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A presente tese teve como objetivo geral preparar, caracterizar e avaliar os efeitos antimicrobianos de um nanocarreador de miconazol (MCZ) à base de nanopartículas magnéticas de óxido de ferro (NPsMOF) funcionalizadas com quitosana (QS). Assim, dois subprojetos (SP1 e SP2) foram desenvolvidos e apresentaram os seguintes objetivos específicos: SP1) Preparar, caracterizar e avaliar os efeitos do nanocarreador NPsMOF-QS-MCZ sobre células planctônicas e biofilmes simples e misto de Candida albicans e Candida glabrata; SP2) Avaliar o efeito do nanocarreador na composição de três diferentes modelos de biofilmes polimicrobianos patogênicos orais (gengivite, prótese total e cárie dentária). A primeira etapa do SP1 consistiu em revestir as NPsMOF com QS e carregar este core-shell com MCZ, a fim de caracterizar este nanocarreador por métodos físico-químicos. As concentrações inibitórias mínimas (CIMs) do nanocarreador foram determinadas pelo método da microdiluição em caldo, usando o índice da concentração inibitória fracionária a fim de avaliar se houve interação sinergística entre os compostos. Ainda, biofilmes simples e mistos de Candida foram formados no fundo de placas de 24 ou 96 poços por 48 h e, em seguida, tratados por 24 h com NPsMOF-QS-MCZ carreando MCZ a 31,2 e 78 µg/ml, na presença e ausência de um campo magnético externo. Os biofilmes foram avaliados quantitativamente por biomassa total, atividade metabólica, contagem de unidades formadoras de colônias (UFCs) e composição da matriz extracelular. Os dados foram analisados por ANOVA a dois fatores, seguida pelo teste de Holm-Sidak (p<0,05). Ainda, a estrutura dos biofilmes foi avaliada qualitativamente por microscopia eletrônica de varredura e microscopia confocal. Os resultados do SP1 mostraram que o nanocarreador apresentou diâmetro menor que 50 nm e valores de CIM menores do que aqueles encontrados para MCZ sozinho, com efeito sinérgico sobre C. albicans. NPsMOF-QS-MCZ a 78 µg/ml foi mais eficaz que MCZ sozinho na redução de UFCs e atividade metabólica de biofilmes misto e simples de C. albicans. A biomassa total dos biofilmes e a matriz extracelular não foram afetadas pelo nanocarreador, e a aplicação de um campo magnético externo não melhorou seu efeito antibiofilme. As imagens de microscopia confirmam que tratamentos com o nanocarreador, principalmente na maior concentração, apresentaram maior atividade antibiofilme. Com relação ao SP2, as CIMs de NPsMOF-QS-MCZ foram determinadas para diferentes espécies microbianas, e todos os biofilmes polimicrobianos foram desenvolvidos por 5 dias e tratados por 24 h com NPsMOF-QS-MCZ a 64 µg/ml. Após o tratamento, os biofilmes foram avaliados quanto à biomassa total, atividade metabólica, contagem de UFCs e análise composicional por PCR quantitativo. Microscopia eletrônica de varredura foi usada para analisar a estrutura do biofilme. As diferenças entre os grupos foram determinadas por teste t não pareado (p<0,05). Os resultados do SP2 mostraram que NPsMOF-QS-MCZ foi mais eficaz que MCZ sozinho contra a maioria das células fúngicas e bacterianas testadas. Ainda, este nanocarreador foi capaz de reduzir a atividade metabólica, biomassa total e UFCs (p<0,05) dos biofilmes, além de alterar a sua ultraestrutura. Por fim, NPsMOF-QS-MCZ afetou a composição dos três biofilmes polimicrobianos avaliados, reduzindo principalmente os números de Streptococcus spp. e alterando a prevalência das espécies nos biofilmes. Em suma, os resultados dos SP1 e SP2 permitiram concluir que o nanocarreador melhorou o efeito antimicrobiano do MCZ, dependendo da espécie e parâmetro de biofilme avaliados. O nanocarreador também mostrou potencial para interferir nas interações sinergísticas entre células fúngicas e bacterianas dentro de biofilmes polimicrobianos(AU)
The present thesis aimed to prepare, characterize and evaluate the antimicrobial effects of a miconazole (MCZ) nanocarrier based on iron oxide magnetic nanoparticles (IONPs) functionalized with chitosan (CS). Thus, two subprojects (SP1 and SP2) were developed and had the following specific objectives: SP1) To prepare, characterize and evaluate the effects of the IONPs-CS-MCZ nanocarrier on planktonic cells and singleand dual-species biofilms of Candida albicans and Candida glabrata; SP2) To evaluate the effect of IONPs-CS-MCZ on the composition of three different models of oral pathogenic biofilms (gingivitis, denture and dental caries). The first step of SP1 was to coat IONPs with CS and to load this core-shell association with MCZ, in order to characterize this nanocarrier by physicochemical methods. The minimum inhibitory concentrations (MICs) of the nanocarrier were determined by the microdilution method, using the fractional inhibitory concentration index in order to assess whether there was synergistic interaction between the compounds.. In addition, single- and dual-species biofilms of Candida species were formed at the bottom of 24- or 96-well plates for 48 h and, in sequence, treated for 24 h with IONPs-CS-MCZ carrying MCZ at 31.2 and 78 µg/ml, in both the presence and absence of an external magnetic field. Biofilms were quantitatively evaluated by total biomass, metabolic activity, counting of colony forming units (CFUs) and extracellular matrix components. Data were analyzed by twoway ANOVA, followed by Holm-Sidak test (p <0.05). In addition, the structure of biofilms was qualitatively evaluated by scanning electron microscopy and confocal microscopy. The results from SP1 showed that IONPs-CS-MCZ presented diameter smaller than 50 nm, and MIC values lower than those found for MCZ alone, with synergistic effect on C. albicans. Moreover, 78 µg/ml IONPs-CS-MCZ was more effective than MCZ alone in reducing CFUs and metabolic activity of single biofilms of C. albicans and dual-species biofilms. Total biofilm biomass and extracellular matrix were not affected by the nanocarrier, and the application of an external magnetic field did not improve the nanocarrier effects. Microscopy images confirm that treatments with the nanocarrier, mainly in the highest concentration, exhibited greater antibiofilm activity. Regarding SP2, the MICs of IONPs-CS-MCZ were determined for different microbial species, and all polymicrobial biofilms were developed for 5 days and treated for 24 h with IONPs-CS-MCZ at 64 µg/ml. After treatment, the biofilms were evaluated for total biomass, metabolic activity, counting of CFUs and quantitative PCR analysis. Scanning electron microscopy was used to analyze the biofilm ultrastructure. Differences between groups were determined by unpaired t-test (p<0.05). Results from SP2 showed that IONPs-CS-MCZ was more effective than MCZ alone against most fungal and bacterial cells tested. Moreover, this nanocarrier was able to reduce the metabolic activity, total biomass and CFUs (P<0.05) of the biofilms, besides altering their ultrastructure. Finally, IONPs-CS-MCZ affected the composition of the three evaluated biofilms, mainly reducing the numbers of Streptococcus spp. and changing the prevalence of species in the biofilms. From the results obtained by SP1 and SP2, it was possible to conclude that the nanocarrier improved the antimicrobial effect of MCZ, depending on the species and biofilm parameter evaluated. Nanocarrier also showed potential to interfere in the synergistic interactions among fungal and bacterial cells within polymicrobial biofilms(AU)
Subject(s)
Biofilms , Dental Prosthesis , Dental PlaqueABSTRACT
BACKGROUND: Iron oxide nanoparticles are a kind of magnetic nanomaterial. Their excellent characteristics make them more and more widely used in medicine, especially in bone tissue regeneration and repair. OBJECTIVE: To review the research progress and application of iron oxide nanoparticles in bone tissue repair and regeneration.METHODS: A computer-based search of CBM, CNKI, PubMed and Elsevier was performed for retrieving articles concerning iron oxide nanoparticles for bone tissue regeneration published from May 2004 to January 2018. The keywords were "bone repair; bone regeneration; iron oxide nanoparticles; magnetic nanoparticles" in Chinese and "IONPs; bone repair; bone regenerate; iron oxide nanoparticles; osteogenesis; stem cell" in English. RESULTS AND CONCLUSION: In recent years, magnetic nanoparticles have been widely used in medical research, such as targeted drug delivery, magnetic resonance imaging, local tissue thermotherapy and tumor therapy, biological separation and biological sensors. The magnetic nanoparticles commonly used at present are iron oxide nanoparticles. Studies have proven that iron oxide nanoparticles have a close relationship with stem cell homing, and they are able to carry drugs into a specific target area, promoting bone tissue regeneration and repair. Although initial results have been achieved in the research on iron oxide nanoparticles in promoting bone tissue repair, the mechanism by which iron oxide nanoparticles promote bone healing has not been fully elucidated, and most studies are only limited to basic research. Future basic and clinical studies on the mechanism of iron oxide nanoparticles in promoting bone tissue healing need to be strengthened.
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Objective To investigate the effect of intravenous ultrafine superparamagnetic iron oxide nanoparticles feraheme (generic name:ferumoxytol) on cerebral infarction volume and inflammatory response in mice with permanent middle cerebral artery occlusion.Methods Thirty C57BL/6J mice were divided into sham operation group,saline control group,and feraheme group by the random number table (n =10 in each group).A permanent right middle cerebral artery occlusion model was induced by the modified suture method in the saline control group and the feraheme group,and no suture was inserted into the mice of the sham operation group.The intervention was performed by tail vein injection at 24 h after modeling.The sham operation group and the feraheme group were injected with 18 mg/kg feraheme,and the saline control group was injected with the same volume of normal saline.The neurobehavioral scores were conducted at 24 h (before the feraheme or saline injection) and 48 h (before the MRI exam) after modeling.MRI scans were performed at 48 h after modeling,and the cerebral infarction volume was calculated according to T2-weighted imaging.After the end of the scan,orbital blood was collected for the detection of serum tumor necrosis factor (TNF)-α,interleukin (IL)-1 β,and IL-6 levels.Then,the mice were sacrificed and the brain tissue was taken for HE staining and Ibal immunohistochemical staining.Results There were no significant differences in the infarct volume and neurological function score between the saline control group and the feraheme group.The serum levels of TNF-α,IL-1β,and IL-6 in the saline control group and the feraheme group were significantly higher than those in the sham operation group (P ＜0.05),but there was no significant difference between the saline control group and the feraheme group.Conclusion Intravenous injection of 18 mg/kg feraheme at 24 h after cerebral ischemia did not affect the infarct volume and inflammatory response,suggesting that this dose of feraheme can be used for molecular imaging studies of inflammatory response after cerebral ischemia.
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Objective To investigate the influence of Fe3 04 magnetic nanoparticles on human keratinocyte cells (HaCaT cells). Methods HaCaT cells were incubated with different concentrations of Fe304magnetic nanoparticles for 4 h at 37°C with 5% C02. Then transmission electron microscopy (TEM) was used to observe the way nanoparticles entering HaCaT cells and the ultrastructure of HaCaT cells. Results The mean diameter of Fe3 04 magnetic nanoparticles was 12 nm. The nanoparticles of different concentrations could enter the HaCaT cells by phagocytosis. After entering the cells the particles were released from phagocytic vesicles and exerted influence on the nearby mitochondria, leading to mitochondria swelling and cristae dissolving. Conclusion Fe3 04 magnetic nanoparticles can damage the ultrastructure of mitochondria near the particles in HaCaT cells, and the effect is in a concentration-dependent manner.
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Objective To develop specific targeted magnetic biomarkers which can selectively mark the senile plaques in Alzheimer' s disease (AD) and verify its feasibility and validity.Methods Aβ1-40 peptide and Tat-PTD ( Tat-protein transduction domain) was binded with dextran-coated ultrasmall superparamagnetic iron oxide ( USPIO) particles.Visualization of plaques in vivo in Alzheimer transgenic mice was investigated at 7.0 Tesla using T2 sequences after intravenous administration of the targeted nanoiron contrast agent and verified by histological staining.Results The targeted nano-iron contrast agent could enter the cultured neural stem cells,and was able to accelerate T2 relaxation rates of water protons in the cells and negatively reinforce the T2 signal intensity in the labeled cells.Plaques were specifically detected in vivo by magnetic resonance imaging ( MRI) and correlated well with histological staining after injection of nano-iron contrast agent into the APP/PS1 mice.Conclusion The targeted nano-iron contrast agent has the ability of selectively labeling the senile plaques in AD brain tissues in vivo,which might enable the early detection of plaques by MRI and can be further applied in the studies of early diagnosis of AD.