Molecular rotors as reporters for viscosity of solutions of collagen like peptides.

We have studied the suitability of using a molecular rotor-based steady-state fluorometric assay for evaluating changes in both the conformation and the viscosity of collagen-like peptide solutions. Our results indicate that a positive charge incorporated on the hydrophobic tail of the BODIPY molecular rotor favours the dye specificity as a reporter for viscosity of these solutions.

Understanding the Kinetics and Spectroscopy of Photoredox Catalysis and Transition-Metal-Free Alternatives.

Over the past decade, the field of photoredox catalysis has gained increasing attention in synthetic organic chemistry because of its wide applicability in sustainable free-radical-mediated processes. Numerous examples have shown that under carefully optimized conditions, efficient and highly selective processes can be developed through excitation of a photosensitizer using inexpensive, readily available light sources. However, despite all of these recent advancements, some generalizations and/or misconceptions have become part of the photoredox culture, and often many of these discoveries lack in-depth investigations into the excited-state kinetics and underlying mechanisms. In this Account, we begin with a tutorial for understanding both the redox properties of excited states and how to measure the kinetics of excited-state processes. We discuss the generalization of direct excitation of closed-shell species to generate more potent reductive or oxidative excited states, using the helium atom as a quantitative example. We also outline how to apply redox potentials to calculate whether the proposed electron transfer events are thermodynamically feasible. In the second half of our tutorial, we discuss how to measure the kinetics of excited-state processes using techniques such as steady-state and time-resolved fluorescence and transient spectroscopy and how to apply the data using Stern-Volmer and kinetic analysis. Then we shift gears to discuss our recent contributions to the field of photoredox catalysis. Our lab focuses on developing transition-metal-free alternatives to ruthenium and iridium bipyridyl complexes for these transformations, with the goal of developing systems in which the reaction kinetics is more favorable. We have found that methylene blue, a member of the thiazine dye family, can be employed in photoredox processes such as oxidative hydroxylations of arylboronic acids to phenols. Interestingly, we were able to demonstrate that methylene blue is more efficient for this reaction than Ru(bpy)3Cl2, which upon further examination using transient spectroscopic techniques we were able to relate to the reductive quenching ability of the aliphatic amine. Recently we were also successful in applying methylene blue for radical trifluoromethylation reactions, which is discussed in detail. Finally, we have also demonstrated that common organic electron donors, such as α-sexithiophene, can be used in photoredox processes, which we demonstrate using the dehalogenation of vic-dibromides as a model system. This is a particularly interesting system because well-defined, long-lived intermediates allowed us to fully characterize the catalytic cycle. Once again, through an in-depth kinetic analysis we were able to gain valuable insights into our reaction mechanism, which demonstrates how powerful a tool proper kinetic analysis can be in the design and optimization of photoredox processes.

Two-Photon Excitation of a Plasmonic Nanoswitch Monitored by Single-Molecule Fluorescence Microscopy.

Visible-light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single-molecule study of the plasmon-assisted isomerization of a photochrome-fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser. The metallic nanostructures significantly enhance the visible light-induced performance of the photoconversion, which normally requires ultraviolet excitation. The resulting ring-open isomer is strongly fluorescent and can also be excited at 633 nm. These stochastic emission events are used to monitor photochromic activation and show quadratic dependence on incident power. The utilization of a single laser wavelength for both photochromic activation and excitation effectively mimics a pseudo two-colours system.

Electron transfer from the benzophenone triplet excited state directs the photochemical synthesis of gold nanoparticles.

The rarely recognized electron donating ability of the benzophenone triplet excited state provides an unusual route for the photochemical synthesis of gold nanoparticles.

Mechanistic insights and kinetic analysis for the oxidative hydroxylation of arylboronic acids by visible light photoredox catalysis: a metal-free alternative.

The photocatalytic hydroxylation of boronic acids with methylene blue as photosensitizer proceeds with high efficiency. Detailed time-resolved studies of the relevant rate constants provide a clear mechanistic understanding of excited-state processes and guided the selection of the photocatalyst and the optimization of experimental conditions.

Development of in situ bioprinting: A mini review.

Bioprinting has rapidly progressed over the past decade. One branch of bioprinting known as in situ bioprinting has benefitted considerably from innovations in biofabrication. Unlike ex situ bioprinting, in situ bioprinting allows for biomaterials to be printed directly into or onto the target tissue/organ, eliminating the need to transfer pre-made three-dimensional constructs. In this mini-review, recent progress on in situ bioprinting, including bioink composition, in situ crosslinking strategies, and bioprinter functionality are examined. Future directions of in situ bioprinting are also discussed including the use of minimally invasive bioprinters to print tissues within the body.

Identification of Radiation-Induced miRNA Biomarkers Using the CGL1 Cell Model System.

MicroRNAs (miRNAs) have emerged as a potential class of biomolecules for diagnostic biomarker applications. miRNAs are small non-coding RNA molecules, produced and released by cells in response to various stimuli, that demonstrate remarkable stability in a wide range of biological fluids, in extreme pH fluctuations, and after multiple freeze-thaw cycles. Given these advantages, identification of miRNA-based biomarkers for radiation exposures can contribute to the development of reliable biological dosimetry methods, especially for low-dose radiation (LDR) exposures. In this study, an miRNAome next-generation sequencing (NGS) approach was utilized to identify novel radiation-induced miRNA gene changes within the CGL1 human cell line. Here, irradiations of 10, 100, and 1000 mGy were performed and the samples were collected 1, 6, and 24 h post-irradiation. Corroboration of the miRNAome results with RT-qPCR verification confirmed the identification of numerous radiation-induced miRNA expression changes at all doses assessed. Further evaluation of select radiation-induced miRNAs, including miR-1228-3p and miR-758-5p, as well as their downstream mRNA targets, Ube2d2, Ppp2r2d, and Id2, demonstrated significantly dysregulated reciprocal expression patterns. Further evaluation is needed to determine whether the candidate miRNA biomarkers identified in this study can serve as suitable targets for radiation biodosimetry applications.

Biosupramolecular complexes of amphiphilic photosensitizers with human serum albumin and cucurbit[7]uril as carriers for photodynamic therapy.

In the present work, we evaluated the supramolecular interactions between three photosensitizers, namely toluidine blue O (TBO, positively charged) and two fatty acid conjugates of 6 and 14 carbon atoms chain lengths (TBOC6 and TBOC14), with human serum albumin (HSA) and the macrocycle cucurbit[7]uril (CB[7]), alone or in combination within a biosupramolecular system as potential carriers of photosensitizers for Photodynamic therapy (PDT). Binding studies were carried out using photophysical and calorimetric techniques and accompanied with molecular docking simulations. Amphiphilic photosensitizers, particularly TBOC14, showed stronger binding to HSA and (CB[7]). Comparing the different delivery systems, (CB[7]) had a marginal effect on cell uptake and phototoxicity in HeLa cells, while HSA showed enhanced cell uptake with phototoxicities that depended on the photosensitizer. Despite low cell uptake, the combination of both (CB[7]) and HSA was the most phototoxic, which illustrates the potential of combining these systems for PDT applications.

A Liquid Hydrogel to Restore Long Term Corneal Integrity After Perforating and Non-Perforating Trauma in Feline Eyes.

Purpose: To evaluate long-term in vivo functionality of corneas regenerated using a cell-free, liquid hydrogel filler (LiQD Cornea) after deep corneal trauma in the feline model. Methods: Two healthy cats underwent 4 mm diameter stepwise 250/450 µm deep surgical corneal ablation with and without needle perforation. The filler comprising 10% (w/w) collagen-like peptide conjugated to polyethylene glycol (CLP-PEG) and 1% fibrinogen and crosslinked with 2% (w/w) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), was applied to the wound bed previously coated with thrombin (250 U/ml). In situ gelation occurred within 5 min, and a temporary tarsorrhaphy was performed. Eyes were examined weekly for 1 month, then monthly over 12 months. Outcome parameters included slit-lamp, Scheimpflug tomography, optical coherence tomography, confocal and specular microscopy, and immunohistochemistry studies. Results: The gelled filler was seamlessly incorporated, supporting smooth corneal re-epithelialization. Progressive in-growth of keratocytes and nerves into the filler corresponding to the mild haze observed faded with time. The regenerated neo-cornea remained stably integrated throughout the 12 months, without swelling, inflammation, infection, neovascularization, or rejection. The surrounding host stroma and endothelium remained normal at all times. Tomography confirmed restoration of a smooth surface curvature. Conclusion: Biointegration of this hydrogel filler allowed stable restoration of corneal shape and transparency in the feline model, with less inflammation and no neovascularization compared to previous reports in the minipig and rabbit models. It offers a promising alternative to cyanoacrylate glue and corneal transplantation for ulcerated and traumatized corneas in human patients.

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

Chiral conducting surfaces via electrochemical oxidation of L-leucine-oligothiophenes.

Polythiophenes bearing a specific chiral center such as L-leucine have been prepared via the electrochemical oxidation of a series of L-leucine functionalized oligothiophenes (monothiophenes and terthiophenes). These oligothiophenes have been prepared through the condensation of L-leucine methyl ester and the corresponding thiophene monomers in the presence of hydroxybenzotriazole (HOBt) and N,N'-dicyclohexylcarbodiimide (DCC) followed by hydrolysis of the esters. The electroactive polymers are electrochemically stable and exhibit excellent adhesive properties on electrode surfaces (platinum, gold, and glassy carbon) as well as interesting optical properties in both doped and undoped states. Hydrogen bonds between a free amino acid (L-leucine, D-leucine, L-alanine, D-alanine, and D/L-alanine) and the L-leucine based polythiophenes (chiral conducting surface) were probed using cyclic voltammetry. Preliminary results show that the capacitive current of a modified L-leucine-polythiophene electrode decreases as a result of the formation of a hydrogen bond barrier on the surface of the chiral conducting surface accompanied with a shift of the oxidation potential. Cyclic voltammetry responses resulting from the interaction of the chiral conducting surface with L and Dfree amino acid isomers are similar. The formation of hydrogen bonds between the chiral conducting surfaces and the free amino acids was characterized by (1)H NMR. A chemical shift was observed for the N-H group in monomer 6 as a result of the hydrogen bond formation between the L-leucine methyl ester (D-leucine methyl ester, D/L-leucine methyl ester) and monomer 6.

Lipoic acid capped silver nanoparticles: a facile route to covalent protein capping and oxidative stability within biological systems.

Covalent attachment of human serum albumin protein to the surface of spherical lipoic acid capped silver nanoparticles results in the generation of stable nanoparticle-protein hybrids with well defined surface composition. Enhanced stability towards oxidation and in the presence of complex media with high ionic strength, holds promise towards the use of these conjugates as therapeutics in biomedical applications and sensing.

BEaTS-α an open access 3D printed device for in vitro electromechanical stimulation of human induced pluripotent stem cells.

3D printing was used to develop an open access device capable of simultaneous electrical and mechanical stimulation of human induced pluripotent stem cells in 6-well plates. The device was designed using Computer-Aided Design (CAD) and 3D printed with autoclavable, FDA-approved materials. The compact design of the device and materials selection allows for its use inside cell incubators working at high humidity without the risk of overheating or corrosion. Mechanical stimulation of cells was carried out through the cyclic deflection of flexible, translucent silicone membranes by means of a vacuum-controlled, open-access device. A rhythmic stimulation cycle was programmed to create a more physiologically relevant in vitro model. This mechanical stimulation was coupled and synchronized with in situ electrical stimuli. We assessed the capabilities of our device to support cardiac myocytes derived from human induced pluripotent stem cells, confirming that cells cultured under electromechanical stimulation presented a defined/mature cardiomyocyte phenotype. This 3D printed device provides a unique high-throughput in vitro system that combines both mechanical and electrical stimulation, and as such, we foresee it finding applications in the study of any electrically responsive tissue such as muscles and nerves.

Nanoengineering the surface of corneal implants: towards functional anti-microbial and biofilm materials.

We report the development and use of a light-mediated in situ grafting technology for the surface modification of biosynthetic corneal implants with peptide-capped nanoparticles (15-65 nm). The resulting materials have antimicrobial properties in bacterial suspension and also reduced the extent of biofilm formation. Our in situ grafting technology offers a rapid route for the introduction of antimicrobial properties to premoulded corneal implants, and potentially other soft implant targets.

LiQD Cornea: Pro-regeneration collagen mimetics as patches and alternatives to corneal transplantation.

Transplantation with donor corneas is the mainstay for treating corneal blindness, but a severe worldwide shortage necessitates the development of other treatment options. Corneal perforation from infection or inflammation is sealed with cyanoacrylate glue. However, the resulting cytotoxicity requires transplantation. LiQD Cornea is an alternative to conventional corneal transplantation and sealants. It is a cell-free, liquid hydrogel matrix for corneal regeneration, comprising short collagen-like peptides conjugated with polyethylene glycol and mixed with fibrinogen to promote adhesion within tissue defects. Gelation occurs spontaneously at body temperature within 5 min. Light exposure is not required-particularly advantageous because patients with corneal inflammation are typically photophobic. The self-assembling, fully defined, synthetic collagen analog is much less costly than human recombinant collagen and reduces the risk of immune rejection associated with xenogeneic materials. In situ gelation potentially allows for clinical application in outpatient clinics instead of operating theaters, maximizing practicality, and minimizing health care costs.

Peptide-Based Functional Biomaterials for Soft-Tissue Repair.

Synthetically derived peptide-based biomaterials are in many instances capable of mimicking the structure and function of their full-length endogenous counterparts. Combine this with the fact that short mimetic peptides are easier to produce when compared to full length proteins, show enhanced processability and ease of modification, and have the ability to be prepared under well-defined and controlled conditions; it becomes obvious why there has been a recent push to develop regenerative biomaterials from these molecules. There is increasing evidence that the incorporation of peptides within regenerative scaffolds can result in the generation of structural recognition motifs that can enhance cell attachment or induce cell signaling pathways, improving cell infiltration or promote a variety of other modulatory biochemical responses. By highlighting the current approaches in the design and application of short mimetic peptides, we hope to demonstrate their potential in soft-tissue healing while at the same time drawing attention to the advances made to date and the problems which need to be overcome to advance these materials to the clinic for applications in heart, skin, and cornea repair.

Light-Activated Peptide-Based Materials for Sutureless Wound Closure.

Using chemically modified extracellular matrix proteins, such as collagen, in combination with light for tissue bonding reduces inflammation and minimizes scarring. However, full length animal or recombinant human collagen proteins are difficult to isolate/produce. Thus, short biomimetic collagen peptides with properties equivalent to collagen at both structural and functional levels may be ideal building blocks for the development of remotely triggered adhesives and fillers. In this work, the conjugation of self-assembling collagen-like peptides to acrylate functionalized polyethylene glycol units yielded adhesive filler materials activated by visible light through the incorporation of a photosensitizer. When tested in a murine skin wound model, the photoactivated adhesives showed reduced scar formation and promoted epithelial regeneration.

Bacterial biofilm formation on implantable devices and approaches to its treatment and prevention.

In living organisms, biofilms are defined as complex communities of bacteria residing within an exopolysaccharide matrix that adheres to a surface. In the clinic, they are typically the cause of chronic, nosocomial, and medical device-related infections. Due to the antibiotic-resistant nature of biofilms, the use of antibiotics alone is ineffective for treating biofilm-related infections. In this review, we present a brief overview of concepts of bacterial biofilm formation, and current state-of-the-art therapeutic approaches for preventing and treating biofilms. Also, we have reviewed the prevalence of such infections on medical devices and discussed the future challenges that need to be overcome in order to successfully treat biofilms using the novel technologies being developed.

Nanoengineered Electroconductive Collagen-Based Cardiac Patch for Infarcted Myocardium Repair.

We have prepared and tested in vivo a novel nanoengineered hybrid electroconductive cardiac patch for treating the infarcted myocardium. Of the prepared and tested patches, only those containing spherical nanogold were able to increase connexin-43 expression in neonatal rat cardiomyocytes cultured under electrical stimulation. In vivo data indicated that only nano-gold-containing patches were able to recover cardiac function. Histological analysis also revealed that connexin-43 levels and blood vessel density were increased, while the scar size was reduced for animals that received the nanogold patch. Thus, our study indicates that the incorporation of electroconductive properties into a collagen-based cardiac patch can improve its therapeutic potential for treating myocardial infarction.

Library of Cationic Organic Dyes for Visible-Light-Driven Photoredox Transformations.

Organic dyes can be excellent catalysts for photoredox chemistry, offering low price, low toxicity, and an exceptional range of available materials. Their use has been limited because in comparison to their transition-metal catalysts the spectroscopic, kinetic, and electrochemical information available is far more limited. To remediate this situation, we have determined the necessary data for 14 readily available dyes with excellent potential as photoredox catalysts. We have also demonstrated the utility of these dyes through visible-light-mediated reductive dehalogenation and Aza-Henry reactions. We envision that this collection of data will lead to an increase in the use of cationic dyes in photoredox processes because users will find the necessary information readily available.