RESUMEN
The goal of this study was to determine whether the guluronate (G) rich alginate OligoG CF-5/20 (OligoG) could detach cystic fibrosis (CF) mucus by calcium chelation, which is also required for normal mucin unfolding. Since bicarbonate secretion is impaired in CF, leading to insufficient mucin unfolding and thereby attached mucus, and since bicarbonate has the ability to bind calcium, we hypothesized that the calcium chelating property of OligoG would lead to detachment of CF mucus. Indeed, OligoG could compete with the N-terminus of the MUC2 mucin for calcium binding as shown by microscale thermophoresis. Further, effects on mucus thickness and attachment induced by OligoG and other alginate fractions of different length and composition were evaluated in explants of CF mouse ileum mounted in horizontal Ussing-type chambers. OligoG at 1.5% caused effective detachment of CF mucus and the most potent alginate fraction tested, the poly-G fraction of about 12 residues, had similar potency compared to OligoG whereas mannuronate-rich (M) polymers had minimal effect. In conclusion, OligoG binds calcium with appropriate affinity without any overt harmful effect on the tissue and can be exploited for treating mucus stagnation.
Asunto(s)
Alginatos/química , Alginatos/farmacología , Calcio/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Moco/efectos de los fármacos , Moco/metabolismo , Alginatos/metabolismo , Alginatos/uso terapéutico , Animales , Quelantes/química , Quelantes/metabolismo , Quelantes/farmacología , Quelantes/uso terapéutico , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácido Glucurónico/farmacología , Ácido Glucurónico/uso terapéutico , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Ácidos Hexurónicos/farmacología , Ácidos Hexurónicos/uso terapéutico , Íleon/efectos de los fármacos , Íleon/metabolismo , Ratones , PolimerizacionRESUMEN
Omega-3 nutraceuticals are extensively used as health supplements worldwide. Various administration forms for delivery of omega-3 are available. However, the niche omega-3 tablets have so far remained unexplored. In this work tablets containing 25-40% (w/w) omega-3 oil as triglycerides or ethyl esters were prepared utilizing a direct compaction grade powder with ß-cyclodextrin as encapsulating agent. It was found that powders with up to 35% (w/w) triglyceride oil and 30% (w/w) ethyl ester oil, respectively, can be directly compressed into tablets of excellent quality. Physical properties of omega-3 containing powders and tablets are described. The powder X-ray diffractograms of the powders and crushed tablets show evidence of the formation of new crystalline phases not present in ß-cyclodextrin. In addition, (1)H NMR data suggest that the ethyl esters form inclusion complexes with ß-cyclodextrin. Compaction of other, commercially available, omega-3 powders was performed as a comparison and deemed unsuccessful.
Asunto(s)
Suplementos Dietéticos/análisis , Ácidos Grasos Omega-3/análisis , Polvos/química , Comprimidos/química , Triglicéridos/análisis , beta-Ciclodextrinas/análisisRESUMEN
Omega-3 fatty acids are used in both nutraceuticals and pharmaceuticals in the form of triglycerides and ethyl esters. Administration forms available for omega-3 include bulk oil, soft gel capsules, emulsions and some powder compositions. Cyclodextrins are substances well known for their ability to encapsulate lipophilic molecules. In the present work, powders loaded with omega-3 oil, ranging from 10 to 40% (w/w), have been prepared by vacuum drying, freeze drying or spray granulation of aqueous mixtures of omega-3 oil and ß-cyclodextrin. The powders were found to be partially crystalline by powder X-ray diffraction and to contain crystalline phases not present in pure ß-cyclodextrin, indicating true complexation. The compactibility of the powders has been explored, revealing that a dry and compactible powder can be prepared from various omega-3 oils and ß-cyclodextrin. Spray granulation was found to be the superior drying method for the preparation of compactible powders.
Asunto(s)
Ácidos Grasos Omega-3/química , beta-Ciclodextrinas/química , Desecación , Composición de Medicamentos , Manipulación de Alimentos , Liofilización , Polvos/química , Difracción de Rayos XRESUMEN
The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.
Asunto(s)
Inovirus/metabolismo , Biblioteca de Péptidos , Secuencia de Bases , Vectores Genéticos/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Transformación GenéticaRESUMEN
Display of peptides on filamentous phage, phage display, is an in vitro selection technique well suited for identification of therapeutic peptide binders for a huge variety of protein targets. The peptides are identified in a process where phage libraries are subjected to affinity selection towards a particular protein target. A successful outcome of an affinity selection is dependent on proper surveillance of the phage life cycle, to make sure that the selection is based on affinity for the target, not on bias in phage propagation rate. In this chapter we present two approaches for protein target presentation and a protocol for phage rescue and propagation, which includes several controls to ensure that all phages initially eluted from the protein target are given equal conditions during the following amplification and selection steps.
Asunto(s)
Cromatografía de Afinidad/métodos , Inovirus/metabolismo , Biblioteca de Péptidos , Secuencia de Aminoácidos , Proteínas Inmovilizadas/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Análisis de Secuencia de Proteína , SolucionesRESUMEN
Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 µm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties.
Asunto(s)
Medios de Contraste/química , Microburbujas , Acústica , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Selectina E/metabolismo , Citometría de Flujo , Humanos , Neovascularización Patológica/diagnóstico por imagen , Fosfolípidos/química , Ultrasonografía , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P(BAD) promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 x 10(9) unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.