Three-dimensional engineered tissue models for experimental nanotheranostics

Nanotechnology is recognized as a powerful tool for investigation and manipulation of a wide of range of biological objects and processes in the life sciences and medicine. In particular, nanoscale structures of dual therapeutic and diagnosis modalities are scrutinised in an emerging discipline of nanotheranostics. Nanoparticles, carrying a payload of drugs or coupled with targeting biomolecules and at the same time detectable in living tissues, portray a typical nanotheranostics agent (NTA), which holds promise for advanced medical applications, such as combined visualization of the nanoparticles' transport or biodistribution with controllable drug delivery, or intrasurgical imaging with local treatment of the revealed pathological foci. The development path of nanotheranostics stems from the existing knowledge base of functional nanomaterial interactions with cells through to their relationship with biological systems at the level of tissues, organs and a whole body, leading to translational clinical research. In vitro two - dimensional (2D) cell cultures give overly simplistic biological context, oftentimes providing apocryphal extrapolations of the impact of functional nanocomplexes to the more complex biological systems. On the other hand, laboratory animals represent cumbersome and expensive models for NTA testing. Extension from the existing in vitro 2D cell cultures towards the more realistic biomimetic three - dimensional (3D) engineered tissues as testing systems for nanotheranostic particles, while avoiding the limitations of the laboratory animal models, represents the main motivation of this PhD project. This thesis addresses the development and validation of the novel 3D biomimetic models of living equivalents of human skin epidermis and metastatic breast cancer by using tissue engineering methodology. The feasibility of the developed tissue engineering constructs (TECs) as testbeds for evaluation of transport, cytotoxic and therapeutic effects of nanotheranostics particles was also investigated. Both normal and tumor TEC models were created by using in - house prepared acellular tissue engineering scaffolds, representing substitutes of organ - specific extracellular matrices of skin derma and liver, seeded with linear keratinocytes (HaCaT) and triple negative breast carcinoma cells (MDA - MB - 231), respectively. The biological effects and in - tissue behaviour of two types of nanotheranostics agents, upconversion nanoparticles (UCNPs) and mesoporous silica nanoparticles loaded with a fluorescent anti - cancer chemotherapeutic drug doxorubicin, were evaluated in these models. In a team of my colleagues, I carried out a systematic study of UCNPs, where these nanoparticles were synthesised and surface - modified with the most representative types of moieties and polymers. Comparative study of these surface - modified UCNPs in terms of their safety and biocompatibility were performed using in vitro 2D cultures of HaCaT keratinocytes and primary human skin fibroblasts. The results show the significant influence of the cell type, surface coating, concentration and exposure time on the overall toxic effects of UCNPs in 2D cell cultures, while predominantly acceptable levels of the cell tolerance to the tested nanomaterials were demonstrated in the tissue - engineered 3D counterpart model of multilayered epithelium. I analysed the validity and feasibility of TECs for modelling of early breast carcinoma metastases to secondary organs aiming for development a new testbed for experimental oncology and nanotheranostics. In particular, the specific patterns of cellular kinetics and histological changes associated with the metastatic colonisation of liver acellular scaffolds by aggressive linear breast cancer cells were observed. The rapid proliferation of cells on the scaffold surface at the initial stage of the cell growth, followed by an invasive stage of in - depth cell penetration prefaced by the cell - induced matrix remodelling was noted. In order to validate the model in terms of the ability to show the cancer - specific behaviour in vivo, breast cancer - liver TECs were grafted on chorioallantoic membranes of chick embryos and the significantly enhanced the angiogenic potential in comparison with that of cellular suspensions and acellular matrices, as controls, was demonstrated. Finally, the cytotoxic effects of free doxorubicin and mesoporous silica nanoparticles loaded with doxorubicin were assayed using MDA - MB - 231 cells' monolayer culture and the metastatic breast cancer TECs. The TECs appeared to support the greater resilience of cells to doxorubicin, despite its comparable cellular internalisation in both 3D and 2D culture models. Drug - loaded mesoporous silica nanoparticles were predicted to have an improved therapeutic efficacy, as benchmarked against that of free drugs in 3D models.