Cassioli, Maria Letizia
(2023)
Encapsulation of metal complex into apoferritin for drug delivery and prodrug activation.
PhD thesis, University of Nottingham.
Abstract
Cancer is one of the principal causes of mortality and the onset of new cases is estimated to increase from 19.3 million new cases in 2020 to 28.4 million by 2040 worldwide. Brain tumours represent a group of diseases characterised by high aggressiveness and poor prognosis. Glioblastoma represents 47% of cases and is currently treated with the anticancer agent temozolomide. This agent can induce tumour regression and promote patients’ survival but is characterised by low specificity and premature conversion into its metabolites causing severe side effects. Recent research has focused on the improvement of the tumour-targeted delivery of drugs such as temozolomide for anticancer chemotherapy exploiting nanocapsules. The protein shell apoferritin is biocompatible, biodegradable, non-immunogenic and pH-responsive and can be preferentially internalised by tumour cells. These features make apoferritin an ideal nanocarrier. However, when small molecules like temozolomide are encapsulated within apoferritin, they can easily leak through the interprotein channels that connect the core of the nanocage with the external environment. This problem can lead to reduced nanoparticle uptake into the brain, consequent decrease in drug efficacy and higher probability of undesired toxicity.
This work aims to exploit the natural affinity of apoferritin for transition metals and their complexes, to improve the retention of temozolomide inside apoferritin when exposed to physiological environments. It was hypothesised that the loading of apoferritin with both Cu2+-phenanthroline complex and temozolomide could promote the coordination of the metal and prevent the escape of the anticancer agent. Furthermore, Cu2+-phenanthroline itself is the precursor of a family of anticancer compounds currently under clinical evaluation. Therefore, Cu2+-phenanthroline has the potential to both prevent temozolomide leakage from apoferritin and contribute to the anticancer activity of the formulation. The work presented in this thesis evaluates the viability of this dual-drug delivery approach by using either horse spleen or human heavy chain apoferritin to achieve temozolomide delivery only in cancer cells and achieve enhanced anticancer activity. The metal affinity of the Apoferritin cage is essential to this approach. Horse spleen apoferritin is employed to evaluate the encapsulation of Cu2+-phenanthroline using the nanoreactor method and its effect on temozolomide loading and retention. With human heavy chain apoferritin, site-specific covalent conjugation of the Cu2+-phenanthroline complex has been undertaken before temozolomide encapsulation given the lower metal accumulation capacity of this apoferritin variant. The nano-formulations are characterised by UV-vis, high resolution transmission electron microscopy (HRTEM), inductively coupled plasma mass spectrometry (ICP-MS), Ellman’s assay, Matrix-assisted Laser Desorption/ionization Time of Flight (MALDI-TOF MS) and tested for in vitro anticancer activity by cell viability 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and clonogenic assays.
The encapsulation of Cu2+-phenanthroline into horse spleen apoferritin led to the formation of different copper(II) species some of them organised into crystal patterns visible by HRTEM and stable for up to 4 months. Upon loading of 339 temozolomide molecules per cage by nanoreactor route, the nano-formulation was able to protect the drug from degradation but the introduction of the metal complex had no effect on drug retention. In vitro results reported enhanced cytotoxicity of the apoferritin encapsulated agents with a 30-fold decrease in GI50 for glioblastoma cell lines compared to temozolomide treatment. The covalent conjugation of Cu2+-phenanthroline to human heavy chain apoferritin was challenging and robust confirmation of bioconjugation could not be achieved due the heterogeneity of the purified reaction mixtures. Consequently, no significant metal accumulation was observed even when 1,10-phenanthroline was detected. Temozolomide was entrapped within human apoferritin with encapsulation efficiency of 43 % of the total temozolomide added (moles) and drug loading of 12.9 % of the total nano-formulation weight (encapsulated drug and protein).
This work illustrates the potential of horse spleen apoferritin as an efficient drug delivery vehicle for the anticancer agent temozolomide, able to protect it from premature degradation and favour intracellular delivery. The anticancer activity of the metallodrug Cu2+-phenanthroline showed promising results for the treatment of glioblastoma multiforme alone and in combination with temozolomide. However, this application would require functionalisation of the apoferritin cage for precise tumour targeting, given the in vitro toxicity caused by the metallodrug to non-tumour cells. In summary, although none of the nano-formulations studied resulted in improved temozolomide retention into apoferritin, this work provided additional evidence to support the use of this protein nanocage as a valuable drug delivery tool.
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