The present work utilizes single molecule methods and analysis to investigate soft and hard substrates. First, the effect of charged hard surfaces on charged probes were evaluated, as the soft surfaces were built upon such a structure. Then, selected polymers were selected according to their importance in smart surface technology. The extent of interaction of the selected probes with the array of soft surfaces gives insight to the potential tunability of these surfaces. The three distinct polymers range from ubiquitous usage to advancements in current technology. The studies presented here are needed to characterize, on the nanoscale, the Coulombic interactions of these polymers.

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<p> The objective of this work was to investigate the potential of poly(vinyl pyrrolidone) (PVP) as a protein resistant biomaterial. Two types of PVP surface were studied: (1) plasma polymerized N-vinyl pyrrolidone monomer on polyethylene (PE), and (2) grafted PVP surfaces formed by reaction of the activated polymer with plasma polymerized allyl amine on PE. Surfaces were also prepared by grafting polyethylene oxide (PEO), a known protein repellent, to plasma polymerized allyl amine and for comparison to PVP. The surfaces were characterized chemically by water contact angle and X-ray photoelectron spectroscopy (XPS). Protein interactions were studied using radiolabeled fibrinogen in PBS buffer. </p> <p> Plasma polymerized N-vinyl pyrrolidone surfaces were prepared in a microwave plasma reactor. Reactions were carried out both at room temperature and at 50°C (increased vapour pressure) in an attempt to increase the extent of plasma polymer deposition. The resulting surfaces showed structures chemically different from conventional linear PVP. XPS analysis suggested the presence of a variety of functional groups, including amines, amides, hydroxyls, carbonyls and urethanes. Mechanisms for the reactions occurring could not be ascertained but it appeared that the monomer was extensively fragmented in the plasma. Although these surfaces were hydrophilic (contact angles of 20 to 30°), they did not resist fibrinogen adsorption: in fact they showed adsorption levels approximately 10% greater than unmodified polyethylene. </p> <p> Methods for direct grafting of polyvinyl pyrrolidone and polyethylene oxide to plasma polymerized allyl amine (PPAA) surfaces were designed on the assumption that the PPAA surfaces would be rich in amino groups for reaction with appropriate polymer chain ends. Although there was 8-12% of nitrogen on the surfaces, the C1 s high resolution showed that amide and urethane functionalities are also present in addition to amines. The hydroxyl end groups of preformed PEO and PVP chains were activated by reaction with either 1-[3- (dimethylamino) propyl], 3-ethylcarbodiimide and N-hydroxy succinimide (EDC/NHS), and N-N-disuccinimidyl carbonate (DSC). NMR spectra of the products of these reactions showed that for PEO, the yields were moderate, and for PVP, the yields were low. Surfaces grafted using polymers activated with EDC/NHS were more hydrophilic than surfaces grafted with DSC-activated polymers. XPS data did not provide clear evidence that significant polymer grafting had occurred in any of the systems. It was concluded that changes in the allyl amine plasma polymer in different environments following plasma polymerization may affect the efficiency of grafting subsequently. XPS data suggested that the allyl amine plasma surfaces undergo oxidation over time in air. Also the films may be partly removed from the polyethylene surface when placed in buffer as suggested by XPS and contact angle data. Various parameters were examined in an attempt to improve the allyl amine plasma polymerization process for greater stability of the film. Increasing the treatment time from 1 0 to 30 minutes gave surfaces that showed a slower change in contact angle when stored in air. </p> <p> Despite the lack of strong chemical evidence of extensive polymer grafting, all of the grafted surfaces were found to be significantly protein repellent, with reductions of 10 to 36 % compared to unmodified polyethylene. The PEO surfaces were more repellent than the PVP, although the differences were not significant. Surfaces grafted using polymers activated with EDC/NHS were more protein repellent than those grafted with DSC-activated polymers. Protein adsorption was not affected by PVP molecular weight in the range 2,500 to 10,000. Since there is considerable overlap of the molecular weight distributions (MWD) of these two polymers, it is speculated that the MWDs of the grafted polymers may be more similar than those of the polymers themselves, possibly due to "selection" of similar, presumably optimal molecular weights. </p> <p> Discussion of the possible reasons for the better protein resistance of PEO compared to PVP is given in terms of chain structure in relation to the steric exclusion and water barrier theories of protein repulsion. </p> / Thesis / Master of Applied Science (MASc)

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Planar supported, polymerized phospholipid bilayers (PPBs) composed of 1,2-bis[10-(2’,4’-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) were generated by a redox polymerization method. The PPBs were supported by a silicon substrate. The PPBs were characterized and tested for uniformity and stability under physiological conditions. The PPBs were analyzed in vitro with murine derived cells that are pertinent to the host response. Cellular attachment and phenotypic changes in RAW 264.7 macrophages and NIH 3T3 fibroblasts were investigated on PPBs and compared to bare silicon controls. Fluorescent and SEM images were used to observe cellular attachment and changes in cellular behavior. The PPBs showed much lower cellular adhesion for both cell lines than bare silicon controls. Of the cells that attached to the PPBs, a very low percentage showed the same morphological expressions as seen on the controls. The hypothesis generated from this work is that defects in the PPBs mediated the cellular attachment and morphological changes that were observed. Finally, a layer-by-layer (LbL) deposition of a poly(acrylic acid) (PAA) and poly(N-vinylpyrrolidone) (PNVP) alternating bilayer was attempted as a proof of concept for future modification of this system.

Planar supported, polymerized phospholipid bilayers (PPBs) composed of 1,2-bis[10-(2’,4’-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) were generated by a redox polymerization method. The PPBs were supported by a silicon substrate. The PPBs were characterized and tested for uniformity and stability under physiological conditions. The PPBs were analyzed in vitro with murine derived cells that are pertinent to the host response. Cellular attachment and phenotypic changes in RAW 264.7 macrophages and NIH 3T3 fibroblasts were investigated on PPBs and compared to bare silicon controls. Fluorescent and SEM images were used to observe cellular attachment and changes in cellular behavior. The PPBs showed much lower cellular adhesion for both cell lines than bare silicon controls. Of the cells that attached to the PPBs, a very low percentage showed the same morphological expressions as seen on the controls. The hypothesis generated from this work is that defects in the PPBs mediated the cellular attachment and morphological changes that were observed. Finally, a layer-by-layer (LbL) deposition of a poly(acrylic acid) (PAA) and poly(N-vinylpyrrolidone) (PNVP) alternating bilayer was attempted as a proof of concept for future modification of this system.

Orientadores: Maria Helena Andrade Santana, Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-11T17:04:37Z (GMT). No. of bitstreams: 1 Martins_MoysesOstDamm_M.pdf: 2259859 bytes, checksum: 461e16fd565ac08cc88d2c5c24c42ac1 (MD5) Previous issue date: 2007 / Resumo: Neste trabalho, foi estudada a detecção da IgG-anticardiolipina presente em soros de pacientes com doenças autoimunes, utilizando lipossomas polimerizados. A vantagem desse sistema é o reconhecimento molecular e transdução de sinal em uma única etapa. O uso de soros reais, ao invés de soros de referência, objetivou avaliar a potencialidade dos lipossomas polimerizados para aplicação em diagnóstico clínico-laboratorial. Os estudos foram realizados com "pools" de soros de pacientes com doenças auto-imunes, os quais foram previamente caracterizados através da dosagem de três níveis de IgG-específica designados como alta (5152,01 mg/mL), média (2030,49 mg/mL) e baixa concentração (13,39 e 14,03 mg/mL). Soro sadio, com concentração de IgG anti-cardiolipina 5,58 mg/mL foi usado como controle. Os lipossomas monoméricos foram compostos do ácido diacetilênico 10,12-tricosadiinóico e cardiolipina. A polimerização foi feita por irradiação de luz ultravioleta com a máxima absorção na região do azul. As interações foram estudadas com a cardiolipina livre e na superfície dos lipossomas polimerizados. A discriminação entre os soros autoimunes e soro sadio foi estudada em lipossomas polimerizados, através da diluição dos soros ou através da sua purificação com gel de sepharose- proteína G, o grau de polimerização e a concentração de cardiolipina. Os resultados mostram que as ligações específicas são predominantes em relação às inespecíficas, porém os componentes não específicos exercem interferência expressiva. Nos lipossomas polimerizados, os padrões de absorbâncias do vermelho e do azul com o tempo, são diferentes para os soros autoimunes e sadio, resultando em sinais espectrais maiores para o soro sadio. A diluição ou purificação muda o padrão de absorbância do azul, a qual decresceu com a interação, intensificando o sinal colorimétrico do vermelho resultante. A polimerização e a concentração de cardiolipina intensificam os sinais colorimétricos, porém não discriminaram a olho nu a diferença entre soro sadio e autoimune. O estudo dos efeitos da diluição e purificação apontam condições onde a discriminação do sinal pode ser maximizada. Esses resultados mostram a potencialidade dos lipossomas polimerizados para a detecção de anticorpos anticardiolipina em soros reais em ensaio de etapa única, e demonstram a factibilidade da análise espectral no estudo de interações moleculares em sistemas complexos. / Abstract: This work studies the detection of anticardiolipin IgG present in the sera o patients with autoimmune diseases , using polymerized liposomes. The advantage of this system is the molecular recognition and transduction of signal in a single step. The use of real sera, instead reference sera, aimed to evaluate the potentiality of polymerized liposomes to application in clinical-laboratorial diagnosis. The studies were carried out using pools of sera from patients with autoimmune diseases, which were previously characterized through the dosage of specific IgG level. Three levels of specific-IgG were selected, which were classified as high (5152,01 mg/mL), medium (2030,49 mg/mL) and low concentration (13,39 e 14,03 mg/mL). Serum from healthy individuals, with 5,58 mg/mL IgG anti-cardiolipin was used as control. The monomeric liposomes were composed by the diacetylenic acid 10,12-tricosadiynoic and cardiolipin. The polymerization was perfomed by irradiation in the UV wave lenght, with the maximum absorption in the blue region. The interactions were studied on the free cardiolipin and on the surface of polymerized liposomes. The discrimination between autoimmune and health sera was studied in polymerized liposomes through the dilution of sera, purification of sera using sepharose- protein G gel, the polymerizatio level and the cardiolipin concentration. The results show that the specific binding are predominant in related to the inspecific ones, but the interference of non-specific components is significant. The patterns of absorbance in red and blue along time in polymerized liposomes are different for the autoimmune sera and healthy serum. The dilution or purification chages the absorbance pattern in the blue, which decreasing as a consequence of the interaction, intensifying the final red signal. The polymerization and the cardiolipin concentration in liposomes intensified the colorimetric signals, but they don't discriminate by naked eye the dfferences between autoimmune and health sera. The study of the effects of dilution and purification pointed out to conditions where the signal discrimination may be maximized. These results show the potentialities of polymerized liposomes to detection of anticardiolipin antibodies in real sera using a single step assay. Furthermore, they demonstrate the factibility of the spectral analysis on the study of molecular interactions in complex systems. / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

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