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Protein-based nanoparticles have gained interest for traversing different biological (e.g., systemic or microenvironmental, etc.) barriers and enabling targeted drug delivery. Silk protein-based nanoparticles are useful and versatile drug delivery systems for sustained and controlled release due to their biocompatibility, biodegradability, accessible chemistries, and ability to stabilize different drugs and other biomolecules. In the present study, silk nanoparticles (SNPs) were engineered using a nanoprecipitation technique with tight control over size (∼45–250 nm diameter) and low polydispersity index by altering variables including stirring speed, reaction bath temperature, silk molecular weight (MW), and silk concentration. Of these variables, stir speed was the most significant contributor toward particle size control. SNPs with positive or negative surface charges and decoration of SNPs with surface antigens were also demonstrated. New mechanistic insights into control of SNP size, cellular uptake using glioblastoma as a model, surface characteristics, and the entrapment of a small-molecule drug (doxorubicin) within the particles were addressed. These insights expand the potential utility of SNPs for medical, environmental, and food applications. [ABSTRACT FROM AUTHOR]

Encapsulation of single cells is a powerful technique used in various fields, such as regenerative medicine, drug delivery, tissue regeneration, cell-based therapies, and biotechnology. It offers a method to protect cells by providing cytocompatible coatings to strengthen cells against mechanical and environmental perturbations. Silk fibroin, derived from the silkworm Bombyx mori , is a promising protein biomaterial for cell encapsulation due to the cytocompatibility and capacity to maintain cell functionality. Here, THP-1 cells, a human leukemia monocytic cell line, were encapsulated with chemically modified silk polyelectrolytes through electrostatic layer-by-layer deposition. The effectiveness of the silk nanocoating was assessed using scanning electron microscopy (SEM) and confocal microscopy and on cell viability and proliferation by Alamar Blue assay and live/dead staining. An analysis of the mechanical properties of the encapsulated cells was conducted using atomic force microscopy nanoindentation to measure elasticity maps and cellular stiffness. After the cells were encapsulated in silk, an increase in their stiffness was observed. Based on this observation, we developed a mechanical predictive model to estimate the variations in stiffness in relation to the thickness of the coating. By tuning the cellular assembly and biomechanics, these encapsulations promote systems that protect cells during biomaterial deposition or processing in general.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni 2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.

Conventional thinking when designing biodegradable materials and devices is to tune the intrinsic properties and morphological features of the material to regulate their degradation rate, modulating traditional factors such as molecular weight and crystallinity. Since regenerated silk protein can be directly thermoplastically molded to generate robust dense silk plastic-like materials, this approach afforded a new tool to control silk degradation by enabling the mixing of a silk-degrading protease into bulk silk material prior to thermoplastic processing. Here we demonstrate the preparation of these silk-based devices with embedded silk-degrading protease to modulate the degradation based on the internal presence of the enzyme to support silk degradation, as opposed to the traditional surface degradation for silk materials. The degradability of these silk devices with and without embedded protease XIV was assessed both in vitro and in vivo. Ultimately, this new process approach provides direct control of the degradation lifetime of the devices, empowered through internal digestion via water-activated proteases entrained and stabilized during the thermoplastic process.

Cultivated meat production is a promising technology to generate meat while reducing the reliance on traditional animal farming. Biomaterial scaffolds are critical components in cultivated meat production, enabling cell adhesion, proliferation, differentiation, and orientation. In the present work, naturally derived glutenin was fabricated into films with and without surface patterning and in the absence of toxic cross-linking or stabilizing agents for cell culture related to cultivated meat goals. The films were stable in culture media for at least 28 days, and the surface patterns induced cell alignment and guided myoblast organization (C2C12s) and served as a substrate for 3T3-L1 adipose cells. The films supported adhesion, proliferation, and differentiation with mass balance considerations (films, cells, and matrix production). Freeze-thaw cycles were applied to remove cells from glutenin films and monitor changes in glutenin mass with respect to culture duration. Extracellular matrix (ECM) extraction was utilized to quantify matrix deposition and changes in the original biomaterial mass over time during cell cultivation. Glutenin films with C2C12s showed mass increases with time due to cell growth and new collagen-based ECM expression during proliferation and differentiation. All mass balances were compared among cell and noncell systems as controls, along with gelatin control films, with time-dependent changes in the relative content of film, matrix deposition, and cell biomass. These data provide a foundation for cell/biomaterial/matrix ratios related to time in culture as well as nutritional and textural features.

Niches with multiple physical and chemical cues can influence the fate of cells and tissues in vivo . Simulating the in vivo niche in the design of bioactive materials is a challenge, particularly to tune multiple cues simultaneously in the same system. Here, an assembly strategy was developed to regulate multiple cues in the same scaffold based on the use of two silk nanofiber components that respond differently during the fabrication processes. An aqueous solution containing the two components, amorphous silk nanofibers (ASNFs) and β-sheet-rich silk nanofibers (BSNFs), was sequentially treated with an electrical field and freeze-drying processes where the BSNFs oriented to the electrical field, while the ASNFs formed stable porous structures during the lyophilization process to impact the mechanical properties. Bioactive cargo, such as deferoxamine (DFO), was loaded on the BSNFs to enrich cell responses with the scaffolds. The in vitro results revealed that the loaded DFO and the anisotropic structures with improved mechanical properties resulted in better vascularization than those of the scaffolds without the anisotropic features. The multiple cues in the scaffolds provided angiogenic niches to accelerate wound healing.

A major challenge of engineering larger macroscale tissues in vitro is the limited diffusion of nutrients and oxygen to the interior. For skeletal muscle, this limitation results in millimeter scale outcomes to avoid necrosis. One method to address this constraint may be to vascularize in vitro -grown muscle tissue, to support nutrient (culture media) flow into the interior of the structure. In this exploratory study, we examine culture conditions that enable myogenic development and endothelial cell survival within tissue engineered 3D muscles. Myoblasts (C2C12s), endothelial cells (HUVECs), and endothelial support cells (C3H 10T1/2s) were seeded into Matrigel-fibrin hydrogels and cast into 3D printed frames to form 3D in vitro skeletal muscle tissues. Our preliminary results suggest that the simultaneous optimization of culture media formulation and cell concentrations is necessary for 3D cultured muscles to exhibit robust myosin heavy chain expression and GFP expression from GFP-transfected endothelial cells. The ability to form differentiated 3D muscles containing endothelial cells is a key step toward achieving vascularized 3D muscle tissues, which have potential use as tissue for implantation in a medical setting, as well as for future foods such as cultivated meats.

Model verification is a critical aspect of scientific accountability, transparency, and learning. Here, we demonstrate an application of a model verification approach for a molecular dynamics (MD) simulation, where the interactions between silica and silk protein were studied experimentally toward understanding biomineralization. Following the ten rules for credible modeling and simulation of biosciences as developed in Erdemir et al., the authors of the original paper collaborated with an external modeling group to verify the key findings of their original simulation model and to document this verification approach. The process resulted in successful replication of the key findings of the original model. Beyond verification, study of the model from a new perspective generated new insight into the basic assumptions. We discuss key learnings for how model validation processes can be improved more generally, specifically through improved documentation methods. We anticipate that this application of our protocol for model verification can be further replicated and improved to verify and validate other simulations.

Fragrances, which are commonly used in food, textiles, consumer products, and medical supplies, are volatile compounds that require stabilization and controlled release due to their sensitivity to environmental conditions such as light, oxygen, temperature, and humidity. Encapsulation in various material matrices is a desired technique for these purposes, and there is a growing interest in using sustainable natural materials to reduce environmental impact. In this study, fragrance encapsulation in microspheres made from silk fibroin (SF) was investigated. Fragrance-loaded silk fibroin microspheres (Fr-SFMSs) were prepared by adding fragrance/surfactant emulsions to silk solutions, followed by mixing them with polyethylene glycol under ambient conditions. The study investigated eight different fragrances, where citral, beta-ionone, and eugenol showed higher binding affinities to silk than the other five fragrances, resulting in better microsphere formation with uniform sizes and higher fragrance loading (10-30%). Citral-SFMSs showed characteristic crystalline β-sheet structures of SF, high thermal stability (initial weight loss at 255 °C), long shelf life at 37 °C (>60 days), and sustained release (∼30% of citral remained after incubation at 60 °C for 24 h). When citral-SFMSs with different sizes were used to treat cotton fabrics, about 80% of the fragrance remained on the fabrics after one wash, and the duration of release from the treated fabrics was significantly longer than that of control samples treated with citral alone (no microspheres). This method of preparing Fr-SFMSs has potential applications in textile finishing, cosmetics, and the food industry.

Natural polymers are extensively utilized as scaffold materials in tissue engineering and 3D disease modeling due to their general features of cytocompatibility, biodegradability, and ability to mimic the architecture and mechanical properties of the native tissue. A major limitation of many polymeric scaffolds is their autofluorescence under common imaging methods. This autofluorescence, a particular challenge with silk fibroin materials, can interfere with the visualization of fluorescently labeled cells and proteins grown on or in these scaffolds, limiting the assessment of outcomes. Here, Sudan Black B (SBB) was successfully used prefixation prior to cell seeding, in various silk matrices and 3D model systems to quench silk autofluorescence for live cell imaging. SBB was also trialed postfixation in silk hydrogels. We validated that multiple silk scaffolds pretreated with SBB (hexafluoro-2-propanol-silk scaffolds, salt-leached sponges, gel-spun catheters, and sponge-gel composite scaffolds) cultured with fibroblasts, adipose tissue, neural cells, and myoblasts demonstrated improved image resolution when compared to the nonpretreated scaffolds, while also maintaining normal cell behavior (attachment, growth, proliferation, differentiation). SBB pretreatment of silk scaffolds is an option for scaffold systems that require autofluorescence suppression.

Structural proteins are the basis of many biomaterials and key construction and functional components of all life. Further, it is well-known that the diversity of proteins' function relies on their local structures derived from their primary amino acid sequences. Here, we report a deep learning model to predict the secondary structure content of proteins directly from primary sequences, with high computational efficiency. Understanding the secondary structure content of proteins is crucial to designing proteins with targeted material functions, especially mechanical properties. Using convolutional and recurrent architectures and natural language models, our deep learning model predicts the content of two essential types of secondary structures, the α-helix and the β-sheet. The training data are collected from the Protein Data Bank and contain many existing protein geometries. We find that our model can learn the hidden features as patterns of input sequences that can then be directly related to secondary structure content. The α-helix and β-sheet content predictions show excellent agreement with training data and newly deposited protein structures that were recently identified and that were not included in the original training set. We further demonstrate the features of the model by a search for de novo protein sequences that optimize max/min α-helix/β-sheet content and compare the predictions with folded models of these sequences based on AlphaFold2 . Excellent agreement is found, underscoring that our model has predictive potential for rapidly designing proteins with specific secondary structures and could be widely applied to biomedical industries, including protein biomaterial designs and regenerative medicine applications.

More than 15% of adults in the United States suffer from some form of chronic kidney disease (CKD). Current strategies for CKD consist of dialysis or kidney transplant, which, however, can take several years. In this light, tissue engineering and regenerative medicine approaches are the key to improving people's living conditions by advancing previous tissue engineering approaches and seeking new targets as intervention methods for kidney repair or replacement. The membrane voltage ( V m ) dynamics of a cell have been associated with cell migration, cell cycle progression, differentiation, and pattern formation. Furthermore, bioelectrical stimuli have been used as a means in the treatment of diseases and wound healing. Here, we investigated the role of V m as a novel target to guide and manipulate in vitro renal tissue models. Human-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1) were cultured on Matrigel to support the formation of 3D proximal tubular-like structures with the incorporation of a voltage-sensitive dye indicator─bis-(1,3-dibutylbarbituric acid)timethine oxonol (DiBAC). The results demonstrated a correlation between the depolarization and the reorganization of human renal proximal tubule cells, indicating V m as a candidate variable to control these events. Accordingly, V m was pharmacologically manipulated using glibenclamide and pinacidil, K ATP channel modulators, and proximal tubule formation and tubule stability over 21 days were assessed. Chronic manipulation of K ATP channels induced changes in the tubular network topology without affecting lumen formation. Thus, a relationship was found between the preluminal tubulogenesis phase and K ATP channels. This relationship may provide future options as a control point during kidney tissue development, treatment, and regeneration goals.

Silk nanofibers are versatile carriers for hydrophobic and hydrophilic drugs, but fall short in terms of effective delivery to cells, which is essential for therapeutic benefits. Here, the size of silk nanofibers was tuned by ultrasonic treatment to improve the cell penetration features without impacting the structural features. The gradual decrease in silk nanofiber length from 1700 to 40 nm resulted in improved cell uptake. The internalized silk nanofiber carriers evaded lysosomes, which facilitated retention in cancer cells in vitro. The smaller sizes also facilitated enhanced penetration of tumor spheroids for improved delivery in vitro. The cytotoxicity of paclitaxel (PTX)-laden nanocarriers increased when the length of the silk nanocarriers decreased. Both the drug loading capacity and delivery of silk nanocarriers with optimized sizes suggest potential utility in cell treatments.

Stem cell fate is determined by specific niches that provide multiple physical, chemical, and biological cues. However, the hierarchy or cascade of impact of these cues remains elusive due to their spatiotemporal complexity. Here, anisotropic silk protein nanofiber-based hydrogels with suitable cell adhesion capacity are developed to mimic the physical microenvironment inside the blastocele. The hydrogels enable mouse embryonic stem cells (mESCs) to maintain stemness in vitro in the absence of both leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs), two critical factors in the standard protocol for mESC maintenance. The mESCs on hydrogels can achieve superior pluripotency, genetic stability, developmental capacity, and germline transmission to those cultured with the standard protocol. Such biomaterials establish an improved dynamic niche through stimulating the secretion of autocrine factors and are sufficient to maintain the pluripotency and propagation of ESCs. The mESCs on hydrogels are distinct in their expression profiles and more resemble ESCs in vivo . The physical cues can thus initiate a self-sustaining stemness-maintaining program. In addition to providing a relatively simple and low-cost option for expansion and utility of ESCs in biological research and therapeutic applications, this biomimetic material helps gain more insights into the underpinnings of early mammalian embryogenesis.