RESUMEN
Mixed thermoreversible gels were successfully fabricated by the addition of a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), to fibrillar nanostructures self-assembled from a short peptide I3K. When the temperature was increased above the lower critical solution temperature of the PNIPAM, the molecules collapsed to form condensed globular particles, which acted as cross-links to connect different peptide nanofibrils and freeze their movements, resulting in the formation of a hydrogel. Since these processes were physically driven, such hydrogels could be reversibly switched between the sol and gel states as a function of temperature. As a model peptide, I3K was formulated with PNIPAM to produce a thermoreversible sol-gel system with a transition temperature of â¼33 °C, which is just below the body temperature. The antibacterial peptide of G(IIKK)3I-NH2 could be conveniently encapsulated in the hydrogel by the addition of the solution at lower temperatures in the sol phase and then increasing the temperature to be above 33 °C for gelation. The hydrogel gave a sustained and controlled linear release of G(IIKK)3I-NH2 over time. Using the peptide nanofibrils as three-dimensional scaffolds, such thermoresponsive hydrogels mimic the extracellular matrix and could potentially be used as injectable hydrogels for minimally invasive drug delivery or tissue engineering.
Asunto(s)
Resinas Acrílicas/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Sistemas de Liberación de Medicamentos , Hidrogeles/farmacología , Resinas Acrílicas/química , Péptidos Catiónicos Antimicrobianos/química , Humanos , Hidrogeles/química , Temperatura , Sensación Térmica , Ingeniería de TejidosRESUMEN
Molecular dynamics (MD) simulations, stochastic optical reconstruction microscopy (STORM), and neutron reflection (NR) were combined to explore how antimicrobial peptides (AMPs) can be designed to promote the formation of nanoaggregates in bacterial membranes and impose effective bactericidal actions. Changes in the hydrophobicity of the designed AMPs were found to have a strong influence on their bactericidal potency and cytotoxicity. G(IIKK)3I-NH2 (G3) achieved low minimum inhibition concentrations (MICs) and effective dynamic kills against both antibiotic-resistant and -susceptible bacteria. However, a G3 derivative with weaker hydrophobicity, KI(KKII)2I-NH2 (KI), exhibited considerably lower membrane-lytic activity. In contrast, the more hydrophobic G(ILKK)3L-NH2 (GL) peptide achieved MICs similar to those observed for G3 but with worsened hemolysis. Both the model membranes studied by Brewster angle microscopy, zeta potential measurements, and NR and the real bacterial membranes examined with direct STORM contained membrane-inserted peptide aggregates upon AMP exposure. These structural features were well supported by MD simulations. By revealing how AMPs self-assemble in microbial membranes, this work provides important insights into AMP mechanistic actions and allows further fine-tuning of antimicrobial potency and cytotoxicity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Materiales Biocompatibles/farmacología , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Tensoactivos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Materiales Biocompatibles/química , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Tamaño de la Partícula , Agregado de Proteínas , Propiedades de Superficie , Tensoactivos/químicaRESUMEN
Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides was formed by attaching an acyl chain to the N-terminus of α-helix-forming peptides with the sequence Cx-G(IIKK)yI-NH2 (CxGy, x = 4-12 and y = 2). CxGy self-assemble into nanofibers above their critical aggregation concentrations (CACs). With increasing x, the CACs decrease and the hydrophobic interactions increase, promoting secondary structure transitions within the nanofibers. Antimicrobial activity, determined by the minimum inhibition concentration (MIC), also decreases with increasing x, but the MICs are significantly smaller than the CACs, suggesting effective bacterial membrane-disrupting power. Unlike conventional antibiotics, both C8G2 and C12G2 can kill Staphylococcus aureus and Escherichia coli after only minutes of exposure under the concentrations studied. C12G2 nanofibers have considerably faster killing dynamics and lower cytotoxicity than their nonaggregated monomers. Antimicrobial activity of peptide aggregates has, to date, been underexploited, and it is found to be a very promising mechanism for peptide design. Detailed evidence for the molecular mechanisms involved is provided, based on superresolution fluorescence microscopy, solid-state nuclear magnetic resonance, atomic force microscopy, neutron scattering/reflectivity, circular dichroism, and Brewster angle microscopy.
Asunto(s)
Antiinfecciosos/química , Lipopéptidos/química , Secuencia de Aminoácidos , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Lipopéptidos/metabolismo , Lipopéptidos/farmacología , Liposomas/química , Liposomas/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente , Nanofibras/química , Conformación Proteica en Hélice alfa , Staphylococcus aureus/efectos de los fármacos , Tensión SuperficialRESUMEN
An optical sensor for quantitative analysis of ultrathin films and adsorbed layers is described. Quantification of both layer thickness and refractive index (density) can be made for in situ and ex-situ coated films. With the use of two polarizations, in situ measurements are made via one path length in a young's interferometer arrangement while ex-situ measurements use multiple path lengths. The multiple path length young's interferometer arrangement is embodied in a solid state waveguide configuration called the multiple path length dual polarization interferometer (MPL-DPI). The technique is demonstrated with ultrathin layers of poly(methylmethacrylate) and human serum albumin.
Asunto(s)
Técnicas Biosensibles/instrumentación , Óptica y Fotónica , Albúmina Sérica/química , Adsorción , Técnicas Biosensibles/métodos , Electroquímica/métodos , Diseño de Equipo/métodos , Humanos , Interferometría/métodos , Fibras Ópticas , Polimetil Metacrilato/química , Dispersión de Radiación , Silicio/química , Resonancia por Plasmón de Superficie/métodosRESUMEN
The complexation behaviour of duplex linear DNA (negatively charged) with amidine functionalised sub-micron latex spheres (positively charged) was studied using dynamic light scattering (DLS) and a PALS interferrometric zeta potential sizer. Four types of DNA-sphere complex were investigated as a function of component concentration by combining amidine functionalised polystyrene microspheres with radii of 10.5 nm and 60 nm, and herring DNA of lengths of 35 nm and 85 nm. At low DNA concentrations (c(DNA)), the undercharged complexes showed a small increase in measured hydrodynamic radius (R(h)) and a decrease in zeta potential with increasing c(DNA). Within a critical DNA concentration range R(h) was seen to peak sharply, and the zeta potentials were approximately 0 mV, corresponding to the formation of unstable neutral complexes. Immediately above this concentration region the measured R(h) values became comparable with those at low c(DNA), and the zeta potential became negative, indicating the formation of stable overcharged complexes. The small and large spheres formed multi-sphere and single sphere overcharged aggregates respectively, which is thought to be determined by the relative magnitude of the chain persistence length (approximately 50 nm) and the sphere radius, switching on or off the DNA bridging interaction.
Asunto(s)
Amidinas/química , ADN/química , Microesferas , Poliestirenos/química , Adsorción , ElectroquímicaRESUMEN
The self-assembly of peptides is explored as an alternative route towards the development of new injectable joint lubricants for osteoarthritis (OA). The versatility of the peptide chemistry allows the incorporation of behavior reminiscent of hyaluronic acid (HA), while the triggered in situ self-assembly provides easy delivery of the samples by injection due to the low viscosity of the peptide solutions (that are initially monomeric). Using design criteria based on the chemical properties of HA, a range of de novo peptides were prepared with systematic alterations of charge and hydrophilicity that self-assembled into nematic fluids and gels in physiological solution conditions. The frictional characteristics of the peptides were evaluated using cartilage on cartilage sliding contacts along with their rheological characteristics. Peptide P(11)-9, whose molecular, mesoscopic, and rheological properties most closely resembled HA was found to be the most effective lubricant amongst the peptides. In healthy static and dynamic friction testing (corresponding to healthy joints) P(11)-9 at 20-40 mg/mL performed similar to HA at 10 mg/mL. In friction tests with damaged cartilage (corresponding to early stage OA) P(11)-9 was a less efficient lubricant than HA, but still the best among all the peptides tested. The results indicate that de novo self-assembling peptides could be developed as an alternate therapeutic lubricant for early stage OA.
Asunto(s)
Lubrificación , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Osteoartritis/tratamiento farmacológico , Secuencia de Aminoácidos , Materiales Biocompatibles , Cartílago/efectos de los fármacos , Fricción , Humanos , Inyecciones , Articulaciones/efectos de los fármacos , Articulaciones/fisiología , Oligopéptidos/administración & dosificaciónRESUMEN
The salivary mucins that include MUC5B (gel-forming) and MUC7 (non-gel-forming) are major contributors to the protective mucus barrier in the oral cavity, and it is possible that dietary components may influence barrier properties. We show how one dietary compound, the green tea polyphenol epigallocatechin gallate (EGCG), can substantially alter the properties of both the polymeric MUC5B network and monomeric MUC7. Using rate-zonal centrifugation, MUC5B in human whole saliva and MUC5B purified from saliva sedimented faster in the presence of EGCG. The faster sedimentation by EGCG was shown to be greater with increasing MUC5B concentration. Particle tracking microrheology was employed to determine the viscosity of purified MUC5B solutions and showed that for MUC5B solutions of 200-1600 µg/mL, EGCG caused a significant increase in mucin viscosity, which was greater at higher MUC5B concentrations. Visualisation of the changes to the MUC5B network by EGCG was performed using atomic force microscopy, which demonstrated increased aggregation of MUC5B in a heterogeneous manner by EGCG. Using trypsin-resistant, high-molecular weight oligosaccharide-rich regions of MUC5B and recombinant N-terminal and C-terminal MUC5B proteins, we showed that EGCG causes aggregation at the protein domains of MUC5B, but not at the oligosaccharide-rich regions of the mucin. We also demonstrated that EGCG caused the majority of MUC7 in human whole saliva to aggregate. Furthermore, purified MUC7 also underwent a large increase in sedimentation rate in the presence of EGCG. In contrast, the green tea polyphenol epicatechin caused no change in the sedimentation rate of either MUC5B or MUC7 in human whole saliva. These findings have demonstrated how the properties of the mucin barrier can be influenced by dietary components. In the case of EGCG, these interactions may alter the function of MUC5B as a lubricant, contributing to the astringency (dry puckering sensation) of green tea.
Asunto(s)
Catequina/análogos & derivados , Catequina/metabolismo , Mucina 5B/metabolismo , Mucinas/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Camellia sinensis , Centrifugación Zonal , Dieta , Humanos , Microscopía de Fuerza Atómica , Polifenoles/metabolismo , Agregado de Proteínas , Estructura Terciaria de Proteína , TéRESUMEN
The parameters that influence the deformation of viscoelastic drops impinging on a viscoelastic bath, and that lead to the detachment of these drops from the surface were investigated. A range of PEO solutions with different viscosities and molecular weights was used and the deformation of each drop during impact was observed using a high speed camera. The use of image analysis to measure the evolution of interfacial areas between the drop and the cavity formed during impact allowed the estimation of the potential and interfacial energies. This gave valuable information for the understanding of drop detachment. The drops need to retain a sufficiently high kinetic energy after impact in order to pass through the surface. It is therefore necessary to limit the deformation of the drop as well as the deformation of the bath (i.e. cavity depth) by increasing the drop viscosity. Reducing the kinetic energy of the drop at the moment of impact also limits deformation and promotes detachment of the drop.
Asunto(s)
Aire , Soluciones/química , Viscosidad , Cinética , Peso Molecular , PolietilenglicolesRESUMEN
We examined a series of novel cationic MPC-based (2-methacryloyloxyethyl phosphorylcholine) copolymers as vectors for gene delivery, with emphasis on the assessment of the effects of the charge ratio (administered via pH variation) on the complex (polyplex) formation and the subsequent transfection efficiency. A combination of electrophoresis, dynamic light scattering, and small angle neutron scattering was used to characterize the structure and charge distribution of the polyplexes formed between the copolymer and the luciferase plasmid DNA. Polymers with larger hydrophobic side chains had lower p K a values and tended to aggregate more strongly. For a given copolymer, electrostatic interaction was the main driving force for the formation of the nanopolyplexes. When the cationic copolymers were in excess, the majority of the polyplexes formed was neutral, and only a small faction of them carried net positive charges. Polyplexes formed under excess copolymer protected the DNA from restriction enzyme digestion. As the copolymers were weak polyelectrolytes, the pH had a distinct effect on the structure and charge distribution of the polyplexes formed. Below the p K a, the copolymers were found to bind with the plasmid DNA in the form of unimers, while above the p K a, the copolymers self-aggregated and complexed with DNA in the form of micelles. It was subsequently found that unimer/DNA polyplexes were far more effective in the transfection of HEK293 cells than micellar DNA polyplexes. The results thus revealed that different hydrophobicities of the side chains in the copolymer series led to different nanostructuring and charge characteristics, which had a consequential effect on the transfection efficiency. This study provided useful insight into the molecular processes underlying polyplex formation and demonstrated a strong link between structural and physical properties of polyplexes and cell transfection efficiency.