Ahmed Shaukat

Biological nanostructures and nanomachines encompass a wide range of natural assemblies from the smallest prokaryotes to viruses, enzymes, and subcellular compartments. Their capabilities are impressive, including replication, locomotion, and catalysis. To be able to design and produce modified or wholly artificial versions of such systems using biological molecules (proteins, nucleic acids, and lipids) is a long-term goal of engineering biology. However, their complexity makes the design and prediction of their properties challenging, while production, purification, and testing can also be difficult. In recent years, new approaches have been developed to facilitate these processes. Here, we review tools for designing biological molecules, highlighting their capabilities and giving examples of their successful application. Finally, we present possible capabilities of future tools and challenges to their development.

The application of fluorescent proteins (FPs) in optoelectronics is hindered by the need for effective protocols to stabilize them under device preparation and operational conditions. Factors such as high temperatures, irradiation, and organic solvent exposure contribute to the denaturation of FPs, resulting in a low device performance. Herein, we focus on addressing the photoinduced heat generation associated with FP motion and rapid heat transfer. This leads to device temperatures of approximately 65 °C, causing FP-denaturation and a subsequent loss of device functionality. We present a FP stabilization strategy involving the integration of electrostatically self-assembled FP-apoferritin cocrystals within a silicone-based color down-converting filter. Three key achievements characterize this approach: (i) an engineering strategy to design positively supercharged FPs (+22) without compromising photoluminescence and thermal stability compared to their native form, (ii) a carefully developed crystallization protocol resulting in highly emissive cocrystals that retain the essential photoluminescence features of the FPs, and (iii) a strong reduction of the device's working temperature to 40 °C, leading to a 40-fold increase in Bio-HLEDs stability compared to reference devices.

Heparin is a widely applied anticoagulant agent. However, in clinical practice, it is of vital importance to reverse its anticoagulant effect to restore the blood-clotting cascade and circumvent side effects. Inspired by protein cages that can encapsulate and protect their cargo from surroundings, we utilize three designed protein copolymers to sequester heparin into inert nanoparticles. In our design, a silk-like sequence provides cooperativity between proteins, generating a multivalency effect that enhances the heparin-binding ability. Protein copolymers complex heparin into well-defined nanoparticles with diameters below 200 nm. We also develop a competitive fluorescent switch-on assay for heparin detection, with a detection limit of 0.01 IU mL^-1 in plasma that is significantly below the therapeutic range (0.2-8 IU mL^-1). Moreover, moderate cytocompatibility is demonstrated by in vitro cell studies. Therefore, such engineered protein copolymers present a promising alternative for neutralizing and sensing heparin, but further optimization is required for in vivo applications.

Supramolecular self‐assembly of biomolecules provides a powerful bottom‐up strategy to build functional nanostructures and materials. Among the different biomacromolecules, protein cages offer various advantages including uniform size, versatility, multi‐modularity, and high stability. Additionally, protein cage crystals present confined microenvironments with well‐defined dimensions. On the other hand, molecular hosts, such as cyclophanes, possess a defined cavity size and selective recognition of guest molecules. However, the successful combination of macrocycles and protein cages to achieve functional co‐crystals has remained limited. In this study, we demonstrate electrostatic binding between cationic pillar[5]arenes and (apo)ferritin cages that results in porous and crystalline frameworks. The electrostatically assembled crystals present a face‐centered cubic (FCC) lattice and have been characterized by means of small‐angle X‐ray scattering and cryo‐TEM. These hierarchical structures result in a multiadsorbent framework capable of hosting both organic and inorganic pollutants, such as dyes and toxic metals, with potential application in water‐remediation technologies.

Nanostructures based on DNA self‐assembly present an innovative way to address the increasing need for target‐specific delivery of therapeutic molecules. Currently, most of the chemotherapeutics being used in clinical practice have undesired and exceedingly high off‐target toxicity. This is a challenge in particular for small molecules, and hence, developing robust and effective methods to lower these side effects and enhance the antitumor activity is of paramount importance. Prospectively, these issues could be tackled with the help of DNA nanotechnology, which provides a route for the fabrication of custom, biocompatible, and multimodal structures, which can, to some extent, resist nuclease degradation and survive in the cellular environment. Similar to widely employed liposomal products, the DNA nanostructures (DNs) are loaded with selected drugs, and then by employing a specific stimulus, the payload can be released at its target region. This review explores several strategies and triggers to achieve targeted delivery of DNs. Notably, different modalities are explained through which DNs can interact with their respective targets as well as how structural changes triggered by external stimuli can be used to achieve the display or release of the cargo. Furthermore, the prospects and challenges of this technology are highlighted.

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.

The strategy of combining biomolecules and synthetic components to develop biohybrids is becoming increasingly popular for preparing highly customized and biocompatible functional materials. Carbon nanotubes (CNTs) benefit from bioconjugation, allowing their excellent properties to be applied to biomedical applications. This study reviews the state‐of‐the‐art research in biomolecule–CNT conjugates and discusses strategies for their self‐assembly into hierarchical structures. The review focuses on various highly ordered structures and the interesting properties resulting from the structural order. Hence, CNTs conjugated with the most relevant biomolecules, such as nucleic acids, peptides, proteins, saccharides, and lipids are discussed. The resulting well‐defined composites allow the nanoscale properties of the CNTs to be exploited at the micro‐ and macroscale, with potential applications in tissue engineering, sensors, and wearable electronics. This review presents the underlying chemistry behind the CNT‐based biohybrid materials and discusses the future directions of the field.

Several bioactive compounds are in use for the treatment of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Historically, willow (salix sp.) bark has been an important source of salisylic acid and other natural compounds with anti-inflammatory, antipyretic and analgesic properties. Among these, picein isolated from hot water extract of willow bark, has been found to act as a natural secondary metabolite antioxidant. The aim of this study was to investigate the unrevealed pharmacological action of picein. In silico studies were utilized to direct the investigation towards the neuroprotection abilities of picein. Our in vitro studies demonstrate the neuroprotective properties of picein by blocking the oxidative stress effects, induced by free radical generator 2-methyl-1,4-naphthoquinone (menadione, MQ), in neuroblastoma SH-SY5Y cells. Several oxidative stress-related parameters were evaluated to measure the protection for mitochondrial integrity, such as mitochondrial superoxide production, mitochondrial activity (MTT), reactive oxygen species (ROS) and live-cell imaging. A significant increase in the ROS level and mitochondrial superoxide production were measured after MQ treatment, however, a subsequent treatment with picein was able to mitigate this effect by decreasing their levels. Additionally, the mitochondrial activity was significantly decreased by MQ exposure, but a follow-up treatment with picein recovered the normal metabolic activity. In conclusion, the presented results demonstrate that picein can significantly reduce the level of MQ-induced oxidative stress on mitochondria, and thereby plays a role as a potent neuroprotectant.

In this communication, electrostatically assembled phthalocyanine (Pc)–DNA origami (DO) complexes are formed and their optical properties are demonstrated. The formation of the complex prevents the Pc aggregation, thus yielding an enhanced optical response and photooxidative resilience towards aggregation in biologically relevant media. Simultaneously, the Pc protects the DO against enzymatic digestion. Both features solve previous drawbacks associated with phthalocyanine photosensitizers and DNA nanocarriers. The studied complexes may find use in technologies related to the photogeneration of singlet oxygen, e.g., photocatalysis, diagnositic arrays and photodynamic therapy.