Investigating the complexation propensity of self-assembling dipeptides with the anticancer peptide-drug Bortezomib: a computational study.

The investigation of potential self-assembled peptides as carriers for the delivery of anticancer drug Bortezomib is the topic of the present study. The self-assembly of Bortezomib in water is examined using all-atom molecular dynamics simulations and corresponding experimental results from FESEM experiments. In addition, a series of dipeptides with a similar chemical formula to Bortezomib with hydrogel-forming ability are being investigated for their propensity to bind to the drug molecule. Dipeptides are divided into two classes, the protected FF (Fmoc-FF and Z-FF) and the LF-based (Cyclo-LF and LF) ones. The thermodynamic stability of the complexes formed in an aqueous environment, as well as key morphological features of the nanoassemblies are investigated at the molecular level. Binding enthalpy between Bortezomib and dipeptides follows the increasing order: LF < Cyclo-LF < Fmoc-FF < Z-FF under both van der Waals and electrostatic contributions. Protected FF dipeptides have a higher affinity for the drug molecule, which will favor its entrapment, giving them an edge over the LF based dipeptides. By evaluating the various measures, regarding both the binding between the two components and the eventual ability of controlled drug release, we conclude that the protected FF class is a more suitable candidate for drug release of Bortezomib, whereas among its two members, Fmoc-FF appears to be more promising. The selection of the optimal candidates based on the present computational study will be a stepping stone for future detailed experimental studies involving the encapsulation and controlled release of Bortezomib both in vitro and in vivo.

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

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.

Corrole and Porphyrin Amino Acid Conjugates: Synthesis and Physicochemical Properties.

A series of conjugates of amino acids with porphyrins and corroles was synthesized. Their self-assembling ability under defined conditions was investigated by scanning electron microscopy. The morphology and photophysical properties of these molecules were studied by absorption and fluorescence spectroscopy in solid, liquid, and self-assembled forms. We observed that both corrole and porphyrin conjugated with the l-phenylalanine-l-phenylalanine peptide to form spherical nanostructures with bathochromic shifts in the emission spectra, indicating the formation of aggregates. These aggregates are characterized by the impressive absorption of light over nearly the whole visible range. The broadening of all bands was particularly strong in the case of corroles. The fluorescence lifetimes of self-assembled species were longer as compared to the solid-state form.

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.

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.

Z-Ala-Ile-OH, a dipeptide building block suitable for the formation of orthorhombic microtubes.

Self-assembling dipeptides have emerged in the last two decades as promising building blocks for the development of novel biomaterials. Among the various classes of dipeptides, aromatic dipeptides and especially diphenylalanine (Phe-Phe), which forms hexagonal nanotubes, have been the most extensively studied. However, aliphatic peptides or mixed aromatic-aliphatic dipeptides seem just as promising, exhibiting various structures ranging from amyloid fibrils to microtubes. Herein we report the single-crystal structure of an aliphatic dipeptide, alanine-isoleucine (Ala-Ile), C17H24N2O5, protected with a benzyloxycarbonyl (Z) group at the N-terminus. The protected dipeptide crystallizes in the orthorhombic space group P212121 and forms hollow microtubes with orthorhombic symmetry upon evaporation on glass surfaces, as shown by field emission scanning electron microscopy (FESEM). These findings provide an increased understanding of the correlation between the single-crystal structure of the peptide building block and its self-assembly mechanism, and expand the library of available building blocks for microtechnological applications.

Self-Assembly of Phenylalanine-Leucine, Leucine-Phenylalanine, and Cyclo(-leucine-phenylalanine) Dipeptides through Simulations and Experiments.

For over two decades, peptide self-assembly has been the focus of attention and a great source of inspiration for biomedical and nanotechnological applications. The resulting peptide nanostructures and their properties are closely related to the information encoded within each peptide building block, their sequence, and their modes of self-organization. In this work. we assess the behavior and differences between the self-association of the aromatic-aliphatic Phe-Leu dipeptide compared to its retro-sequence Leu-Phe and cyclic Cyclo(-Leu-Phe) counterparts, using a combination of simulation and experimental methods. Detailed all-atom molecular dynamics (MD) simulations offer a quantitative prediction at the molecular level of the conformational, dynamical and structural properties of the peptides' self-assembly, while field emission scanning electron microscopy (FESEM) experiments allow microscopic observation of the self-assembled end-structures. The complementarity and qualitative agreement between the two methods not only highlights the differences between the self-assembly propensity of cyclic and linear retro-sequence peptides but also sheds light on underlying mechanisms of self-organization. The self-assembling propensity was found to follow the order: Cyclo(-Leu-Phe) > Leu-Phe > Phe-Leu.

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.

A Doubly Fmoc-Protected Aspartic Acid Self-Assembles into Hydrogels Suitable for Bone Tissue Engineering.

Hydrogels have been used as scaffolds for biomineralization in tissue engineering and regenerative medicine for the repair and treatment of many tissue types. In the present work, we studied an amino acid-based material that is attached to protecting groups and self-assembles into biocompatible and stable nanostructures that are suitable for tissue engineering applications. Specifically, the doubly protected aspartic residue (Asp) with fluorenyl methoxycarbonyl (Fmoc) protecting groups have been shown to lead to the formation of well-ordered fibrous structures. Many amino acids and small peptides which are modified with protecting groups display relatively fast self-assembly and exhibit remarkable physicochemical properties leading to three-dimensional (3D) networks, the trapping of solvent molecules, and forming hydrogels. In this study, the self-assembling fibrous structures are targeted toward calcium binding and act as nucleation points for the binding of the available phosphate groups. The cell viability, proliferation, and osteogenic differentiation of pre-osteoblastic cells cultured on the formed hydrogel under various conditions demonstrate that hydrogel formation in CaCl2 and CaCl2-Na2HPO4 solutions lead to calcium ion binding onto the hydrogels and enrichment with phosphate groups, respectively, rendering these mechanically stable hydrogels osteoinductive scaffolds for bone tissue engineering.

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.

Self-assembly into spheres of a hybrid diphenylalanine-porphyrin: increased fluorescence lifetime and conserved electronic properties.

A series of protected phenylalanine and diphenylalanine derivatives have been coupled through a peptide bond to a monoaminoporphyrin to form new materials. A comparative study in solution and in the solid state has been performed and confirmed new and interesting properties for the self-assembled hybrid materials while conserving the electronic properties of the chromophore. Thus, they are powerful candidates for use in dye-sensitized solar cells.

Neutron fibre diffraction studies of amyloid using H2O/D2O isotopic replacement.

The first neutron fibre diffraction studies of an amyloid system are presented. The techniques used to prepare the large samples needed are described, as well as the procedures used to isotopically replace H2O in the sample by D2O. The results demonstrate the feasibility of this type of approach for the pursuit of novel structural analyses that will strongly complement X-ray fibre diffraction studies and probe aspects of amyloid structure that to date have remained obscure. The approach is demonstrated using an amyloid form of the peptide NSGAITIG, but is equally applicable for the study of other systems such as Alzheimer's Aß peptide.

Combined effect of shear stress and laser-patterned topography on Schwann cell outgrowth: synergistic or antagonistic?

Although the peripheral nervous system exhibits a higher rate of regeneration than that of the central nervous system through a spontaneous regeneration after injury, the functional recovery is fairly infrequent and misdirected. Thus, the development of successful methods to guide neuronal outgrowth, in vitro, is of great importance. In this study, a precise flow controlled microfluidic system with specific custom-designed chambers, incorporating laser-microstructured polyethylene terephthalate (PET) substrates comprising microgrooves, was fabricated to assess the combined effect of shear stress and topography on Schwann cells' behavior. The microgrooves were positioned either parallel or perpendicular to the direction of the flow inside the chambers. Additionally, the cell culture results were combined with computational flow simulations to calculate accurately the shear stress values. Our results demonstrated that wall shear stress gradients may be acting either synergistically or antagonistically depending on the substrate groove orientation relative to the flow direction. The ability to control cell alignment in vitro could potentially be used in the fields of neural tissue engineering and regenerative medicine.

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.

Supramolecular Nanodrugs Constructed by Self-Assembly of Peptide Nucleic Acid-Photosensitizer Conjugates for Photodynamic Therapy.

Two hybrid materials were designed by conjugating peptide nucleic acids (PNAs) to porphyrin or boron-dipyrromethene, generating PNA-porphyrin (PNA-TPP) and PNA-BODIPY (PNA-BDP) conjugates, respectively. Because of the combination of the supramolecular characteristics of PNAs and photosensitizers, the two hybrid conjugates readily self-assemble in aqueous solutions and produce well-defined nanoparticles with uniform particle sizes. The resulting two kinds of nanoparticles show good stability in biological solutions and upon dilution. Importantly, the nanoparticles can efficiently interact with cancer cells and the internalized nanoparticles are mainly distributed in the cytoplasm without discernible cytotoxicity in the dark, enabling them to be applied as photodynamic nanoagents for selective killing cells. Hence, self-assembly of PNA-photosensitizer conjugates may hold promise for advancing the rational design and construction of photodynamic nanoagents for cancer therapy.

Self-assembly of Alanine-Isoleucine and Isoleucine-Isoleucine Dipeptides through Atomistic Simulations and Experiments.

A detailed investigation of the structural and conformational properties of alanine-isoleucine (Ala-Ile) and isoleucine-isoleucine (Ile-Ile) dipeptides is presented in water and in methanol solvents. We propose a consistent combination of complementary simulation and experimental methods, covering a broad range of length and time scales, from the very short (i.e., atomic level), via all-atom molecular dynamics (MD) simulations, up to the macroscopic one, via scanning electron microscopy (SEM) experiments. The examined samples from both simulations and experiment cover a board range of concentrations since these are usually in different concentration windows (i.e., high values in simulations vs low values in experiments). In the present study, there is an overlapping concentration regime and a qualitative agreement between simulation and experimental results is observed. The effect of temperature on the formed structures is found to be small, from both simulation and experiments, when temperature varies from 278 to 300 K. Furthermore, the differences of Ala-Ile and Ile-Ile dipeptides from dialanine (Ala-Ala) and diphenylalanine (Phe-Phe) dipeptides in similar conditions are highlighted. Based on various measures, the strength of the self-assembly propensity of the four dipeptides in aqueous solutions attains the following order: Phe-Phe > Ala-Ile > Ala-Ala > Ile-Ile.

Self-assembly of aliphatic dipeptides coupled with porphyrin and BODIPY chromophores.

In this work, the self assembly ability of chromophores covalently linked to aliphatic dipeptides is described. Altering various parameters such as the protecting group, the solvent mixture, the dipeptide and the chromophore resulted in different nanostructures. Interestingly, a peptide-porphyrin hybrid is capable of forming a hydrogel in HFIP-water solvent mixture.

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.