Bouzinab, Kaouthar
(2021)
Apoferritin delivery of imidazotetrazine agents for targeted brain tumour therapy.
PhD thesis, University of Nottingham.
Abstract
Glioblastoma multiforme (GBM) is an aggressive grade IV astrocytoma. The standard of care for GBM includes surgery, radiotherapy and temozolomide (TMZ; DNA alkylating) chemotherapy. Poor TMZ accumulation at the tumour site, resistance and toxicity, limit the success of this treatment. Some modes of TMZ resistance includes drug efflux by P-glycoprotein 1 (Pgp), overexpression of O6-methylguanine DNA-methyltransferase (MGMT; removes cytotoxic O6-methylated guanine (O6-MeG) lesions), deficiency in base excision repair (BER; e.g. poly (ADP-ribose) polymerase (PARP); removes N7-methylguanine (N7-MeG) and N3-methyladenine (N3-MeA) lesions)) and/or deficiency in mismatch repair (MMR; leads to tolerance of O6-MeG lesions).
Herein, we employed a nano drug delivery system (DDS), to combat the limitations associated with TMZ. Apoferritin (AFt) has been investigated as a DDS for the delivery of various anti-cancer agents, due to its biocompatibility and its capacity to bind to transferrin receptor 1 (TfR1). TfR1 binding enables AFt (along with the drug load) to cross the blood brain barrier (BBB) and accumulate in iron hungry cancer cells that overexpress this receptor; minimising unwanted toxicity. Also, to thwart TMZ resistance, we utilised TMZ analogues such as N3-propargyl (N3P) and C8-thiazole (T25) analogues, which impart excellent anti-cancer activity irrespective of the MGMT and/or MMR status of cancer cells, and explored combinations of TMZ with inhibitors of MGMT (e.g., O6-Benzylguanine; O6-BeG) or PARP-1 (e.g., niraparib; NRP), to enhance the number of methylated lesions and cancer cell death.
Our encapsulation process via the ‘nanoreactor’ method garnered > 510 TMZ, N3P, T25 or O6-BeG molecules and > 80 molecules of NRP per AFt cage, with encapsulation efficiencies > 60%. In addition, the protein remained intact after test agent encapsulation, with comparable size, charge, and molecular weight to AFt alone (prior to encapsulation). Moreover, in vitro test agent release studies for AFt-TMZ, AFt-N3P and AFt-T25, at pH 7.4, demonstrated slower test agent release in the first 3 h compared to at pH 5.5.
In vitro cytotoxicity assays revealed intriguing results with AFt-TMZ, which demonstrated significantly lower GI50 values in TMZ resistant U373M (MGMT +ve; 50% growth inhibition (GI50) = 0.768 μM) compared to naked TMZ treatment (GI50 = 376 μM). Supporting studies demonstrated greater O6-MeG adducts, cell cycle perturbation and DNA double-strand breaks with AFt-TMZ compared to naked TMZ treatment. Additionally, environmental scanning electron microscopy (ESEM) and confocal microscopy revealed that GBM cells appeared more shrunken, with obvious blebbing (signs of apoptosis), following treatment with AFt-TMZ over TMZ. Furthermore, the T25 analogue demonstrated even greater potency when delivered inside AFt (in U373M: GI50 = 0.077 μM), as well as the combination of AFt-TMZ with AFt-NRP (in U373M: GI50 = 0.072 μM). Alone, AFt was shown to be non-toxic and imparted selective activity to the test agent, with greater anti-cancer activity seen in cancer over non-cancer cells. This was likely due to differences in TfR1 expression, where all cancer cells but the non-tumorigenic MRC-5 cells expressed TfR1.
In conclusion, test agents delivered by AFt demonstrated enhanced potency over test agent alone, in a selective and cancer specific manner. Tackling resistance with the use of TMZ analogues or inhibitors of MGMT or PARP-1 further potentiated the anti-cancer activity in TMZ resistant GBM. By overcoming TMZ resistance and toxicity, we aim to prolong patient’s survival and quality of life.
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