Carbon dots-loaded layer-by-layer capsules as multifunctional nanotheranostic delivery systems to treat osteosarcoma

Abstract

Nanotheranostics, an emerging field which encompasses nanotechnology used theranostically, is the combination of diagnostics and therapeutic techniques. It exploits the unique properties of nanoparticles, particularly large surface area to volume ratio, optical and magnetic properties, low melting point and mechanical strength. Theranostic strategies have shown potential in monitoring targeted delivery of drugs. In this thesis, carbon dots were combined with the layer-by-layer (LBL) technique to create a platform for drug delivery that could be easily tracked via fluorescent imaging. This type of treatment has the potential to be less harsh and more effective than current treatments, such as chemo and radiotherapy which have very extreme side effects including pain, nausea, and hair loss. Specifically, carbon dots (synthesised from renewable and sustainable sources e.g. spent coffee ground, a food waste) are nanoparticles that have interesting physical and optical properties that make them a good candidate for fluorescent bioimaging, replacing conventional toxic, metal-based nanomaterials, whereas the LBL technique applies layers of alternately charged polyelectrolytes by self-assembly to create a thin film or nanocapsules. The technique is cheap, simple and has a high degree of control over layer size. Carbon dot (CD) syntheses were performed using hydrothermal processes with conventional and microwave assisted heating approaches. Hydrothermal processes are simple and non- hazardous using water as a process medium at subcritical conditions (180 and 300 °C, with corresponding pressures 10−88 bar) to synthesise CDs as they are simple to operate and do not require further modification steps. The effects of synthesising conditions such as pH, types of catalysts, ratio of feedstock material and water, reaction time and temperature on properties of CDs were examined. The surface properties of CDs were characterised with Fourier transform infrared (FT-IR) spectroscopy and X-Ray photoelectron spectroscopy (XPS). The results showed that the CDs had an increased ratio of oxygen (40-70%) against carbon and nitrogen, when compared to starting spent coffee grounds (~10%). This is due to the introduction of oxygen-containing functional groups to the surface of the carbon dots such as carboxylic acid and alcohol. The size of the CDs was between 1 and 14nm and was correlated with reaction conditions such as retention time and catalyst choice. Regardless of synthesising conditions, the CDs showed blue emissions between 300 and 450nm with UV (ultraviolet) excitation energies 250-300nm and quantum yields up to 13%. The CDs that were synthesised in tannic acid and aqueous conditions were chosen for biocompatibility testing (PrestoBlue), using fibroblast cells. The dots synthesised using aqueous conditions showed at least 80% cell viability with the highest concentration of 1mg/ml. These CDs are suitable for application in this work as imaging agents due to their optical properties and biocompatibility. For the development of the nanocapsule, calcium phosphate nanoparticles were chosen as a template because they are simple to synthesise and have a charged surface, meaning that they are easy to functionalise with a LBL technique (involving stacking layers of polyelectrolytes with alternating charges). This technique can be done in aqueous conditions at atmospheric temperature and pressure. The dipping technique was applied because it is fast and requires no specialised equipment. Initially LBL capsules were created using polyethyleneimine as the first layer followed by the main polyelectrolytes using renewable, non-toxic chitosan and alginate. The transmission electron microscopy (TEM), FT-IR and XPS showed the use of sodium acetate buffer partially dissolved the calcium phosphate core and destabilised the layers. Cell tests against Saos-2 and U2OS cell lines showed little therapeutic effect and encapsulation efficiency tests showed that drug encapsulation was unsuccessful, however incorporation of carbon dots into the layers showed potential in photoluminescence spectroscopy with a 380% increase in photoluminescence intensity when compared against nanoparticles with no carbon dots. A second LBL synthesis was attempted on poly(allylamine hydrochloride) (PAH) functionalised calcium phosphate nanoparticles using aqueous conditions and chitosan and chondroitin sulphate as the polyelectrolyte layers. These devices performed well in terms of drug encapsulation, release, and in-vitro cell tests against Saos-2 and U2OS cell lines where the nanoparticles showed particular therapeutic effect against Saos-2 cells. Incorporation of carbon dots had little detrimental effect on the performance of the particles further suggesting that the combination of carbon dots and the LBL technique should be a candidate for future research for the nanotheranostic treatment of osteosarcoma.