the infusion was performed by UHPLC-Q-TOF and TXRF. Bioactivity was assayed by antioxidant capacity, cell cytotoxicity, and cell oxidative stress assays. Inhibition of both Aβ aggregation for Alzheimer's disease and gastrointestinal enzymes for metabolic syndromes were evaluated. The results show that the infusion is rich in hydroxycinnamic acids and other bioactive compounds. The infusion does not contain toxic metals or cytotoxicity activity. The infusion can reduce intracellular reactive oxygen species in HUVEC cells and showed a reduction in the Aβ aggregation being a potential beverage for Alzheimer's prevention. Finally, the infusion had in-vitro hypoglycemic and hypolipidemic effects. These results support the usage of Berberis microphylla G Forst leaves as a new functional beverage.

Polyphenolic extracts from Diospyros kaki and Vitis vinifera by-products stimulate cytoprotective effects in bacteria-cell host interactions by mediation of transcription factor Nrf2

BackgroundThe intestinal and skin epithelium play a strong role against bacterial stimuli which leads to inflammation and oxidative stress when overwhelmed. Polyphenols from fruit-rich diets and by-products show promise against bacterial deleterious effects; however, their antibacterial and health-promoting effects remain understudied. PurposeThis study aimed to assess the impact of polyphenolic extracts of grape (GrPE), persimmon (PePE) and pomegranate (PoPE) by-products on bacterial pathogen-host interactions, focusing beyond growth inhibition to explore their effects on bacterial adhesion, invasion, and modulation of host responses. MethodsThe microdilution method, as well as the tetrazolium based MTT cell proliferation and cytotoxicity assay with crystal violet staining were used to identify extracts sub-inhibitory concentrations that interfere with bacterial adhesion, invasion or lipopolysaccharides (LPS) effect on cell hosts without compromising host viability. The cytoprotective effects of extracts were assessed in a knock-down model of nuclear factor erythroid 2-related factor 2 (Nrf2). ResultsAll extracts demonstrated significant reductions in pathogen adhesion to Caco-2 and HaCaT cells while preserving cellular integrity. Notably, PePE exhibited specific efficacy against Salmonella enterica adhesion, attributed mostly to its gallic acid content, whereas PoPE reduced S. enterica invasion in Caco-2 cells. The extracts supported the prevalence of non-pathogenic and commensal strains of intestinal and skin surfaces, selectively reducing pathogenic adhesion. The extracts mitigated the oxidative stress, enhanced the barrier function, and modulated the pro-inflammatory cytokines in LPS-challenged cells. GrPE, rich in anthocyanins, and PePE were found to mediate their protective effects through Nrf2 activation, while PoPE exerted multifaceted actions independent of Nrf2. ConclusionOur results highlight the therapeutic potential of GrPE, PePE, and PoPE in shaping bacterial-host interactions, endorsing their utility as novel nutraceuticals for both oral and topical applications to prevent potential bacterial infections through innovative mechanisms.

Cell Proliferation and Cytotoxicity Assays, The Fundamentals for Drug Discovery

Review Cell Proliferation and Cytotoxicity Assays, The Fundamentals for Drug Discovery Jingyi Niu†, Minai Li† and Ying Wang* State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao 999078, China * Correspondence: emilyywang@um.edu.mo † These authors contributed equally to this work Received: 6 May 2024; Revised: 19 June 2024; Accepted: 21 June 2024; Published: 20 August 2024 Abstract: Cell proliferation and cytotoxicity assays are fundamental to drug discovery. This review summarizes prevalent methodologies for assessing cell proliferation and cytotoxicity, including direct cell count, metabolic activity, luminescent labeling, and tri-color viability imaging. The critical determinants that can significantly impact these assay outcomes, such as cellular doubling time, transitional states like quiescence and autophagy, cell cycle stages, metabolic enzyme functions, and genetic variability, are also explored. It is necessary to integrate the commonly used assays with additional analytical techniques to achieve precision in drug discovery. A multi-tiered approach that combines cellular assays with molecular analyses can improve screening processes, reduce false negatives, and increase confidence in the therapeutic potential of lead compounds.

Design, Synthesis and Bioactivity Evaluation of Ag(I)-, Au(I)- and Au(III)-Quinoxaline-Wingtip N-Heterocyclic Carbene Complexes Against Antibiotic Resistant Bacterial Pathogens.

Intending to homogenize the biological activities of both quinoxaline and imidazole moieties, the proligand, 1-methyl-3-quinoxaline-imidazolium hexaflurophosphate (1.HPF6), and [Ag(1)2][PF6], (2); [Au(1)2][PF6], (3); and [Au(1)Cl3], (4) NHC complexes were synthesized. All the synthesized compounds were characterized by elemental analysis, NMR, and UV-Vis spectroscopy. Finally, single crystal X-ray structures revealed a linear geometry for complex 2 whereas a square planar geometry for complex 4. The formation of complex 3 was confirmed and supported by its MS spectra. The antibacterial activities of all the synthesized complexes were investigated against gram-positive bacteria and gram-negative bacteria. The Au(III)-NHC complex, 4 showed the highest antibacterial activity with extremely low MIC values against both the bacterial strains (0.24 μg mL-1). Monitoring of zeta potential supports the higher activity of complex 4 compared to 2 and 3. ROS production by complex 4 has also been measured in vitro in the CT26 cancer cell lines, which is directly responsible for targetting and killing the bacterial pathogens. Cell cytotoxicity assay using 293T cell lines has been performed to investigate the biocompatibility nature of complex 4. Also, an excellent hemocompatibility was assigned to it from its hemolytic studies, which provide valuable insights into the design of novel antibacterial agents.

Meldonium, as a potential neuroprotective agent, promotes neuronal survival by protecting mitochondria in cerebral ischemia–reperfusion injury

BackgroundStroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown.MethodsA rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium’s neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen–glucose deprivation reperfusion was used to study meldonium’s protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting.ResultsMeldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia–reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3β signaling pathway to inhibit mitochondria-dependent neuronal apoptosis.ConclusionOur study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis.

New Analogues of the Nicotinamide Phosphoribosyltransferase Inhibitor FK866 as Potential Anti-Pancreatic Cancer Agents.

During the past two decades, many nicotinamide phosphoribosyltransferase (NAMPT) inhibitors were prepared and tested because this enzyme is overexpressed in pancreatic cancer. Although FK866 is a well-known, strong NAMPT inhibitor, it suffers severe drawbacks. Our work aimed to synthesize efficient NAMPT inhibitors featuring better pharmacokinetic properties than the pyridine-containing FK866. To this aim, the new anticancer agents were based on benzene, pyridazine, or benzothiazole moieties as a cap group instead of the pyridine unit found in FK866 and other NAMPT inhibitors. The new compounds, prepared exploiting standard heterocycle chemistry and coupling reactions (e.g., formation of amides, ureas, and cyanoguanidines, copper-mediated azide-alkyne cycloaddition), have been fully characterized using NMR and HRMS analyses. Their activity has been evaluated using cytotoxicity and intracellular NAD depletion assays in the human pancreatic cancer cell line MiaPaCa-2. Among the 14 products obtained, compound 28, bearing a pyridazine unit as the cap group and a thiophene moiety as the tail group, showed 6.7 nanomolar inhibition activity in the intracellular NAD depletion assay and 43 nanomolar inhibition in the MiaPaCa-2 cells cytotoxicity assay, comparable to that observed for FK866. The positive results observed for some newly synthesized molecules, particularly those carrying a thiophene unit as a tail group, indicate that they could act as in vivo anti-pancreatic cancer agents.

Transformation of Metal-Organic Framework from Kinetic to Thermodynamic Product for Controlled Delivery of Vitamin C.

A biocompatible metal-organic framework (MOF), named HSTC-4, constructed using the flexible 4,4'-oxybis(benzoic acid) (OBA), was developed to enable efficient loading and controlled release of vitamin C (VC) through a combination of strategies involving ligand length, structure design, and metal selection. The kinetic product HSTC-4 demonstrates a propensity for transforming into the thermodynamically stable HSTC-5 under external stimuli, such as photoillumination and vacuum heating, as witnessed by single-crystal to single-crystal transformation. Density functional theory (DFT) calculations reveal that the VC guest molecules exhibit stronger binding affinity with HSTC-5 due to its narrower pores compared to HSTC-4, resulting in a slower release of VC from VC@HSTC-5. Furthermore, precise control over VC release can be achieved by introducing surface modifications involving SiO2 onto the structure of VC@HSCT-5, while simultaneously adjusting environmental factors such as pH and temperature conditions. Preliminary cell culture experiments and cytotoxicity assays highlight the biocompatibility of HSTC-5, suggesting that it is a promising platform for sustained drug delivery and diverse biomedical applications.

T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway

BackgroundBone defects, resulting from substantial bone loss that exceeds the natural self-healing capacity, pose significant challenges to current therapeutic approaches due to various limitations. In the quest for alternative therapeutic strategies, bone tissue engineering has emerged as a promising avenue. Notably, excretory proteins from Toxoplasma gondii (TgEP), recognized for their immunogenicity and broad spectrum of biological activities secreted or excreted during the parasite’s lifecycle, have been identified as potential facilitators of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Building on our previous findings that TgEP can enhance osteogenic differentiation, this study investigated the molecular mechanisms underlying this effect and assessed its therapeutic potential in vivo.MethodsWe determined the optimum concentration of TgEP through cell cytotoxicity and cell proliferation assays. Subsequently, hBMSCs were treated with the appropriate concentration of TgEP. We assessed osteogenic protein markers, including alkaline phosphatase (ALP), Runx2, and Osx, as well as components of the BMP/Smad signaling pathway using quantitative real-time PCR (qRT-PCR), siRNA interference of hBMSCs, Western blot analysis, and other methods. Furthermore, we created a bone defect model in Sprague-Dawley (SD) male rats and filled the defect areas with the GelMa hydrogel, with or without TgEP. Microcomputed tomography (micro-CT) was employed to analyze the bone parameters of defect sites. H&E, Masson and immunohistochemical staining were used to assess the repair conditions of the defect area.ResultsOur results indicate that TgEP promotes the expression of key osteogenic markers, including ALP, Runx2, and Osx, as well as the activation of Smad1, BMP2, and phosphorylated Smad1/5—crucial elements of the BMP/Smad signaling pathway. Furthermore, in vivo experiments using a bone defect model in rats demonstrated that TgEP markedly promoted bone defect repair.ConclusionOur results provide compelling evidence that TgEP facilitates hBMSC osteogenic differentiation through the BMP/Smad signaling pathway, highlighting its potential as a therapeutic approach for bone tissue engineering for bone defect healing.

Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

Study of different heterocycles showing significant anti-severe acute respiratory syndrome 2 activity in vitro and in vivo.

With the emergence of severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), antiviral drug development has gained increased significance due to the high incidence and potentially severe complications of the resulting coronavirus infection. Heterocycle compounds, acting as antimetabolites of DNA and RNA monomers, rank among the most effective antiviral drugs. These compounds' antiviral effects on various SARS-CoV-2 isolates, as found in existing data collections, form the basis for further research. The aim of this study was to examine the possible antiviral effect of some originally synthesized heterocyclic compounds. The main methods were cell culturing, cytotoxicity assay, qRT-PCR assay, tissue and blood cells analysis, and micro-computed tomography (micro-CT) imaging. In both in vitro and in vivo conditions, the elimination of SARS-Cov-2 occurred significantly earlier after administration of the compounds compared to the control group. In hamsters, the primary symptoms of coronavirus disease disappeared following administration of heterocycle compounds. Using delta and omicron strains of the SARS-CoV-2 virus, newly created heterocycle compound analogs dramatically reduced SARS-CoV-2 multiplication, resulting in a drop in viral RNA load in the supernatant under in vitro conditions. Improvements in pathological manifestations in the blood, bone marrow, and internal organs of hamsters demonstrated that heterocycle compounds inhibited SARS-CoV-2 replication both in vitro and in vivo.

Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

NK cell transfer overcomes resistance to PD-(L)1 therapy in aged mice

BackgroundCancer is the leading cause of death among older adults. Although the integration of immunotherapy has revolutionized the therapeutic landscape of cancer, the complex interactions between age and immunotherapy efficacy remain incompletely defined. Here, we aimed to elucidate the relationship between aging and immunotherapy resistance.MethodsFlow cytometry was performed to evaluate the infiltration of immune cells in the tumor microenvironment (TME). In vivo T cell proliferation, cytotoxicity and migration assays were performed to evaluate the antitumor capacity of tumor antigen-specific CD8+ T cells in mice. Real-time quantitative PCR (qPCR) was used to investigate the expression of IFN-γ-associated gene and natural killer (NK)-associated chemokine. Adoptive NK cell transfer was adopted to evaluate the effects of NK cells from young mice in overcoming the immunotherapy resistance of aged mice.ResultsWe found that elderly patients with advanced non-small cell lung cancer (aNSCLC) aged ≥ 75 years exhibited poorer progression-free survival (PFS), overall survival (OS) and a lower clinical response rate after immunotherapy. Mechanistically, we showed that the infiltration of NK cells was significantly reduced in aged mice compared to younger mice. Furthermore, the aged NK cells could also suppress the activation of tumor antigen-specific CD8+ T cells by inhibiting the recruitment and activation of CD103+ dendritic cells (DCs). Adoptive transfer of NK cells from young mice to aged mice promoted TME remodeling, and reversed immunotherapy resistance.ConclusionOur findings revealed the decreased sensitivity of elderly patients to immunotherapy, as well as in aged mice. This may be attributed to the reduction of NK cells in aged mice, which inhibits CD103+ DCs recruitment and its CD86 expression and ultimately leads to immunotherapy resistance.

QbD Enabled Development and Evaluation of Pazopanib Loaded Nanoliposomes for PDAC Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the highly fatal types of cancer with high mortality/incidence. Considering the crucial role of vascular endothelial growth factor (VEGF) in PDAC progression, its inhibition can be a viable strategy for the treatment. Pazopanib, a second-generation VEGF inhibitor, is approved for the treatment of various oncological conditions. However, due to associated limitations like low oral bioavailability (14-39%), high inter/intra-subject variability, stability issues, etc., high doses (800mg) are required, which further lead to non-specific toxicities and also contribute toward cancer resistance. Thus, to overcome these challenges, pazopanib-loaded PEGylated nanoliposomes were developed and evaluated against pancreatic cancer cell lines. The nanoliposomes were prepared by thin-film hydration method, followed by characterization and stability studies. This QbD-enabled process design successfully led to the development of a suitable pazopanib liposomal formulation with desirable properties. The % entrapment of PZP-loaded non-PEGylated and PEGylated nanoliposomes was found to be 75.2% and 84.9%, respectively, whereas their particle size was found to be 129.7nm and 182.0nm, respectively. The developed liposomal formulations exhibited a prolonged release and showed desirable physicochemical properties. Furthermore, these liposomal formulations were also assessed for in vitro cell lines, such as cell cytotoxicity assay and cell uptake. These studies confirm the effectiveness of developed liposomal formulations against pancreatic cancer cell lines. The outcomes of this work provide encouraging results and a way forward to thoroughly investigate its potential for PDAC treatment.

Delivery of Chlorambucil to the Brain Using Surface Modified Solid Lipid Nanoparticles.

The delivery of drugs to the brain in the therapy of diseases of the central nervous system (CNS) remains a continuing challenge because of the lack of delivery systems that can cross the blood-brain barrier (BBB). Therefore, there is a need to develop an innovative delivery method for the treatment of CNS diseases. Thus, we have investigated the interaction of γ-aminobutyric acid (GABA) and S-(-)-γ-amino-α-hydroxybutyric acid (GAHBA) with the GABA receptor by performing a docking study. Both GABA and GAHBA show comparable binding affinities toward the receptor. In this study, we developed surface-modified solid lipid nanoparticles (SLNs) using GAHBA-derived lipids that can cross the BBB. CLB-loaded SLNs were characterized by a number of methods including differential scanning calorimetry, dynamic light scattering, UV-vis spectroscopy, and transmission electron microscopy. The blank and CLB-loaded SLN suspensions were found to exhibit good storage stability. Also, the SLNs showed a higher encapsulation efficiency for CLB drugs. In vitro release kinetics of CLB at physiological temperature was also investigated. The results of the in vitro cell cytotoxicity assay and flow cytometry studies in the human glioma U87MG cell line and human prostate cancer PC3 cell line suggested a higher efficacy of the GAHBA-modified CLB-loaded SLNs in U87MG cells. The transcription level of GABA receptor expression in the target organ and cell line was analyzed by a reverse transcription polymerase chain reaction study. The in vivo biodistribution and brain uptake in C57BL6 mice and SPECT/CT imaging in Wistar rats investigated using 99mTc-labeled SLN and autoradiography suggest that the SLNs have an increasing brain uptake. We have demonstrated the delivery of the anticancer drug chlorambucil (CLB) to glioma.

3D printed kombucha biomaterial as a tissue scaffold and L929 cell cytotoxicity assay

AbstractTissue engineering includes the construction of tissue‐organ scaffold. The advantage of three‐dimensional scaffolds over two‐dimensional scaffolds is that they provide homeostasis for a longer time. The microbial community in Symbiotic culture of bacteria and yeast (SCOBY) can be a source for kombucha (kombu tea) production. In this study, it was aimed to investigate the usage of SCOBY, which produces bacterial cellulose, as a biomaterial and 3D scaffold material. 3D printable biomaterial was obtained by partial hydrolysis of oolong tea and black tea kombucha biofilms. In order to investigate the usage of 3D kombucha biomaterial as a tissue scaffold, “L929 cell line 3D cell culture” was created and cell viability was tested in the biomaterial. At the end of the 21st day, black tea showed 51% and oolong tea 73% viability. The cytotoxicity of the materials prepared by lyophilizing oolong and black tea kombucha beverages in fibroblast cell culture was determined. Black tea IC50 value: 7.53 mg, oolong tea IC50 value is found as 6.05 mg. Fibroblast viability in 3D biomaterial + lyophilized oolong and black tea kombucha beverages, which were created using the amounts determined to these values, were investigated by cell culture Fibroblasts in lyophilized and 3D biomaterial showed viability of 58% in black tea and 78% in oolong tea at the end of the 7th day. In SEM analysis, it was concluded that fibroblast cells created adhesion to the biomaterial. 3D biomaterial from kombucha mushroom culture can be used as tissue scaffold and biomaterial.

‘Charge Reverse’ Halogen Bonding Contacts in Metal-Organic Multi-Component Compounds: Antiproliferative Evaluation and Theoretical Studies

Two new metal–organic multi-component compounds of Ni(II) and Co(II), viz. [Ni(3-CNpy)2(H2O)4]ADS·2.75H2O (1) and [Co(3-CNpy)2(H2O)4](4-ClbzSO3)2 (2) (3-CNpy = 3-cyanopyridine, ADS = anthraquinone-1,5-disulfonate, 4-ClbzSO3 = 4-chlorobenzenesulfonate), were synthesized and characterized using single crystal XRD, TGA, spectroscopic (IR, electronic) and elemental analyses. Both the compounds crystallize as multi-component compounds of Ni(II) and Co(II), with uncoordinated ADS and 4-ClbzSO3 moieties in the crystal lattice, respectively. Crystal structure analyses revealed the presence of antiparallel nitrile···nitrile and π-stacked assemblies involving alternate coordinated 3-CNpy and uncoordinated ADS and 4-ClbzSO3 moieties. Moreover, unconventional charge reverse Cl∙∙∙N halogen bonding contacts observed in compound 2 provide additional reinforcement to the crystal structure. Theoretical calculations confirm that the H-bonding interactions, along with anion–π(arene) and anion–π(CN) in 1 and π–π, antiparallel CN···CN and charge reverse Cl···N halogen bonds in 2, play crucial roles in the solid state stability of the compounds. In vitro anticancer activities observed through the trypan blue cell cytotoxicity assay reveal that the compounds induce significant concentration dependent cytotoxicity in Dalton’s lymphoma (DL) cancer cells, with nominal effects in normal healthy cells. Molecular docking studies reveal that the compounds can effectively bind with the active sites of anti-apoptotic proteins, which are actively involved in cancer progression.