<p>Improved materials for implantation as intraocular lens (IOL) devices are needed to minimize the occurrence of posterior capsule opacification (PCO). In this work, novel polydimethylsiloxane (PDMS) loaded with matrix metalloprotease inhibitors (MMPI) were developed as model IOL materials.</p> <p>PDMS was chosen as silicones are currently used successfully as IOLs. Inhibitor release rates and amount of initial burst of drug-loaded PDMS could be controlled by changing solvent when loading into elastomer base, as well as drug loading method, and release buffer.</p> <p>Two lens epithelial cell lines were characterized for in vitro tests: FHL124 and HLE B3. These cell lines produce different combinations of extracellular matrix proteins when grown on various biomaterial surfaces. Significant differences between the two cell lines were observed both in collagen VIII and α-smooth muscle actin levels, both when cells were unstimulated, and as a result of epithelial to mesenchymal transition induced by treatment with transforming growth factor β2. FHL124 cells were selected in further tests due to their consistent expression of extracellular matrix components when exposed to different materials.</p> <p>Solutions of synthetic MMPI GM6001 and MMP 2/9 Inhibitor II, known to mitigate anterior subcataract formation, were released from PDMS and found to protect in a modest but significant way against protein profile changes and to delay migration. Due to the Zn²⁺ dependence of MMPs, chelators, including EDTA, TPEN and 1-10 phenanthroline were examined as alternative inhibitors. Only the latter was found to have a significant effect on cell migration rates in vitro. Sulfadiazine, due to its chemical resemblance to synthetic MMPI was determined to be the most efficient at reducing migration rates as well as to have the lowest toxicity.</p> <p>Overall, sulfadiazine was determined in this work to be a potentially effective solution to mitigating PCO. These results indicate that releasing MMPI molecules from PDMS as a model IOL is a promising way to mitigate aspects of PCO.</p> / Thesis / Doctor of Philosophy (PhD)

A presença de inibidores no plasma e soro de animais resistentes ao veneno de serpentes, assim como no plasma ou soro de serpentes imunes a seu próprio veneno, é amplamente conhecida. Um grupo destes inibidores são os inibidores de metaloproteases do veneno de serpente (SVMPs), eles têm sido caracterizados do ponto de vista físico químico, porém a falta de informações estruturais e termodinâmicas acerca da sua inibição sobre as SVMPs, tem permanecido como um grande obstáculo para sua aplicação terapêutica ou desenvolvimento como ferramentas moleculares. Por tanto, isolamos um novo inibidor de metaloproteases a partir do soro de C. d. terrificus, por nós denominado crotaini, e realizamos a caracterização cinética de sua interação com a bothropasina, uma das principais metaloproteases do veneno de B. jararaca, e da mesma forma, caracterizamos cineticamente a interação desta metaloprotease com dos inibidores já conhecidos, DM40 e DM43, isolados do soro de D. marsupialis. Determinando as constantes cinéticas mediante cinética enzimática e ressonância plasmônica de superfície (SPR) no Biacore T200. Os valores obtidos permitiram estabelecer que a interação entre o crotaini e a bothropasina, é de alta afinidade, além de evidenciar a sua ligação de forma equimolar e estável. Assim mesmo, o crotaini apresentou uma constante de inibição menor às constantes determinadas para o DM40 e DM43. Adicionalmente, estudos de interação após tratamento quelante da enzima permitem prever que existem diferenças respeito aos domínios estruturais envolvidos na interação do crotaini com a bothropasina, em comparação aos inibidores DM40 e DM43. / The presence of inhibitors in plasma or serum from snake venom - resistant animals is well known. These inhibitors are also present in plasma and serum of snakes that are immune to their own venom. A group of these molecules are snake venom metalloprotease inhibitors (SVMPIs). Although the physicochemical properties of these inhibitors are well established, the lack of structural and thermodynamic information has been a bottleneck for their therapeutic use and development as molecular tools. In this work a new metalloprotease inhibitor was isolated from C. d. terrificus, and named crotaini. The kinetic interaction of crotaini with bothropasin, one of the main metalloproteases from the B. jararaca venom, was investigated. Similarly, the kinetic interactions of crotaini with the inhibitors DM40 and DM43, isolated from D. marsupialis serum, were also studied. The kinetic constants were determined by enzymatic kinetics and surface plasmonic resonance (SPR) in a Biacore T200. The obtained values enabled us to demonstrate the high affinity of the inhibitors toward the metalloprotease, achieved through an equimolar and stable complex formation. In addition, studies of interactions of the metaloprotease bothropasin with its inhibitors in the presence of chelant agents revealed differences between that established with crotaini as compared with those made with DM40 and DM43.