Delivery of Temozolomide and N3-Propargyl Analog to Brain Tumors Using an Apoferritin Nanocage.

Kaouthar Bouzinab,Pavel Gershkovich,Lyudmila Turyanska,Malcolm F G Stevens,Neil R Thomas,Nicola Weston,Tracey D Bradshaw,Marianne B Ashford,Christopher J Moody,Helen S Summers

doi:10.1021/acsami.0c01514

Abstract

Glioblastoma multiforme (GBM) is a grade IV astrocytoma, which is the most aggressive form of brain tumor. The standard of care for this disease includes surgery, radiotherapy and temozolomide (TMZ) chemotherapy. Poor accumulation of TMZ at the tumor site, tumor resistance to drug, and dose-limiting bone marrow toxicity eventually reduce the success of this treatment. Herein, we have encapsulated >500 drug molecules of TMZ into the biocompatible protein nanocage, apoferritin (AFt), using a "nanoreactor" method (AFt-TMZ). AFt is internalized by transferrin receptor 1-mediated endocytosis and is therefore able to facilitate cancer cell uptake and enhance drug efficacy. Following encapsulation, the protein cage retained its morphological integrity and surface charge; hence, its cellular recognition and uptake are not affected by the presence of this cargo. Additional benefits of AFt include maintenance of TMZ stability at pH 5.5 and drug release under acidic pH conditions, encountered in lysosomal compartments. MTT assays revealed that the encapsulated agents displayed significantly increased antitumor activity in U373V (vector control) and, remarkably, the isogenic U373M (MGMT expressing TMZ-resistant) GBM cell lines, with GI50 values <1.5 μM for AFt-TMZ, compared to 35 and 376 μM for unencapsulated TMZ against U373V and U373M, respectively. The enhanced potency of AFt-TMZ was further substantiated by clonogenic assays. Potentiated G2/M cell cycle arrest following exposure of cells to AFt-TMZ indicated an enhanced DNA damage burden. Indeed, increased O6-methylguanine (O6-MeG) adducts in cells exposed to AFt-TMZ and subsequent generation of γH2AX foci support the hypothesis that AFt significantly enhances the delivery of TMZ to cancer cells in vitro, overwhelming the direct O6-MeG repair conferred by MGMT. We have additionally encapsulated >500 molecules of the N3-propargyl imidazotetrazine analog (N3P), developed to combat TMZ resistance, and demonstrated significantly enhanced activity of AFt-N3P against GBM and colorectal carcinoma cell lines. These studies support the use of AFt as a promising nanodelivery system for targeted delivery, lysosomal drug release, and enhanced imidazotetrazine potency for treatment of GBM and wider-spectrum malignancies.

Highlights

Glioblastoma multiforme (GBM), a grade IV astrocytoma, is the most prevalent and aggressive adult central nervous system tumor, presenting heterogeneous, highly angiogenic, invasive, and migratory characteristics.[1−4] GBM cells infiltrate healthy areas of the brain and are surrounded by a blood−brain tumor barrier and blood−brain barrier (BBB).[5]
transferrin receptor 1 (TfR1) has been shown to be overexpressed by GBM and present on BBB endothelial cells, but not peripheral endothelium.[30]
We evaluated the loading of TMZ into AFt for GBM targeting, with the goal to achieve enhanced transport of the molecules across the BBB, delivery to, and accumulation within cancer cells

Summary

Introduction

Glioblastoma multiforme (GBM), a grade IV astrocytoma, is the most prevalent and aggressive adult central nervous system tumor, presenting heterogeneous, highly angiogenic, invasive, and migratory characteristics.[1−4] GBM cells infiltrate healthy areas of the brain and are surrounded by a blood−brain tumor barrier and blood−brain barrier (BBB).[5]. TMZ therapy causes dose-limiting bone marrow toxicity, presenting an additional barrier to successful treatment.[10]. Intracellular drug resistance mechanisms further exacerbate efficacy. The active methyldiazonium cation is released and reacts with DNA purine bases, methylating N3-adenine, O6-, and N7-guanine.[11] O6-Methylguanine (O6-MeG) is the most cytotoxic product produced.[12,13] The mechanism of action of TMZ has been established.[14−16] It is accepted that deficiency in DNA mismatch repair (MMR) leading to Received: January 25, 2020 Accepted: February 19, 2020 Published: February 19, 2020

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Results

Conclusion