Investigating potential nano- and micro-drug delivery systems toward the non-invasive treatment of glioblastoma brain tumours

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Habra, K. ORCID: 0000-0001-8272-6048, 2023. Investigating potential nano- and micro-drug delivery systems toward the non-invasive treatment of glioblastoma brain tumours. PhD, Nottingham Trent University.

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Abstract

Glioblastoma is one of the most aggressive and fatal brain tumours and is uncurable in most cases. The complete removal of glioblastoma brain tumours (GBM) is impossible by surgery alone. Despite aggressive chemotherapy and radiotherapy treatments following surgery, tumour cells continue to grow, leading to the death of patients within 15 months after diagnosis. The carnosine dipeptide is an attractive option for treating GBM, with growing numbers of studies now demonstrating its tumour accessibility, resulting in improved survival in pre-clinical GBM models. Several attempts at carnosine treatments have been developed and tested in GBM patients, however, these trials have not progressed due to the short lifetime of carnosine as a result of its enzymatic degradation in the presence of the naturally occurring carnosinase enzyme in the brain. This project aims to investigate the potential of using nano- and micro-drug delivery systems for non-invasive treatment of GBM for applying carnosine as a complementary therapy.

Carnosine was successfully embedded within a carrier that can be externally triggered to release its full oncological treatment potential of the dipeptide in situ. The drug delivery device was comprised of novel nano-rod-shaped superparamagnetic iron oxide nanoparticles (ca. 86 × 19 × 11 nm) capped with a branched polyethyleneimine, capable of loading carnosine. The therapeutic agent was released by the drug delivery carrier in the presence of heat or a rotating magnetic field, as an external trigger. The new drug delivery system was characterised using electron microscopy, dynamic light scattering, elemental analysis, and magnetic resonance imaging (MRI) techniques. In addition, the cytotoxicity studies were also investigated which enabled the determination of the safety margin of applying the coated iron oxide nanorods on U87 MG cells.

To determine the effectiveness of the carnosine delivery systems as a treatment for glioblastoma, the coated iron oxide nanorods were screened in vitro using the U87 MG human glioblastoma astrocytoma cell line. Labile carnosine (100 mM) was determined to suppress the proliferation and mobility of U87 MG cells within 48 hours, significantly reducing migration and potential metastasis. The cytotoxicity studies enabled calculating the half maximal inhibitory concentration (IC50) and the half maximal effective concentration (EC50) of the carnosine. The active carnosine was found to be fully released from the carrier, with only mild hyperthermia conditions at 40 ˚C being necessary. This is achievable in clinical applications, for both sustained and triggered release treatment of glioblastoma brain tumours, utilising the paramagnetic properties of iron oxide nanorods. This demonstrates the potential to inhibit post-surgery metastasis with the benefit of non-invasive monitoring via MRI scanning.

The controlled release of carnosine treatment was also inspected by applying external trigger. Therefore, the nano-rod-shaped superparamagnetic iron oxide and the carnosine were encapsulated inside poly(lactic-co-glycolic acid) beads (10 μm) using a hydrodynamic microfluidic flow focusing system, and the formulation was characterised by scanning and transmission electron microscopy (SEM, TEM) and Fourier-transform infrared spectroscopy (FT-IR). A non-heating rotating magnetic field (Halbach magnet array, 1 Tesla, 20 Hz, 30 min) was utilised to stimulate the release of carnosine from the polymeric beads by rotating the nano rods from distance. Additional potential treatment in intranasal application was also investigated via a spray device. The microfluidic flow focusing system was utilised to encapsulate the carnosine therapeutic inside a liposome (ca. 300 nm diameter) matrix with a stability profile of 30 days. The sub-microscale size of the liposomes and the mucoadhesive properties were expected to enhance the nasal bioavailability of carnosine over a prolonged time. The liposomal formulation was optimised to load the carnosine (75% w/w). The characterisation of the liposomes was confirmed via microscopic imaging, dynamic light scattering (DLS), Liquid chromatography–mass spectrometry (LC-MS), and Fourier-transform infrared spectroscopy (FT-IR). The stability and sustained release profiles were investigated using physicochemical studies of membrane dialysis over time. The carnosine-loaded liposomes were stable (30 days at 8 ˚C) as a ready-to-use suspension for intranasal spray application. Overall, these results provide a promising optimised formula for complementary carnosine treatment, which is recommended to be studied in vivo in rat models in the near future.

Spheroids, a complex 3-dimensional (3D) structure, resemble in vivo tumour growth more closely. As part of this project, a protocol has been developed towards a rapid and high throughput method for the generation of single spheroids using various cancer cell lines, including different cancer cells (U87 MG, SEBTA-027, SF188), prostate cancer cells (DU-145, TRAMP-C1) and breast cancer cells (BT-549, Py230) in 96-round bottom well plates. The proposed method was associated with significantly low costs per plate without the need for refining or transferring. The homogeneous compact spheroid morphology was evidenced within one day after following this new protocol. Proliferating cells, on the surface of the spheroid, were traced using confocal microscopy and the Incucyte® live cell analysis system. In contrast, dead cells were found to be located inside the core region of the spheroid. Hematoxylin and eosin (H&E) staining of spheroid sections was utilised to investigate the tightness of the cell packaging. This method enabled the determination of the EC50 of the anti-cancer dipeptide carnosine on a U87 MG 3D culture. This new protocol allows for the robust generation of various uniform spheroids that show 3D morphological characteristics. As such, further studies will be developed towards an in vivo animal model to demonstrate the potential clinical viability of this work on various cancer types, such as brain and prostate tumours.

Item Type:	Thesis
Creators:	Habra, K.
Date:	June 2023
Rights:	This work is the intellectual copyright of the author. You may copy up to five per cent of this work for private study, or personal, non-commercial research. Any reuse of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the first instance to the owner of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	09 Aug 2023 09:43
Last Modified:	09 Aug 2023 09:43
URI:	https://irep.ntu.ac.uk/id/eprint/49532