Experimental and computational investigation of the kinetic evolution of the glutaminolysis pathway and its interplay with the glycolysis pathway.

Exploring cellular responses necessitates studying real-time metabolic pathway kinetics, considering the adaptable nature of cells. Glycolysis and glutaminolysis are interconnected pathways fundamental to driving cellular metabolism, generating both energy and essential biosynthetic molecules. While prior studies explored glycolysis tracking, this research focuses on monitoring the kinetics of the glutaminolysis pathway by evaluating the effect of glutamine availability on glycolytic kinetics and by investigating the impact of a stimulator (oligomycin) and inhibitor (2DG) on the glycolytic flux in the presence of glutamine. Additionally, we adapted a rate equation model to provide improved understanding of the pathway kinetics. The experimental and simulated results indicate a significant reduction in extracellular lactate production in the presence of glutamine, reflecting a shift from glycolysis towards oxidative phosphorylation, due to the additional contribution of glutamine to energy production through the ETC (electron transport chain), reducing the glycolytic load. Oligomycin, an ETC inhibitor, increases lactate production to the original glycolytic level, despite the presence of glutamine. Nevertheless, its mechanism is influenced by the presence of glutamine, as predicted by the model. Conversely, 2DG notably reduces lactate production, affirming its glycolytic origin. The gradual increase in lactate production under the influence of 2DG implies increased activation of glutaminolysis as an alternative energy source. The model also simulates the varying metabolic responses under varying carbon/modulator concentrations. In conclusion, the kinetic model described here contributes to the understanding of changes in intracellular metabolites and their interrelationships in a way which would be challenging to obtain solely through kinetic assays.

Kinetic modelling of the cellular metabolic responses underpinning in vitro glycolysis assays.

This study aims to demonstrate the benefits of augmenting commercially available, real-time, in vitro glycolysis assays with phenomenological rate equation-based kinetic models, describing the contributions of the underpinning metabolic pathways. To this end, a commercially available glycolysis assay, sensitive to changes in extracellular acidification (extracellular pH), was used to derive the glycolysis pathway kinetics. The pathway was numerically modelled using a series of ordinary differential rate equations, to simulate the obtained experimental results. The sensitivity of the model to the key equation parameters was also explored. The cellular glycolysis pathway kinetics were determined for three different cell-lines, under nonmodulated and modulated conditions. Over the timescale studied, the assay demonstrated a two-phase metabolic response, representing the differential kinetics of glycolysis pathway rate as a function of time, and this behaviour was faithfully reproduced by the model simulations. The model enabled quantitative comparison of the pathway kinetics of three cell lines, and also the modulating effect of two known drugs. Moreover, the modelling tool allows the subtle differences between different cell lines to be better elucidated and also allows augmentation of the assay sensitivity. A simplistic numerical model can faithfully reproduce the differential pathway kinetics for three different cell lines, with and without pathway-modulating drugs, and furthermore provides insights into the cellular metabolism by elucidating the underlying mechanisms leading to the pathway end-product. This study demonstrates that augmenting a relatively simple, real-time, in vitro assay with a model of the underpinning metabolic pathway provides considerable insights into the observed differences in cellular systems.

Monitoring and modelling the glutamine metabolic pathway: a review and future perspectives.

BACKGROUND: Analysis of the glutamine metabolic pathway has taken a special place in metabolomics research in recent years, given its important role in cell biosynthesis and bioenergetics across several disorders, especially in cancer cell survival. The science of metabolomics addresses the intricate intracellular metabolic network by exploring and understanding how cells function and respond to external or internal perturbations to identify potential therapeutic targets. However, despite recent advances in metabolomics, monitoring the kinetics of a metabolic pathway in a living cell in situ, real-time and holistically remains a significant challenge. AIM: This review paper explores the range of analytical approaches for monitoring metabolic pathways, as well as physicochemical modeling techniques, with a focus on glutamine metabolism. We discuss the advantages and disadvantages of each method and explore the potential of label-free Raman microspectroscopy, in conjunction with kinetic modeling, to enable real-time and in situ monitoring of the cellular kinetics of the glutamine metabolic pathway. KEY SCIENTIFIC CONCEPTS: Given its important role in cell metabolism, the ability to monitor and model the glutamine metabolic pathways are highlighted. Novel, label free approaches have the potential to revolutionise metabolic biosensing, laying the foundation for a new paradigm in metabolomics research and addressing the challenges in monitoring metabolic pathways in living cells.

What We Need to Know about Liposomes as Drug Nanocarriers: An Updated Review.

Liposomes have been attracted considerable attention as phospholipid spherical vesicles, over the past 40 years. These lipid vesicles are valued in biomedical application due to their ability to carry both hydrophobic and hydrophilic agents, high biocompatibility and biodegradability. Various methods have been used for the synthesis of liposomes, so far and numerous modifications have been performed to introduce liposomes with different characteristics like surface charge, size, number of their layers, and length of circulation in biological fluids. This article provides an overview of the significant advances in synthesis of liposomes via active or passive drug loading methods, as well as describes some strategies developed to fabricate their targeted formulations to overcome limitations of the "first-generation" liposomes.

Novel and efficient method for loading aptamer-conjugated liposomes with arsenic trioxide for targeting cancer cells.

Although the therapeutic effect of liposomal arsenic trioxide arsenic trioxide (ATO) in the treatment of solid tumours has been confirmed, its dose-limiting loading is a challenging issue. To solve the problems in the preparation of liposomal ATO, different loading strategies were evaluated and compared. In addition, liposomes decorated with anti-nucleolin aptamers were developed as a novel formulation for targeted delivery with high loading efficiency and sustained releasing property in order to treat solid tumours. The liposomes were prepared by a thin-film method exploiting the passive loading strategy of Co(II) hydrogen arsenite (CHA). The structural characteristics of the liposomes were also investigated by Fourier transform infra-red spectroscopy (FT-IR), dynamic light scattering (DLS), zeta potentiometry, field emission scanning electron microscopy (FESEM), and Energy Dispersive X-ray Diffraction (EDX) techniques. To evaluate the potential cytotoxicity of this liposomal drug vehicle in vitro, MTT assay was performed on HT-29 cancer cell line. The results showed that the synthesised liposomes loaded with CHA exhibited high entrapment efficiency (77%). MTT assays showed a significant difference between the percentage of viable cells when HT -29 cells were treated with free ATO and liposomal formulation which can be corresponded to the sustained release of the drug from the liposomes. The results of this study may lead to a promising strategy for the effective treatment of solid tumours.