Large-Scale, Proteinaceous Nanotube Arrays with Programmable Hydrophobicity, Oleophilicity, and Gas Permeability.

Nanotubular structures possess remarkable advantages in a broad range of areas, such as catalysis, sensing, microencapsulation, selective mass transport, filtration, and drug delivery. While the fields of carbon nanotubes and nanotubes made of several noncarbon materials (e.g., metals, oxides, semiconductors) have been progressing rapidly, proteinaceous nanotubes remained largely underexplored. Here, by retrofitting a template wetting approach with multiple silk-based suspensions, we present a rapidly scalable and robust technology for fabricating large arrays (e.g., 20 × 20 cm2) of well-aligned 1D nanostructures made of silk proteins. Benefiting from the polymorphic nature of silk, precise control over the size, density, aspect ratio, and morphology (tubes versus pillars) of silk nanostructures is achieved, which then allows for programmable modulation of the end materials' functions and properties (e.g., hydrophobicity, oleophilicity, and gas permeability). The silk nanotube arrays fabricated present great utility as antifouling coatings against marine algae and in oil extraction from oil-water mixtures.

Charge, Aspect Ratio, and Plant Species Affect Uptake Efficiency and Translocation of Polymeric Agrochemical Nanocarriers.

An incomplete understanding of how agrochemical nanocarrier properties affect their uptake and translocation in plants limits their application for promoting sustainable agriculture. Herein, we investigated how the nanocarrier aspect ratio and charge affect uptake and translocation in monocot wheat (Triticum aestivum) and dicot tomato (Solanum lycopersicum) after foliar application. Leaf uptake and distribution to plant organs were quantified for polymer nanocarriers with the same diameter (∼10 nm) but different aspect ratios (low (L), medium (M), and high (H), 10-300 nm long) and charges (-50 to +15 mV). In tomato, anionic nanocarrier translocation (20.7 ± 6.7 wt %) was higher than for cationic nanocarriers (13.3 ± 4.1 wt %). In wheat, only anionic nanocarriers were transported (8.7 ± 3.8 wt %). Both low and high aspect ratio polymers translocated in tomato, but the longest nanocarrier did not translocate in wheat, suggesting a phloem transport size cutoff. Differences in translocation correlated with leaf uptake and interactions with mesophyll cells. The positive charge decreases nanocarrier penetration through the leaf epidermis and promotes uptake into mesophyll cells, decreasing apoplastic transport and phloem loading. These results suggest design parameters to provide agrochemical nanocarriers with rapid and complete leaf uptake and an ability to target agrochemicals to specific plant organs, with the potential to lower agrochemical use and the associated environmental impacts.

Microencapsulation of High-Content Actives Using Biodegradable Silk Materials.

There is a compelling need across several industries to substitute non-degradable, intentionally added microplastics with biodegradable alternatives. Nonetheless, stringent performance criteria in actives' controlled release and manufacturing at scale of emerging materials hinder the replacement of polymers used for microplastics fabrication with circular ones. Here, the authors demonstrate that active microencapsulation in a structural protein such as silk fibroin can be achieved by modulating protein protonation and chain relaxation at the point of material assembly. Silk fibroin micelles' size is tuned from several to hundreds of nanometers, enabling the manufacturing-by retrofitting spray drying and spray freeze drying techniques-of microcapsules with tunable morphology and structure, that is, hollow-spongy, hollow-smooth, hollow crumpled matrices, and hollow crumpled multi-domain. Microcapsules degradation kinetics and sustained release of soluble and insoluble payloads typically used in cosmetic and agriculture applications are controlled by modulating fibroin's beta-sheet content from 20% to near 40%. Ultraviolet-visible studies indicate that burst release of a commonly used herbicide (i.e., saflufenacil) significantly decreases from 25% to 0.8% via silk fibroin microencapsulation. As a proof-of-concept for agrochemicals applications, a 6-day greenhouse trial demonstrates that saflufenacil delivered on corn plants via silk microcapsules reduces crop injury when compared to the non-encapsulated version.

A bioinspired approach to engineer seed microenvironment to boost germination and mitigate soil salinity.

Human population growth, soil degradation, and agrochemical misuse are significant challenges that agriculture must face in the upcoming decades as it pertains to global food production. Seed enhancement technologies will play a pivotal role in supporting food security by enabling germination of seeds in degraded environments, reducing seed germination time, and boosting crop yields. So far, a great effort has been pursued in designing plants that can adapt to different environments and germinate in the presence of abiotic stressors, such as soil salinity, heat, and drought. The technology proposed here seeks a different goal: To engineer the microenvironment of seeds by encapsulation, preservation, and precise delivery of biofertilizers that can boost seed germination and mitigate abiotic stressors. In particular, we developed a biomaterial based on silk fibroin (S) and trehalose that can be mixed with rhizobacteria and applied on the surface of seeds, retrofitting currently used techniques for seed coating, i.e., dip coating or spray drying. A micrometer thick transparent robust coating is formed by material assembly. The combination of a polymorphic protein as S and of a disaccharide used by living systems to tolerate abiotic stressors provides a beneficial environment for the survival of nonspore forming rhizobacteria outside the soil and in anhydrous conditions. Using Rhizobium tropici CIAT 899 and Phaseolus vulgaris as working models, we demonstrated that rhizobacteria delivered in the soil after coating dissolution infect seedlings' roots, form root nodules, enhance yield, boost germination, and mitigate soil salinity.

Self-Perpetuating Carbon Foam Microwave Plasma Conversion of Hydrocarbon Wastes into Useful Fuels and Chemicals.

White wastes (unseparated plastics, face masks, textiles, etc.) pose a serious challenge to sustainable human development and the ecosystem and have recently been exacerbated due to the surge in plastic usage and medical wastes from COVID-19. Current recycling methods such as chemical recycling, mechanical recycling, and incineration require either pre-sorting and washing or releasing CO2. In this work, a carbon foam microwave plasma process is developed, utilizing plasma discharge to generate surface temperatures exceeding ∼3000 K in a N2 atmosphere, to convert unsorted white wastes into gases (H2, CO, C2H4, C3H6, CH4, etc.) and small amounts of inorganic minerals and solid carbon, which can be buried as artificial "coal". This process is self-perpetuating, as the new solid carbon asperities grafted onto the foam's surface actually increase the plasma discharge efficiency over time. This process has been characterized by in situ optical probes and infrared sensors and optimized to handle most of the forms of white waste without the need for pre-sorting or washing. Thermal measurement and modeling show that in a flowing reactor, the device can achieve locally extremely high temperatures, but the container wall will still be cold and can be made with cheap materials, and thus, a miniaturized waste incinerator is possible that also takes advantage of intermittent renewable electricity.

Programming function into mechanical forms by directed assembly of silk bulk materials.

We report simple, water-based fabrication methods based on protein self-assembly to generate 3D silk fibroin bulk materials that can be easily hybridized with water-soluble molecules to obtain multiple solid formats with predesigned functions. Controlling self-assembly leads to robust, machinable formats that exhibit thermoplastic behavior consenting material reshaping at the nanoscale, microscale, and macroscale. We illustrate the versatility of the approach by realizing demonstrator devices where large silk monoliths can be generated, polished, and reshaped into functional mechanical components that can be nanopatterned, embed optical function, heated on demand in response to infrared light, or can visualize mechanical failure through colorimetric chemistries embedded in the assembled (bulk) protein matrix. Finally, we show an enzyme-loaded solid mechanical part, illustrating the ability to incorporate biological function within the bulk material with possible utility for sustained release in robust, programmably shapeable mechanical formats.

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds.

Light-induced material phase transitions enable the formation of shapes and patterns from the nano- to the macroscale. From lithographic techniques that enable high-density silicon circuit integration, to laser cutting and welding, light-matter interactions are pervasive in everyday materials fabrication and transformation. These noncontact patterning techniques are ideally suited to reshape soft materials of biological relevance. We present here the use of relatively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and 3D multiscale patterns in soft silk protein hydrogels without exogenous or chemical cross-linkers. We find that high-resolution features can be generated within bulk hydrogels through nearly 1 cm of material, which is 1.5 orders of magnitude deeper than other biocompatible materials. Examples illustrating the materials, results, and the performance of the machined geometries in vitro and in vivo are presented to demonstrate the versatility of the approach.

Silk-based resorbable electronic devices for remotely controlled therapy and in vivo infection abatement.

A paradigm shift for implantable medical devices lies at the confluence between regenerative medicine, where materials remodel and integrate in the biological milieu, and technology, through the use of recently developed material platforms based on biomaterials and bioresorbable technologies such as optics and electronics. The union of materials and technology in this context enables a class of biomedical devices that can be optically or electronically functional and yet harmlessly degrade once their use is complete. We present here a fully degradable, remotely controlled, implantable therapeutic device operating in vivo to counter a Staphylococcus aureus infection that disappears once its function is complete. This class of device provides fully resorbable packaging and electronics that can be turned on remotely, after implantation, to provide the necessary thermal therapy or trigger drug delivery. Such externally controllable, resorbable devices not only obviate the need for secondary surgeries and retrieval, but also have extended utility as therapeutic devices that can be left behind at a surgical or suturing site, following intervention, and can be externally controlled to allow for infection management by either thermal treatment or by remote triggering of drug release when there is retardation of antibiotic diffusion, deep infections are present, or when systemic antibiotic treatment alone is insufficient due to the emergence of antibiotic-resistant strains. After completion of function, the device is safely resorbed into the body, within a programmable period.

Highly tunable elastomeric silk biomaterials.

Elastomeric, fully degradable and biocompatible biomaterials are rare, with current options presenting significant limitations in terms of ease of functionalization and tunable mechanical and degradation properties. We report a new method for covalently crosslinking tyrosine residues in silk proteins, via horseradish peroxidase and hydrogen peroxide, to generate highly elastic hydrogels with tunable properties. The tunable mechanical properties, gelation kinetics and swelling properties of these new protein polymers, in addition to their ability to withstand shear strains on the order of 100%, compressive strains greater than 70% and display stiffness between 200 - 10,000 Pa, covering a significant portion of the properties of native soft tissues. Molecular weight and solvent composition allowed control of material mechanical properties over several orders of magnitude while maintaining high resilience and resistance to fatigue. Encapsulation of human bone marrow derived mesenchymal stem cells (hMSC) showed long term survival and exhibited cell-matrix interactions reflective of both silk concentration and gelation conditions. Further biocompatibility of these materials were demonstrated with in vivo evaluation. These new protein-based elastomeric and degradable hydrogels represent an exciting new biomaterials option, with a unique combination of properties, for tissue engineering and regenerative medicine.

Precise and High-Throughput Delivery of Micronutrients in Plants Enabled by Pollen-Inspired Spiny and Biodegradable Microcapsules.

Decarbonizing food production and mitigating agriculture's environmental impact require new technologies for precise delivery of fertilizers and pesticides to plants. The cuticle, a waxy barrier that protects the surface of leaves, causes 60%-90% runoff of fertilizers and pesticides, leading to the wastage of intensive resources, soil depletion, and water bodies pollution. Solutions to mitigate runoff include adding chemicals (e.g., surfactants) to decrease surface tension and enhance cuticles' permeability but have low efficacy. In this study, vapor-induced synergistic differentiation (VISDi) is used to nanomanufacture echinate pollen-like, high payload content (≈50 wt%) microcapsules decorated with robust spines that mechanically disrupt the cuticle and adhere to the leaf. VISDi induces a core-shell structure in the spines, enabling the release of agrochemicals from the microparticles' body into the leaf. As proof of concept, precise and highthroughput delivery of iron fertilizer in Fe-deficient spinach plants is demonstrated. Spray of spiny microparticles improves leaf adhesion by mechanical interlocking, reduces wash-off by an ≈12.5 fold, and enhances chlorophyll content by ≈7.3 times compared to the application of spherical counterparts. Together, these results show that spiny microparticles can mitigate agricultural runoff and provide a high-throughput tool for precise plant drug delivery.

Directed Assembly of Proteinaceous-Polysaccharide Nanofibrils to Fabricate Membranes for Emerging Contaminant Remediation.

Emerging contaminants, including per- and polyfluoroalkyl substances and heavy metals, are threatening the health of humans and ecosystems. Their removal from the environment remains challenging. Here, we designed silk fibroin-cellulose nanocrystal (silk-CNC) nanofibrillar and nanoporous membranes for emerging contaminant remediation. The protein-polysaccharide nanofibrils were fabricated by templating the assembly of silk fibroin using CNCs. Silk fibroin polymorphic nature combined with surface charge modulation of CNCs produced cationic silk-CNC(+) and anionic silk-CNC(-) nanofibrils that can target a broad spectrum of contaminants. Silk-CNC(+) nanofibrils and membranes exhibited antimicrobial properties and captured both short-chain heptafluorobutyric acid, perfluorobutanesulfonic acid, and long-chain perfluorooctanoic acid by virtue of hydrophobic attraction from ß-sheeted silk fibroin and electrostatic interactions with CNC(+). Silk-CNC(-) provided the opportunity to target cations such as heavy metal cocontaminants. The nanofabrication of biopolymer-based membranes combines high performance with environmentally benign and cost-effective removal of emerging contaminants for water purification, wastewater treatment, and remediation.

Tardigrade secretory proteins protect biological structures from desiccation.

Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focus on the proteins belonging to the secreted family of tardigrade proteins termed secretory-abundant heat soluble ("SAHS") proteins, and investigate their ability to protect diverse biological structures. Recombinantly expressed SAHS proteins prevent desiccated liposomes from fusion, and enhance desiccation tolerance of E. coli and Rhizobium tropici upon extracellular application. Molecular dynamics simulation and comparative structural analysis suggest a model by which SAHS proteins may undergo a structural transition upon desiccation, in which removal of water and solutes from a large internal cavity in SAHS proteins destabilizes the beta-sheet structure. These results highlight the potential application of SAHS proteins as stabilizing molecules for preservation of cells.

Polymeric Nanocarriers Autonomously Cross the Plant Cell Wall and Enable Protein Delivery for Stress Sensing.

Delivery of proteins in plant cells can facilitate the design of desired functions by modulation of biological processes and plant traits but is currently limited by narrow host range, tissue damage, and poor scalability. Physical barriers in plants, including cell walls and membranes, limit protein delivery to desired plant tissues. Herein, a cationic high aspect ratio polymeric nanocarriers (PNCs) platform is developed to enable efficient protein delivery to plants. The cationic nature of PNCs binds proteins through electrostatic. The ability to precisely design PNCs' size and aspect ratio allowed us to find a cutoff of ≈14 nm in the cell wall, below which cationic PNCs can autonomously overcome the barrier and carry their cargo into plant cells. To exploit these findings, a reduction-oxidation sensitive green fluorescent protein (roGFP) is deployed as a stress sensor protein cargo in a model plant Nicotiana benthamiana and common crop plants, including tomato and maize. In vivo imaging of PNC-roGFP enabled optical monitoring of plant response to wounding, biotic, and heat stressors. These results show that PNCs can be precisely designed below the size exclusion limit of cell walls to overcome current limitations in protein delivery to plants and facilitate species-independent plant engineering.

Determination of multiphoton absorption of silk fibroin using the Z-scan technique.

The multiphoton absorption of silk fibroin at 810 nm was determined by open-aperture Z-scan. Three-photon absorption was confirmed at this wavelength and the three-photon cross section of silk fibroin was measured. Silk fibroin of varying molecular weight was tested and the cross section was found to increase exponentially with increasing molecular weight. The confirmation of a relatively large three-photon absorption cross section in silk will help lay the groundwork for future investigation of direct laser writing of three-dimensional structures within a silk matrix.

Integrating biopolymer design with physical unclonable functions for anticounterfeiting and product traceability in agriculture.

Smallholder farmers and manufacturers in the Agri-Food sector face substantial challenges because of increasing circulation of counterfeit products (e.g., seeds), for which current countermeasures are implemented mainly at the secondary packaging level, and are generally vulnerable because of limited security guarantees. Here, by integrating biopolymer design with physical unclonable functions (PUFs), we propose a cryptographic protocol for seed authentication using biodegradable and miniaturized PUF tags made of silk microparticles. By simply drop casting a mixture of variant silk microparticles on a seed surface, tamper-evident PUF tags can be seamlessly fabricated on a variety of seeds, where the unclonability comes from the stochastic assembly of spectrally and visually distinct silk microparticles in the tag. Unique, reproducible, and unpredictable PUF codes are generated from both Raman mapping and microscopy imaging of the silk tags. Together, the proposed technology offers a highly secure solution for anticounterfeiting and product traceability in agriculture.

Unbiased in silico design of pH-sensitive tetrapeptides.

We used coarse-grain molecular dynamics simulations to screen all possible histidine-bearing tetrapeptide sequences, finding novel peptide sequences with pH-tunable assembly properties. These tetrapeptides could be used for various biological applications, such as triggered delivery of bioactive molecules.

Drug Delivery in Plants Using Silk Microneedles.

New systems for agrochemical delivery in plants will foster precise agricultural practices and provide new tools to study plants and design crop traits, as standard spray methods suffer from elevated loss and limited access to remote plant tissues. Silk-based microneedles can circumvent these limitations by deploying a known amount of payloads directly in plants' deep tissues. However, plant response to microneedles' application and microneedles' efficacy in deploying physiologically relevant biomolecules are unknown. Here, it is shown that gene expression associated with Arabidopsis thaliana wounding response decreases within 24 h post microneedles' application. Additionally, microinjection of gibberellic acid (GA3 ) in A. thaliana mutant ft-10 provides a more effective and efficient mean than spray to activate GA3 pathways, accelerating bolting and inhibiting flower formation. Microneedle efficacy in delivering GA3 is also observed in several monocot and dicot crop species, i.e., tomato (Solanum lycopersicum), lettuce (Lactuca sativa), spinach (Spinacia oleracea), rice (Oryza Sativa), maize (Zea mays), barley (Hordeum vulgare), and soybean (Glycine max). The wide range of plants that can be successfully targeted with microinjectors opens the doors to their use in plant science and agriculture.

Temperature-Responsive Bottlebrush Polymers Deliver a Stress-Regulating Agent In Vivo for Prolonged Plant Heat Stress Mitigation.

Anticipated increases in the frequency and intensity of extreme temperatures will damage crops. Methods that efficiently deliver stress-regulating agents to crops can mitigate these effects. Here, we describe high aspect ratio polymer bottlebrushes for temperature-controlled agent delivery in plants. The foliar-applied bottlebrush polymers had near complete uptake into the leaf and resided in both the apoplastic regions of the leaf mesophyll and in cells surrounding the vasculature. Elevated temperature enhanced the in vivo release of spermidine (a stress-regulating agent) from the bottlebrushes, promoting tomato plant (Solanum lycopersicum) photosynthesis under heat and light stress. The bottlebrushes continued to provide protection against heat stress for at least 15 days after foliar application, whereas free spermidine did not. About 30% of the ∼80 nm short and ∼300 nm long bottlebrushes entered the phloem and moved to other plant organs, enabling heat-activated release of plant protection agents in phloem. These results indicate the ability of the polymer bottlebrushes to release encapsulated stress relief agents when triggered by heat to provide long-term protection to plants and the potential to manage plant phloem pathogens. Overall, this temperature-responsive delivery platform provides a new tool for protecting plants against climate-induced damage and yield loss.

Design of Biodegradable, Climate-Specific Packaging Materials That Sense Food Spoilage and Extend Shelf Life.

The AgriFood systems in tropical climates are under strain due to a rapid increase in human population and extreme environmental conditions that limit the efficacy of packaging technologies to extend food shelf life and guarantee food safety. To address these challenges, we rationally designed biodegradable packaging materials that sense spoilage and prevent molding. We nanofabricated the interface of 2D covalent organic frameworks (COFs) to reinforce silk fibroin (SF) and obtain biodegradable membranes with augmented mechanical properties and that displayed an immediate colorimetric response (within 1 s) to food spoilage, using packaged poultry as an example. Loading COF with antimicrobial hexanal also mitigated biotic spoilage in high-temperature and -humidity conditions, resulting in a four-order of magnitude decrease in the total amount of mold growth in soybeans packaged in silk-COF, when compared to cling film (i.e., polyethylene). Together, the integration of sensing, structural reinforcement, and antimicrobial agent delivery within a biodegradable nanocomposite framework defines climate-specific packaging materials that can decrease food waste and enhance food safety.

Biomaterials for boosting food security.

Renewable silk-protein technologies promote plant growth and reduce food waste.