Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546137
Title: An investigation of the use of dendrimer-based carrier to cross cellular barrier
Author: Teow, Hueyminn
Awarding Body: University of Central Lancashire
Current Institution: University of Central Lancashire
Date of Award: 2011
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Abstract:
Dendrimers are a novel class of highly branched polymers with a high degree of uniformity and monodispersity. As a result of the unique properties and characteristics of dendrimers, they have found a wide range of pharmaceutical applications. This study investigated the ability of polyamidoamine (PAMAM) dendrimer-based drug delivery systems to enhance the permeability of a drug that is a substrate of P-glycoprotein (P-gp) efflux transporter with low water solubility. This thesis described the syntheses, characterisations and biological evaluations of the dendrimer-based drug delivery system to overcome the cellular barriers. G3 PAMAM dendrimers were conjugated with lauryl chains via a carbamate bond. Paclitaxel, a low water solubility P-gp substrate and anti-tumour drug, was conjugated to G3 and/or lauryl-G3 PAMAM dendrimer conjugates via a glutaric anhydride linker. Unmodified G3 PAMAM dendrimer and all the dendrimer conjugates (lauryl-G3 and G3-drug conjugates) were labelled with fluorescein isothiocyanate (FITC) for quantitative detection by spectrofluorimetry in permeability studies. These conjugates were characterised using various techniques including thin layer chromatography, 1H and 13C NMR, ESI-MS and dynamic light scattering. Chemical stability studies showed that the tested dendrimer conjugate (FITC-G3L6-glu-pac) was stable at all test pHs (1.2, 7.4, and 8.5) after 48 h of incubation at 37oC. The ester bond of the conjugate was stable under several chemical conditions after 10 days of incubation. Biological evaluation of the dendrimer conjugates was initially conducted using Caco-2 cells. Lactate dehydrogenase (LDH) release assay showed that conjugation of lauryl chains and paclitaxel molecules on the surface of G3 PAMAM dendrimer significantly (p < 0.05) increased the cytotoxicity in Caco-2 cells. The permeation of G3 PAMAM dendrimer and drug conjugates was investigated by measuring the apparent permeability coefficient (Papp) in both apical to basolateral AàB and BàA directions across Caco-2 cell monolayers at 37oC. The BàA Papp of paclitaxel was found to be significantly (p < 0.05) higher than the AàB Papp, indicating active function of P-gp efflux transporter system in the cell model. Covalent conjugation of paclitaxel to G3 PAMAM dendrimer via a glutaric anhydride linker significantly (p < 0.05) increased its AàB Papp through Caco-2 cell monolayers. A more pronounced increase of paclitaxel permeation was observed when surface modified G3 PAMAM dendrimers with six lauryl chains were used as the carrier. L6-G3-glu-pac was found with highest permeability across the Caco-2 cell monolayers. The results suggested that G3 PAMAM dendrimer-based drug delivery systems enhance the permeability of paclitaxel and bypass P-gp efflux transporter system in Caco-2 cell monolayers, thereby overcoming the intestinal barrier. Further biological evaluation was carried out using porcine brain endothelial cells (PBECs) as a blood-brain barrier (BBB) cell model to examine the potential of G3 PAMAM dendrimer as a carrier for paclitaxel (P-gp substrate) to bypass the BBB. Cell culture conditions of PBECs were monitored and examined to establish optimal conditions for cell growth. PBECs were successfully cultured with characteristic elongated spindle-like morphology. Integrity of the cell monolayers was evaluated by measuring the transendothelial electrical resistance (TEER) across the cell monolayers. Successfully cultured cell monolayers that achieved TEER values of higher than 200 Ω.cm2 were used for permeability studies. Elevation of TEER was observed when PBEC monolayers were cultured in the serum-free medium with hydrocortisone and treated with the cAMP/RO-20-1724 solution mixture. G3 PAMAM dendrimer was found to be relatively non-toxic to PBECs compared to all other conjugates. Conjugation of lauryl chains and paclitaxel molecules on the surface of G3 PAMAM dendrimer significantly (p < 0.05) increased the cytotoxicity in PBECs, as assessed by LDH assay. Permeability studies of paclitaxel across the PBEC monolayers showed a similar transport profile to that of Caco-2 cell. The significant higher BàA Papp of paclitaxel compared to the AàB Papp indicated active function of P-gp efflux transporter system in the cell model. The AàB Papp of L6-G3-glu-pac was found to be approximately 12 fold greater than that of free paclitaxel across the PBEC monolayers, where lauryl chains were acting as permeability enhancer. The cytotoxicity and permeability results using PBECs were found to be in good agreement with the findings when Caco-2 cells were used as the cellular barrier cell model. From these studies, it can be concluded that G3 PAMAM dendrimers-based drug delivery systems are potential nanocarriers for low solubility and P-gp substrate drug to bypass the P-gp efflux transporter system and overcome cellular barriers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.546137  DOI: Not available
Keywords: Q Science (General) ; R Medicine (General) ; QD Chemistry
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