Polymeric micelle nanomedicines for monitoring therapeutic efficacy

Abstract

The development of ‘smart’ nanomedicines capable of visualising stimuli-responsive delivery of therapeutics within complex biological environments continues to provide a promising outlook for the future treatment of cancer. Technological advances of in vivo molecular imaging technology, such as MRI, PET and optical imaging, have increased our ability to successfully visualise and monitor the delivery and effect of nanomedicines in the body. However, understanding the fate of these nanomaterials in delivering therapeutic agents homogenously across a heterogeneous tumour mass remains elusive, largely due to the lack of suitable technologies for probing these complex systems.In order to further advance the development of nanomaterials for cancer therapy, visualisation of the complex bio-nano interactions, such as cell death as a result of therapeutic treatment, occurring within the tumour microenvironment is crucial. Recently, an alternative in vivo imaging technique, multispectral optoacoustic tomography (MSOT), has been shown to reveal important pharmacokinetic and pharmacodynamic information about nanomedicines. Combining the high contrast of optical imaging and the high resolution of ultrasound, MSOT surpasses the capabilities of its constituent techniques providing superior deep tissue imaging through sensitive detection of endogenous, exogenous and switchable probes.This thesis aims to demonstrate the synthesis, characterisation and application of redox‑responsive polymeric micelles loaded with a cancer therapeutic and labelled with an apoptosis‑sensitive imaging probe to investigate the in vivo therapeutic efficacy of these materials across the tumour mass. Using MSOT, these micelles can provide real-time monitoring of therapeutic effect upon delivery and uptake into tumour tissue in vivo, offering insights into the fate of nanomedicines within the tumour microenvironment.Chapter 1 provides an overview of the field of cancer-based nanomedicines highlighting the advances made to date in the design and visualisation of stimuli-responsive materials within biological systems. The current limitations of these platforms as cancer therapeutics are highlighted throughout with a focus on the need for in vivo imaging to provide further insights into the biological fate of these materials. The role of molecular imaging in combination with polymeric micelles as a model system is described as a step forward in informing the future design of cancer nanomedicines.In Chapter 2, a review article provides the reader with additional information on the recent state of the field involving polymeric systems which have successfully been imaged in vivo. The review explores the various molecular imaging modalities being used for in vivo imaging and describes the recent endeavours to develop switchable imaging probes capable of responding to specific biological stimuli to investigate the biological fate of theranostic nanomaterials further.To add to the current understanding, Chapter 3 focuses on the development and synthesis of a drug-loaded, reversibly crosslinked polymeric micelle platform for targeted therapeutic delivery to cancer-cells. Comprising disulfide crosslinks which can be selectively cleaved in response to the reductive environment found within cancer cells, different sized drug-loaded micelles were successfully characterised for the controlled release of the cancer therapeutic doxorubicin. In vitro cell viability and cellular uptake studies using flow cytometry and live-cell microscopy were performed following the incorporation of a bispecific antibody onto the micelle nanomedicines which enabled enhanced targeted uptake of these materials into breast cancer cells to deliver a therapeutic payload and generate a cytotoxic effect.Chapter 4 describes the synthesis and characterisation of an apoptosis-sensitive imaging probe designed to provide real-time visualisation of therapeutic efficacy using both fluorescence and optoacoustic imaging. Here the synthesis and activation of the fluorescence resonance energy transfer between the fluorophore Cy5.5 and a quencher chromophore linked by a caspase-3-cleavable tetrapeptide motif are explored as a stimuli-responsive imaging probe to monitor cell apoptosis in response to the therapeutic doxorubicin. The change in fluorescence and optoacoustic signal upon exposure to caspase-3 using an enzymatic assay and in drug-treated cells was fully characterised to identify the spectral properties of the probe to be used for in vivo imaging.Chapter 5 brings together the components developed in the previous two chapters to demonstrate the application of the developed polymeric micelles labelled with the apoptosis-sensitive imaging probe to monitor the real-time therapeutic efficacy of these materials both in vitro and in vivo. Monitoring of the in vivo fluorescence and optoacoustic signal following intratumoural delivery of the probe-labelled polymeric micelles enabled real-time visualisation of the probe activation in response to therapeutic delivery within the tumour. This chapter also demonstrates the challenges of applying this new technique to complex biological systems.In summary, this thesis describes the development of a novel nanomedicine platform capable of visualising and monitoring therapeutic efficacy within tumour-bearing mice in vivo. It demonstrates the application of optoacoustic imaging as an emerging molecular imaging technique to visualise complex biological events and barriers that currently hinder the future success of cancer nanomedicines. This new knowledge plays a fundamental role in the future progression and rational design of nanomedicines towards clinical translation.