Periodic Reporting for period 1 - FluoroDendriNostic (Combined fluorinated polymer and poly-L-lysine dendrimer as new potential contrast agents for magnetic resonance imaging 19F)
Période du rapport: 2016-09-01 au 2018-08-31
Early diagnosis of diseases, including tumors, is the ultimate goal of biomedical imaging. Magnetic resonance imaging (MRI) is a technique for non-destructive and non-invasive diagnosis of a number of diseases including cancer. However, early detection of such diseases requires techniques with high sensitivity, while maintaining high specificities for cancerous or diseased cells. The use of contrast agents improves the image quality and hence leads to a significant improvement in image analysis which in turns leads to more reliable/sensitive diagnosis. Recently, contrast agents bearing 19F have been introduced as an attractive alternative to purely hydrogenated compounds, because of their unique spectroscopic signature which leads to a high signal to noise ratio. However promising these new fluorinated compounds are, their tendency to aggregate and the low mobility of fluorinated chains in physiological media have known to limit the sensitivity of 19F MRI for clinical applications. Recently, hyperbranched fluoropolymers demonstrate to be a promising platform for in vivo imaging by 19F magnetic resonance. There is significant interest in such hyperbranched polymers for use in nanomedicine applications. This project aimed to design, synthesize and characterize a series of novel fluorinated structures and examine their behavior in aqueous media and to assess 19F MRI capability of these new structure. Fluoropolymers, in this project, are polymers which are containing 19F that are soluble in water and can be readily functionalized to enable targeting. This project represented a key objective in the advancement of MRI research. This program of work had also developed a new and exciting collaboration between the host, Benoit Couturaud and collaborator (A/Prof Kristofer Thurecht) and had place this new team at the leading edge of this important field.
The Marie Curie fellow conducted the proposed research in Prof. Rachel O’Reilly’s group. The research in this group is highly interdisciplinary yet has a strong polymer synthesis focus. In particular her group is well-known in the fields of polymer synthesis with controlled architectures and functionalities, and their supramolecular self-assembly into precision nanostructures targeted with potential applications in catalysis and nanotechnology devices. The Marie Curie fellow has been trained to use various facilities and instruments (scattering, microscopy etc) in the O’Reilly group as well as in the wider University. In particular, he was trained on the investigation of nano-objects’ morphologies via a range of transmission electron microscopy methods, which is a very important skill for visualizing the nanostructures of polymer. Therefore, through this fellowship, the fellow has improved his interdisciplinary expertise whilst also gain exposure to related industrial polymer research ongoing in the University.
The first part of the project involved the synthesis of polymers using well established controlled radical polymerization methods. Specifically, the work begun with the synthesis of polymers using RAFT polymerization (reversible addition-fragmentation chain transfer) using a library of new synthesized bifunctional fluorinated monomers. This approach allows for the fluorinated group to be readily solubilize to counter balance fluorine intrinsic hydrophobicity and enhance fluorine density in the resultant polymer – this was key for realization and design of the proposed nanostructures.
These new fluorinated monomers were polymerized using a RAFT agent. This allowed the preparation of a series of completely novel hydrophilic hyperbanched polymers. Given the novel compositional and morphological properties of these hyperbranched polymers a key study explored the influence of fluorine density on the resultant morphology/size and solution properties of the nanostructure.
The nanostructures were studied by NMR spectroscopy, light scattering (dynamic and static to obtain size and shape information) and electron microscopy (TEM). These are all methods routinely used by the host group and provided the Marie Curie fellow with invaluable new advanced laboratory skills. Relaxation times of 19F MRI spin-lattice and spin-spin (T1 and T2) were also examined by 19F NMR in various aqueous media. The concentration of 19F atom (spin density) and the T2 relaxation time of the polymer (molecular mobility) were studied by 19F NMR to optimize the effectiveness of the contrast agent. New materials were examined, using standard MTT cytotoxicity assays in order to confirm the non-toxicity of synthesized structures. To further optimise the application of these new materials we have focused on enhancing the specificity of the new contrast agent towards different immortalized cancer cell lines.
Binding on several methoxy group on the hyperbranched nanostructure with cancer cell-targeting ligands was performed to improve their ability to enhance contrast accumulation in cancer cells. An in vitro uptake study into a cancer cell models was performed to examine the selectivity of the modified contrast agent. Afterward, promising material was studied by MRI using an in vivo study (in mice) to determine the quality of the new type of contrast agent. In vivo studies were initiated when the Marie Curie fellow was on a short visit at the collaborator, University of Queensland. The collaborator group are the only group in the world pioneering the use of such nanostructures for MRI. Fluorinated nanostructures were also modified with Cyanine 5 in order to perform confocal fluorescence imaging of individual cells in vitro and also fluorescence imaging in vivo. These innovative in vivo studies in mice (performed by the collaborator) demonstrated potential of generated nanostructured as a powerful platform for the detection of diseases using 19F MRI.
The first part of the project involved the synthesis of polymers using well established controlled radical polymerization methods. Specifically, the work begun with the synthesis of polymers using RAFT polymerization (reversible addition-fragmentation chain transfer) using a library of new synthesized bifunctional fluorinated monomers. This approach allows for the fluorinated group to be readily solubilize to counter balance fluorine intrinsic hydrophobicity and enhance fluorine density in the resultant polymer – this was key for realization and design of the proposed nanostructures.
These new fluorinated monomers were polymerized using a RAFT agent. This allowed the preparation of a series of completely novel hydrophilic hyperbanched polymers. Given the novel compositional and morphological properties of these hyperbranched polymers a key study explored the influence of fluorine density on the resultant morphology/size and solution properties of the nanostructure.
The nanostructures were studied by NMR spectroscopy, light scattering (dynamic and static to obtain size and shape information) and electron microscopy (TEM). These are all methods routinely used by the host group and provided the Marie Curie fellow with invaluable new advanced laboratory skills. Relaxation times of 19F MRI spin-lattice and spin-spin (T1 and T2) were also examined by 19F NMR in various aqueous media. The concentration of 19F atom (spin density) and the T2 relaxation time of the polymer (molecular mobility) were studied by 19F NMR to optimize the effectiveness of the contrast agent. New materials were examined, using standard MTT cytotoxicity assays in order to confirm the non-toxicity of synthesized structures. To further optimise the application of these new materials we have focused on enhancing the specificity of the new contrast agent towards different immortalized cancer cell lines.
Binding on several methoxy group on the hyperbranched nanostructure with cancer cell-targeting ligands was performed to improve their ability to enhance contrast accumulation in cancer cells. An in vitro uptake study into a cancer cell models was performed to examine the selectivity of the modified contrast agent. Afterward, promising material was studied by MRI using an in vivo study (in mice) to determine the quality of the new type of contrast agent. In vivo studies were initiated when the Marie Curie fellow was on a short visit at the collaborator, University of Queensland. The collaborator group are the only group in the world pioneering the use of such nanostructures for MRI. Fluorinated nanostructures were also modified with Cyanine 5 in order to perform confocal fluorescence imaging of individual cells in vitro and also fluorescence imaging in vivo. These innovative in vivo studies in mice (performed by the collaborator) demonstrated potential of generated nanostructured as a powerful platform for the detection of diseases using 19F MRI.
This project addressed new synthetic methods for fluoropolymers synthesis, as well as develop entirely new nanostructured materials and test their efficacy as contrast agents for 19F MRI. The development of these novel contrast agents with smart nanostructures and unique characteristics including tunable size and morphology and excellent biocompatibility represent a new platform technology for application in areas of medicine and more specifically imaging. Given the importance of contrast agents in medicine in our daily lives the dissemination of the research results is likely to have significant impact globally in both academia and industry. Nanostructured materials also have significant interest for a range of applications beyond those proposed herein. Communication to the polymer science and biology material science fields enables follow-up research to realize new innovations for the utilization of the new materials developed in the program as the next generation of diagnosis nanomaterials. Additionally, the results already leads to two high impact publications and two others publications are in preparation, ensuring the communication of the results to a wide range of interested researchers. The Marie Curie fellow had actively seeks out all chances to communicate with the diversity of the scientific academic community, as well as industry engineers and clinicians to widely disseminate the research results during four conferences during this fellowship.