Dynein functions in galectin-3 mediated processes of clathrin-independent endocytosis.

Multiple endocytic processes operate in cells in tandem to uptake multiple cargoes involved in diverse cellular functions, including cell adhesion and migration. The best-studied clathrin-mediated endocytosis (CME) involves the formation of a well-defined cytoplasmic clathrin coat to facilitate cargo uptake. According to the glycolipid-lectin (GL-Lect) hypothesis, galectin-3 (Gal3) binds to glycosylated membrane receptors and glycosphingolipids (GSLs) to drive membrane bending and tubular membrane invaginations that undergo scission to form a morphologically distinct class of uptake structures, termed clathrin-independent carriers (CLICs). Which components from cytoskeletal machinery are involved in the scission of CLICs remains to be explored. In this study, we propose that dynein is recruited onto Gal3-induced tubular endocytic pits and provides the pulling force for friction-driven scission. The uptake of Gal3 and its cargoes (CD98/CD147) is significantly dependent on dynein activity, whereas only transferrin (CME marker) is slightly affected upon dynein inhibition. Our study reveals that Gal3 and Gal3-dependent (CD98 and CD147) clathrin-independent cargoes require dynein for the clathrin-independent endocytosis.

Additive-anchored thermoresponsive nanoscale self-assembly generation in normal and reverse Tetronics®.

Self-assembly of ethylene oxide (EO)-propylene oxide (PO)-based star-shaped block copolymers (BCPs) in the presence of different kinds of additives is investigated in an aqueous solution environment. Commercially available four-armed BCPs, namely Tetronics® (normal: T904 with EO as the terminal end block; and reverse: T90R4 with PO as the terminal end block), each with 40%EO, are used. The effect of various additives such as electrolytes (NaCl and Na2SO4), nonelectrolyte polyols (glucose and sorbitol), and ionic surfactants (viz. anionic-sodium dodecyl sulfate (SDS), cationic-dodecyltrimethylammonium bromide (DTAB) and zwitterionic dodecyldimethylammonium propane sulfonate (C12PS)) on these BCPs is examined to observe their influence on micellization behaviour. The presence of salts and polyols displayed interesting phase behaviour, i.e., the cloud point (CP) was decreased, the water structure was affected and the micelles were dehydrated by expelling water molecules, and thus they were likely to promote micelle formation/growth. In contrast, ionic surfactants in small amounts interacted with the BCPs and showed an increase in CPs thereby forming mixed micelles with increasing charges and decreasing micellar sizes, finally transforming to small surfactant-rich mixed micelles. Molecular interactions such as electrostatic and hydrogen bonding involved within the examined entities are put forth employing a computational simulation approach using the Gaussian 09 window for calculation along with the GaussView 5.0.9 programming software using the (DFT)/B3LYP method and 3-21G basis set. The hydrodynamic diameter (Dh) of the micelles is examined using dynamic light scattering (DLS), while the various micellar parameters inferring the shape/geometry are obtained using small-angle neutron scattering (SANS) by the best fitting of the structure factors. It is observed that 10 w/v% T904 remains as spherical micelles with some micellar growth under physiological conditions (37 °C), while 10 w/v% T90R4 remains as unimers and forms spherical micelles in the presence of additives at 37 °C. Furthermore, the additive-induced micellar systems are tested as developing nanovehicles for anticancer (curcumin, Cur) drug solubilization using UV-vis spectroscopy, which shows a prominent increase in absorbance with enhanced solubilization capacity. Additionally, the cytotoxic effect of Cur loaded on the BCP micelles in HeLa cells is studied through confocal microscopy by capturing fluorescence images that depict HeLa cell growth inhibition under the influence of additive-induced micellar systems.

Structural DNA nanotechnology at the nexus of next-generation bio-applications: challenges and perspectives.

DNA nanotechnology has significantly progressed in the last four decades, creating nucleic acid structures widely used in various biological applications. The structural flexibility, programmability, and multiform customization of DNA-based nanostructures make them ideal for creating structures of all sizes and shapes and multivalent drug delivery systems. Since then, DNA nanotechnology has advanced significantly, and numerous DNA nanostructures have been used in biology and other scientific disciplines. Despite the progress made in DNA nanotechnology, challenges still need to be addressed before DNA nanostructures can be widely used in biological interfaces. We can open the door for upcoming uses of DNA nanoparticles by tackling these issues and looking into new avenues. The historical development of various DNA nanomaterials has been thoroughly examined in this review, along with the underlying theoretical underpinnings, a summary of their applications in various fields, and an examination of the current roadblocks and potential future directions.

Glycosylated Porphyrin Derivatives for Sonodynamic Therapy: ROS Generation and Cytotoxicity Studies in Breast Cancer Cells.

Sonodynamic therapy (SDT) is a promising alternative to photodynamic therapy for achieving site-specific cytotoxic therapy. Porphyrin derivative molecules have been reported extensively in photodynamic therapy. We have previously shown that the glycosylation of porphyrin-based sonosensitizers can enhance their cellular uptake. However, the sonodynamic potential of these water-soluble glycosylated porphyrins has not been investigated. In this study, we characterized the sonodynamic response of two water-soluble glycosylated porphyrin derivatives. Ultrasound (US) exposure was performed (1 MHz frequency, intensities of 0.05-1.1 W/cm2) for 0-3 min in continuous mode. Reactive oxygen species (ROS) generation was quantified via ultraviolet-visible (UV-vis) spectrophotometry. MTT assay was used to quantify cytotoxicity caused by sonodynamic effects from these derivatives in the human mammary carcinoma (SUM-159) cell line in vitro. ROS generation from the porphyrin derivatives was demonstrated at a concentration of 15 µM. No significant cytotoxic effects were observed with the sonosensitizer alone or US exposure alone over the tested range of intensities and duration. The free base porphyrin derivative caused 60-70% cell death, whereas the zinc-porphyrin derivative with Zn metal conjugation caused nearly 50% cytotoxicity when exposed at 0.6 W/cm2 intensity for 3 min. These studies demonstrate the potential of anticancer SDT with soluble glycosylated porphyrins.

ß-Carotene laden antibacterial and antioxidant gelatin/polyglyceryl stearate nano-hydrogel system for burn wound healing application.

Worldwide, burn wounds are severe health issues prone to bacterial infections and challenging to treat with traditional wound dressings. Therefore, a highly desirable biological macromolecules-based wound dressing with good antioxidant, antibacterial, biocompatible, and a large surface area is required. Herein, aim to develop a biological macromolecules-based physically cross-linked gelatin/polyglyceryl stearate/graphene oxide (GPGO) hydrogel to treat burn wounds. Four sets of hydrogels were prepared by varying GO concentrations. FT-IR, FE-SEM, viscosity analysis, mechanical and thermal stability confirmed the successful preparation of hydrogels with desired properties. Further, ß-carotene (0.5 mg/mL) was encapsulated in hydrogels to enhance the antioxidant activity, and a cumulative release as well as kinetics at pH 6.4 and 7.4 was performed. With an increase in GO concentration, hydrogels showed sustained release of ß-carotene. Among all, GPGO-3 ß hydrogel showed the highest antioxidant potency (57.75 %), hemocompatible (<5 %), cytocompatible (viable with NIH 3T3 cells), cell migration, proliferation, and in vitro wound healing. Also, GPGO-3 ß hydrogel showed efficient antibacterial activity (%inhibition of 85.5 % and 80.2 % and zone of 11 mm and 9.8 mm against S. aureus and E. coli). These results demonstrated the ability of GPGO-3 ß hydrogel as a promising candidate for burn wound healing applications.

Pathophysiology to advanced intra-articular drug delivery strategies: Unravelling rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most prevalent life-long autoimmune diseases with an unknown genesis. It primarily causes chronic inflammation, pain, and synovial joint-associated cartilage and bone degradation. Unfortunately, limited information is available regarding the etiology and pathogenesis of this chronic joint disorder. In the last few decades, an improved understanding of RA pathophysiology about key immune cells, antibodies, and cytokines has inspired the development of several anti-rheumatic drugs and biopharmaceuticals to act on RA-affected joints. However, life-long frequent systemic high doses of commercially available drugs are currently a limiting factor in the efficient management of RA. To address this issue, various single and double-barrier intra-articular drug delivery systems (IA-DDSs) such as nanocarriers, microparticles, hydrogels, and particles-hybrid hydrogel composite have been developed which can exclusively target the RA-affected joint cavity and release the precisely controlled therapeutic drug concentration for prolonged time whilst avoiding the systemic toxicity. This review provides a comprehensive overview of the pathogenesis of RA and discusses the rational design and development of biomaterials-based novel IA-DDs, ranging from conventional to advanced systems, for improved treatment of RA. Therefore, this review aims to unravel the pathophysiology of rheumatoid arthritis and explore cutting-edge IA-DD strategies exploiting biomaterials. It offers researchers a consolidated and up-to-date resource platform to analyze existing knowledge, identify research gaps, and contribute to the scientific literature.

Hypoxia Modulates Cellular Endocytic Pathways and Organelles with Enhanced Cell Migration and 3D Cell Invasion.

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.

Synthesis of novel zinc porphyrins with bioisosteric replacement of Sorafenib: Efficient theranostic agents for anti-cancer application.

Novel zinc porphyrins (trans-A2B2 and A3B type) are reported containing pharmacophoric groups derived from Sorafenib at the meso-positions. The pharmacophoric and bioisosteric modification of Sorafenib was done with 2-methyl-4-nitro-N-phenylaniline. The in-vitro photo-cytotoxicity studies of zinc porphyrins on HeLa cells revealed excellent PDT based autophagy inhibition of cancer cells, with IC50 values between 6.2 to 15.4 µM. The trans-A2B2 type zinc porphyrin with two bioisosteric groups gave better cytotoxicity than A3B type. Molecular docking studies revealed excellent binding with mTOR protein kinase of the designed porphyrins. The confocal studies indicated significant ER localization of trans-A2B2 type zinc porphyrin in HeLa cells along with ROS generation. trans-A2B2 type zinc porphyrin induced ER stress in cancer cells, thereby causing elevation of Ca+2 ions in cytoplasm, which led to cancer cell death via autophagy pathway. The studies suggested that trans-A2B2 and A3B type zinc porphyrins can be developed as theranostic agents for anti-cancer applications.

Red emitting carbon dots: surface modifications and bioapplications.

Quantum dots (QDs), and carbon quantum dots (CDs) in particular, have received significant attention for their special characteristics. These particles, on the scale of several nanometers, are often produced using simple and green methods, with naturally occurring organic precursors. In addition to facile production methods, CDs present advantageous applications in the field of medicine, primarily for bioimaging, antibacterial and therapeutics. Also, CDs present great potential for surface modification through methods like doping or material mixing during synthesis. However, the bulk of current literature focuses on CDs emitting in the blue wavelengths which are not very suitable for biological applications. Red emitting CDs are therefore of additional interest due to their brightness, photostability, novelty and deeper tissue penetration. In this review article, red CDs, their methods of production, and their biological applications for translational research are explored in depth, with emphasis on the effects of surface modifications and doping.

Endocytic pathways of pathogenic protein aggregates in neurodegenerative diseases.

Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aß, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species.

Introduction to emerging concepts in nucleic acids: structures, functions and applications.

An introduction to the Nanoscale, Nanoscale Advances and Physical Chemistry Chemical Physics (PCCP) themed collection on DNA and RNA nanotechnology, featuring a selection of excellent articles that highlight the potential of nucleic acids for various applications.

Reprogramming M1-to-M2 Phenotype to Alleviate Inflammation: Using Liposomal Curcumin as a Tool to Redefine Macrophage Functionality.

The versatile nature of macrophages and their ability to switch between various activation states plays a pivotal role in both promoting and inhibiting inflammatory processes. In pathological inflammatory conditions, classically activated M1 macrophages are often associated with initiating and maintaining inflammation, while alternatively activated M2 macrophages are linked to the resolution of chronic inflammation. Achieving a favorable equilibrium between M1 and M2 macrophages is crucial for mitigating inflammatory environments in pathological conditions. Polyphenols are known to have strong inherent antioxidative capabilities, and curcumin has been found to reduce macrophage inflammatory reactions. However, its therapeutic efficacy is compromised due to its poor bioavailability. The present study aims to harness the properties of curcumin by loading it in nanoliposomes and enhancing the M1-to-M2 macrophage polarization. A stable liposome formulation was achieved at 122.1 ± 0.08 nm, and a sustained kinetic release of curcumin was observed within 24 h. The nanoliposomes were further characterized using TEM, FTIR, and XRD, and the morphological changes in macrophage cells, RAW264.7, were observed in SEM, indicating a distinct M2-type phenotype after the treatment with liposomal curcumin. ROS may partially control macrophage polarization and be observed to decrease after treatment with liposomal curcumin. The nanoliposomes were able to successfully internalize in the macrophage cells, and an enhanced expression of ARG-1 and CD206 with a decrease in iNOS, CD80, and CD86 levels suggested the polarization of LPS-activated macrophages toward the M2 phenotype. Also, liposomal curcumin treatment dose-dependently inhibited TNF-α, IL-2, IFN-γ, and IL-17A at secretory levels and simultaneously increased the levels of cytokines like IL-4, IL-6, and IL-10.

Spatiotemporal dynamics of DNA nanocage uptake in zebrafish embryos for targeted tissue bioimaging applications.

Three-dimensional DNA nanocages have attracted significant attention for various biomedical applications including targeted bioimaging in vivo. Despite the numerous advantages, the use and in vivo exploration of DNA nanocages are limited as the cellular targeting and intracellular fate of these DNA nanocages within various model systems have not been explored well. Herein, using a zebrafish model system, we provide a detailed understanding of time-, tissue- and geometry-dependent DNA nanocage uptake in developing embryos and larvae. Of all the geometries tested, tetrahedrons showed significant internalization in 72 hours post-fertilized larvae upon exposure, without disturbing the expression of genes involved in embryo development. Our study provides a detailed understanding of the time and tissue-specific uptake of DNA nanocages in the zebrafish embryos and larvae. These findings will provide valuable insights into the internalization and biocompatible potential of DNA nanocages and will help to predict their candidature for biomedical applications.

An Isothiazolanthrone-Based Self-Assembling Anticancer Color-Changing Dye for Concurrent Imaging and Monitoring of Cell Viability.

We report the photophysical properties, self-assembly and biological evaluation of an isothiazolanthrone-based dye, 7-amino-6H-anthra[9,1-cd]isothiazol-6-one (AAT), which reveals anticancer properties and can be potentially used as dye for monitoring cell viability. The solvent-dependent photophysical studies suggest that the emission of AAT is sensitive to environment polarity due to which interesting changes in the colored emission may be observed owing to the charge transfer (CT) processes. AAT also self-assembles to tree-like branched morphologies and produce, a greenish emission inside the cells when imaged after short interval (15 mins) of incubation while a red fluorescence could be noted after 24 h. Interestingly, AAT also produce differential emission inside mouse normal cells as compared to its cancer cell lines since it possess anticancer activity. The experimental observations were also validated theoretically via computational modeling.

Tissue-Derived Primary Cell Type Dictates the Endocytic Uptake Route of Carbon Quantum Dots and In Vivo Uptake.

Carbon quantum dots (CQDs) require systemic biological delivery to advance their applications in drug delivery, biosensing, and bioimaging. We describe the endocytic pathways of green-emitting fluorescent carbon quantum dots (GCQDs) with sizes varying from 3 to 5 nm in mouse tissue-derived primary cells, tissues, and zebrafish embryos. The GCQDs demonstrated cellular internalization into mouse kidney and liver primary cells via a clathrin-mediated pathway. Using imaging, we were able to identify and reinforce the animal's body features in terms of different tissues exhibiting differential affinity for these CQDs, which will be extremely beneficial in the development of next-generation bioimaging and therapeutic scaffolds based on carbon-based quantum dots.

pH-Responsive and Reversible A-Motif-Based DNA Hydrogel: Synthesis and Biosensing Application.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Mechanistic insight into the structure, thermodynamics and dynamics of equilibrium gels of multi-armed DNA nanostars.

The unique sequence specificity rule of DNA makes it an ideal molecular building block for constructing periodic arrays and devices with nanoscale accuracy and precision. Here, we present the self-assembly of DNA nanostars having three, four and five arms into a gel phase using a simplistic coarse-grained bead-spring model developed by Z. Xing, C. Ness, D. Frenkel and E. Eiser (Macromolecules, 2019, 52, 504-512). Our simulations show that the DNA nanostars form a thermodynamically stable fully bonded gel phase from an unstructured liquid phase with the lowering of temperature. We characterize the phase transition by calculating several structural features such as the radial distribution function and structure factor. The thermodynamics of gelation is quantified by the potential energy and translational pair-entropy of the system. The phase transition from an arrested gel phase to an unstructured liquid phase has been modelled using a two-state theoretical model. We find that this transition is enthalpy driven, and loss of configuration and translational entropy is counterpoised by enthalpic interaction of the DNA sticky-ends, which gives rise to a gel phase at low temperature. The absolute rotational and translational entropy of the systems, measured using a two-phase thermodynamic model, also substantiates the gel transition. The slowing down of the dynamics upon approaching the transition temperature from a high temperature demonstrates the phase transition to a gel phase. A detailed numerical simulation study of the morphology, dynamics and thermodynamics of DNA gelation can provide guidance for future experiments, is easily extensible to other polymeric systems, and is expected to help in understanding the physics of self-assembly.

Graphene Oxide-Carbamoylated Chitosan Hydrogels with Tunable Mechanical Properties for Biological Applications.

Chitosan (CH)-based hydrogels have been extensively researched in numerous biological applications, including drug delivery, biosensing, wound healing, and tissue engineering, to name a few. Previously, modified CH hydrogels by carbamoylation, using potassium cyanate (KCNO) as the cross-linker, have shown improvement in viscoelastic properties and biocompatibility. In this study, graphene oxide (GO) nanofillers are added to carbamoylated CH to form a nanocomposite hydrogel and study the influence of CH molecular weight (Mw) and GO loading concentrations on hydrogel properties. The physical properties (swelling, degradation, and porous structure) of the hydrogels can be tuned as required for cell attachment and spreading by varying both the GO concentration and the Mw of CH. Rheological characterization showed an improvement in the mechanical properties (storage modulus, yield stress, and viscosity) of the synthesized CH-GO hydrogels with an increase in the Mw of CH and the GO concentration. Human retinal pigmented epithelial-1 (RPE-1) cells seeded onto the prepared hydrogel scaffolds showed good cell viability, adhesion, and cell spreading, confirming their cytocompatibility, with dependence on both Mw of CH and GO loading.

Self-Assembled DNA Nanocages Promote Cell Migration and Differentiation of Human Umbilical Vein Endothelial Cells.

DNA nanocages have been explored for abilities to influence cellular behavior and functions. Recent times have seen the development of new emergent functionalities of DNA nanodevices as a class of biomaterials with an immense capacity to interface with biological systems and with vast potential in disease diagnosis and therapeutics. Being chemically robust and biocompatible in nature, DNA nanocages have been surface modified and structurally fine-tuned to find emerging applications in the field of stem-cell therapy and tissue regeneration. DNA nanocages can be used for therapeutic angiogenesis that involves the induction of blood vessel formation and can be used to treat ischemic diseases like stroke or heart failure. This work addresses the effect of DNA nanocages' structural topology on their capacity to stimulate endothelial cell angiogenesis. We tested a panel of four DNA nanocage geometries and checked their potential on the differentiation of human umbilical vein endothelial cells (HUVECs). While different DNA nanocage geometries showed successful induction of angiogenesis and cell migration in HUVECs, tetrahedral DNA cages showed the maximum uptake and angiogenesis potential, thus indicating that not only the composition of materials, but also the 3D arrangement of ligands might play role in stimulating angiogenesis.

Cationic lipid modification of DNA tetrahedral nanocages enhances their cellular uptake.

Self-assembled DNA nanocages are among the most promising candidates for bioimaging and payload delivery into cells. DNA nanocages have great potential to efficiently address drug resistance and nucleic acid delivery problems due to precise control of their shape and size, and excellent biocompatibility. Although DNA nanostructures demonstrate some cellular uptake, because they bear a highly negative charge, the uptake of tetrahedral nanostructures is hindered by electrostatic repulsion. In this study, we describe a method to enhance the cellular uptake of DNA nanostructures using a binary system containing DNA and a positively charged head group with a hydrophobic lipid chain containing lipids for cellular internalization. Here we represent the functionalization of a model cage, DNA tetrahedron (TD) with a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Atomic force microscopy (AFM) and other standard characterization techniques were used to explore the co-assembly of the DNA tetrahedron and DOTMA. We revealed a simple confocal microscopy-based approach to show the enhancement in the cellular uptake of DNA nanocages. This new method will find multiple applications in delivery applications such as gene transfection, drug delivery and targeted bioimaging.