Polymer-Quantum Dot hybrid nanotheranostics for Osteosarcoma treatment

Abstract

Osteosarcoma is a type of cancer which makes up approximately 3% of cancers in children aged 0-19, most often diagnosed between people aged 10-30. The prognosis for survival can be up to 68% with a 5+ year survival rate, but as low as 27% if it has spread to other parts of the body. However, there are limitations in current therapy such as invasive and damaging characteristics towards non cancerous cells. This has driven the research field to the use of nanotherapy, an emerging field, in particular metal-based quantum dots (QDs), which are fluorescent nanoparticles exhibiting great potential in bioimaging. Despite this, these QDs still have limitations such as their toxicity upon accumulation inside the body (damaging human cells). Carbon-based quantum dots (CQDs) (size less than 10nm), exhibiting similar behaviour and properties (fluorescence) and bioimaging to conventional metallic QDs, have demonstrated as promising candidates due to their advantages such as low toxicity to human cells, excellent water solubility and exceptional photoluminescence which is required for bioimaging. The aim of this PhD project was to develop new nanotheranostics by synthesis of nanocapsules incorporating CQDs alongside cancer drugs that will target the cancer cells. This PhD focused on the conversion of the biomass waste such as chitin and walnut shell into CQDs and their subsequent application in the development of cancer drug containing nanocapsules for biomedical applications for osteosarcoma. The synthesis of CQDs from the biomass wastes was achieved by employment of a bottom-up 2-step method ‘pyrolysis-carbonization’, which generated CQDs with a size range of 2- 10nm and quantum yield (QY) of approximately 10%. Moreover, a novelty discovered was the employment of the bio-oil previously seen as the waste material of the pyrolysis step of this method, to generate CQDs which displayed all the characteristics that CQDs possess but had a QY of around 20% (a particle size distribution of 1.0-4.5nm for CQDs derived from chitin bio-oil and a particle size distribution of 2.0-6.5nm for CQDs derived from walnut shell bio-oil). The overall analysis of these CQDs confirmed the presence of the desired characteristics such as C=O and C=C bonds which give rise to p-p* interactions and ensure the CQDs emit fluorescence in the 200-300nm UV-Visible region. This analysis provided a significant breakthrough in the synthesis of CQDs by use of biomass waste, as the novel use of bio-oil to synthesise CQDs to increase the sustainability as well as the overall yield of the process. The utilisation of these CQDs helped to generate an overall nanocapsule by a process called Layer-by Layer (LbL). This entailed using the CQDs as the core of the capsule, and around the CQDs were layers of alternating charge, with the drugs Doxorubicin and Docetaxel (conventional utilised drugs in osteosarcoma therapy) incorporated in the layers. The morphology and size/composition were studied to test its compatibility with the cancer cells. This finding was significant due to the outcome that CQDs could be combined with the cancer drugs doxorubicin and docetaxel in a LbL process. These novel nanocapsules of size around 150-160 nm are hence deemed to be successful in biomedical applications as they successfully were employed in this process. The size of these nanocapsules was deemed to be small enough to target and accumulate into cancer sites whilst retaining the drugs and CQDs. The drug release of these nanocapsules and interaction with cancer cells was tested in vitro by analysing the actual drug released into physiological solution. Computational models were developed using COMSOL software and validated to predict the in vitro drug release, which when plotted to match the experimentally determined in vitro drug release behaviour, was further beneficial as it allowed for the simultaneous determination of the nanocapsules parameters. The experimentally determined drug release behaviour indicated a burst release in the first 24 hours upon interaction with the cells, akin to established nanocapsule behaviour, which was followed by a controlled drug release over a 28 day period enabling drugs to reach the intended target site. The design of these computational models enabled the study of the time of drug release from the nanocapsule, to be visualised and therefore the conditions of the nanocapsule which enable this release were scrutinised. This will assist future tests’ conditions on drug release be explored and predict the drug release behaviour based on the parameters. In vitro tests confirmed the interaction of these nanocapsules with cancer cells and showed successful drug release into these cells, with the confirmation of this interaction determined by simultaneous PrestoBlue and Live/Dead assays, which outlined the metabolic activity of the cells upon interaction with the nanocapsule. Not only that, but a fluorescence staining assay was carried out in order to detect the specific proteins within the cells that have interacted with the nanocapsule, and hence determine if the nanocapsule had performed its intended therapeutic action. The Live/Dead assay was made possible due to the fluorescence of the CQDs, establishing this nanocapsule as a feasible employer for cancer therapy. At each assembly stage, multiple analytical techniques verified the success of the synthesis of the overall nanocapsule. This work provides novel insights for furthering a cleaner safer cancer therapy by employing biomass waste with less waste accumulated and a higher yielding process. Not only that, the validation of employment of CQDs to generate a drug containing nanocapsule over traditional metal-based QDs, which have previously been employed in the synthesis of nanocapsules, further ensures that this is a cleaner safe cancer therapy system.