Mitigating diabetes associated with reactive oxygen species (ROS) and protein aggregation through pharmacological interventions.

Diabetes mellitus, a complex metabolic disorder, presents a growing global health challenge. In 2021, there were 529 million diabetics worldwide. At the super-regional level, Oceania, the Middle East, and North Africa had the highest age-standardized rates. The majority of cases of diabetes in 2021 (>90.0%) were type 2 diabetes, which is largely indicative of the prevalence of diabetes in general, particularly in older adults (K. L. Ong, et al., Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021, Lancet, 2023, 402(10397), 203-234). Nowadays, slowing the progression of diabetic complications is the only effective way to manage diabetes with the available therapeutic options. However, novel biomarkers and treatments are urgently needed to control cytokine secretion, advanced glycation end products (AGEs) production, vascular inflammatory effects, and cellular death. Emerging research has highlighted the intricate interplay between reactive oxygen species (ROS) and protein aggregation in the pathogenesis of diabetes. In this scenario, the main aim of this paper is to provide a comprehensive review of the current understanding of the molecular mechanisms underlying ROS-induced cellular damage and protein aggregation, specifically focusing on their contribution to diabetes development. The role of ROS as key mediators of oxidative stress in diabetes is discussed, emphasizing their impact on cellular components and signaling. Additionally, the involvement of protein aggregation in impairing cellular function and insulin signaling is explored. The synergistic effects of ROS and protein aggregation in promoting ß-cell dysfunction and insulin resistance are examined, shedding light on potential targets for therapeutic intervention.

Antimicrobial Potency of Fmoc-Phe-Phe Dipeptide Hydrogels with Encapsulated Porphyrin Chromophores Is a Promising Alternative in Antimicrobial Resistance.

Antimicrobial resistance (AMR) poses a significant global health risk as a consequence of misuse of antibiotics. Owing to the increasing antimicrobial resistance, it became imperative to develop novel molecules and materials with antimicrobial properties. Porphyrins and metalloporphyrins are compounds which present antimicrobial properties especially after irradiation. As a consequence, porphyrinoids have recently been utilized as antimicrobial agents in antimicrobial photodynamic inactivation in bacteria and other microorganisms. Herein, we report the encapsulation of porphyrins into peptide hydrogels which serve as delivery vehicles. We selected the self-assembling Fmoc-Phe-Phe dipeptide, a potent gelator, as a scaffold due to its previously reported biocompatibility and three different water-soluble porphyrins as photosensitizers. We evaluated the structural, mechanical and in vitro degradation properties of these hydrogels, their interaction with NIH3T3 mouse skin fibroblasts, and we assessed their antimicrobial efficacy against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) bacteria. We found out that the hydrogels are cytocompatible and display antimicrobial efficiency against both strains with the zinc porphyrins being more efficient. Therefore, these hydrogels present a promising alternative for combating bacterial infections in the face of growing AMR concerns.

Delivery of Porphyrins Through Self-Assembling Peptide Hydrogels for Accelerated Healing of Experimental Skin Defects In Vivo.

INTRODUCTION: The care and healing of skin defects resulting from different causes has been the object of research to achieve rapid and complete skin regeneration. Hydrogels have been used for their ability to maintain hydration during wound healing, absorb wound exudate, and cover the underlying tissue without adherence while being transparent. In this study, we evaluated the efficacy of a hydrogel (H) with encapsulated porphyrin (H+P) on a rat model of surgically-induced skin defects. METHODS: Four round 6 mm diameter skin defects were performed under general anesthesia on the dorsal area of 24 three-month-old "Young" and 24 twelve-month-old "Mature" male rats. Each age group was separated into the Control, H, and H+P groups, n=8 each, where no therapy, H, or H+P was respectively applied daily for 20 days. Digital photographs and skin biopsies were taken on the third, seventh, 10th, and 20th postoperative days and evaluated by planimetry, histology, and immunohistochemistry. RESULTS: Planimetry results demonstrated significantly decreased perimeter, diameter, and area measurements (p<0.005) of group H+P compared to Control and H groups on days 10 and 20 in the young rats, while in the mature rats, the significant differences were evident earlier (perimeter third day p<0.05; diameter and area seventh day p<0.05 and p<0.005, respectively vs. H). Granulation and scar tissue formation were also reduced in the H+P groups although they were not statistically significant. CONCLUSIONS: The application of H+P on the skin defects benefited the healing process in both young and mature animal groups, as evidenced by the statistically significant findings of planimetry. The beneficial healing process was more pronounced in the mature animals, both in the level of statistical significance as well as regarding time (evident already on the third day of healing), probably due to porphyrin assisting the reduced healing rate, which is observed in organisms of advanced age.

Stimuli-Responsive Polysaccharide Hydrogels and Their Composites for Wound Healing Applications.

There is a growing concern about wound care, since traditional dressings such as bandages and sutures can no longer meet existing needs. To address the demanding requirements, naturally occurring polymers have been extensively exploited for use in modern wound management. Polysaccharides, being the most abundant biopolymers, have some distinct characteristics, including biocompatibility and biodegradability, which render them ideal candidates for wound healing applications. Combining them with inorganic and organic moieties can produce effective multifunctional composites with the desired mechanical properties, high wound healing efficiencies and excellent antibacterial behavior. Recent research endeavors focus on the development of stimuli-responsive polysaccharide composites for biomedical applications. Polysaccharide composites, being sensitive to the local environment, such as changes of the solution temperature, pH, etc., can sense and react to the wound conditions, thus promoting an effective interaction with the wound. This review highlights the recent advances in stimuli-responsive polysaccharide hydrogels and their composites for use in wound healing applications. The synthetic approaches, physical, chemical, and biochemical properties as well as their function in wound healing will be discussed.

Remdesivir-loaded bis-MPA hyperbranched dendritic nanocarriers for pulmonary delivery.

Remdesivir is the only clinically available antiviral drug for the treatment of COVID-19. However, its very limited aqueous solubility confines its therapeutic activity and the development of novel inhaled nano-based drug delivery systems of remdesivir for enhanced lung tissue targeting and efficacy is internationally pursued. In this work 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) hyperbranched dendritic nano-scaffolds were employed as nanocarriers of remdesivir. The produced nano-formulations, empty and loaded, consisted of monodisperse nanoparticles with spherical morphology and neutral surface charge and sizes ranging between 80 and 230 nm. The entrapment efficiency and loading capacity of the loaded samples were 82.0% and 14.1%, respectively, whereas the release of the encapsulated drug was complete after 48 h. The toxicity assays in healthy MRC-5 lung diploid fibroblasts and NR8383 alveolar macrophages indicated their suitability as potential remdesivir carriers in the respiratory system. The novel nano-formulations are non-toxic in both tested cell lines, with IC50 values higher than 400 µΜ after 72 h treatment. Moreover, both free and encapsulated remdesivir exhibited very similar IC50 values, at the range of 80-90 µM, while its aqueous solubility was increased, overall presenting a suitable profile for application in inhaled delivery of therapeutics.

Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61-392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin-streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

Design and Synthesis of Porphyrin-Nitrilotriacetic Acid Dyads with Potential Applications in Peptide Labeling through Metallochelate Coupling.

The need to detect and monitor biomolecules, especially within cells, has led to the emerging growth of fluorescent probes. One of the most commonly used labeling techniques for this purpose is reversible metallochelate coupling via a nitrilotriacetic acid (NTA) moiety. In this study, we focus on the synthesis and characterization of three new porphyrin-NTA dyads, TPP-Lys-NTA, TPP-CC-Lys-NTA, and Py 3 P-Lys-NTA composed of a porphyrin derivative covalently connected with a modified nitrilotriacetic acid chelate ligand (NTA), for possible metallochelate coupling with Ni2+ ions and histidine sequences. Emission spectroscopy studies revealed that all of the probes are able to coordinate with Ni2+ ions and consequently can be applied as fluorophores in protein/peptide labeling applications. Using two different histidine-containing peptides as His6-tag mimic, we demonstrated that the porphyrin-NTA hybrids are able to coordinate efficiently with the peptides through the metallochelate coupling process. Moving one step forward, we examined the ability of these porphyrin-peptide complexes to penetrate and accumulate in cancer cells, exploring the potential utilization of our system as anticancer agents.

Evaluation of the Hemocompatibility and Anticancer Potential of Poly(ε-Caprolactone) and Poly(3-Hydroxybutyrate) Microcarriers with Encapsulated Chrysin.

In this work, novel chrysin-loaded poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers were synthesized according to a modified oil-in-water single emulsion/solvent evaporation method, utilizing poly(vinyl alcohol) surfactant as stabilizer and dispersing agent for the emulsification, and were evaluated for their physico-chemical and morphological properties, loading capacity and entrapment efficiency and in vitro release of their load. The findings suggest that the novel micro-formulations possess a spherical and relatively wrinkled structure with sizes ranging between 2.4 and 24.7 µm and a highly negative surface charge with z-potential values between (-18.1)-(-14.1) mV. The entrapment efficiency of chrysin in the poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers was estimated to be 58.10% and 43.63%, whereas the loading capacity was found to be 3.79% and 15.85%, respectively. The average release percentage of chrysin was estimated to be 23.10% and 18.01%, respectively. The novel micromaterials were further biologically evaluated for their hemolytic activity through hemocompatibility studies over a range of hematological parameters and cytoxicity against the epithelial human breast cancer cell line MDA-MB 231. The poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers reached an IC50 value with an encapsulated chrysin content of 149.19 µM and 312.18 µM, respectively, and showed sufficient blood compatibility displaying significantly low (up to 2%) hemolytic percentages at concentrations between 5 and 500 µg·mL-1.

Designer Amyloid Cell-Penetrating Peptides for Potential Use as Gene Transfer Vehicles.

Cell-penetrating peptides are used extensively to deliver molecules into cells due to their unique characteristics such as rapid internalization, charge, and non-cytotoxicity. Amyloid fibril biomaterials were reported as gene transfer or retroviral infection enhancers; no cell internalization of the peptides themselves is reported so far. In this study, we focus on two rationally and computationally designed peptides comprised of ß-sheet cores derived from naturally occurring protein sequences and designed positively charged and aromatic residues exposed at key residue positions. The ß-sheet cores bestow the designed peptides with the ability to self-assemble into amyloid fibrils. The introduction of positively charged and aromatic residues additionally promotes DNA condensation and cell internalization by the self-assembled material formed by the designed peptides. Our results demonstrate that these designer peptide fibrils can efficiently enter mammalian cells while carrying packaged luciferase-encoding plasmid DNA, and they can act as a protein expression enhancer. Interestingly, the peptides additionally exhibited strong antimicrobial activity against the enterobacterium Escherichia coli.

A self-assembly study of PNA-porphyrin and PNA-BODIPY hybrids in mixed solvent systems.

In this work a peptide nucleic acid (PNA) was covalently connected with two different chromophores, namely porphyrin and boron-dipyrromethene. To the best of our knowledge, this is the first example in the literature where a PNA unit is covalently linked to such chromophores. The self-assembly properties of the hybrids were examined through electron microscopy experiments by adopting the "good-bad" solvent self-assembly protocol. For both hybrids (PNA-TPP and PNA-BDP) we were able to observe distinctive supramolecular architectures. During these studies we investigated the influence of the solvent system, the concentration and the deposition method on the morphology of the formed nanostructures. In the case of PNA-TPP under all examined conditions well-formed nanospheres were obtained. Interestingly, in the PNA-BDP hybrid by simply altering the solvent mixture, self-assemblies of two different morphologies were formed (spherical and flake shaped). Absorption and emission studies suggested the formation of J-aggregates in all the obtained nanostructures. The nano-architectures assembled by PNA conjugates are capable of light-harvesting and producing hydrogen using Pt nanoparticles as a photocatalyst.

Computational Design of Functional Amyloid Materials with Cesium Binding, Deposition, and Capture Properties.

Amyloid materials are gaining increasing attention as promising materials for applications in numerous fields. Computational methods have been successfully implemented to investigate the structures of short amyloid-forming peptides, yet their application in the design of functional amyloid materials is still elusive. Here, we developed a computational protocol for the design of functional amyloid materials capable of binding to an ion of interest. We applied the protocol in a test case involving the design of amyloid materials with cesium ion deposition and capture properties. As part of the protocol, we used an optimization-based design model to introduce mutations at non-ß-sheet residue positions of an amyloid designable scaffold. The designed amino acids introduced to the scaffold mimic how amino acids bind to cesium ions according to experimentally resolved structures and also aim at energetically stabilizing the bound conformation of the pockets. The optimum designs were computationally validated using a series of simulations and structural analysis to select the top designed peptides, which are predicted to form fibrils with cesium ion binding properties for experimental testing. Experiments verified the amyloid-forming properties of the selected top designed peptides, as well as the cesium ion deposition and capture properties by the amyloid materials formed. This study demonstrates the first, to the best of our knowledge, computational design protocol to functionalize amyloid materials for ion binding properties and suggests that its further advancement can lead to novel, highly promising functional amyloid materials of the future.

A novel amyloid designable scaffold and potential inhibitor inspired by GAIIG of amyloid beta and the HIV-1 V3 loop.

The GAIIG sequence, common to the amyloid beta peptide (residues 29-33) and to the HIV-1 gp120 (residues 24-28 in a typical V3 loop), self-assembles into amyloid fibrils, as suggested by theory and the experiments presented here. The longer YATGAIIGNII sequence from the V3 loop also self-assembles into amyloid fibrils, of which the first three and the last two residues are outside the amyloid GAIIG core. We postulate that this sequence, with suitably selected modifications at the flexible positions, can serve as a designable scaffold for novel amyloid-based materials. Moreover, we report the single crystal X-ray structure of the beta-breaker peptide GAIPIG at 1.05 Å resolution. The structural information provided in this study could serve as the basis for structure-based design of potential inhibitors of amyloid formation.

Self-assembly study of nanometric spheres from polyoxometalate-phenylalanine hybrids, an experimental and theoretical approach.

Herein, we report on the study of supramolecular assemblies based on polyoxometalates (POMs) upon their modification with amino acids. Two POM-amino acid hybrids were synthesized by coupling a functionalized Keggin type polyoxoanion [PW11O39{Sn(C6H4)C[triple bond, length as m-dash]C(C6H4)COOH}]4- with carboxyl-protected (methyl-ester) phenylalanine or diphenylalanine peptides. Surprisingly, all compounds, including the initial POM, formed supramolecular nanospheres in different solvent mixtures, which were examined by scanning electron microscopy (SEM). Molecular dynamics (MD) simulations for the POM-amino acid species revealed that the hydrophobic forces are mainly responsible for the initial aggregation into incipient micelle type structures, in which the organic arms are buried inside the aggregate while POM polar heads are more exposed to the solvent with tetrabutyl-ammonium counter cations acting as linkers.

Self-assembly of (boron-dipyrromethane)-diphenylalanine conjugates forming chiral supramolecular materials.

Herein, we present the synthesis of a series of boron-dipyrromethane (BDP) derivatives bearing diphenylalanine (FF) at their meso position via amide bond coupling. The BDP-FF bioconjugates are able to form self-assembled materials with different morphologies. By altering various parameters such as the protecting group of the FF peptide or the solvent system of the self-assembly process, we were able to obtain either fibrillar or spherical nanostructures. Furthermore, we confirmed that both the formation as well as the dissociation of the self-assemblies is a reversible procedure that can be achieved by simply altering the solvent mixture. Electronic circular dichroism (ECD) studies demonstrated a characteristic mirror image relationship regarding the FLFL and FDFD enantiomers, revealing the chiral nature of the obtained materials. Interestingly, an intense excitonic bisignate signal was observed in the ECD spectrum of the fibrillar structures, whereas the spherical assemblies remained ECD silent. What is more, the electronic circular dichroism studies were supported by quantum chemical calculations.

Oleylamine as a beneficial agent for the synthesis of CoFe2O4 nanoparticles with potential biomedical uses.

The multifunctional role of oleylamine (OAm) as a versatile and flexible reagent in synthesis as well as a desired surface ligand for the synthesis of CoFe2O4 nanoparticles (NPs) is described. CoFe2O4 NPs were prepared by a facile, reproducible and scalable solvothermal approach in the presence of pure OAm. By monitoring the volume of OAm, different shapes of NPs, spherical and truncated, were formed. The syntheses led to high yields of monodispersed and considerably small (9-11 nm) CoFe2O4 NPs with enhanced magnetization (M(s) = 84.7-87.5 emu g(-1)). The resulting hydrophobic CoFe2O4 NPs were easily transferred to an aqueous phase through the formation of reverse micelles between the hydrophobic chains of OAm and cetyltrimethylammonium bromide (CTAB) and transverse relaxivities (r2) were measured. The spherical NPs had a greater effect on water proton relaxivity (r2 = 553 mM(-1) s(-1)) at an applied magnetic field of 11.7 T. The NPs became fluorescent probes by exploiting the presence of the double bond of OAm in the middle of the molecule; a thiol-ene "click" reaction with the fluorophore bovine serum albumin (FITC-BSA) was achieved. The labeled/biofunctionalized CoFe2O4 NPs interacted with cancer (HeLa and A549) and non-cancer cell lines (MRC5 and dental MSCS) and cell viability was estimated. A clear difference of toxicity between the cancer and non-cancer cells was observed while low cytotoxicity in living cells was supported. Confocal laser microscopy showed that NPs entered the cell membranes and were firstly localized close to them provoking a membrane expansion and were further accumulated perinuclearly without entering the nuclei.