Jearranaiprepame, S
(2025)
Hydrogel-based local delivery loaded with Poly(β-Amino Ester) (PBAE)-siRNA polyplexes for glioblastoma therapy.
PhD thesis, University of Nottingham.
Abstract
Glioblastoma (GBM) is an aggressive and highly heterogeneous primary brain tumour known for its resistance to standard therapies and frequent recurrence, particularly during the critical gap period between surgical resection and the initiation of chemoradiotherapy. Gene therapies using small interfering RNA (siRNA) have emerged as a potential approach to selectively silence genes involved in cancer progression. However, the clinical application of siRNA remains limited due to its instability, inefficient delivery to target tissues, off-target effects, and the difficulty of achieving delivery within the brain cavity. To overcome these limitations, this study aims to develop and evaluate a hydrogel-based local delivery system incorporating poly(β-amino ester) (PBAE) polyplexes for targeted and sustained siRNA delivery, to control GBM progression during the critical therapeutic gap window. In this study, both linear and branched PBAEs were synthesised via Michael addition reactions using various acrylate and amine monomers. Among the formulations, the branched BF-TMPTA20 PBAE demonstrated the most promising performance. Gel permeation chromatography (GPC) analysis revealed a molar mass of 10200 Da, and NMR confirmed successful end-capping. When complexed with scrambled siRNA, BF-TMPTA20 polyplexes maintained a positive surface charge and particle sizes below 200.0 nm across all polymer:siRNA w/w ratios, with the strongest binding affinity observed at a 64:1 ratio. The polyplexes also exhibited non-toxicity in GIN31-fluc cells and slightly higher transfection efficiency compared to Lipofectamine-3000 at the optimal w/w ratio. Cellular internalisation studies in both 2D and 3D cell culture revealed efficient intracellular delivery and escape from endosomal/lysosomal compartments, indicating BF-TMPTA20 as a potential carrier for siRNA delivery to GBM cells. This polymer was subsequently complexed with therapeutic siRNAs targeting CDC42BPA and CDC42BPB, genes known to be upregulated in invasive GBM phenotypes.
Knockdown of CDC42BPA and CDC42BPB either individually or in combination significantly reduced GBM cell proliferation and invasion, induced apoptosis, and altered the tumour microenvironment. Dual-siRNA treatment produced significantly greater biological effects than single-siRNA treatments, likely due to complementary or overlapping gene silencing effects. To apply as a local delivery system, the BF-TMPTA20 polyplexes were incorporated into a thermoresponsive mPEG-PCL-HMDI-PCL-mPEG (PECE) triblock copolymer hydrogel. The hydrogel exhibited prolonged siRNA release over a two-week period while maintaining transfection efficiency and minimal cytotoxicity. In conclusion, the BF-TMPTA20 polyplex-loaded PECE hydrogel system offers a promising approach for localised siRNA delivery in GBM. It addresses key limitations in gene therapy, including cellular uptake, endosomal escape, and sustained delivery, and may serve as an effective system to prevent tumour recurrence during the critical post-surgical treatment period.
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