RESUMEN
The adhesion of alginate hydrogels to solid surfaces was probed by atomic force microscopy (AFM) in the sphere/plane geometry. For this purpose a novel approach has been developed for the immobilization of soft colloidal probes onto AFM-cantilevers, which is inspired by techniques originating from cell biology. The aspiration and consecutive manipulation of hydrogel beads by micropipettes allows the entire manipulation sequence to be carried-out in situ. Hence, any alteration of the hydrogel beads upon drying can be excluded. The adhesive behaviour of alginate hydrogels was first evaluated by determining the distribution of pull-off forces on self-assembled monolayers (SAMs) terminating in different functional groups (-CH3, -OH, -NH2, -COOH). It was demonstrated that solvent exclusion plays practically no role in the adhesion process, in clear difference to solid colloidal probes. The adhesion of alginate beads is dominated by chemical interactions rather than solvent exclusion, in particular in the case of amino-terminated SAMs. The data set acquired on the SAMs provided the framework to relate the adhesion of alginate beads on recombinant spider silk protein films to specific functional groups. The preparation of soft colloidal probes and the presented approach in analysing the adhesive behaviour is not limited to alginate hydrogel beads but can be generally applied for probing and understanding the adhesion behaviour of hydrogels on a wide range of substrates, which would be relevant for various applications such as biomedical surface modification or tissue engineering.
RESUMEN
Medical therapies are often accompanied by un-wanted side-effects or, even worse, targeted cells can develop drug resistance leading to an ineffective treatment. Therefore, drug delivery systems are under investigation to lower the risk thereof. Drug carriers should be biocompatible, biodegradable, nontoxic, non-immunogenic, and should show controllable drug loading and release properties. Previous studies qualified spider silk particles as drug delivery carriers, however, cellular uptake was only tested with unloaded spider silk particles. Here, the effect of drug loading on cellular uptake of previously established spider silk-based particles made of eADF4(C16), eADF4(C16)RGD, eADF4(C16)R8G and eADF4(κ16) was investigated. Fluorescently labelled polyethylenimine was used as a model substance for loading eADF4(C16), eADF4(C16)RGD or eADF4(C16)R8G particles, and fluorescently labelled ssDNA was used for loading eADF4(κ16) particles. Upon loading polyanionic eADF4(C16) and eADF4(C16)RGD particles with polycationic polyethylenimine the cellular uptake efficiency was increased, while the uptake of eADF4(C16)R8G and polycationic eADF4(κ16) particles was decreased upon substance loading. The latter could be circumvented by coating substance-loaded eADF4(κ16) particles with an additional layer of eADF4(κ16) (layer-by-layer coating). Further, it could be shown that eADF4(C16)RGD and eADF4(κ16) uptake was based on clathrin-mediated endocytosis, whereas macropinocytosis was more important in case of eADF4(C16) and eADF4(C16)R8G particle uptake. Finally, it was confirmed that drugs, such as doxorubicin, can be efficiently delivered into and released within cells when spider silk particles were used as a carrier.
Asunto(s)
Portadores de Fármacos/química , Liberación de Fármacos , Nanopartículas/química , Seda/química , Animales , Supervivencia Celular , Clatrina/química , ADN/química , Doxorrubicina/química , Endocitosis , Colorantes Fluorescentes/química , Células HeLa , Humanos , Nanomedicina , Tamaño de la Partícula , Poliaminas/química , Polielectrolitos , Polietileneimina/química , Rodaminas/química , ArañasRESUMEN
Nature reveals a great variety of inorganic-organic composite materials exhibiting good mechanical properties, high thermal and chemical stability, and good barrier properties. One class of natural bio-nanocomposites, e.g. found in mussel shells, comprises protein matrices with layered inorganic fillers. Inspired by such natural bio-nanocomposites, the cationic recombinant spider silk protein eADF4(κ16) was processed together with the synthetic layered silicate sodium hectorite in an all-aqueous setup. Drop-casting of this bio-nanocomposite resulted in a thermally and chemically stable film reflecting a one-dimensional crystal. Surprisingly, this bio-nanocomposite coating was, though produced in an all-aqueous process, completely water insoluble. Analyzing the structural details showed a low inner free volume due to the well-oriented self-assembly/alignment of the spider silk proteins on the nanoclay surface, yielding high oxygen and water vapor barrier properties. The here demonstrated properties in combination with good biocompatibility qualify this new bio-nanocomposite to be used in packaging applications.
Asunto(s)
Seda/química , Nanocompuestos , Vapor , Volatilización , AguaRESUMEN
Spider silk fibers have a sophisticated hierarchical structure composed of proteins with highly repetitive sequences. Their extraordinary mechanical properties, defined by a unique combination of strength and extensibility, are superior to most man-made fibers. Therefore, spider silk has fascinated mankind for thousands of years. However, due to their aggressive territorial behavior, farming of spiders is not feasible on a large scale. For this reason, biotechnological approaches were recently developed for the production of recombinant spider silk proteins. These recombinant proteins can be assembled into a variety of morphologies with a great range of properties for technical and medical applications. Here, the different approaches of biotechnological production and the advances in material processing toward various applications will be reviewed.
Asunto(s)
Biotecnología/métodos , Fibroínas/biosíntesis , Arañas/metabolismo , Animales , Fibroínas/química , Fibroínas/genética , Proteínas Recombinantes/biosíntesisRESUMEN
Drug delivery carriers stabilize drugs and control their release, expanding the therapeutic window, and avoiding side effects of otherwise freely diffusing drugs in the human body. Materials used as carrier vehicles have to be biocompatible, biodegradable, nontoxic, and nonimmunogenic. Previously, particles made of the recombinant spider silk protein eADF4(C16) could be effectively loaded with positively and neutrally charged model substances. Here, a new positively charged variant thereof, named eADF4(κ16), has been engineered. Its particle formation is indistinguishable to that of polyanionic eADF4(C16), but in contrast polycationic eADF4(κ16) allows incorporation of negatively charged substances. Both high-molecular-weight substances, such as nucleic acids, and low-molecular-weight substances could be efficiently loaded onto eADF4(κ16) particles, and release of nucleic acids was shown to be well controlled.