The self-assembly of lipopeptide (peptide amphiphile) molecules bearing single linear lipid chains has been widely studied, as has their diverse range of bioactivities. Here, we introduce lipopeptides bearing one or two cycloalkane chains (cycloheptadecyl or cyclododecyl) conjugated to the collagen-stimulating pentapeptide KTTKS used in Matrixyl formulations. The self-assembly of all four molecules is probed using fluorescence probe measurements to detect the critical aggregation concentration (CAC), and cryogenic-TEM and small-angle X-ray scattering (SAXS) to image the nanostructure. The peptide conformation is studied using circular dichroism (CD) and FTIR spectroscopies. All the cycloalkane lipopeptides show excellent compatibility with dermal fibroblasts. The compounds bearing one or two cyclododecyl chains (denoted as DKT and DDKT, respectively) show wound healing in diabetic rats, the improvement being markedly enhanced for DDKT. Interestingly, the revival of hair follicles and blood vessels in the dermis were observed, which are the critical markers of effective wound repair. Analysis of H&E-stained tissue images (from a rat model) shows that the rat groups treated with DDKT and DKT displayed a significantly increased amount of regenerated hair follicles, indicating a faster healing process for DDKT compared to the control group. Collagen deposition was also enhanced, especially for DDKT, and by day 20, the DDKT-treated groups had developed a dense collagen network accompanied by a regenerated epidermis. At the same time, the number of blood vessels in DDKT-treated diabetic wounds was significantly higher than in control groups and neovascularization was substantially enhanced, as assayed using α-SMA (a marker for vascular smooth muscle cells) and CD31 (a marker specific to vascular endothelial cells). These results suggest that the lead lipopeptide DDKT exhibits a remarkable pro-vascularization capability and shows great promise for future application as a wound-healing biomaterial.
A histidine-based amphiphilic peptide (P) has been found to form an injectable transparent hydrogel in phosphate buffer solution over a pH range from 7.0 to 8.5 with an inherent antibacterial property. It also formed a hydrogel in water at pH = 6.7. The peptide self-assembles into a nanofibrillar network structure which is characterized by high-resolution transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy, small-angle X-ray scattering, Fourier-transform infrared spectroscopy, and wide-angle powder X-ray diffraction. The hydrogel exhibits efficient antibacterial activity against both Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). The minimum inhibitory concentration of the hydrogel ranges from 20 to 100 μg/mL. The hydrogel is capable of encapsulation of the drugs naproxen (a non-steroidal anti-inflammatory drug), amoxicillin (an antibiotic), and doxorubicin, (an anticancer drug), but, selectively and sustainably, the gel releases naproxen, 84% being released in 84 h and amoxicillin was released more or less in same manner with that of the naproxen. The hydrogel is biocompatible with HEK 293T cells as well as NIH (mouse fibroblast cell line) cells and thus has potential as a potent antibacterial and drug releasing agent. Another remarkable feature of this hydrogel is its magnification property like a convex lens.
ABSTRACT 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.
The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers' disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly "nanodisc" structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.
The confined crystallization of poly(ethylene oxide) (PEO) in predominantly spherical microdomains formed by several diblock copolymers was studied and compared. Two polybutadiene-b-poly(ethylene oxide) diblock copolymers were prepared by sequential anionic polymerization (with approximately 90 and 80 wt % polybutadiene (PB)). These were compared to equivalent samples after catalytic hydrogenation that produced double crystalline polyethylene-b-poly(ethylene oxide) diblock copolymers. Both systems are segregated into microdomains as indicated by small-angle X-ray scattering (SAXS) experiments performed in the melt and at lower temperatures. However, the PB-b-PEO systems exhibited a higher degree of order in the melt. A predominantly spherical morphology of PEO in a PB or a PE matrix was observed by both SAXS and transmission electron microscopy, although a possibly mixed morphology (spheres and cylinders) was formed when the PEO composition was close to the cylinder−sphere domain transitional composition as indicated by SAXS. Differential scanning calorimetry experiments showed that a fractionated crystallization process for the PEO occurred in all samples, indicating that the PE cannot nucleate PEO in these diblock copolymers. A novel result was the observation of a subsequent fractionated melting that reflected the crystallization process. Sequential isothermal crystallization experiments allowed us to thermally separate at least three different crystallization and melting peaks for the PEO microdomains. The lowest melting point fraction was the most important in terms of quantity and corresponded to the crystallization of isolated PEO spheres (or cylinders) that were either superficially or homogeneously nucleated. This was confirmed by Avrami index values of approximately 1. The isothermal crystallization results indicate that the PE matrix restricts the crystallization of the covalently bonded PEO to a higher degree compared to PB.
A diblock copolymer of propylene oxide and ethylene oxide, denoted P94E316, was prepared by sequential anionic polymerization of the two monomers. 13C NMR spectroscopy was used to obtain the absolute number-average molar mass and overall composition (whence the molecular formula), and gel permeation chromatography was used to confirm a narrow chain-length distribution. A number of techniques (light scattering, dye solubilization with DPH (1,6-diphenyl-1,3,5-hexatriene), surface tension) were used to confirm micellization in dilute aqueous solution and to determine the temperature dependence of the critical micelle concentration. Light scattering was also used to determine the temperature dependence of micellar association number and radius. Copolymer solutions of concentration 10 and 20 g dm-3 copolymer were used to solubilize a nematic liquid crystal mixture with a wide nematic range, coded BL002. The extent of solubilization in the temperature range 20−40 °C could be correlated with the extent of micellization of the copolymer, leading to an upper limit at 40 °C of ca. 50 mg (g of copolymer)-1, i.e., 180 mg (g of hydrophobe)-1. It was noted that BL002 solubilization could be adapted to provide a method for determining the critical micelle temperatures of aqueous solutions of the copolymer.
Lipopolysaccharides (LPSs) based on lipid A produced by bacteria are of interest due to their bioactivity in stimulating immune responses, as are simpler synthetic components or analogues. Here, the self-assembly in water of two monodisperse lipid A derivatives based on simplified bacterial LPS structures is examined and compared to that of a native Escherichia coli LPS using small-angle X-ray scattering and cryogenic transmission electron microscopy. The critical aggregation concentration is obtained from fluorescence probe experiments, and conformation is probed using circular dichroism spectroscopy. The E. coli LPS is found to form wormlike micelles, whereas the synthetic analogues bearing six lipid chains and with four or two saccharide head groups (Kdo2-lipid A and monophosphoryl lipid A) self-assemble into nanosheets or vesicles, respectively. These observations are rationalized by considering the surfactant packing parameter.
The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 ± 4.5 Å and minor axial radius of 40.4 ± 0.18 Å. These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5–11) and at elevated temperatures (20–65 °C). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-β-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a.
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