Designed Spider Silk-Based Drug Carrier for Redox- or pH-Triggered Drug Release.

Targeted drug delivery and controlled drug release can be obtained using specifically designed polymers as carriers. Due to their biocompatibility and biodegradability and especially the lack of an immune response, materials made of spider silk proteins are promising candidates for use in such applications. Particles made of recombinant spider silk proteins have previously been shown to be suitable drug and gene carriers as they could readily be loaded with various drug substances or biologicals, and subsequent release was observed over a defined period of time. However, the respective substances were bound non-covalently via hydrophobic or charge-charge interactions, and hence, the release of loaded substances could not be spatio-temporally controlled. Here, we present a setup of chemically modified recombinant spider silk protein eADF4 and variants thereof, combining their well-established biocompatible properties with covalent drug binding and triggered release upon changes in the pH or redox state, respectively. The usefulness of the spider silk platform technology was shown with model substances and cytostatic drugs bound to spider silk particles or films via a pH-labile hydrazine linker as one option, and the drugs could be released from the spider silk carriers upon acidification of the environment as seen, e.g., in tumorous tissues or endo/lysosomes. Sulfhydryl-bearing spider silk variants allowed model substance release if exposed to intracellular GSH (glutathione) levels as a second coupling option. The combination of non-immunogenic, nontoxic spider silk materials as drug carriers with precisely triggerable release chemistry presents a platform technology for a wide range of applications.

Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. RESULTS: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. CONCLUSION: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

First Cases of Natural Infections with Borrelia hispanica in Two Dogs and a Cat from Europe.

Canine cases of relapsing fever (RF) borreliosis have been described in Israel and the USA, where two RF species, Borrelia turicatae and Borrelia hermsii, can cause similar clinical signs to the Borrelia persica in dogs and cats reported from Israel, including fever, lethargy, anorexia, thrombocytopenia, and spirochetemia. In this report, we describe the first clinical cases of two dogs and a cat from Spain (Cordoba, Valencia, and Seville) caused by the RF species Borrelia hispanica. Spirochetes were present in the blood smears of all three animals, and clinical signs included lethargy, pale mucosa, anorexia, cachexia, or mild abdominal respiration. Laboratory findings, like thrombocytopenia in both dogs, may have been caused by co-infecting pathogens (i.e., Babesia vogeli, confirmed in one dog). Anemia was noticed in one of the dogs and in the cat. Borrelia hispanica was confirmed as an infecting agent by molecular analysis of the 16S rRNA locus. Molecular analysis of housekeeping genes and phylogenetic analyses, as well as successful in vitro culture of the feline isolate confirmed the causative agent as B. hispanica.

Enhanced Antibacterial Activity of Se Nanoparticles Upon Coating with Recombinant Spider Silk Protein eADF4(κ16).

PURPOSE: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. METHODS: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. RESULTS: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. CONCLUSION: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.

Surface Modification of Spider Silk Particles to Direct Biomolecular Corona Formation.

In recent years, spider silk-based materials have attracted attention because of their biocompatibility, processability, and biodegradability. For their potential use in biomaterial applications, i.e., as drug delivery systems and implant coatings for tissue regeneration, it is vital to understand the interactions between the silk biomaterial surface and the biological environment. Like most polymeric carrier systems, spider silk material surfaces can adsorb proteins when in contact with blood, resulting in the formation of a biomolecular corona. Here, we assessed the effect of surface net charge of materials made of recombinant spider silk on the biomolecular corona composition. In-depth proteomic analysis of the biomolecular corona revealed that positively charged spider silk materials surfaces interacted predominantly with fibrinogen-based proteins. This fibrinogen enrichment correlated with blood clotting observed for both positively charged spider silk films and particles. In contrast, negative surface charges prevented blood clotting. Genetic engineering allows the fine-tuning of surface properties of the spider silk particles providing a whole set of recombinant spider silk proteins with different charges or peptide tags to be used for, for example, drug delivery or cell docking, and several of these were analyzed concerning the composition of their biomolecular corona. Taken together this study demonstrates how the surface net charge of recombinant spider silk surfaces affects the composition of the biomolecular corona, which in turn affects macroscopic effects such as fibrin formation and blood clotting.

Engineered hybrid spider silk particles as delivery system for peptide vaccines.

The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable.

In Vivo Coating of Bacterial Magnetic Nanoparticles by Magnetosome Expression of Spider Silk-Inspired Peptides.

Magnetosomes are natural magnetic nanoparticles with exceptional properties that are synthesized in magnetotactic bacteria by a highly regulated biomineralization process. Their usability in many applications could be further improved by encapsulation in biocompatible polymers. In this study, we explored the production of spider silk-inspired peptides on magnetosomes of the alphaproteobacterium Magnetospirillum gryphiswaldense. Genetic fusion of different silk sequence-like variants to abundant magnetosome membrane proteins enhanced magnetite biomineralization and caused the formation of a proteinaceous capsule, which increased the colloidal stability of isolated particles. Furthermore, we show that spider silk peptides fused to a magnetosome membrane protein can be used as seeds for silk fibril growth on the magnetosome surface. In summary, we demonstrate that the combination of two different biogenic materials generates a genetically encoded hybrid composite with engineerable new properties and enhanced potential for various applications.

Applicability of biotechnologically produced insect silks.

Silks are structural proteins produced by arthropods. Besides the well-known cocoon silk, which is produced by larvae of the silk moth Bombyx mori to undergo metamorphosis inside their silken shelter (and which is also used for textile production by men since millennia), numerous further less known silk-producing animals exist. The ability to produce silk evolved multiple independent times during evolution, and the fact that silk was subject to convergent evolution gave rise to an abundant natural diversity of silk proteins. Silks are used in air, under water, or like honey bee silk in the hydrophobic, waxen environment of the bee hive. The good mechanical properties of insect silk fibres together with their non-toxic, biocompatible, and biodegradable nature renders these materials appealing for both technical and biomedical applications. Although nature provides a great diversity of material properties, the variation in quality inherent in materials from natural sources together with low availability (except from silkworm silk) impeded the development of applications of silks. To overcome these two drawbacks, in recent years, recombinant silks gained more and more interest, as the biotechnological production of silk proteins allows for a scalable production at constant quality. This review summarises recent developments in recombinant silk production as well as technical procedures to process recombinant silk proteins into fibres, films, and hydrogels.

Properties of Engineered and Fabricated Silks.

Silk is a protein-based material which is predominantly produced by insects and spiders. Hundreds of millions of years of evolution have enabled these animals to utilize different, highly adapted silk types in a broad variety of applications. Silk occurs in several morphologies, such as sticky glue or in the shape of fibers and can, depending on the application by the respective animal, dissipate a high mechanical energy, resist heat and radiation, maintain functionality when submerged in water and withstand microbial settling. Hence, it's unsurprising that silk piqued human interest a long time ago, which catalyzed the domestication of silkworms for the production of silk to be used in textiles. Recently, scientific progress has enabled the development of analytic tools to gain profound insights into the characteristics of silk proteins. Based on these investigations, the biotechnological production of artificial and engineered silk has been accomplished, which allows the production of a sufficient amount of silk materials for several industrial applications. This chapter provides a review on the biotechnological production of various silk proteins from different species, as well as on the processing techniques to fabricate application-oriented material morphologies.

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.