Oxygen releasing patches based on carbohydrate polymer and protein hydrogels for diabetic wound healing: A review.

Diabetic wounds are among the major healthcare challenges, consuming billions of dollars of resources and resulting in high numbers of morbidity and mortality every year. Lack of sufficient oxygen supply is one of the most dominant causes of impaired healing in diabetic wounds. Numerous clinical and experimental studies have demonstrated positive outcomes as a result of delivering oxygen at the diabetic wound site, including enhanced angiogenesis, antibacterial and cell proliferation activities. However, prolonged and sustained delivery of oxygen to improve the wound healing process has remained a major challenge due to rapid release of oxygen from oxygen sources and limited penetration of oxygen into deep skin tissues. Hydrogels made from sugar-based polymers such as chitosan and hyaluronic acid, and proteins such as gelatin, collagen and hemoglobin have been widely used to deliver oxygen in a sustained delivery mode. This review presents an overview of the recent advances in oxygen releasing hydrogel based patches as a therapeutic modality to enhance diabetic wound healing. Various types of oxygen releasing wound healing patch have been discussed along with their fabrication method, release profile, cytocompatibility and in vivo results. We also briefly discuss the challenges and prospects related to the application of oxygen releasing biomaterials as wound healing therapeutics.

Lipoprotein-induced cell growth and hemocyanin biosynthesis in rhogocytes.

Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the scientists despite hemocyanin's importance as a carrier protein and complex molecule with anti-viral activity. Although a hypothetical model of hemocyanin release from the rhogocytes lacunae was proposed based on colloid-osmotic pressure mechanism, lack of in vitro studies limits further validation of this model. In this study, we aim to investigate the impact of cell culture conditions and nature of hemocyanin biosynthesis of rhogocyte cells dissociated from Haliotis laevigata mantle tissue. Population of cells with different hemocyanin expression levels was profiled using flow cytometry, while hemocyanin concentrations in the media were elucidated by ELISA assay. We demonstrated that addition of lipoprotein supplement into the media resulted in a burst secretion of hemocyanin into the culture media. Over 7 days of culture, the population of cells tagged with hemocyanin antibody increased steadily while hemocyanin release in the media decreased significantly. Variation of culture medium, temperature, growth supplement type and concentration also impacted the cell growth and hemocyanin biosynthesis. These results indicated the possibility of an active process triggered by the addition of supplement to synthesise the protein at the highest amount during the first hour. The current study provides a glimpse of the hemocyanin biosynthesis by rhogocyte that may be significant to understand the cell ability to synthesise supramolecular protein and secretion through lacunae.

3D-printed bioresorbable poly(lactic-co-glycolic acid) and quantum-dot nanocomposites: Scaffolds for enhanced bone mineralization and inbuilt co-monitoring.

Multifunctional 3D-printed nanocomposites based on poly(lactic-co-glycolic acid), that is, PLGA (RESOMER® LG857S) were developed for simultaneous monitoring of cells and scaffold as a function of time and spectral responses. These were achieved by impregnating carbon quantum dots (CQDs) on PLGA using melt-blending, plasticating extrusion, and 3D-printing. The nanocomposites enabled enhanced bio-affinity and cellular interactions for bone tissue engineering (TE). PLGA (control) and PLGA-CQD scaffolds were used for growing human adipose-derived-stem-cells (ADSCs) and tested for cell biocompatibility, cellular adhesion, growth, and osteogenesis. CQDs were found to enhance the hydrophilicity of nanocomposites and promote cellular nesting. MTS assays confirmed that CQDs on PLGA act as cell anchoring sites, thereby enhancing seeding efficiency and cell proliferation. Alkaline phosphate tests showed increased osteogenesis and Alizarin assays confirmed enhanced bone mineralization on PLGA-CQD. The qPCR tests based on selected mRNA expressions showed that the incorporation of CQDs significantly enhanced osteogenesis of ADSCs during all three phases of cell differentiation. The intrinsic luminescence of the composites allowed label-free monitoring of cell proliferation and bone mineralization on the scaffolds. Thus, the CQDs facilitated significant enhancements in composite processability with customized fabrication of 3D printed scaffolds, bone tissue osteoconductivity, and monitoring of cell-scaffold activities, offering multifunctional benefits for bone TE.

Additive-Free All-Carbon Composite: A Two-Photon Material System for Nanopatterning of Fluorescent Sub-Wavelength Structures.

The major bottleneck in fabrication of engineered 3D nanostructures is the choice of materials. Adding functionality to these nanostructures is a daunting task. In order to mitigate these issues, we report a two-photon patternable all carbon material system which can be used to fabricate fluorescent 3D micro/nanostructures using two-photon lithography, with subwavelength resolution. The synthesized material system eliminates the need to use conventional two-photon absorbing materials such as two-photon dyes or two-photon initiators. We have used two different trifunctional acrylate monomers and carbon dots, synthesized hydrothermally from a polyphenolic precursor, to formulate a two-photon processable resin. Upon two-photon excitation, photogenerated electrons in the excited states of the carbon dots facilitate the free radical formation at the surface of the carbon dots. These radicals, upon interaction with vinyl moieties, enable cross-linking of acrylate monomers. Free-radical induced two-photon polymerization of acrylate monomers without any conventional proprietary two-photon absorbing materials was accomplished at an ultrafine subwavelength resolution of 250 nm using 800 nm laser excitation. The effect of critical parameters such as average laser power, carbon dot concentration, and radiation exposure were determined for the fabrication of one-, two-, and three-dimensional functional nanostructures, applicable in a range of domains where fluorescence and toxicity are of the utmost importance. A fabrication speed as high as 100 mm/s was achieved. The ability to fabricate functional 3D micro-/nanostructures is anticipated to instigate a paradigm shift in various areas such as metamaterials, energy storage, drug delivery, and optoelectronics to name a few.

Franz cells for facile biosensor evaluation: A case of HRP/SWCNT-based hydrogen peroxide detection via amperometric and wireless modes.

Reducing animal use in biosensor research requires broader use of in vitro methods. In this work, we present a novel application of Franz cells suitable for biosensor development and evaluation in vitro. The work describes how Franz cell can be equipped with electrodes enabling characterization of biosensors in close proximity to skin. As an example of a sensor, hydrogen peroxide biosensor was prepared based on horseradish peroxidase (HRP)/single-walled carbon nanotube (SWCNT)-modified textile. The electrode exhibited lower detection limit of 0.3 µM and sensitivity of 184 µA mM-1 cm-2. The ability of this biosensor to monitor H2O2 penetration through skin and dialysis membranes was evaluated in Franz cell setup in amperometric and wireless modes. The results also show that catalase activity present in skin is a considerable problem for epidermal sensing of H2O2. This work highlights opportunities and obstacles that can be addressed by assessment of biosensors in Franz cell setup before progressing to their testing in animals and humans.

Two-photon active nucleus-targeting carbon dots: enhanced ROS generation and photodynamic therapy for oral cancer.

Novel conjugated carbon dots (CDs) were synthesized as two-photon active photosensitisers to unleash lethal reactive oxygen species (ROS) for nucleus-targeting photodynamic therapy (PDT). To enhance the therapeutic efficiency and preclude non-specific CD uptake, we employed a combination of folic acid and curcumin for two-photon NIR-triggered ROS generation and enhanced internalization in the nucleus. Consequently, enhanced destruction of cancer cells occurred by directly attacking the DNA. The intrinsic ROS generation and nucleus-targeting ability of CDs introduced multifunctional two-photon active nanoprobes within a single platform for enhanced PDT in oral cancer theranostics.

Interactions at scaffold interfaces: Effect of surface chemistry, structural attributes and bioaffinity.

Effective regenerative medicine relies on understanding the interplay between biomaterial implants and the adjoining cells. Scaffolds contribute by presenting sites for cellular adhesion, growth, proliferation, migration, and differentiation which lead to regeneration of tissues over desired periods of time. The fabrication and recruitment of scaffolds often fail to consider the interactions that occur at the interfaces, thereby risking rejection. This lack of knowledge on interfacial microenvironments and related exchanges often causes reduced cellular interactions, poor cell survival and intervention failure. Successful regenerative therapy requires scaffolds with bespoke biocompatibility, optimum pore structure, and cues for cell attachments. These factors determine the development of cellular affinity in scaffolds. For biomedical applications, a detailed understanding of scaffolds and their interfaces is required for better tuning of biomaterials to suit the microenvironments. In this review, we discuss the role of biointerfaces with a focus on surface chemistry, pore structure, scaffold hydro-affinity and their biointeractions. An understanding of the effect of scaffold interfacial properties is crucial for enhancing the progress of tissue engineering towards clinical applications.

Conjugated carbon quantum dots: Potent nano-antibiotic for intracellular pathogens.

A common theme in the persistence of microbial infections involves intracellular survival of microbial pathogens within host cells where they stay sheltered from attack by antimicrobial agents. In order to improve antimicrobial access inside host cells, we developed nanoparticles intracellular delivery of antibiotics. Using an intracellular infection model with the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), we demonstrated significantly enhanced intracellular microbicidal activity with the standard antibiotic metronidazole (MET) through its conjugation onto 1-5 nm biocompatible nano-carrier, carbon quantum dot, which was derived from chlorophyll (cCQD). The conjugated cCQD-MET were rapidly internalized into the cultured cells, reaching almost 90% uptake within 3 h of the challenge. Our results consistently showed enhanced antimicrobial activity of the conjugate compared to MET alone. Even at concentrations as low as 0.26 µM, the conjugate showed 72% enhancement compared to the drug alone, resulting in significantly increased inhibition of intracellular P. gingivalis at lower antibiotic dosages. We achieved a high drug payload (80% w/w) on cCQD without affecting the potency of metronidazole as determined by cytotoxicity assays, cellular uptake of metronidazole, P. gingivalis invasion and elimination assays. The synthesized cCQD also displayed high fluorescence with 56% quantum yield at an absorbance peak of 380 nm and an emission peak of 480 nm, thus, allowing for fluorescence tracking and quantification of the drug intracellularly. A similar strategy may be used to repurpose other antibiotics for the treatment of intracellular bacterial infections.

Two-Photon Active Boron Nitride Quantum Dots for Multiplexed Imaging, Intracellular Ferric Ion Biosensing, and pH Tracking in Living Cells.

Nanoparticles are key vehicles for targeted therapies because they can pass through biological barriers, enter into cells, and distribute within cell structures. We investigated the synthesis of blue and green emissive hexagonal boron nitride quantum dots (hBNQDs) using a liquid-exfoliation technique followed by hydrothermal treatment. A distinct shift from blue to bright-green emission was observed upon surface passivating the dots using poly(ethylene glycol) or PEG200 under the same UV irradiation. The quantum yield of the hBNQDs increased with the surface passivation. Multiplexed imaging was accomplished using the hBNQDs in conjunction with organic dyes. The hBNQDs provided images with distinctive emission wavelengths and fluorescence lifetimes. Although the fluorescence signals of blue- and green-emissive hBNQDs overlap spectrally with those of the emission wavelengths of the organic dyes, the fluorescence lifetime data were resolved temporally using software-based time gates. The blue-emissive hBNQD-b quantum dots were validated as sensitive platforms for detecting intracellular ferric ions with a low limit of detection (20.6 nM). The green-emissive hBNQD-g quantum dots successfully identified intracellular variations in pH, and the localization in human breast cancer cells was determined during their life cycles via fluorescence lifetime imaging.

Doped graphene/Cu nanocomposite: A high sensitivity non-enzymatic glucose sensor for food.

An amperometric non-enzymatic glucose sensor was developed based on nitrogen-doped graphene with dispersed copper nanoparticles (Cu-NGr). The sensing element was tested in conjunction with a modified glassy carbon electrode for glucose detection. The Cu-NGr composite was prepared by one pot synthesis from a mixture of graphene oxide, copper nitrate and uric acid, followed by thermal annealing at 900°C for 1h. Detailed characterizations showed homogeneous copper nanoparticle dispersion and the presence of significant proportion of graphitic nitrogen. The developed electrode presented high electrocatalytic activity towards glucose through synergetic effect of copper nanoparticles and nitrogen-doped graphene. Amperometric analysis confirmed high glucose sensitivity and ultra-low detection of 10nM glucose over a linear range. The sensor was tested for direct application to detect glucose in food samples for which the sensor displayed high selectivity with excellent reproducibility and recovery in complex food materials.

Abalone Hemocyanin Blocks the Entry of Herpes Simplex Virus 1 into Cells: a Potential New Antiviral Strategy.

A marine-derived compound, abalone hemocyanin, from Haliotis rubra was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections. In vitro assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED50) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors. However, hemocyanin had no effect on postentry events and did not block infection by binding to cellular receptors for HSV. By the use of different mutants of gD and gB and a competitive heparin binding assay, both protein charge and conformation were shown to be the driving forces of the interaction between hemocyanin and viral glycoproteins. These findings also suggested that hemocyanin may have different motifs for binding to each of the viral glycoproteins B and D. The dimer subunit of hemocyanin with a 10-fold-smaller molecular mass exhibited similar binding to viral surface glycoproteins, showing that the observed inhibition did not require the entire multimer. Therefore, a small hemocyanin analogue could serve as a new antiviral candidate for HSV infections.

Co-Doping of Activated Graphene for Synergistically Enhanced Electrocatalytic Oxygen Reduction Reaction.

Doping of graphene has emerged as a key strategy to improve the electrocatalytic performance of the oxygen reduction reaction (ORR). Activated graphene co-doped with iodine and nitrogen atoms (NIG) was developed in this work using a facile scalable approach. The onset potential, current density, and four-electron reduction pathway of the newly developed catalyst were significantly improved. The charge-transfer resistance of co-doped NIG was found to be much lower than nitrogen-doped graphene (NG); furthermore, the stability of NIG and its resistance to methanol crossover were also improved. The synergistically enhanced ORR performance of NIG was found to be a result of a high strain and size advantage of the larger iodine atom clusters (compared to nitrogen), which facilitate the simultaneous enrichment of anode electrons and O2 and H2 O molecule transport at catalytic sites, inducing four-electron transfer in a single step. These results are promising for application in alkaline fuel cells.

Distribution and characterization of rhogocyte cell types in the mantle tissue of Haliotis laevigata.

Molluscan rhogocytes are known to be the only cells able to synthesize hemocyanin that is one of the largest respiratory proteins in nature. However, investigation of rhogocyte cells in vitro is limited due to difficulty in isolating and establishing marine cell culture. The aim of this study was to investigate the nature and distribution of rhogocyte cells of Haliotis laevigata in the mantle tissue with respect to the expression of the two known isoforms of hemocyanin. Rhogocyte cells were identified using immunofluorescence-fluorescence in situ hybridization (IF-FISH) that involved simultaneous staining of localized hemocyanin by a polyclonal antibody while the mRNA was hybridized with FISH probes. The distribution of rhogocyte cells was demonstrated using flow cytometry, followed by cell sorting with fluorescence-activated cell sorter (FACS) and confocal microscope imaging for further characterization. Our results suggested that the mantle tissue is dominated by two distinct populations of rhogocyte cells that synthesize hemocyanin type 1. Observation with confocal microscopy of both populations revealed hemocyanin localization in the periphery of the cell membrane. Cell population with higher antibody signal had irregular and elongated cell morphology with punctate mRNA probe signals. The second population with lower antibody signal had ovoid morphology and wide distribution of mRNA probe signals. We suggest that these populations represent two distinct phases of hemocyanin biosynthesis of a single isoform, which is closely related to Haliotis tuberculata type 1 hemocyanin (HtH1). The knowledge acquired in this study enhances the understanding of the biology of rhogocyte cells and biosynthesis of hemocyanin.

Edge-enriched graphene quantum dots for enhanced photo-luminescence and supercapacitance.

Graphene quantum dots (GQDs) with their edge-bound nanometer-size present distinctive properties owing to quantum confinement and edge effects. We report a facile ultrasonic approach with chemical activation using KOH to prepare activated GQDs or aGQDs enriched with both free and bound edges. Compared to GQDs, the aGQDs we synthesized had enhanced BET surface area by a factor of about six, the photoluminescence intensity by about four and half times and electro-capacitance by a factor of about two. Unlike their non-activated counterparts, the aGQDs having enhanced edge states emit enhanced intense blue luminescence and exhibit electrochemical double layer capacitance greater than that of graphene, activated or not. Apart from their use as part of electrodes in a supercapacitor, the superior luminescence of aGQDs holds potential for use in biomedical imaging and related optoelectronic applications.

Formulation of abalone hemocyanin with high antiviral activity and stability.

Hemocyanin has been shown to have potential antiviral activity against herpes simplex virus type-1. However, current liquid formulations have short shelf life and high risk of bacterial contamination. The aim of our study was to develop a stable functional formulation. Analytical techniques (nano-differential scanning calorimetry and spectroscopy) and biological assays (cytotoxicity and plaque reduction) were employed to measure the effect of sugar addition on the physical properties and shelf life of the solid formulated hemocyanin. Sucrose improved thermal stability significantly by both increasing the aggregation onset temperature (70°C to>78 °C) and enhancing the activation energy (18%). Lyophilisation without trehalose caused degradation and unfolding of the α-helices of hemocyanin. However, the addition of an optimal proportion of trehalose:protein (5:1 by weight) prevented the degradation and unfolding during lyophilisation, hence maintained the protein solubility. The estimated ED50 values of the formulated solid (0.43±0.1) and liquid samples (0.37±0.06) were similar in magnitude, and were significantly lower than the respective controls; thus, confirming enhanced antiviral activity of the formulation. Formulated compounds were stable for six months at 5 °C storage. The enhanced shelf life and stable antiviral activity of the formulation offers its significant potential as effective therapeutic agent in future clinical applications.

High-yield aqueous phase exfoliation of graphene for facile nanocomposite synthesis via emulsion polymerization.

Aqueous phase exfoliation was developed for producing high-yield graphene nanosheets from expanded graphite (EG). The process included ultrasonication with sodium dodecyl sulfate (SDS) emulsion in aqueous phase. The high throughput exfoliation process was characterized by UV-vis spectroscopy, transmission electron microscopy (TEM) and electrical impedance spectroscopy (EIS). Controlled sonication experiments revealed that optimum exfoliation corresponds to maxima in UV-vis spectra. TEM results showed that the exfoliated graphene comprised nanoflakes having ≤5 layers (~60%) and ≤10 layers for 90% of the product. The potential use of this highly dispersed graphene was demonstrated by one-pot synthesis of graphene/polymer composite via in situ emulsion polymerization with styrene. The integrated role of SDS included adsorption and exfoliation of graphite, dispersion of graphene produced and assisting with micelle formation in emulsion. The high surface area graphene nanosheets as dispersed phase in polymeric nanocomposites showed significant improvement in thermal stability and electrical conductivity.