Tian, Bowen; (2010) Liposome-QD Hybrids & the Development of Targeted Theranostic Modalities. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The construction of theranostic nanoscale constructs integrating imaging and therapy into one unit has attracted enormous interest for various biomedical applications. Quantum dots have been explored as fluorescent probes for optical imaging with superior fluorescent properties to traditionally-used organic dyes. Liposomes are one of the most clinically established nanometer-scale delivery systems. In the present work, we report the construction of liposome-quantum dot (L-QD) hybrid systems (100 nm in diameter) and the engineering of multifunctional theranostic modalities for integrated targeted imaging and therapy against cancer. This was accomplished stepwise, as follows: (a) the incorporation of hydrophobic TOPO-capped, CdSe/ZnS QD (< 5 nm) into the lipid bilayer of liposomes; (b) the loading of doxorubicin (an anthracycline) using the osmotic gradient technique in the internal liposome aqueous phase; (c) the design of targeted antibody-PEG-DSPE (antibody-conjugated lipids) inserted into the liposome surface by the post-insertion technique. The resulting L-QD systems were systematically characterised by dynamic light scattering (DLS, size and zeta potential), atomic force microscopy (AFM), cryogenic electron microscopy (cryo-EM), fluorescent microscopy and surface plasmon resonance (SPR). Structural elucidation using AFM in air and water revealed that the incorporation of QD took place by hydrophobic self-association within the lipid membranes. To further equip L-QD hybrids with a therapeutic capability, doxorubicin (Dox) was loaded into L-QD using the osmotic gradient technique and loading efficiencies above 95%. Structural elucidation by cryo-EM showed that Dox formed crystal-like stuctures inside of L-QD, as reported for liposomes, and was also confirmed by AFM. MUC1 antibody-PEG-DSPE micelles were post-inserted into the L-QD surface and the specific binding to MUC1 receptors was evidenced by SPR, demonstrating irreversible binding to the epitope due to a multivalent effect. In biological studies, MUC1 antibody-targeted L-QD could target MUC1 positive cells (MCF7 and DU 145), leading to significant enhancement in cellular uptake compared to MUC1 negative cells (Calu6). Antibody-targeted, Dox-loaded L-QD hybrids significantly improved cell kill of MCF7 and DU 145 compared to Calu6. The IVIS camera was used to image QD fluorescence following intratumoral (i.t.) administration of L-QD and L-QD-Dox in DU145 tumours. Overall, this project has demonstrated a strategy for the engineering of a targeted theranostic system based on liposome-quantum dot hybrid vesicles for tumour imaging and therapy in vitro and in vivo.

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