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Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release.

ABSTRACT: Hydrogels are excellent scaffolds to accommodate sensitive enzymes in a protective environment. However, the lack of suitable immobilization techniques on substrates and the lack of selectivity to anchor a biocatalyst are major drawbacks preventing the use of hydrogels in bioanalytical devices. Here, nanofilm coatings on surfaces were made of a recombinant spider silk protein (rssp) to induce rssp self-assembly and thus the formation of fibril-based nanohydrogels. To functionalize spider silk nanohydrogels for bioselective binding of proteins, two different antithrombin aptamers were chemically conjugated with the rssp, thereby integrating the target-binding function into the nanohydrogel network. Human thrombin was selected as a sensitive model target, in which the structural integrity determines its activity. The chosen aptamers, which bind various exosites of thrombin, enabled selective and cooperative embedding of the protein into the nanohydrogels. The change of the aptamer secondary structure using complementary DNA sequences led to the release of active thrombin and confirmed the addressable functionalization of spider silk nanohydrogels.

SUBMITTER: Humenik M

PROVIDER: S-EPMC7096766 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release.

Humenik Martin M Preiß Tamara T Gödrich Sebastian S Papastavrou Georg G Scheibel Thomas T

Materials today. Bio 20200224

Hydrogels are excellent scaffolds to accommodate sensitive enzymes in a protective environment. However, the lack of suitable immobilization techniques on substrates and the lack of selectivity to anchor a biocatalyst are major drawbacks preventing the use of hydrogels in bioanalytical devices. Here, nanofilm coatings on surfaces were made of a recombinant spider silk protein (rssp) to induce rssp self-assembly and thus the formation of fibril-based nanohydrogels. To functionalize spider silk na ...[more]

PMID: 32259099

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Project description:Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.

Project description:BACKGROUND: It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. METHODOLOGY/PRINCIPAL FINDINGS: We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. CONCLUSIONS: Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.

Dataset Information

Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release.

Publications

Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release.

Similar Datasets

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