Facile synthesis of well-defined hydrophilic methacrylic macromonomers using ATRP and click chemistry

A range of well-defined hydrophilic methacrylic macromonomers has been synthesized by the judicious combination of atom transfer radical polymerization (ATRP) and copper-catalyzed 1,3-dipolar cycloaddition (azide-alkyne click chemistry). An azido a-functionalized ATRP initiator was used to produce well-defined homopolymers with terminal azide functionality via ATRP in protic media at 20 °C, with generally good control being achieved over both target molecular weight and final polydispersity (Mw/Mn = 1.10-1.35). Suitable methacrylic monomers include 2-aminoethyl methacrylate hydrochloride, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-(methacryloyloxy)ethyl phosphorylcholine, glycerol monomethacrylate, potassium 3-sulfopropyl methacrylate, and quaternized 2-(dimethylamino)ethyl methacrylate. These homopolymer precursors were then efficiently clicked using either propargyl methacrylate or propargyl acrylate to yield near-monodisperse (meth)acrylate-capped macromonomers with either cationic, anionic, nonionic, or zwitterionic character. Moreover, this generic route to well-defined hydrophilic macromonomers is also suitable for “one-pot” syntheses, as exemplified for 2-hydroxyethyl methacrylate and glycerol monomethacrylate-based macromonomers.

Transition metal ion coordination in hydrophilic polymer membranes

The research described within this thesis is concerned with the investigation of transition metal ion complexation within hydrophilic copolymer membranes. The membranes are copolymers of 4-methyl-4'-vinyl-2,2'-bipyridine, the 2-hydroxyethyl ester of 4,4'- dicarboxy-2,2'-bipyridine & bis-(5-vinylsalicylidene)ethylenediamine with 2-hydroxyethyl methacrylate. The effect of the polymer matrix on the formation and properties of transition metal iron complexes has been studied, specifically Cr(III) & Fe(II) salts for the bipyridyl- based copolymer membranes and Co(II), Ni(II) & Cu(II) salts for the salenH2- based copolymer membranes. The concomitant effect of complex formation on the properties of the polymer matrix have also been studied, e.g. on mechanical strength. A detailed body of work into the kinetics and thermodynamics for the formation of Cu(II) complexes in the salenH2- based copolymer membranes has been performed. The rate of complex formation is found to be very slow while the value of K for the equilibrium of complex formation is found to be unexpectedly small and shows a slight anion dependence. These phenomena are explained in terms of the effects of the heterogeneous phase provided by the polymer matrix. The transport of Cr(III) ions across uncomplexed and Cr(III)-pre-complexed bipyridyl-based membranes has been studied. In both cases, no Cr(III) coordination occurs within the time-scale of an experiment. Pre-complexation of the membrane does not lead to a change in the rate of permeation of Cr(III) ions. The transport of Co(II), Ni(II) & Cu(II) ions across salenH2- based membranes shows that there is no detectable lag-time in transport of the ions, despite independent evidence that complex formation within the membranes does occur. Finally, the synthesis of a number of functionalised ligands is described. Although they were found to be non-polymerisable by the methods employed in this research, they remain interesting ligands which provide a startmg pomt for further functionalisation.

The effect of certain hydrophilic polymers on suspension stability

The adsorption of nonionic surface active agents of polyoxyethylene glycol monoethers of n hexadecanols on polystyrene latex and nonionic cellulose polymers of hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose on polystyrene latex and ibuprofen drug particles have been studied. The adsorbed layer thicknesses were determined by means of microelectrophoretic and viscometric methods. The conformation of the adsorbed molecules at the solid-liquid interface was deduced from the molecular areas and the adsorbed layer thicknesses. Comparison of the adsorption results obtained from polystyrene latex and ibuprofen particles was made to explain the conformation difference between these two adsorbates. Sedimentation volumes and redispersibility values were the main criteria used to evaluate suspension stability. At low concentrations of surface active agents, hard caked suspensions were found, probably due to the attraction between the uncoated areas or, the mutual adsorption of the adsorbed molecules on the bare surface of the particles in the sediment. At high concentrations of hydroxypropyl cellulose and hydroxypropyl methylcellulose, heavily caked sediments were attributed to network structure formation by the adsorbed molecules. An attempt was made to relate the characteristics of the suspensions to the potential energy of interaction curves. Generally, the agreement between theory and experiment was good, but for hydroxyethyl cellulose-ibuprofen systems discrepancies were found. Experimental studies showed that hydroxyethyl cellulose flocculated polystyrene latex over a rather wide range of concentrations; similarly, hydroxyethyl cellulose-ibuprofen suspensions were also flocculated. Therefore, it ls suggested that a term to account for flocculation energy of the polymer should be added to the total energy of interaction. A rheometric method was employed to study the flocculation energy of the polymer.

Visual performance of the human eye with soft hydrophilic contact lenses

The recording of visual acuity using the Snellen letter chart is only a limited measure of the visual performance of an eye wearing a refractive aid. Qualitative in addition to quantitative information is required to establish such a parameter: spatial, temporal and photometric aspects must all be incorporated into the test procedure. The literature relating to the correction of ametropia by refractive aids was reviewed. Selected aspects of a comparison between the correction provided by spectacles and contact lenses were considered. Special attention was directed to soft hydrophilic contact lenses. Despite technological advances which have produced physiologically acceptable soft lenses, there still remain associated with this recent form of refractive aid unpredictable visual factors. Several techniques for vision assessment were described, and previous studies of visual performance were discussed. To facilitate the investigation of visual performance in a clinical environment, a new semi-automated system was described: this utilized the presentation of broken ring test stimuli on a television screen. The research project comprised two stages. Initial work was concerned with the validation of the television system, including the optimization of its several operational variables. The second phase involved the utilization of the system in an investigation of visual performance aspects of the first month of regular daily soft contact lens wear by experimentally-naive subjects. On the basis of the results of this work an ‘homoeostatic’ model has been proposed to represent the strategy which an observer adopts in order to optimize his visual performance with soft contact lenses.

Comparison of hydrophilic and hydrophobic active molecules release from polymer-phospholipid complexes

Hypercoiling polymers can be suited for application to living systems because they are similar in structure to the protein-based lipid assemblies found at fluid interfaces within the body. This leads to a range of exciting possibilities, not only in membrane transport applications but also in biosensors, drug delivery and mechanistic studies of biological membrane function. This study is focused in the study of the stability and suitability of nanostructures made of a hypercoiling polymer for drug delivery applications. The polymer poly (styrene-maleic acid) (PSMA) was combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) to form amphiphilic nanostructures. The stability and suitability of these polymer-phospholipid nanocarriers for hydrophobic and hydrophilic molecules load and release was analyzed by several techniques. It was found that several of the studied molecules had a substantial effect on the surface charge and stability of the nanocarrier. It was also demonstrated that two types of nanocarriers, chemically modified and unmodified, were able to control the release of the molecules, especially in the case of hydrophobic compounds. In addition, as the hydrophobicity increased the release slowed down. These clear nanocarriers have the potential to behave very favorably at interfaces such as the tear lipid film were transparency is a requirement, giving a new way of controlled drug release in the eye.

Transition metal ion co-ordination in hydrophilic polymer membrane

This thesis is concerned with the investigation of transition metal (TM) ion complexation with hydrophilic membranes composed of copolymers of 4-vinyl pyridine & 4-methyl-4'vinyl- 2,2'-bipyridine with 2-hydroxyethyl methacrylate. The Cu(II), CoCII) & Fe(II) complexes with these coordinating membranes were characterised by a variety of techniques, in order to assess the effect of the polymer on the properties of the complex, and vice versa. A detailed programme of work was instigated into the kinetics of formation for the polymer-bound tris(bipyridyl) iron(II) complex; the rate and extent of complex formation was found to be anion-dependent. This is explained in terms of the influence of the anion on the transport properties and water content of the membrane, the controlling factor in the development of the tris-complex being the equilibrium concentration of Fe(II) in the gel matrix. A series of transport studies were performed with a view to the potential application of complexing hydrogel membranes for aqueous TM ion separations. A number of salts were studied individually and shown to possess a range of permeabilities; the degree of interaction between particular metal-ion:ligand combinations is given by the lag-time observed before steady-state permeation is achieved. However, when two TM salts that individually display different transport properties were studied in combination, they showed similar lag-times & permeabilities, characteristic of the more strongly coordinating metal ion. This 'anti-selective' nature thus renders the membrane systems unsuitable for TM ion separations. Finally, attempts were made to synthesise and immobilise a series of N ,0-donor macrocyclic ligands into hydrogel membranes. Although the functionalisation reactions failed, limited transport data was obtained from membranes in which the ligands were physically entrapped within the polymer matrix.

Double hydrophilic polyphosphoester containing copolymers as efficient templating agents for calcium carbonate microparticles

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Crocin and β-Carotene bleaching assays as analytical tools in the optimization of the extraction of hydrophilic and lipophilic antioxidants from tomato

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop worldwide and a rich source of hydrophilic (H) and lipophilic (L) antioxidants. The H fraction is constituted mainly by ascorbic acid and soluble phenolic compounds, while the L fraction contains carotenoids (mostly lycopene), tocopherols, sterols and lipophilic phenolics [1,2]. To obtain these antioxidants it is necessary to follow appropriate extraction methods and processing conditions. In this regard, this study aimed at determining the optimal extraction conditions for H and L antioxidants from a tomato surplus. A 5-level full factorial design with 4 factors (extraction time (I, 0-20 min), temperature (T, 60-180 •c), ethanol percentage (Et, 0-100%) and solid/liquid ratio (S/L, 5-45 g!L)) was implemented and the response surface methodology used for analysis. Extractions were carried out in a Biotage Initiator Microwave apparatus. The concentration-time response methods of crocin and P-carotene bleaching were applied (using 96-well microplates), since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively [3]. Measurements were carried out at intervals of 3, 5 and 10 min (initiation, propagation and asymptotic phases), during a time frame of 200 min. The parameters Pm (maximum protected substrate) and V m (amount of protected substrate per g of extract) and the so called IC50 were used to quantify the response. The optimum extraction conditions were as follows: r~2.25 min, 7'=149.2 •c, Et=99.1 %and SIL=l5.0 giL for H antioxidants; and t=l5.4 min, 7'=60.0 •c, Et=33.0% and S/L~l5.0 g/L for L antioxidants. The proposed model was validated based on the high values of the adjusted coefficient of determination (R2.wi>0.91) and on the non-siguificant differences between predicted and experimental values. It was also found that the antioxidant capacity of the H fraction was much higher than the L one.

Optimization of microwave-assisted extraction of hydrophilic and lipophilic antioxidants from a surplus tomato crop by response surface methodology

Tomato is the second most important vegetable crop worldwide and a rich source of industrially interesting antioxidants. Hence, the microwave-assisted extraction of hydrophilic (H) and lipophilic (L) antioxidants from a surplus tomato crop was optimized using response surface methodology. The relevant independent variables were temperature (T), extraction time (t), ethanol concentration (Et) and solid/liquid ratio (S/L). The concentration-time response methods of crocin and β-carotene bleaching were applied, since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively. The optimum operating conditions that maximized the extraction were as follows: t, 2.25 min; T, 149.2 ºC; Et, 99.1 %; and S/L, 45.0 g/L for H antioxidants; and t, 15.4 min; T, 60.0 ºC; Et, 33.0 %; and S/L, 15.0 g/L for L antioxidants. This industrial approach indicated that surplus tomatoes possess a high content of antioxidants, offering an alternative source for obtaining natural value-added compounds. Additionally, by testing the relationship between the polarity of the extraction solvent and the antioxidant activity of the extracts in H and L media (polarity-activity relationship), useful information for the study of complex natural extracts containing components with variable degrees of polarity was obtained.

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300 nm. In terms of drug loading, microfluidics production promoted high loading within ∼100 nm vesicles for both the water soluble drug (20–25% of initial amount added) and the bilayer embedded drug (40–42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

PDMS spreading morphological patterns on substrates of different hydrophilicity in air vacuum and water

In paper has been to investigate the morphological patterns and kinetics of PDMS spreading on silicon wafer using combination of techniques like ellipsometry, atomic force microscope (AFM), scanning electron microscope (SEM) and optical microscopy. A macroscopic silicone oil drops as well as PDMS water based emulsions were studied after deposition on a flat surface of silicon wafer in air, water and vacuum. our own measurements using an imaging ellipsometer, which also clearly shows the presence of a precursor film. The diffusion constant of this film, measured with a 60 000 cS PDMS sample spreading on a hydrophilic silicon wafer, is Df = 1.4  10-11 m2/s. Regardless of their size, density and method of deposition, droplets on both types of wafer (hydrophilic and hydrophobic) flatten out over a period of many hours, up to 3 days. During this process neighbouring droplets may coalesce, but there is strong evidence that some of the PDMS from the droplets migrates into a thin, continuous film that covers the surface in between droplets. The thin film appears to be ubiquitous if there has been any deposition of PDMS. However, this statement needs further verification. One question is whether the film forms immediately after forced drying, or whether in some or all cases it only forms by spreading from isolated droplets as they slowly flatten out.