Enhanced cellular uptake of engineered spider silk particles.

Drug delivery systems allow tissue/cell specific targeting of drugs in order to reduce total drug amounts administered to an organism and potential side effects upon systemic drug delivery. Most drug delivery systems are polymer-based, but the number of possible materials is limited since many commercially available polymers induce allergic or inflammatory responses or lack either biodegradability or the necessary stability in vivo. Spider silk proteins represent a new class of (bio)polymers that can be used as drug depots or drug delivery systems. The recombinant spider silk protein eADF4(C16), which can be processed into different morphologies such as particles, films, or hydrogels, has been shown to fulfil most criteria necessary for its use as biomaterial. Further, eADF4(C16) particles have been shown to be well-suited for drug delivery. Here, a new method was established for particle production to reduce particle size and size distribution. Importantly, cellular uptake of these particles was shown to be poor in HeLa cells. Therefore, variants of eADF4(C16) with inversed net charge or incorporated cell penetrating peptides and receptor interacting motifs were tested, showing much better cellular uptake. Interestingly, uptake of all silk variant particles was mainly achieved by clathrin-mediated endocytosis.

Enzymatic Degradation of Films, Particles, and Nonwoven Meshes Made of a Recombinant Spider Silk Protein.

The performance of biomaterials in vivo is largely influenced by their stability and the rate and extent to which they degrade. Materials for tissue engineering applications, for example, have to be mechanically stable to support cell adhesion and proliferation without collapsing. On the other hand they need to be replaced gradually by native extracellular matrix and have to be (slowly) biodegradable. Therefore, it is of critical importance to be able to tune the degradation behavior of a biomaterial. Recombinantly produced spider silk proteins have been shown to be versatile biopolymers for medical applications. They can be processed into a variety of morphologies, and by chemical or genetic modification the properties can be adjusted to specific demands. Furthermore, in vivo experiments confirmed the lack of immunological reactions toward certain spider silks. In this study the degradation behavior of the recombinant spider silk protein eADF4(C16) in solution as well as processed into particles, films and nonwoven meshes was analyzed, and the impact of cross-linking of the scaffolds was assessed thereon. In addition to two bacterial proteolytic model enzymes, protease type XIV from Streptomyces griseus (PXIV) and collagenase type IA from Clostridium histolyticum (CHC) used in all experiments, several recombinant human matrix metalloproteinases (MMPs) and one elastase were used in studying degradation of soluble eADF4(C16). For soluble eADF4(C16) all degradation kinetics were similar. In case of eADF4(C16) scaffolds significant differences were observable between PXIV and CHC. All scaffolds were more stable toward proteolytic degradation in the presence of CHC than in the presence of PXIV. Further, particles were degraded significantly faster than films, and nonwoven meshes showed the highest proteolytic stability. Chemical cross-linking of the scaffolds led to a decrease in both degradation rate and extent.