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Acetylcholinesterase (AChE) plays a pivotal role in the cholinergic system, and its inhibition is sought after in a wide range of applications, from insect control to Alzheimer's disease treatment. While the primary physiological isoforms of AChE are membrane-bound proteins, most assays for discovering new, safer, and potent inhibitors are conducted using commercially available soluble isoforms, such as the electric eel AChE (eeAChE). In this study, we conducted a comparative analysis of the activity and selectivity to phenolic inhibitors of recombinant human AChE, eeAChE and a mutant variant of human AChE known as dAChE4. Despite numerous mutations, dAChE4 closely resembles its parental protein and serves as a suitable model for monomeric human AChE. We also established an in vitro system of membrane-bound AChE to create a model that closely mimics the physiological isoforms. This system ensures the proper work of the enzyme and allowed us to control the exact concentration of enzyme and lipids per assay.

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Psoriasis is an immune-mediated chronic inflammatory disease that causes major psychosocial impact. Topical corticosteroids represent the standard pharmacological treatment for mild-to-moderate disease, but their local and systemic adverse effects reinforce the need for treatment innovations. Here we developed lamellar phase-based formulations for topical delivery of a hybrid dexamethasone and hydrogen sulfide (H2S) donor molecule (Dexa-TBZ), aiming to potentiate the effects of the glucocorticoid with H2S. They offer the possibility to obtain precursor formulations free of water that originate lamellar phases upon water addition, preventing drug hydrolysis during storage. Two groups of formulations were developed varying the surfactants and oil phase types and content. Systems containing 20 and 70 % of water formed, respectively, bulk lamellar phase and a more fluid formulation consisting of dispersed droplets (< 1000 nm) stabilized by lamellar phase. Both presented pseudoplastic behavior. Dexa-TBZ was incorporated at 1 %, remaining stable for 8 h. Drug content decreased to ∼80 % after 1 week in precursor formulations free of water, but remained stable after that. Without causing changes to the cutaneous barrier function ex vivo or to the histological structure of the skin in vivo, the formulation containing phosphatidylcholine as surfactant and 70 % of water promoted 1.8- and 2.7-fold increases in Dexa-TBZ penetration in the stratum corneum and epidermis+dermis, respectively, compared to a control solution, demonstrating their potential applicability as topical delivery systems.

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Graphene nanoplatelets (UGZ-1004) are emerging as a promising biomaterial in regenerative medicine. This study comprehensively evaluates UGZ-1004, focusing on its physical properties, cytotoxicity, intracellular interactions, and, notably, its effects on mesenchymal stem cells (MSCs). UGZ-1004 was characterized by lateral dimensions and layer counts consistent with ISO standards and demonstrated a high carbon purity of 0.08%. Cytotoxicity assessments revealed that UGZ-1004 is non-toxic to various cell lines, including 3T3 fibroblasts, VERO kidney epithelial cells, BV-2 microglia, and MSCs, in accordance with ISO 10993-5:2020/2023 guidelines. The study focused on MSCs and revealed that UGZ-1004 supports their gene expression alterations related to self-renewal and proliferation. MSCs exposed to UGZ-1004 maintained their characteristic surface markers. Importantly, UGZ-1004 promoted significant upregulation of genes crucial for cell cycle regulation and DNA repair, such as CDK1, CDK2, and MDM2. This gene expression profile suggests that UGZ-1004 can enhance MSC self-renewal capabilities, ensuring robust cellular function and longevity. Moreover, UGZ-1004 exposure led to the downregulation of genes associated with tumor development, including CCND1 and TFDP1, mitigating potential tumorigenic risks. These findings underscore the potential of UGZ-1004 to not only bolster MSC proliferation but also enhance their self-renewal processes, which are critical for effective regenerative therapies. The study highlights the need for continued research into the long-term impacts of graphene nanoplatelets and their application in MSC-based regenerative medicine.

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In the pharmaceutical sector, solid lipid nanoparticles (SLN) are vital for drug delivery incorporating a lipid core. Chondroitin sulfate (CHON) is crucial for cartilage health. It is often used in osteoarthritis (OA) treatment. Due to conflicting results from clinical trials on CHON's efficacy in OA treatment, there has been a shift toward exploring effective topical systems utilizing nanotechnology. This study aimed to optimize a solid lipid nanoparticle formulation aiming to enhance CHON permeation for OA therapy. A 3 × 3 × 2 Design of these experiments determined the ideal parameters: a CHON concentration of 0.4 mg/mL, operating at 20,000 rpm speed, and processing for 10 min for SLN production. Transmission electron microscopy analysis confirmed the nanoparticles' spherical morphology, ensuring crucial uniformity for efficient drug delivery. Cell viability assessments showed no significant cytotoxicity within the tested parameters, indicating a safe profile for potential clinical application. The cell internalization assay indicates successful internalization at 1.5 h and 24 h post-treatment. Biopharmaceutical studies supported SLNs, indicating them to be effective CHON carriers through the skin, showcasing improved skin permeation and CHON retention compared to conventional methods. In summary, this study successfully optimized SLN formulation for efficient CHON transport through pig ear skin with no cellular toxicity, highlighting SLNs' potential as promising carriers to enhance CHON delivery in OA treatment and advance nanotechnology-based therapeutic strategies in pharmaceutical formulations.

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Despite the numerous studies on biocompatibility with nano-biomaterials, the biological effects of strontium-substituted HA nanoparticles (nSrHA) need to be better understood. So, we conducted an embryotoxicity test using zebrafish (Danio rerio) according to the OECD 236 guideline, a model that represents a viable alternative that bridges the gap between in vitro and mammalian models. Zebrafish embryos were exposed for 120 h to microspheres containing nSrHA nanoparticles with low and high crystallinity, synthesized at temperatures of 5°C (nSrHA5) and 90°C (nSrHA90). We evaluated lethality, developmental parameters, and reactive oxygen species (ROS) production. The larval behavior was assessed at 168 hpf to determine if the biomaterials affected motor responses and anxiety-like behavior. The results showed that the survival rate decreased significantly for the nSrHA5 group (low crystalline particles), and an increase in ROS was also observed in this group. However, none of the biomaterials caused morphological changes indicative of toxicity during larval development. Additionally, the behavioral tests did not reveal any alterations in all experimental groups, indicating the absence of neurotoxic effects from exposure to the tested biomaterials. These findings provide valuable insights into the biosafety of modified HA-based nanostructured biomaterials, making them a promising strategy for bone tissue repair. As the use of hydroxyapatite-based biomaterials continues to grow, it is crucial to ensure rigorous control over the quality, reliability, and traceability of these materials.

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The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) play important roles not only in maintaining physiological functions, but also in pathological conditions and events. Importantly, these molecules show a complex interplay in cancer biology, demonstrating both tumor-promoting and anti-tumor activities depending on their concentration, flux, and the environmental redox state. Additionally, various cell types respond differently to NO and H2S. These gasotransmitters can be synergistically combined with traditional anticancer treatments such as radiotherapy, immunotherapy, chemotherapy, and phototherapy. Notably, NO, and more recently H2S, have been shown to reverse multidrug resistance. Nanomaterials to deliver NO donors and, to a lesser extent, H2S donors, have emerged as a promising approach for targeted delivery of these gasotransmitters. Nanotechnology has advanced the delivery of anticancer drugs, enhancing efficiency and reducing side effects on non-cancerous cells. This review highlights recent progress in the design of NO and H2S-releasing nanomaterials for anticancer effects. It also explores the interactions between NO and H2S, which are crucial for developing combined therapies and nanomedicines with minimal side effects.

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Photoelectrochemical (PEC) nanobiosensors integrate molecular (bio)recognition elements with semiconductor/plasmonic photoactive nanomaterials to produce measurable signals after light-induced reactions. Recent advancements in PEC nanobiosensors, using light-matter interactions, have significantly improved sensitivity, specificity, and signal-to-noise ratio in detecting (bio)analytes. Tunable nanomaterials activated by a wide spectral radiation window coupled to electrochemical transduction platforms have further improved detection by stabilizing and amplifying electrical signals. This work reviews PEC biosensors based on nanomaterials like metal oxides, carbon nitrides, quantum dots, and transition metal chalcogenides (TMCs), showing their superior optoelectronic properties and analytical performance for the detection of clinically relevant biomarkers. Furthermore, it highlights the innovative role of red light and NIR-activated PEC nanobiosensors in enhancing charge transfer processes, protecting them from biomolecule photodamage in vitro and in vivo applications. Overall, advances in PEC detection systems have the potential to revolutionize rapid and accurate measurements in clinical diagnostic applications. Their integration into miniaturized devices also supports the development of portable, easy-to-use diagnostic tools, facilitating point-of-care (POC) testing solutions and real-time monitoring.

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The post COVID-19 pandemic era has emerged with more efficient vaccines, all based on genetic materials. However, to expand the use of nucleic components as vaccines, a new generation of nanosystems particularly constructed to increase RNA/DNA stability, half-life and facilitate administration are still required. This review highlights novel developments in mRNA and pDNA vaccines formulated into nanostructures exclusively composed by biopolymeric materials. Recent advances suggest that a new generation of vaccines may arise by adapting the structural features of biopolymers with the effectiveness of nucleic acids. The advantages offered by biopolymers, such as increased stability and targeting ability may cause a revolution in the immunization field for offering promptly adaptable and effective formulations for worldwide distribution.

[Box: see text].

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BACKGROUND: Implementing encapsulation techniques is pivotal in safeguarding bioactive molecules against environmental conditions for drug delivery systems. Moreover, the food-grade nanocarrier is a delivery system and food ingredient crucial in creating nutraceutical foods. Nano α-lactalbumin has been shown to be a promissory nanocarrier for hydrophobic molecules. Furthermore, the nanoprotein can enhance the tecno-functional properties of food such as foam and emulsion. The present study investigated the nanostructured α-lactalbumin protein (nano α-la) as a delivery and controlled release system for bioactive molecules in a gastric-intestinal in vitro mimic system. RESULTS: The nano α-la was synthesized by a low self-assembly technique, changing the solution ionic strength by NaCl and obtaining nano α-la 191.10 ± 21.33 nm and a spherical shape. The nano α-la showed higher encapsulation efficiency and loading capacity for quercetin than riboflavin, a potential carrier for hydrophobic compounds. Thermal analysis of nano α-la resulted in a ΔH of -1480 J g-1 for denaturation at 57.44 °C. The nanostructure formed by self-assembly modifies the foam volume increment and stability. Also, differences between nano and native proteins in emulsion activity and stability were noticed. The release profile in vitro showed that the nano α-la could not hold the molecules in gastric fluid. The Weibull and Korsmeyer-Peppas model better fits the release profile behavior in the studied fluids. CONCLUSION: The present study shows the possibility of nano α-la as an alternative to molecule delivery systems and nutraceutical foods' formulation because of the high capacity to encapsulate hydrophobic molecules and the improvement of techno-functional properties. However, the nanocarrier is not perfectly suitable for the sustainable delivery of molecules in the gastrointestinal fluid, demanding improvements in the nanocarrier. © 2024 Society of Chemical Industry.

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The presence of phenazopyridine in water is an environmental problem that can cause damage to human health and the environment. However, few studies have reported the adsorption of this emerging contaminant from aqueous matrices. Furthermore, existing research explored only conventional modeling to describe the adsorption phenomenon without understanding the behavior at the molecular level. Herein, the statistical physical modeling of phenazopyridine adsorption into graphene oxide is reported. Steric, energetic, and thermodynamic interpretations were used to describe the phenomenon that controls drug adsorption. The equilibrium data were fitted by mono, double, and multi-layer models, considering factors such as the numbers of phenazopyridine molecules by adsorption sites, density of receptor sites, and half saturation concentration. Furthermore, the statistical physical approach also calculated the thermodynamic parameters (free enthalpy, internal energy, Gibbs free energy, and entropy). The maximum adsorption capacity at the equilibrium was reached at 298 K (510.94 mg g-1). The results showed the physical meaning of adsorption, indicating that the adsorption occurs in multiple layers. The temperature affected the density of receptor sites and half saturation concentration. At the same time, the adsorbed species assumes different positions on the adsorbent surface as a function of the increase in the temperature. Meanwhile, the thermodynamic functions revealed increased entropy with the temperature and the equilibrium concentration.

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This work presents a study on the effects of periodic boundary conditions (PBC) on the energetic/structural properties and hydrogen bond dynamics (HB) using molecular dynamics (MD) simulations of peptide membranes composed of alanine and histidine. Our results highlight that simulations using small surface areas for the peptide membrane may result in nonconvergent values for membrane properties, which are only observed in regions simulated at a certain distance from the PBCs. Specifically, regarding hydrogen bonds, a property pervasive in peptide membranes, our findings indicate a significant increase in the lifetime of these interactions, reaching values ∼19% higher when observed in structures free from PBCs. For peptide mobility in these nanomembranes, our results compare regions simulated directly under the influence of PBCs with regions free from these conditions, emphasizing greater mobility of amino acid psi/phi angles in the latter model.

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Foodborne pathogens are a grave concern for the for food, medical, environmental, and economic sectors. Their ease of transmission and resistance to treatments, such as antimicrobial agents, make them an important challenge. Food tainted with these pathogens is swiftly rejected, and if ingested, can result in severe illnesses and even fatalities. This review provides and overview of the current status of various pathogens and their metabolites transmitted through food. Despite a plethora of studies on treatments to eradicate and inhibit these pathogens, their indiscriminate use can compromise the sensory properties of food and lead to contamination. Therefore, the study of detection methods such as electrochemical biosensors has been proposed, which are devices with advantages such as simplicity, fast response, and sensitivity. However, these biosensors may also present some limitations. In this regard, it has been reported that nanomaterials with high conductivity, surface-to-volume ratio, and robustness have been observed to improve the detection of foodborne pathogens or their metabolites. Therefore, in this work, we analyze the detection of pathogens transmitted through food and their metabolites using electrochemical biosensors based on nanomaterials.

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Bone tissue engineering is a promising alternative to repair wounds caused by cellular or physical accidents that humans face daily. In this sense, the search for new graphene oxide (GO) nanofillers related to their degree of oxidation is born as an alternative bioactive component in forming new scaffolds. In the present study, three different GOs were synthesized with varying degrees of oxidation and studied chemically and tissue-wise. The oxidation degree was determined through infrared (FTIR), X-ray diffraction (XRD), X-ray photoelectron (XPS), and Raman spectroscopy (RS). The morphology of the samples was analyzed using scanning electron microscopy (SEM). The oxygen content was deeply described using the deconvolution of RS and XPS techniques. The latter represents the oxidation degree for each of the samples and the formation of new bonds promoted by the graphitization of the material. In the RS, two characteristic bands were observed according to the degree of oxidation and the degree of graphitization of the material represented in bands D and G with different relative intensities, suggesting that the samples have different crystallite sizes. This size was described using the Tuinstra-Koenig model, ranging between 18.7 and 25.1 nm. Finally, the bone neoformation observed in the cranial defects of critical size indicates that the F1 and F2 samples, besides being compatible and resorbable, acted as a bridge for bone healing through regeneration. This promoted healing by restoring bone and tissue structure without triggering a strong immune response.

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Nanostructured lipid carriers (NLC) have emerged as innovative drug delivery systems, offering distinct advantages over other lipid-based carriers, such as liposomes and solid lipid nanoparticles. Benzocaine (BZC), the oldest topical local anesthetic in use, undergoes metabolism by pseudocholinesterase, leading to the formation of p-aminobenzoic acid, a causative agent for allergic reactions associated with prolonged BZC usage. In order to mitigate adverse effects and enhance bioavailability, BZC was encapsulated within NLC. Utilizing a 23 factorial design, formulations comprising cetyl palmitate (solid lipid), propylene glycol monocaprylate (liquid lipid), and Pluronic F68 as surfactants were systematically prepared, with variations in the solid/liquid lipid mass ratios (60:40-80:20%), total lipid contents (15-25%), and BZC concentrations (1-3%). The optimized formulation underwent characterization by dynamic light scattering, differential scanning calorimetry, Raman imaging, X-ray diffraction, small-angle neutron scattering, nanotracking analysis, and transmission electron microscopy (TEM)/cryo-TEM, providing insights into the nanoparticle structure and the incorporation of BZC into its lipid matrix. NLCBZC exhibited a noteworthy encapsulation efficiency (%EE = 96%) and a 1 year stability when stored at 25 °C. In vitro kinetic studies and in vivo antinociceptive tests conducted in mice revealed that NLCBZC effectively sustained drug release for over 20 h and prolonged the anesthetic effect of BZC for up to 18 h. We therefore propose the use of NLCBZC to diminish the effective anesthetic concentration of benzocaine (from 20 to 3% or less), thus minimizing allergic reactions that follow the topical administration of this anesthetic and, potentially, paving the way for new routes of BZC administration in pain management.

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Smart nanocarrier-based bioactive delivery systems are a current focus in nanomedicine for allowing and boosting diverse disease treatments. In this context, the design of hybrid lipid-polymer particles can provide structure-sensitive features for tailored, triggered, and stimuli-responsive devices. In this work, we introduce hybrid cubosomes that have been surface-modified with a complex of chitosan-N-arginine and alginate, making them pH-responsive. We achieved high-efficiency encapsulation of acemannan, a bioactive polysaccharide from Aloe vera, within the nanochannels of the bioparticle crystalline structure and demonstrated its controlled release under pH conditions mimicking the gastric and intestinal environments. Furthermore, an acemannan-induced phase transition from Im3m cubic symmetry to inverse hexagonal HII phase enhances the bioactive delivery by compressing the lattice spacing of the cubosome water nanochannels, facilitating the expulsion of the encapsulated solution. We also explored the bioparticle interaction with membranes of varying curvatures, revealing thermodynamically driven affinity towards high-curvature lipid membranes and inducing morphological transformations in giant unilamellar vesicles. These findings underscore the potential of these structure-responsive, membrane-active smart bioparticles for applications such as pH-triggered drug delivery platforms for the gastrointestinal tract, and as modulators and promoters of cellular internalization.

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New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

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A better understanding of how emulsifier type could differently influence the behavior of nanostructured lipid carriers (NLC) under the gastrointestinal digestion process, as well as at the cellular level, is of utmost importance for the NLC-based formulations' optimization and risk assessment in the food field. In this study, NLC composed by fully hydrogenated soybean and high-oleic sunflower oils were prepared using soy lecithin (NLC Lß) or Tween 80 (NLC Tß) as an emulsifier. ß-Carotene was entrapped within NLC developed as a promising strategy to overcome ß-carotene's low bioavailability and stability. The effect of emulsifier type on the digestibility of ß-carotene-loaded NLC was evaluated using an in vitro dynamic digestion model mimicking peristalsis motion. The influence of ß-carotene-loaded NLC on cell viability was assessed using Caco-2 cells in vitro. NLC Tß remained stable in the gastric compartment, presenting particle size (PS) similar to the initial NLC (PS: 245.68 and 218.18 nm, respectively), while NLC Lß showed lower stability (PS > 1000 nm) in stomach and duodenum phases. NLC Tß also provided high ß-carotene protection and delivery capacity (i.e., ß-carotene bioaccessibility increased 10-fold). Based on the results of digestion studies, NLC Tß has shown better physical stability during the passage through the in vitro dynamic gastrointestinal system than NLC Lß. Moreover, the developed NLC did not compromise cell viability up to 25 µg/mL of ß-carotene. Thus, the NLC developed proved to be a biocompatible structure and able to incorporate and protect ß-carotene for further food applications. PRACTICAL APPLICATION: The findings of this study hold significant implications for industrial applications in terms of developing nanostructured lipid carriers from natural raw materials widely available and used to produce other lipid-based products in the food industry, as an alternative to synthetic ones. In this respect, the ß-carotene-loaded NLC developed in this study would find a great industrial application in the food industry, which is in constant search to develop functional foods capable of increasing the bioavailability of bioactive compounds.

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Biocompounds are metabolites synthesized by plants, with clinically proven capacity in preventing and treating degenerative diseases in humans. Carbon-based nanomaterials (CNMs) are atomic structures that assume different hybridization and shape. Due to the reactive property, CNMs can induce the synthesis of metabolites, such as biocompounds in cells and various plant species, by generating reactive oxygen species (ROS). In response, plants positively or negatively regulate the expression of various families of genes and enzymes involved in physiological and metabolomic pathways of plants, such as carbon and nitrogen metabolism, which are directly involved in plant development and growth. Likewise, ROS can modulate the expression of enzymes and genes related to the adaptation of plants to stress, such as the glutathione ascorbate cycle, the shikimic acid, and phenylpropanoid pathways, from which the largest amount of biocompounds in plants are derived. This document exposes the ability of three CNMs (fullerene, graphene, and carbon nanotubes) to positively or negatively regulate the activity of enzymes and genes involved in various plant species' primary and secondary metabolism. The mechanism of action of CNMs on the production of biocompounds and the effect of the translocation of CNMs on the growth and content of primary metabolites in plants are described. Adverse effects of CNMs on plants, prospects, and possible risks involved are also discussed. The use of CNMs as inducers of biocompounds in plants could have implications and relevance for human health, crop quality, and plant adaptation and resistance to biotic and abiotic stress.

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Bifunctional electrocatalysts are the attractive research in the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER) in the overall water-splitting reactions. The design and development of the cost-effective OER/HER bifunctional electrocatalysts with superior catalytic activity are still remaining as the big challenges. Herein, we have developed the CuO-ZnO nanocomposite as a bifunctional OER/HER electrocatalyst via simple chemical precipitation method. The nanocomposite was investigated for its crystalline structure, surface morphology and the functions of elements using XRD, FT-IR, SEM, TEM and XPS characterization techniques, respectively. The nanocomposite exhibited the excellent activity for the overall water-splitting in an alkaline medium. The CuO-ZnO nanocomposite showed the less onset potential of 1.4 and 0.15 V versus RHE in 1M KOH (Tafel slopes value of 0.180 and 0.400 V dec-1) for OER and HER, respectively. Hence, the as-prepared bifunctional electrocatalyst displayed the high stability for 10 h in the water electrolysis processes.

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