The Future Is Bright for Polyoxometalates

Polyoxometalates (POMs) are clusters of units of oxoanions of transition metals, such as Mo, W, V and Nb, that can be formed upon acidification of neutral solutions. Once formed, some POMs have shown to persist in solution, even in the neutral and basic pH range. These inorganic clusters, amenable of a variety of structures, have been studied in environmental, chemical, and industrial fields, having applications in catalysis and macromolecular crystallography, as well as applications in biomedicine, such as cancer, bacterial and viral infections, among others. Herein, we connect recent POMs environmental applications in the decomposition of emergent pollutants with POMs’ biomedical activities and effects against cancer, bacteria, and viruses. With recent insights in POMs being pure, organic/inorganic hybrid materials, POM-based ionic liquid crystals and POM-ILs, and their applications in emergent pollutants degradation, including microplastics, are referred. It is perceived that the majority of the POMs studies against cancer, bacteria, and viruses were performed in the last ten years. POMs’ biological effects include apoptosis, cell cycle arrest, interference with the ions transport system, inhibition of mRNA synthesis, cell morphology changes, formation of reaction oxygen species, inhibition of virus binding to the host cell, and interaction with virus protein cages, among others. We additionally refer to POMs’ interactions with various proteins, including P-type ATPases, aquoporins, cinases, phosphatases, among others. Finally, POMs’ stability and speciation at physiological conditions are addressed.

Polyelectrolyte Encapsulation and Confinement within Protein Cage-Inspired Nanocompartments

Protein cages are nanocompartments with a well-defined structure and monodisperse size. They are composed of several individual subunits and can be categorized as viral and non-viral protein cages. Native viral cages often exhibit a cationic interior, which binds the anionic nucleic acid genome through electrostatic interactions leading to efficient encapsulation. Non-viral cages can carry various cargo, ranging from small molecules to inorganic nanoparticles. Both cage types can be functionalized at targeted locations through genetic engineering or chemical modification to entrap materials through interactions that are inaccessible to wild-type cages. Moreover, the limited number of constitutional subunits ease the modification efforts, because a single modification on the subunit can lead to multiple functional sites on the cage surface. Increasing efforts have also been dedicated to the assembly of protein cage-mimicking structures or templated protein coatings. This review focuses on native and modified protein cages that have been used to encapsulate and package polyelectrolyte cargos and on the electrostatic interactions that are the driving force for the assembly of such structures. Selective encapsulation can protect the payload from the surroundings, shield the potential toxicity or even enhance the intended performance of the payload, which is appealing in drug or gene delivery and imaging.

Bioengineered Ferritin Nanocarriers for Cancer Therapy

Ferritin naturally exists in most organisms and can specifically recognize the transferrin 1 receptor (TfR1), which is generally highly expressed on various types of tumor cells. The pH dependent reversible assembling and disassembling property of ferritin renders it as a suitable candidate for encapsulating a variety of anticancer drugs and imaging probes. Ferritins external surface is chemically and genetically modifiable which can serve as attachment site for tumor specific targeting peptides or moieties. Moreover, the biological origin of these protein cages makes it a biocompatible nanocarrier that stabilizes and protects the enclosed particles from the external environment without provoking any toxic or immunogenic responses. Recent studies, further establish ferritin as a multifunctional nanocarrier for targeted cancer chemotherapy and phototherapy. In this review, we introduce the favorable characteristics of ferritin drug carriers, the specific targeted surface modification and a multifunctional nanocarriers combined chemotherapy with phototherapy for tumor treatment. Taken together, ferritin is a potential ideal base of engineered nanoparticles for tumor therapy and still needs to explore more on its way.

ASAXS measurements on ferritin and apoferritin at the bioSAXS beamline P12 (PETRA III, DESY)

Small-angle X-ray scattering is widely utilized to study biological macromolecules in solution. For samples containing specific (e.g. metal) atoms, additional information can be obtained using anomalous scattering. Here, measuring samples at different energies close to the absorption edges of relevant elements provides specific structural details. However, anomalous small-angle X-ray scattering (ASAXS) applications to dilute macromolecular solutions are challenging owing to the overall low anomalous scattering effect. Here, pilot ASAXS experiments from dilute solutions of ferritin and cobalt-loaded apoferritin are reported. These samples were investigated near the resonance X-ray K edges of Fe and Co, respectively, at the EMBL P12 bioSAXS beamline at PETRA III, DESY. Thanks to the high brilliance of the P12 beamline, ASAXS experiments are feasible on dilute protein solutions, allowing one to extract the Fe- or Co-specific anomalous dispersion terms from the ASAXS data. The data were subsequently used to determine the spatial distribution of either iron or cobalt atoms incorporated into the ferritin/apoferritin protein cages.

Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

Encapsulin based protein cages are nanoparticles with different biomedical applications, such as targeted drug delivery or imaging agents. These particles are biocompatible and can be produced in bacteria, allowing large scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore the potential of their surface modification and optimize their production. In this study we design and show new surface modifications of the Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on Tm encapsulin with a His-tag insertion after the residue 64 and the residue 127, and the modification of the C-terminal on Bl encapsulin, are reported. The multi-modification of the Tm encapsulin enables up to 240 different functionalities on the cage surface, resulting from 4 potential modifications per protein subunit. We furthermore report an improved protocol giving a better stability and providing a notable increase of the production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a function loop, allow for further study of the use of these protein nanocages in biomedical applications.

The OP Protein Cage: A Versatile Molecular Delivery Platform

Well-defined containers constructed from multiple protein subunits are a unique class of nanomaterial useful in supramolecular chemistry and biology. These protein cages are widespread in nature, where they are responsible for a diversity of important tasks. As such, producing our own designer protein cages, complete with bespoke functionalities, is a promising avenue to new nanodevices, biotechnology and therapies. Herein, we describe how an artificial, computationally designed protein cage can be rationally engineered using supramolecular intuition to produce new functional capsules. Positive supercharging the interior cavity of this porous protein cage enables the efficient encapsulation of oligonucleotides by electrostatically-driven self-assembly. Moreover, the resulting cargo-loaded cages enter mammalian cells and release their cargo, for example siRNA which modulates gene expression. To expand the cargo scope of this proteinaceous container, a higher level of supramolecular complexity can also be introduced. Encapsulation of anionic surfactants affords protein-scaffolded micelles, which are capable of sequestering poorly water-soluble small molecules within their hydrophobic cores. These hybrid particles stably carry bioactive cargo and deliver it intracellularly, thereby increasing potency. Further development of these genetically-encoded materials is ongoing towards specific applications ranging from cell biology to medicine.

A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures

Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence–structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.