Multivalency in healable supramolecular polymers: the effect of supramolecular cross-link density on the mechanical properties and healing of noncovalent polymer networks

Polymers with the ability to heal themselves could provide access to materials with extended lifetimes in a wide range of applications such as surface coatings, automotive components and aerospace composites. Here we describe the synthesis and characterisation of two novel, stimuli-responsive, supramolecular polymer blends based on π-electron-rich pyrenyl residues and π-electron-deficient, chain-folding aromatic diimides that interact through complementary π–π stacking interactions. Different degrees of supramolecular “cross-linking” were achieved by use of divalent or trivalent poly(ethylene glycol)-based polymers featuring pyrenyl end-groups, blended with a known diimide–ether copolymer. The mechanical properties of the resulting polymer blends revealed that higher degrees of supramolecular “cross-link density” yield materials with enhanced mechanical properties, such as increased tensile modulus, modulus of toughness, elasticity and yield point. After a number of break/heal cycles, these materials were found to retain the characteristics of the pristine polymer blend, and this new approach thus offers a simple route to mechanically robust yet healable materials.

The effect of polyoxyethylene polymers on the transport of ranitidine in Caco-2 cell monolayers.

Previous in vivo studies using PEG 400 showed an enhancement in the bioavailability of ranitidine. This study investigated the effect of PEG 200, 300 and 400 on ranitidine transport across Caco-2 cells. The effect of PEG polymers (20%, v/v) on the bi-directional flux of (3)H-ranitidine across Caco-2 cell monolayers was measured. The concentration dependence of PEG 400 effects on ranitidine transport was also studied. A specific screen for P-glycoprotein (P-gp) activity was used to test for an interaction between PEG and P-gp. In the absence of PEG, ranitidine transport showed over 5-fold greater flux across Caco-2 monolayers in the secretory than the absorptive direction; efflux ratio 5.38. PEG 300 and 400 significantly reduced this efflux ratio (p<0.05), whereas PEG 200 had no effect (p>0.05). In concordance, PEG 300 and 400 showed an interaction with the P-gp transporter, whereas PEG 200 did not. Interestingly, with PEG 400 a non-linear concentration dependence was seen for the inhibition of the efflux ratio of ranitidine, with a maxima at 1%, v/v (p<0.05). The inhibition of ranitidine efflux by PEG 300 and 400 which interact with P-gp provides a mechanism that may account for the observations of ranitidine absorption enhancement by PEG 400 in vivo.

Hyperbranched polymers containing oxazoline monomers andsuccinic anhydride: applications in fast drying, low solvent coating formulations

tMelt-polycondensation of succinic acid anhydride with oxazoline-based diol monomers gave hyper-branched polymers with carboxylicacids terminal groups.1H NMR and quantitative13C NMRspectroscopy coupled with DEPT-13513C NMR experiment showed high degrees of branching (over 60%).Esterification of the acid end groups by addition of citronellol at 160◦C produced novel white spirit solubleresins which were characterized by Fourier transform-infrared (FTIR) spectroscopy, gel permeation chro-matography (GPC), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Blendsof the new hyperbranched materials with commercial alkyd resins resulted in a dramatic, concentrationdependent drop in viscosity. Solvent-borne coatings were formulated containing the hyperbranchedpolymers. Dynamic mechanical analysis studies revealed that the air drying rates of the new coatingsystems were enhanced compared with identical formulations containing only commercial alkyd resins.

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase.

Dynamics of entangled polymers

In this book chapter, we focus on the basic questions on dynamics of entangled polymers that faced the earliest researchers, and see how their answers stand up in light of what has been learned in the last 40 years. In the process, we hope to provide the novice with a basic understanding of the physical concepts without burdening the reader with the mathematics, or with detailed data that can easily be found in the books referred to above. At the same time, we hope to update the knowledgeable non-specialist with the latest developments in the area. For the expert, we hope to provoke new ways of thinking about an old, but incompletely solved, problem area.

Probing the mucoadhesive interactions between porcine gastric mucin and some water-soluble polymers

This study investigates the structural features of porcine gastric mucin (PGM) in aqueous dispersions and its interactions with water-soluble polymers (poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(ethylene oxide), and poly(ethylene glycol)) using isothermal titration calorimetry, turbidimetric titration, dynamic light scattering, and transmission electron microscopy. It is established that PAA (450 kDa) and PMAA (100 kDa) exhibit strong specific interactions with PGM causing further aggregation of its particles, while PAA (2 kDa), poly(ethylene oxide) (1 000 kDa), and poly(ethylene glycol) (10 kDa) do not show any detectable effects on mucin. Sonication of mucin dispersions prior to their mixing with PAA (450 kDa) and PMAA (100 kDa) leads to more pronounced intensity of interactions.

Investigating the influence of extracellular matrix and glycolytic metabolism on muscle stem cell migration on their native fibre environment

The composition of the extracellular matrix (ECM) of skeletal muscle fibres is a unique environment that supports the regenerative capacity of satellite cells; the resident stem cell population. The impact of environment has great bearing on key properties permitting satellite cells to carry out tissue repair. In this study, we have investigated the influence of the ECM and glycolytic metabolism on satellite cell emergence and migration- two early processes required for muscle repair. Our results show that both influence the rate at which satellite cells emerge from the sub-basal lamina position and their rate of migration. These studies highlight the necessity of performing analysis of satellite behaviour on their native substrate and will inform on the production of artificial scaffolds intended for medical uses.

Molecular design of a discrete chain-folding polyimide for controlled inkjet deposition of supramolecular polymers

A supramolecular polymer based upon two complementary polymer components is formed by sequential deposition from solution in THF, using a piezoelectric drop-on-demand inkjet printer. Highly efficient cycloaddition or ‘click’ chemistry afforded a well-defined poly(ethylene glycol) featuring chain-folding diimide end groups, which possesses greatly enhanced solubility in THF relative to earlier materials featuring random diimide sequences. Blending the new polyimide with a complementary poly(ethylene glycol) system bearing pyrene end groups (which bind to the chain-folding diimide units) overcomes the limited solubility encountered previously with chain-folding polyimides in inkjet printing applications. The solution state properties of the resulting polymer blend were assessed via viscometry to confirm the presence of a supramolecular polymer before depositing the two electronically complementary polymers by inkjet printing techniques. The novel materials so produced offer an insight into ways of controlling the properties of printed materials through tuning the structure of the polymer at the (supra)molecular level.

Regulation of cell migration in cancer: investigation into the regulation of cancer cell blebbing in the extracellular matrix

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.

Temperature regulation of extracellular proteases in ectomycorrhizal fungi (Hebeloma spp.) grown in axenic culture

Strains of Hebeloma representative of different climatic zones were grown in axenic culture at either 2 °C and 22° or 6° and 22°. Culture filtrates were assayed for proteolytic activity using FITC labelled BSA as a substrate. Assays were run between 0–37°. Growth at low temperature induced greater proteolytic activity (g−1 D.W. mycelium). Many of the strains produced protease(s) which retained significant activity at temperatures as low as 0°, and a thermal optimum between 0–6° with a second optimum at higher temperature. The results are discussed in relation the nutrient acquisition potential of ectomycorrhizal fungi at low temperature and the contribution such cold active proteases might make to the soil enzyme pool.

Syncytiotrophoblast extracellular vesicles from pre-eclampsia placentas differentially affect platelet function

Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.

Enzymatically-triggered, isothermally responsive polymers: re-programming poly(oligoethylene glycols) to respond to phosphatase

Polymers which can respond to externally applied stimuli have found much application in the biomedical field due to their (reversible) coil–globule transitions. Polymers displaying a lower critical solution temperature are the most commonly used, but for blood-borne (i.e., soluble) biomedical applications the application of heat is not always possible, nor practical. Here we report the design and synthesis of poly(oligoethylene glycol methacrylate)-based polymers whose cloud points are easily varied by alkaline phosphatase-mediated dephosphorylation. By fine-tuning the density of phosphate groups on the backbone, it was possible to induce an isothermal transition: A change in solubility triggered by removal of a small number of phosphate esters from the side chains activating the LCST-type response. As there was no temperature change involved, this serves as a model of a cell-instructed polymer response. Finally, it was found that both polymers were non cytotoxic against MCF-7 cells (at 1 mg·mL–1), which confirms promise for biomedical applications.

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.