Rozprawy doktorskie na temat „Gamma-sterilization”

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Laboratory analyses were conducted on synthetic iron oxides to assess the use of gamma (γ-) irradiation as an efficient sterilization technique to remove microorganisms present in natural bacteriogenic iron oxides (BIOS) and to determine if the technique induces mineralogical changes within the Fe-rich minerals. Fe-oxides (ferrihydrite, lepidocrocite, and goethite) were synthesized with and without alginate (as a proxy for exopolysaccharides) and microbial reductions were carried out using the bacterium Shewanella putrefaciens CN32. A total of 18 Fe-oxide minerals were subjected to microbial reduction to assess redox stability, alteration due to varying levels of gamma irradiation (0, 5, and 28 kGy), and the addition of the exopolysaccharide alginate. Iron reduction rates varied for each Fe-oxide with faster Fe (III) reduction rates observed for the amorphous poorly-sorted 2-line ferrihydrite and slower Fe (III) reduction for the more crystalline Fe-oxides lepidocrocite and goethite. There was no significant impact to the Fe (III) reduction rates due to gamma irradiation (p> 0.05), which was confirmed using a t-test for statistical variance between gamma irradiated samples. However, the addition of alginate enabled lepidocrocite and goethite to achieve maximum Fe (III) reduction by an average of 7 days faster when compared to the Fe-oxides synthesized without the exopolysaccharide.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

A esterilidade é um requisito indispensável para a maioria dos biomateriais. Devido à sensibilidade dos hidrogéis, a sua esterilização apresenta-se como um desafio, sobretudo quando estão em causa nanoestruturas ou a presença de fármacos. O objetivo deste trabalho foi estudar os principais efeitos de diversos métodos de esterilização nas propriedades de diferentes tipos de sistemas à base de hidrogel. Além dos métodos convencionais (calor húmido e radiação gama) foi aferida a aplicabilidade da esterilização por ozono, um processo que embora se revele recente e ainda em fase de desenvolvimento, não deixa, no entanto, de ser bastante promissor. Estudou-se uma formulação de hidrogel natural nanoparticulado à base de quitosano (nanogel), com um largo espectro de possíveis aplicações, e duas formulações de hidrogéis sintéticos para aplicações oftálmicas (modelos de lentes de contato convencional e da nova geração de silicone-hidrogel), com e sem fármacos incorporados. Avaliaram-se alterações nas principais propriedades vitais ao bom desempenho e integridade dos materiais. No caso do nanogel: tamanho médio de partícula, potencial zeta, índice de polidispersão, absorvância, morfologia, estrutura química e citotoxicidade. No caso dos hidrogéis sintéticos: intumescimento, propriedades ópticas, permeabilidade iónica, molhabilidade, morfologia e topografia, estrutura química, propriedades mecânicas, citotoxicidade e perfil de libertação dos fármacos. Para garantir a melhor eficácia dos métodos de esterilização, realizaram-se testes de esterilidade após a contaminação intencional das amostras com diferentes cargas dos respetivos indicadores biológicos. A esterilização por calor húmido revelou-se o método mais agressivo, no caso do nanogel, e o menos agressivo para os hidrogéis sintéticos, quer na presença quer na ausência de fármacos. Relativamente à irradiação gama, para o nanogel, verificou-se que a sua resistência à irradiação aumenta consideravelmente na presença de açucares protetores. Já os hidrogéis sintéticos mostram-se resistentes à técnica, apresentando sinais de degradação evidente apenas para a dose mais elevada (25 kGy). Na presença de fármacos, a irradiação gama mostrou-se inadequada devido à sensibilidade dos mesmos. Os resultados obtidos são promissores no que diz respeito à aplicabilidade da esterilização por ozono, tanto para o nanogel como para os hidrogéis sintéticos, sem fármacos incorporados. Os fármacos sofrem, na generalidade, degradação quando expostos a este agente oxidante. Em suma, face à evidente complexidade dos fatores envolvidos (e.g. natureza, composição e propriedades dos materiais, estabilidade dos fármacos, condições e parâmetros dos processos esterilização), torna-se difícil generalizar os efeitos e prever o resultado dos métodos de esterilização. A escolha do processo mais adequado deve, portanto, ser feita caso a caso.
Sterility is mandatory requirement for most biomaterials. Because of their known sensitivity, hydrogel sterilization poses as a challenge, particularly when it comes to nanostructures or when drugs are incorporated. The main goal of this work was to study the key effects of different sterilization methods on the properties of different types of hydrogel-based systems. In addition to the conventional methods (steam heat and gamma radiation) the applicability of a promising new ozone method was assessed. Two formulations of synthetic hydrogels for ophthalmic applications (representing conventional contact lenses and new generation silicone-hydrogel contact lens), unloaded and loaded with ophthalmic drugs, were studied; and a natural chitosan-based nanostructured hydrogel (nanogel), with a broad spectrum of possible applications. Changes in the essential properties were evaluated. For synthetic hydrogels: swelling, optical properties, ionic permeability, wettability, morphology and topography, chemical structure, mechanical properties, cytotoxicity and drug release profile. For the nanogel: average particle size, zeta potential, polydispersity index, absorbance, morphology, chemical structure and cytotoxicity. In order to ensure the effectiveness of the sterilization methods, sterility tests were carried out after purposely contaminating the samples with different loads of biological indicators. Seam heat sterilization proved to be the most aggressive method for the nanogel, while being the least aggressive for the synthetic hydrogels in the presence and absence of drugs. Regarding gamma irradiation, the nanogel resistance to irradiation increased considerably in the presence of protective sugars. The synthetic hydrogels were resistant to this technique, showing signs of severe degradation only at the highest dose (25 kGy). In the presence of drugs, gamma irradiation proved to be inadequate due to drug degradation. The obtained results were encouraging with regard to the applicability of ozone sterilization for both the nanogel and the unloaded synthetic hydrogels. The drugs generally undergo degradation when exposed to this oxidizing agent. In view of the evident complexity of the factors involved (e.g. nature, composition and properties of materials, drug stability, conditions and parameters of sterilization processes), it is difficult to generalize the effects and predict the outcome of the sterilization methods. The selection of the most suitable procedure must be made on a case-by-case basis.

Em 1967, uma carta enviada ao British Medical Journal relatou que a re-esterilização em Óxido de Etileno (EO) do Cloreto de Polivinil (PVC) previamente gamairradiado forma grandes quantidades de etileno cloridrina, um composto altamente tóxico. Mesmo não apresentando dados mensurados, esta carta iniciou uma polêmica que até hoje divide a opinião de pesquisadores. Dirimir esta dúvida é relevante, uma vez que produtos de PVC esterilizados em Radiação Gama são habitualmente reesterilizados em EO em caso de vencimento do período de validade da esterilização, que é determinado pelo fabricante. Este estudo objetivou evidenciar a toxicidade de tubos de PVC por meio do teste de citotoxicidade pelo método da difusão em ágar em culturas celulares NCTC clone 929. Foram criados quatro grupos experimentais com 81 unidades de análise: G1, constituído de tubos in natura; G2 constituído de tubos esterilizados em Radiação Gama; G3 constituído de tubos esterilizados em EO e G4 constituído de tubos esterilizados em Raios Gama e re-esterilizados em Óxido de Etileno. Os testes foram realizados em triplicata e cada tubo foi testado de forma a representar as superfícies internas, externas e massa. Após a mensuração do halo incolor, as unidades de análise foram graduadas de acordo com os graus de reatividade biológica descritos na norma ISO 10993-5:2009. Foram consideradas citotóxicas apenas as unidades análise que obtiveram grau três ou acima. Os resultados revelaram toxicidade celular apenas no grupo G3, no qual foram observadas placas com morte de todas as células, fato que demandou repetições, conforme a metodologia adotada. Na primeira repetição houve persistência de unidades de análise capazes de causar a morte de todas as células da placa. Na segunda repetição, nenhuma unidade de análise foi classificada como citotóxica. Inferiu-se que houve falhas no processo de aeração no grupo G3 e na primeira repetição. O resultado da cromatografia gasosa dos grupos G3 e G4 atestou que os materiais de ambos os grupos estariam seguros para uso. O valor máximo dos resíduos encontrados em ambos os grupos foi idêntico, contradizendo os resultados do teste de citotoxicidade. Concluiu-se que, nas condições deste experimento, os materiais de PVC esterilizados em Radiação Gama e, consecutivamente, reesterilizados em EO não são citotóxicos e a cromatografia gasosa, utilizada isoladamente, pode produzir resultados questionáveis quanto à segurança dos materiais esterilizados em EO.
In 1967, a letter sent to the British Medical Journal reported that the re-sterilization with Ethylene Oxide (EtO) of previously irradiated Polyvinyl Chloride (PVC) formed large amounts of ethylene chlorohydrin, a highly toxic compound. Even though it did not present measured data, this letter initiated a controversy that still divide the opinion of researchers to date. To solve this doubt is relevant, as PVC products submitted to gamma radiation are usually re-sterilized with EtO, when the sterilization validity has expired, which is determined by the manufacturer. The present study aimed at assessing the toxicity of PVC tubes through the cytotoxicity test, using the NCTC clone 929 cell culture agar diffusion test. Four experimental groups were created with 81 analysis units: G1, consisting of tubes in natura; G2, consisting of tubes submitted to gamma radiation; G3, consisting of tubes submitted to EtO sterilization and G4, consisting of tubes submitted to gamma-radiation and re-sterilized with EtO. The tests were carried out in triplicate and each tube was tested in order to represent its internal and external surfaces, as well as its mass. After the measurement of the colorless halo, the analysis units were graded according to the degrees of biological reactivity described in the ISO 10993-5:2009. Only the analysis units that were considered grade 3 and above were considered cytotoxic. The results showed evidence of cell toxicity only in G3, which disclosed plaques that presented death of all cells, a fact that necessitated repetition of the experiment, according to the adopted methodology. The first repetition showed the persistence of the analysis units to cause the death of all cells in the plaque. At the second repetition, none of the analysis units was classified as being cytotoxic. It was inferred that there were failures in the process of aeration in group G3 and in the first repetition. The results of the gas chromatography of groups G3 and G4 demonstrated that the materials from both groups would be safe for use. The maximum value of residues found in both groups was identical, in disagreement with the cytotoxicity test results. It was concluded that, according to the conditions of this experiment, the PVC materials submitted to gamma-radiation and consecutively sterilized by EtO are not cytotoxic and that the gas chromatography, when used alone, can yield debatable results regarding the safety of materials sterilized with EtO.

A radiação gama é uma das tecnologias aplicadas para a esterilização de sistemas de embalagens contendo produtos para a saúde. No processo de esterilização, é fundamental que as propriedades das embalagens sejam mantidas. Neste estudo, duas amostras de embalagens comerciais, no formato de envelope compostas por papel grau cirúrgico de um dos lados e filme plástico laminado do outro, foram irradiadas com raios gama com doses de 25 kGy (taxa de dose de 1,57 kGy/h) e 50 kGy (taxa de dose de 1,48 kGy/h). Uma das amostras de embalagem era constituída por papel de fibras de coníferas e filme plástico laminado de poli(tereftalato de etileno) (PET)/polietileno (PE) e a outra por papel de fibras de coníferas e de folhosas e filme plástico laminado de poli(tereftalato de etileno) (PET)/polipropileno (PP). Os efeitos da radiação nas propriedades físicas e químicas dos papéis e dos filmes plásticos, assim como nas propriedades da embalagem foram estudados. O papel foi o material mais sensível à radiação, sendo a alvura, o pH e as resistências ao rasgo, ao arrebentamento e à tração os parâmetros nos quais foram observadas maiores modificações, em ambas as amostras. Todavia, dos dois tipos de papel, o efeito foi mais pronunciado para a amostra com fibras de conífera e de folhosas. A porosidade dos papéis foi alterada com 50 kGy. No caso dos filmes plásticos, a propriedade com maior modificação foi a resistência à tração, em ambas as amostras. No caso das embalagens, a irradiação diminuiu a resistência da selagem. Os efeitos observados para a dose de 50 kGy foram em média mais pronunciados quando comparados com as modificações nos valores das amostras tratadas com 25 kGy, que é a dose usualmente empregada para esterilização de produtos para a saúde. A dosimetria deste estudo foi realizada nos materiais irradiados com 25 kGy, 40 kGy e 50 kGy, demonstrando sua importância à medida que a variação média para as três doses estudadas foi de 20 %.
Gamma radiation is one of the technologies applied for the sterilization of packaging systems containing products for health. During sterilization process it is critical that the properties of packages are maintained. In this study two samples of commercial pouch packaging comprised of surgical grade paper on one side and the other side multilayer plastic film were irradiated with gamma rays. The following doses were applied 25 kGy (1,57 kGy/h) and 50 kGy (1,48 kGy/h). One packaging sample was paper formed by softwood fibers and multilayer plastic film based on poly(ethylene terephthalate) (PET)/polyethylene (PE). The second type of paper sample was made by a mixture of softwood and hardwood fibers and multilayer plastic film based on polyethylene terephthalate (ethylene) (PET)/polypropylene (PP). The effects of radiation on the physical and chemical properties of papers and multilayer plastic films, as well as the properties of the package were studied. The paper was the more radiation sensitive among the studied materials and radiation effects were more pronounced at brightness, pH, tearing resistance, bursting strength and tensile strength. Nonetheless, worst comparatively effects were noted on the sample made by a mixture of softwood and hardwood fibers. The porosity of paper was enhanced by 50 kGy. In the case of plastic films, radiation effects on tensile strength was the most pronounced property for both samples. In the case of the packaging the sealing resistance decreased with radiation. The effects observed for the treatment at 50 kGy were more pronounced when compared to 25 kGy. This last is the dose which is usually applied to sterilize health products. A dosimetry study was performed during irradiation at 25 kGy, 40 kGy and 50 kGy and its importance may be reported by the average dose variation 20 %.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

In allogeneic bone transplantation, the transmission of viral and non-viral infectious pathogens is the most severe undesirable concomitant phenomenon. The investigations published were examined regarding the inactivating capacity of inactivation procedures that are presently performed in bone banks (peracetic acid/ethanol, gamma irradiation, moist heat) against clinically relevant pathogens (aiming at a virus titer reduction of at least 4 log10 TCID50/ml or titer reduction of non-viral micro-organisms of at least 5 log10 cfu/ml). In the suspension experiments, treatment with peracetic acid/ethanol (peracetic acid 2%, ethanol 96%, aqua ad iniectabilia 2:1:1, 4 hours, 200 mbar, agitation) achieved a titer reduction of > 4 log10 already after 5 minutes for a number of viruses (PSR, PV, BVDV). HIV-2 was also inactivated within 5 minutes below the level of detection ( 4 log10 TCID50/ml was only reached after 4 hours. The results mentioned could be confirmed in the carrier test (contaminated spongiosa cuboids used as ‘worst case’ scenario). In the suspension experiment as well as in the carrier test, the HAV titer was reduced after 4 hours by only 3.7 log10 and 2.87 log10, respectively. The preceding step of defatting the spongiosa tissue by chloroform/ethanol was validated using cell-associated HAV and showed an HAV titer reduction of 7 log10. In the investigations regarding non-viral pathogens, all test organisms were completely inactivated by more than 5 log 10 steps (cfu/ml). Gamma irradiation was the second procedure examined. D10 values (irradiation dose required to reduce 90% of the pathogen titers by one log10 step) that were determined in inactivation kinetics experiments (irradiation conditions: –30°C, 60Co source) corresponded to data published so far. In order to provide for maximal safety, an irradiation dose of 34 kGy was recommended for allogeneic bone transplants using BPV and a diaphysis model from human femurs. The ‘Marburg bone bank system’ was the third procedure examined (thermal disinfection, guaranteed temperature of at least 82.5°C for a minimum of 15 min) using centrally contaminated human femoral heads. All viruses were completely inactivated and their titer reduced by more than 4 log10 steps. Vegetative bacteria and fungi were also completely inactivated (>= 6 log10 in the supernatant). As expected, spores and spore-forming pathogens were not sufficiently inactivated and not inactivated, respectively (titer reduction of less than 2 log10 cfu/ml). However, the latter group can be disregarded, since femoral heads are procured in the operation room under sterile conditions and the following production process rules out a secondary contamination with spores. It could be shown in the investigations presented that all three procedures examined guarantee an inactivation of the viruses investigated according to the recommendations by the senior federal authorities. The three treatment procedures offer additional biosafety by a comprehensive inactivation of non-viral pathogens.