Intracellular Sites Research Articles

MiR158a negatively regulates plant resistance to Phytophthora parasitica by repressing AtTN7 that requires EDS1-PAD4-ADR1 complex in Arabidopsis thaliana.

Small RNAs are involved in diverse cellular processes, including plant immunity to pathogens. Here, we report that miR158a negatively regulates plant immunity to the oomycete pathogen Phytophthora parasitica in Arabidopsis thaliana. By performing real-time quantitative PCR, transient expression, and RNA ligase-mediated 5' rapid amplification of cDNA ends assays, we demonstrate that miR158a downregulates AtTN7 expression by cleaving its 3'-untranslated region. AtTN7 positively affects plant immunity and encodes a truncated intracellular nucleotide-binding site and leucine-rich repeat receptor containing the Toll/interleukin-1 receptor. AtTN7 can degrade oxidized forms of nicotinamide adenine dinucleotide (NAD+). Further genetic and molecular analyses reveal that the Enhanced Disease Susceptibility 1-Phytoalexin Deficient 4-Activated Disease Resistance 1 complex is required for AtTN7-mediated immunity. ADR1-dependent Ca2+ influx is crucial for activating salicylic acid signaling to condition AtTN7-triggered immunity. Our study uncovers the immune roles and regulatory mechanisms of miR158a and its target AtTN7. Both miR158a-downregulation and AtTN7-overexpression lead to enhanced plant resistance to P. parasitica without affecting plant growth phenotypes, suggesting their application potentials and the utilization of miRNAs in identifying novel immune genes for the development of plant germplasm resources with enhanced disease resistance.

Secretase promotes AD progression: simultaneously cleave Notch and APP.

Alzheimer's disease (AD) involves complex pathological mechanisms. Secretases include membrane protein extracellular structural domain proteases and intramembrane proteases that cleave the topology to type I or type II. Secretases can effectively regulate the activation of Notch and amyloid precursor protein (APP), key factors in the progression of AD and cancer. This article systematically summarizes the intracellular localization, cleavage sites and products, and biological functions of six subtypes of secretases (α-secretase, β-secretase, γ-secretase, δ-secretase, ε-secretase, and η-secretase), and for the first time, elucidates the commonalities and differences between these subtypes of secretases. We found that each subtype of secretase primarily cleaves APP and Notch as substrates, regulating Aβ levels through APP cleavage to impact the progression of AD, while also cleaving Notch receptors to affect cancer progression. Finally, we review the chemical structures, indications, and research stages of various secretase inhibitors, emphasizing the promising development of secretase inhibitors in the fields of cancer and AD.

An ultrasensitive lysosome-targeting NIR fluorescence probe for detection of hydroxyl radical during ferroptosis and cuproptosis

Hydroxyl radical (•OH) is one of the most destructive reactive oxygen species in biosystems, mainly produced by the participation of transition metals. Lysosomes, the major metabolic center of intracellular metals, may accelerate the generation of •OH by metal-participated reactions due to their acidic microenvironment, which may make lysosomes a major intracellular site for •OH production. In this work, an ultrasensitive NIR lysosome-targeting •OH fluorescence probe, Lyso-OH, was developed for in situ detection of •OH in lysosomes. The reaction of Lyso-OH and •OH leads to a large spectroscopic red-shift and a significant NIR fluorescence response at 655 nm. Such large changes in π-conjugation and spectroscopic properties can achieve an extremely low background fluorescence, which is helpful to obtain a high-contrast and ultrasensitive NIR fluorescence response, enabling Lyso-OH to detect the trace •OH produced by autoxidation of low-valent metals (e.g. Fe2+ and Cu+). Lyso-OH has good lysosome-targeting ability and has been used for the fluorescence imaging of two transition metal-dependent regulated cell death processes, ferroptosis and cuproptosis. The results revealed that both ferroptosis and cuproptosis were accompanied by lysosomal •OH level increase. Moreover, Lyso-OH was also capable of monitoring the •OH level fluctuations in mice models of inflammation and tumor. The good analytical performance of Lyso-OH may allow it to be widely used in more •OH-associated physiological and pathological processes.

N-[4-(Benzyloxy)-3-methoxybenzyl)]adamantane-1-amine (DZH2), a dual CCR5 and CXCR4 inhibitor as a potential agent against triple negative breast cancer.

DZH2, a dual inhibitor of the chemokine receptors CCR5 and CXCR4, was discovered from virtual screening for CCR5 antagonists. In specific Ca2+ chemokine signaling assays, DZH2 displayed low micromolar IC50 values at both chemokine receptors. Its binding to intracellular allosteric binding sites of CCR5 and CXCR4 was confirmed by MD simulations and binding free-energy calculations. DZH2 is superior to the CCR5 antagonist maraviroc in terms of its inhibitory activity on the growth of two breast cancer cell lines. In MCF7 and MDA-MB-231 cells, DZH2 was a >100-fold more potent inhibitor of cell viability compared to maraviroc. DZH2 (6.7 µM) reduced migration of MDA-MB-231 cells to 4% compared to 50% inhibition of migration caused by maraviroc (780 µM). Also, DZH2 was a significantly more potent inhibitor of colony formation in MDA-MB-231 cells than maraviroc. In MCF10 cells, DZH2 caused no alteration in the gene expression with respect to cellular pathways mediating cell death, indicating its selectivity to breast cancer cells.

Dopamine internalization via Uptake2 and stimulation of intracellular D5-receptor-dependent calcium mobilization and CDP-diacylglycerol signaling.

Dopamine stimulates CDP-diacylglycerol biosynthesis through D1-like receptors, particularly the D5 subtype most of which is intracellularly localized. CDP-diacylglycerol regulates phosphatidylinositol-4,5-bisphosphate-dependent signaling cascades by serving as obligatory substrate for phosphatidylinositol biosynthesis. Here, we used acute and organotypic brain tissues and cultured cells to explore the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol. Dopamine stimulated CDP-diacylglycerol in organotypic and neural cells lacking the presynaptic dopamine transporter, and this action was selectively mimicked by D1-like receptor agonists SKF38393 and SKF83959. Dopaminergic CDP-diacylglycerol stimulation was blocked by decynium-22 which blocks Uptake2-like transporters and by anti-microtubule disrupters of cytoskeletal transport, suggesting transmembrane uptake and guided transport of the ligands to intracellular sites of CDP-diacylglycerol regulation. Fluorescent or radiolabeled dopamine was saturably transported into primary neurons or B35 neuroblastoma cells expressing the plasmamembrane monoamine transporter, PMAT. Microinjection of 10-nM final concentration of dopamine into human D5-receptor-transfected U2-OS cells rapidly and transiently increased cytosolic calcium concentrations by 316%, whereas non-D5-receptor-expressing U2-OS cells showed no response. Given that U2-OS cells natively express PMAT, bath application of 10μM dopamine slowly increased cytosolic calcium in D5-expressing cells. These observations indicate that dopamine is actively transported by a PMAT-implicated Uptake2-like mechanism into postsynaptic-type dopaminoceptive cells where the monoamine stimulates its intracellular D5-type receptors to mobilize cytosolic calcium and promote CDP-diacylglycerol biosynthesis. This is probably the first demonstration of functional intracellular dopamine receptor coupling in neural tissue, thus challenging the conventional paradigm that postsynaptic dopamine uptake serves merely as a mechanism for deactivating spent or excessive synaptic transmitter.

Highly integrated, self-powered and activatable bipedal DNA nanowalker for imaging of base excision repair in living cells

DNA walkers have attracted considerable attention in biosensing and bioimaging. Compared with the conventional single leg-based DNA walker, the bipedal DNA walker has remarkable advantages, with improved sensitivity and fast kinetics, and can work efficiently in a crowded cellular environment. However, most reported bipedal DNA walkers are powered by exogenous supplementation, and elaborate DNA sequence designs, auxiliary additives or extra carriers are often needed. A highly integrated bipedal DNA walker that can address robustness, sensitivity and consistency issues in a single system is highly desirable but remains a great challenge. We herein report a novel bipedal DNA nanowalker system through simple assembly of a DNA substrate, hairpin functionalized-AuNPs (AuNPs-H2), and a blocked Mn2+-dependent DNAzyme hairpin (H1) on degradable MnO2 nanosheets, which holds great potential for living cell operation. Highly integrated features enable the simultaneous delivery of core components of the bipedal DNA walker, including a walking track (AuNPs-H2), a walking strand (H1 cleaved by APE1), and a driving force (Mn2+-dependent DNAzyme cleavage) as a whole, thereby enhancing the control of the spatiotemporal distribution of these components at the intracellular target sites. The redox reaction between the MnO2 nanosheets and GSH inside the cells not only consumed the intracellular GSH to improve the biostability of the walking track but also generated abundant Mn2+ as a cofactor of the DNAzyme. As a proof of concept, the developed nanowalker was demonstrated to work efficiently for monitoring base excision repair (BER)-related human apurinic/apyrimidinic endonuclease 1 (APE1) in living cells, highlighting the great potential of the bipedal DNA nanowalker in biological systems.Graphical abstract

Synthesis of a novel adapter lipid using Fc-region mediated antibody modification for post-insert preparation of transferrin receptor targeted messenger RNA-loaded lipid nanoparticles

Lipid nanoparticles (LNPs) are promising delivery systems with the ability to deliver small interfering RNA (siRNA) and messenger RNA (mRNA) in diseased tissues and intracellular sites of action. However, delivery to non-hepatic tissues via systemic administration remains challenging. Antibody modification of LNPs is a hopeful approach for improving their selectivity to target tissues. The conventional method of antibody modification via thiol–maleimide linkage is concerned with reduced recognition efficiency of the disease-related target molecules owing to variations in antibody orientation on the surface of the LNPs. In this study, we developed a novel adapter lipopeptide for antibody modification of LNPs via the Fc-region. Here, we selected RI7-217, an anti-transferrin receptor antibody, as the ligand. Through optimization of spacer peptides, we found a FcBP-EKGG-lipid exhibits high water-dispersibility for post-insertion method to LNPs. We prepared RI7-217-modified LNPs by modifying LNPs with FcBP-EKGG-lipids and mixing the antibodies. We found that the luciferase protein expression of RI7-217-modified LNPs was significantly enhanced in an antibody-specific manner against transferrin receptor-expressing U-87 MG cells. This information would be valuable in the development of antibody-modified LNPs for cell-selective targeting.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle.

Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1.

In this study, we describe the structure-based development of the first fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor type 1 (CCR1), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in inflammation and immune diseases. Starting from previously reported intracellular allosteric modulators of CCR1, tetramethylrhodamine (TAMRA)-labeled ligands were designed, synthesized, and tested for their suitability as fluorescent tracers to probe binding to the IABS of CCR1. In the course of these studies, we developed LT166 (12) as a highly versatile fluorescent CCR1 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonradioactive and high-throughput manner. Besides the detection of intracellular allosteric ligands by direct competition with 12, we were also able to monitor the binding of extracellular antagonists due to their positive cooperative binding with 12. Thereby, we provide a straightforward and nonradioactive method to easily distinguish between ligands binding to the IABS of CCR1 and extracellular negative modulators. Further, we applied 12 for the identification of novel chemotypes for intracellular CCR1 inhibition that feature high binding selectivity for CCR1 over CCR2. For one of the newly identified intracellular CCR1 ligands (i.e., 23), we were able to show CCR1 over CCR2 selectivity also on a functional level and demonstrated that this compound inhibits basal β-arrestin recruitment to CCR1, thereby acting as an inverse agonist. Thus, our fluorescent CCR1 ligand 12 represents a highly promising tool for future studies of CCR1-targeted pharmacology and drug discovery.

Brain Delivery of Protein Therapeutics by Cell Matrix-Inspired Biomimetic Nanocarrier.

Protein therapeutics are anticipated to offer significant treatment options for central nervous system (CNS) diseases. However, the majority of proteins are unable to traverse the blood-brain barrier (BBB) and reach their CNS target sites. Inspired by the natural environment of active proteins, the cell matrix components hyaluronic acid (HA) and protamine (PRTM) are used to self-assemble with proteins to form a protein-loaded biomimetic core and then incorporated into ApoE3-reconstituted high-density lipoprotein (rHDL) to form a protein-loaded biomimetic nanocarrier (Protein-HA-PRTM-rHDL). This cell matrix-inspired biomimetic nanocarrier facilitates the penetration of protein therapeutics across the BBB and enables their access to intracellular target sites. Specifically, CAT-HA-PRTM-rHDL facilitates rapid intracellular delivery and release of catalase (CAT) via macropinocytosis-activated membrane fusion, resulting in improved spatial learning and memory in traumatic brain injury (TBI) model mice (significantly reduces the latency of TBI mice and doubles the number of crossing platforms), and enhances motor function and prolongs survival in amyotrophic lateral sclerosis (ALS) model mice (extended the median survival of ALS mice by more than 10 days). Collectively, this cell matrix-inspired nanoplatform enables the efficient CNS delivery of protein therapeutics and provides a novel approach for the treatment of CNS diseases.

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm2/s), slow (∼0.04 μm2/s), and fast (∼0.14 μm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1.

The membrane protein family of G protein-coupled receptors (GPCRs) represents a major class of drug targets. Over the last years, the presence of additional intracellular binding sites besides the canonical orthosteric binding pocket has been demonstrated for an increasing number of GPCRs. Allosteric modulators harnessing these pockets may represent valuable alternatives when targeting the orthosteric pocket is not successful for drug development. Starting from SBI-553, a recently discovered intracellular allosteric modulator for neurotensin receptor subtype 1 (NTSR1), we developed the fluorescent molecular probe 14. Compound 14 binds to NTSR1 with an affinity of 0.68 μM in the presence of the agonist NT(8-13). NanoBRET-based ligand binding assays with 14 were established to derive the affinity and structure-activity relationships for allosteric NTSR1 modulators in a direct and nonisotopic manner, thereby facilitating the search for and optimization of novel allosteric NTSR1 ligands. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists. Moreover, employing 14 as a probe in a drug library screening, we identified novel chemotypes as binders for the intracellular allosteric SBI-553 binding pocket of NTSR1 with single-digit micromolar affinity. These hits may serve as interesting starting points for the development of novel intracellular allosteric ligands for NTSR1 as a highly interesting yet unexploited drug target in the fields of pain and addiction disorder therapy.

Aggregation-induced emission fluorogenic probes for wash-free imaging of lipid droplets in living cells

As essential subcellular organelles, lipid droplets (LDs) are involved in various the pathophysiological processes of metabolic diseases, including fatty liver and diabetes. Therefore, detecting LDs is conducive for studying the pathogeny and the early diagnosis of associated diseases. In this work, a series of fluorescent probes L1-L3 based on triphenylamine derivatives were designed and synthesized for tracking LDs in living cells. Due to the different terminal moieties of the three probes, L1 and L3 are aggregation-induced emission (AIE)-active, while L2 is a dual-state emission (DSE) molecule. In addition, confocal cell imaging experiments showed that probes L1-L3 specifically target intracellular LDs sites without the post washing. Importantly, probe L3 allows real-time monitoring of the dynamic processes and changes in the number of LDs. In summary, probe L3 is an ideal probe for studying LDs-related pathophysiological processes, and this work provides more ideas for the future design and synthesis of high-performance fluorescent probes for targeting LDs.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Optical and thermo-plasmonic properties of spherical and hollow gold nanoparticles injected in cell organelles

The advent of nanotechnology has profoundly altered many traditional treatment approaches, due to the beneficial physicochemical and functional characteristics of gold nanoparticles (AuNPs). These gold nanostructures are attracting particular interest in a wide range of biomedical applications, such as thermotherapy, diagnostics, detection, and drug delivery. Due to their exceptional characteristics, AuNPs have long been considered as a potential means of combating cancer. These features include surface plasmon resonance (SPR), surface chemistry and multi-functionalization. In addition to targeting tissues and cancer cells, AuNPs with the ability to specifically target organelles are attracting growing interest and are seen as the next evolutionary step in nanomedicine, forming the third generation of this technology. Subcellular targeting offers unique advantages over free targeting, as it enables specific delivery of encapsulated NPs to the intracellular site of action. This translates into greater therapeutic efficacy with a reduced dose and, above all, the ability to overcome immune resistance. AuNPs act as small heat sources taking advantage of SPR phenomenon, enabling targeted heating of specific organelles. The thermoplasmonic effect disrupts organelle function in targeted cancer cells, leading to their destruction. In this work, we used the finite element method (FEM) to examine the properties associated with the SPR of both spherical and hollow AuNPs when they are embedded in the main cellular components, including the lysosome, cell membrane, mitochondria, nucleus, cytoplasm and extracellular medium. In order to predict the thermal destructive power of solid spheres (SAuNPs) and hollow (HAuNPs) gold nanoparticles embedded in these different organelles, we simulated their absorption capacity, field enhancement effect and temperature distribution. The results obtained show that the thermoplasmonic effect is very important for HAuNPs compared with SAuNPs. In addition, the optical and thermoplasmonic properties of SAuNPs and HAuNPs show differential behavior for different cell organelles.

A fluorescent probe for detecting H2O2 and delivering H2S in lysosomes and its application in maintaining the redox environments

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that can be used as a marker for the occurrence of oxidative stress in the organism. Lysosomes serve as intracellular digestive sites, and when the concentration of H2O2 in them is abnormal, lysosomal function is often impaired, leading to the development of diseases. Hydrogen sulfide (H2S) acts as a gaseous signaling molecule that scavenges H2O2 from cells and tissues, thereby maintaining the redox environment of the body. However, most of the reported hydrogen peroxide fluorescent probes so far can only detect H2O2, but cannot maintain the intracellular redox environment. In this paper, an H2O2 fluorescent probe LN-HOD with lysosomal targeting properties was designed and synthesized by combining the H2O2 recognition site with a naphthylamine fluorophore via a thiocarbamate moiety. The probe has the advantages of large Stokes shift (110 nm), high sensitivity and good H2S release capability. The probe LN-HOD can be used to detect H2O2 in cells, zebrafish and plant roots. In addition, LN-HOD detects changes in the concentration of H2O2 in plant roots when Arabidopsis is stressed by cadmium ion (Cd2+). And through its ability to release H2S, it can help to remove excess H2O2 and maintain the redox environment in cells, zebrafish and plant roots. The present work provides new ideas for the detection and assisted removal of H2O2, which contributes to the in-depth study of the cellular microenvironment in organisms.

Understanding Sorafenib-Induced Cardiovascular Toxicity: Mechanisms and Treatment Implications.

Tyrosine kinase inhibitors (TKIs) have been recognized as crucial agents for treating various tumors, and one of their key targets is the intracellular site of the vascular endothelial growth factor receptor (VEGFR). While TKIs have demonstrated their effectiveness in solid tumor patients and increased life expectancy, they can also lead to adverse cardiovascular effects including hypertension, thromboembolism, cardiac ischemia, and left ventricular dysfunction. Among the TKIs, sorafenib was the first approved agent and it exerts anti-tumor effects on hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) by inhibiting angiogenesis and tumor cell proliferation through targeting VEGFR and RAF. Unfortunately, the adverse cardiovascular effects caused by sorafenib not only affect solid tumor patients but also limit its application in curing other diseases. This review explores the mechanisms underlying sorafenib-induced cardiovascular adverse effects, including endothelial dysfunction, mitochondrial dysfunction, endoplasmic reticulum stress, dysregulated autophagy, and ferroptosis. It also discusses potential treatment strategies, such as antioxidants and renin-angiotensin system inhibitors, and highlights the association between sorafenib-induced hypertension and treatment efficacy in cancer patients. Furthermore, emerging research suggests a link between sorafenib-induced glycolysis, drug resistance, and cardiovascular toxicity, necessitating further investigation. Overall, understanding these mechanisms is crucial for optimizing sorafenib therapy and minimizing cardiovascular risks in cancer patients.