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Title: Studies of diffusion in cells using a self-fluorescent dendrimer
Author: Ruenraroengsak, Pakatip
ISNI:       0000 0004 2671 0705
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2008
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Dendrimer synthesis and potential applications of the lysine-based dendrimers as gene carrier systems and nano-fluorescent probes have been explored. An intrinsically fluorescent polylysine dendrimer (DM1), (Gly)(Lys)63(NH2)64, was synthesized by both Fmoc- (30% overall yield) and Boc-SPPS (64% overall yield) by divergent method and characterized by NMR, MS and RP-HPLC. Boc-SPPS is a recommended method for synthesizing this compound which provided higher yield and purer product. Diffusibility of the DM1 in various types of media and in cells has revealed the difference between in vitro and in vivo transport. In concentrated and more complex media (20-60%(v/v) glycerol solutions, HPMC and actin gels) when beyond the limits of PCS, the FRAP technique allowed the diffusion coefficients (D) of DM1 to be accessed. For DNA delivery, DM1, DM2 ((C18)(Lys)7(NH2)8) and DM3 ((C18)(Lys)7(NH2)7(RGD)1) were capable of condensing DNA. The DNA was better condensed by DM2 and DM3 possibly due to the synergistic effects between the electrostatic and hydrophobic interactions. At charge ratios of 20:1 (DM1) and 10:1 (DM2 and DM3) (+/-) a 4h incubation period produced optimal transfection. Confocal microscopy and luciferase assay indicated transfection efficiencies of the dendrimers in descending order: DM3 > DM2 > LPP(Lipofectamine-PlusTM) > DM1 > pDNA. As a nano-fluorescent probe (DM1), the method for studying the dynamic uptake of this fluorescent probe has been established and allowed the D values to be calculated. Values were found in the range from 5.99x10-11 cm2 s-1 (in SK/MES-1 cells) to 9.82x10-11 cm2 s-1 (in Caco-2 cells) for the dendrimer and 2.36x10-11 cm2 s-1 (in Caco-2 cells) for the dendriplexes. The difference implied variation in the intracellular architecture of the cell types and the effect of particle size. Using the DM1 with other fluorescent dyes the excitation/emission spectra of the dyes and the concentration used of the DM1 must be considered to avoid the contribution of the DM 1 in emission spectrum of the dyes. The uptake of both dendrimer and dendriplexes entailed endocytosis which was reduced by the effect of medium flow. The DM1 dendrimer clearly interacted with actin filaments via the electrostatic interaction as confirmed by the TEM, SEM and SDS-PAGE results. Actin formed reversible insoluble complexes with DM1 indicating none of chemical bonding within the complexes reinforced by results from Western blots. DM1 exhibited biphasic effects on the actin polymerisation process depending on its concentration. It displayed inhibitor and activator behaviour, respectively, at low and high concentration. It was concluded that DM1, DM2 and DM3 serve as potential tools for gene delivery whereas their unique architectures still allow them to be engineered for indefinite applications in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available