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Title: Investigation into synthetic amphiphilic polymers for intracellular delivery
Author: Roebuck, Deborah Anne
ISNI:       0000 0004 7233 1060
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Delivery of siRNA therapeutics to their target site within the cell interior is a challenge that hinders their effective use in disease treatment. PP-75, a pH-responsive polymer, demonstrates potential to enhance intracellular siRNA-delivery by overcoming endosomal entrapment. PP-75 also provides a promising platform for development of targeted delivery, following conjugation of DARPin targeting ligands. Novel PP-75 cross-linker derivatives, PP-75-aminofluorescein, PP-75-siRNA and PP-75-DARPin conjugates have been developed and characterised for in vitro application. PP-75 delivery to SK-BR-3 (Her2+) and MDA-MB-231 (Her2-) breast cancer cells has been demonstrated for the first time. The membrane-lytic activity of PP-75 was limited at physiological pH but effective within the pH range typical of early endosomes. PP-75 did not demonstrate cytoxicity, with cells tolerating treatments up to 2.5 mg/mL over 72 h. Cellular internalisation and endosomal escape of PP-75 aminofluorescein (AFC) was confirmed via confocal microscopy, demonstrated by diffuse cytoplasmic delivery. Flow cytometry confirmed cellular internalisation of PP-75 AFC was via endocytosis. As reporter cells expressing firefly and Renilla luciferases, the breast cancer lines offered a robust assay read out capable of distinguishing between target specific and non-specific gene knockdown. The functionality of novel siRNA payloads that targeted firefly luciferase was confirmed by mRNA and protein knockdown and provided the foundation for delivery of PP-75 siRNA conjugates. Target specific DARPin affinity was confirmed for the SK-BR-3 (Her2+) cells and not MDA-MD-231 (Her2-) cells, demonstrating selective binding to the extracellular Her2 protein. The expression of a novel but structurally comparable negative control DARPin (5K D1) demonstrated no affinity for either cell line. The introduction of a free cysteine residue to the DARPin sequences facilitated attachment onto PP-75. PP-75 therefore has the potential to demonstrate intracellular delivery of siRNA payloads, capable of delivery to specific cell populations via DARPin targeting.
Supervisor: Chen, Rongjun Sponsor: Biotechnology and Biological Sciences Research Council ; MedImmune Inc
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral