Colpan, Reyhan Dilsu
(2025)
Theranostic apoferritin nanocages for brain tumours: dual delivery of temozolomide and lead sulfide quantum dots.
PhD thesis, University of Nottingham.
Abstract
Glioblastoma (GBM) is the most common and aggressive (grade 4) primary brain tumour, arising from malignant transformation of glial cells, a consequence of genetic and epigenetic alterations. Its global incidence is 3-5 cases per 100, 000 person-years. GBM remains a clinical challenge, a consequence of treatment resistance, poor delivery because of restrictive barriers such as the blood brain barrier (BBB), ATP-binding cassette proteins, and tumour localisation. Median survival, with standard of care treatment which includes temozolomide (TMZ), is ~16 months. However, TMZ chemotherapy is limited by toxicity, drug resistance, chemical properties of TMZ, and poor drug accumulation at the tumour site. Resistance mechanisms include overexpression of O6-methylguanine-DNA methyltransferase (MGMT), drug efflux transporters (P-glycoprotein (P-gp)), base excision repair (BER), and mismatch repair (MMR) deficiency.
GBM imaging is essential for tumour characterisation, localisation, and surgery, particularly in addressing GBM-specific challenges such as anatomical barriers and deeply located tumours. Therefore, deep tissue imaging with near infrared region II (NIR-II, 1000-1700 nm) rather than near infrared region I (NIR-I, 650-900 nm) has gained attention. In this context, quantum dots (QDs), particularly lead sulfide quantum dots (PbS QDs), can be used in NIR-II/ short wave infrared (SWIR) probes for cancer imaging applications due to their advantageous properties, including stable optical emission when compared to traditional fluorescent agents. However, their potential toxicity is the major limitation for their use in cancer imaging.
This PhD project investigated a novel theranostic formulation for the treatment and monitoring of GBM, addressing the need for new targeted drug delivery systems (DDS) using nanoparticles (NPs) to overcome both diagnostic and therapeutic limitations. Therefore, the combination of diagnostic and therapeutic approach in one construct, called as theranostics, has gained attention. Among various DDS, apoferritin (AFt) is a promising candidate for improving delivery to the brain by exploiting overexpression of transferrin receptor 1 (TfR1) on GBM and BBB endothelial cells. Herein, we developed a dual function theranostic platform based on horse spleen AFt nanocages co-encapsulated with TMZ and PbS QDs, AFt-PbS-TMZ, for simultaneous TfR1 targeted delivery of therapeutic and imaging agents. AFt-PbS-TMZ (12.1 ± 0.6 nm in diameter from high resolution transmission microscopy) demonstrated high encapsulation efficiencies (74.4 ± 11.25% for TMZ, one QD for each cage) and pH-dependent TMZ release (70% at pH 5.5, 33% at pH 7.4; after 24 h at 37°C).
In two-dimensional (2D) in vitro cultures, PrestoBlue (PB) assay results with AFt-PbS-TMZ demonstrated enhanced inhibitory effects with lower IC50 values (3.5 ± 0.1 µM for U373M, 8.5 ± 0.7 µM for U373V, 10.9 ± 2.7 µM for U87MG) for GBM cells, whereas non-cancerous MRC-5 fibroblasts and human astrocytes exhibited IC50 > 30 µM (p < 0.001) post-treatment with 6 days, demonstrating GBM selectivity related to higher TfR1 expression in GBM cells, confirmed by western blot and flow cytometry methods. In addition, AFt delivery overcame TMZ resistance-mediated by MGMT; AFt-TMZ and AFt-PbS-TMZ overwhelmed the MGMT suicide repair protein, rapidly depleting its expression in MGMT-positive U373M cells.
To enable more predictive translation of 2D culture results into in vivo models, three-dimensional (3D) in vitro GBM models were utilised. In 3D spheroid cultures, AFt-PbS-TMZ leveraged AFt’s co-delivery advantages to achieve enhanced inhibition, IC50 was 5.0 ± 0.5 µM, and it significantly (p < 0.0001) reduced spheroid volume over 6 days. This was likely due to enhanced TfR1 expression in spheroid cultures compared to 2D monolayers. In addition, deep tissue photoluminescence (PL) studies confirmed that PbS QDs signalling in U87MG spheroid cultures post-treatment with AFt-PbS-TMZ for 6 days and can be imaged in vivo.
In conclusion, these findings highlight the potential of AFt-PbS-TMZ as an inherent TfR1 targeted theranostic agent, combining GBM therapy and imaging abilities in a single nano-sized (12 nm) platform. The pH-responsive TMZ release and NIR-II emission from PbS QDs have a potential to address both diagnostic and therapeutic needs of GBM.
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