Antimicrobial Nitric Oxide-Releasing Electrospun Dressings for Wound Healing Applications.

Nonhealing and chronic wounds represent a major problem for the quality of life of patients and have cost implications for healthcare systems. The pathophysiological mechanisms that prevent wound healing are usually multifactorial and relate to patient overall health and nutrition, vascularity of the wound bed, and coexisting infection/colonization. Bacterial infections are one of the predominant issues that can stall a wound, causing it to become chronic. Successful wound healing often depends on weeks or months of antimicrobial therapy, but this is problematic given the rise in multidrug-resistant bacteria. As such, alternatives to antibiotics are desperately needed to aid the healing of chronic, and even acutely infected wounds. Nitric oxide (NO) kills bacteria through a variety of mechanisms, and thus, bacteria have shown no tendency to develop resistance to NO as a therapeutic agent and therefore can be a good alternative to antibiotic therapy. In this paper, we report on the development of NO-releasing electrospun membranes fabricated from polycaprolactone (PCL)/gelatin blends and optimized to reduce bacterial infection. The NO payload in the membranes was directly related to the number of amines (and hence the amount of gelatin) in the blend. Higher NO payloads corresponded with a higher degree of antimicrobial efficacy. No cytotoxicity was observed for electrospun membranes, and an in vitro wound closure assay demonstrated closure within 16 h. The results presented here clearly indicate that these NO-releasing electrospun membranes hold significant promise as wound dressings due to their antimicrobial activity and biocompatibility.

Antimicrobial Nitric Oxide Releasing Contact Lens Gels for the Treatment of Microbial Keratitis.

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against Pseudomonas aeruginosa and Staphylococcus aureus was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.

Ice-recrystallization inhibiting polymers protect proteins against freeze-stress and enable glycerol-free cryostorage.

Proteins are ubiquitous in molecular biotechnology, biotechnology and as therapeutics, but there are significant challenges in their storage and distribution, with freezing often required. This is traditionally achieved by the addition of cryoprotective agents such as glycerol (or trehalose) or covalent modification of mutated proteins with cryoprotectants. Here, ice recrystallization inhibiting polymers, inspired by antifreeze proteins, are used synergistically with poly(ethylene glycol) as an alternative to glycerol. The primary mechanism of action appears to be preventing irreversible aggregation due to ice growth. The polymer formulation is successfully used to cryopreserve a range of important proteins including insulin, Taq DNA polymerase and an IgG antibody. The polymers do not require covalent conjugation, nor modification of the protein and are already used in a wide range of biomedical applications, which will facilitate translation to a range of biologics.

Artificial cell membrane binding thrombin constructs drive in situ fibrin hydrogel formation.

Cell membrane re-engineering is emerging as a powerful tool for the development of next generation cell therapies, as it allows the user to augment therapeutic cells to provide additional functionalities, such as homing, adhesion or hypoxia resistance. To date, however, there are few examples where the plasma membrane is re-engineered to display active enzymes that promote extracellular matrix protein assembly. Here, we report on a self-contained matrix-forming system where the membrane of human mesenchymal stem cells is modified to display a novel thrombin construct, giving rise to spontaneous fibrin hydrogel nucleation and growth at near human plasma concentrations of fibrinogen. The cell membrane modification process is realised through the synthesis of a membrane-binding supercationic thrombin-polymer surfactant complex. Significantly, the resulting robust cellular fibrin hydrogel constructs can be differentiated down osteogenic and adipogenic lineages, giving rise to self-supporting monoliths that exhibit Young's moduli that reflect their respective extracellular matrix compositions.

Functionalized Triblock Copolymer Vectors for the Treatment of Acute Lymphoblastic Leukemia.

The chemotherapeutic Parthenolide is an exciting new candidate for the treatment of acute lymphoblastic leukemia, but like many other small-molecule drugs, it has low aqueous solubility. As a consequence, Parthenolide can only be administered clinically in the presence of harmful cosolvents. Accordingly, we describe the synthesis, characterization, and testing of a range of biocompatible triblock copolymer micelles as particle-based delivery vectors for the hydrophobic drug Parthenolide. The drug-loaded particles are produced via an emulsion-to-micelle transition method, and the effects of introducing anionic and cationic surface charges on stability, drug sequestration, biocompatibility, and efficacy are investigated. Significantly, we demonstrate high levels of efficacy in the organic solvent-free systems against human mesenchymal stem cells and primary T-acute lymphoblastic leukemia patient cells, highlighting the effectiveness of the delivery vectors for the treatment of acute lymphoblastic leukemia.

Regulation of Scaffold Cell Adhesion Using Artificial Membrane Binding Proteins.

The rapid pace of development in biotechnology has placed great importance on controlling cell-material interactions. In practice, this involves attempting to decouple the contributions from adhesion molecules, cell membrane receptors, and scaffold surface chemistry and morphology, which is extremely challenging. Accordingly, a strategy is presented in which different chemical, biochemical, and morphological properties of 3D biomaterials are systematically varied to produce novel scaffolds with tuneable cell affinities. Specifically, cationized and surfactant-conjugated proteins, recently shown to have non-native membrane affinity, are covalently attached to 3D scaffolds of collagen or carboxymethyl-dextran, yielding surface-functionalized 3D architectures with predictable cell immobilization profiles. The artificial membrane-binding proteins enhance cellular adhesion of human mesenchymal stem cells (hMSCs) via electrostatic and hydrophobic binding mechanisms. Furthermore, functionalizing the 3D scaffolds with cationized or surfactant-conjugated myoglobin prevents a slowdown in proliferation of seeded hMSCs cultured for seven days under hypoxic conditions.

Enhanced non-vitreous cryopreservation of immortalized and primary cells by ice-growth inhibiting polymers.

Cell cryopreservation is an essential tool in modern biotechnology and medicine. The ability to freeze, store and distribute materials underpins basic cell biology and enables storage of donor cells needed for transplantation and regenerative medicine. However, many cell types do not survive freezing and the current state-of-the-art involves the addition of significant amounts of organic solvents as cryoprotectants, which themselves can be cytotoxic, or simply interfere with assays. A key cause of cell death in cryopreservation is ice recrystallization (growth), which primarily occurs during thawing. Here it is demonstrated that the addition of ice recrystalization inhibiting polymers to solutions containing low (non vitrifying) concentrations of DMSO enhance cell recovery rates by up to 75%. Cell functionality is also demonstrated using a placental cell line, and enhanced cryopreservation of primary rat hepatocytes is additionally shown. The crucial role of the polymers architecture (chain length) is shown, with shorter polymers being more effective than longer ones.

Using molecular rotors to probe gelation.

A series of fluorescent probes, including a number of molecular rotors, have been used to follow the self-assembly of dipeptide-based low molecular weight gelators. We show that these probes can be used to gain an insight into the assembly process. Thioflavin T, a commonly used stain for ß-sheets, appears to act as a molecular rotor in these gelling systems, with the fluorescence data closely matching that of other rotors. The molecular rotor was incorporated into an assay system with glucose oxidase to enable glucose-concentration specific gelation and hence generating a fluorescent output. Applying this system to urine from patients with various levels of glycosuria (a symptom of diabetes), it was found to provide excellent correlation with different clinical assessments of diabetes. This demonstrates a new concept in gelation-linked biosensing for a real clinical problem.

Glycerol-Free Cryopreservation of Red Blood Cells Enabled by Ice-Recrystallization-Inhibiting Polymers.

Cryopreservation is fundamental in prolonging the viabilities of cells and tissues of clinical and biotechnological relevance ex vivo. Furthermore, there is an increasing need to address storage at more easily accessible temperatures in the developing world because of limited resources. Here, the cryopreservation of erythrocytes (red blood cells) with storage at -20 °C using hydroxyethyl starch (HES) and the ice recrystallization inhibitor poly(vinyl alcohol) (PVA), which is a biomimetic of naturally occurring antifreeze (glyco)proteins (AF(G)Ps), is described. This strategy eliminates the need for high concentrations of membrane penetrating solvents such as glycerol or dimethyl sulfoxide (DMSO). The addition of only 0.1-0.5 wt % PVA to the polymeric cryoprotectant, HES, significantly enhances cell recovery under conditions that promote damage due to ice recrystallization. The comparative ease with which the addition and removal of both HES and PVA can be attained is an additional attractive quality. Coupled with the benefits attained by the ice recrystallization inhibition activity of PVA, this methodology therefore offers a strategy that could aid the storage and distribution of biological materials.

Synthesis and characterisation of glucose-functional glycopolymers and gold nanoparticles: study of their potential interactions with ovine red blood cells.

Carbohydrate-protein interactions can assist with the targeting of polymer- and nano-delivery systems. However, some potential protein targets are not specific to a single cell type, resulting in reductions in their efficacy due to undesirable non-specific cellular interactions. The glucose transporter 1 (GLUT-1) is expressed to different extents on most cells in the vasculature, including human red blood cells and on cancerous tissue. Glycosylated nanomaterials bearing glucose (or related) carbohydrates, therefore, could potentially undergo unwanted interactions with these transporters, which may compromise the nanomaterial function or lead to cell agglutination, for example. Here, RAFT polymerisation is employed to obtain well-defined glucose-functional glycopolymers as well as glycosylated gold nanoparticles. Agglutination and binding assays did not reveal any significant binding to ovine red blood cells, nor any haemolysis. These data suggest that gluco-functional nanomaterials are compatible with blood, and their lack of undesirable interactions highlights their potential for delivery and imaging applications.

Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing.

The cryopreservation of cells, tissue and organs is fundamental to modern biotechnology, transplantation medicine and chemical biology. The current state-of-the-art method of cryopreservation is the addition of large amounts of organic solvents such as glycerol or dimethyl sulfoxide, to promote vitrification and prevent ice formation. Here we employ a synthetic, biomimetic, polymer, which is capable of slowing the growth of ice crystals in a manner similar to antifreeze (glyco)proteins to enhance the cryopreservation of sheep and human red blood cells. We find that only 0.1 wt% of the polymer is required to attain significant cell recovery post freezing, compared with over 20 wt% required for solvent-based strategies. These results demonstrate that synthetic antifreeze (glyco)protein mimics could have a crucial role in modern regenerative medicine to improve the storage and distribution of biological material for transplantation.

Uptake of poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles in microvascular endothelial cells and transport across the blood-brain barrier.

The facile and modular functionalization of gold nanoparticles makes them versatile tools in nanomedicine, for instance, photothermal therapy, contrast agents or as model nanoparticles to probe drug-delivery mechanisms. Since endothelial cells from various locations in the body exhibit unique phenotypes we quantitatively examined the amount of different sized poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles internalized into primary human dermal endothelial cells or human brain endothelial cells (hCMEC/D3) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and visualized the nanoparticles using light and electron microscopy. Poly(2-hydroxypropylmethacrylamide)-coated gold nanoparticles exhibited high uptake into brain endothelial cells and were used to examine transport mechanisms across the blood-brain barrier using a well-established in vitro model of the blood-brain barrier. Our results demonstrate that 35 nm-sized gold nanoparticles were internalized best into human brain endothelial cells by a flotillin-dependent endocytotic pathway. The uptake into the cells is not correlated with transport across the blood-brain barrier. We demonstrated that the surface modification of gold nanoparticles impacts the internalization process in different cells. In addition, to evaluating toxicity and uptake potential of nanoparticles into cells, the transport properties across cell barriers are important criteria to classify nanoparticle properties regarding targeted delivery of drugs.

Ice recrystallisation inhibition by polyols: comparison of molecular and macromolecular inhibitors and role of hydrophobic units.

The ability of polyols to act as ice recrystallisation inhibitors (IRI), inspired by antifreeze (glyco)proteins are studied. Poly(vinyl alcohol), PVA, a known IRI active polymer was compared to a panel of mono and polysaccharides, with the aim of elucidating why some polyols are active and others show no activity. When corrected for total hydroxyl concentration all the carbohydrate-based polyols displayed near identical activity with no significant influence of molecular weight. Conversely, PVA was several orders of magnitude more active and its activity displays significant dependence on molecular-weight implying that its mechanism of action is not identical to that of carbohydrates. In a second step, the role of hydrophobicity was studied and it is observed that monosaccharide IRI activity is enhanced by alkylation. Dye-quenching assays demonstrated that PVA is able to present a hydrophobic surface without self-aggregation. Therefore, the ability to present a hydrophobic domain is hypothesised to be essential to obtain high IRI activity, which has many biotechnological applications.

Exploiting thermoresponsive polymers to modulate lipophilicity: interactions with model membranes.

Upon heating above their lower critical solution temperature (LCST) poly[oligo(ethyleneglycol)methacrylate]s (POEGMA) were shown to undergo a shift in their partition coefficient triggering aqueous to organic phase transfer, which indicated their potential to partition into cell membranes upon application of an external stimulus. Fluorescence-based assays indicated that the LCST transition did not induce lysis of model phospholipid vesicles but did promote fusion, as confirmed by dynamic light scattering. Membrane perturbation assays and linear dichroism spectroscopy investigations suggest that POEGMAs above their transition temperatures can interact with, or insert into, membranes. These findings will help develop the application of responsive polymers in drug delivery.