Intracellular Environment Research Articles

Differential local ordering of mixed crowders determines effective size and stability of ss-DNA capped gold nanoparticle.

Understanding the influence of a crowded intracellular environment on the structure and solvation of DNA functionalized gold nanoparticles (ss-DNA AuNP) is necessary for designing applications in nanomedicine. In this study, the effect of single (Gly, Ser, Lys) and mixture of amino acids (Gly+Ser, Gly+Lys, Ser+Lys) at crowded concentrations is examined on the structure of the ss-DNA AuNP using molecular dynamics simulations. Using the structural estimators such as pair correlation functions and ligand shell positional fluctuations, the solvation entropy is estimated. Combining the AuNP-solvent interaction energy with the solvation entropy estimates, the free energy of solvation of the AuNP in crowded solutions is computed. The solvation entropy favours the solvation free energy which becomes more favourable for larger effective size of AuNP in crowded solutions relative to that in water. The effective size of AuNP depends on the different propensity of the crowders to adsorb on Au surface, with the smallest crowder (Gly) having the highest propensity inducing the least effective AuNP size as compared to other single crowder solutions. In mixed crowded solutions of amino acids of variable size and chemistry, distinctive local adsorption of the crowders on the gold surface is observed that controls the additive or non-additive crowding effects which govern an increase (in Gly+Ser) or decrease (in Gly+Lys) in nanoparticle effective size respectively. The results shed light into the fundamental understanding of the influence of intracellular crowding on structure of ss-DNA AuNP and plausible employability of crowding as a tool to design programmable self-assembly of functionalized nanoparticles.

Real-Time Monitoring of RAS Activity Using In Vitro and In-Cell NMR Spectroscopy.

The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.

Functionalities of pH-responsive DNA nanostructures in tumor-targeted strategies.

Nanostructures integrating pH-sensitive DNA motifs have emerged as versatile platforms for active tumor targeting, owing to their ability to undergo conformation changes in response to the common acidic environment of the tumor extracellular matrix and endocytosis pathway. This review summarizes the latest advances in the design and application of various pH-responsive DNA nanostructures for tumor-targeted strategies, including tumor recognition, cell imaging, dynamic nanocarrier construction, and controlled drug release. A comprehensive framework for pH-controlled multi-stage tumor targeting is introduced, addressing the divergences in targeting strategies for extracellular and intracellular environments. The unique attributes, practical performance and application challenges of pH-responsive DNA nanostructures are also critically discussed to provide guidance for future development in this field.

Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde.

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs in vitro using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.

Advancements in molecular disassembly of optical probes: a paradigm shift in sensing, bioimaging, and therapeutics.

The majority of self-assembled fluorescent dyes suffer from aggregation-caused quenching (ACQ), which detrimentally affects their diagnostic and therapeutic effectiveness. While aggregation-induced emission (AIE) active dyes offer a promising solution to overcome this limitation, they may face significant challenges as the intracellular environment often prevents aggregation, leading to disassembly and posing challenges for AIE fluorogens. Recent progress in signal amplification through the disassembly of ACQ dyes has opened new avenues for creating ultrasensitive optical sensors and enhancing phototherapeutic outcomes. These advances are well-aligned with cutting-edge technologies such as single-molecule microscopy and targeted molecular therapies. This work explores the concept of disaggregation-induced emission (DIE), showcasing the revolutionary capabilities of DIE-based dyes from their design to their application in sensing, bioimaging, disease monitoring, and treatment in both cellular and animal models. Our objective is to provide an in-depth comparison of aggregation versus disaggregation mechanisms, aiming to stimulate further advancements in the design and utilization of ACQ fluorescent dyes through DIE technology. This initiative is poised to catalyze scientific progress across a broad spectrum of disciplines.

A highly selective probe engineered to detect polarity and distinguish normal cells and tumor cells in tissue sections.

Early diagnostics and therapies for diseases such as cancer are limited by the fact that the inducing factors for the development of cytopathies are not clear. The stable polarity of lipid droplets is a potential biomarker for tumor cells; however, the complex intracellular biological environment poses great difficulties for specific detection of the polarity. Therefore, to meet this pressing challenge, we designed a highly selective fluorescent probe, DCI-Cou-polar, which used the ICT mechanism to differentiate normal cells and tumor cells in tissue sections by detecting changes in the polarities of intracellular lipid droplets. The introduction of a cyclic amine at the 7-position of coumarin (benzoquinolizine coumarin) reduced its ability to donate electrons compared with the diethylamino group, which increased the probe selectivity while retaining the sensitivity to polarity. With NIR emission and large Stokes shifts, DCI-Cou-polar has high sensitivity to polarity, excellent photostability, and biocompatibility, and it tracks lipid droplets with high fidelity. Therefore, we believe that this polarity-sensitive probe provides information on the connection between the polarity of lipid droplets and tumors while improving the development of highly selective polarity probes.

Thermomechanical properties of confined magnetic nanoparticles in electrospun polyacrylonitrile nanofiber matrix exposed to a magnetic environment: structure, morphology, and stabilization (cyclization).

Electrospun metal oxide-polymer nanofiber composites hold promise for revolutionizing biomedical applications due to their unique combination of electronic and material properties and tailorable functionalities. An investigation into the incorporation of Fe-based nanofillers for optimizing the polyacrylonitrile matrix was conducted, where the systematic and organized arrangement of inorganic components was achieved through non-covalent bonding. These carefully dispersed nanomaterials exhibit the intrinsic electronic characteristics of the polymers and concurrently respond to external magnetic fields. Electrospinning was utilized to fabricate polyacrylonitrile nanofibers blended with Fe2O3 and MnZn ferrite nanoparticles, which were thermomechanically, morphologically, and spectroscopically characterized in detail. With the application of an external magnetic field in the course of dynamic mechanical measurements under tension, the storage modulus of the glass transition T g of PAN/Fe2O3 rises at the expense of the loss modulus, and a new peak emerges at ∼350 K. For the PAN/MnZn ferrite nanofibers a relatively larger shift in T g (from ∼367 K to ∼377 K) is observed, emphasizing that in comparison to Fe2O3, Mn2+ ions in particular enhance the material's magnetic response in MnZn Ferrite. The magnetic oxide particles are homogenously dispersed in polyacrylonitrile, corroborated by high-resolution scanning electron microscopy. Both nanopowder additions lead to a slight shift of the peak towards larger angles, related to the shrinkage of the polymer. Produced nanofibers with high mechanical and heating efficiency can optimize the influence of the intracellular environment, magnetic refrigeration systems and sensors/actuators by their magnetic behavior and heat generation.

Immobilising an acid-cleavable dimeric phthalocyanine on gold nanobipyramids for intracellular pH detection and photodynamic elimination of cancer cells.

An acetal-linked dimeric phthalocyanine has been synthesised and immobilised on the surface of gold nanobipyramids. The resulting nanocomposite serves as a highly sensitive probe for intracellular pH through its acid-responsive fluorescence and surface-enhanced Raman scattering signals. The phthalocyanine units released in the acidic intracellular environment can also effectively eliminate the cancer cells upon light irradiation, rendering this simple fabricated nanosystem a bimodal and bifunctional theranostic agent.

Control over membrane fluidity and biophysical properties of synthetic terpolymer stabilized complex coacervates

Cell membranes are vital barriers that regulate the composition of the intracellular environment and facilitate communication processes essential for cellular function and survival. In comparison to lipid membranes, artificial polymeric...

NIR-II light powered hydrogel nanomotor for intravesical instillation with enhanced bladder cancer therapy.

Intravesical instillation is the common therapeutic strategy for bladder cancer. Besides chemo drugs, nanoparticles are used as intravesical instillation reagents, offering appealing therapeutic approaches for bladder cancer treatment. Metal oxide nanoparticle based chemodynamic therapy (CDT) converts tumor intracellular hydrogen peroxide to ROS with cancer cell-specific toxicity, which makes it a promising approach for the intravesical instillation of bladder cancer. However, the limited penetration of nanoparticle based therapeutic agents into the mucosa layer of the bladder wall poses a great challenge for the clinical application of CDT in intravesical instillation. Herein, we developed a 1064 nm NIR-II light driven hydrogel nanomotor for the CDT for bladder cancer via intravesical instillation. The hydrogel nanomotor was synthesized via microfluidics, wrapped with a lipid bilayer, and encapsulates CuO2 nanoparticles as a CDT reagent and core-shell structured Fe3O4@Cu9S8 nanoparticles as a fuel reagent with asymmetric distribution in the nanomotor (LipGel-NM). An NIR-II light irradiation of 1064 nm drives the active motion of LipGel-NMs, thus facilitating their distribution in the bladder and deep penetration into the mucosa layer of the bladder wall. After FA-mediated endocytosis in bladder cancer cells, CuO2 is released from LipGel-NMs due to the acidic intracellular environment for CDT. The NIR-II light powered active motion of LipGel-NMs effectively enhances CDT, providing a promising strategy for bladder cancer therapy.

Two-photon excited-state dynamics of mEGFP-linker-mScarlet-I crowding biosensor in controlled environments.

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L-1) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet-I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

Nonpolar selective emission (NPSE) of carbonyl-bridged rhodols.

Polarity-responsive luminophores (PRLs), whose emission properties change in response to the polarity of the surrounding environment, are used for the fluorescence sensing of intracellular environments and various chemical compounds. Herein, we propose a concept called nonpolar selective emission (NPSE) for the development of a new PRL family. Unlike the conventional emission of PRLs, the NPSE luminophore can switch to a completely non-emissive state upon a slight increase in solvent polarity. The NPSE concept offers a new means of distinguishing between nonpolar and low-polarity environments. Moreover, the NPSE property is little affected by the viscosity of the surrounding medium. We demonstrate that NPSE dyes can be used as emission sensors for molecular gases. Furthermore, we discovered the potential use of NPSE dyes as a time-dependent security ink triggered by the volatilization of polar molecules.

Unraveling Ros Conversion Through Enhanced Enzyme-Like Activity with Copper-Doped Cerium Oxide for Tumor Nanocatalytic Therapy.

Nanozyme catalytic therapy for cancer treatments has become one of the heated topics, and the therapeutic efficacy is highly correlated with their catalytic efficiency. In this work, three copper-doped CeO2 supports with various structures as well as crystal facets are developed to realize dual enzyme-mimic catalytic activities, that is superoxide dismutase (SOD) to reduce superoxide radicals to H2 O2 and peroxidase (POD) to transform H2 O2 to ∙OH. The wire-shaped CeO2 /Cu-W has the richest surface oxygen vacancies, and a low level of oxygen vacancy (Vo) formation energy, which allows for the elimination of intracellular reactive oxygen spieces (ROS) and continuous transformation to ∙OH with cascade reaction. Moreover, the wire-shaped CeO2 /Cu-W displays the highest toxic ∙OH production capacity in an acidic intracellular environment, inducing breast cancer cell death and pro-apoptotic autophagy. Therefore, wire-shaped CeO2 /Cu nanoparticles as an artificial enzyme system can have great potential in the intervention of intracellular ROS in cancer cells, achieving efficacious nanocatalytic therapy.

CO2-Responsive Water-Soluble Conjugated Polymers as a Multifunctional Fluorescent Probe for Bioimaging Applications.

We describe important progress in the synthesis and development of gas-responsive water-soluble conjugated polymers (WSCPs) with potential as multifunctional fluorescent materials for biomedical imaging and probes. A water-soluble WSCP (I-PT) composed of a hydrophobic fluorescent polythiophene backbone and a hydrophilic imidazole side chain was successfully prepared through a facile and efficient two-step synthetic route. Owing to the repulsive force between the hydrophilic and hydrophobic segments and the highly sensitive carbon dioxide (CO2)- and nitrogen (N2)-responsive imidazole groups in its structure, I-PT can spontaneously self-assemble into spherical-like nanoparticles in an aqueous environment, and thus exhibits unique light absorption and fluorescence properties as well as rapid responsiveness to CO2 and N2. In addition, its structure, optical absorption/fluorescence behavior, and surface potential can be quickly turned on and off through alternating cycles of CO2 and N2 bubbling and exhibit controllable cyclic switching stability, thereby allowing effective manipulation of its hierarchical structure and chemical-physical characteristics. More importantly, a series of in vitro cell experiments confirmed that, compared to the significant cytotoxicity of pristine and N2-treated I-PT nanoparticles, CO2-treated I-PT nanoparticles exhibit extremely low cytotoxicity in normal and cancer cells and undergo greatly accelerated cellular uptake, resulting in a significant increase in the intensity and stability of their fluorescence signal in the intracellular environment. Overall, this newly discovered CO2/N2-responsive system provides new insights to effectively enhance the biocompatibility, cellular internalization, and intracellular fluorescence characteristics of WSCPs and holds great potential for biomedical imaging/sensing applications.

MLOsMetaDB, a meta-database to centralize the information on liquid-liquid phase separation proteins and membraneless organelles.

Over the past few years, there has been a focus on proteins that create separate liquid phases in the intracellular liquid environment, known as membraneless organelles (MLOs). These organelles allow for the spatiotemporal associations of macromolecules that dynamically exchange within the cellular milieu. They provide a form of compartmentalization crucial for organizing key functions in many cells. Metabolic processes and signaling pathways in both the cytoplasm and nucleus are among the functions performed by MLOs, which are facilitated by diverse combinations of proteins and nucleic acids. However, disruptions in these liquid-liquid phase separation processes (LLPS) may lead to several diseases, such as neurodegenerative disorders and cancer, among others. To foster the study of this process and MLO function, we present MLOsMetaDB (http://mlos.leloir.org.ar), a comprehensive resource of information on MLO- and LLPS-related proteins. Our database integrates and centralizes available information for every protein involved in MLOs, which is otherwise disseminated across a plethora of different databases. Our manuscript outlines the development and features of MLOsMetaDB, which provides an interactive and user-friendly environment with modern biological visualizations and easy and quick access to proteins based on LLPS role, MLO location, and organisms. In addition, it offers an advanced search for making complex queries to generate customized information. Furthermore, MLOsMetaDB provides evolutionary information by collecting the orthologs of every protein in the same database. Overall, MLOsMetaDB is a valuable resource as a starting point for researchers studying the many processes driven by LLPS proteins and membraneless organelles.

Desulfurization of thiosemicarbazones: the role of metal ions and biological implications.

Thiosemicarbazones are biologically active substances whose structural formula is formed by an azomethine, an hydrazine, and a thioamide fragments, to generate a R2C=N-NR-C(=S)-NR2 backbone. These compounds often act as ligands to generate highly stable metal-organic complexes. In certain experimental conditions, however, thiosemicarbazones undergo reactions leading to the cleavage of the chain. Sometimes, the breakage involves desulfurization processes. The present work summarizes the different chemical factors that influence the desulfurization reactions of thiosemicarbazones, such as pH, the presence of oxidant reactants or the establishment of redox processes as those electrochemically induced, the effects of the solvent, the temperature, and the electromagnetic radiation. Many of these reactions require coordination of thiosemicarbazones to metal ions, even those present in the intracellular environment. The nature of the products generated in these reactions, their detection in vivo and in vitro, together with the relevance for the biological activity of these compounds, mainly as antineoplastic agents, is discussed.

Unleashing the Power of Proenzyme Delivery for Targeted Therapeutic Applications Using Biodegradable Lipid Nanoparticles.

Proenzymes, functioning as inactive precursor forms of enzymes, hold significant promise for regulating essential biological processes. Their inherent property of latency, remaining inert until they arrive at the intended site of action, positions them as particularly promising candidates for the development of targeted therapeutics. Despite this potential, the therapeutic potential of proenzymes is challenged by designing proenzymes with excellent selectivity for disease cells. This limitation is further exacerbated by the inability of proenzymes to spontaneously cross the cell membrane, a biological barrier that impedes the cellular internalization of exogenous macromolecules. Therefore, efficacious intracellular delivery is paramount to unlocking the full therapeutic potency of proenzymes.In this Account, we first elucidate our recent advancements made in designing biodegradable lipid nanoparticles (LNPs) for the cell-specific delivery of biomacromolecules, including proteins and nucleic acids. Using a strategy of parallel synthesis, we have constructed an extensive library of ionizable lipids, each integrated with different biodegradable moieties. This combinatorial approach has led to the identification of LNPs that are particularly efficacious for the delivery of biomacromolecules specifically to tumor cells. This innovation capitalizes on the unique intracellular environment of cancer cells to control the degradation of LNPs, thereby ensuring the targeted release of therapeutics within tumor cells. Additionally, we discuss the structure-activity relationship governing the delivery efficacy of these LNPs and their applicability in regulating tumor cell signaling, specifically through the delivery of bacterial effector proteins.In the second segment, we aim to provide an overview of our recent contributions to the field of proenzyme design, where we have chemically tailored proteins to render them responsive to the unique milieu of tumor cells. Specifically, we elaborate on the chemical principles employed to modify proteins and DNAzymes, thereby priming them for activation in the presence of NAD(P)H:quinone oxidoreductase 1 (NQO1), an enzyme that is prevalently upregulated within tumor cells. We summarize the methodologies for intracellular delivery of these proenzymes using biodegradable LNPs, both in vitro and in vivo. The concomitant intracellular delivery and activation of proenzymes are examined in the context of enhanced therapeutic outcomes and targeted CRISPR/Cas9 genome editing.In conclusion, we offer a perspective on the chemical principles that could be leveraged to optimize LNPs for tissue-specific delivery of proenzymes. We also explore chemical strategies for the irreversible modulation of proenzyme activity within living cells and in vivo. Through this discussion, we provide insights into potential avenues for overcoming existing limitations and enhancing the delivery of proenzymes using LNPs, particularly for developing tumor-targeted therapies and genome editing applications.

Synergetic effects of conductive materials and bacterial population in inoculum on mixed-culture biohydrogen production

This study examined the effect of conductive materials (softwood pellet biochar pyrolyzed at 550 °C and 700 °C, powdered activated carbon and stainless-steel) on the two series of batch biohydrogen production inoculated with three types of heated-treated anaerobic digester sludge. Biochar pyrolyzed at 550 °C and stainless-steel exhibited the highest enhanced hydrogen yield up to 117 % in the first and second series of experiments, respectively. Importantly, the enhancement effect by the introduction of conductive materials was significantly affected by the microbial population in inoculum, particularly the relative abundance of electroactive H2-producing Clostridium sp. Metabolic flux analysis revealed that the enhanced H2 yield was concurrent with the decrease of lactate production and the increase of H2-producing acetogenesis. Conductive materials also improved electron transport system activity and NADH/NAD+ ratio, which are related with the metabolic activity. This study shows that introducing conductive material can control intracellular redox environment, thereby reinforcing the metabolic activity selectively.

Endogenous mRNA-Powered and Spatial Confinement-Derived DNA Nanomachines for Ultrarapid and Sensitive Imaging of Let-7a.

DNA nanostructure-based signal amplifiers offer new tools for imaging intracellular miRNA. However, the inadequate kinetics and susceptibility to enzymatic hydrolysis of these amplifiers, combined with a deficient cofactor concentration within the intracellular environment, significantly undermine their operational efficiency. In this study, we address these challenges by encapsulating a localized target strand displacement assembly (L-SD) and a toehold-exchange endogenous-powered component (R-mRNA) within a framework nucleic acid (FNA) structure─20 bp cubic DNA nanocage (termed RL-cube). This design enables the construction of an endogenous-powered and spatial-confinement DNA nanomachine for ratiometric fluorescence imaging of intracellular miRNA Let-7a. The R-mRNA is designed to be specifically triggered by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an abundant cellular enzyme, and concurrently releases a component that can recycle the target Let-7a. Meanwhile, L-SD reacts with Let-7a to release a stem-loop beacon, generating a FRET signal. The spatial confinement provided by the framework, combined with the ample intracellular supply of GAPDH, imparts remarkable sensitivity (7.57 pM), selectivity, stability, biocompatibility, and attractive dynamic performance (2240-fold local concentration, approximately four times reaction rate, and a response time of approximately 7 min) to the nanomachine-based biosensor. Consequently, this study introduces a potent sensing approach for detecting nucleic acid biomarkers with significant potential for application in clinical diagnostics and therapeutics.

Effects of exogenous amino acids on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1

How to improve the freeze-drying survival rate of Lactic acid bacteria (LAB) by increasing its resistance is a problem that is worth paying attention to. We evaluated the effect of exogenous amino acids on freeze-drying resistance of strain using Lactiplantibacillus plantarum LIP-1 as model probiotics. We found that the survival rate of L. plantarum LIP-1 after freeze-dried could be significantly enhanced by adding 0.04 g/L glycine to medium (p < 0.05), compare with other amino acids. Mechanistic studies showed that the content of peptidoglycan was increased to improve the integrity of the cell wall via metabolizing glycine; By metabolizing glycine, the content of unsaturated fatty acid was increased, such as C19cyc11, to promote membrane fluidity. In addition, the metabolism of glycine could neutralize the acidic intracellular environment to reduce DNA damage, thus improve the survival rate of freeze-dried strain. These findings may provide a new idea for industrialization of LAB powder.