Single-Response Duplexing of Electrochemical Label-Free Biosensor from the Same Tag.

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors - Benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy.

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

ZnO-Based Electrochemical Immunosensor to Assess Vaccine-Induced Antibody-Mediated Immunity against Wild-Type and Gamma SARS-CoV-2 Strains.

The evaluation of serological responses to COVID-19 is crucial for population-level surveillance, developing new vaccines, and evaluating the efficacy of different immunization programs. Research and development of point-of-care test technologies remain essential to improving immunity assessment, especially for SARS-CoV-2 variants that partially evade vaccine-induced immune responses. In this work, an impedimetric biosensor based on the immobilization of the recombinant trimeric wild-type spike protein (S protein) on zinc oxide nanorods (ZnONRs) was employed for serological evaluation. We successfully assessed its applicability using serum samples from spike-based COVID-19 vaccines: ChAdOx1-S (Oxford-AstraZeneca) and BNT162b2 (Pfizer-BioNTech). Overall, the ZnONRs/ spike-modified electrode displayed accurate results for both vaccines, showing excellent potential as a tool for assessing and monitoring seroprevalence in the population. A refined outcome of this technology was achieved when the ZnO immunosensor was functionalized with the S protein from the P.1 linage (Gamma variant). Serological responses against samples from vaccinated individuals were acquired with excellent performance. Following studies based on traditional serological tests, the ZnONRs/spike immunosensor data reveal that ChAdOx1-S vaccinated individuals present significantly less antibody-mediated immunity against the Gamma variant than the BNT162b2 vaccine, highlighting the great potential of this point-of-care technology for evaluating vaccine-induced humoral immunity against different SARS-CoV-2 strains.

Electrochemical Immunosensors Based on Zinc Oxide Nanorods for Detection of Antibodies Against SARS-CoV-2 Spike Protein in Convalescent and Vaccinated Individuals.

Even after over 2 years of the COVID-19 pandemic, research on rapid, inexpensive, and accurate tests remains essential for controlling and avoiding the global spread of SARS-CoV-2 across the planet during a potential reappearance in future global waves or regional outbreaks. Assessment of serological responses for COVID-19 can be beneficial for population-level surveillance purposes, supporting the development of novel vaccines and evaluating the efficacy of different immunization programs. This can be especially relevant for broadly used inactivated whole virus vaccines, such as CoronaVac, which produced lower titers of neutralizing antibodies. and showed lower efficacy for specific groups such as the elderly and immunocompromised. We developed an impedimetric biosensor based on the immobilization of SARS-CoV-2 recombinant trimeric spike protein (S protein) on zinc oxide nanorod (ZnONR)-modified fluorine-doped tin oxide substrates for COVID-19 serology testing. Due to electrostatic interactions, the negatively charged S protein was immobilized via physical adsorption. The electrochemical response of the immunosensor was measured at each modification step and characterized by scanning electron microscopy and electrochemical techniques. We successfully evaluated the applicability of the modified ZnONR electrodes using serum samples from COVID-19 convalescent individuals, CoronaVac-vaccinated with or without positive results for SARS-CoV-2 infection, and pre-pandemic samples from healthy volunteers as controls. ELISA for IgG anti-SARS-CoV-2 spike protein was performed for comparison, and ELISA for IgG anti-RBDs of seasonal coronavirus (HCoVs) was used to test the specificity of immunosensor detection. No cross-reactivity with HCoVs was detected using the ZnONR immunosensor, and more interestingly, the sensor presented higher sensitivity when compared to negative ELISA results. The results demonstrate that the ZnONRs/spike-modified electrode displayed sensitive results for convalescents and vaccinated samples and shows excellent potential as a tool for the population's assessment and monitoring of seroconversion and seroprevalence.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Looking into a crystal ball: printing and patterning self-assembled peptide nanostructures.

The solution processability of organic semiconductors and conjugated polymers along with the advent of nanomaterials as conducting inks have revolutionized next-generation flexible consumer electronics. Another equally important class of nanomaterials, self-assembled peptides, heralded as next-generation materials for bioelectronics, have a lot of potential in printed technology. In this minireview, we address the self-assembly process in dipeptides, their application in electronics, and recent progress in three-dimensional printing. The prospect of a generalizable path for nanopatterning self-assembled peptides using ice lithography and its challenges are further discussed.

Interaction between glyphosate pesticide and amphiphilic peptides for colorimetric analysis.

The large-scale use of glyphosate pesticides in food production has attracted attention due to environmental damage and toxicity risks. Several regulatory authorities have established safe limits or concentrations of these pesticides in water and various food products consumed daily. The irreversible inhibition of acetylcholinesterase (AChE) activity is one of the strategies used for pesticide detection. Herein, we found that lipopeptide sequences can act as biomimetic microenvironments of AChE, showing higher catalytic activities than natural enzymes in an aqueous solution, based on IC50 values. These biomolecules contain in the hydrophilic part the amino acids l-proline (P), l-arginine (R), l-tryptophan (W), and l-glycine (G), covalently linked to a hydrophobic part formed by one or two long aliphatic chains. The obtained materials are referred to as compounds 1 and 2, respectively. According to fluorescence assays, 2 is more hydrophobic than 1. The circular dichroism (CD) data present a significant difference in the molar ellipticity values, likely related to distinct conformations assumed by the proline residue in the lipopeptide supramolecular structure in solution. The morphological aspect was further characterized using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM), which showed that compounds 1 and 2 self-assembly into cylindrical and planar core-shell structures, respectively. The mimetic AchE behaviour of lipopeptides was confirmed by Ellman's hydrolysis reaction, where the proline residue in the peptides act as a nucleophilic scavenger of organophosphate pesticides. Moreover, the isothermal titration calorimetry (ITC) experiments revealed that host-guest interactions in both systems were dominated by enthalpically-driven thermodynamics. UV-vis kinetic experiments were performed to assess the inhibition of the lipopeptide catalytic activity and the IC50 values were obtained, and we found that the detection limit correlated with the increase in hydrophobicity of the lipopeptides, implying the micellization process is more favorable.

Modular Label-Free Electrochemical Biosensor Loading Nature-Inspired Peptide toward the Widespread Use of COVID-19 Antibody Tests.

Limitations of the recognition elements in terms of synthesis, cost, availability, and stability have impaired the translation of biosensors into practical use. Inspired by nature to mimic the molecular recognition of the anti-SARS-CoV-2 S protein antibody (AbS) by the S protein binding site, we synthesized the peptide sequence of Asn-Asn-Ala-Thr-Asn-COOH (abbreviated as PEP2003) to create COVID-19 screening label-free (LF) biosensors based on a carbon electrode, gold nanoparticles (AuNPs), and electrochemical impedance spectroscopy. The PEP2003 is easily obtained by chemical synthesis, and it can be adsorbed on electrodes while maintaining its ability for AbS recognition, further leading to a sensitivity 3.4-fold higher than the full-length S protein, which is in agreement with the increase in the target-to-receptor size ratio. Peptide-loaded LF devices based on noncovalent immobilization were developed by affording fast and simple analyses, along with a modular functionalization. From studies by molecular docking, the peptide-AbS binding was found to be driven by hydrogen bonds and hydrophobic interactions. Moreover, the peptide is not amenable to denaturation, thus addressing the trade-off between scalability, cost, and robustness. The biosensor preserves 95.1% of the initial signal for 20 days when stored dry at 4 °C. With the aid of two simple equations fitted by machine learning (ML), the method was able to make the COVID-19 screening of 39 biological samples into healthy and infected groups with 100.0% accuracy. By taking advantage of peptide-related merits combined with advances in surface chemistry and ML-aided accuracy, this platform is promising to bring COVID-19 biosensors into mainstream use toward straightforward, fast, and accurate analyses at the point of care, with social and economic impacts being achieved.

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.

In Situ Nanocoating on Porous Pyrolyzed Paper Enables Antibiofouling and Sensitive Electrochemical Analyses in Biological Fluids.

Electrochemical detection in complex biofluids is a long-standing challenge as electrode biofouling hampers its sensing performance and commercial translation. To overcome this drawback, pyrolyzed paper as porous electrode coupled with the drop casting of an off-the-shelf polysorbate, that is, Tween 20 (T20), is described here by taking advantage of the in situ formation of a hydrophilic nanocoating (2 nm layer of T20). The latter prevents biofouling while providing the capillarity of samples through paper pores, leveraging redox reactions across both only partially fouled and fresh electrodic surfaces with increasing detection areas. The nanometric thickness of this blocking layer is also essential by not significantly impairing the electron-transfer kinetics. These phenomena behave synergistically to enhance the sensibility that further increases over long-term exposures (4 h) in biological fluids. While the state-of-the-art antibiofouling strategies compromise the sensibility, this approach leads to peak currents that are up to 12.5-fold higher than the original currents after 1 h exposure to unprocessed human plasma. Label-free impedimetric immunoassays through modular bioconjugation by directly anchoring spike protein on gold nanoparticles are also allowed, as demonstrated for the COVID-19 screening of patient sera. The scalability and simplicity of the platform combined with its unique ability to operate in biofluids with enhanced sensibility provide the generation of promising biosensing technologies toward real-world applications in point-of-care diagnostics, mass testing, and in-home monitoring of chronic diseases.

Structure optimization of lipopeptide assemblies for aldol reactions in an aqueous medium.

Four amphiphilic peptides were synthesized, characterized, and evaluated regarding their efficiency in the catalysis of direct aldol reactions in water. The lipopeptides differ by having a double lipid chain and a guanidinium pyrrole group functionalizing one Lys side chain. All the samples are composed of the amino acids l-proline (P), l-arginine (R), or l-lysine (K) functionalized with the cationic guanidiniocarbonyl pyrrole unit (GCP), l-tryptophan (W), and l-glycine (G), covalently linked to one or two long aliphatic chains, leading to surfactant-like designs with controlled proline protonation state and different stereoselectivity. Critical aggregation concentrations (cac) were higher in the presence of the GCP group, suggesting that self-assembly depends on charge distribution along the peptide backbone. Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS) showed a rich polymorphism including spherical, cylindrical, and bilayer structures. Molecular dynamics simulations performed to assess the lipopeptide polymorphs revealed an excellent agreement with core-shell arrangements derived from SAXS data and provided an atomistic view of the changes incurred by modifying head groups and lipid chains. The resulting nanostructures behaved as excellent catalysts for aldol condensation reactions, in which superior conversions (>99%), high diastereoselectivities (ds = 94 : 6), and enantioselectivities (ee = 92%) were obtained. Our findings contribute to elucidate the effect of nanoscale organization of lipopeptide assemblies in the catalysis of aldol reactions in an aqueous environment.

Peptide-Tetrapyrrole Supramolecular Self-Assemblies: State of the Art.

The covalent and noncovalent association of self-assembling peptides and tetrapyrroles was explored as a way to generate systems that mimic Nature's functional supramolecular structures. Different types of peptides spontaneously assemble with porphyrins, phthalocyanines, or corroles to give long-range ordered architectures, whose structure is determined by the features of both components. The regular morphology and ordered molecular arrangement of these systems enhance the photochemical properties of embedded chromophores, allowing applications as photo-catalysts, antennas for dye-sensitized solar cells, biosensors, and agents for light-triggered therapies. Chemical modifications of peptide and tetrapyrrole structures and control over the assembly process can steer the organization and influence the properties of the resulting system. Here we provide a review of the field, focusing on the assemblies obtained from different classes of self-assembling peptides with tetrapyrroles, their morphologies and their applications as innovative functional materials.

Amyloid Formation by Short Peptides in the Presence of Dipalmitoylphosphatidylcholine Membranes.

The aggregation of two short peptides, [RF] and [RF]4 (where R = arginine and F = phenylalanine), at dipalmitoylphosphatidylcholine (DPPC) model membranes was investigated at the air-water interface using the Langmuir technique and vesicles in aqueous solutions. The molar ratio of the peptide and lipid components was varied to provide insights into the peptide-membrane interactions, which might be related to their cytotoxicity. Both peptides exhibited affinity to the DPPC membrane interface and rapidly adopted ß-sheet-rich structures upon adsorption onto the surface of the zwitterionic membrane. Results from adsorption isotherm and small-angle X-ray scattering experiments showed changes in the structural and thermodynamic parameters of the membrane with increasing peptide concentration. Using atomic force microscopy, we showed the appearance of pores through the bilayer membranes and peptide aggregation at different interfaces, suggesting that the hydrophobic residues might have an effect on both pore size and layer structure, facilitating the membrane disruption and leading to different cytotoxicity effects.

Sono-Assembly of the [Arg-Phe]4 Octapeptide into Biofunctional Nanoparticles.

High-frequency ultrasound treatment is found to be a one-pot green technique to produce peptide-based nanostructures by ultrasound assisted self-assembly of oligopeptides. [Arg-Phe]4 octapeptides, consisting of alternating arginine (Arg/R) and phenylalanine (Phe/F) sequences, were subjected to 430 kHz ultrasound in aqueous solution in the absence of any external agents, to form [RF]4 nanoparticles ([RF]4-NPs), ~220 nm in diameter. A comprehensive analysis of the obtained nanoparticles demonstrated that the aromatic moieties of the oligopeptides can undergo oxidative coupling to form multiple oligomeric species, which then self-assemble into well-defined fluorescent nanoparticles. [RF]4-NPs were functionalized with polyethylene glycol (PEGylated) to improve their colloidal stability. Unlike the parent peptide, the PEGylated [RF]4-NPs showed limited cytotoxicity towards MDA-MB-231 cells. Furthermore, the intracellular trafficking of PEGylated [RF]4-NPs was investigated after incubation with MDA-MB-231 cells to demonstrate their efficient endo-lysosomal escape. This work highlights that the combined use of ultrasonic technologies and peptides enables easy fabrication of nanoparticles, with potential application in drug delivery.

Polymorphism of asymmetric catalysts based on amphiphilic lipopeptides in solution.

The self-assembly of model [P]RWG lipopeptides (P: l-proline, R: l-arginine, W: l-tryptophan, G: l-glycine), containing one or two aliphatic octadecyl (C18) chains in water and cyclohexanone/water solutions was examined. The self-assembly of mixtures of these RWG and PRWG lipopeptides was also investigated. These materials presented a similar critical aggregation concentration of ∼4.0 × 10-4 wt% and were characterized by unordered secondary structures with some ß-sheet content. TEM and cryo-TEM revealed the presence of mainly nanotape structures with micelles observed for systems rich in PRWG(C18H37). Analysis of detailed SAXS form factor measurements revealed the presence of bilayers 3-4 nm thick while the PRWG(C18H37) micelles have a core radius of approximately 3 nm, and a shell thickness of 2 nm. For the cyclohexanone/water systems polymorphs containing cluster aggregates (with radius of 0.25 nm to 0.50 nm) and some elongated structures (with radius of 5.7 nm to 26.1 nm) were seen. Longer structures were formed with the increase of the proline-containing lipopeptide content. The catalytic activity of these peptides was assessed using a model nitro-aldol reaction. The concentration of water in the reaction system influenced the conversion, higher content promoted better efficiency for the water systems, but the opposite was observed for the cyclohexanone/water samples.

Self-assembled gold nanoparticles and amphiphile peptides: a colorimetric probe for copper(II) ion detection.

Morphological, spectroscopic and scattering studies of the self-assembly and aggregation process of hybrids containing gold nanoparticles (AuNPs) and the amyloid peptides [RF]4 and P[RF]4 (where R = arginine; F = phenylalanine; P = proline) in aqueous solution were performed. Two methodologies were tested for the AuNP nucleation, using sodium borohydride (NaBH4) or epigallocatechin gallate (EGCG) as a reducing agent. This led to remarkable distinct modes of assembly, AuNP decorated fibrils with NaBH4 reduction or isolated AuNPs with EGCG reduction. For both methodologies, the presence of spherical AuNPs was observed by plasmonic resonance bands in absorption spectra at ∼520 nm. Zeta potential measurements confirmed stable systems, with a similar aggregation state. Circular dichroism spectra revealed an antiparallel ß-sheet conformation of the peptides. The transmission electron microscopy (TEM) images showed the coexistence of nanometer fibers and globular nanoparticles with 20 nm size. The small-angle X-ray scattering (SAXS) results show that the NaBH4 systems presented large cylindrical structures, while with increasing P[RF]4 content, a decrease in radius was observed. However, the EGCG-AuNPs were characterized by spherical particles, with a radius of 10-20 nm. Also, the colorimetric efficiency of the hybrids in the capture of Cu2+ ions in solution was monitored. Raman spectroscopy data confirmed the conformation/structure of self-assembled samples. Moreover, there are indications for a surface-enhanced Raman spectroscopy (SERS) effect for Cu2+ sites. The set of results indicates that these systems could act as a promising sensitive metal concentration probes.

Influence of Preparation Methodology on the Photocatalytic Activity of Nitrogen Doped Titanate and TiO2 Nanotubes.

Titanate and TiO2 nanotubes (TiNTs and TiO2NTs) are known as good photocatalysts under UV light irradiation, due to their high band gap energy. The nitrogen-doping process is one of the most studied methods to make these materials sensitive to visible light. However, most of these studies are focused on how calcination temperature affects the crystalline structure and, consequently, the photocatalytic efficiency, and few are investigated about the choice of the doping method. Here we report the synthesis of nitrogen-doped TiNTs and TiO2NTs (NTiNTs and NTiO2NT) through two different methods, and the comparison of the obtained materials properties. The photocatalytic efficiency was evaluated under visible light and correlated to the nature of nitrogen doping, which is a consequence of the doping method chosen. We found that the NTiNT's lamellar structure and its more negative zeta potential shall facilitate its interaction with the dye to be degraded. Moreover, the adsorbed nitrogen plays an important role over the degradation process.

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.

Amyloid Peptide Mixtures: Self-Assembly, Hydrogelation, Nematic Ordering, and Catalysts in Aldol Reactions.

Morphological, spectroscopic, and scattering studies of the self-assembly and aggregation of mixtures of [RF]4 and P[RF]4 peptides (where R = arginine; F = phenylalanine; P = proline), in solution and as hydrogels, were performed to obtain information about polymorphism. CD data confirmed a ß-sheet secondary structure in aqueous solution, and TEM images revealed nanofibers with diameters of ∼10 nm and micrometer lengths. SAXS curves were fitted using a mass fractal-component and a long cylinder shell form factor for the liquid samples, and only a long cylinder shell form factor for the gels. Increasing the P[RF]4 content in the systems leads to a reduction in cylinder radius and core scattering density, suggesting an increase in packing of the peptide molecules; however, the opposite effect is observed for the gels, where the scattering density is higher in the shell for the systems containing higher P[RF]4 content. These compounds show potential as catalysts in the asymmetric aldol reactions, with cyclohexanone and p-nitrobenzaldehyde in aqueous media. A moderate conversion (36.9%) and a good stereoselectivity (69:31) were observed for the system containing only [RF]4. With increasing P[RF]4 content, a considerable decrease of the conversion was observed, suggesting differences in the self-assembly and packing factor. Rheological measurements were performed to determine the shear moduli for the soft gels.