Spectroscopic and computational investigations of organometallic complexation of group 12 transition metals by methanobactins from Methylocystis sp. SB2.

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as "methanobactins," which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

Rapid Buildup Arrays with Orthogonal Biochemistry Gradients via Light-Induced Thiol-Ene "Click" Chemistry for High-Throughput Screening of Peptide Combinations.

The concept of high-throughput screening sheds new light on fabrication and analysis of materials. Herein, a combinatorial surface-modified platform with biochemical gradients was developed through thiol-ene "click" chemistry by adjusting the intensity of ultraviolet (UV) irradiation. Contact angle, X-ray photoelectron spectroscopy, and ellipsometry measurement results demonstrated that the sulfhydryl molecules including polyethylene glycol and RGD (arginine-glycine-aspartic acid) and REDV (arginine-glutamic acid-aspartic acid-valine) peptides can be directly attached onto alkene-modified substrates, in which the graft density can be well controlled by the intensity of UV irradiation. The multistep attachment of different molecules onto substrates is archived via the multistep UV-initiated thiol-ene "click" reaction. The high-throughput arrays with the gradient density of single ligand and the orthogonal gradient density of two ligands were rapidly fabricated via the one-step UV gradient irradiation and the two-step orthogonal UV gradient-initiated thiol-ene "click" reaction. Endothelial cells (ECs) and smooth muscle cells (SMCs) were cocultured on the array with the orthogonal gradient density of RGD and REDV to screen the peptide combination with high EC selectivity, which is essential for in situ endothelialization during stent implant. From 64, 8 × 8, combinations investigated, a special combinatorial surface representing the really high competitiveness of ECs over SMCs was screened. This platform puts forward a facile, high-throughput method to study the combinatorial variation of biochemical signals to cell behavior.

Intracellular antioxidant activity and apoptosis inhibition capacity of PEF-treated KDHCH in HepG2 cells.

The effect of pulsed electric field (PEF) treatment on the intracellular antioxidant and apoptotic activity of the peptide Lys-Asp-His-Cys-His (KDHCH) was examined using model HepG2 cells. First, PEF treatment conditions specific for the antioxidant peptide were optimized, and it was found that PEF treatment could enhance DPPH, ABTS and hydroxyl radical scavenging capacity of KDHCH. Second, KDHCH subjected to PEF treatment at 1800 Hz and 15 kV/cm was investigated using various intracellular antioxidant assays. PEF treatment decreased the EC50 value and increased the protective ability of oxidative stress inhibition and reactive oxygen species (ROS) scavenging activity of KDHCH. Furthermore, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and glutathione reductase (GR) activities of KDHCH-pre-treated HepG2 cells increased significantly compared with those of the H2O2 damaged group, whereas lactate dehydrogenase (LDH) and malonaldehyde (MDA) content were decreased. PEF-treated KDHCH exhibited an increased capacity to maintain the stability of mitochondrial membrane potential (MMP) and reduced the level of caspase-3. These results indicate that PEF treatment can enhance the intracellular antioxidant activity of KDHCH, which can inhibit the effect of H2O2 oxidation on HepG2 cells by inhibiting the accumulation of intracellular ROS, regulating antioxidant related enzymes, and blocking the apoptotic mitochondrial pathways activated by ROS.

Two-Photon Probes for Golgi Apparatus: Detection of Golgi Apparatus in Live Tissue by Two-Photon Microscopy.

We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) from 6-(benzo[ d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[ d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi-network peptide (SDYQRL) as the Golgi-apparatus-targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon-excited fluorescence at 462 and 560 nm, respectively, with effective two-photon-action cross-section values of 1860 and 1600 × 10-50 cm4·s/photon, respectively. The probes can detect the Golgi apparatus in live cells and deep inside live tissue via two-photon microscopy at widely separated wavelength regions with high selectivity and minimal pH interference, and they are photostable and have low cytotoxicity.

Hollow-Core-Photonic-Crystal-Fiber-Based Miniaturized Sensor for the Detection of Aggregation-Induced-Emission Molecules.

A miniature sensor for detection of aggregation-induced-emission (AIE) molecules is proposed in this work. The sensing head is fabricated by use of hollow-core photonic crystal fiber with a core diameter of about 4.8 µm. The cladding holes are sealed with a fusion splicing technique, and the central hole remains open to allow the filtration of solution with AIE molecules. When the solution is excited by an ultraviolet lamp, the fluorescence is received by a fiber-optic spectrometer. The fluorescence intensity is associated with the concentration of AIE molecules and the infiltrated-core length. In the whole process of the experiments, the output-peak wavelength is stable, which indicates that the existing forms of AIE particles are stable, and the fluorescence reabsorption can be neglected. The experimental results obtained are in accordance with traditional microplate-spectrophotometer methods. The most exciting result is that the amount of sample measured can be as low as 0.36 nL, which allows the detection of AIE molecules at only 0.02 pmol. In addition, the miniature sensor was successfully applied to the detection of an AIE-based bioprobe for evaluating the activity of the dipeptidyl-peptidase 4 (DPP-4) inhibitor sitagliptin with an IC50 of 59.80 ± 3.06 nM. The advantages of small device size and nanoliter-scale sample volumes suggest that the proposed sensor is promising for many biosensing applications.

Enzyme-triggered self-assembly of gold nanoparticles for enhanced retention effects and photothermal therapy of prostate cancer.

A peptide-modified gold nanoparticle was developed for tumour-targeted therapy. Triggered by alkaline phosphatase, the CREKA-YPFFK(Nph) peptide can self-assemble and further result in accumulation of gold nanoparticles in tumour cells. The large-sized gold nanoparticle aggregates cannot escape from the tumour tissue, therefore realizing the goal of tumour-specific targeting, enhanced retention and photothermal effects.

Tailored photocleavable peptides: fragmentation and neutralization pathways in high vacuum.

Photocleavable tags (PCTs) have the potential for excellent spatio-temporal control over the release of subunits of complex molecules. Here, we show that electrosprayed oligopeptides, functionalized by a tailored ortho-nitroarylether can undergo site-specific photo-activated cleavage under UV irradiation (266 nm) in high vacuum. The comparison of UV photodissociation (UVPD) and collision-induced dissociation (CID) points to the thermal nature of the cleavage mechanism, a picture corroborated by the temperature dependence of the process. Two competing photodissociation pathways can be identified. In one case a phenolate anion is separated from a neutral zwitterion. In the other case a neutral phenol derivative leaves a negatively charged peptide behind. To understand the factors favoring one channel over the other, we investigate the influence of the peptide length, the nature of the phenolic group and the position of the nitro-group (ortho vs. para). The observed gas phase cleavage of a para-nitro benzylic ether markedly differs from the established behavior in solution.

Photo-responsive Bioactive Surfaces Based on Cucurbit[8]uril-Mediated Host-Guest Interactions of Arylazopyrazoles.

A photoswitchable arylazopyrazole (AAP) derivative binds with cucurbit[8]uril (CB[8]) and methylviologen (MV2+ ) to form a 1:1:1 heteroternary host-guest complex with a binding constant of Ka =2×103 m-1 . The excellent photoswitching properties of AAP are preserved in the inclusion complex. Irradiation with light of a wavelength of 365 and 520 nm leads to quantitative E- to Z- isomerization and vice versa, respectively. Formation of the Z-isomer leads to dissociation of the complex as evidenced using 1 H NMR spectroscopy. AAP derivatives are then used to immobilize bioactive molecules and photorelease them on demand. When Arg-Gly-Asp-AAP (AAP-RGD) peptides are attached to surface bound CB[8]/MV2+ complexes, cells adhere and can be released upon irradiation. The heteroternary host-guest system offers highly reversible binding properties due to efficient photoswitching and these properties are attractive for designing smart surfaces.

Hierarchical supramolecular hydrogels: self-assembly by peptides and photo-controlled release via host-guest interaction.

A hierarchical supramolecular hydrogel was self-assembled from a Fmoc-RGDS tetrapeptide and showed photo-controlled release directed by host-guest interaction. Multiple payloads, including vesicles, were successively released from a single peptide hydrogel.

Switchable photooxygenation catalysts that sense higher-order amyloid structures.

Proteins can misfold into amyloid structures that are associated with diseases; however, the same proteins often have important biological roles. To degrade selectively the amyloid form without affecting the fraction of functional protein is, therefore, an attractive goal. Here we report target-state-dependent photooxygenation catalysts that are active only when bound to the cross-ß-sheet structure that is characteristic of pathogenic aggregated amyloid proteins. We show these catalysts can selectively oxygenate the amyloid form of amyloid ß-protein (Aß) 1-42 in the presence of non-amyloid off-target substrates. Furthermore, photooxygenation with a catalyst that bears an Aß-binding peptide attenuated the Aß pathogenicity in the presence of cells. We also show that selective photooxygenation is generally applicable to other amyloidogenic proteins (amylin, insulin, ß2-microglobulin, transthyretin and α-synuclein) and does not affect the physiologically functional non-aggregate states of these proteins. This is the first report of an artificial catalyst that can be selectively and reversibly turned on and off depending on the structure and aggregation state of the substrate protein.

Phototriggered fibril-like environments arbitrate cell escapes and migration from endothelial monolayers.

Cell detachment and migration from the endothelium occurs during vasculogenesis and also in pathological states. Here, we use a novel approach to trigger single cell release from an endothelial monolayer by in-situ opening of adhesive, fibril-like environment using light-responsive ligands and scanning lasers. Cell escapes from the monolayer were observed on the fibril-like adhesive tracks with 3-15 µm width. The frequency of endothelial cell escapes increased monotonically with the fibril width and with the density of the light-activated adhesive ligand. Interestingly, treatment with VEGF induced cohesiveness within the cell layer, preventing cell leaks. When migrating through the tracks, cells presented body lateral reduction and nuclear deformation imposed by the line width and dependent on myosin contractility. Cell migration mode changed from mesenchymal to amoeboid-like when the adhesive tracks narrowed (≤5 µm). Moreover, cell nucleus was shrunk showing packed DNA on lines narrower than the nuclear dimensions in a mechanisms intimately associated with the stress fibers. This platform allows the detailed study of escapes and migratory transitions of cohesive cells, which are relevant processes in development and during diseases such as organ fibrosis and carcinomas.

A photo-responsive peptide- and asparagine-glycine-arginine (NGR) peptide-mediated liposomal delivery system.

The conjugation of tunable peptides or materials with nanocarriers represents a promising approach for drug delivery to tumor cells. In this study, we report the development of a novel liposomal carrier system that exploits the cell surface binding synergism between photo-sensitive peptides (PSPs) and targeting ligands. The positive charges of the lysine residues on the cell-penetrating peptides (CPPs) were temporarily caged by the photolabile-protective groups (PG), thereby forming a PSP. Furthermore, this PSP enhances specific uptake into cancer cells after rapidly uncaging the PG via near-infrared (NIR) light illumination. In the circulatory system, the cell penetrability of PSP was hindered. In contrast, the asparagine-glycine-arginine (NGR) peptide moieties, selectively bind to CD13-positive tumors, were attached to the nanocarrier to facilitate the active accumulation of this liposomal carrier in tumor tissue. The dual-modified liposomes (PSP/NGR-L) were prepared by emulsification method, and the concentrations of DSPE-PEG2000-psCPP and DSPE-PEG5000-NGR in the liposomes were chosen to be 4% and 1% (molar ratio), respectively. The mean particle size of the PSP/NGR-L was about 95 nm, and the drug entrapment efficiency was more than 90%. Cellular uptake results demonstrated that the proposed PSP/NGR-L had an enhancement of cancer cell recognition and specific uptake. Furthermore, the PSP/NGR-L demonstrated a stronger antitumor efficacy in the HT-1080 tumor model in nude mice with the aid of NIR illumination.

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications.

The use of tantalum as biomaterial for orthopedic applications is gaining considerable attention in the clinical practice because it presents an excellent chemical stability, body fluid resistance, biocompatibility, and it is more osteoconductive than titanium or cobalt-chromium alloys. Nonetheless, metallic biomaterials are commonly bioinert and may not provide fast and long-lasting interactions with surrounding tissues. The use of short cell adhesive peptides derived from the extracellular matrix has shown to improve cell adhesion and accelerate the implant's biointegration in vivo. However, this strategy has been rarely applied to tantalum materials. In this work, we have studied two immobilization strategies (physical adsorption and covalent binding via silanization) to functionalize tantalum surfaces with a cell adhesive RGD peptide. Surfaces were used untreated or activated with either HNO3 or UV/ozone treatments. The process of biofunctionalization was characterized by means of physicochemical and biological methods. Physisorption of the RGD peptide on control and HNO3-treated tantalum surfaces significantly enhanced the attachment and spreading of osteoblast-like cells; however, no effect on cell adhesion was observed in ozone-treated samples. This effect was attributed to the inefficient binding of the peptide on these highly hydrophilic surfaces, as evidenced by contact angle measurements and X-ray photoelectron spectroscopy. In contrast, activation of tantalum with UV/ozone proved to be the most efficient method to support silanization and subsequent peptide attachment, displaying the highest values of cell adhesion. This study demonstrates that both physical adsorption and silanization are feasible methods to immobilize peptides onto tantalum-based materials, providing them with superior bioactivity.

Photo-responsive and NGR-mediated multifunctional nanostructured lipid carrier for tumor-specific therapy.

A novel nanostructured lipid carrier (NLC) modified with photon-sensitive cell penetrating peptides (psCPP) and Asn-Gly-Arg (NGR) was designed to enhance paclitaxel (PTX)-targeted delivery and antitumor effect. The NGR moiety selectively binds to CD13-positive tumors. On other hand, the psCPP moiety enhance specific cancer cellular uptake after rapidly cleaving the two-photon excitation-responsive protective group, in this case, illumination in the presence of near-IR (NIR) light at the tumor site. The dual-modified NLC (psCPP/NGR-NLC) were prepared by emulsification method, and the concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000-psCPP (DSPE-PEG2000 -psCPP) and DSPE-PEG5000 -NGR in the NLC were chosen to be 4% and 1% (molar ratio), respectively. The mean particle size of the psCPP/NGR-NLC was about 100 nm, and the drug entrapment efficiency was more than 90%. Stability study showed that the prepared NLCs were physically and chemically stable at 2°C-8°C up to 1 month. Cellular uptake results demonstrated that the proposed psCPP/NGR-NLC had an enhancement of cancer cell recognition and specific uptake. Pharmacokinetic study showed that the prepared psCPP/NGR-NLC possessed the long-circulation characteristic with the t1/2 of 6.112 ± 0.304 h. Pharmacodynamics results confirmed that, with the aid of NIR illumination and NGR, the tumor inhibition ratio of psCPP/NGR-NLC group was significantly higher than the other PTX groups.

Extracellular-matrix-based and Arg-Gly-Asp-modified photopolymerizing hydrogels for cartilage tissue engineering.

Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application.

Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Stability of an anti-stroke peptide: driving forces and kinetics in chemical degradation.

NR2B9c (Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val) is a 9-amino acid peptide that has been illustrated to be a potential anti-stroke drug. For more effective treatment, suitable drug delivery systems should be developed. However, little is known about the stability of NR2B9c which is essential to its formulation. In this study, a reversed-phase high-performance liquid chromatography (HPLC) was applied to study the forced degradation behavior and stability of NR2B9c. HPLC studies were performed with an C8 column using a mobile phase consisting of acetonitrile (14.5:85.5, v/v) and aqueous solution (0.1% trifluoroacetic acid (TFA) and 0.05 M KH2PO4). The flow rate and the wavelength set during HPLC detection were 1.0 mL/min and 205 nm, respectively. The degradation pattern of NR2B9c aqueous solution followed pseudo first-order kinetics. The degradation rate at pH 7.5 was the slowest according to the plotting V-shaped pH-rate profile. The influence of temperature on the rate of reactions was interpreted in terms of Arrhenius equation (r(2)>0.98). Thermodynamic parameters were calculated based on Eyring equation (r(2)>0.98). The concentrations of drug, buffer species, buffer concentrations, oxidation and organic solvents have noticeable effects on the degradation of NR2B9c while ultrasound shows little impact under the experimental conditions. In a word, this study may give a detailed description of stability of NR2B9c.

Hydrogel cell patterning incorporating photocaged RGDS peptides.

As we aim towards enhancing our knowledge of complex cell behaviors and developing intricate cell-based devices and improved therapeutics, it becomes imperative that we be able to control and manipulate the spatial localization of cells. Here we have developed a novel strategy to pattern cells using a hyaluronic acid hydrogel material and photocaged RGDS (Arg-Gly-Asp-Ser) peptides. In this report, we discuss the chemical synthesis and photoactive properties of the caged peptides as well as the subsequent binding of these peptides to our hydrogel base. We further demonstrate the ability of this modified hydrogel material to pattern fibroblast cells on the micron scale using near-UV light exposure through a patterned photomask to selectively switch areas of the hydrogel surface from cell non-adhesive to cell adhesive. The cells are found to adhere and proliferate along the developed line patterns for at least 2.5 days, demonstrating significantly enhanced pattern longevity in comparison with previously reported studies.

A novel biomimetic photochemical switch at work: design of a photomodulable peptide.

We report the outcomes of our recent computational and experimental work for the development of a novel biomimetic molecular switch. Furthermore, we present the new results on the design and computational characterization of a "functional" cyclic peptidomimetic formed by the switch conjugated to a biologically active peptide: the RGD sequence involved in the control of cell adhesion. Structural properties of the construct are investigated in aqueous solution using molecular dynamics (MD) simulations. Analysis of MD trajectories reveals that, for each diastereoisomer of the switch (E or Z), different conformations are stabilized. Electrostatic and spectroscopic properties of such conformers are evaluated by means of ab initio multiconfiguration quantum chemical method implemented in a quantum-mechanical/molecular-mechanic (CASPT2//CASSCF/6-31G*/AMBER) scheme.

RNA aptamers that reversibly bind to photoresponsive peptide.

Modulation of biological network constituted by diverse interactions among biologically active molecules has provided innovative biotechnologies. Here, we report RNA aptamers that bind to photoresponsive peptide (KRAzR; Lys-Arg-Azobenzene-Arg) containing azobenzene chromophore, which can change its geometrical structure by phohtoirradiation. Aptamers were identified by 10 cycles of in vitro selection procedure from DNA library containing 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation. Upon irradiation of 360 nm light over the KRAzR-immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm light restored the aptamer binding ability of the surface. We also observed that direct photoirradiation of aptamer-peptide complex on a gold surface actively promoted dissociation of the complex.