All-aqueous Process Research Articles

Modulating metal-centered dimerization of a lanthanide chaperone protein for separation of light lanthanides

Elucidating details of biology's selective uptake and trafficking of rare earth elements, particularly the lanthanides, has the potential to inspire sustainable biomolecular separations of these essential metals for myriad modern technologies. Here, we biochemically and structurally characterize Methylobacterium (Methylorubrum) extorquens LanD, a periplasmic protein from a bacterial gene cluster for lanthanide uptake. This protein provides only four ligands at its surface-exposed lanthanide-binding site, allowing for metal-centered protein dimerization that favors the largest lanthanide, LaIII. However, the monomer prefers NdIII and SmIII, which are disfavored lanthanides for cellular utilization. Structure-guided mutagenesis of a metal-ligand and an outer-sphere residue weakens metal binding to the LanD monomer and enhances dimerization for PrIII and NdIII by 100-fold. Selective dimerization enriches high-value PrIII and NdIII relative to low-value LaIII and CeIII in an all-aqueous process, achieving higher separation factors than lanmodulins and comparable or better separation factors than common industrial extractants. Finally, we show that LanD interacts with lanmodulin (LanM), a previously characterized periplasmic protein that shares LanD's preference for NdIII and SmIII. Our results suggest that LanD's unusual metal-binding site transfers less-desirable lanthanides to LanM to siphon them away from the pathway for cytosolic import. The properties of LanD show how relatively weak chelators can achieve high selectivity, and they form the basis for the design of protein dimers for separation of adjacent lanthanide pairs and other metal ions.

Multicellular Layered Nanofibrous Poly(Oligo Ethylene Glycol Methacrylate) (POEGMA)-Based Hydrogel Scaffolds via Reactive Cell Electrospinning.

While hydrogels are demonstrated to be effective scaffolds for soft tissue engineering, existing fabrication techniques pose limitations in terms of being able to reproduce both the micro/nanofibrous structures of native extracellular matrix as well as the spatial arrangement of different cell types inherent of more complex tissues. Herein, a reactive cell electrospinning strategy is described using hydrazide and aldehyde-functionalized poly(oligoethylene glycol methacrylate) precursor polymers that can create nanofibrous hydrogel scaffolds with controllable local cell gradients using a sequential all-aqueous process that does not require additives or external energy. Cells can be encapsulated directly during the fabrication process in different layers within the scaffold, enabling localized segregation of different cell types within the structures without compromising their capacity to proliferate (≈4-fold increase in cell density over a 14 day incubation period). This sequential reactive electrospinning approach thus offers promise to generate coculture fibrous hydrogel networks in which both the nanoscale architecture and the cell distribution can be controlled, as it is essential to recreate more complex types of tissues.

High-capacity semi-organic polymer batteries: From monomer to battery in an all-aqueous process

Aqueous Zinc-batteries comprising organic cathode materials represent interesting candidates for sustainable, safe, environmentally friendly, and highly flexible secondary energy storage system. Here, we demonstrate that such an oxygen-tolerant, semi-organic setup can conveniently be prepared in an all-aqueous process, including all steps from the initial polymerization to the coin cell assembly without the need of any intermediate purification steps, and still reach high energy densities of 2.55 mAh cm−2. The TEMPO-modified and crosslinked poly(methacrylamide) cathode facilitates an excellent compatibility to the aqueous electrolyte and an efficient accessibility of the redox-active species due to the increased polarity of the material compared to the common poly(TEMPO-methacrylate) (PTMA). The material exhibits excellent stability (capacity retention of >99% after 1000 cycles at 5C) with low self-discharge and features high rate capabilities. The casting process from the aqueous slurry further appeared to be suitable for the preparation of ultra-thick but still homogeneous binder-free electrodes and exhibit total device capacities of approximately 4.5 mAh in a 2032-type coin cell. Since the overall process avoids harsh conditions or harmful solvents, is scalable, and relies on straightforward processing steps, it represents a promising step towards cost-effective and efficient rechargeable Zinc-based semi-organic batteries with competing capacities if further optimized.

Biomimetic Natural Silk Nanofibrous Microspheres for Multifunctional Biomedical Applications.

Silk nanofibrils (SNFs) extracted from natural silkworm silk represent a class of high-potential protein nanofiber material with unexplored biomedical applications. In this study, a SNF-assembled microsphere with extracellular matrix (ECM)-mimicking architecture and high specific surface area was developed. The SNFs were exfoliated from silkworm silks through an all-aqueous process and used as the building blocks for constructing the microspheres. Inspired by the structure and bioactive composition of ECM, hyaluronic acid (HA) was used as a bio-glue to regulate SNF assembly. With the assistance of HA, the SNF microspheres with stable fluffy nanofibrous structures were synthesized through electrospray. The biomimetic structure and nature derived composition endow the microspheres with excellent biocompatibility and enhanced osteogenic differentiation-inducing ability to mesenchymal stem cells. As proof of versatility, the SNF microspheres were further functionalized with other molecules and nanomaterials. Taking the advantages of the excellent blood compatibility and modifiability from the molecular level to the nanoscale of SNF microspheres, we demonstrated their versatile applications in protease detection and blood purification. On the basis of these results, we foresee that this natural silk-based nanofibrous microsphere may serve as a superior biomedical material for tissue engineering, early disease diagnosis, and therapeutic devices.

Comparative Toxicity, Biodistribution and Excretion of Ultra-Small Gold Nanoclusters with Different Emission Wavelengths.

The exponentially increased use of gold nanoclusters in diagnosis and treatment has raised serious concern about their potential threat to living organisms. However, the mechanisms of toxicity of gold nanoclusters in vitro and in vivo remain poorly understood. In this work, comparative toxicity studies, including biodistribution and excretion, were carried out with mildly and chemically synthesized ultra-small L-histidine-protected and bovine serum albumin (BSA)-protected gold nanoclusters in an all-aqueous process. These nanoclusters did not induce a remarkable impact on cell viability, even at relatively high concentrations (100 μg/mL). The haemolytic assay demonstrated that the gold nanoclusters could not destroy blood cell at 600 μg/mL. After intravenous injection with mice, the biocompatibility, biodistribution, and excretion were determined. Quantitative analysis results showed that accumulation varied in the liver, spleen, kidney, and lung, though primarily in the liver and spleen. They were excreted in urine and faeces, but mainly excreted through urine. In our study, no obvious abnormalities were found in body weight, behavioral changes, blood and serum biochemical indicators, and histopathology. These findings suggested that both gold nanoclusters showed similar effects in vivo and were safe and biocompatible, laying the foundation for safe biomedical application in the future.

Interfacial Photo-Cross-Linking: Simple but Powerful Approach for Fabricating Capsule Polymer Particles with Tunable pH-Responsive Controlled Release Capability.

Herein, we describe capsule polymer particles with precisely controlled pH-responsive release properties prepared directly via the interfacial photo-cross-linking of spherical poly(2-diethylaminoethyl methacrylate-co-2-cinnamoylethyl methacrylate) (P(DEAEMA-CEMA)) particles. In the interfacial photo-cross-linking, photoreactive cinnamoyl groups in the polymer particles were cross-linked via [2π + 2π] cycloaddition reactions at the polymer/water interface, showing that the shell-cross-linked hollow polymer particles can be directly prepared from spherical polymer particles. The approach has fascinating advantages such as using minimal components, simplicity, and not requiring sacrificial template particles and toxic solvents. The following important observations are made: (I) encapsulated materials were stably retained in the capsule particles under neutral pH conditions; (II) encapsulated materials were released from the capsule particles under acidic pH conditions; (III) the release kinetics of encapsulated materials were controlled by the pH conditions; i.e., immediate and sustained release was achieved by varying the acidity of the aqueous media; (IV) the photoirradiation time did not significantly affect the release kinetics under different pH conditions; and (V) the pH-responsive release properties were regulated by changing the polymer composition in P(DEAEMA-CEMA). Furthermore, by exploiting the pH-responsiveness, capsule particles are successfully obtained via an all-aqueous process from spherical polymer particles. The advantages of the all-aqueous encapsulation process allowed the water-soluble biomacromolecules such as DNA and saccharides to be successfully encapsulated in the P(DEAEMA-CEMA) hollow particles. With this simple interfacial photo-cross-linking strategy, we envision the ready synthesis of sophisticated particulate materials for broad application in advanced research fields.

All-Aqueous-Processed Injectable In Situ Forming Macroporous Silk Gel Scaffolds for Minimally Invasive Intracranial and Osteological Therapies.

Hydrogels are widely utilized in regenerative medicine for drug delivery and tissue repair due to their superior biocompatibility and high similarity to the extracellular matrix. For minimally invasive therapies, in situ forming gel scaffolds are desirable, but technical challenges remain to be overcome to achieve the balance between tissue-like strength and cell-sized porosity, especially for intracranial and osteological therapies. Here, a new method-inspired by the liquid crystalline spinning process in natural silk fibers-is reported for preparing injectable silk gel scaffolds with favorable preclinical efficacy and unique characteristics including 1) in situ gelling for minimally invasive surgeries, 2) controllable porosity for efficient cellular infiltration and desirable degradation, 3) resilient and tunable mechanical properties that are compatible with the modulus regime of native soft tissues, and 4) all-aqueous processing that avoids toxic solvents and enables facile loading of bioactive agents. Moreover, hierarchically structured heterogeneous silk gel scaffolds with variable porosity and bioactive agent gradients within 3D matrices can be achieved for sustained drug release and guided tissue regeneration. Preclinical efficacy studies in rodent models show efficient bacterium and glioma inhibition and positive effects on bone regeneration and vascularization.

Physically crosslinked silk fibroin/hyaluronic acid scaffolds

Combining the properties of natural protein and polysaccharide is a promising strategy to generate bioactive biomaterials with controlled structure. Here, a new method of preparing water‐insoluble silk fibroin/hyaluronic acid (SF/HA) scaffolds with tunable performances using an all-aqueous process is reported. Freezing-induced assembly was used to form silk I crystallization in the SF/HA blends. Silk I crystallization enhanced the stability of SF/HA scaffolds in water by forming silk I crystal networks to entrap blended HA without chemical cross-linking. Increasing HA content significantly enhanced the flexibility and water binding capacity of porous scaffolds, but high amount of HA reduced the water-stability of porous scaffolds due to insufficient silk I crystal cross-links. The enzymatic degradation behavior of the SF/HA scaffolds was investigated, revealing that the regulation ability of HA in the SF scaffolds. This novel nonchemically cross-linked protein/polysaccharide scaffold may be useful for soft tissue engineering due to excellent biocompatibility and tunable performances.

From Silk Spinning to 3D Printing: Polymer Manufacturing using Directed Hierarchical Molecular Assembly.

Silk spinning offers an evolution-based manufacturing strategy for industrial polymer manufacturing, yet remains largely inaccessible as the manufacturing mechanisms in biological and synthetic systems, especially at the molecular level, are fundamentally different. The appealing characteristics of silk spinning include the sustainable sourcing of the protein material, the all-aqueous processing into fibers, and the unique material properties of silks in various formats. Substantial progress has been made to mimic silk spinning in artificial manufacturing processes, despite the gap between natural and artificial systems. This report emphasizes the universal spinning conditions utilized by both spiders and silkworms to generate silk fibers in nature, as a scientific and technical framework for directing molecular assembly into high-performance structures. The preparation of regenerated silk feedstocks and mimicking native spinning conditions in artificial manufacturing are discussed, as is progress and challenges in fiber spinning and 3D printing of silk-composites. Silk spinning is a biomimetic model for advanced and sustainable artificial polymer manufacturing, offering benefits in biomedical applications for tissue scaffolds and implantable devices.

Aqueous electrospinning of recombinant spider silk proteins.

There has been a significant increase in the use of sensitive biological components, e.g., growth factors or enzymes, in implanted scaffolds/devices. To prevent diffusion away from the targeted area and to maximize access of the biological agent to the desired target, it is necessary to provide a supportive substrate to immobilize and protect biological agents from the environment. For this purpose, nanofiber fabrics are highly promising due to their high porosity, capacity for solution flow-through and high surface-to-volume ratio. However, electrospinning often requires harsh processing conditions, such as the use of volatile solutions, which can result in loss of activity of the incorporated biological components. In this study we developed a mild process for electrospinning of eADF4(C16), a recombinant spider silk protein. eADF4(C16) is non-cytotoxic, displays excellent stability against hydrolytic and enzymatic degradation and opens the opportunity for genetic addition of bioactive factors. Therefore, an aqueous spinning dope of eADF4(C16) was loaded with either green fluorescence protein (GFP) or the recombinant fusion protein GFP-eADF4(C16). The fluorescence activity of GFP is dependent on its proper folding, which does not occur in organic solvents, making it an attractive model protein. We were able to demonstrate the usability as well as the significance of the all-aqueous processing conditions for the activity of GFP in electrospun spider silk scaffolds.

One-Step Formation of Microporous Hydrogel Sponges Encapsulating Living Cells by Utilizing Bicontinuous Dispersion of Aqueous Polymer Solutions.

With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.

Helicobacter pylori Ribosomal Protein-A2 Peptide/Silk Fibroin Nanofibrous Composites as Potential Wound Dressing.

Although different kinds of antibacterial formulas have been explored against bacterial infections, the development of both biocompatible and efficiently antibacterial matrices is still a challenge. In this study, we report a novel HPRP-A2 antimicrobial peptide/silk fibroin (SF) composite nanofibrous matrix fabricated by an all-aqueous electrospinning process (HPRP-A2 is an antibacterial peptide originated from Helicobacter pylori). The HPRP-A2/SF composite nanofibers had a round and smooth morphology. The incorporation of HPRP-A2 had little influence on both the morphology and biocompatibility of the SF nanofibers. Interestingly, the composite nanofibrous matrices showed an impressive antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, the HPRP-A2/SF composite nanofibers showed excellent performance on accelerating healing of wound according to the data of animal experiment. Considering the facile and all-aqueous process, the HPRP-A2/SF composite nanofibrous matrices could be a promising candidate for antibacterial or wound management applications.

Facile incorporation of REDV into porous silk fibroin scaffolds for enhancing vascularization of thick tissues

Rapid neovascularization within scaffolds is critical for the regeneration of thick complex tissues. The surface immobilization of peptides and other active molecules have been explored to improve the vascularization capacity of implants. However, the rapid degradation of these molecules, the reaction conditions and cross-linking usually result in decreased vascularization capability. Here, we introduced a temperate, all-aqueous process to achieve bulk porous silk fibroin (SF) scaffolds. A temperature controlled process was used to induce the water stable structure by SF self-assembly. Arg-Glu-Asp-Val (REDV) peptides were added into SF solution and fixed within SF scaffolds during the self-assembly process. The results showed that the functionalized scaffolds markedly promoted the adhesion of endothelial cells in vitro. Moreover, the in vivo studies indicated enhanced cell infiltration in the bulk functionalized SF scaffolds and impressive vascularization at 4 weeks post-implantation. The functionalized scaffolds demonstrated excellent vascularization capability, providing an exciting biomaterial option for thick tissue regeneration.

Simultaneous Separation of Actinium and Radium Isotopes from a Proton Irradiated Thorium Matrix

A new method has been developed for the isolation of 223,224,225Ra, in high yield and purity, from a proton irradiated 232Th matrix. Herein we report an all-aqueous process using multiple solid-supported adsorption steps including a citrate chelation method developed to remove >99.9% of the barium contaminants by activity from the final radium product. A procedure involving the use of three columns in succession was developed, and the separation of 223,224,225Ra from the thorium matrix was obtained with an overall recovery yield of 91 ± 3%, average radiochemical purity of 99.9%, and production yields that correspond to physical yields based on previously measured excitation functions.

Natural Fiber-Anchored Few-Layer Graphene Oxide Nanosheets for Ultrastrong Interfaces in Poly(lactic acid)

The development of high-performance graphene oxide (GO) nanocomposites is challenged by the lack of a feasible and effective route to disperse and exfoliate GO nanosheets, as well as technical gaps in providing precision control over the interfacial interactions. Here we disclose an all-aqueous processing method to immobilize few-layer extended GO nanosheets at ramie fiber driven by hydrogen bonding. The reinforcing efficacy of GO-functionalized ramie (GOFR) was examined in a poly(lactic acid) (PLA) matrix, wherein the GOFR provided large active surfaces to induce chain ordering and lamellar organization. It permitted the preferable formation of well-organized PLA transcrystallinity at GOFR, in contrast to calabash-like and less-ordered transcrystallinity induced by pristine ramie due to the inferior nucleation activity. The transcrystallization kinetics and lamellar orientation degree were significantly facilitated by the addition of chain mobility accelerator in PLA, permitting the formation of prevaili...

Silk Fibroin Microneedles for Transdermal Vaccine Delivery.

Microneedles represent an exciting departure from the existing parenteral injection paradigm for drug delivery, particularly for the administration of vaccines. While the benefit of delivering vaccine antigens to immunocompetent tissue in the skin has been established, there have been varying degrees of success using microneedles to do so. Here, we investigate the use of silk fibroin protein to produce microneedles and evaluate their ability to deliver vaccines against influenza, Clostridium difficile, and Shigella. Fibroin protein from the silkworm Bombyx mori possesses suitable properties for use in a microneedle system, including all-aqueous processing, mechanical strength in dried formats, biocompatibility, and the ability to temperature stabilize biomacromolecules. As such, this biomaterial combines the processing and biocompatibility advantages of a dissolving microneedle system with the product stability and mechanical strength of coated insoluble microneedles. Through successful vaccination of mice against three separate antigens, we establish that silk fibroin is well-suited for use as a solid-coated microneedle delivery system and offers long-term potential similar to that of the leading microneedle biomaterials.

Investigate the Effect of Thawing Process on the Self-Assembly of Silk Protein for Tissue Applications.

Biological self-assembly is a process in which building blocks autonomously organize to form stable supermolecules of higher order and complexity through domination of weak, noncovalent interactions. For silk protein, the effect of high incubating temperature on the induction of secondary structure and self-assembly was well investigated. However, the effect of freezing and thawing on silk solution has not been studied. The present work aimed to investigate a new all-aqueous process to form 3D porous silk fibroin matrices using a freezing-assisted self-assembly method. This study proposes an experimental investigation and optimization of environmental parameters for the self-assembly process such as freezing temperature, thawing process, and concentration of silk solution. The optical images demonstrated the possibility and potential of −80ST48 treatment to initialize the self-assembly of silk fibroin as well as controllably fabricate a porous scaffold. Moreover, the micrograph images illustrate the assembly of silk protein chain in 7 days under the treatment of −80ST48 process. The surface morphology characterization proved that this method could control the pore size of porous scaffolds by control of the concentration of silk solution. The animal test showed the support of silk scaffold for cell adhesion and proliferation, as well as the cell migration process in the 3D implantable scaffold.

Regenerated silk materials for functionalized silk orthopedic devices by mimicking natural processing

Silk fibers spun by silkworms and spiders exhibit exceptional mechanical properties with a unique combination of strength, extensibility and toughness. In contrast, the mechanical properties of regenerated silk materials can be tuned through control of the fabrication process. Here we introduce a biomimetic, all-aqueous process, to obtain bulk regenerated silk-based materials for the fabrication of functionalized orthopedic devices. The silk materials generated in the process replicate the nano-scale structure of natural silk fibers and possess excellent mechanical properties. The biomimetic materials demonstrate excellent machinability, providing a path towards the fabrication of a new family of resorbable orthopedic devices where organic solvents are avoided, thus allowing functionalization with bioactive molecules to promote bone remodeling and integration.

Structural Insights into Water-Based Spider Silk Protein-Nanoclay Composites with Excellent Gas and Water Vapor Barrier Properties.

Nature reveals a great variety of inorganic-organic composite materials exhibiting good mechanical properties, high thermal and chemical stability, and good barrier properties. One class of natural bio-nanocomposites, e.g. found in mussel shells, comprises protein matrices with layered inorganic fillers. Inspired by such natural bio-nanocomposites, the cationic recombinant spider silk protein eADF4(κ16) was processed together with the synthetic layered silicate sodium hectorite in an all-aqueous setup. Drop-casting of this bio-nanocomposite resulted in a thermally and chemically stable film reflecting a one-dimensional crystal. Surprisingly, this bio-nanocomposite coating was, though produced in an all-aqueous process, completely water insoluble. Analyzing the structural details showed a low inner free volume due to the well-oriented self-assembly/alignment of the spider silk proteins on the nanoclay surface, yielding high oxygen and water vapor barrier properties. The here demonstrated properties in combination with good biocompatibility qualify this new bio-nanocomposite to be used in packaging applications.

Silk Biomaterials with Vascularization Capacity.

Functional vascularization is critical for the clinical regeneration of complex tissues such as kidney, liver or bone. The immobilization or delivery of growth factors has been explored to improve vascularization capacity of tissue engineered constructs, however, the use of growth factors has inherent problems such as the loss of signaling capability and the risk of complications such as immunological responses and cancer. Here, a new method of preparing water-insoluble silk protein scaffolds with vascularization capacity using an all aqueous process is reported. Acid was added temporally to tune the self-assembly of silk in lyophilization process, resulting in water insoluble scaffold formation directly. These biomaterials are mainly noncrystalline, offering improved cell proliferation than previously reported silk materials. These systems also have appropriate softer mechanical property that could provide physical cues to promote cell differentiation into endothelial cells, and enhance neovascularization and tissue ingrowth in vivo without the addition of growth factors. Therefore, silk-based degradable scaffolds represent an exciting biomaterial option, with vascularization capacity for soft tissue engineering and regenerative medicine.