Improving bone healing using polymeric nanoparticle mediated delivery of wnt agonists

Bone fractures and their complications are prevalent medical issues with high financial costs and loss in quality of life. Pharmaceutical treatments to aid bone fracture repair are not yet available in clinic, despite their potential for improving fracture outcomes. Therapeutic candidates such as Wnt agonists may improve the quality and rate of healing by boosting key signalling involved in fracture repair. However, these therapies are limited by off-target effects and poor bioavailability at the injury site. Polymeric nanoparticles (polymersomes; PMs) may provide a solution by enabling controlled spatio-temporal drug delivery. This research aimed to develop a PM drug delivery therapeutic for the controlled and localised delivery of a Wnt agonist to a bone injury site, in order to promote bone repair and ultimately improve fracture healing outcomes. In this study, the efficiency of PMs for encapsulating Wnt agonists BIO and CHIR and achieving in vitro induction of a Wnt signalling response was assessed using techniques of UVVis spectroscopy and a luciferase reporter cell line. Here PMs were successfully produced with a sufficient dose of BIO and CHIR to induce Wnt signalling. In contrast to free compounds, PM-encapsulated BIO and CHIR prevented cytotoxicity when incubated with primary bone cells. This study also assessed the in vivo localisation of PMs using an IVIS imaging method which semi-quantified a fluorescent PM payload distribution within a mouse bone injury model. It was found that following injury, either immediate or a 7- day delayed IV administration of PMs resulted in passive accumulation at a target bone injury site. Additionally, a preliminary study using a clodronate liposome pre-treatment to deplete the macrophage population of MPS organs found no significant change in PM accumulation to a target bone injury site. Finally, this study tests the hypothesis that PM-encapsulated BIO can induce Wnt signalling activity and osteogenic differentiation in vivo. RT-qPCR analysis of tissues of high PM accumulation extracted 24 or 48 hours following IV treatment of PM-BIO revealed disparate in vivo gene expression of Wnt signalling inhibitors (Axin2 and Dkk1) depending on the presence of a bone injury, as well as the induction of early osteogenic gene expression (Runx2) at a target injured bone. Overall the findings presented in this thesis demonstrate that PM delivery of a Wnt agonist provides a viable bone-promoting therapeutic which could improve fracture healing outcomes

Dataset for Thesis entitled 'Improving bone healing using polymeric nanoparticle mediated delivery of Wnt agonists.'

This dataset supports the thesis entitled: Improving Bone Healing Using Polymeric Nanoparticle Mediated Delivery of Wnt Agonists For a description of how data was collected please refer to the Thesis Methods and Materials Chapter 2. Specialist Software required: Graph pad prism for .pzfx files Malvern Zetasizer Softwear for .dts and .zmes files Applied Biosystems 7500 Real-Time PCR System Software for .eds files Files labelled according to the related figure published in thesis</span

Polymersome nanoparticles for delivery of Wnt-activating small molecules

Spatiotemporal control of drug delivery is important for a number of medical applications and may be achieved using polymersome nanoparticles (PMs). Wnt signalling is a molecular pathway activated in various physiological processes, including bone repair, that requires precise control of activation. Here, we hypothesise that PMs can be stably loaded with a small molecule Wnt agonist, 6-bromoindirubin-3′-oxime (BIO), and activate Wnt signalling promoting the osteogenic differentiation in human primary bone marrow stromal cells (BMSCs). We showed that BIO-PMs induced a 40% increase in Wnt signaling activation in reporter cell lines without cytotoxicity induced by free BIO. BMSCs incubated with BIO-PMs showed a significant up-regulation of the Wnt target gene AXIN2 (14 ± 4 fold increase, P &lt; 0.001) and a prolonged activation of the osteogenic gene RUNX2. We conclude that BIO-PMs could represent an innovative approach for the controlled activation of Wnt signaling for promoting bone regeneration after fracture.</p

Antibiotic-loaded polymersomes for clearance of intracellular burkholderia thailandensis

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM) encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study, we developed PMs composed of widely available poly(ethylene oxide)-polycaprolactone block copolymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM colocalization with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.</p