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.

A Versatile Magnetic Nanoplatform for Plug-and-Play Functionalization: Genetically Programmable Cargo Loading to Bacterial Magnetosomes by SpyCatcher "Click Biology".

Bacterial magnetosomes ("MAGs") represent a promising class of magnetic iron oxide nanoparticles with exceptional material characteristics and high application potential in the biomedical and biotechnological field. For the surface functionalization of MAGs with different protein cargos, their enveloping membrane can be addressed by genetic means. However, the expression of foreign polypeptides as translational fusion to magnetosome membrane proteins is still laborious and lacks versatility as the generated particles are monospecific and thus restricted to predetermined functions. Utilizing the SpyTag-SpyCatcher (ST-SC) bioconjugate system, we here establish a flexible platform for the targeted nanoassembly of multifunctional MAGs that combines the rapidity of chemical coupling (e.g., by cross-linking reactions) and the unmatched selectivity and controllability of in vivo functionalization. MAGs genetically engineered to display either SC- or ST-connectors are shown to efficiently bind a variety of complementary tagged (protein) cargo. Specifically, we cover a broad spectrum of representative functional moieties and foreign cargo (such as enzymes, antibodies, fluorophores, and silica beads) with relevance in biotechnology and biomedicine and demonstrate the interchangeability of the MAGs-adapted ST-SC system. For the controlled generation of artificial shells surrounding the particles, SC-MAGs are effectively coated by protein-corona proteins. The potential of the here-provided toolkit is even more enhanced by using SC-MAGs as an affinity tool for selective protein pulldown in vitro and in vivo. Overall, this innovative technology turns bacterial MAGs into a flexible magnetic nanoscaffold for the targeted plug-and-play display of virtually unlimited additional functionalities, thereby generating a multitude of magnetic hybrid materials that can be used in many applications.

A Magnetosome-Based Platform for Flow Biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein "cargo" can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space-time yield of 49.2 mmol L-1 h-1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.

Biocompatibility, uptake and subcellular localization of bacterial magnetosomes in mammalian cells.

Magnetosomes represent biogenic, magnetic nanoparticles biosynthesized by magnetotactic bacteria. Subtle biological control on each step of biomineralization generates core-shell nanoparticles of high crystallinity, strong magnetization and uniform shape and size. These features make magnetosomes a promising alternative to chemically synthesized nanoparticles for many applications in the biotechnological and biomedical field, such as their usage as biosensors in medical diagnostics, as drug-delivery agents, or as contrast agents for magnetic imaging techniques. Thereby, the particles are directly applied to mammalian cells or even injected into the body. In the present work, we provide a comprehensive characterization of isolated magnetosomes as potential cytotoxic effects and particle uptake have not been well studied so far. Different cell lines including cancer cells and primary cells are incubated with increasing particle amounts, and effects on cell viability are investigated. Obtained data suggest a concentration-dependent biocompatibility of isolated magnetosomes for all tested cell lines. Furthermore, magnetosome accumulation in endolysosomal structures around the nuclei is observed. Proliferation rates are affected in the presence of increasing particle amounts; however, viability is not affected and doubling times can be restored by reducing the magnetosome concentration. In addition, we evidence magnetosome-cell interactions that are strong enough to allow for magnetic cell sorting. Overall, our study not only assesses the biocompatibility of isolated magnetosomes, but also evaluates effects on cell proliferation and the fate of internalized magnetosomes, thereby providing prerequisites for their future in vivo application as biomedical agents.