Light-Activated, Bioadhesive, Poly(2-hydroxyethyl methacrylate) Brush Coatings.

Rapid adhesion between tissue and synthetic materials is relevant to accelerate wound healing and to facilitate the integration of implantable medical devices. Most frequently, tissue adhesives are applied as a gel or a liquid formulation. This manuscript presents an alternative approach to mediate adhesion between synthetic surfaces and tissue. The strategy presented here is based on the modification of the surface of interest with a thin polymer film that can be transformed on-demand, using UV-light as a trigger, from a nonadhesive into a reactive and tissue adhesive state. As a first proof-of-concept, the feasibility of two photoreactive, thin polymer film platforms has been explored. Both of these films, colloquially referred to as polymer brushes, have been prepared using surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate (HEMA). In the first part of this study, it is shown that direct UV-light irradiation of PHEMA brushes generates tissue-reactive aldehyde groups and facilitates adhesion to meniscus tissue. While this strategy is very straightforward from an experimental point of view, a main drawback is that the generation of the tissue reactive aldehyde groups uses the 250 nm wavelength region of the UV spectrum, which simultaneously leads to extensive photodegradation of the polymer brush. The second part of this report outlines the synthesis of PHEMA brushes that are modified with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (TFMDA) moieties. UV-irradiation of the TFMDA containing brushes transforms the diazirine moieties into reactive carbenes that can insert into C-H, N-H, and O-H bonds and mediate the formation of covalent bonds between the brush surface and meniscus tissue. The advantage of the TFMDA-modified polymer brushes is that these can be activated with 365 nm wavelength UV light, which does not cause photodegradation of the polymer films. While the work presented in this manuscript has used silicon wafers and fused silica substrates as a first proof-of-concept, the versatility of SI-ATRP should enable the application of this strategy to a broad range of biomedically relevant surfaces.

Structure-Activity Relationships of Voltaglue Organic Blends.

Voltage-activated, one-pot adhesives are an emerging platform with many potential advantages, but require multicomponent grafting of electrochemical donors and acceptors for operation in organic environments. This formulation strategy reduces throughput efficiency, organic solubility, and requires additional purification of the grafted dendrimers. A more advanced strategy is proposed for setting up the donor-acceptor conductive network by exploiting a flexible blending design, providing faster throughput of structure-activity analyses with less synthetic investment. The blend method investigates the ampere-dependent storage modulus and gelation time as a function of both donor and acceptor concentration. This blend strategy allows a rapid evaluation of donor-acceptor parameters involved in voltage-activated adhesive formulations.

In Vitro Biocompatibility of Diazirine-Grafted Biomaterials.

Photoactivation of aryl-diazirines is an emerging method of rapid, covalent crosslinking under ambient conditions. These attributes make those compounds candidates for grafting onto inert polymer backbones in order to produce stimuli-sensitive biomaterials. However, no risk assessments are available to gauge the toxicity of the leachable components after crosslinking activation. Herein, a stimuli-sensitive biomaterial is formulated from diazirine-grafted polycaprolactone tetrol. Also known as CaproGlu, this biomaterial undergoes UVA-activated crosslinking, with many positive attributes toward bioadhesive applications; hydrophobic, solvent-free, liquid at room temperature, and transitions into a foam biorubber after mild UVA illumination. As a model diazirine-grafted biomaterial, hydrolyzed CaproGlu leachates are evaluated for genotoxicity and skin sensitization, namely, Ames test, direct peptide reactivity, and ARE-Nrf2 luciferase assays. The degradation products of diazirine-mediated crosslinking observe little to no risk of in vitro genotoxicity or skin sensitization.

Metabolic reduction of resazurin; location within the cell for cytotoxicity assays.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity.

Nonthrombogenic Hydrogel Coatings with Carbene-Cross-Linking Bioadhesives.

Bioadhesives are a current unmet clinical need for mending of blood contacting soft tissues without inducing thrombosis. Recent development of carbene precursor bioadhesives with the advantages of on-demand curing, tuneable modulus, and wet adhesion have been synthesized by grafting diazirine onto poly (amidoamine) (PAMAM-G5) dendrimers. Herein, the structure activity relationships of platelet adhesion and activation is evaluated for the first time on the cured PAMAM-g-diazirine bioadhesives. Three strategies were employed to prevent healthy human donor platelets from adhering and activating on light-cured bioadhesive surfaces: (1) Attenuation of cationic surface charge, (2) antifouling composites by incorporating heparin and alginate in uncured formulation, and (3) heparin wash of cured bioadhesive surface. Topographical imaging of cured and ethanol dehydrated bioadhesive surfaces was used to quantify the adhered and activated platelets with scanning electron microscopy, whose resolution allowed identification of round senescent, short dendritic, and long dendritic platelets. Cured surfaces of PAMAM-g-diazirine (15%) had 10300 ± 500 adhered platelets mm-2 with 99.7% activation into short/long dendritic cells. Reduction of primary amines by higher degree of diazirine grafting or capping of free amines by acetylation reduces platelet adherence (2400 ± 200 vs 3000 ± 300, respectively). Physical incorporation of heparin and alginate in the formulations reduced the activated platelet; 1300 ± 300 and 300 ± 50, activated platelets mm-2, in comparison with additive free adhesive formulation. Similarly, heparin rinse of the surface of additive free bioadhesive reduced the activated platelet to platelets of heparin composites at 600 ± 100 platelets mm-2. PAMAM-g-diazirine (15%) bioadhesive retained the photocured mechanical properties and lap shear adhesion despite the addition of heparin and alginate additives.

Theoretical and Experimental Studies of N,N-Dimethyl-N'-Picryl-4,4'-Stilbenediamine.

N,N-dimethyl-N'-picryl-4,4'-stilbenediamine (DMPSDA) was prepared, purified and crystallised in a form of black lustrous crystals, and its absorption and fluorescence spectra were recorded in cyclohexane, acetonitrile and dimethyl sulfoxide. Non-emissive intramolecular charge transfer state (ICT) was clearly observed in this molecule in all three solvents. Theoretical calculations demonstrating a betaine electronic structure of the trinitrophenyl group in the ground state of the molecule and a charge transfer nature of the long wavelength transition S0 → S1 supported the experimental observations of the ICT formation in the molecule.

On-Demand Bioadhesive Dendrimers with Reduced Cytotoxicity.

Tissue adhesives based on polyamidoamine (PAMAM) dendrimer, grafted with UV-sensitive aryldiazirine (PAMAM-g-diazirine) are promising new candidates for light active adhesion on soft tissues. Diazirine carbene precursors form interfacial and intermolecular covalent crosslinks with tissues after UV light activation that requires no premixing or inclusion of free radical initiators. However, primary amines on the PAMAM dendrimer surface present a potential risk due to their cytotoxic and immunological effects. PAMAM-g-diazirine formulations with cationic pendant amines converted into neutral amide groups were evaluated. In vitro toxicity is reduced by an order of magnitude upon amine capping while retaining bioadhesive properties. The in vivo immunological response to PAMAM-g-diazirine formulations was found to be optimal in comparison to standard poly(lactic-co-glycolic acid) (PLGA) thin films.

Addressing Unmet Clinical Needs with UV Bioadhesives.

The invasive practice of suturing for wound closure has persisted for millennia; with the rate of medical development, it is staggering that there are few viable alternatives to invasive mechanical fasteners. Biocompatible and biodegradable polymers are attractive candidates for versatile bioadhesives and could revolutionize surgical procedures. Bioadhesives can be broadly placed into two groups: activated and instant. Almost all commercially available bioadhesives are instant, which cross-link by mixing two components or on contact with moisture. Activated bioadhesives, on the other hand, allow control of when and where a bioadhesive cross-links and, in some cases, the extent of cross-linking. Despite significant progress, there has been little translation of activated bioadhesives to clinical use. This review discusses recent developments in UV-activated bioadhesives toward addressing unmet clinical needs.

Stimulation and Inhibition of Anaerobic Digestion by Nickel and Cobalt: A Rapid Assessment Using the Resazurin Reduction Assay.

Stimulation of anaerobic digestion by essential trace metals is beneficial from a practical point of view to enhance the biodegradability and degradation rate of wastes. Hence, a quick method to determine which metal species, and at what concentration, can optimize anaerobic digestion is of great interest to both researchers and operators. In this present study, we investigated the effect of nickel(II), cobalt(II), and their mixture, on the anaerobic digestion of synthetic municipal wastewater. Using a volumetric method, that is, measuring methane production over time, revealed that anaerobic digestion was stimulated by the addition of 5 mg L-1 nickel(II), and cobalt(II), and their mixture in day(s). However, using a novel resazurin reduction assay, and based on its change in rate over time, we evaluated both inhibition at 250 mg L-1 nickel(II) and cobalt(II), and also the stimulatory effect of 5 mg L-1 nickel(II), and cobalt(II), and their mixture, in just 6 h. By investigating the dynamic distribution of these metals in the liquid phase of the anaerobic system and kinetics of resazurin reduction by nickel spiked anaerobic sludge, the concentration of nickel(II) on anaerobic digestion performance was profiled. Three critical concentrations were determined; stimulation starting (around 1 mg L-1), stimulation ending (around 100 mg L-1) and stimulation maximizing (around 10 mg L-1). Hence, we propose that the resazurin reduction assay is a novel and quick protocol for studying the stimulation of anaerobic bioprocesses by bioavailable essential trace metals.

Tunable chemical release from polyester thin film by photocatalytic zinc oxide and doped LiYF4 upconverting nanoparticles.

Once manufactured or implanted, polyester release kinetics tend to be fixed with little modulation possible for optimal local chemical concentrations. Here, a typical implantable polyester was fabricated into thin films (∼50 µm thick) with additives of photocatalytic ZnO nanoparticles, lanthanide-doped LiYF4 nanoparticle upconverting nanoparticles, or a combination thereof and irradiated with either 6 mW ultraviolet (365 nm) light emitting diodes or 50 mW near-infrared (980 nm) laser diodes to induce polymer photooxidation. Irradiated polyester films with the aforementioned photoadditives had enhanced release kinetics up to 30 times more than nonirradiated, neat films with extended release times of 28 days. Near-infrared, ZnO-mediated photocatalysis had the highest light on/light off ratio release kinetics of 15.4, while doped LiYF4 upconversion nanoparticles paired with ZnO nanoparticles had the highest linear R(2) correlation of 0.98 with respect to duty cycle and release kinetics. Future applications of the technology will aim toward modulation of previously developed polymeric reagents/drugs for real-time, feedback-optimized release.

Modeling and Application of a Rapid Fluorescence-Based Assay for Biotoxicity in Anaerobic Digestion.

The sensitivity of anaerobic digestion metabolism to a wide range of solutes makes it important to be able to monitor toxicants in the feed to anaerobic digesters to optimize their operation. In this study, a rapid fluorescence measurement technique based on resazurin reduction using a microplate reader was developed and applied for the detection of toxicants and/or inhibitors to digesters. A kinetic model was developed to describe the process of resazurin reduced to resorufin, and eventually to dihydroresorufin under anaerobic conditions. By modeling the assay results of resazurin (0.05, 0.1, 0.2, and 0.4 mM) reduction by a pure facultative anaerobic strain, Enterococcus faecalis, and fresh mixed anaerobic sludge, with or without 10 mg L(-1) spiked pentachlorophenol (PCP), we found it was clear that the pseudo-first-order rate constant for the reduction of resazurin to resorufin, k1, was a good measure of "toxicity". With lower biomass density and the optimal resazurin addition (0.1 mM), the toxicity of 10 mg L(-1) PCP for E. faecalis and fresh anaerobic sludge was detected in 10 min. By using this model, the toxicity differences among seven chlorophenols to E. faecalis and fresh mixed anaerobic sludge were elucidated within 30 min. The toxicity differences determined by this assay were comparable to toxicity sequences of various chlorophenols reported in the literature. These results suggest that the assay developed in this study not only can quickly detect toxicants for anaerobic digestion but also can efficiently detect the toxicity differences among a variety of similar toxicants.

New Photochrome Probe Allows Simultaneous pH and Microviscosity Sensing.

4-N,N'-dimethylamino-4'-N'-stilbenemaleamic acid (DASMA), a unique molecular photochrome probe that exhibits solubility and retains trans-cis photoisomerisation in a wide range of organic solvents and aqueous pH environments, was prepared, purified and chemically characterised. Absorption, fluorescence excitation and emission spectra and constant-illumination fluorescence decay were measured in acetonitrile, dimethyl sulfoxide, ethanol, propylene carbonate, and aqueous glycerol mixtures. The pseudo-first-order fluorescence decay rates were found to be strongly dependent on the medium viscosity. In addition, the molecule exhibited the pH-dependent fluorescence and photoisomerisation kinetics.

Novel Photochrome Aptamer Switch Assay (PHASA) for adaptive binding to aptamers.

A novel Photochrome-Aptamer Switch Assay (PHASA) for the detection and quantification of small environmentally important molecules such as toxins, explosives, drugs and pollutants, which are difficult to detect using antibodies-based assays with high sensitivity and specificity, has been developed. The assay is based on the conjugation of a particular stilbene-analyte derivative to any aptamer of interest. A unique feature of the stilbene molecule is its reporting power via trans-cis photoisomerisation (from fluorescent trans-isomer to non-fluorescent cis-isomer) upon irradiation with the excitation light. The resulting fluorescence decay rate for the trans-isomer of the stilbene-analyte depends on viscosity and spatial freedom to rotate in the surrounding medium and can be used to indicate the presence of the analyte. Quantification of the assay is achieved by calibration of the fluorescence decay rate for the amount of the tested analyte. Two different formats of PHASA have been recently developed: direct conjugation and adaptive binding. New stilbene-maleimide derivatives used in the adaptive binding format have been prepared and characterised. They demonstrate effective binding to the model thiol compound and to the thiolated Malachite Green aptamer.

Tougher Bioadhesives through Dual Stimulation Strategies.

Carbene-based bioadhesives have favourable attributes for tissue adhesion, including non-specific bonding to wet and dry tissues, but suffer from relatively weak fracture strength after photocuring. Light irradiation of carbene-precursor (diazirine) also creates inert side products that are absent under thermal activation. Herein, a dual activation method combines light irradiation at elevated temperatures for the evaluation of diazirine depletion and effects on cohesive properties. A customized photo/thermal-rheometer evaluates viscoelastic properties, correlated to the kinetics of carbene:diazoalkane ratios via 19F NMR). The latter exploits the sensitive -CF3 functional group to determine joule-based light/temperature kinetics on trifluoroaryl diazirine consumption. The combination of heat and photoactivation produced bioadhesives that are 3× tougher compared to control. Dual thermal/light irradiation may be a strategy to improve viscoelastic dissipation and toughness of photo-activated adhesive resins.

Color changing bioadhesive barrier for peripherally inserted central catheters.

Bacteria migration at catheter insertion sites presents a serious complication (bacteraemia) with high mortality rates. One strategy to mediate bacteraemia is a physical barrier at the skin-catheter interface. Herein a colorimetric biosensor adhesive (CathoGlu) is designed and evaluated for both colorimetric detection of bacterial infection and application as a bacteria barrier. The design intent combines viscous, hydrophobic bioadhesive with an organic pH indicator (bromothymol blue). Visual observation can then distinguish healthy skin at pH = ∼5 from an infected catheter insertion site at pH = ∼8. The liquid-to-biorubber transition of CathoGlu formulation occurs via a brief exposure to UVA penlight, providing an elastic barrier to the skin flora. Leachates from CathoGlu demonstrate no genotoxic and skin sensitization effect, assessed by OECD-recommended in vitro and in chemico assays. The CathoGlu formulation was found non-inferior against clinically approved 2-octyl-cyanoacrylate (Dermabond™), and adhesive tape (Micropore™) within an in vivo porcine model. CathoGlu skin adhesive provides new opportunities to prevent sepsis in challenging clinical situations.

Collagen-cellulose composite thin films that mimic soft-tissue and allow stem-cell orientation.

Mechanical properties of collagen films are less than ideal for biomaterial development towards musculoskeletal repair or cardiovascular applications. Herein, we present a collagen-cellulose composite film (CCCF) compared against swine small intestine submucosa in regards to mechanical properties, cell growth, and histological analysis. CCCF was additionally characterized by FE-SEM, NMR, mass spectrometry, and Raman Microscopy to elucidate its physical structure, collagen-cellulose composition, and structure activity relationships. Mechanical properties of the CCCF were tested in both wet and dry environments, with anisotropic stress-strain curves that mimicked soft-tissue. Mesenchymal stem cells, human umbilical vein endothelial cells, and human coronary artery smooth muscle cells were able to proliferate on the collagen films with specific cell orientation. Mesenchymal stem cells had a higher proliferation index and were able to infiltrate CCCF to a higher degree than small intestine submucosa. With the underlying biological properties, we present a collagen-cellulose composite film towards forthcoming biomaterial-related applications.

Synthesis and Evaluation of Metal Lipoate Adhesives.

The development of new bioadhesives with integrated properties remains an unmet clinical need to replace staples or sutures. Current bioadhesives do not allow electronic activation, which would allow expansion into laparoscopic and robotic surgeries. To address this deficiency, voltage-activated adhesives have been developed on both carbene- and catechol-based chemical precursors. Herein, a third platform of voltage-activated adhesive is evaluated based on lipoic acid, a non-toxic dithiolane found in aerobic metabolism and capable of ring-opening polymerization. The electro-rheological and adhesive properties of lithium, sodium, and potassium salts of lipoic acid are applied for wet tissue adhesion. At ambient conditions, potassium lipoate displays higher storage modulus than lithium or sodium salt under similar conditions. Voltage stimulation significantly improves gelation kinetics to Na- and K-lipoates, while Li-lipoate is found to not require voltage stimulation for gelation. Lap shear adhesion strength on wetted collagen substrates reveals that the synthetic metal lipoates have comparable adhesion strength to fibrin sealants without viral or ethical risks.

Bacterial cellulose adhesive patches designed for soft mucosal interfaces.

The wet environment in the oral cavity is challenging for topical disease management approaches. The compromised material properties leading to weak adhesion and short retention (<8 h) in such environment result in frequent reapplication of the therapeutics. Composites of bacterial cellulose (BC) and carbene-based bioadhesives attempt to address these shortcomings. Previous designs comprised of aqueous formulations. The current design, for the first time, presents dry, shelf-stable cellulose patches for convenient ready-to-use application. The dry patches simultaneously remove tissue surface hydration while retaining carbene-based photocuring and offers on-demand adhesion. The dry patch prototypes are optimized by controlling BC/adhesive mole ratios and dehydration technique. The adhesion strength is higher than commercial denture adhesives on soft mucosal tissues. The structural integrity is maintained for a minimum of 7 days in aqueous environment. The patches act as selective nanoporous barrier against bacteria while allowing permeation of proteins. The results support the application of BC-based adhesive patches as a flexible platform for wound dressings, drug depots, or combination thereof.

Fibrillated bacterial cellulose liquid carbene bioadhesives for mimicking and bonding oral cavity surfaces.

Topical treatments for oral wounds and infections exhibit weak adhesion to wet surfaces which results in short retention duration (6-8 hours), frequent dosing requirement and patient incompatibility. To address these limitations, aqueous composites made of fibrillated bacterial cellulose and photoactive bioadhesives are designed for soft epithelial surfaces. The aqueous composites crosslink upon photocuring within a minute and exhibit a transition from viscous to elastic adhesive hydrogels. The light-cured composites have shear moduli mimicking oral mucosa and other soft tissues. The tunable adhesion strength ranges from 3 to 35 kPa on hydrated tissue-mimicking surfaces (collagen film). The results support the application of bacterial cellulose hydrogel systems for potential treatment of mucosal wounds.