Comparison of the self-assembly and cytocompatibility of conjugates of Fmoc (9-fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids.

The self-assembly in aqueous solution of three Fmoc-amino acids with hydrophobic (aliphatic or aromatic, alanine or phenylalanine) or hydrophilic cationic residues (arginine) is compared. The critical aggregation concentrations were obtained using intrinsic fluorescence or fluorescence probe measurements, and conformation was probed using circular dichroism spectroscopy. Self-assembled nanostructures were imaged using cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Fmoc-Ala is found to form remarkable structures comprising extended fibril-like objects nucleating from spherical cores. In contrast, Fmoc-Arg self-assembles into plate-like crystals. Fmoc-Phe forms extended structures, in a mixture of straight and twisted fibrils coexisting with nanotapes. Spontaneous flow alignment of solutions of Fmoc-Phe assemblies is observed by SAXS. The cytocompatibility of the three Fmoc-amino acids was also compared via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] mitochondrial activity assays. All three Fmoc-amino acids are cytocompatible with L929 fibroblasts at low concentration, and Fmoc-Arg shows cell viability up to comparatively high concentration (0.63 mM).

Self-Assembly and Cytocompatibility of Amino Acid Conjugates Containing a Novel Water-Soluble Aromatic Protecting Group.

There has been considerable interest in peptides in which the Fmoc (9-fluorenylmethoxycarbonyl) protecting group is retained at the N-terminus, since this bulky aromatic group can drive self-assembly, and Fmoc-peptides are biocompatible and have applications in cell culture biomaterials. Recently, analogues of new amino acids with 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting groups have been developed for water-based peptide synthesis. Here, we report on the self-assembly and biocompatibility of Smoc-Ala, Smoc-Phe and Smoc-Arg as examples of Smoc conjugates to aliphatic, aromatic, and charged amino acids, respectively. Self-assembly occurs at concentrations above the critical aggregation concentration (CAC). Cryo-TEM imaging and SAXS reveal the presence of nanosheet, nanoribbon or nanotube structures, and spectroscopic methods (ThT fluorescence circular dichroism and FTIR) show the presence of ß-sheet secondary structure, although Smoc-Ala solutions contain significant unaggregated monomer content. Smoc shows self-fluorescence, which was used to determine CAC values of the Smoc-amino acids from fluorescence assays. Smoc fluorescence was also exploited in confocal microscopy imaging with fibroblast cells, which revealed its uptake into the cytoplasm. The biocompatibility of these Smoc-amino acids was found to be excellent with zero cytotoxicity (in fact increased metabolism) to fibroblasts at low concentration.

Harnessing Efficient ROS Generation in Carbon Dots Derived from Methyl Red for Antimicrobial Photodynamic Therapy.

The emergence of drug-resistant pathogenic microorganisms has become a public health concern, with demand for strategies to suppress their proliferation in healthcare facilities. The present study investigates the physicochemical and antimicrobial properties of carbon dots (CD-MR) derived from the methyl red azo dye. The morphological and structural analyses reveal that such carbon dots present a significant fraction of graphitic nitrogen in their structures, providing a wide emission range. Based on their low cytotoxicity against mammalian cells and tunable photoluminescence, these carbon dots are applied to bioimaging in vitro living cells. The possibility of using CD-MR to generate reactive oxygen species (ROS) is also analyzed, and a high singlet oxygen quantum efficiency is verified. Moreover, the antimicrobial activity of CD-MR is analyzed against pathogenic microorganisms Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans. Kirby-Bauer susceptibility tests show that carbon dots synthesized from methyl red possess antimicrobial activity upon photoexcitation at 532 nm. The growth inhibition of C. neoformans from CD-MR photosensitization is investigated. Our results show that N-doped carbon dots synthesized from methyl red efficiently generate ROS and possess a strong antimicrobial activity against healthcare-relevant pathogens.

DNA-templated self-assembly of bradykinin into bioactive nanofibrils.

Bradykinin (BK) is a peptide hormone that plays a crucial role in blood pressure control, regulates inflammation in the human body, and has recently been implicated in the pathophysiology of COVID-19. In this study, we report a strategy for fabricating highly ordered 1D nanostructures of BK using DNA fragments as a template for self-assembly. We have combined synchrotron small-angle X-ray scattering and high-resolution microscopy to provide insights into the nanoscale structure of BK-DNA complexes, unveiling the formation of ordered nanofibrils. Fluorescence assays hint that BK is more efficient at displacing minor-groove binders in comparison with base-intercalant dyes, thus, suggesting that interaction with DNA strands is mediated by electrostatic attraction between cationic groups at BK and the high negative electron density of minor-grooves. Our data also revealed an intriguing finding that BK-DNA complexes can induce a limited uptake of nucleotides by HEK-293t cells, which is a feature that has not been previously reported for BK. Moreover, we observed that the complexes retained the native bioactivity of BK, including the ability to modulate Ca2+ response into endothelial HUVEC cells. Overall, the findings presented here demonstrate a promising strategy for the fabrication of fibrillar structures of BK using DNA as a template, which keep bioactivity features of the native peptide and may have implications in the development of nanotherapeutics for hypertension and related disorders.

Self-assembled aggregates based on cationic amphiphilic peptides: structural insight.

Short and ultra-short peptides have recently emerged as suitable building blocks for the fabrication of self-assembled innovative materials. Peptide aggregation is strictly related to the amino acids composing the sequence and their capability to establish intermolecular interactions. Additional structural and functional properties can also be achieved by peptide derivatization (e.g. with polymeric moieties, alkyl chains or other organic molecules). For instance, peptide amphiphiles (PAs), containing one or more alkyl tails on the backbone, have a propensity to form highly ordered nanostructures like nanotapes, twisted helices, nanotubes and cylindrical nanostructures. Further lateral interactions among peptides can also promote hydrogelation. Here we report the synthesis and the aggregation behaviour of four PAs containing cationic tetra- or hexa-peptides (C19-VAGK, C19-K1, C19-K2 and C19-K3) derivatized with a nonadecanoic alkyl chain. In their acetylated (Ac-) or fluorenylated (Fmoc-) versions, these peptides previously demonstrated the ability to form biocompatible hydrogels potentially suitable as extracellular matrices for tissue engineering or diagnostic MRI applications. In the micromolar range, PAs self-assemble in aqueous solution into nanotapes, or small clusters, resulting in high biocompatibility on HaCat cells up to 72 hours of incubation. Moreover, C19-VAGK also forms a gel at a concentration of 5 wt%.

Modification of the Linker Amino Acid in the Cell-Penetrating Peptide NickFect55 Leads to Enhanced pDNA Transfection for In Vivo Applications.

Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a "kinked" structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.

Cell Adhesion Motif-Functionalized Lipopeptides: Nanostructure and Selective Myoblast Cytocompatibility.

The conformation and self-assembly of four lipopeptides, peptide amphiphiles comprising peptides conjugated to lipid chains, in aqueous solution have been examined. The peptide sequence in all four lipopeptides contains the integrin cell adhesion RGDS motif, and the cytocompatibility of the lipopeptides is also analyzed. Lipopeptides have either tetradecyl (C14, myristyl) or hexadecyl (C16, palmitoyl) lipid chains and peptide sequence WGGRGDS or GGGRGDS, that is, with either a tryptophan-containing WGG or triglycine GGG tripeptide spacer between the bioactive peptide motif and the alkyl chain. All four lipopeptides self-assemble above a critical aggregation concentration (CAC), determined through several comparative methods using circular dichroism (CD) and fluorescence. Spectroscopic methods [CD and Fourier transform infrared (FTIR) spectroscopy] show the presence of ß-sheet structures, consistent with the extended nanotape, helical ribbon, and nanotube structures observed by cryogenic transmission electron microscopy (cryo-TEM). The high-quality cryo-TEM images clearly show the coexistence of helically twisted ribbon and nanotube structures for C14-WGGRGDS, which highlight the mechanism of nanotube formation by the closure of the ribbons. Small-angle X-ray scattering shows that the nanotapes comprise highly interdigitated peptide bilayers, which are also present in the walls of the nanotubes. Hydrogel formation was observed at sufficiently high concentrations or could be induced by a heat/cool protocol at lower concentrations. Birefringence due to nematic phase formation was observed for several of the lipopeptides, along with spontaneous flow alignment of the lyotropic liquid crystal structure in capillaries. Cell viability assays were performed using both L929 fibroblasts and C2C12 myoblasts to examine the potential uses of the lipopeptides in tissue engineering, with a specific focus on application to cultured (lab-grown) meat, based on myoblast cytocompatibility. Indeed, significantly higher cytocompatibility of myoblasts was observed for all four lipopeptides compared to that for fibroblasts, in particular at a lipopeptide concentration below the CAC. Cytocompatibility could also be improved using hydrogels as cell supports for fibroblasts or myoblasts. Our work highlights that precision control of peptide sequences using bulky aromatic residues within "linker sequences" along with alkyl chain selection can be used to tune the self-assembled nanostructure. In addition, the RGDS-based lipopeptides show promise as materials for tissue engineering, especially those of muscle precursor cells.

Nanostructure Formation and Cell Spheroid Morphogenesis of a Peptide Supramolecular Hydrogel.

Peptide-based hydrogels have attracted much attention due to their extraordinary applications in biomedicine and offer an excellent mimic for the 3D microenvironment of the extracellular matrix. These hydrated matrices comprise fibrous networks held together by a delicate balance of intermolecular forces. Here, we investigate the hydrogelation behavior of a designed decapeptide containing a tetraleucine self-assembling backbone and fibronectin-related tripeptides near both ends of the strand. We have observed that this synthetic peptide can produce hydrogel matrices entrapping >99% wt/vol % water. Ultrastructural analyses combining atomic force microscopy, small-angle neutron scattering, and X-ray diffraction revealed that amyloid-like fibrils form cross-linked networks endowed with remarkable thermal stability, the structure of which is not disrupted up to temperatures >80 °C. We also examined the interaction of peptide hydrogels with either NIH3T3 mouse fibroblasts or HeLa cells and discovered that the matrices sustain cell viability and induce morphogenesis into grape-like cell spheroids. The results presented here show that this decapeptide is a remarkable building block to prepare highly stable scaffolds simultaneously endowed with high water retention capacity and the ability to instruct cell growth into tumor-like spheroids even in noncarcinoma lineages.

Methods for Small-Angle Scattering Measurements on Peptiplexes of DNA with Cell-Penetrating Peptides.

Peptiplexes are soft biomaterials formed through the noncovalent association between cell-penetrating peptides and nucleic acids. Although internalization often involves electrostatic anchoring followed by endocytosis, the mode of action of these transporters remains elusive in many cases, and proper understanding of mechanisms behind their penetrating capabilities necessarily entails structural data at the nanoscopic scale. In this chapter, we examine the structural landscape of peptiplexes, emphasizing the complex behavior of these polyelectrolyte self-assemblies and how supramolecular order impacts their translocation efficiency. We discuss experimental tools commonly used to investigate the structure of peptiplexes and pay special attention to small-angle X-ray scattering (SAXS) as a suitable method for unveiling their nanoscale organization. A roadmap for standard SAXS measurements in CPP/DNA samples is presented alongside a selection of observations from our own experience dealing with SAXS applied to the investigation of CPPs.

Tools for the investigation of adverse events: scoping review / Herramientas para investigación de eventos adversos: revisión de alcance / Ferramentas para investigação de eventos adversos: revisão de escopo*

ABSTRACT Objective: To map, in the literature, the risk management tools aimed at investigating health adverse events. Method: Scoping review according to the Joanna Brigss Institute, with acronym PCC (Population: hospitalized patients, Concept: tools for the investigation of adverse events, and Context: health institutions) carried out in MEDLINE (OVID), EMBASE, LILACS, Scopus, CINAHL, and gray literature. Results: The search totaled 825 scientific productions, 31 of which met the objective of the study, which consisted of 27 scientific articles and 4 expert consensus. It was possible to carry out a synthesis of the necessary steps for the investigation of adverse events and use of the tools according to the extent of damage. Conclusion: The practice of investigating adverse events should be guided by a thorough understanding of contributing factors, a fair culture, and the involvement of senior leadership.

RESUMEN Objetivo: Mapeo en la literatura de las herramientas de la gestión de riesgo con énfasis en la investigación de eventos adversos en salud. Método: Revisión de alcance según Joanna Brigss Institute con el acrónimo PCC (Población: pacientes ingresados, Concepto: herramientas para la investigación de eventos adversos y Contexto: instituciones de salud) realizada en las bases de datos MEDLINE (OVID), EMBASE, LILACS, Scopus, CINAHL y literatura gris. Resultados: La búsqueda llegó a un total de 825 producciones científicas, siendo que 31 lograron el objetivo del estudio, el cual fue compuesto por 27 artículos científicos y 4 consensos de expertos. Fue posible realizar una síntesis de las etapas necesarias para la investigación de eventos adversos y utilización de las herramientas de acuerdo con el grado del daño. Conclusión: La práctica de investigación de eventos adversos deberá pautarse en la comprensión exhaustiva de los factores contribuyentes, cultura justa e involucramiento de alto liderazgo.

RESUMO Objetivo: Mapear na literatura as ferramentas da gestão de risco voltadas para investigação de eventos adversos na saúde. Método: Revisão de escopo segundo o Joanna Brigss Institute, com acrônimo PCC (População: pacientes internados, Conceito: ferramentas para a investigação de eventos adversos e Contexto: instituições de saúde), realizada nas bases MEDLINE (OVID), EMBASE, LILACS, Scopus, CINAHL e literatura cinzenta. Resultados: A busca totalizou 825 produções científicas, sendo que 31 atenderam o objetivo do estudo, sendo composta por 27 artigos científicos e 4 consensos de especialistas. Foi possível realizar uma síntese das etapas necessárias para a investigação de eventos adversos e utilização das ferramentas de acordo com o grau do dano. Conclusão: A prática de investigação de eventos adversos deverá ser pautada na compreensão exaustiva dos fatores contribuintes, cultura justa e envolvimento da alta liderança.

Microfluidic encapsulation of nanoparticles in alginate microgels gelled via competitive ligand exchange crosslinking.

Efficient delivery of nanometric vectors complexed with nanoparticles at a target tissue without spreading to other tissues is one of the main challenges in gene therapy. One means to overcome this problem is to confine such vectors within microgels that can be placed in a target tissue to be released slowly and locally. Herein, a conventional optical microscope coupled to a common smartphone was employed to monitor the microfluidic production of monodisperse alginate microgels containing nanoparticles as a model for the encapsulation of vectors. Alginate microgels (1.2%) exhibited an average diameter of 125 ± 3 µm, which decreased to 106 ± 5 µm after encapsulating 30 nm fluorescent nanoparticles. The encapsulation efficiency was 70.9 ± 18.9%. In a 0.1 M NaCl solution, 55 ± 5% and 92 ± 4.7% of nanoparticles were released in 30 minutes and 48 hours, respectively. Microgel topography assessment by atomic force microscopy revealed that incorporation of nanoparticles into the alginate matrix changes the scaffold's interfacial morphology and induces crystallization with the appearance of oriented domains. The high encapsulation rate of nanoparticles, alongside their continuous release of nanoparticles over time, makes these microgels and the production unit a valuable system for vector encapsulation for gene therapy research.

Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids.

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain ß-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Model self-assembling arginine-based tripeptides show selective activity against Pseudomonas bacteria.

Three model arginine-rich tripeptides RXR (X = W, F or non-natural residue 2-napthylalanine) were investigated as antimicrobial agents, with a specific focus to target Pseudomonas aeruginosa through membrane lysis. Activity against biofilms was related to binding of the second messenger molecule, nucleotide bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Strong selective activity against P. aeruginosa in planktonic form was observed for RFR and RWR.

Nanoscopic Structure of Complexes Formed between DNA and the Cell-Penetrating Peptide Penetratin.

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNA condensation is accompanied by random-to-ß-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin ß-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into ß-sheets are key steps for organization is proposed.