Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

Therapeutic Polymer-Based Cannabidiol Formulation: Tackling Neuroinflammation Associated with Ischemic Events in the Brain.

Cannabidiol (CBD) is the most relevant nonpsychostimulant phytocompound found in Cannabis sativa. CBD has been extensively studied and has been proposed as a therapeutic candidate for neuroinflammation-related conditions. However, being a highly lipophilic drug, it has several drawbacks for pharmaceutical use, including low solubility and high permeability. Synthetic polymers can be used as drug delivery systems to improve CBD's stability, half-life, and biodistribution. Here, we propose using a synthetic polymer as a nanoparticulate vehicle for CBD (NPCBD) to overcome the pharmacological drawbacks of free drugs. We tested the NPCBD-engineered system in the context of ischemic events in a relevant oxygen and glucose deprivation (OGD) model in primary cortical cells (PCC). Moreover, we have characterized the inflammatory response of relevant cell types, such as THP-1 (human monocytes), HMC3 (human microglia), and PCC, to NPCBD and observed a shift in the inflammatory state of the treated cells after the ischemic event. In addition, NPCBD exhibited a promising ability to restore mitochondrial function after OGD insult in both HMC3 and PCC cells at low doses of 1 and 0.2 µM CBD. Taken together, these results suggest the potential for preclinical use.

Protein nitration induced by Hemin/NO: A complementary mechanism through the catalytic functions of hemin and NO-scavenging.

Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (●NO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the ●NO-binding affinity of hemin has been studied along with the influence of ●NO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and ●NO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its ●NO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of ●NO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and ●NO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.

Interactions between Nitric Oxide and Hyaluronan Implicate the Migration of Breast Cancer Cells.

Nitric oxide (•NO) is one of the prominent free radicals, playing a pivotal role in breast cancer progression. Hyaluronic acid (HA) plays an essential role in neutralizing free radicals in tumor tissues. However, its interactions with nitric oxide have not been thoroughly investigated. Hence, this study attempts to understand the mechanism of these interactions and the different effects on the intracellular •NO levels and migration of breast cancer cells. The affinity of HA to scavenge •NO was investigated alongside the accompanying changes in specific physico-chemical properties and the further effects on the •NO-induced attachment and migration of the breast cancer cell lines, MDA-MB-231 and HCC1806. The reaction of the nitrogen dioxide radical, formed via •NO/O2 interactions, with HA initiated a series of oxidative reactions, which, in the presence of •NO, induce the fragmentation of the polymeric chains. Furthermore, these interactions were found to hinder the NO-induced migration of cancer cells. However, the NO-induced HA modification/fragmentation was inhibited in the presence of hemin, a NO-scavenging compound. Collectively, these results help toward understanding the involvement of HA in the •NO-induced cell migration and suggest the possible modification of HA, used as one of the main materials in different biomedical applications.

The role of extracellular matrix in tumour angiogenesis: the throne has NOx servants.

The extracellular matrix (ECM) dynamics in tumour tissue are deregulated compared to the ECM in healthy tissue along with disorganized architecture and irregular behaviour of the residing cells. Nitric oxide (NO) as a pleiotropic molecule exerts different effects on the components of the ECM driving or inhibiting augmented angiogenesis and tumour progression and tumour cell proliferation and metastasis. These effects rely on the concentration of NO within the tumour tissue, the nature of the surrounding microenvironment and the sensitivity of resident cells to NO. In this review article, we summarize the recent findings on the correlation between the levels of NO and the ECM components towards the modulation of tumour angiogenesis in different types of cancers. These are discussed principally in the context of how NO modulates the expression of ECM proteins resulting in either the promotion or inhibition of tumour growth via tumour angiogenesis. Furthermore, the regulatory effects of individual ECM components on the expression of the NO synthase enzymes and NO production were reviewed. These findings support the current efforts for developing effective therapeutics for cancers.

Hyaluronan-Based Hydrogels for 3D Modeling of Tumor Tissues.

Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.

Engineering hemin-loaded hyaluronan needle-like microparticles with photoprotective properties against UV-induced tissue damage.

This study aimed to develop hyaluronan (HA)-based hydrogel microparticles (MPs) loaded with hemin to address the limitations of traditional macroscale hydrogels. The objective is to design MPs such that they can modulate their physicochemical properties. Given the widespread use of ultraviolet C (UVC) light in various industries and the need for protective measures against accidental exposure, this study evaluated the potential of hemin-loaded MPs to protect human dermal fibroblasts from oxidative stress and cell death caused by UVC exposure. Multiple MP formulations were developed and analysed for size, surface charge, swelling behaviour, degradation rate, and radical scavenging capabilities, both with and without hemin loading. The most promising formulations were tested against UVC-exposed cells to assess cell viability, intracellular nitric oxide (˙NO) and reactive oxygen species levels, and protein carbonylation. The fabricated particles were in the form of microneedles, and the degree of their crosslinking and the role of hemin in the chemical crosslinking reaction were found to influence the surface charge and hydrodynamic diameter of the MPs. Increased crosslinking resulted in reduced swelling, slower degradation, and decreased hemin release rate. MPs with a higher degree of swelling were capable of releasing hemin into the culture medium, leading to enhanced bilirubin generation in dermal fibroblasts following cellular uptake. Pre-treatment with these MPs protected the cells from UVC-induced cell death, nitrosative stress, and protein carbonylation. These findings highlight the potential of the studied MPs to release hemin and to minimise the harmful effects of UVC on dermal fibroblasts.

Iron protoporphyrin IX-hyaluronan hydrogel-supported luminol chemiluminescence for the detection of nitric oxide in physiological solutions.

Due to its role as a free radical signal-transducing agent with a short lifespan, precise measurement of nitric oxide (●NO) levels presents significant challenges. Various analytical techniques offer distinct advantages and disadvantages for ●NO detection. This research aims to simplify the detection process by developing a hydrogel system using iron(III)-protoporphyrin IX (hemin)-loaded hyaluronan for the detection of ●NO in solution. Various hydrogel formulations were created, and the effects of their components on hydrogel-supported luminol chemiluminescence (CL) kinetics, radical scavenging, and physicochemical properties were analysed through factorial analysis. The candidate formulations were then evaluated using two ●NO donors. An increase in the degree of crosslinking in unloaded formulations enhanced interactions with the CL reaction components, hydrogen peroxide (H2O2) and luminol, thereby affecting light generation. However, hemin loading negated these effects, resulting in more prominent luminescence kinetics in formulations with lower crosslinking degrees. Similarly, ●NO influenced the kinetics differently, interacting with both the CL reaction and hydrogel components. Hemin-loaded formulations exhibited enhanced signal propagation when exposed to ●NO, followed by H2O2 and luminol, whereas reversing the order of addition inhibited this propagation. The magnitude of these luminescence changes depended on the type and concentration of the ●NO donor, demonstrating greater sensitivity to ●NO levels compared to amperometric sensing. These findings suggest that the studied hydrogel platform has potential for the facile and accurate detection of ●NO in solution, requiring minimal sample sizes.

Examining Hemin and its Derivatives: Induction of Heme-Oxygenase-1 Activity and Oxidative Stress in Breast Cancer Cells through Collaborative Experimental Analysis and Molecular Dynamics Simulations.

Hemin triggers intracellular reactive oxygen species (ROS) accumulation and enhances heme oxygenase-1 (HOX-1) activity, indicating its potential as an anticancer agent, though precise control of its intracellular levels is crucial. The study explores the impact of hemin and its derivatives, hemin-tyrosine, and hemin-styrene (H-Styr) conjugates on migration, HOX-1 expression, specific apoptosis markers, mitochondrial functions, and ROS generation in breast cancer cells. Molecular docking and dynamics simulations were used to understand the interactions among HOX-1, heme, and the compounds. Hemin outperforms its derivatives in inducing HOX-1 expression, exhibiting pro-oxidative effects and reducing cell migration. Molecular simulations show that heme binds favorably to HOX-1, followed by the other compounds, primarily through van der Waals and electrostatic forces. However, only van der Waals forces determine the H-Styr complexation. These interactions, influenced by metalloporphyrin characteristics, provide insights into HOX-1 regulation and ROS generation, potentially guiding the development of breast cancer therapies targeting oxidative stress.

Cold plasma treatment of tannic acid as a green technology for the fabrication of advanced cross-linkers for bioactive collagen/gelatin hydrogels.

Tannic acid (TA) is a natural compound studied as the cross-linker for biopolymers due to its ability to form hydrogen bonds. There are different methods to improve its reactivity and effectiveness to be used as a modifier for biopolymeric materials. This work employed plasma to modify tannic acid TA, which was then used as a cross-linker for fabricating collagen/gelatin scaffolds. Plasma treatment did not cause any significant changes in the structure of TA, and the resulting oxidized TA showed a higher antioxidant activity than that without treatment. Adding TA to collagen/gelatin scaffolds improved their mechanical properties and stability. Moreover, the obtained plasma-treated TA-containing scaffolds showed antibacterial properties and were non-hemolytic, with improved cytocompatibility towards human dermal fibroblasts. These results suggest the suitability of plasma treatment as a green technology for the modification of TA towards the development of advanced TA-crosslinked hydrogels for various biomedical applications.

Nitric Oxide-Scavenging, Anti-Migration Effects, and Glycosylation Changes after Hemin Treatment of Human Triple-Negative Breast Cancer Cells: A Mechanistic Study.

The enhanced expression of nitric oxide (•NO) synthase predicts triple-negative breast cancer outcome and its resistance to different therapeutics. Our earlier work demonstrated the efficiency of hemin to scavenge the intra- and extracellular •NO, proposing its potency as a therapeutic agent for inhibiting cancer cell migration. In continuation, the present work evaluates the effects of •NO on the migration of MDA-MB-231 cells and how hemin modulates the accompanied cellular behavior, focusing on the corresponding expression of cellular glycoproteins, migration-associated markers, and mitochondrial functions. We demonstrated for the first time that while •NO induced cell migration, hemin contradicted that by •NO-scavenging. This was in combination with modulation of the •NO-enhanced glycosylation patterns of cellular proteins with inhibition of the expression of specific proteins involved in the epithelial-mesenchymal transition. These effects were in conjunction with changes in the mitochondrial functions related to both •NO, hemin, and its nitrosylated product. Together, these results suggest that hemin can be employed as a potential anti-migrating agent targeting •NO-scavenging and regulating the expression of migration-associated proteins.

A Multimodal Scaffold for SDF1 Delivery Improves Cardiac Function in a Rat Subacute Myocardial Infarct Model.

Ischemic heart disease is one of the leading causes of death worldwide. The efficient delivery of therapeutic growth factors could counteract the adverse prognosis of post-myocardial infarction (post-MI). In this study, a collagen hydrogel that is able to load and appropriately deliver pro-angiogenic stromal cell-derived factor 1 (SDF1) was physically coupled with a compact collagen membrane in order to provide the suture strength required for surgical implantation. This bilayer collagen-on-collagen scaffold (bCS) showed the suitable physicochemical properties that are needed for efficient implantation, and the scaffold was able to deliver therapeutic growth factors after MI. In vitro collagen matrix biodegradation led to a sustained SDF1 release and a lack of cytotoxicity in the relevant cell cultures. In vivo intervention in a rat subacute MI model resulted in the full integration of the scaffold into the heart after implantation and biocompatibility with the tissue, with a prevalence of anti-inflammatory and pro-angiogenic macrophages, as well as evidence of revascularization and improved cardiac function after 60 days. Moreover, the beneficial effect of the released SDF1 on heart remodeling was confirmed by a significant reduction in cardiac tissue stiffness. Our findings demonstrate that this multimodal scaffold is a desirable matrix that can be used as a drug delivery system and a scaffolding material to promote functional recovery after MI.

NUIG4: A biocompatible pcu metal-organic framework with an exceptional doxorubicin encapsulation capacity.

Metal-organic frameworks (MOFs) are promising multifunctional porous materials for biomedical and environmental applications. Here, we report synthesis and characterization of a new MOF based on the tetrahedral secondary building unit [Zn4O(CBAB)3]n (NUIG4), where CBABH2 = 4-((4-carboxybenzylidene)amino)benzoic acid. NUIG4 belongs to the family of MOFs with primitive cubic pcu topology, being a rare example with 4-fold interpenetration. The pore architecture enables unprecedentedly high doxorubicin (DOX) loading capacity (1955 mg DOX/g NUIG4) with pH-controlled release. Solid-state NMR and ab initio modeling confirmed formation of aromatic π-π stacking interactions between DOX and the framework. Preliminary cell-line experiments suggested a protective effect of NUIG4 on healthy HDF cells against DOX toxicity. NUIG4 also displays potential for adsorptive small-molecule gas separation, with a BET surface area of 1358 m2 g-1 and high selectivity of 2.75 for C2H2 over CO2.

Protocol for in vitro skin fibrosis model to screen the biological effects of antifibrotic compounds.

Controversies remain over the standard procedures for the modeling of skin fibrosis and its use in in vitro testing of different drugs. Here, we report a reproducible protocol for producing a skin fibrosis model using human dermal fibroblasts seeded in collagen hydrogel. Detailed procedures for the fabrication of cell/hydrogel constructs, fibrosis induction, protein extraction for western blotting analysis are presented along with how this model can be employed for investigating the possible anti-fibrotic functions of certain chemical compounds.

Recent Advances in the Design and Sensing Applications of Hemin/Coordination Polymer-Based Nanocomposites.

The fabrication of biomimetic catalysts as substituents for enzymes is of critical interest in the field due to the problems associated with the extraction, purification, and storage of enzymes in sensing applications. Of these mimetics, hemin/coordination polymer-based nanocomposites, mainly hemin/metal-organic frameworks (MOF), have been developed for various biosensing applications because of the unique properties of each component, while trying to mimic the normal biological functions of heme within the protein milieu of enzymes. This critical review first discusses the different catalytic functions of heme in the body in the form of enzyme/protein structures. The properties of hemin dimerization are then elucidated with the supposed models of hemin oxidation. After that, the progress in the fabrication of hemin/MOF nanocomposites for the sensing of diverse biological molecules is discussed. Finally, the challenges in developing this type of composites are examined as well as possible proposals for future directions to enhance the sensing performance in this field further.

Plasma & Microwaves as Greener Options for Nanodiamond Purification: Insight Into Cytocompatibility.

The potential biomedical applications of nanodiamond have been considered over the last few decades. However, there is still uncertainty regarding the extent to which the surface characteristics of this material can influence potential applications. The present study investigated the effects of surface characteristics alongside the prospective of improving nanodiamond production using cold plasma and microwave technologies for the surface tailoring of the nanocarbons. Numerous approaches were applied to purify, refine and modify a group of nanosized diamonds at each step of their production cycle: from the detonation soot as the initial raw material to already certified samples. The degree of surface changes were deliberately performed slowly and kept at different non-diamond carbon presence stages, non-carbon elemental content, and amount converted superficial moieties. In total, 21 treatment procedures and 35 types of nanosize diamond products were investigated. In addition cultures of human fibroblast cells showed enhanced viability in the presence of many of the processed nanodiamonds, indicating the potential for dermal applications of these remarkable nanomaterials.

Physical preparation of alginate/chitosan polyelectrolyte complexes for biomedical applications.

Polyelectrolyte complexes represent a special class of polymeric compounds consisting of stoichiometric equivalents of oppositely charged polyions interacting together spontaneously to yield a complex in different forms. The present study aimed at preparing coacervates of alginate and chitosan polymers ready for casting as wound dressing films. This was based on controlling the pH of solutions and the reactions speed through controlling the rate of mixing of the polymers solutions together without using any water-miscible solvents. Alginate was modified with radiation and oxidation, and the interactions of the resulting chains and chitosan chains were tested with FTIR spectroscopy and scanning of the resulting films with SEM. This work showed the ability to prepare a complex of highly connected polymeric chains for further biomedical applications. This complex in the form of hydrogel could enhance the proliferation of cells in vitro and the healing efficiency with accelerating the wound closure rate as evidenced through the histological observations.