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1.
Biomacromolecules ; 21(12): 4904-4912, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33249826

RESUMO

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.


Assuntos
Liberação Controlada de Fármacos , Seda , Aranhas , Animais , Materiais Biocompatíveis , Portadores de Fármacos , Concentração de Íons de Hidrogênio , Oxirredução , Proteínas Recombinantes
2.
Biomacromolecules ; 19(3): 962-972, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29357230

RESUMO

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.


Assuntos
Nanopartículas de Magnetita , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Biossíntese Peptídica , Peptídeos , Seda/biossíntese , Aranhas/genética , Animais , Magnetossomos/genética , Magnetossomos/ultraestrutura , Magnetospirillum/genética , Magnetospirillum/ultraestrutura , Seda/genética
3.
Z Naturforsch C J Biosci ; 72(9-10): 365-385, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28746045

RESUMO

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.


Assuntos
Biotecnologia/métodos , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Seda/metabolismo , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Bioengenharia/métodos , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Humanos , Hidrogéis/química , Proteínas de Insetos/química , Proteínas de Insetos/genética , Seda/química , Seda/genética
4.
ACS Appl Mater Interfaces ; 12(22): 24635-24643, 2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32369330

RESUMO

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.


Assuntos
Coroa de Proteína/metabolismo , Seda/química , Aranhas/química , Adsorção , Sequência de Aminoácidos , Animais , Fibrinogênio/metabolismo , Humanos , Ligação Proteica , Engenharia de Proteínas , Seda/genética , Seda/metabolismo , Eletricidade Estática , Propriedades de Superfície
5.
Biomaterials ; 172: 105-115, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29723755

RESUMO

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.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Seda/química , Aranhas/química , Vacinas de Subunidades Antigênicas/farmacologia , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Antígenos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/citologia , Liberação Controlada de Fármacos , Feminino , Humanos , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Ovalbumina/química , Tamanho da Partícula , Proteínas Recombinantes/química , Propriedades de Superfície , Linfócitos T Citotóxicos , Distribuição Tecidual
6.
Biomater Sci ; 3(3): 543-51, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26222296

RESUMO

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.


Assuntos
Materiais Biocompatíveis/química , Fibroínas/metabolismo , Proteínas Recombinantes/química , Aranhas/química , Animais , Sistemas de Liberação de Medicamentos , Fibroínas/química , Humanos , Tamanho da Partícula , Proteínas Recombinantes/metabolismo
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