School of Chemistry - Theses

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    The interaction kinetics of a melittin derivative with a phospholipid membrane
    A deeper understanding about the lipid-peptide interactions contributes significantly to the development of drug delivery systems. The utilization of a model to scrutinize the lipid-peptide interactions helps to overcome the resistance of anti-microbial agents and the unselectiveness of the anti-cancer agents. Cytolytic peptides, the peptides that able to lyse various bacteria or mammal cells, become one of the anti-microbial agent and anti-cancer agent candidates to overcome those problems. A fluorescently labeled melittin derivative and 1,2 dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) were used as a model to study the lipid-peptide interaction. One of the cytolytic peptide, melittin, is an α-helical peptide which has 6 positive charge in physiological condition. Melittin is labeled with Alexa 430. Using the steady-state fluorescence spectroscopy and Fluorescence Lifetime Imaging Microscopy (FLIM), the information on the melittin microenvironment changes through the spectral characteristic and the lifetime of Alexa 430, can be monitored. DPPC SUVs behavior was observed through the Rayleigh light scattering intensity change. The data shows that the interaction between melittin with DPPC SUVs is dependent upon the lipid-peptide ratio. At lipid-peptide ratio of 100:1, or high lipid-peptide ratio, melittin is associated with the vesicle without vesicles size change. This is indicated by the increase of Alexa 430 lifetime and quantum yield with negligible light scattering change. At lipid-peptide ratios of 50:1, 40:1 and 30:1; or the medium lipid-peptide ratio, Alexa 430 lifetime and quantum yield raise significantly followed by the increase of light scattering that be sign of further melittin insertion accompanied by the vesicles fusion. The data implies the pore formation without involving melittin insertion to the hydrocarbon chain structure. At lipid-peptide ratios of 20:1 and 10:1, or the low lipid-peptide ratio, light scattering data shows a decrease in vesicle size, which is attributed to vesicles micellization by melittin. Furthermore, the different time scale of the kinetic progress parameters; the fluorescence intensity, the lifetime and the Rayleigh light scattering intensity; signify the multi steps process which takes place during the interaction. In high lipid-peptide ratio, melittin associate with the vesicles rapidly without further vesicles size changes. In medium lipid-peptide ratio, the data implies the formation of Toroidal-pore followed by slow vesicles fusion. Meanwhile, at low lipid-peptide ratio, membrane micellization occurs in a very short time indicating the existence of the Carpet model. However, the limitation of the observation time frame gives uncertainty whether Toroidal-pore and Carpet model is the most suitable model to illustrate the lipid-peptide interaction. Hence, this research proves that melittin disrupt the zwitterionic membrane through a complex mechanism depends on the lipid-peptide ratio which need further investigation.