Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.563577
Title: Strategies for microsphere-mediated cellular delivery
Author: Cardenas-Maestre, Juan Manuel
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2011
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Abstract:
Amino-functionalised polystyrene microspheres are promising candidates as delivery systems due to their unique features, tunable surface functionalities, and controllable release of the cargo. Herein several strategies for the conjugation of biologically relevant cargoes to these microspheres and their biological evaluation are described. Firstly, dispersion and suspension polymerisation methods were applied for the synthesis of these devices. Subsequently, these polymeric particles were employed in multistep solid phase synthesis to conjugate a broad range of cargoes. The capability of the resulting constructs to cross the cell membrane and deliver the desired cargo was evaluated by flow cytometry and confocal microscopy. Additionally, the effect of these particles on cell viability was determined. Moreover a chemical strategy for dual fuctionalisation allowed the production of microspheres capable of carrying two cargos simultaneously (e.g. a biologically relevant cargo and a tracking fluorophore). Several strategies were used to transport biomolecules such as peptides and oligonucleotides inside cells. Cell-impermeable peptides with neuroprotective activity were conjugated to microspheres to facilitate their internalisation and they were efficiently delivered into neuroblastom cells (SH-SY5Y) without affecting their therapeutic activity. In addition, several microsphere-mediated oligonucleotide delivery strategies were investigated. As a first approach, siRNA was successfully attached to microspheres via thiol linkage or via electrostatic interaction (by formation of polycationated microspheres-siRNA microplexes). Using both strategies EGFP expression was efficiently down-regulated in cervical cancer cells permanently expressing EGFP (HeLa-EGFP) following beadfection. Additionally embryonic stem (ES) cells were beadfected with siRNA linked to microspheres by amide formation and essential transcription factors implicated in cell renewal and differentiation were successfully silenced, exceeding the silencing capabilities of commercially available lipofection products. Furthermore, a novel approach for the intracellular delivery of plasmid DNA was designed. Following an easy protocol for the linearisation and functionalisation of the plasmid DNA, this was covalently coupled to beads and cells were homogeneously ‘beadfected’. Finally, the coupling of fluorogenic substrates for caspase-3 to microspheres allowed the in situ monitoring and quantification of apoptotic process within cells. In conclusion, these small particles are excellent devices for the efficient intracellular delivery of a broad range of cargoes.
Supervisor: Sanchez-Martin, Rosario. ; Bradley, Mark. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.563577  DOI: Not available
Keywords: polystyrene ; intracellular delivery ; microspheres
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