Development of an iron overload HepG2 cell model using ferrous ammonium citrate.

Cell-based iron overload models provide tremendous utility for the investigations into the pathogenesis of different diseases as well as assessing efficacy of various therapeutic strategies. In the literature, establishing such models vary widely with regards to cell lines, iron source, iron treatment conditions and duration. Due to this diversity, researchers reported significant differences in the measured outcomes, either in cellular function or response to a stimulus. Herein, we report the process required to establish an iron overload HepG2 cell model to achieve a consistent and reproducible results such that the literature can strive towards a consensus. Iron loading in cells was achieved with 50 µM of iron every 24 h for 2 days, followed by an additional 24 h of maintenance in fresh media. We demonstrated that iron overloaded cells had significantly increased ROS generation, labile and total iron whilst having various cellular functions resemble cells without iron overload. The present report addresses key pitfalls with regards to the lack of consensus currently present in the literature.

Autonomous macroscopic signal deciphering the geometric self-sorting of pillar[n]arenes.

In this communication, we have deciphered the geometric self-sorting of pillar[n]arenes by analyzing the fluid flow pattern obtained during the self-assembly of complementary pillar[n]arenes on the surface. The concept was further extended to demonstrate flow manipulation inside a microchannel where multiple sites were available for self-sorting, and the resultant flow velocity was tuned by the feeding ratio of the complementary pairs.

Discrimination of enantiomers and constitutional isomers by self-generated macroscopic fluid flow.

The amplification of weak molecular signals to visible output could provide a gateway to the macroscopic world. In this context, supramolecular interfaces were designed by depositing macrocyclic "host" molecules in a multilayer film that can be utilized to discriminate isomers by their fluid flow response upon "host-guest" molecular recognition.

A visible-light activated ROS generator multilayer film for antibacterial coatings.

The current scenario of antibiotic-resistant bacteria and pandemics caused by viruses makes research in the area of antibacterial and antiviral materials and surfaces more urgent than ever. In this regard, salicylideneimine based tetracoordinate boron-containing organic compounds are emerging as a new class of photosensitizers for singlet oxygen generation. However, the inherent inability of small organic molecules to be processed limits their potential use in functional coatings. Here we show the synthesis of a novel polymer functionalized with diiodosalicylideneimine-boron difluoride (PEI-BF2) and its utility for surface coating inside glass vials via layer-by-layer (LbL) assembly. The multilayer thin films are characterized using AFM and UV-Vis spectroscopy and the resultant coatings display excellent stability. The multilayer coating could be activated using visible light, and owing to the photocatalytic activity of the incorporated PEI-BF2, the surface coating is able to generate singlet oxygen efficiently upon light irradiation. Further, the multilayer coated surfaces exhibit remarkable antimicrobial activity towards both Gram-positive and Gram-negative bacteria under a variety of conditions. Thus, owing to the simple synthesis and the convenient methodology adopted for the preparation of multilayer coatings, the material reported here could pave the way for the development of sunlight activated large area self-sterile surfaces.

Active transport nanochelators for the reduction of liver iron burden in iron overload.

Liver dysfunction and failure account for a major portion of premature deaths in patients suffering from various iron associated pathogeneses, particularly primary and secondary iron overload disorders, despite intensive treatment. The liver is a central player in iron homeostasis and a major iron storage organ, and currently, there are no active approaches for the excretion of excess liver iron. Herein, we report a new method for the rapid reduction of iron burden in iron overload diseases by developing a new class of liver targeted nanochelators with favorable pharmacokinetics and biodistribution. The new nanochelators bypass the reticuloendothelial system and specifically target hepatocytes without non-specific accumulation in other organs. The targeted nanochelators bound and neutralized excess iron in the liver and from the vasculature and, eventually leading to rapid hepatobiliary excretion of labile iron. Further, these rapidly excreted nanochelators did not induce toxicity in the liver, were highly cytocompatible in both iron overload and non-loaded conditions, and were promising in mitigating iron triggered free radical oxidative damage. These studies provide key insights into the development of organ targeted nanochelating systems and the rapid reduction of iron burden in vivo. This methodology allows for further development of nanotherapeutics for specific iron overload diseases.

Polymer multilayer films regulate macroscopic fluid flow and power microfluidic devices via supramolecular interactions.

Self-powered supramolecular micropumps could potentially provide a solution for powerless microfluidic devices where the fluid flow can be manipulated via modulating non-covalent interactions. An attempt has been made to fabricate thin-film-based micropumps by depositing a ß-cyclodextrin ('host') functionalized polymer on a glass slide via layer-by-layer assembly. These supramolecular micropumps turned on the fluid flow upon addition of 'guest' molecules to the multilayer films. The flow velocity was tuned using the concentration of the guest molecules as well as the number of host layers inside the multilayer films. Numerical modelling reveals that the solutal buoyancy, which originates from host-guest complexation, is primarily responsible for the fluid flow. In view of its potential application in self-powered devices, the thin-film-based micropump was integrated into a microfluidic device to show molecular and colloidal transport over long distances.

Anisotropic Compartmentalization of the Liquid-Liquid Interface using Dynamic Imine Chemistry.

The liquid-liquid interface offers a fascinating avenue for generating hierarchical compartments. Herein, the dynamic imine chemistry is employed at the oil-water interface to investigate the effect of dynamic covalent bonds for modulating the droplet shape. The imine bond formation between oil-soluble aromatic aldehydes and water-soluble polyethyleneimine greatly stabilized the oil-water interface by substantially lowering the interfacial tension. The successful jamming of imine-mediated assemblies was observed when a compressive force was applied to the droplet. Thus, the anisotropic compartmentalization of the liquid-liquid interface was created, and it was later altered by changing the pH of the surrounding environment. Finally, a proof-of-concept demonstration of a pH-triggered cargo release across the interfacial membrane confirmed the feasibility of stimuli-responsive behavior of dynamic imine assemblies.

Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750 UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750 UHRA-8 and 32 in mPEG750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

Inhibitory effect of nucleotides on acetylcholine esterase activity and its microflow-based actuation in blood plasma.

The inhibitory effect of nucleotides on the catalytic activity of acetylcholine esterase (AChE) was rationalized and a similar inhibition trend was observed when analyzing the macroscopic fluid flow generated by surface immobilized AChE. Additionally, the demonstration of enzymatic micropumping by showing adenine-nucleotide responsive AChE actuated fluid flow from blood plasma paved the way for designing future lab-on-a-chip devices in complex biological environments with potential clinical applications.

Fluid actuation and buoyancy driven oscillation by enzyme-immobilized microfluidic microcapsules.

Mimicking microorganism's locomotion and actuation under fluid is difficult to realize. To better comprehend the motility in non-living matter, self-propelled synthetic systems are being developed as a fast-growing area of research. Inspired by the self-powered enzyme micropumps where the enzyme catalysis was harnessed to create motion, herein, enzyme-immobilized microfluidic microcapsules (MCs) were used as a microscale engine to maneuver the fluid flow. The fluid actuation was tuned by various parameters such as substrate concentration, reaction rate, diameter of MCs and the population of the MCs inside the flow chamber. The same MCs, when suspended in a solution, showed buoyancy driven motility by creating oxygen bubbles via an enzymatic reaction and the velocity of the MCs was directly dependent on the number of nucleated oxygen bubbles generated on the MC surface.

Modulation of liquid structure and controlling molecular diffusion using supramolecular constructs.

The non-equilibrium liquid structure was achieved by interfacial jamming of pillar[5]arene carboxylic acid (P[5]AA) mediated by hydrogen bonding interactions. The assembly was reversibly modulated via jamming to unjamming transition thus dynamically shaping the liquid droplets. Interestingly, these supramolecular constructs showed pH-switchable gated diffusion of encapsulants, hence showcasing a next generation smart release system.

Rapid Assembly of Infection-Resistant Coatings: Screening and Identification of Antimicrobial Peptides Works in Cooperation with an Antifouling Background.

Bacterial adhesion and the succeeding biofilm formation onto surfaces are responsible for implant- and device-associated infections. Bifunctional coatings integrating both nonfouling components and antimicrobial peptides (AMPs) are a promising approach to develop potent antibiofilm coatings. However, the current approaches and chemistry for such coatings are time-consuming and dependent on substrates and involve a multistep process. Also, the information is limited on the influence of the coating structure or its components on the antibiofilm activity of such AMP-based coatings. Here, we report a new strategy to rapidly assemble a stable, potent, and substrate-independent AMP-based antibiofilm coating in a nonfouling background. The coating structure allowed for the screening of AMPs in a relevant nonfouling background to identify optimal peptide combinations that work in cooperation to generate potent antibiofilm activity. The structure of the coating was changed by altering the organization of the hydrophilic polymer chains within the coatings. The coatings were thoroughly characterized using various surface analytical techniques and correlated with the efficiency to prevent biofilm formation against diverse bacteria. The coating method that allowed the conjugation of AMPs without altering the steric protection ability of hydrophilic polymer structure results in a bifunctional surface coating with excellent antibiofilm activity. In contrast, the conjugation of AMPs directly to the hydrophilic polymer chains resulted in a surface with poor antibiofilm activity and increased adhesion of bacteria. Using this coating approach, we further established a new screening method and identified a set of potent surface-tethered AMPs with high activity. The success of this new peptide screening and coating method is demonstrated using a clinically relevant mouse infection model to prevent catheter-associated urinary tract infection (CAUTI).

Role of Iron in the Molecular Pathogenesis of Diseases and Therapeutic Opportunities.

Iron is an essential mineral that serves as a prosthetic group for a variety of proteins involved in vital cellular processes. The iron economy within humans is highly conserved in that there is no proper iron excretion pathway. Therefore, iron homeostasis is highly evolved to coordinate iron acquisition, storage, transport, and recycling efficiently. A disturbance in this state can result in excess iron burden in which an ensuing iron-mediated generation of reactive oxygen species imparts widespread oxidative damage to proteins, lipids, and DNA. On the contrary, problems in iron deficiency either due to genetic or nutritional causes can lead to a number of iron deficiency disorders. Iron chelation strategies have been in the works since the early 1900s, and they still remain the most viable therapeutic approach to mitigate the toxic side effects of excess iron. Intense investigations on improving the efficacy of chelation strategies while being well tolerated and accepted by patients have been a particular focus for many researchers over the past 30 years. Moreover, recent advances in our understanding on the role of iron in the pathogenesis of different diseases (both in iron overload and iron deficiency conditions) motivate the need to develop new therapeutics. We summarized recent investigations into the role of iron in health and disease conditions, iron chelation, and iron delivery strategies. Information regarding small molecule as well as macromolecular approaches and how they are employed within different disease pathogenesis such as primary and secondary iron overload diseases, cancer, diabetes, neurodegenerative diseases, infections, and in iron deficiency is provided.

Valveless flow reversal by a pH responsive supramolecular micropump.

A valveless micropump was designed via dynamic supramolecular interaction between beta-cyclodextrin (ß-CD) and benzimidazole (BzI). It shows flow reversal in response to the pH change. An L-shaped microchannel was used to demonstrate the flow reversibility over long distances.

Monitoring, Managing, and Communicating Risk of Harmful Algal Blooms (HABs) in Recreational Resources across Canada.

Globally, harmful algal blooms (HABs) are on the rise, as is evidence of their toxicity. The impacts associated with blooms, however, vary across Nation states, as do the strategies and protocols to assess, monitor, and manage their occurrence. In Canada, water quality guidelines are standardized nationally, but the management strategies for HABs are not. Here, we explore current strategies to understand how to better communicate risks associated with HABs to the public. Our team conducted an environmental scan on provincial and territorial government agency protocols around HABs. Results suggest that there are variations in the monitoring, managing, and communicating of risk to the public: British Columbia, Manitoba, New Brunswick, and Quebec have well-established inter-agency protocols, and most provinces report following federal guidelines for water quality. Notably, 3 northern territories have no HABs monitoring or management protocols in place. More populous provinces use a variety of information venues (websites, social media, on site postings, and radio) to communicate risks associated with HABs, whereas others' communications are limited. To induce more collaboration on HABs monitoring and management and reduce the associated risks, creating a coherent system with consistent messaging and inter-agency communication is suggested.

A facile colorimetric method for the quantification of labile iron pool and total iron in cells and tissue specimens.

Quantification of iron is an important step to assess the iron burden in patients suffering from iron overload diseases, as well as tremendous value in understanding the underlying role of iron in the pathophysiology of these diseases. Current iron determination of total or labile iron, requires extensive sample handling and specialized instruments, whilst being time consuming and laborious. Moreover, there is minimal to no overlap between total iron and labile iron quantification methodologies-i.e. requiring entirely separate protocols, techniques and instruments. Herein, we report a unified-ferene (u-ferene) assay that enables a 2-in-1 quantification of both labile and total iron from the same preparation of a biological specimen. We demonstrate that labile iron concentrations determined from the u-ferene assay is in agreement with confocal laser scanning microscopy techniques employed within the literature. Further, this assay offers the same sensitivity as the current gold standard, inductively coupled plasma mass spectrometry (ICP-MS), for total iron measurements. The new u-ferene assay will have tremendous value for the wider scientific community as it offers an economic and readily accessible method for convenient 2-in-1 measurement of total and labile iron from biological samples, whilst maintaining the precision and sensitivity, as compared to ICP-MS.

Self-Limiting Mussel Inspired Thin Antifouling Coating with Broad-Spectrum Resistance to Biofilm Formation to Prevent Catheter-Associated Infection in Mouse and Porcine Models.

Catheter-associated urinary tract infections (CAUTIs) are one of the most commonly occurring hospital-acquired infections. Current coating strategies to prevent catheter-associated biofilm formation are limited by their poor long-term efficiency and limited applicability to diverse materials. Here, the authors report a highly effective non-fouling coating with long-term biofilm prevention activity and is applicable to diverse catheters. The thin coating is lubricous, stable, highly uniform, and shows broad spectrum prevention of biofilm formation of nine different bacterial strains and prevents the migration of bacteria on catheter surface. The coating method is adapted to human-sized catheters (both intraluminal and extraluminal) and demonstrates long-term biofilm prevention activity over 30 days in challenging conditions. The coated catheters are tested in a mouse CAUTI model and demonstrate high efficiency in preventing bacterial colonization of both Gram-positive and Gram-negative bacteria. Furthermore, the coated human-sized Foley catheters are evaluated in a porcine CAUTI model and show consistent efficiency in reducing biofilm formation by Escherichia coli (E. coli) over 95%. The simplicity of the coating method, the ability to apply this coating on diverse materials, and the high efficiency in preventing bacterial adhesion increase the potential of this method for the development of next generation infection resistant medical devices.

Maneuvering Fluid Motion and Flow-Induced Detection of Toxins by Enzyme Multilayer Films.

In view to develop an autonomous lab-on-a-chip device for detection of toxins without using any spectroscopic or electrochemical equipment, self-powered enzyme micropumps were fabricated via layer-by-layer assembly of enzymes and polyelectrolytes. The thin film-based enzyme micropumps turned on fluid flow in the presence of respective substrates in a concentration-dependent manner, and the rate of the enzymatic reaction was the key for maneuvering the fluid flow. Furthermore, the newly engineered enzyme-based micropumps were able to detect toxic metals and organophosphorus pesticides by modulating the fluid flow speed as the rate of the enzymatic reaction was altered by the presence of inhibitors. Thus, by regulating fluid flow in a micropump, low concentrations of analytes (e.g., target biomarkers and inhibitors) in biological fluids can be quantitatively identified for testing in a resource-constrained environment.

Polyglycerol-Based Macromolecular Iron Chelator Adjuvants for Antibiotics To Treat Drug-Resistant Bacteria.

Iron is an essential micronutrient for life. Its redox activity is a key component in a plethora of vital enzymatic reactions that take place in processes such as drug metabolism, DNA synthesis, steroid synthesis, gene regulation, and cellular respiration (oxygen transport and the electron transport chain). Bacteria are highly dependent on iron for their survival and growth and have specific mechanisms to acquire iron. Limiting the availability of iron to bacteria, thereby preventing their growth, provides new opportunities to treat infection in the era of the persistent rise of antibiotic-resistant bacteria. In this work, we have developed macromolecular iron chelators, conjugates of a high-affinity iron chelator (HBEDS) with polyglycerol, in an attempt to sequester iron uptake by bacteria to limit their growth in order to enhance antibiotic activity. The new macromolecular chelators are successful in slowing the growth of Staphylococcus aureus and worked as an efficient bacteriostatic against S. aureus. Further, these cytocompatible macrochelators acted as effective adjuvants to prevent bacterial growth when used in conjunction with antibiotics. The adjuvant activity of the macrochelators depends on their molecular weight and the chelator density on these molecules. These selective macro iron(III) chelators are highly efficient in growth inhibition and killing of methicillin-resistant S. aureus in conjunction with a low concentration of rifampicin.

Design of Safe Nanotherapeutics for the Excretion of Excess Systemic Toxic Iron.

Chronic transfusion of red blood cells (RBCs) to patients with ß-thalassemia, sickle cell disease, and other acquired anemic disorders generates significant amounts of bioactive iron deposits in the body. The inactivation and excretion of redox active iron(III) from the blood pool and organs are critical to prevent organ damage, and are the focus of iron chelation therapy (ICT) using low molecular weight Fe(III) specific chelators. However, the current ICT is suboptimal because of the short circulation time of chelators, toxicity, severe side effects, difficult regime of administration, and patient noncompliance. To address this issue, we have designed long circulating and biodegradable nanoconjugates with enhanced circulation time and well-defined biodegradability to improve iron excretion and avoid nonspecific organ accumulation. A series of iron chelating nanoconjugates were generated with deferoxamine (DFO) as the iron(III) specific chelator using polymer scaffolds containing structurally different acidic pH sensitive ketal groups. The type of degradation linkages used in the polymer scaffold significantly influenced the vascular residence time, biodistribution, and mode of excretion of chelators in mice. Remarkably, the conjugate, BGD-60 (140 kDa; R h, 10.6 nm; cyclic ketal), exhibited the long circulation half-life (t 1/2ß, 64 h), a 768-fold increase compared to DFO, and showed minimal polymer accumulation in major organs. The nanoconjugates were found to be nontoxic and excreted iron significantly better than DFO in iron overloaded mice. BGD-60 showed greater iron mobilization from plasma (p = 0.0390), spleen (p < 0.0001), and pancreas (p < 0.0001) whereas BDD-200 (340 kDa; R h, 13.7 nm; linear ketal) mobilized iron significantly better from the spleen, liver, and pancreas (p < 0.0001, p < 0.0001, and p < 0.0001, respectively) compared to DFO at equivalent doses. The nanoconjugate's favorable long blood circulation time, biodegradability, and iron excretion profiles highlight their potential for future clinical translation.