Nanoparticles for the selective delivery of photosensitisers for photodynamic cancer therapy

Photodynamic therapy (PDT) requires a photosensitiser, light and oxygen to generate reactive oxygen species and cell death. The hydrophobicity of photosensitisers can be overcome using nanoparticles. Furthermore, nanoparticles can be functionalised with cancer-specific ligands to increase selectivity towards tumours. The aim of this thesis was to investigate the use of nanoparticles for PDT of breast cancer. Gold nanoparticles (AuNPs) were functionalised with polyethylene glycol (PEG) and a zinc phthalocyanine photosensitiser (Pc). Two Pcs differing in the length of the carbon chain that connects the Pc to the gold, three (C3Pc) or eleven (C11Pc) carbon atoms, were explored. Fluorescence emission intensity was higher for free C11Pc. Conversely, on the surface of the AuNPs, it was higher for C3Pc. The higher fluorescence emission intensity of C3Pc-PEG-AuNPs correlated with an increased production of singlet oxygen (1O2). SK-BR-3 cells internalised both nanosystems but cell death was enhanced with C3Pc-PEG-AuNPs (80 %), due to metal-enhanced fluorescence, as compared to C11Pc-PEG-AuNPs (10 %). The conjugation of the AuNPs with a breast cancer-specific antibody improved the internalisation and PDT efficacy of both nanosystems. The potential use of a carbohydrate, lactose, to target the galectin-1 receptor on breast cancer cells was studied. Lactose and either C3Pc or C11Pc were conjugated to AuNPs. Two breast cancer cell lines, SK-BR-3 and MDA-MB-231, were used. While lactose-C11Pc-AuNPs only induced phototoxicity to SK-BR-3 cells, lactose-C3Pc-AuNPs induced effective PDT for both cell lines. Evidence of targeting galectin-1 was only observed for MDA-MB-231 cells. The use of upconverting nanoparticles (UCNPs) functionalised with the photosensitiser Rose Bengal (RB) for near-infrared PDT was investigated. Energy transfer between the UCNPs and RB upon excitation at 980 nm allowed the generation of 1O2. SK-BR-3 cells successfully internalised the UCNPs and induced effective PDT at 15 μg/mL, leading to minimal dark toxicity and effective cell death following irradiation

Towards optimisation of surface enhanced photodynamic therapy of breast cancer cells using gold nanoparticle-photosensitiser conjugates

Gold nanoparticles (AuNPs; ca. 4 nm) were synthesised and functionalised with a mixed monolayer of polyethylene glycol (PEG) and one of two zinc phthalocyanines (ZnPcs), the difference between the two molecules was the length of the carbon chain that connects the Pc to the gold core. The chain was composed of either three (C3Pc) or eleven (C11Pc) carbon atoms. The C11Pc photosensitiser displayed higher fluorescence emission intensity than the C3Pc in solution. By contrast, the C3Pc photosensitiser exhibited higher fluorescence when bound to the surface of the AuNPs than the C11Pc, despite the shorter carbon chain which was expected to quench the fluorescence. In addition, the C3Pc nanoparticle conjugates exhibited an enhancement in the production of singlet oxygen (1O2). The metal-enhanced 1O2 production led to a remarkable photodynamic efficacy for the treatment of human breast cancer cells

Photosensitiser functionalised luminescent upconverting nanoparticles for efficient photodynamic therapy of breast cancer cells

Photodynamic therapy (PDT) is a well-established treatment of cancer in which cell toxic reactive oxygen species, including singlet oxygen (1O2), are produced by a photosensitiser drug following irradiation of a specific wavelength. Visible light is commonly used as the excitation source in PDT, although these wavelengths do have limited tissue penetration. In this research, upconverting nanoparticles (UCNPs) functionalised with the photosensitiser Rose Bengal (RB) have been designed and synthesised for PDT of breast cancer cells. The use of UCNPs shifts the required excitation wavelength for the production of 1O2 to near infrared light (NIR) thus allowing deeper tissue penetration. The system was designed to maximise the production of 1O2via efficient Förster resonance energy transfer (FRET) from the UCNPs to the photosensitiser. Highly luminescent NaYF4:Yb,Er,Gd@NaYF4 core–shell UCNPs were synthesised that exhibited two main anti-Stokes emission bands at 541 and 652 nm following 980 nm irradiation. RB was chosen as the photosensitiser since its absorption band overlaps with the green emission of the UCNPs. To achieve efficient energy transfer from the nanoparticles to the photosensitiser, the functionalised UCNPs included a short L-lysine linker to attach the RB to the nanocore yielding RB-lysine functionalised UCNPs. The efficient FRET from the UCNPs to the RB was confirmed by luminescence lifetime measurements. The light emitted by the UCNPs at 541 nm, following excitation at 980 nm, generates the 1O2via the RB. Multi-photon and confocal laser scanning microscopies confirmed the internalisation of the RB-lysine-UCNPs by SK-BR-3 breast cancer cells. Cell viability studies revealed that the RB-lysine-UCNPs induced low dark toxicity in cells prior to PDT treatment. Importantly, following irradiation at 980 nm, high levels of cell death were observed in cells loaded with the RB-lysine-UCNPs. Cell death following PDT treatment was also confirmed using propidium iodide and confocal microscopy. The high drug loading capacity (160 RB/nanoparticle) of the UCNPs, the efficient FRET from the UCNPs to the photosensitiser, the high level of accumulation inside the cells and their PDT cell kill suggest that the RB-lysine-UCNPs are promising for NIR PDT and hence suitable for the treatment of deep-lying cancer tumours

A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162 ng mL-1 achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrates the effectiveness and robustness of the assay. The immunoassay presents a simple and cost-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations

Correction to: Cluster identification, selection, and description in Cluster randomized crossover trials: the PREP-IT trials

An amendment to this paper has been published and can be accessed via the original article

Implementing stakeholder engagement to explore alternative models of consent: An example from the PREP-IT trials

Introduction: Cluster randomized crossover trials are often faced with a dilemma when selecting an optimal model of consent, as the traditional model of obtaining informed consent from participant's before initiating any trial related activities may not be suitable. We describe our experience of engaging patient advisors to identify an optimal model of consent for the PREP-IT trials. This paper also examines surrogate measures of success for the selected model of consent. Methods: The PREP-IT program consists of two multi-center cluster randomized crossover trials that engaged patient advisors to determine an optimal model of consent. Patient advisors and stakeholders met regularly and reached consensus on decisions related to the trial design including the model for consent. Patient advisors provided valuable insight on how key decisions on trial design and conduct would be received by participants and the impact these decisions will have. Results: Patient advisors, together with stakeholders, reviewed the pros and cons and the requirements for the traditional model of consent, deferred consent, and waiver of consent. Collectively, they agreed upon a deferred consent model, in which patients may be approached for consent after their fracture surgery and prior to data collection. The consent rate in PREP-IT is 80.7%, and 0.67% of participants have withdrawn consent for participation. Discussion: Involvement of patient advisors in the development of an optimal model of consent has been successful. Engagement of patient advisors is recommended for other large trials where the traditional model of consent may not be optimal