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Polymeric nanoparticles loaded with a Wnt agonist for enhancing bone fracture healing

Polymeric nanoparticles loaded with a Wnt agonist for enhancing bone fracture healing
Polymeric nanoparticles loaded with a Wnt agonist for enhancing bone fracture healing
In the UK, over 2 million people suffer a bone fracture every year. 10% of bone fractures will not heal adequately and require surgical intervention and, as yet, there is no approved systemic drug that is effective in promoting and accelerating fracture healing. Wnt signalling activation is a promising therapeutic target to address this paucity of treatments. Wnt is a molecular pathway that controls bone homeostasis and repair. However, its activation can have both positive and negative effects on bone cell function depending on the timing and site of delivery. Polymersomes (PMs) are polymeric nanoparticles that allow for a spatio-temporal controlled delivery of molecules, including Wnt agonists. This study addresses the hypothesis that PMs loaded with a Wnt agonist can be used as a novel systemic treatment to accelerate bone fracture healing fracture. The aims were: to assess cellular uptake of PMs and to quantify the amount of payload released intracellularly from PMs, to determine the ability of Wnt agonist loaded PMs to promote the osteogenic differentiation of human bone marrow stromal cells (BMSCs), and to assess the distribution of PMs in vivo following systemic injection in a mouse model of bone fracture. Cellular uptake was demonstrated using fluorescein as a model payload. By combining microscopy and flow cytometry, it was demonstrated that PMs are internalised by different cell types, including skeletal stem cells (SSCs), and real-time intracellular release of fluorescein was quantified at a single-cell level. Activation of Wnt signalling was achieved loading PMs with the Wnt agonist 6-bromoindirubin-3’-oxime (BIO), and demonstrated using a luciferase assay and RT-qPCR. In a reporter cell line, BIO-PMs induced a significant activation of the Wnt pathway without cytotoxicity, differently from free BIO. In BMSCs, BIO PMs induced a significant increase in the expression of the Wnt target gene AXIN2 (p<0.05) and in the expression of the early osteogenic marker RUNX2 (p<0.05). Biodistribution in vivo was assessed loading PMs with a fluorescent dye (DiR) and using IVIS and histological analysis. PMs localised in the fractured bone within 24 hours after systemic administration in mice with a femoral drill defect and reached the maximum of accumulation after 48 hours. Histological sectioning confirmed the presence of PMs in the defect area post injection. Preliminary results demonstrated that BIO-PMs injected systemically have the ability to promote bone formation after injury. This project demonstrated that PMs are internalised by SSCs, which are the ideal cellular targets for bone regenerative approaches. When loaded with Wnt agonists, PMs induce a controlled activation of the pathway, promoting osteogenic differentiation of BMSCs. Upon systemic injection in vivo, PMs accumulate at the fracture site and were able to promote bone formation. Overall, the novel and exciting findings presented in this project showed that PMs loaded with Wnt agonists could represent an effective pharmacological treatment to promote bone regeneration after fracture.
University of Southampton
Scarpa, Edoardo
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Scarpa, Edoardo
e7d094b2-e64f-4b4c-a452-d478a912b171
Evans, Nicholas
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Newman, Tracey
322290cb-2e9c-445d-a047-00b1bea39a25
Oreffo, Richard
ff9fff72-6855-4d0f-bfb2-311d0e8f3778

Scarpa, Edoardo (2016) Polymeric nanoparticles loaded with a Wnt agonist for enhancing bone fracture healing. University of Southampton, Doctoral Thesis, 295pp.

Record type: Thesis (Doctoral)

Abstract

In the UK, over 2 million people suffer a bone fracture every year. 10% of bone fractures will not heal adequately and require surgical intervention and, as yet, there is no approved systemic drug that is effective in promoting and accelerating fracture healing. Wnt signalling activation is a promising therapeutic target to address this paucity of treatments. Wnt is a molecular pathway that controls bone homeostasis and repair. However, its activation can have both positive and negative effects on bone cell function depending on the timing and site of delivery. Polymersomes (PMs) are polymeric nanoparticles that allow for a spatio-temporal controlled delivery of molecules, including Wnt agonists. This study addresses the hypothesis that PMs loaded with a Wnt agonist can be used as a novel systemic treatment to accelerate bone fracture healing fracture. The aims were: to assess cellular uptake of PMs and to quantify the amount of payload released intracellularly from PMs, to determine the ability of Wnt agonist loaded PMs to promote the osteogenic differentiation of human bone marrow stromal cells (BMSCs), and to assess the distribution of PMs in vivo following systemic injection in a mouse model of bone fracture. Cellular uptake was demonstrated using fluorescein as a model payload. By combining microscopy and flow cytometry, it was demonstrated that PMs are internalised by different cell types, including skeletal stem cells (SSCs), and real-time intracellular release of fluorescein was quantified at a single-cell level. Activation of Wnt signalling was achieved loading PMs with the Wnt agonist 6-bromoindirubin-3’-oxime (BIO), and demonstrated using a luciferase assay and RT-qPCR. In a reporter cell line, BIO-PMs induced a significant activation of the Wnt pathway without cytotoxicity, differently from free BIO. In BMSCs, BIO PMs induced a significant increase in the expression of the Wnt target gene AXIN2 (p<0.05) and in the expression of the early osteogenic marker RUNX2 (p<0.05). Biodistribution in vivo was assessed loading PMs with a fluorescent dye (DiR) and using IVIS and histological analysis. PMs localised in the fractured bone within 24 hours after systemic administration in mice with a femoral drill defect and reached the maximum of accumulation after 48 hours. Histological sectioning confirmed the presence of PMs in the defect area post injection. Preliminary results demonstrated that BIO-PMs injected systemically have the ability to promote bone formation after injury. This project demonstrated that PMs are internalised by SSCs, which are the ideal cellular targets for bone regenerative approaches. When loaded with Wnt agonists, PMs induce a controlled activation of the pathway, promoting osteogenic differentiation of BMSCs. Upon systemic injection in vivo, PMs accumulate at the fracture site and were able to promote bone formation. Overall, the novel and exciting findings presented in this project showed that PMs loaded with Wnt agonists could represent an effective pharmacological treatment to promote bone regeneration after fracture.

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Published date: September 2016
Organisations: University of Southampton, Human Development & Health

Identifiers

Local EPrints ID: 408720
URI: http://eprints.soton.ac.uk/id/eprint/408720
PURE UUID: 6d08e5bc-e1f9-453b-b515-02f242729dad
ORCID for Tracey Newman: ORCID iD orcid.org/0000-0002-3727-9258
ORCID for Richard Oreffo: ORCID iD orcid.org/0000-0001-5995-6726

Catalogue record

Date deposited: 27 May 2017 04:02
Last modified: 14 Mar 2019 01:51

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Contributors

Author: Edoardo Scarpa
Thesis advisor: Nicholas Evans
Thesis advisor: Tracey Newman ORCID iD
Thesis advisor: Richard Oreffo ORCID iD

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