Measuring the Accessible Surface Area within the Nanoparticle Corona Using Molecular Probe Adsorption.

The corona phase-the adsorbed layer of polymer, surfactant, or stabilizer molecules around a nanoparticle-is typically utilized to disperse nanoparticles into a solution or solid phase. However, this phase also controls molecular access to the nanoparticle surface, a property important for catalytic activity and sensor applications. Unfortunately, few methods can directly probe the structure of this corona phase, which is subcategorized as either a hard, immobile corona or a soft, transient corona in exchange with components in the bulk solution. In this work, we introduce a molecular probe adsorption (MPA) method for measuring the accessible nanoparticle surface area using a titration of a quenchable fluorescent molecule. For example, riboflavin is utilized to measure the surface area of gold nanoparticle standards, as well as corona phases on dispersed single-walled carbon nanotubes and graphene sheets. A material balance on the titration yields certain surface coverage parameters, including the ratio of the surface area to dissociation constant of the fluorophore, q/KD, as well as KD itself. Uncertainty, precision, and the correlation of these parameters across different experimental systems, preparations, and modalities are all discussed. Using MPA across a series of corona phases, we find that the Gibbs free energy of probe binding scales inversely with the cube root of surface area, q. In this way, MPA is the only technique to date capable of discerning critical structure-property relationships for such nanoparticle surface phases. Hence, MPA is a rapid quantitative technique that should prove useful for elucidating corona structure for nanoparticles across different systems.

Rational Design Principles for the Transport and Subcellular Distribution of Nanomaterials into Plant Protoplasts.

The ability to control the subcellular localization of nanoparticles within living plants offers unique advantages for targeted biomolecule delivery and enables important applications in plant bioengineering. However, the mechanism of nanoparticle transport past plant biological membranes is poorly understood. Here, a mechanistic study of nanoparticle cellular uptake into plant protoplasts is presented. An experimentally validated mathematical model of lipid exchange envelope penetration mechanism for protoplasts, which predicts that the subcellular distribution of nanoparticles in plant cells is dictated by the particle size and the magnitude of the zeta potential, is advanced. The mechanism is completely generic, describing nanoparticles ranging from quantum dots, gold and silica nanoparticles, nanoceria, and single-walled carbon nanotubes (SWNTs). In addition, the use of imaging flow cytometry to investigate the influence of protoplasts' morphological characteristics on nanoparticle uptake efficiency is demonstrated. Using DNA-wrapped SWNTs as model nanoparticles, it is found that glycerolipids, the predominant lipids in chloroplast membranes, exhibit stronger lipid-nanoparticle interaction than phospholipids, the major constituent in protoplast membrane. This work can guide the rational design of nanoparticles for targeted delivery into specific compartments within plant cells without the use of chemical or mechanical aid, potentially enabling various plant engineering applications.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

A Nanobionic Light-Emitting Plant.

The engineering of living plants for visible light emission and sustainable illumination is compelling because plants possess independent energy generation and storage mechanisms and autonomous self-repair. Herein, we demonstrate a plant nanobionic approach that enables exceptional luminosity and lifetime utilizing four chemically interacting nanoparticles, including firefly luciferase conjugated silica (SNP-Luc), d-luciferin releasing poly(lactic-co-glycolic acid) (PLGA-LH2), coenzyme A functionalized chitosan (CS-CoA) and semiconductor nanocrystal phosphors for longer wavelength modulation. An in vitro kinetic model incorporating the release rates of the nanoparticles is developed to maximize the chemiluminescent lifetimes to exceed 21.5 h. In watercress (Nasturtium officinale) and other species, the nanoparticles circumvent limitations such as luciferin toxicity above 400 µM and colocalization of enzymatic reactions near high adenosine triphosphate (ATP) production. Pressurized bath infusion of nanoparticles (PBIN) is introduced to deliver a mixture of nanoparticles to the entire living plant, well described using a nanofluidic mathematical model. We rationally design nanoparticle size and charge to control localization within distinct tissues compartments with 10 nm nanoparticles localizing within the leaf mesophyll and stomata guard cells, and those larger than 100 nm segregated in the leaf mesophyll. The results are mature watercress plants that emit greater than 1.44 × 1012 photons/sec or 50% of 1 µW commercial luminescent diodes and modulate "off" and "on" states by chemical addition of dehydroluciferin and coenzyme A, respectively. We show that CdSe nanocrystals can shift the chemiluminescent emission to 760 nm enabling near-infrared (nIR) signaling. These results advance the viability of nanobionic plants as self-powered photonics, direct and indirect light sources.

Lipid Exchange Envelope Penetration (LEEP) of Nanoparticles for Plant Engineering: A Universal Localization Mechanism.

Nanoparticles offer clear advantages for both passive and active penetration into biologically important membranes. However, the uptake and localization mechanism of nanoparticles within living plants, plant cells, and organelles has yet to be elucidated.1 Here, we examine the subcellular uptake and kinetic trapping of a wide range of nanoparticles for the first time, using the plant chloroplast as a model system, but validated in vivo in living plants. Confocal visible and near-infrared fluorescent microscopy and single particle tracking of gold-cysteine-AF405 (GNP-Cys-AF405), streptavidin-quantum dot (SA-QD), dextran and poly(acrylic acid) nanoceria, and various polymer-wrapped single-walled carbon nanotubes (SWCNTs), including lipid-PEG-SWCNT, chitosan-SWCNT and 30-base (dAdT) sequence of ssDNA (AT)15 wrapped SWCNTs (hereafter referred to as ss(AT)15-SWCNT), are used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within the organelle, despite the negative zeta potential of the envelope. We develop a mathematical model of this lipid exchange envelope and penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. The theory predicts a critical particle size below which the mechanism fails at all zeta potentials, explaining why nanoparticles are critical for this process. LEEP constitutes a powerful particulate transport and localization mechanism for nanoparticles within the plant system.

A Ratiometric Sensor Using Single Chirality Near-Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring.

Advances in the separation and functionalization of single walled carbon nanotubes (SWCNT) by their electronic type have enabled the development of ratiometric fluorescent SWCNT sensors for the first time. Herein, single chirality SWCNT are independently functionalized to recognize either nitric oxide (NO), hydrogen peroxide (H(2)O(2)), or no analyte (remaining invariant) to create optical sensor responses from the ratio of distinct emission peaks. This ratiometric approach provides a measure of analyte concentration, invariant to the absolute intensity emitted from the sensors and hence, more stable to external noise and detection geometry. Two distinct ratiometric sensors are demonstrated: one version for H(2)O(2), the other for NO, each using 7,6 emission, and each containing an invariant 6,5 emission wavelength. To functionalize these sensors from SWCNT isolated from the gel separation technique, a method for rapid and efficient coating exchange of single chirality sodium dodecyl sulfate-SWCNT is introduced. As a proof of concept, spatial and temporal patterns of the ratio sensor response to H(2)O(2) and, separately, NO, are monitored in leaves of living plants in real time. This ratiometric optical sensing platform can enable the detection of trace analytes in complex environments such as strongly scattering media and biological tissues.

Chemically-modulated photoluminescence of graphene oxide for selective detection of neurotransmitter by "turn-on" response.

Designing artificial nanomaterials capable of selectively detecting targets without the use of expensive and fragile antibodies is of great interest in the applications of nanomedicine. Here, we show that the photoluminescence (PL) of graphene oxide (GO) was chemically modulated for the selective detection of a neurotransmitter without the use of antibodies. GO was functionalized with nitrotriacetic acid (NTA) on which four different metal ions were chelated (M-NTA-GO), which led to its different PL responses to neurotransmitters. In particular, the Cu-NTA-GO hybrid was able to selectively detect norepinephrine at nanomolar concentrations in a simple manner via its "turn-on" PL. Moreover, it was successfully applied to the selective detection of norepinephrine secreted from living PC-12 cells.

Antioxidant activity of caffeoyl-prolyl-histidine amide and its effects on PDGF-induced proliferation of vascular smooth muscle cells.

Caffeic acid (CA) is one of the antioxidants found in plants, which protects vascular cells against vascular injuries from oxidative stress. In our previous study, caffeoyl-prolyl-histidine amide (CA-L-Pro-L-His-NH2; CA-PH; a CA derivative) was synthesized, which exhibited a strong antioxidant activity with sufficient stability. In this study, we investigated the role of CA-PH in vascular smooth muscle cells (VSMCs) and confirmed the enhanced antioxidant activity of CA-PH compared with that of CA. In in vitro tube assays, CA-PH showed a higher free-radical-scavenging activity and lipid-peroxidation-inhibition activity than those of CA. In VSMCs, CA-PH significantly reduced hydrogen peroxide-induced ROS generation and increased the expression of heme oxygenase-1. Moreover, CA-PH effectively inhibited the platelet-derived growth factor-induced cellular proliferation of VSMCs, which was confirmed by a decrease in the expression of the proliferating cell nuclear antigen and the phosphorylation of Akt.

Effect of alpha-resorcylic acid-L-phenylalanine amide on collagen synthesis and matrix metalloproteinase expression in fibroblasts.

Regulation of collagen synthesis and matrix metalloproteinases (MMPs) expression levels has been an important issue in medicinal, pharmaceutical and cosmetic industries. Herein, α-Resorcylic acid-phenylalanine amide (α-RA-F) was prepared and its biological activities were observed. We found that α-RA-F boosted collagen synthesis and reduced MMPs expression levels in human fibroblasts without cytotoxicity.

Chemical modulation of bioactive compounds via oligopeptide or amino acid conjugation.

Conjugation of an oligopeptide or an amino acid to bioactive compounds is one of the simplest chemical modifications to modulate the biological functions of the compounds. Recently, numerous methods have been proposed for the modification of their properties, including the alteration of their chemical and physical properties, and of their original bioactivities. We review the current knowledge of the adaptability of oligopeptide or amino acid conjugation for modulating the biological activities of biomolecules.

Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase inhibitor and antioxidant.

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.

Single-Molecule Methane Sensing Using Palladium-Functionalized nIR Fluorescent Single-Walled Carbon Nanotubes.

There has been considerable interest in detecting atmospheric and process-associated methane (CH4) at low concentrations due to its potency as a greenhouse gas. Nanosensor technology, particularly fluorescent single-walled carbon nanotube (SWCNT) arrays, is promising for such applications because of their chemical sensitivities at single-molecule detection limits. However, the methodologies for connecting the stochastic molecular fluctuations from gas impingement on such sensors require further development. In this work, we synthesize Pd-conjugated ss(GT)15-DNA-wrapped SWCNTas near-infrared (nIR) fluorescent, single-molecule sensors of CH4. The complexes are characterized using X-ray photoelectron spectroscopy (XPS) and spectrophotometry, demonstrating spectral changes between the Pd2+ and Pd0 oxidation states. The nIR fluctuations generated upon exposure from 8 to 26 ppb of CH4 were separated into high- and low-frequency components. Aggregating the low-frequency components for an array of sensors showed the most consistent levels of detection with a limit of 0.7 ppb. These results advance the hardware and computational methods necessary to apply this approach to the challenge of environmental methane sensing.

Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation.

Decellularized extracellular matrix scaffold, widely utilized for organ engineering, often undergoes matrix decomposition after transplantation and produces byproducts that cause inflammation, leading to clinical failure. Here we propose a strategy using nano-graphene oxide to modify the biophysical properties of decellularized liver scaffolds. Notably, we demonstrate that scaffolds crosslinked with nano-graphene oxide show high resistance to enzymatic degradation via direct inhibition of matrix metalloproteinase activity and increased mechanical rigidity. We find that M2-like macrophage polarization is promoted within the crosslinked scaffolds, which reduces graft-elicited inflammation. Moreover, we show that low activities of matrix metalloproteinases, attributed to both nano-graphene oxide and tissue inhibitors of metalloproteinases expressed by M2c, can protect the crosslinked scaffolds against in vivo degradation. Lastly, we demonstrate that bioengineered livers fabricated with the crosslinked scaffolds remain functional, thereby effectively regenerating damaged livers after transplantation into liver failure mouse models. Overall, nano-graphene oxide crosslinking prolongs allograft survival and ultimately improves therapeutic effects of bioengineered livers, which offer an alternative for donor organs.

Single-Walled Carbon Nanotube-Guided Topical Skin Delivery of Tyrosinase to Prevent Photoinduced Damage.

When the skin is exposed to ultraviolet radiation (UV), it leads to the degradation of the extracellular matrix (ECM) and results in inflammation. Subsequently, melanocytes are triggered to induce tyrosinase-mediated melanin synthesis, protecting the skin. Here, we introduce a proactive approach to protect the skin from photodamage via the topical delivery of Streptomyces avermitilis-derived tyrosinase (SaTy) using single-walled carbon nanotube (SWNT). Utilizing a reverse electrodialysis (RED) battery, we facilitated the delivery of SaTy-SWNT complexes up to depths of approximately 300 µm, as analyzed by using confocal Raman microscopy. When applied to ex vivo porcine skin and in vivo albino mouse skin, SaTy-SWNT synthesized melanin, resulting in 4-fold greater UV/vis absorption at 475 nm than in mice without SaTy-SWNT. The synthesized melanin efficiently absorbed UV light and alleviated skin inflammation. In addition, the densification of dermal collagen, achieved through SaTy-mediated cross-linking, reduced photoinduced wrinkles by 66.3% in the affected area. Our findings suggest that SWNT-mediated topical protein delivery holds promise in tissue engineering applications.

In vivo surface-enhanced Raman scattering nanosensor for the real-time monitoring of multiple stress signalling molecules in plants.

When under stress, plants release molecules to activate their defense system. Detecting these stress-related molecules offers the possibility to address stress conditions and prevent the development of diseases. However, detecting endogenous signalling molecules in living plants remains challenging due to low concentrations of these analytes and interference with other compounds; additionally, many methods currently used are invasive and labour-intensive. Here we show a non-destructive surface-enhanced Raman scattering (SERS)-based nanoprobe for the real-time detection of multiple stress-related endogenous molecules in living plants. The nanoprobe, which is placed in the intercellular space, is optically active in the near-infrared region (785 nm) to avoid interferences from plant autofluorescence. It consists of a Si nanosphere surrounded by a corrugated Ag shell modified by a water-soluble cationic polymer poly(diallyldimethylammonium chloride), which can interact with multiple plant signalling molecules. We measure a SERS enhancement factor of 2.9 × 107 and a signal-to-noise ratio of up to 64 with an acquisition time of ~100 ms. To show quantitative multiplex detection, we adopted a binding model to interpret the SERS intensities of two different analytes bound to the SERS hot spot of the nanoprobe. Under either abiotic or biotic stress, our optical nanosensors can successfully monitor salicylic acid, extracellular adenosine triphosphate, cruciferous phytoalexin and glutathione in Nasturtium officinale, Triticum aestivum L. and Hordeum vulgare L.-all stress-related molecules indicating the possible onset of a plant disease. We believe that plasmonic nanosensor platforms can enable the early diagnosis of stress, contributing to a timely disease management of plants.

Structural and Electronic Modulations of Imidazolium Covalent Organic Framework-Derived Electrocatalysts for Oxygen Redox Reactions in Rechargeable Zn-Air Batteries.

Covalent organic frameworks (COFs) are promising candidates for the controllable design of electrocatalysts. However, bifunctional electrocatalytic activities for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) remain challenging in COFs. In this study, imidazolium-rich COFs (IMCOFs) with well-defined active sites and characteristic three-dimensional assembly structures were readily prepared, and their electronic structures were tuned by Co incorporation to elicit bifunctional electrocatalytic activities for the ORR and OER. The Co nanoparticle-incorporated spherical IMCOF-derived electrocatalyst (CoNP-s-IMCOF) exhibited lower overpotentials for the ORR and OER compared with the atomic Co-incorporated planar IMCOF-derived electrocatalyst (Co-p-IMCOF). Computational simulations revealed that the imidazole carbon sites of CoNP-s-IMCOF rather than the triazine carbons were the active sites for the ORR and OER, and its p-band center downshifted via charge transfer, facilitating the chemisorption of oxygen intermediates during the reactions. A Zn-air battery with CoNP-s-IMCOF exhibited a small voltage gap of 1.3 V with excellent durability for 935 cycles. This approach for control over the three-dimensional assembly and electronic structures of IMCOFs can be extended to the development of diverse catalytic nanomaterials for applications of interest.

Dual effects of caffeoyl-amino acidyl-hydroxamic acid as an antioxidant and depigmenting agent.

Anti-aging and depigmentation have both been an important subject of study for skin disease and the cosmetic industry. Caffeic acid (CA) has shown synergistically enhanced antioxidant activity when conjugated with amino acids. Hydroxamic acid (NHOH) is a well-known metal chelator, potentially having both tyrosinase inhibitory activity and free radical scavenging activity. We prepared caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) and found that caffeoyl-prolyl-hydroxamic acid (CA-Pro-NHOH) contained excellent antioxidant activity and tyrosinase inhibitory activity by various bioassay systems. Also, CA-Pro-NHOH showed mild melanogenesis inhibitory activity in Mel-Ab cells.

Kojic acid-amino acid amide metal complexes and their melanogenesis inhibitory activities.

Tyrosinase plays a critical role in the early stages of the melanin synthetic pathway by catalyzing the oxidation of the substrate. Therefore, tyrosinase inhibitors have been intensively studied in both cosmetic and food industries to develop hypopigmentary agents and prevent enzymatic browning in food. Previously, we reported that kojic acid-amino acid amide (KA-AA-NH(2)) showed enhanced tyrosinase inhibitory activity compared with kojic acid alone, but this was not observed in a cell test because of poor cell permeability. To enhance cell permeability, we prepared copper and zinc complexes of KA-AA-NH(2) and characterized them using FT-IR spectroscopy, ESI-MS spectrometry, and inductively coupled plasma analysis. We then showed that KA-AA-NH(2) copper complexes exhibited melanogenesis inhibitory activity in Mel-Ab cells.

Graphene oxide film guided skeletal muscle differentiation.

Engineered muscle tissues can be used for the regeneration or substitution of irreversibly damaged or diseased muscles. Recently, graphene oxide (GO) has been shown to improve the adsorption of biomolecules through its biocompatibility and intrinsic π-π interactions. The possibility of producing various GO modifications may also provide additional functionality as substrates for cell culture. In particular, substrates fabricated from pristine GO have been shown to improve cellular functions and influence stem cell differentiation. In this study, we fabricated tunable GO substrates with various physical and chemical properties and demonstrated the ability of the substrate to support myogenic differentiation. Higher cellular adhesion affinity with unique microfilament anchorage was observed for GO substrates with increased GO concentrations. In addition, amino acid (AA)-conjugated GO (GO-AA) substrates were fabricated to modify GO chemical properties and study the effects of chemically modified GO substrates on myogenic differentiation. Our findings demonstrate that minor tuning of GO significantly influences myogenic differentiation.

Antioxidative activities of histidine containing caffeic acid-dipeptides.

Antioxidants have been utilized in both the food and cosmetics industries to neutralize the activities of reactive oxygen species (ROS) and free radicals. Histidine-containing peptides are powerful antioxidants that exist in nature. Additionally, hydroxycinnamic acid (HCA)-peptide conjugates exhibit a synergistically enhanced antioxidative activity. Thus, caffeic acid (CA), a natural antioxidant, was conjugated to histidine-containing dipeptides (His dipeptides) in order to develop better antioxidants. The antioxidative activities were measured using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and lipid peroxidation test with ferric thiocyanate method. Some of the CA-His dipeptides exhibited better radical scavenging activities than CA, and all of the CA-His dipeptides showed enhanced lipid peroxidation inhibitory activities. His dipeptide enhanced the antioxidative activity of CA, and the position of histidine also affected the antioxidative activity of the compounds. CA-proline-histidine amide (CA-Pro-His-NH(2)) exhibited the highest activity in both the free radical scavenging test and the lipid peroxidation inhibition test.