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|Title:||Cellular interactive mechanisms of antimicrobial protein Bmattacin2 on bacteria and human cells||Authors:||Liu, Xuan||Degree:||Ph.D.||Issue Date:||2015||Abstract:||The goal of this research is to establish a theoretical framework and to study potential mechanisms of the interactions between a recombinant insect AMP (Antimicrobial peptides), Bmattacin2 and different organisms including bacteria and human normal cells and cancerous cells for the purpose of antimicrobial application and cancer treatment. Based on critical analysis of the molecular features of Bmattacin2, the potential functions and mechanisms of the interactions were hypothesized. To validate potential mechanism of how Bmattacin2 interacts with bacteria, a series of experiments were conducted to evaluate its antibacterial activity. A broth micro-dilution assay was adopted to determine the minimal inhibition concentration (MIC) of Bmattacin2 for Gram-positive and Gram-negative bacteria. The membrane integrity of bacteria was observed by scanning electron microscopy. The electrostatic attraction between Bmattacin2 and bacteria cell was examined in terms of zeta potential measurement. The experimental results revealed that Bmattacin2 expressed a broad antibacterial spectrum which is active in relation to both Gram-positive and Gram-negative bacteria. The MIC values for each category were both in the micro-molar range. The membrane structure of bacteria was severely disrupted, indicating that it might serve as a main target of attack by Bmattacin2. The neutralization of the negative surface charge of bacteria is correlated with the inhibition of bacterial growth. Secondly, the biocompatibility of Bmattacin2 in relation to normal human cells was assessed by employing a cell viability assay (MTS). Membrane integrity and intracellular structure of cells were observed by scanning electron microscopy and fluorescence microscopy. Zeta potential and transmembrane potential of cells in the absence and presence of Bmattacin2 were measured to evaluate the electrostatic interaction between AMP and human cell membrane. Results showed that Bmattacin2 is biocompatible for HFF-1 and FHC cells under 12μM. The surface and intracellular morphology of both cells were unaffected under the same dosage. Zeta potential of FHC cells were barely interrupted by Bmattacin2, while the surface charge neutralization of HFF-1 cells was triggered, indicating that the electrostatic attraction between HFF-1 cell and Bmattacin2 is not effective enough to initiate adverse effect.
Thirdly, similar methods have been adopted to examine the cellular interaction of Bmattacin2 with cancerous human cells. The cytotoxicity of Bmattacin2 exhibited significant differences between cancerous cells and normal cells from skin and colon tissue under a range of concentrations (2-12μM). Bmattacin2 (2μM) altered the surface morphology of cancerous cells while showing no effect on normal cells under the same concentration. Together with the interference of nucleus/cytoskeleton structures of cells, the above results indicated that cancerous cells are more sensitive to Bmattacin2 compared with normal cells. Zeta potential and transmembrane potential of cancer cells (HCT116 and A375) were measured consequently. Surface charge neutralization processes for both cancer cells were found more significant than their normal cell counterparts, implying that the electrostatic attraction between Bmattacin2 and cell membrane contributed to the anticancer activity of Bmattacin2. Apoptosis and intracellular cell cycle alteration of HCT116 cells under low concentrations further suggested an alternative killing pathway other than membrane attack. As Bmattacin2 is bioactive in relation to bacteria and human cells and will degrade when it is placed in vivo, the therapeutic effects could be seriously reduced in clinical application. Therefore, it would be desirable to encapsulate them in the fibers of scaffolds and provide a localized controlled release to the targeting cells in the surgical operational process. Cellular viability measurement and morphological observation demonstrated that PLLA/Bmattacin2 fibrous scaffolds expressed prolonged inhibition effect on the growth of HCT116 cancer cells while the growth of HFF-1 normal cells was not affected after long-term cultivation. Further, Bmattacin2 also showed a significant synergetic effect with 5-FU in inhibition of cancer cells. In conclusion, it was confirmed that the strong electrostatic attraction contributed to the cellular interactive mechanisms between Bmattacin2 and targeted cells. Due to the differences of cell membranes among bacteria, human normal cells and cancer cells, Bmattacin2 has a broad spectrum of antibacterial effect and anticancer activity, but minor effects on human normal cells, which shows promising potential biomedical applications in tissue engineering and cancer therapy.
|Subjects:||Anti-infective agents -- Testing.
Bacterial cell walls -- Synthesis -- Inhibitors.
Hong Kong Polytechnic University -- Dissertations
|Pages:||xix, 189 leaves : color illustrations ; 30 cm|
|Appears in Collections:||Thesis|
View full-text via https://theses.lib.polyu.edu.hk/handle/200/7991
Citations as of May 28, 2023
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