Regiocontrol of the Bulk Polymerization of Lysine Ethyl Ester by the Selection of Suitable Immobilized Enzyme Catalysts.

The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.

Cell Wall-Denaturing Molecules for Plant Gene Modification.

Zwitterionic molecules, such as zwitterionic liquids (ZILs) and polypeptides (ZIPs), are attracting attention for application in new methods that can be used to loosen tight cell wall networks in a biocompatible manner. These novel methods can enhance the cell wall permeability of nanocarriers and increase their transfection efficiency into targeted subcellular organelles in plants. Herein, we provide an overview of the recent progress and future perspectives of such molecules that function as boosters for cell wall-penetrating nanocarriers.

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle-Complex-Mediated DNA Delivery to Specific Plant Organelles.

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants.

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification.

Direct delivery of enzymes into intact plants using cell-penetrating peptides (CPPs) is an attractive approach for modifying plant functions without genetic modification. However, by conventional methods, it is difficult to maintain the enzyme activity for a long time because of proteolysis of the enzymes under physiological conditions. Here, we developed a novel enzyme delivery system using polyion complex vesicles (PICsomes) to protect the enzyme from proteases. We created PICsome-bearing reactive groups at the surface by mixing an anionic block copolymer, alkyne-TEG-P(Lys-COOH), and a cationic peptide, P(Lys). The PICsome encapsulated neomycin phosphotransferase II (NPTII), a kanamycin resistance enzyme, and protected NPTII from proteases in vitro. A CPP-modified PICsome delivered NPTII into the root hair cells of Arabidopsis thaliana seedlings and provided kanamycin resistance in the seedlings that lasted for 7 days. Thus, the PICsome-mediated enzyme delivery system is a promising method for imparting long-term transient traits to plants without genetic modification.

Dual Peptide-Based Gene Delivery System for the Efficient Transfection of Plant Callus Cells.

Owing to their diverse functions and tunable physicochemical properties, peptides are promising alternatives to the conventional gene delivery tools that are available for plant systems. However, peptide-mediated gene delivery is limited by low transfection efficiency in plants because of the insufficient cytosolic translocation of DNA cargo. Here, we report a dual peptide-based gene delivery system for the efficient transfection of plant callus cells. This system is based on the combination of an artificial peptide composed of cationic cell-penetrating and hydrophobic endosomal escape domains with a gene carrier peptide composed of amphiphilic cell-penetrating and cationic DNA-binding domains. Cellular internalization and transfection studies revealed that this dual peptide-based system enables more efficient transfection of callus cells than does a carrier peptide alone by enhancing the endocytic uptake and subsequent cytosolic translocation of a carrier peptide/DNA complex. The present strategy will expand the utility of peptide-mediated plant gene delivery for a wide range of applications and basic research.

Zwitterionic Polypeptides: Chemoenzymatic Synthesis and Loosening Function for Cellulose Crystals.

A polypeptide with a GlyHisGly repeating sequence containing zwitterionic structures that effectively interact with cellulose was synthesized for dissociation of cellulose crystals. Polypeptide with the GlyHisGly sequence was synthesized by chemoenzymatic polymerization and postfunctionalization of the His residues was performed to afford imidazolium butyrate on the side chains. The resulting zwitterionic polypeptide effectively dissociated bundles of tunicate cellulose nanocrystals, even when the conditions were mild and the concentration of the polypeptide was as low as 1-2 mg mL-1. Polypeptide treatment also affected the morphology of the cell walls in cultured plant cells, and the cellulose microfibril networks and amorphous polysaccharide layer were dissociated according to atomic force microscopy (AFM). The zwitterionic polypeptide treatment did not change the crystal structure of the cellulose nanocrystals. Analysis of the mechanical properties of the cellulose nanocrystals by force curve measurements using AFM revealed that the elastic modulus of the cellulose nanocrystals increased after treatment with the zwitterionic polypeptide, indicating that the amorphous part of the cellulose nanocrystals was removed by interactions with the polypeptide. At a concentration of the polypeptide that enabled the dissociation of the cellulose network, the zwitterionic polypeptide showed negligible cytotoxicity to the plant cells. The mild and noncytotoxic technique for loosening cellulose microfibrils/nanocrystals that was developed in this study has tremendous significance for the modification of cellulose in terms of polymer chemistry, material science, and plant biotechnology.

Development of Reactive Oxygen Species-Triggered Degradable Nanoparticles Using Oligoproline-Containing Peptides.

Oligoproline-containing peptides, GPPG and GPPPG, were designed and developed for nanoparticle-based delivery platforms, and their degradation is triggered by reactive oxygen species (ROS). Peptides containing more than two consecutive proline residues were found to be cleavable in 1 mM of ROS generated by hydrogen peroxide in the presence of CuSO4, which corresponds to plant cells under photosynthetic conditions. The nanoparticles formed by the peptides were also ROS-degradable and efficiently encapsulated a hydrophobic dye. The hydrophobic cargo in the peptide nanoparticles was released into the cytosol of plant leaf cells in response to the ROS generated in chloroplasts by light irradiation. Furthermore, local laser irradiation enabled the peptide nanoparticles to release their cargo at only the irradiated cell, promising site-selective cargo release triggered by irradiation.

Block Copolymer/Plasmid DNA Micelles Postmodified with Functional Peptides via Thiol-Maleimide Conjugation for Efficient Gene Delivery into Plants.

Introducing exogenous genes into plant cells is essential for a wide range of applications in agriculture and plant biotechnology fields. Cationic peptide carriers with cell-penetrating and DNA-binding domains successfully deliver exogenous genes into plants. However, their cell-penetrating activity may be attenuated by undesired electrostatic interactions between the cell-penetrating peptide (CPP) domain and DNA cargo, resulting in limited gene delivery efficiency. Here, we developed the block copolymer maleimide-conjugated tetra(ethylene glycol) and poly(l-lysine) (MAL-TEG-PLL). Through electrostatic interactions with plasmid DNA (pDNA), MAL-TEG-PLL formed a micelle that presented maleimide groups on its surface. The micelle enabled postmodification with cysteine-containing functional peptides, including a CPP (BP100-Cys) and nuclear localization signal (Cys-NLS) via thiol-maleimide conjugation, thereby avoiding undesired interactions. According to a comparison of gene delivery efficiencies among the peptide-postmodified micelles, the amount of BP100-Cys on the micelle surface was key for efficient gene delivery. The BP100-postmodified micelle showed more efficient delivery compared with that of the BP100-premodified micelle. Thus, postmodification of polymeric micelles with functional peptides opens the door to designing highly efficient plant gene delivery systems.

Molecular Interactions and Toughening Mechanisms in Silk Fibroin-Epoxy Resin Blend Films.

Natural silkworm silks have been applied to reinforce epoxy resin to achieve sub-ambient and impact toughness in the composite. However, the molecular interactions at the silk fiber-matrix interface of the composite are poorly understood. In this work, silk fibroin extracted from Bombyx mori silk is blended with an epoxy resin polymer system to study the molecular interactions between silk fibroin, epoxy compounds, and hardeners. The effects of chemical crosslinks between epoxy groups and hardeners or silk fibroin, as well as physical crosslinks in the ß-sheet structure of silk fibroin, were discussed on the thermal stability, glass transition behavior, and mechanical properties of the blend films. A relationship between the crosslinking structure and mechanical properties for the films is proposed to enlighten on the toughening mechanisms. The findings would provide insights into forming strong and tough silk fibroin material as well as understanding the interface interactions of silk-epoxy composites.

Tensile Reinforcement of Silk Films by the Addition of Telechelic-Type Polyalanine.

An appropriate modification technique for silk materials is needed to effectively improve their physical properties for specific applications. A telechelic-type polyalanine (T-polyA) was synthesized by papain-catalyzed polymerization as a novel reinforcing agent for silk materials. A silk fibroin obtained from Bombyx mori was homogeneously doped with T-polyA, and casting a solution of silk fibroin and T-polyA in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) resulted in a robust and transparent film. Tensile deformation studies of the silk composite film containing T-polyA with prestretching revealed that the tensile strength and toughness were enhanced relative to those of a silk-only film. To determine the capability of T-polyA to reinforce the tensile property of silk films, the secondary structure in the silk composite film was characterized by wide-angle X-ray diffraction (WAXD) analysis. Antiparallel ß-sheet structures of T-polyA and GAGAGS motifs of silk fibroin formed independently in the prestretched composite film. In addition, measuring the tensile deformation and performing WAXD analysis simultaneously demonstrated that the ß-sheet structures of both T-polyA and the silk fibroin were aligned along the stretching direction and that T-polyA had no significant effect on the final morphology of the silk crystal domains. The silk film was toughened by the addition of T-polyA because of the generation of the T-polyA ß-sheet in the amorphous region of the composite film. This work provides novel insight into the design and development of tough silk materials with controlled and aligned ß-sheet structures.

The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization.

The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

Ampholytic Peptides Consisting of an Alternating Lysine/Glutamic Acid Sequence for the Simultaneous Formation of Polyion Complex Vesicles.

Nanoarchitectures such as micelles and vesicles that self-assemble via electrostatic interactions between their charged polymeric components have been widely used as material delivery platforms. In this work, ampholytic peptides with a sequence of alternating lysine and glutamic acid residues were designed and synthesized via chemoenzymatic polymerization. This alternating sequence was achieved by trypsin-catalyzed polymerization of a dipeptide monomer. Due to the electrostatic interaction between the anionic and cationic residues, the prepared ampholytic peptides spontaneously formed nanosized assemblies with a size of 100-200 nm in water. Modification with tetra(ethylene glycol) (TEG) at the N-terminus of these ampholytic alternating peptides resulted in the formation of stable nanosized assemblies, while peptides consisting of random sequences of lysine and glutamic acid formed large aggregates with deteriorated stability even with TEG modification. Morphological observations using a field-emission scanning electron microscope and an atomic force microscope revealed that the obtained assemblies were spherical and hollow, indicating the spontaneous formation of vesicles from the TEG-modified ampholytic alternating peptides. These vesicles were able to encapsulate a model fluorescent protein within their hollow structures without structural collapse causing loss of fluorescence, demonstrating the potential of these nanocarriers for use in material delivery systems.

Replicating shear-mediated self-assembly of spider silk through microfluidics.

The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of ß-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that ß-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a ß-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.

Spectral multitude and spectral dynamics reflect changing conjugation length in single molecules of oligophenylenevinylenes.

Single-molecule study of phenylenevinylene oligomers revealed distinct spectral forms due to different conjugation lengths which are determined by torsional defects. Large spectral jumps between different spectral forms were ascribed to torsional flips of a single phenylene ring. These spectral changes reflect the dynamic nature of electron delocalization in oligophenylenevinylenes and enable estimation of the phenylene torsional barriers.

Chemoenzymatic Polymerization of l-Serine Ethyl Ester in Aqueous Media without Side-Group Protection.

Poly(l-serine) (polySer) has tremendous potential as a polypeptide-based functional material due to the utility of the hydroxyl group on its side chain; however, tedious protection/deprotection of the hydroxyl groups is required for its synthesis. In this study, polySer was synthesized by the chemoenzymatic polymerization (CEP) of l-serine ethyl ester (Ser-OEt) or l-serine methyl ester (Ser-OMe) using papain as a catalyst in an aqueous medium. The CEP of Ser-OEt proceeded at basic pH ranging from 7.5 to 9.5 and resulted in the maximum precipitate yield of polySer at an optimized pH of 8.5. A series of peaks detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the formed precipitate consisted of polySer with a degree of polymerization ranging from 5 to 22. Moreover, infrared spectroscopy, circular dichroism spectroscopy, and synchrotron wide-angle X-ray diffraction measurements indicated that the obtained polySer formed a ß-sheet/strand structure. This is the first time the synthesis of polySer was realized by CEP in aqueous solution without protecting the hydroxyl group of the Ser monomer.

Polymer-coated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria.

The delivery of genetic material into plants has been historically challenging due to the cell wall barrier, which blocks the passage of many biomolecules. Carbon nanotube-based delivery has emerged as a promising solution to this problem and has been shown to effectively deliver DNA and RNA into intact plants. Mitochondria are important targets due to their influence on agronomic traits, but delivery into this organelle has been limited to low efficiencies, restricting their potential in genetic engineering. This work describes the use of a carbon nanotube-polymer hybrid modified with functional peptides to deliver DNA into intact plant mitochondria with almost 30 times higher efficiency than existing methods. Genetic integration of a folate pathway gene in the mitochondria displays enhanced plant growth rates, suggesting its applications in metabolic engineering and the establishment of stable transformation in mitochondrial genomes. Furthermore, the flexibility of the polymer layer will also allow for the conjugation of other peptides and cargo targeting other organelles for broad applications in plant bioengineering.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.

1000 spider silkomes: Linking sequences to silk physical properties.

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

All-Peptide-Based Polyion Complex Vesicles: Facile Preparation and Encapsulation of the Protein in Active Form.

The polyion complex vesicle (PICsome) is a promising platform for bioactive molecule delivery as well as nanoreactor systems. In addition to anionic and cationic charged blocks, a hydrophilic poly(ethylene glycol) (PEG) block is mostly employed for PICsome formation; however, the long-term safety of the PEG component in vivo is yet to be clarified. In this study, we developed novel PEG-free PICsome comprising all peptide components. Instead of the PEG block, we selected the sarcosine (Sar) oligomer as a hydrophilic block and fused it with anionic oligo(l-glutamic acid). Mixing the Sar-containing anionic peptide with cationic oligo(l-lysine) resulted in the formation of stable vesicles. The peptide-based PICsome was able to encapsulate a model protein in its hollow structure. After modification of the surface with a cell-penetrating peptide, the protein-encapsulated PICsome was successfully delivered into plant cells, indicating its promised for application as a biocompatible carrier for protein delivery.