Orientador: Eloi da Silva Feitosa / Banca: Leo Degreve / Banca: Luiz Carlos Gomide Freitas / Banca: José Roberto Ruggiero / Banca: Marinômio Lopes Cornélio / Resumo: O Poli(etileno glicol) (PEG) é um polímero sintético cujas características tem despertado grande interesse em diversas áreas. Suas aplicações podem ser vistas nas mais variadas áreas, desde biotecnologia e medicina até aplicações industriais e cosméticos. Alguns aspectos físicos como a estrutura adotada por esse polímero em diferentes solventes e detalhes sobre a interação entre essas moléculas ainda necessitam de maiores esclarecimentos, o que o torna objeto de intensa investigação. Essa tese visa desenvolver um modelo para moléculas de PEG, que possa ser utilizado em experimentos de dinâmica molecular. Resultados de simulações com esse modelo foram comparados a dados experimentais presentes na literatura, de forma a verificar o comportamento do modelo em diferentes condições, avaliando assim sua adequação. Os valores de densidade, obtidos dos sistemas simulados, apresentaram erro máximo de 1,14% para concentrações de até 50% de PEG400. A densidade do sistema em função da temperatura concorda com os dados da literatura, mantendo um erro fixo de 0,35%, que está relacionado com a concentração de 50% utilizada nessa simulação. A estrutura helicoidal, apresentada pelas moléculas de PEG ao final do processo de preparação dos modelos, é perdida rapidamente em todas as diferentes condições em que o sistema foi simulado, indicando que tal estrutura é energeticamente desfavorável em água. Com o aumento da concentração de PEG, as seguintes estruturas foram encontradas: moléculas de PEG livres em solução em concentrações inferiores a 5%, aglomerados de PEG entre 5 e 50%, com uma transição gradual entre uma estrutura e outra. Os resultados obtidos para concentrações acima de 50% não são conclusivos. Seguindo o procedimento aplicado ao modelo inicial, de PEG400, foi desenvolvido ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Poly(ethylene glycol) (PEG) is a synthetic polymer whose characteristics have attracted great interest in different fields. It has been applied in very different areas, from biotechnology and medicine to industry and cosmetics. Physical aspects like the structure PEG assumes in different solvent and details on the interaction between these polymers still lack clarity, make PEG an object of intense investigation. This Thesis aims do develop a model for PEG molecules that can be used in molecular dynamic simulations. Results of simulations using this model were compared to published experimental data, in order to investigate the behavior of the model under different conditions to evaluate its validity. The density values obtained from the simulations exhibit a maximum error of 1.14% for PEG400 concentrations up to 50%. The system density as a function of temperature agrees with experimental data from the literature within an error of 0.35% for the 50% PEG in the simulation. The helicoidal structure assumed by the PEG molecules at the end of the procedure of the model preparation is quickly lost under every simulation condition, thus indicating that the helicoidal structure is not energetically favorable for PEG in water. As PEG concentration is increased, the following structures were found: free PEG molecules below ca 5%, PEG clusters from ca 5-50%, with a gradual transition from one structure to another. The results for concentrations higher than 50% are not conclusive. Following the procedure applied to the initial PEG400 model, a second model was developed, almost four times larger, and used to investigate possible molecular effects capable to induce phase thermoseparation. The transition from different system states took place on average temperatures between 423.3 K and 424.1 K at the average pressure of 8.98 Bar. / Doutor

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Predložená diplomová práce se zabývá syntézou biodegradabilních polyuretanu (bio-PU) na bázi polyethylenglykolu (PEG-u) a polykaprolaktonu (PCL) pro využití k medicínským úcelum. Cílem práce bylo vyvinout metodiku prípravy elastomerního polyuretanu, ze kterého by se dal v budoucnu pripravit skafold (nosic bunek) použitelný v tkánovém inženýrství pro humánní regenerativní medicínu. Teoretická cást práce shrnuje informace o materiálech využitelných pro zamýšlenou aplikaci. Zahrnuty jsou také techniky prípravy skafoldu, jejich biokompatibilita a charakterizace vlastností výsledného materiálu. Praktická cást je pak zamerena na navržení vhodné metodiky pro prípravu funkcních vzorku obsahujících PEG, PCL, hexamethylen-diisokyanát (HMDI) a ethylhexanoát cínatý jako katalyzátor. U pripravených vzorku byl zjištován vliv jejich složení (predevším ruzný obsah a molekulová hmotnost PEG-u) na botnání a hydrolytickou stabilitu spolecne s testováním mechanických vlastností, sledováním kinetiky sítování a stanovením stupne konverze. Morfologie vzorku byla sledována pomocí optické mikroskopie, chemické složení bylo potvrzeno infracervenou spektroskopií a tepelné vlastnosti byly urceny diferencní kompenzacní kalorimetrií. Vývoj v metodice prípravy ukázal, že je nezbytné odstranit pred syntézou z výchozích látek veškeré necistoty, predevším vodu a kyslík, jinak vznikaly nedokonale zreagované vzorky s velkým obsahem póru ruzných velikostí. Syntéza všech vzorku probíhala pod dusíkovou atmosférou ve dvou krocích, kdy v prvním se pouze homogenizovaly oba polyoly (PEG i PCL) na vakuové lince pri 130 °C a ve druhém pak vznikaly výsledné bio-PU za pridání HMDI pri 65 °C v inertní atmosfére manipulacního boxu. Vzorky pak byly dopolymerovány ve forme pri 65 °C pod dobu 48 hodin v sušárne. Ruzné fyzikální podmínky metodiky vedly k príprave jak nepruhledných bílých filmu, tak i vzorku s viditelnou separací fází až po flexibilní transparentní filmy. Pozorování optickým mikroskopem vzorku s fázovou separací prokázalo prítomnost sférolitické struktury krystalické fáze PCL v amorfní fázi matrice PEG-u. Sledování kinetiky sítování pomocí soxhletovy extrakce potvrdilo maximální konverzi polymerace (96 %) již za 4 h dopolymerování v sušárne. Testování zkouškou v tahu ukázalo, že výrazný vliv na pevnost v tahu má isokyanátový index (pomer NCO/OH skupin). Molekulová hmotnost PEG-u ovlivnovala pevnost v tahu jen u vzorku zcela zesítovaných. Merení botnání vzorku ve vode prokázalo, že s rustem molekulové hmotnosti PEG-u roste i obsah vody (od cca 28 až po 58 hm.%) ve vzorcích. Také degradace filmu ve vode pri 37 °C probíhala dle predpokladu rychleji u vzorku s vyšší molekulovou hmotností PEG-u. V predložené diplomové práci bylo potvrzeno, že množstvím a molekulovou hmotností polyethylenglykolu je možné rídit hydrolytickou stabilitu výsledných biodegradabilních polyuretanových elastomeru.

Předkládaná dizertační práce se zabývá novým způsobem přípravy biodegradabilních polyuretanů (PU) a jejich modifikací biologicky aktivními celulózovými nanokrystaly. Literární rešerše se zaměřuje na bioresorbovatelné PU v tkáňovém inženýrství. Shrnuje příklady těchto PU elastomerů, skafoldů (nosičů buněk) i injektovatelných PU společně se způsoby biodegradace na netoxické produkty. Poslední část je zaměřena na nanocelulózu, která si získala pozornost díky svým pozoruhodným fyzikálním (velký specifický povrch, mechanické vlastnosti) a biologickým (biokompatibilita, biodegradabilita a nízká toxicita) vlastnostem jako materiál pro biomedicínu. V experimentální části byly charakterizovány amfifilní biodegradovatelné polyuretanové filmy (bio-PU) syntetizované bez použití rozpouštědla polyadiční reakcí z hydrofilního poly(ethylenglykolu) (PEG) a hydrofobního poly(e-kaprolaktonu) (PCL) jako makrodiolů společně s hexamethylen diizokyanátem. Připravené bio-PU filmy byly charakterizovány pro různé poměry jak mezi PEG/PCL, tak i mezi NCO/OH reagujícími skupinami (izokyanátový poměr). Bio-PU filmy projevily markantní nárůst mechanických vlastností při hmotnostním poměru PEG/PCL rovnému nebo menšímu než 20/80 díky vzniku krystalických domén PCL. Přítomnost PEGu zvyšovala schopnost bio-PU filmu absorbovat vodu i urychlila jeho hydrolytickou degradaci. Oproti tomu nižší absorpční schopnost a delší čas hydrolytické degradace materiálu způsobil vyšší izokyanátový poměr, a tedy i vyšší síťová hustota. Třetí část práce se zabývá přípravou polyuretanových nanokompozitů unikátní metodou bez použití rozpouštědla za využití bio-PU matrice a celulózových nanokrystalů buď nemodifikovaných, nebo povrchově roubovaných PEGem. Strukturní analýza prokázala, že přítomnost tyčinkovitých nanočástic způsobuje imobilizaci polymerních segmentů, v důsledku čehož se zvýšila tuhost a křehkost materiálu. Nastavením vhodného poměru mezi PEG/PCL, množstvím izokyanátu, či přídavkem modifikovaného nanoplniva může být bio-PU materiál "ušit na míru" s vhodnými mechanickými (houževnatost, tažnost) a fyzikálními (botnání, degradace) vlastnostmi. Díky přípravě bez použití rozpouštědla by mohly být připravené materiály využity v regenerativní medicíně např. jako cévní štěpy.

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Des copolymères à blocs polylactide-poly(éthylène glycol) (PLA-PEG) ont été synthétisés par polymérisation par ouverture de cycle du L- ou D-lactide en présence de PEG mono- ou dihydroxylé. La masse molaire, la composition, la cristallisation et les propriétés thermiques des copolymères ont été caractérisées à l'aide de la DSC, NMR, GPC, etc. Des micelles biorésorbables ont été obtenues par la méthode dissolution directe sans utiliser de solvants organiques. Des mesures de tension de surface ont permis de déterminer la concentration micellaire critique des copolymères. Les résultats montrent que les micelles mélange L/D sont plus stables que les micelles seules en raison de l'effet stéréocomplexation entre les blocs L-PLA et D-PLA. Les comportements d'agrégation des micelles en milieu aqueux ont été étudiés en détail. Il apparaît que les micelles mélange présentent un nombre d'agrégation inférieur à celui des micelles seules. La dégradation hydrolytique des micelles a été étudiée dans les conditions in vitro. Le paclitaxel a été utilisé comme modèle de principes actifs pour évaluer les comportements in vitro et in vivo des micelles. Les méthodes dissolutio n directe et dialyse conduisent à des résultats comparables en ce qui concerne l'efficacité d'encapsulation et la teneur en principe actif. Les micelles mélange L/D présentent une meilleure capacité d'encapsulation que les micelles seules. Les expériences in vivo montrent que le paclitaxel est distribué dans tous les organes avec concentrations élevées après injection des micelles chargées. En comparaison avec la formulation clinique et les micelles par dialyse, les micelles chargées en paclitaxel par dissolution directe présentent un effet antitumeur plus important. / A series of polylactide-poly(ethylene glycol) (PLA-PEG) block copolymers were synthesized by ring-opening polymerization of L- or D-lactide in the presence of mono- or dihydroxyl PEG. The molar mass, composition, crystallization and thermal properties were characterized by using DSC, NMR, GPC, etc. Bioresorbable micelles were obtained by direct dissolution method without using any organic solvents. Surface tension measurements were used to determine the critical micellar concentration of the copolymers. The results show that L/D mixed micelles are more stable than single ones due to strong stereocomplexation effect between L-PLA and D-PLA blocks. The aggregation behavior of micelles in aqueous medium was investigated in detail. It appears that mixed micelles present lower aggregation number than single ones. The hydrolytic degradation of the micelles was investigated under in vitro conditions. Paclitaxel was used as a model drug to investigate the in vitro and in vivo behavior of micelles as drug delivery system. The direct dissolution method yields comparable drug encapsulation efficiency and loading content as the traditional dialysis method. The drug encapsulation ability is higher for L/D mixed copolymer micelles than single micelles due to stereocomplexation. In vivo experiments show that paclitaxel is widely distributed and kept at high concentration levels in various tissues after administration of drug-loaded micelles. Compared with the current clinical formulation and micelles by dialysis, paclitaxel-loaded micelles by direct dissolution exhibit the highest antitumor ability.

Crystalline polymer/salt complexes can conduct, in contrast to the view held for 30 years. The alpha-phase of the crystalline poly(ethylene oxide)₆:LiPF₆ is composed of tunnels formed from pairs of (CH₂-CH₂-O)ₓ chains, within which the Li⁺ ions reside and along which the latter migrate.¹ When a polydispersed polymer is used, the tunnels are composed of 2 strands, each built from a string of PEO chains of varying length. It has been suggested that the number and the arrangement of the chain ends within the tunnels affects the ionic conductivity.² Using polymers with uniform chain length is important if we are to understand the conduction mechanism since monodispersity results in the chain ends occurring at regular distances along the tunnels and imposes a coincidence of the chain ends between the two strands.² Since each Li⁺ is coordinated by 6 ether oxygens (3 oxygens from each of the two polymeric strands forming a tunnel), monodispersed PEOs with the number of ether oxygen being a multiple of 3 (NO = 3n) can form either “all-ideal” or “all-broken” coordination environments at the end of each tunnel, while for both NO = 3n-1 and NO = 3n+1 complexes, both “ideal” and “broken” coordinations must occur throughout the structure. A synthetic procedure has been developed and a series of 6 consecutive (increment of EO unit) monodispersed molecular weight PEOs have been synthesised. The synthesis involves one end protection of a high purity glycol, functionalisation of the other end, ether coupling reaction (Williamson's type ether synthesis³), deprotection and reiteration of ether coupling. The parameters of the process and purification methods have been strictly controlled to ensure unprecedented level of monodispersity for all synthesised samples. Thus obtained high purity polymers have been used to study the influence of the individual chain length on the structure and conductivity of the crystalline complexes with LiPF₆. The results support the previously suggested model of the chain-ends arrangement in the crystalline complexes prepared with monodispersed PEO² over a range of consecutive chain lengths. The synthesised complexes constitute a series of test samples for establishing detailed mechanism of ionic conductivity. Such series of monodispersed crystalline complexes have been studied and characterised here (PXRD, DSC, AC impedance) for the first time. References: 1. G. S. MacGlashan, Y. G. Andreev, P. G. Bruce, Structure of the polymer electrolyte poly(ethylene oxide)₆:LiAsF₆. Nature, 1999, 398(6730): p. 792-794. 2. E. Staunton, Y. G. Andreev, P. G. Bruce, Factors influencing the conductivity of crystalline polymer electrolytes. Faraday Discussions, 2007, 134: p. 143-156. 3. A. Williamson, Theory of Aetherification. Philosophical Magazine, 1850, 37: p. 350-356.

Une série de copolymères à blocs amphiphiles polylactide/poly(éthylène glycol) (PLA/PEG) a été synthétisée par polymérisation par ouverture de cycles, chimie clic, ou couplage des homopolymères PLA et PEG avec des extrémités de chaînes fonctionnels (OH, COOH ou NH2). Différentes configurations de PLA (PLLA et PDLA) ont été considérées pour élucider l'effet de stéréocomplexation sur les propriétés physico-chimiques des copolymères. Les copolymères présentent différentes structures de chaînes, y compris dibloc PEG-PLA, tribloc PLA-PEG-PLA du type ABA ou PEG-PLA-PEG asymétrique du type BAB-type. La masse molaire des copolymères varies de 2 000 à 20 000, et le rapport EO/LA de 1.0 to 7.0 environ. Différents agrégats formés par auto-assemblage tels que des nanotubes, polymersomes, filomicelles, micelles anisotropiques et micelles sphériques ont été obtenus par dissolution directe de copolymères, mélange de différents copolymères, ou mélange de copolymères avec surfactant dans une solution aqueuse. Des modèles théoriques ont été proposés pour simuler la formation des agrégats et expliquer l'évolution de leur taille et morphologie en fonction du rapport EO/LA, longueur de bloc, etc. / A series of amphiphilic polylactide/poly(ethylene glycol) (PLA/PEG) block copolymers was synthesized by ring-opening polymerization, click chemistry, or coupling PLA and PEG homopolymers with functional end groups (OH, COOH or NH2). Different configurations of PLA (PLLA and PDLA) were considered to elucidate the effect of stereocomplexation on the physico-chemical properties of copolymers. The obtained copolymers exhibit diverse chain structures, including PEG-PLA diblock, PLA-PEG-PLA ABA-type or asymmetric PEG-PLA-PEG BAB-type triblock structures. The molar mass of copolymers ranges from c.a. 2,000 to 20,000, and the EO/LA ratio from c.a. 1.0 to 7.0. Diverse self-assembled aggregates, including nanotubes, polymersomes, filomicelles, anisotropic micelles and spherical micelles, were obtained by dissolving the resulting copolymers, mixing different copolymers, or mixing copolymers with surfactant in aqueous solution. Theoretical models are proposed to simulate the formation of aggregates and to explain the evolution of their size and morphology as a function of EO/LA ratio, block length, etc.

Nanofabrication techniques were used to manufacture nanometer scale protein templates. The fabrication approach employs electron beam lithography (EBL) patterning on poly(ethylene glycol) (PEG) thiol (CH3O(CH2CH2O)17NHCO(CH2)2SH) self-assembled monolayers (SAM) on Au. The PEG SAM prevented protein surface adhesion and binding sites for protein were created in the SAM by EBL. Subsequent to EBL, the patterns in the PEG SAM were backfilled with 40-nm NeutrAvidin-coated fluorescent spheres (FluoSpheres). The spontaneous and directed immobilization of the spheres from a solution to the patterns resulted in high resolution protein patterns. The FluoSpheres could be arranged in any arbitrary pattern with ultimately only one or a few FluoSpheres at each binding site. Growth dynamics and SAM morphology of PEG on Au were studied by atomic force microscopy (AFM). PEG SAMs on three types of Au with different microstructure were examined: thermally evaporated granular Au and two types of Au films produced by hydrogen flame annealing of granular Au, Au(111) and "terraced" Au (crystal orientation unknown). The different Au surfaces' substructure affected the morphology and mechanical properties of the PEG SAM. On Au(111), AFM imaging revealed monolayer formation through three distinct steps: island nucleation, island growth, and coalescence. The fine-structure of the SAM revealed dendritic island formation - an observation which can be explained by attractive intermolecular interactions and diffusion-limited aggregation. Island growth was not observed on the "terraced" Au. AFM studies of EBL patterned PEG SAMs on Au(111) revealed two different patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature depth is directly dose dependent. At higher doses electron beam induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. In addition to PEG SAMs, fibronectin monolayers on SiO2 surfaces were patterned by EBL. The areas exposed with EBL lose their functionality and do not bind anti-fibronectin. With this approach we constructed fibronectin templates and used them for cell studies demonstrating pattern dependent cell geometries and cell adhesion. / QC 20101008