膠質母細胞瘤 (GBM) 是最致命的癌症形式，其特徵是不受約束的惡性腫瘤。由於預後不佳，5 年生存率低於 5%，因此迫切需要開發一種有效的治療材料。自過去幾十年以來，支架一直被廣泛使用，並且已被證明是一種具有潛力遞送媒介，可持續和延長納米粒子的釋放。利用3D列印技術所製造的支架不僅可生產所需形狀和尺寸的支架，更可提高支架的精度，從而為建構具專一性的植入物生產開闢了一條新途徑。支架進一步塗佈有機金屬框架 MOFs–NH2MIL88B，用以啟動納米催化活性。MOFs的添加，不僅可在癌細胞中引發 Fenton 反應，也能與自噬抑制分子一起在腫瘤切除位置誘導免疫反應，從而提高治療效率。

Glioblastoma (GBM) is the most lethal form of cancer characterized by unconstrained malignant neoplasm. With the 5-year survival rate being less than 5% due to penurious prognosis there is a desperate need to develop a potent therapeutic material. Extensive research has been carried out in the past decade for the inhibition of glioblastoma recurrence with most of the results being up to par in few cases with very low survival predictability. Scaffolds have proven to be a potent delivery agent for sustained and prolonged release of therapeutic drugs and nanoparticles. 3D printing of scaffold allows for the production of scaffolds with desired shape and size thereby opening a pathway for the construction of patient specific implant production. Various forms of nanoparticle have been studied and scrutinized for the delivery of drugs which include lipid-based nanoparticles, polymer-based nanoparticles, metallic nanoparticles and viral vectors. A new class of nanoparticles named metal organic frameworks (MOFs) have been bringing significant change with dramatic experimental results in the field of nanotechnology. MOFs are superior class of nanoparticles and their porous structure makes them a suitable candidate for loading and carrying drugs, proteins, and antigens with minimal loss for sustained release.
In this study we fabricated MOFs for efficient cancer therapy. We further successfully surface modified 3D printed scaffold with MOFs nanoparticle. The MOFs NH2-MIL-88B is an Fe ion-based MOF with high catalytic ability. The SRNP MOFs nanoparticle possessed a length of 160 nm and a width of 59 nm whereas the LRNP MOFs possessed a minimal length and width equivalent to 175 nm and 76 nm respectively. Both SRNP and LRNP exhibited a charge of -1.6 mV and 14.3 mV. The MOFs coated scaffold exhibited slow and sustained release of MOF for a period of 11 days. A total of 465 g and 598 g of nanoparticle was released by SNS and LNS scaffolds respectively for a period of 11 days.
We utilized glioblastoma cell line for in-vivo analysis in order to determine the toxicity. We utilized both nanoparticles and MOFs coated scaffolds for in-vitro assays. Both nanoparticles SRNP and LRNP exhibited 50% cell viability at a very low concentration of 10 g. The scaffolds SNS and LNS exhibited cell viability of 41.7% and 32% respectively. The cellular uptake of nanoparticles was determined using fluorescence imaging and flow cytometry. Autophagosome accumulation in cytoplasm was also carried out for MOFs nanoparticles and MOFs nanoparticle coated scaffolds. In the current study we incorporated chloroquine (CQ), the results obtained revealed higher accumulation of autophagosomes for CQ treated groups due to lysosomal deacidification.
In-vivo analysis were carried out using C57BL6 mice, which were injected with ALTS1C1 cells. After 16 days of inoculation the mice were treated with SNS, LNS and Blank HBA/ F127 scaffolds in combination with CQ. Amplification of cytotoxic T cells was observed in CQ treated groups due to ROS mediated immunogenic cell death. The infiltration of T cells was quantified using flow cytometry analysis for lymph nodes and spleen along with immunofluorescence imaging of brain slices. Both flow cytometry and immunofluorescence imaging results were used to interpret the infiltration of T cell. The percentage of CD8+ in SNS, LNS and HBA/ F127 scaffolds treated with CQ accounts to 32%, 35.5% and 40.2% which was much higher when compared with control group (21.4%). Addition of MOFs not only initiated Fenton Reaction in the cancer cells but it also induced immunogenic response at the tumour resected cavity Thus, making surface modified scaffolds with Fenton nano-agent MOF a potential candidate for glioblastoma therapy.

中文摘要
Abstract
Table of Contents
List of Schemes
Chapter 1 Introduction 1
Chapter 2 Literature Review and Theory 5
2.1 Introduction to Glioblastoma 5
2.2 Post Operative Therapy 8
2.3 Immunotherapy for Glioblastoma 13
2.4 Scaffolds for Glioblastoma Therapy 17
2.5 Metal Organic Frameworks (MOFs) 22
Chapter 3 Materials and Methods 30
3.1 Materials 30
3.1.1 Materials used in making 3D printed scaffold: 30
3.1.2 Materials used in synthesis and coating of MOF nanoparticle (NH2MIL-88B): 30
3.1.3 Materials used in Fenton reaction experiment: 30
3.1.4 Materials used for in-vitro studies: 30
3.1.5 Materials used in in-vivo studies 31
3.2 Apparatus and Instruments: 32
3.3 Methods 34
3.3.1 Synthesis of NH2MIL-88B nanoparticles (SRNP): 34
3.3.2 Synthesis of NH2MIL-88B nanoparticles (LRNP): 34
3.3.3 Synthesis of HBA/ F-127 resin mixture for 3D printing: 35
3.3.4 Designing of octahedral shaped scaffold: 35
3.3.5 3D printing of octahedral scaffold and treatment: 36
3.3.6 Synthesis of small nanoparticle coated scaffold (SNS): 37
3.3.7 Synthesis of large nanoparticle coated scaffold (LNS): 37
3.3.8 Preparation of CQ solution for in-vitro experiments: 38
3.3.9 Preparation of CQ solution for in-vivo experiments: 38
3.4 Material Characterisation: 39
3.4.1 SEM sample preparation for nanoparticle: 39
3.4.2 SEM sample preparation for 3D printed scaffolds: 39
3.4.3 TEM sample preparation for nanoparticles: 39
3.4.4 Fourier infrared transform spectroscopy (FTIR) for nanoparticles: 40
3.4.5 Fourier infrared transform spectroscopy (FTIR) for 3D printed scaffolds: 40
3.4.6 Powder X-ray diffraction (XRD) for nanoparticles: 40
3.4.7 Dynamic light scattering (DLS) for MOF nanoparticle: 40
3.4.8 Dynamic light scattering (DLS) for MOF nanoparticle coated scaffold: 40
3.4.9 X-ray Photoelectron Spectrometer (XPS) for nanoparticles: 41
3.4.10 Cumulative MOF nanoparticle release from scaffold: 41
3.4.11 SEM images for MOF dissolve test: 41
3.4.12 Fenton reaction experiment using UV-Vis for nanoparticles: 41
3.4.13 Fenton reaction experiment using UV-Vis for nanoparticles coated scaffold: 42
3.5 In-Vitro Experiments: 42
3.5.1 Cell culture: 42
3.5.2 Cell viability assay of nanoparticles: 42
3.5.3 Cell viability assay of nanoparticles coated scaffold: 43
3.5.4 Cellular uptake (Nanoparticle): 43
3.5.5 LC3B autophagy expression for MOF nanoparticles: 44
3.5.6 LC3B autophagy expression in nanoparticle coated scaffold: 44
3.5.7 Live/ Dead assay: 45
3.6 In-Vivo Experiments: 46
3.6.1 Immunohistochemistry staining: 46
3.6.2 Flow cytometer for immune response: 47
Chapter 4 Result and Discussion 49
4.1 Characterisation of MOF Nanoparticle and MOF Coated Scaffold: 49
4.1.1 SEM, TEM and EDS spectra for MOF nanoparticle: 49
4.1.2 Fourier transform infrared spectroscopy (FTIR) for MOF nanoparticle: 50
4.1.3 X-ray diffraction for MOF nanoparticle: 51
4.1.4 X-ray photoelectron spectroscopy (XPS) for MOF nanoparticle: 51
4.1.5 Dynamic light scattering analysis for MOF nanoparticles: 52
4.1.6 Catalytic performance (Fenton reaction) of MOF nanoparticle: 53
4.1.7 SEM for MOF nanoparticle coated scaffold: 54
4.1.8 MOF dissolve test for MOF coated scaffold: 56
4.1.9 FTIR spectrum for MOF nanoparticle coated scaffold: 56
4.1.10 Cumulative MOF release from the scaffold: 57
4.1.11 Dynamic light scattering analysis for MOF nanoparticle coated scaffold: 58
4.1.12 Catalytic performance (Fenton reaction) of MOF nanoparticle coated scaffold: 60
4.2 In-vitro Assays for MOF Nanoparticle and MOF Coated Scaffolds: 60
4.2.1 Cellular viability assay for nanoparticle: 60
4.2.2 Cellular uptake of MOF nanoparticles fluorescence images: 61
63
4.2.3 Cellular uptake flow cytometry: 64
4.2.4 LC3B expression for nanoparticles treated glioblastoma cells: 64
4.2.5 Cellular viability assay for MOF nanoparticle coated scaffolds: 66
4.2.6 LC3B expression for MOF nanoparticles coated scaffold treated glioblastoma cells: 68
4.2.7 LIVE/ Dead assay for scaffolds coated with MOF nanoparticle: 70
4.3 In-vivo Studies for MOF Coated Scaffolds: 71
4.3.1 Post operative immune response confocal images: 71
4.3.2 Quantification of immune cells using flow cytometry: 77
Chapter 5 Conclusion 80
Reference: 82

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