Photo-reactive polymers for the immobilisation of epidermal growth factors.

Photo-reactive polymers are important for biomaterials, including devices with a 3D-structure. Here, different types of photo-reactive polymers were prepared and utilised for immobilisation of growth factors. They were synthesised by conjugation of gelatin with the azidophenyl group or by copolymerisation of the azidophenyl group-coupled methacrylate with poly(ethylene glycol) methacrylate. The azidophenyl content and the zeta potential of the prepared polymers were measured. After spin coating of polymers, the thickness and the water contact angle of coated layers were measured. The amount of the immobilised epidermal growth factor (EGF) was determined using fluorescence labelling. Cell adhesion responded to the nature of photo-reactive polymers but did not depend on the immobilised EGF. However, cell growth was dependent on the amount of immobilised EGF and was significantly affected by the nature of photo-reactive polymers. The study shows that the properties of the photo-immobilisation matrix significantly influence the biological activity.

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Using molecular dynamics simulations to prioritize and understand AI-generated cell penetrating peptides.

Cell-penetrating peptides have important therapeutic applications in drug delivery, but the variety of known cell-penetrating peptides is still limited. With a promise to accelerate peptide development, artificial intelligence (AI) techniques including deep generative models are currently in spotlight. Scientists, however, are often overwhelmed by an excessive number of unannotated sequences generated by AI and find it difficult to obtain insights to prioritize them for experimental validation. To avoid this pitfall, we leverage molecular dynamics (MD) simulations to obtain mechanistic information to prioritize and understand AI-generated peptides. A mechanistic score of permeability is computed from five steered MD simulations starting from different initial structures predicted by homology modelling. To compensate for variability of predicted structures, the score is computed with sample variance penalization so that a peptide with consistent behaviour is highly evaluated. Our computational pipeline involving deep learning, homology modelling, MD simulations and synthesizability assessment generated 24 novel peptide sequences. The top-scoring peptide showed a consistent pattern of conformational change in all simulations regardless of initial structures. As a result of wet-lab-experiments, our peptide showed better permeability and weaker toxicity in comparison to a clinically used peptide, TAT. Our result demonstrates how MD simulations can support de novo peptide design by providing mechanistic information supplementing statistical inference.

Strategy to Immobilize Peptide Probe Selected through In Vitro Ribosome Display for Electrochemical Aptasensor Application.

In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.

Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.

Disulfide-Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA.

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.

Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds.

Redox-active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl-functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo-excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6-fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo-excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal-complexes.

Controlled quercetin release from high-capacity-loading hyperbranched polyglycerol-functionalized graphene oxide.

PURPOSE: An efficient drug-delivery system was prepared based on graphene oxide using a facile and one-step strategy for controlling the release of anticancer drugs. METHODS: Fabrication of single-layer graphene oxide (GO) sheets was carried out by both modified and improved Hummers method. Biocompatible hyperbranched polyglycerol (HPG) was grafted on the surface of GO through the ring-opening hyperbranched polymerization of glycidol. Various ratios of GO and glycidol were used for polymer grafting. An anticancer drug, quercetin (Qu), was loaded into modified GO via noncovalent interactions. RESULTS: Polymer grafting on the surface of GO sheets was confirmed by results obtained from Fourier-transform infrared and Raman spectroscopy, thermogravimetric analysis, energy-dispersive X-ray and X-ray spectroscopy, scanning electron microscopy, and atomic force microscopy. It was revealed that polymerization increased d-spacing between the basal planes. In addition, as a hydrophilic polymer, HPG improved the stability and dispersion of GO sheets in biological solutions and endowed extra drug-loading capacity for the sheets. The effect of hyperbranched structure on drug loading and release was investigated by comparing drug loading and release for HPG-modified GO and linear PPO-modified GO. Our experiments indicated high drug-loading capacity (up to 185%), and excellent encapsulation efficiency (up to 93%) for HPG-GO compared to linear PO-grafted GO. The release profile of Qu under various pH levels exhibited controlled and sustained drug release without an initial burst effect for HPG-GO, suggesting that an acidic solution could facilitate drug release. HPG-GO did not show any cytotoxicity on the MCF7 cell line in different concentrations during 72 hours' incubation. Uptake and entrance of HPG-GO into the cells were verified by determining the intracellular amount of Qu by high-performance liquid chromatography. CONCLUSION: A combination of the unique properties of GO and the biodegradable polymer polyglycerol revealed high drug-loading capacity, pH-dependent drug release, and cytocompatibility with HPG-GO, thus introducing it as a promising nanocarrier for anticancer drug delivery.

Correction: In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection.

Correction for 'In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection' by Tara Bahadur K. C. et al., Chem. Commun., 2018, 54, 5201-5204.

In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection.

An electrosensitive peptide probe has been developed from an in vitro selection technique using biorthogonal tRNA prepared with an electroreactive non-natural amino acid, 3,4-ethylenedioxythiophene-conjugated aminophenylalanine. The selected probe quantitatively detected the influenza virus based on a signal "turn-on" mechanism. The developed strategy could be used to develop electrochemical biosensors toward a variety of targets.

Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group.

To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.

Two site genetic incorporation of varying length polyethylene glycol into the backbone of one peptide.

Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.

A dual functional peptide carrying in vitro selected catalytic and binding activities.

When minimal functional sequences are used, it is possible to integrate multiple functions on a single peptide chain, like a "single stroke drawing". Here a dual functional peptide was designed by combining in vitro selected catalytic and binding activities. For catalytic activity, we performed in vitro selection for a peptide aptamer binding to hemin by using ribosome display and isolated a peptide that had peroxidase activity in the presence of hemin. By combining the selected catalytic peptide with a peptide antigen, which can be recognized by an antibody, an enzyme-antibody conjugate-like peptide was obtained. This study demonstrates a successful strategy to create dual functionalized peptide chains for use in immunoassays.

In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.

Photoresponsive peptide aptamer to glutathione-immobilized microbeads was in vitro selected using ribosome display incorporated with tRNA carrying an amino acid coupled with an azobenzene.

pH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity.

To demonstrate the potential of pH-sensitive carbonate apatite (CO3Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO3Ap complexes, or pCI-neo/CO3Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO3Ap (10 µg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 µg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO3Ap (100 µg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 µg of pCI-neo-sOVA delivered by CO3Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO3Ap nanoparticles for DNA vaccine delivery.

Mussel-inspired human gelatin nanocoating for creating biologically adhesive surfaces.

Recombinant human gelatin was conjugated with dopamine using carbodiimide as a surface modifier. This dopamine-coupled human gelatin (D-rhG) was characterized by (1)H-nuclear magnetic resonance, mass spectroscopy, and circular dichroism. D-rhG-coated surface properties were analyzed by physicochemical methods. Additionally, cell attachment and growth on the modified surfaces was assessed using human umbilical endothelial cells. Binding of gelatin onto titanium was significantly enhanced by dopamine conjugation. The thickness of the D-rhG coating depended on the treatment pH; thicker layers were formed at higher pH values, with a maximum thickness of 30 nm. D-rhG enhanced the binding of collagen-binding vascular endothelial growth factor and cell adhesion as compared with gelatin alone, even at the same surface concentration. The D-rhG surface modifier enhanced substrate binding by creating an adhesive nanointerface that increased specific protein binding and cell attachment.

Direct in vitro selection of titanium-binding epidermal growth factor.

Epidermal growth factor (EGF) with affinity to TiO2 surfaces was obtained by direct in vitro selection. A random peptide library was generated for fusion to the N-terminal of EGF, and polypeptides exhibiting affinity were selected in vitro by ribosome display. The best-performing polypeptide sequence was selected for synthesis using a solid-phase method and showed high affinity to TiO2 after refolding. Molecular dynamic simulations indicated that the interaction of the selected peptide segment with the TiO2 surface was comparable to that of a previously reported titanium-binding peptide, TBP-1. The hydroxyl groups in the selected peptide segment were found to be critical for the binding interaction. NIH3T3 cell culture for two days in the presence of the TiO2-binding EGF showed that it was able to enhance cell proliferation as much as unmodified EGF in solution. As a result, the selected EGF construct was able to induce cell proliferation on titanium surfaces. This direct in vitro selection technique should extend the possibilities for the design of other surface-binding growth factors.

A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.

A peptide that binds and emits fluorescence in response to conformational change in a target protein was developed by in vitro selection using tRNA carrying a fluorogenic amino acid. This technology could prove to be useful for the development of separation-free immunoassays and bio-imaging analyses.

An epidermal growth factor derivative with binding affinity for hydroxyapatite and titanium surfaces.

An epidermal growth factor (EGF) derivative with affinity for apatite and titanium surfaces was designed using a peptide moiety derived from salivary statherin, a protein that adheres to hydroxyapatite. Since the active sequence has two phosphoserine residues, the EGF derivative was prepared by organic synthesis, and a 54 residue peptide was successfully prepared using this method. Circular dichroism spectra indicated that the conformation of EGF was not significantly altered by the addition of the affinity peptide sequence and the mitogenic activity was only slightly reduced when compared with the wild-type protein. However, the binding affinity of the modified EGF to hydroxyapatite and titanium was significantly higher than the unmodified EGF. The phosphate groups in the affinity sequence contributed to the affinity of modified EGF to both apatite and titanium. The modified EGF significantly enhanced the growth of cells on hydroxyapatite and titanium. It was also demonstrated that the bound EGF enhanced the signal transduction for longer periods than unbound EGF. In conclusion, the modified EGF had significantly higher binding affinity for apatite and titanium than soluble EGF, and the bound EGF significantly enhanced cell growth by long-lasting activation of intracellular signal transduction.

Bio-orthogonal and combinatorial approaches for the design of binding growth factors.

Merrifield chemistry enables the convenient synthesis of oligonucleotides and peptides, while recombinant DNA technology has facilitated protein engineering. Recently, protein engineering has been extended into bio-orthogonal protein engineering by the development of specific chemical or enzymatic modification technologies. The combinatorial approach of molecular evolutionary engineering (or in vitro selection) has also provided a new design tool for functional peptides. These methodologies have enabled the development of various new proteinaceous materials for biological and medical applications. Here, we will discuss recent progress in the molecular design of proteins with respect to the preparation of binding growth factors, which are of increasing importance in the biomaterials field.