Delivery of temozolomide and N3-propargyl analog to brain tumors using an apoferritin nanocage

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 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

Relaxation of mitochondrial hyperfusion in the diabetic retina via N6-furfuryladenosine confers neuroprotection regardless of glycaemic status

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.</p

Apoferritin delivery of imidazotetrazine agents for targeted brain tumour therapy

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

Chemosensitization of Temozolomide-Resistant Pediatric Diffuse Midline Glioma Using Potent Nanoencapsulated Forms of a N(3)-Propargyl Analogue

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O6-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether β-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ~630 vs ~21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC50 = 14.6 (Lip-N3P) vs 32.8 μM (N3P); DIPG-IV) and (IC50 = 101.8 (AFt-N3P) vs 111.9 μM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT+ DIPG-VI (100 μM) and mismatch repair deficient DIPG-XIX (50 μM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (89Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation

In search of effective therapies to overcome resistance to Temozolomide in brain tumours

Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease

Relaxation of mitochondrial hyperfusion in the diabetic retina via N6-furfuryladenosine confers neuroprotection regardless of glycaemic status

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.</p