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Lipoic acid is a biocompatible compound with antioxidant activity that is of considerable interest in cosmetic formulations, and the disulfide group in the N-terminal ring confers redox activity. Here, we study the self-assembly and aspects of the bioactivity of a lipopeptide (peptide amphiphile) comprising the KTTKS collagen-stimulating pentapeptide sequence conjugated to an N-terminal lipoic acid chain, lipoyl-KTTKS. Using SAXS, SANS and cryo-TEM, lipoyl-KTTKS is found to form a population of curly fibrils (wormlike micelles) above a critical aggregation concentration. Upon chemical reduction, the fibrils (and β-sheet structure) are disrupted because of the breaking of the disulfide bond, which produces dihydrolipoic acid. Lipoyl-KTTKS also undergoes photo-degradation in the presence of UV radiation. Through cell assays using fibroblasts, we found that lipoyl-KTTKS has excellent cytocompatibility across a wide concentration range, stimulates collagen production, and enhances the rate of cell coverage in a simple in vitro scratch assay of 'wound healing'. Lipoyl-KTTKS thus has several notable properties that may be useful for the development of cosmetics, cell scaffolds or tissue engineering materials., (© 2025 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Short bioactive peptide sequences are of great interest in biomaterials development. We investigate the self-assembly of a lipopeptide containing both the highly cationic CSK 4 toll-like receptor agonist hexapeptide sequence and RGDS integrin-binding motif, i.e., C 16 -CSK 4 RGDS, as well as the control containing a scrambled terminal sequence C 16 -CSK 4 GRDS. Both lipopeptides are found to form micelles, as revealed by small-angle X-ray scattering and cryogenic transmission electron microscopy, and modeled using atomistic molecular dynamics simulations. We carefully examined methods to probe the aggregation of the molecules, i.e. to obtain the critical micelle concentration (CMC). Fluorescent probe assays using 1-anilino-8-naphthalenesulfonate (ANS) reveal low CMC values, 1-2 μM, which contrast with consistent values more than 2 orders of magnitude larger obtained from surface tension and electrical conductivity as well as unexpected UV/vis absorption spectra discontinuities and fluoresccence probe assays using Nile red. The anomalous results obtained from an ANS fluorescence probe are ascribed to the effect of ANS binding to the cationic (lysine and arginine) residues in the lipopeptide, which leads to a conformational change, as shown by circular dichroism, even at low concentrations below the actual CMC. Despite the small change in the peptide sequence (swapping of G and R residues), there is surprisingly a significant difference in the aggregation propensity and association number, both of which are greater for C 16 -CSK 4 GRDS. Both lipopeptides are cytocompatible (with fibroblasts and myoblasts) at low concentration, although cytotoxicity is noted at higher concentration.

Peptide-polymer systems hold strong potential for applications in nanotherapeutics. Desmopressin, a synthetic analogue of the antidiuretic hormone arginine vasopressin, may serve as a valuable case of study in this context since it is a first-line treatment for disorders affecting water homeostasis, including diabetes insipidus. It also has an established use as a hemostatic agent in von Willebrand disease, and recently, its repurposing has been suggested as a neoadjuvant in the treatment of certain types of cancer. Despite its well-documented clinical uses, studies on the supramolecular organization of desmopressin and its association with polymers remain scarce, limiting the therapeutic benefits of these nanostructured arrays. Here, we investigate the self-assembly of desmopressin and its association with sodium polystyrene sulphonate (NaPSS), a potassium-binding polymer used to treat hyperkalemia. Using structural techniques such as small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and atomic force microscopy combined with infrared nanospectroscopy (AFM-IR), we identified that desmopressin associates with NaPSS to form hybrid fibrillar nanoassemblies characterized by β-turn enriched domains and the appearance of β-sheet content. In vitro cytotoxicity assays conducted on breast cancer cell lines MCF-7 and MDA-MB-231 showed that NaPSS/desmopressin complexes are well-tolerated by the non-metastatic MCF-7 cells while displaying inhibitory effects against the metastatic MDA-MB-231 cells. The findings presented here, which demonstrate the successful association between two clinically validated drugs and the ability of the hybrid matrix to modulate cell interactions, potentially contribute to the design of peptide-polymer therapeutic systems.

The interaction of the surfactant-like peptide (SLP) R 3 L 12 bearing three cationic arginine residues with model liposomes is investigated in aqueous solution at various pH values, under conditions for which the SLP self-assembles into nanotubes. The structure of liposomes of model anionic lipid DPPG [1,2-dipalmitoyl- sn -glycero-3-phospho-rac-(1-glycerol)], or zwitterionic lipid DPPE [1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine] is probed using small-angle X-ray scattering and cryogenic-transmission electron microscopy. The unilamellar vesicles of DPPG are significantly restructured in the presence of R 3 L 12 , especially at low pH, and multilamellar vesicles of DPPE are also restructured under these conditions. The SLP promotes the release of cargo encapsulated in the vesicles as probed by calcein fluorescence, with notably higher release for anionic DPPG vesicles. Laurdan fluorescence experiments to probe membrane fluidity (lipid chain ordering) show that R 3 L 12 destabilizes the lipid gel phase, especially for anionic DPPG. This model nanotube-forming SLP has promise as a pH-sensitive release system for vesicle-encapsulated cargo.

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.

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 Ca 2+ 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.

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.

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.

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 β-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.

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., (© 2021 Wiley Periodicals LLC.)

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.

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-β-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 β-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 β-sheets are key steps for organization is proposed.