Functional Ligand-Enabled Particle Assembly for Bio-Nano Interactions.

Functional ligands consist of a wide range of small or large molecules that exhibit a spectrum of physical, chemical, and biological properties. A suite of small molecules (e.g., peptides) or macromolecular ligands (e.g., antibodies and polymers) have been conjugated to particle surfaces for specific applications. However, postfunctionalization of ligands often presents challenges in controlling the surface density and may require the chemical modification of ligands. As an alternative option to postfunctionalization, our work has focused on using functional ligands as building blocks to assemble particles while maintaining their intrinsic (functional) properties. Through self-assembly or template-mediated assembly strategies, we have developed a range of protein-, peptide-, DNA-, polyphenol-, glycogen-, and polymer-based particles. This Account discusses the assembly of such nanoengineered particles, which includes self-assembled nanoparticles, hollow capsules, replica particles, and core-shell particles, according to three categories of functional ligands (i.e., small molecules, polymers, and biomacromolecules) that are used as building blocks for their formation. We discuss a range of covalent and noncovalent interactions among ligand molecules that have been explored to facilitate the assembly of particles. The physicochemical properties of the particles, including size, shape, surface charge, permeability, stability, thickness, stiffness, and stimuli-responsiveness, can be readily controlled by varying the ligand building block or by tuning the assembly method. By selecting specific ligands as building blocks, the bio-nano interactions (i.e., stealth, targeting, and cell trafficking) can also be modulated. For instance, particles composed mainly of low-fouling polymers (i.e., poly(ethylene glycol)) exhibit an extended blood circulation time (half-life > 12 h), while antibody-based nanoparticles demonstrate that a trade-off between stealth and targeting may be required when designing targeting nanoparticle systems. Small molecular ligands, such as polyphenols, have been used as building blocks for particle assembly as they can interact with various biomacromolecules through multiple noncovalent interactions, retain the function of biomacromolecules within the assembly, enable pH-responsive disassembly when coordinating with metal ions, and facilitate endosomal escape of nanoparticles. A perspective is provided on the current challenges associated with the clinical translation of ligand-based nanoparticles. This Account is also expected to serve as a reference to guide the fundamental research and development of functional particle systems assembled from various ligands for diverse applications.

Laser-assisted synthesis and modification of 2D materials.

Two-dimensional materials with unique physical, electronic, and optical properties have been intensively studied to be utilized for the next-generation electronic and optical devices, and the use of laser energy in the synthesis and modification of 2D materials is advantageous due to its convenient and fast fabrication processes as well as selective, controllable, and cost-effective characteristics allowing the precise control in materials properties. This paper summarizes the recent progress in utilizations of laser technology in synthesizing, doping, etching, transfer and strain engineering of 2D materials, which is expected to provide an insight for the future applications across diverse research areas.

Particle Engineering via Supramolecular Assembly of Macroscopic Hydrophobic Building Blocks.

Tailoring the hydrophobicity of supramolecular assembly building blocks enables the fabrication of well-defined functional materials. However, the selection of building blocks used in the assembly of metal-phenolic networks (MPNs), an emerging supramolecular assembly platform for particle engineering, has been essentially limited to hydrophilic molecules. Herein, we synthesized and applied biscatechol-functionalized hydrophobic polymers (poly(methyl acrylate) (PMA) and poly(butyl acrylate) (PBA)) as building blocks to engineer MPN particle systems (particles and capsules). Our method allowed control over the shell thickness (e.g., between 10 and 21 nm), stiffness (e.g., from 10 to 126 mN m-1 ), and permeability (e.g., 28-72 % capsules were permeable to 500 kDa fluorescein isothiocyanate-dextran) of the MPN capsules by selection of the hydrophobic polymer building blocks (PMA or PBA) and by controlling the polymer concentration in the MPN assembly solution (0.25-2.0 mM) without additional/engineered assembly processes. Molecular dynamics simulations provided insights into the structural states of the hydrophobic building blocks during assembly and mechanism of film formation. Furthermore, the hydrophobic MPNs facilitated the preparation of fluorescent-labeled and bioactive capsules through postfunctionalization and also particle-cell association engineering by controlling the hydrophobicity of the building blocks. Engineering MPN particle systems via building block hydrophobicity is expected to expand their use.

Metal-Phenolic-Mediated Assembly of Functional Small Molecules into Nanoparticles: Assembly and Bioapplications.

Small molecules, including therapeutic drugs and tracer molecules, play a vital role in biological processing, disease treatment and diagnosis, and have inspired various nanobiotechnology approaches to realize their biological function, particularly in drug delivery. Desirable features of a delivery system for functional small molecules (FSMs) include high biocompatibility, high loading capacity, and simple manufacturing processes, without the need for chemical modification of the FSM itself. Herein, we report a simple and versatile approach, based on metal-phenolic-mediated assembly, for assembling FSMs into nanoparticles (i.e., FSM-MPN NPs) under aqueous and ambient conditions. We demonstrate loading of anticancer drugs, latency reversal agents, and fluorophores at up to ~80 % that is mostly facilitated by π and hydrophobic interactions between the FSM and nanoparticle components. Secondary nanoparticle engineering involving coating with a polyphenol-antibody thin film or sequential co-loading of multiple FSMs enables cancer cell targeting and combination delivery, respectively. Incorporating fluorophores into FSM-MPN NPs enables the visualization of biodistribution at different time points, revealing that most of these NPs are retained in the kidney and heart 24 h post intravenous administration. This work provides a viable pathway for the rational design of small molecule nanoparticle delivery platforms for diverse biological applications.

Crystalline Metal-Organic Framework Coatings Engineered via Metal-Phenolic Network Interfaces.

Crystalline metal-organic frameworks (MOFs) have garnered extensive attention owing to their highly ordered porous structure and physicochemical properties. However, their practical application often requires their integration with various substrates, which is challenging because of their weakly adhesive nature and the diversity of substrates that exhibit different properties. Herein, we report the use of amorphous metal-phenolic network coatings to facilitate the growth of crystalline MOF coatings on various particle and planar substrates. Crystalline MOFs with different metal ions and morphologies were successfully deposited on substrates (13 types) of varying sizes, shapes, and surface chemistries. Furthermore, the physicochemical properties of the coated crystalline MOFs (e.g., composition, thickness) could be tuned using different synthesis conditions. The engineered MOF-coated membranes demonstrated excellent liquid and gas separation performance, exhibiting a high H2 permeance of 63200 GPU and a H2/CH4 selectivity of 10.19, likely attributable to the thin nature of the coating (~180 nm). Considering the vast array of MOFs available (>90,000) and the diversity of substrates, this work is expected to pave the way for creating a wide range of MOF composites and coatings with potential applications in diverse fields.

Synthesis of Thermoresponsive, Catechol-Rich Poly(ethylene glycol) Brush Polymers for Attenuating Cellular Oxidative Stress.

Herein, we report a platform to integrate customizable quantities of catechol units into polymers by reacting caffeic acid carbonic anhydride with polymers having pendant amine groups. Brush poly(ethylene glycol)-caffeamide (PEG-CAF) copolymers based on oligo(ethylene glycol)methyl ether methacrylate (OEGMA500) were obtained with a catechol content of approximately 30, 40, and 50 mol % (vs OEGMA content). Owing to the hydrophobicity of the introduced CAF groups, the catechol copolymers exhibited cloud points in the range of 23-46 °C and were used to fabricate thermoresponsive FeIII metal-phenolic network capsules. Polymers with the highest CAF content (50 mol %) proved most effective for attenuating reactive oxygen species levels in vitro, in co-cultured fibroblasts, and breast cancer cells, even in the presence of an exogenous oxidant source. The reported approach to synthesize customizable catechol materials could be generalized to other amine-functional polymers, with potential biomedical applications such as adhesives or stimuli-responsive drug delivery systems.

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation.

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.

Engineering Flexible Metal-Phenolic Networks with Guest Responsiveness via Intermolecular Interactions.

Flexible metal-organic materials are of growing interest owing to their ability to undergo reversible structural transformations under external stimuli. Here, we report flexible metal-phenolic networks (MPNs) featuring stimuli-responsive behavior to diverse solute guests. The competitive coordination of metal ions to phenolic ligands of multiple coordination sites and solute guests (e.g., glucose) primarily determines the responsive behavior of the MPNs, as revealed experimentally and computationally. Glucose molecules can be embedded into the dynamic MPNs upon mixing, leading to the reconfiguration of the metal-organic networks and thus changes in their physicochemical properties for targeting applications. This study expands the library of stimuli-responsive flexible metal-organic materials and the understanding of intermolecular interactions between metal-organic materials and solute guests, which is essential for the rational design of responsive materials for various applications.

Macromolecular Engineering of Thermoresponsive Metal-Phenolic Networks.

Dynamic nanostructured materials that can react to physical and chemical stimuli have attracted interest in the biomedical and materials science fields. Metal-phenolic networks (MPNs) represent a modular class of such materials: these networks form via coordination of phenolic molecules with metal ions and can be used for surface and particle engineering. To broaden the range of accessible MPN properties, we report the fabrication of thermoresponsive MPN capsules using FeIII ions and the thermoresponsive phenolic building block biscatechol-functionalized poly(N-isopropylacrylamide) (biscatechol-PNIPAM). The MPN capsules exhibited reversible changes in capsule size and shell thickness in response to temperature changes. The temperature-induced capsule size changes were influenced by the chain length of biscatechol-PNIPAM and catechol-to-FeIII ion molar ratio. The metal ion type also influenced the capsule size changes, allowing tuning of the MPN capsule mechanical properties. AlIII-based capsules, having a lower stiffness value (10.7 mN m-1), showed a larger temperature-induced size contraction (∼63%) than TbIII-based capsules, which exhibit a higher stiffness value (52.6 mN m-1) and minimal size reduction (<1%). The permeability of the MPN capsules was controlled by changing the temperature (25-50 °C)─a reduced permeability was obtained as the temperature was increased above the lower critical solution temperature of biscatechol-PNIPAM. This temperature-dependent permeability behavior was exploited to encapsulate and release model cargo (500 kDa fluorescein isothiocyanate-tagged dextran) from the capsules; approximately 70% was released over 90 min at 25 °C. This approach provides a synthetic strategy for developing dynamic and thermoresponsive-tunable MPN systems for potential applications in biological science and biotechnology.

DNA-Grafted Poly(acrylic acid) for One-Step DNA Functionalization of Iron Oxide Nanoparticles.

Here, we report one-step DNA functionalization of hydrophobic iron oxide nanoparticles (IONPs) using DNA-grafted poly(acrylic acid) (PAA- g-DNA). PAA- g-DNA was synthesized by coupling PAA to amine-modified oligonucleotides via solid-phase amide chemistry, which yielded PAA grafted with multiple DNA strands with high graft efficiencies. Synthesized PAA- g-DNA was utilized as a phase-transfer and DNA functionalization agent for hydrophobic IONPs, taking advantage of unreacted carboxylic acid groups. The resulting DNA-modified IONPs were well dispersed in aqueous solutions and possessed DNA binding properties characteristic of polyvalent DNA nanostructures, showing that this approach provides a simple one-step method for DNA functionalization of hydrophobic IONPs.

Multimodal Shape Transformation of Dual-Responsive DNA Block Copolymers.

Herein, we report the self-assembly and multimodal shape transformation of dual-responsive DNA di- and triblock copolymers. Dual-responsive DNA diblock copolymer was synthesized by coupling a thermoresponsive polymer, poly(N-isopropylacrylamide (PNIPAM), and an oligonucleotide. DNA-b-PNIPAM possesses thermoresponsive properties of PNIPAM as well as molecular recognition properties of DNA. Thus, they undergo reversible temperature-triggered transition at lower critical solution temperature (LCST) between molecular DNA and polymer micelles with high density DNA corona. The hybridization of DNA-b-PNIPAM and DNA-modified nanoparticles generates functional nanoparticles showing unique temperature-dependent aggregation and disaggregation behaviors due to the dual-responsive nature of DNA-b-PNIPAM. DNA triblock copolymers of DNA-b-PNIPAM-b-PMA were synthesized by introducing a hydrophobic block, poly(methyl acrylate) (PMA), to DNA/PNIPAM block copolymers, which form spherical micelles at room temperature. They are capable of nanoscale shape transformation through the combination of thermal trigger and DNA binding. DNA-b-PNIPAM-b-PMA micelles undergo sphere-to-cylinder shape changes above LCST due to the conformational change of PNIPAM. The shape change is reversible, and fast cylinder-to-sphere transition occurs when the temperature is lowered below LCST. The low temperature spherical morphology can also be accessed while keeping the temperature above LCST by introducing complementary DNA strands with single stranded overhang regions. These results demonstrate the multidimensional shape changing capability of DNA-b-PNIPAM-b-PMA enabled by the dual-responsive property.

Gold nanoparticles-based colorimetric assay for cathepsin B activity and the efficiency of its inhibitors.

Cathepsin B has been suggested to be a prognostic marker of melanoma, glioma, and a variety of cancers such as brain, breast, colon, esophageal, gastric, lung, ovarian, and thyroid cancers. Cathepsin B inhibitors have also been considered as anticancer drug candidates; hence, there has been a growing need for a probe which enables the selective and simple detection of cathepsin B and its inhibitors. For the purpose of selective assay, a cathepsin B-specific substrate, N,N'-diBoc-dityrosine-glycine-phenylalanine-3-(methylthio)propylamine (DBDY-Gly-Phe-MTPA) was synthesized in this study. Phe-MTPA, which was produced via cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA, allowed aggregation of gold nanoparticles (AuNPs) leading to a color change from red to blue. When tested for cathepsins B, L, and S, this assay method exhibited AuNPs color change only in reaction to cathepsin B. The limits of detection for cathepsin B was 10 and 5 nM in the 1 and 2 h hydrolysis reactions, respectively. The efficiency of cathepsin B inhibitors such as leupeptin, antipain, and chymostatin was easily compared by the degree of color change. Moreover, IC50 values of leupeptin, antipain, and chymostatin were found to be 0.11, 0.48, and 1.78 µM, respectively, which were similar to the results of previous studies. Therefore the colorimetric assay of cathepsin B and cathepsin B inhibitors using DBDY-Gly-Phe-MTPA and AuNPs allowed not only the selective but also the simple assay of cathepsin B and its inhibitors, which was possible with the naked eye.

Chicken Embryo Fibroblast Viability and Trans-Differentiation Potential for Cultured Meat Production Across Passages.

This study was conducted to analyze the viability of primary chicken embryo fibroblasts and the efficiency of adipogenic trans-differentiation for cultured meat production. In isolating chicken embryo fibroblasts (CEFs) from a heterogeneous cell pool containing chicken satellite cells (CSCs), over 90% of CEFs expressed CD29 and vimentin. The analysis of the proliferative capabilities of CEFs revealed no significant differences in EdU-positive cells (%), cumulative cell number, doubling time, and growth rate from passage 1 to passage 9 (p > 0.05). This indicates that CEFs can be isolated by 2 h of pre-plating and survive stably up to passage 9, and that primary fibroblasts can serve as a valuable cell source for the cultured meat industry. Adipogenic trans-differentiation was induced up to passage 9 of CEFs. As passages increased, lipid accumulation and adipocyte size significantly decreased (p < 0.05). The reduced differentiation rate of primary CEFs with increasing passages poses a major challenge to the cost and efficiency of cultured meat production. Thus, effective cell management and the maintenance of cellular characteristics for a long time are crucial for ensuring stable and efficient cultured fat production in the cultured meat industry.

Development and Comparative Evaluation of Imitated Fiber from Different Protein Sources Using Wet-Spinning.

Texture is a major challenge in addressing the need to find sustainable meat alternatives, as consumers desire alternative meat to have a sensory profile like meat. In this study, the fabrication of imitated muscle fiber (IMF) is performed by introducing different kinds of protein sources, with an effective bottom-up technique- wet spinning. Herein, the protein sources (pea protein isolate, wheat protein, and myofibrillar paste) were combined with sodium alginate to stimulate the bonding with the coagulation solution for fabrication. It has been found that the fabrication of IMF is possible using all the protein sources, however, due to the difference in protein structure, a significant difference was observed in quality characteristics compared to conventional meat. Additionally, combination of wheat protein and pea protein isolate has given similar values as conventional meat in terms of some of the texture profiles and Warner-Bratzler shear force. In general, the optimization of protein sources for wet spinning can provides a novel way for the production of edible fiber of alternative meat.

Scaffolding fundamentals and recent advances in sustainable scaffolding techniques for cultured meat development.

In cultured meat (CM) production, Scaffolding plays an important role by aiding cell adhesion, growth, differentiation, and alignment. The existence of fibrous microstructure in connective and muscle tissues has attracted considerable interest in the realm of tissue engineering and triggered the interest of researchers to implement scaffolding techniques. A wide array of research efforts is ongoing in scaffolding technologies for achieving the real meat structure on the principality of biomedical research and to replace serum free CM production. Scaffolds made of animal-derived biomaterials are found efficient in replicating the extracellular matrix (ECM), thus focus should be paid to utilize animal byproducts for this purpose. Proper identification and utilization of plant-derived scaffolding biomaterial could be helpful to add diversified options in addition to animal derived sources and reduce in cost of CM production through scaffolds. Furthermore, techniques like electrospinning, modified electrospinning and 3D bioprinting should be focused on to create 3D porous scaffolds to mimic the ECM of the muscle tissue and form real meat-like structures. This review discusses recent advances in cutting edge scaffolding techniques and edible biomaterials related to structured CM production.

Trends in Hybrid Cultured Meat Manufacturing Technology to Improve Sensory Characteristics.

The projected growth of global meat production over the next decade is attributed to rising income levels and population expansion. One potentially more pragmatic approach to mitigating the adverse externalities associated with meat production involves implementing alterations to the production process, such as transitioning to cultured meat, hybrid cultured meat, and meat alternatives. Cultured meat (CM) is derived from animal stem cells and undergoes a growth and division process that closely resembles the natural in vivo cellular development. CM is emerging as a widely embraced substitute for traditional protein sources, with the potential to alleviate the future strain on animal-derived meat production. To date, the primary emphasis of cultured meat research and production has predominantly been around the ecological advantages and ethical considerations pertaining to animal welfare. However, there exists substantial study potential in exploring consumer preferences with respect to the texture, color, cuts, and sustainable methodologies associated with cultured meat. The potential augmentation of cultured meat's acceptance could be facilitated through the advancement of a wider range of cuts to mimic real muscle fibers. This review examines the prospective commercial trends of hybrid cultured meat. Subsequently, the present state of research pertaining to the advancement of scaffolding, coloration, and muscle fiber development in hybrid cultured meat, encompassing plant-based alternatives designed to emulate authentic meat, has been deliberated. However, this discussion highlights the obstacles that have arisen in current procedures and proposes future research directions for the development of sustainable cultured meat and meat alternatives, such as plant-based meat production.

Effect of Chicken Age on Proliferation and Differentiation Abilities of Muscle Stem Cells and Nutritional Characteristics of Cultured Meat Tissue.

This study aimed to investigate effects of chicken age on proliferation and differentiation capacity of muscle satellite cells (MSCs) and to determine total amino acid contents of cultured meat (CM) produced. Chicken MSCs (cMSCs) were isolated from hindlimb muscles of broiler chickens at 5-week-old (5W) and 19-embryonic-day (19ED), respectively. Proliferation abilities (population doubling time and cell counting kit 8) of cMSCs from 19ED were significantly higher than those from 5W (p<0.05). Likewise, both myotube formation area and expression of myosin heavy chain heavy of cMSCs from 19ED were significantly higher than those from 5W (p<0.05). After cMSCs were serially subcultured for long-term cultivation in 2D flasks to produce cultured meat tissue (CMT), total amino acid contents of CMT showed no significant difference between 5W and 19ED chickens (p>0.05). This finding suggests that cMSCs from chicken embryos are more suitable for improving the production efficiency of CM than those derived from young chickens.

New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material.

The increasing number of reports for disease-related proteases has necessitated materials for the fast, sensitive, and specific assessment of protease activities. The purpose of this study was to synthesize and test a dityrosine-based substrate for the selective assay of a specific cysteine cathepsin. DBDY-Gly-INH)2 was synthesized from the conjugation of N,N'-diBoc-dityrosine (DBDY) with two molecules of glycine and isoniazid (INH) for this purpose. The fluorescence of DBDY (λex=284-320nm, λem=400-420nm) disappeared due to the quenching effect of INH. However, the protease-catalyzed hydrolysis resulted in the release of INH and recovered the fluorescence of DBDY. When reacted with 13 proteases, DBDY-Gly-INH)2 was hydrolyzed by the cysteine proteases only. Meeting the growing need to discriminate cysteine cathepsins (e.g., cathepsins B, L, and S found at high levels in various cancers), DBDY-Gly-INH)2 was tested as a substrate for cathepsins B, L, and S. Only cathepsin B catalyzed the hydrolysis reaction among the three cathepsins. The reaction rate followed the Michaelis-Menten kinetics, and the KM and kcat/KM values were 2.88µM and 3.87×10(3)M(-1)s(-1), respectively, which were comparable to those for the materials reported for the selective assay of cathepsin B. Considering the simple preparation of DBDY-(Gly-INH)2, DBDY-(Gly-INH)2 is believed to be valuable for the sensitive and selective assay of cathepsin B activity.

Aging mechanism for improving the tenderness and taste characteristics of meat.

Tenderness and taste characteristics of meat are the key determinants of the meat choices of consumers. This review summarizes the contemporary research on the molecular mechanisms by which postmortem aging of meat improves the tenderness and taste characteristics. The fundamental mechanism by which postmortem aging improves the tenderness of meat involves the operation of the calpain system due to apoptosis, resulting in proteolytic enzyme-induced degradation of cytoskeletal myofibrillar proteins. The improvement of taste characteristics by postmortem aging is mainly explained by the increase in the content of taste-related peptides, free amino acids, and nucleotides produced by increased hydrolysis activity. This review improves our understanding of the published research on tenderness and taste characteristics of meat and provides insights to improve these attributes of meat through postmortem aging.

A Comparative Study on the Taste Characteristics of Satellite Cell Cultured Meat Derived from Chicken and Cattle Muscles.

This study investigated the amino acid and nucleotide-related compound composition and taste characteristics of cultured muscle tissue (CMT) obtained by culturing satellite cells isolated from chicken and cattle and compared them to those of traditional meat (TM). The content of all amino acids except valine and tyrosine was significantly different between CMT and TM (p<0.05). The amount of glutamic acid was not significantly different between CMT and TM in cattle, but the glutamic acid in chicken CMT was lower than that of TM (p<0.05). Among the nucleotide-related compounds, only the content of inosine-5'-monophosphate (IMP) was significant, and the amount of IMP in CMT derived from chicken and cattle was significantly lower than that of TM (p<0.05). There were significant differences in the taste characteristics assessed by an electronic tongue system, and the umami, bitterness, and sourness values of CMT were significantly lower than those of TM from both chicken and cattle (p<0.05). The results of the present study suggest that it is necessary to develop a satellite cell culture method that could increase the umami and bitterness intensity of CMT and adjust the composition of the growth medium to produce cultured meat with a taste similar to that of TM.