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.

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.

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.

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.

Self-assembly and intracellular delivery of DNA by a truncated fragment derived from the Trojan peptide Penetratin.

Penetratin is a short Trojan peptide that attracts great interest in biomedical research for its capacity to translocate biological membranes. Herein, we study in detail both self-assembly and intracellular delivery of DNA by the heptamer KIWFQNR, a truncated peptide derived from Penetratin. This shortened sequence possesses a unique design with bolaamphiphilic characteristics that preserves the longest noncationic amino acid portion found in Penetratin. These features convey amphipathicity to assist self-assembly and make it a suitable model for exploring the role of hydrophobic residues for peptide interaction and cell uptake. We show that the fragment forms peptiplexes (i.e., peptide-DNA complexes), and aggregates into long nanofibers with clear ß-sheet signature. The supramolecular structure of nanofibers is likely composed of DNA cores surrounded by a peptide shell to which the double helix behaves as a template and induces fibrillization. A nucleation and growth mechanism proceeding through liquid-liquid phase separation of coacervates is proposed for describing the self-assembly of peptiplexes. We also demonstrate that peptiplexes deliver double-stranded 200 bp DNA into HeLa cells, indicating its potential for preparing non-viral vectors for oligonucleotides through noncovalent strategies. Since the main structural features of native Penetratin are conserved in this simpler fragment, our findings also highlight the role of uncharged amino acids for structuration, and thus for the ability of Penetratin to cross cell membranes.

Amphipathic design dictates self-assembly, cytotoxicity and cell uptake of arginine-rich surfactant-like peptides.

Amphiphilicity is the most critical parameter in the self-assembly of surfactant-like peptides (SLPs), regulating the way by which hydrophobic attraction holds peptides together. Its effects go beyond supramolecular assembly and may also trigger different cell responses of bioactive peptide-based nanostructures. Herein, we investigate the self-assembly and cellular effects of nanostructures based on isomeric SLPs composed by arginine (R) and phenylalanine (F). Two amphipathic designs were studied: a diblock construct F4R4 and its bolaamphiphile analog R2F4R2. A strong sequence-dependent polymorphism emerges with appearance of globules and vesicle-like assemblies, or flat nanotapes and cylindrical micelles. The diblock construct possesses good cell penetrating capabilities and effectiveness to kill SK-MEL-28 melanoma tumor cells, in contrast to reduced intracellular uptake and low cytotoxicity exhibited by the bolaamphiphilic form. Our findings demonstrate that amphipathic design is a relevant variable for self-assembling SLPs to modulate different cellular responses and may assist in optimizing the production of nanostructures based on arginine-enriched sequences in cell penetrating and antimicrobial peptides.

ß-sheet assembly in amyloidogenic glutamic acid nanostructures: Insights from X-ray scattering and infrared nanospectroscopy.

Glutamic acid-rich peptides are crucial to a variety of biological processes, including glutamatergic neurotransmission and immunological defense. Glutamic acid sequences often exhibit unusual organization into ß2 -type sheets, where bifurcated H bonds formed between glutamic acid side chains and NH in amide bonds on adjacent ß-strands play a paramount role for stabilizing the molecular assembly. Herein, we investigate the self-assembly and supramolecular structure of simplified models consisting of alternating glutamic acid/phenylalanine residues. Small-angle X-ray scattering and atomic force microscopy show that the aggregation pathway is characterized by the formation of small oligomers, followed by coalescence into nanofibrils and nanotapes. Amyloidogenic features are further demonstrated through fiber X-ray diffraction, which reveal molecular packing according to cross-ß patterns, where ß-strands appear perpendicularly oriented to the long axis of nanofibrils and nanotapes. Nanoscale infrared spectroscopy from individual nanoparticles on dried samples shows a remarkable decrease of ß2 -sheet content, accompanied by growth of standard ß-sheet fractions, indicating a ß2 -to-ß1 transition as a consequence of the release of solvent from the interstices of peptide assemblies. Our findings highlight the key role played by water molecules in mediating H-bond formation in ß2 -sheets commonly found in amyloidogenic glutamic acid-rich aggregates.

Comparative Cytogenetics of Omophoita abbreviata and O. aequinoctialis (Coleoptera, Chrysomelidae, Alticini) from the Adolpho Ducke Forest Reserve in Brazilian Amazonia: Intrapopulation Variation in Karyotypes.

The chromosomes of 2 flea beetle species from central Amazonia, Omophoita abbreviata and O. aequinoctialis (Alticini), were investigated through analysis of meiotic and mitotic cells. These species belong to the subtribe Oedionychina, a taxon that has unique cytogenetic features, such as giant sex chromosomes which are aligned at a distance during meiosis I (asynaptic). O. abbreviata and O. aequinoctialis have a meiotic formula of 10II + X + y, which is predominant in this subtribe. While the species of the genus Omophoita possess a relatively stable karyotype, a typical feature for Oedionychina, the present study identified inter- and intrapopulational variation in chromosome morphology, constitutive heterochromatin, and the presence and number of B chromosomes in O. aequinoctialis. In addition, FISH mapping of telomeric sequences revealed signals in the collochores, raising several questions on the chromosomal evolution in this group.