Permselectivity of Silk Fibroin Hydrogels for Advanced Drug Delivery Neurotherapies.

A promising trend in tissue engineering is using biomaterials to improve the control of drug concentration in targeted tissue. These vehicular systems are of specific interest when the required treatment time window is higher than the stability of therapeutic molecules in the body. Herein, the capacity of silk fibroin hydrogels to release different molecules and drugs in a sustained manner was evaluated. We found that a biomaterial format, obtained by an entirely aqueous-based process, could release molecules of variable molecular weight and charge with a preferential delivery of negatively charged molecules. Although the theoretical modeling suggested that drug delivery was more likely to be driven by Fickian diffusion, the external media had a considerable influence on the release, with lipophilic organic solvents such as acetonitrile-methanol (ACN-MeOH) intensifying the release of hydrophobic molecules. Second, we found that silk fibroin could be used as a vehicular system to treat a variety of brain disorders as this biomaterial sustained the release of different factors with neurotrophic (brain-derived neurotrophic factor) (BDNF), chemoattractant (C-X-C motif chemokine 12) (CXCL12), anti-inflammatory (TGF-ß-1), and angiogenic (VEGF) capacities. Finally, we demonstrated that this biomaterial hydrogel could release cholesteronitrone ISQ201, a nitrone with antioxidant capacity, showing neuroprotective activity in an in vitro model of ischemia-reoxygenation. Given the slow degradation rate shown by silk fibroin in many biological tissues, including the nervous system, our study expands the restricted list of drug delivery-based biomaterial systems with therapeutic capacity for both short- and especially long-term treatment windows and has merit for use with brain pathologies.

Identification of Individual Target Molecules Using Antibody-Decorated DeepTipTM Atomic-Force Microscopy Probes.

A versatile and robust procedure is developed that allows the identification of individual target molecules using antibodies bound to a DeepTipTM functionalized atomic-force microscopy probe. The model system used for the validation of this process consists of a biotinylated anti-lactate dehydrogenase antibody immobilized on a streptavidin-decorated AFM probe. Lactate dehydrogenase (LDH) is employed as target molecule and covalently immobilized on functionalized MicroDeckTM substrates. The interaction between sensor and target molecules is explored by recording force-displacement (F-z) curves with an atomic-force microscope. F-z curves that correspond to the genuine sensor-target molecule interaction are identified based on the following three criteria: (i) number of peaks, (ii) value of the adhesion force, and (iii) presence or absence of the elastomeric trait. The application of these criteria leads to establishing seven groups, ranging from no interaction to multiple sensor-target molecule interactions, for which force-displacement curves are classified. The possibility of recording consistently single-molecule interaction events between an antibody and its specific antigen, in combination with the high proportion of successful interaction events obtained, increases remarkably the possibilities offered by affinity atomic-force microscopy for the characterization of biological and biomimetic systems from the molecular to the tissue scales.

Impact of environmental factors on spider silk properties.

Spider orb webs have evolved to stop flying prey, fast and slow alike. One of the main web elements dissipating impact energy is the radial fibers, or major ampullate silks, which possess a toughness surpassing most man-made materials. Orb webs are extended phenotypes, and as such their architectural elements, including major ampullate silks, have been selected to optimize prey capture under the respective environmental conditions. In this study, we investigated the correlation of three landscape scales and three microhabitat characteristics with intrinsic silk properties (elastic modulus, yield stress, tensile strength, extensibility, and toughness) to understand underlying ecological patterns. For this purpose, we collected and mechanically tested major ampullate silks from 50 spider species inhabiting large altitudinal and climatic gradients in Colombia. Using regression analysis and model selection, we investigated the environmental drivers of inter- and intra-specific patterns of major ampullate silk properties, taking into account phylogenetic relatedness based on newly sequenced mitochondrial genomes. We found that the total amount of energy absorbed, i.e., toughness and tensile strength, is higher for fibers from species inhabiting regions where heavy rainfall is common. Interestingly, we observe the same general trend between individuals of the same species, stressing the importance of this environmental driver. We also observe a phylogenetic conservation in the relation of environmental variables with silk tensile strength and yield stress. In conclusion, the increase in major ampullate silk tensile strength and toughness may reflect an adaptation to prevent frequent rain damage to orb webs and the associated energetic loss.

Statistical Study of Low-Intensity Single-Molecule Recognition Events Using DeepTipTM Probes: Application to the Pru p 3-Phytosphingosine System.

The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics.