Intracellular Fate Research Articles

Shape Effect of Polymer-Based Multilayer Microcapsules on Cellular Internalization.

The intracellular fate of drug carriers had received extensive attention, which was profoundly influenced by the shapes of carriers. However, it has not been fully addressed due to the lack of effective strategies to prepare carriers with different shapes and the interference of other parameters (such as stiffness and chemistry of the shaped particle and the different cell lines). In this work, polymer-based microcapsules with different shapes (spherical, peanut, dumbbell, and cubic) but the same surface chemistry were engineered through the alternative deposition of polyethylenimine (PEI) and polyethylene glycol (PEG) onto the sacrificial CaCO3 templates with different well-defined shapes. Various techniques (SEM, CLSM, AFM, FTIR, and XPS) were utilized to determine the shapes and chemical composition of these microcapsules. The effect of microcapsule shape on cellular internalization kinetics and the endocytosis mechanism was thoroughly studied, and dumbbell and cubic microcapsules showed greater internalization rates and amounts than spherical and peanut microcapsules. These microcapsules were internalized through micropinocytosis, and the shapes had no obvious effect on the endocytosis mechanism. This work provides a wealth of information about the relationship between the shape of microcapsules and their performance in cellular internalization, which will be of great help in the development of related drug carriers.

When subcellular chemical imaging enlightens our understanding on intestinal absorption, intracellular fate and toxicity of PFOA in vitro

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant that accumulates in the human body, leading to major health issues. Upon oral uptake, the gastrointestinal tract is the first biological barrier against PFOA. However, the localization of PFOA and its impact on the intestinal wall are largely unknown. Here we achieve a breakthrough in the knowledge of intestinal absorption, intracellular fate and toxicity of PFOA using in vitro assays combined with novel analytical imaging techniques. For the first time, we localized PFOA in the cytosol of Caco-2 cells after acute exposure using high spatial resolution mass spectrometry imaging, and we estimated the PFOA cytosolic concentration. Knowing that PFOA enters and accumulates in the intestinal cells, we also performed common toxicity assays assessing cell metabolic activity, membrane integrity, oxidative stress response, and cell respiration. This study integrating powerful analytical techniques with widely used toxicology assays provides insightful information to better understand potential negative impacts of PFOA and opens new opportunities in toxicology and life science in general.

Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstractα‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2. The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.

Elucidating siRNA Cellular Delivery Mechanism Mediated by Quaternized Starch Nanoparticles.

Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20kHz) application may have induced siRNA release resulting in faster gene silencing kinetics.

Mind the Particle Rigidity: Blooms the Bioavailability via Rapidly Crossing the Mucus Layer and Alters the Intracellular Fate of Curcumin.

Overcoming intestinal epithelial barriers to enhance bioavailability is a major challenge for oral delivery systems. Desirable nanocarriers should simultaneously exhibit rapid mucus penetration and efficient epithelial uptake; however, they two generally require contradictory structural properties. Herein, we proposed a strategy to construct multiperformance nanoparticles by modifying the rigidity of amphiphilic nanostructures originating from soy polypeptides (SPNPs), where its ability to overcome multibarriers was examined from both in vitro and in vivo, using curcumin (CUR) as a model cargo. Low-rigidity SPNPs showed higher affinity to mucin and were prone to getting stuck in the mucus layer. When they reached epithelial cells, they tended to be endocytosed through the clathrin and macropinocytosis pathways and further transferred to lysosomes, showing severe degradation and lower transport of CUR. Increased particle rigidity generally improved the absorption of CUR, with medium-rigidity SPNPs bloomed maximum plasma concentration of CUR by 80.62-fold and showed the highest oral bioavailability. Results from monocultured and cocultured cell models demonstrated that medium-rigidity SPNPs were least influenced by the mucus layer and changes in rigidity significantly influenced the endocytosis and intracellular fate of SPNPs. Those with higher rigidity preferred to be endocytosed via a caveolae-mediated pathway and trafficked to the ER and Golgi, facilitating their whole transcytosis, and avoiding intracellular metabolism. Moreover, rigidity modulation efficiently induces the reversible opening of intercellular tight junctions, which synergistically improves the transport of CUR into blood circulation. This study suggested that rigidity regulation on food originated amphiphilic peptides could overcome multiple physiological barriers, showing great potential as natural building block toward oral delivery.

Protein corona alleviates adverse biological effects of nanoplastics in breast cancer cells.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs (e.g. oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs. To this end, here we use pristine and human-plasma pre-coated polystyrene (PS) nanoparticles (NPs) and investigate them in cultured breast cancer cells both in terms of internalization and cell biochemical response to the exposure. It is found that pristine NPs tend to stick to the cell membrane and inhibit HER-2-driven signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are associated with cancer cell survival and growth. By contrast, the formation of a protein corona around the same NPs can promote their uptake by endocytic vesicles and final sequestration within lysosomes. Of note is that such intracellular fate of PS-NPs is associated with mitigation of the biochemical alterations of the phosphorylated AKT (pAKT)/AKT and phosphorylated ERK (pERK)/ERK levels. These findings provide the distribution of NPs in human breast cancer cells, may broaden our understanding of the interactions between NPs and breast cancer cells and underscore the crucial role of the protein corona in modulating the impact of MNPs on human health.

Size of lipid emulsion droplets influences metabolism in human CD4+ T cells

ScopeTriglyceride-based lipid emulsions are critical for total parenteral nutrition (TPN), but their long-term use has adverse effects, such as severe liver dysfunction necessitating improved formulations. This study compares the uptake mechanism and intracellular fate of novel glycerol-stabilized nano-sized lipid emulsions with conventional emulsions in CD4+ T cells, focusing on their impact on cellular metabolism. Methods and resultsNanoemulsions were formulated with increased glycerol content. Uptake of emulsions in primary human CD4+ T cells was investigated using different endocytic blockers, then quantified by flow cytometry, and visualized by confocal microscopy. To investigate emulsion intracellular fate, fatty acids in membrane phospholipids were quantified by GC-MS/MS and cellular metabolism was assessed by Seahorse technology. Results show T cells internalize both conventional and nano-sized emulsions using macropinocytosis. Fatty acids from emulsions are stored as neutral lipids in intracellular vesicles and are incorporated into phospholipids of cellular membranes. However, only nanoemulsions additionally use clathrin-mediated endocytosis and deliver fatty acids to mitochondria for increased β-oxidation. ConclusionsSize of lipid emulsion droplets significantly influences their uptake and subsequent metabolism in CD4+ T cells. Our results highlight the potential for improved nutrient utilization with nanoemulsions in TPN formulations possibly leading to less adverse effects.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

(Invited) An in Vitro Model of Mechanical Strain Enhances Cellular Uptake of Single-Walled Carbon Nanotubes

Single-walled carbon nanotubes (SWCNTs) are ideal optical biosensors due to their intrinsic near-infrared (NIR) emission, which is highly sensitive, easily tunable, and indefinitely photostable. Due to such desirable properties coupled with appropriate functionalization strategies, SWCNTs have been used in numerous in vitro, live-cell, and in vivo applications. Depending on the type of functionalization as well as cell type, SWCNTs can be internalized by live cells via demonstrated energy-dependent endocytosis processes where they are retained within the endosomal pathway or are expelled by the cells. While this uptake and intracellular processing of SWCNTs has been recently examined, these studies are usually performed with standard cell culture techniques in petri dishes or 3D cultures. Unlike these cultures, cells in living organisms are continuously exposed to a wide variety of stimuli, e.g., periodic mechanical strain, during both normal and disease-related conditions. Thus, it is important to investigate how cells under physiological stretching compare to those grown in standard cultures. Herein, using a novel cell stretching platform to mimic physiological conditions, we investigate the uptake of SWCNTs into different human cancer cells.Our results indicate that cells exposed to uniform radial stretching display significantly higher rates of nanoparticle uptake, which can be correlated to their increased level of cellular activity. This approach recognizes the importance of studying cellular responses in environments that more closely resemble the complex conditions found in living organisms. It suggests a potential connection between mechanical stimuli and cellular processes related to SWCNT uptake. This finding has implications for understanding how nanotube uptake differs in different cellular stress environments and how cells respond to their dynamic and ever-changing microenvironment.Investigating how the mechanical strain influences the endocytosis processes and subsequent intracellular fate of SWCNTs can provide valuable insights into the interplay between nanomaterials and living cells under more realistic conditions. Further aiding us in the development of more accurate intracellular biosensors and effective drug delivery platforms using SWCNTs.

The Extracellular Metallometabolome: Metallophores, Metal Ionophores, and Other Chelating Agents as Natural Products

Natural products include inorganic as well as organic compounds. Living organisms face constant challenges in acquiring essential metal ions and getting rid of non-essential ones with toxic actions. They employ an extracellular biochemistry in these tasks and use it to engage in a chemical warfare against invaders and competitors by either increasing or decreasing the availability of metal ions for maintaining their welfare in aquatic or terrestrial ecological niches. To control mutualistic, cambialistic or parasitic symbiosis with other organisms they use a remarkably rich suite of secreted bioactive molecules with ligand donor atoms for metal binding. This overview discusses the interactions of these extracellular natural products with a multitude of metal ions in the periodic system of the elements. It focuses mainly on metallophores and metal ionophores secreted from bacteria, fungi, and plants, but metal-carrying cofactors and other chelating agents will also be mentioned in the context of related functions and with an intent to categorize. The intracellular fate of the metal ions and the controlled pathways for the biosynthesis, secretion, uptake, biodegradation or recycling of the secreted natural products that interact with metal ions will not be covered. Metallophores make extracellular metal ions available via delivery to specific transporters and unavailable to competing organisms, especially pathogens, though some invaders have developed ways to compete efficiently for metal ions. The classic concept of siderophores, carriers of iron(III) ions, is extended here to specific and broad-band metallophores for metal ions such as copper (chalkophores), zinc (zincophores), and yet others. Metal ionophores, in contrast, transport metal ions through biological membranes. There is a wide variety of chemical structures for either metallophores or metal ionophores. Together with physicochemical investigations of metal complexation und conditions mimicking the natural environment, “omics” mining and mapping the diversity of chemotypes is an on-going effort with analytic, genetic, and bioinformatic tools and comes together in defining the metallometabolome, which combines the metabolome and the metallome. Investigations are highly multidisciplinary, include an important, but academically infrequently crossed bridge between the biosciences (biochemistry) and the earth sciences (geochemistry), define significant applications in the pharmaceutical/medical sciences regarding immune modulation and the control of virulence at the host-pathogen interface, and have implications for the nutritional/toxicological and environmental/ecological sciences.

In vivo Fate of Targeted Drug Delivery Carriers.

This review aimed to systematically investigate the intracellular and subcellular fate of various types of targeting carriers. Upon entering the body via intravenous injection or other routes, a targeting carrier that can deliver therapeutic agents initiates their journey. If administered intravenously, the carrier initially faces challenges presented by the blood circulation before reaching specific tissues and interacting with cells within the tissue. At the subcellular level, the car2rier undergoes processes, such as drug release, degradation, and metabolism, through specific pathways. While studies on the fate of 13 types of carriers have been relatively conclusive, these studies are incomplete and lack a comprehensive analysis. Furthermore, there are still carriers whose fate remains unclear, underscoring the need for continuous research. This study highlights the importance of comprehending the in vivo and intracellular fate of targeting carriers and provides valuable insights into the operational mechanisms of different carriers within the body. By doing so, researchers can effectively select appropriate carriers and enhance the successful clinical translation of new formulations.

Differentially Charged Liposomes Stimulate Dendritic Cells with Varying Effects on Uptake and Processing When Used Alone or in Combination with an Adjuvant.

Liposomes carrying differential charges have been extensively studied for their role in stimulating dendritic cells (DCs), major antigen-presenting cells, known to serve as a pivotal bridge between innate and adaptive immunity. However, the impact of the differentially charged liposomes on activating DCs remains to be understood. In this study, we have investigated the impact of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based neutral, anionic, and cationic liposomes on the uptake, immunostimulation, and intracellular fate in mouse bone-marrow-derived DCs. We observed that liposomes could induce phenotypic maturation of DCs by inducing the expression of costimulatory molecules (CD40 and CD86) and production of cytokines tumor necrosis factor-α, interleukin-12,and nitric oxide. Interestingly, admixing monophosphoryl lipid A with charged liposomes further enhances the expression of the costimulatory molecules and production of cytokines, with preferential activation by positively charged liposomes. Fluorometric analysis using a pH-sensitive dye and flow-cytometry-based pathway inhibition assays revealed that cationic liposomes were taken up more efficiently by DCs through endocytosis and transported to neutral compartments for further processing, whereas anionic and neutral liposomes were inclined to accumulate in acidic compartments. These findings therefore endorse the use of cationic DSPC liposomes as a preferred option for vaccine delivery vehicles over neutral and negatively charged liposomes, particularly for the preferential activation of DCs.

Mitochondrial iron deficiency triggers cytosolic iron overload in PKAN hiPS-derived astrocytes

Disease models of neurodegeneration with brain iron accumulation (NBIA) offer the possibility to explore the relationship between iron dyshomeostasis and neurodegeneration. We analyzed hiPS-derived astrocytes from PANK2-associated neurodegeneration (PKAN), an NBIA disease characterized by progressive neurodegeneration and high iron accumulation in the globus pallidus. Previous data indicated that PKAN astrocytes exhibit alterations in iron metabolism, general impairment of constitutive endosomal trafficking, mitochondrial dysfunction and acquired neurotoxic features. Here, we performed a more in-depth analysis of the interactions between endocytic vesicles and mitochondria via superresolution microscopy experiments. A significantly lower number of transferrin-enriched vesicles were in contact with mitochondria in PKAN cells than in control cells, confirming the impaired intracellular fate of cargo endosomes. The investigation of cytosolic and mitochondrial iron parameters indicated that mitochondrial iron availability was substantially lower in PKAN cells compared to that in the controls. In addition, PKAN astrocytes exhibited defects in tubulin acetylation/phosphorylation, which might be responsible for unregulated vesicular dynamics and inappropriate iron delivery to mitochondria. Thus, the impairment of iron incorporation into these organelles seems to be the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, triggering mitochondrial dysfunction. Overall, the data elucidate the mechanism of iron accumulation in CoA deficiency, highlighting the importance of mitochondrial iron deficiency in the pathogenesis of disease.

Engineering W/O/W pickering emulsion for the enhanced antigen delivery and immune responses

Tumor immunotherapy, particularly cancer vaccines, holds promise for combating cancer by harnessing tailored immune responses against malignant cells. However, conventional approaches face challenges in efficiently delivering antigens for optimal immune activation. Emulsion adjuvants, like MF59, enhance cellular uptake but struggle to induce robust CD8+ T cell responses. Here, we introduce a novel strategy employing a water-in-oil-in-water (W/O/W) multiple Pickering emulsion (mPE) for antigen delivery. The mPE, utilizing biocompatible, pH-sensitive particles, encapsulates antigens within the inner water phase, ensuring enhanced intracellular processing and dictating the intracellular fate of antigens for improved cross-presentation. In vitro and in vivo studies demonstrated that mPEs induced robust dendritic cells activation and antigen cross-presentation, leading to a significantly enhanced immune response. Notably, calcium phosphate-stabilized mPE (CaP-mPE) illustrated the more robust IFN-γ+ T cell responses. In comparison with traditional surfactant-stabilized multiple emulsions, CaP-mPE significantly inhibit tumor growth and effectively prolong the survival of tumor-bearing mice. This innovative approach offers a promising avenue for the development of effective cancer vaccines with potent cellular immune responses.

Pathways for macrophage uptake of cell-free circular RNAs

Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA, which may comprise cellular debris and pathogen genomes. Here, we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, Toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

Spatiotemporal tracking of intracellular nanoparticles using complementary imaging systems reveals acute ferroptosis triggered by burst reduction of ferric ions

Uptake and intracellular trafficking of nanoparticles are tightly regulated by their interactions with cellular organelles and physiological microenvironment. Although the dynamic physicochemical reactions at the interface of nanoparticles and cells ultimately determine the intracellular distribution and fate, microscopic tracing and quantitative analysis of the nanoparticles have been hampered by the limited resolution associated with individual nanoparticle trafficking. Herein, we report spatiotemporal investigations on autophagic clearance of biodegradable iron oxide-silica core-shell nanoparticles in terms of intracellular trafficking and ionic dissolution at a single cell level using multimodal imaging systems. By combining transmission electron microscopy and super-resolution confocal laser scanning microscopy with fluorescence correlation spectroscopy, the complementary imaging analysis exclusively shows the intracellular uptake, endosomal fusion and biodegradative clearance, leading to identify the step-by-step endocytic transport pathway and autophagic degradation pathways. Tracing the intracellular trafficking of nanoparticles reveals that they are spontaneously transported from endosomes to lysosomes, and transiently stimulate autophagy while maintaining cell viability. While protecting iron oxide core, the silica shell is gradually degraded during endocytosis and autophagic clearance, resulting in ionic dissolution of iron oxide in acidic environment. Moreover, burst reduction of ferric ions by adding ascorbic acid readily triggers acute ferroptosis owing to rapid supplement of ferrous ions and Fenton reaction in cancer cells. The complementary imaging strategy provides insights into the design of biocompatible nanomedicines for cellular delivery and the degradative mechanisms beyond the intracellular fate.

Monitoring the Intracellular Fate of Molecular Beacons: The Challenge of False Positive Signals

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time‐stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta‐cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant.

Mycobacterium tuberculosis hijacks host macrophages-derived interleukin 16 to block phagolysosome maturation for enhancing intracellular growth

ABSTRACT The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of Mycobacterium tuberculosis (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with M. marinum and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.

Modular design of cyclic peptide – polymer conjugate nanotubes for delivery and tunable release of anti-cancer drug compounds

High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.