Tuning and targeting semiconducting polymer nanoparticles to enhance in vivo photoacoustic imaging

Photoacoustic imaging (PAI) is a promising imaging modality which combines high spatial resolution with excellent contrast generation. To fulfil the potential of using PAI in clinical settings for cancer detection, the development of novel contrast agents with strong absorptions in the near infrared and high tumour specificity in vivo is required. Semiconducting polymer nanoparticles (SPNs) encapsulating organic semiconducting polymers in lipid nanoparticles have emerged as excellent candidates for photoacoustic (PA) contrast generation: they retain the polymer’s ability to generate high PA contrast, and the lipid formulation grants SPNs excellent physiological properties. However, these SPNs rely so far on the enhanced retention and permeability (EPR) effect for tumour accumulation. The reliability of this passive mode of accumulation in clinical settings has been recently called into question. To address this, the formulation of targeted SPNs using EGFR-targeting peptides was explored. SPNs based on novel semiconducting polymers as well as commercially available polymers were formulated via the mini-emulsion and nanoprecipitation methods. Lipid formulations included PEGylated lipids as well as functional PEG lipids on which N-terminal Cysteine-modified EGFR-targeting peptides were conjugated via the thiol-maleimide Michael addition. The synthetic accessibility of both the pre- and post-formulation functionalisation strategies was assessed in Chapter 3. To quantify surface functionalisation, a novel NMR characterisation strategy for the routine quantification of maleimide moieties tethered to the surface of nanoparticles was proposed. To compare the targeting efficiency of EGFR-targeting peptides A-R, D4 and GE11, a library of peptide-dye conjugates was constructed using fluorescein-5-maleimide. This library included scrambled controls and short PEG spacers introduced between the targeting sequence and the cysteine residue. The synthesis of these conjugates is described in Chapter 4. While synthesising these peptides, preliminary data supporting the intramolecular transcyclisation of the thiol-maleimide adducts was obtained. The intramolecular transcyclisation of thiol-maleimide adducts is underreported in the literature and clinically relevant. The thiazine rearrangement products are protected from thiol-exchange which, in physiological conditions, can lead to the partial loss of functionality of targeting ligands synthesised using the thiol-maleimide reaction

EGFR-targeted semiconducting polymer nanoparticles for photoacoustic imaging

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV–visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles

EGFR-targeted semiconducting polymer nanoparticles for photoacoustic imaging

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV-visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles

The CDR1 and other regions of immunoglobulin light chains are hot spots for amyloid aggregation

Immunoglobulin light chain-derived (AL) amyloidosis is a debilitating disease without known cure. Almost nothing is known about the structural factors driving the amyloidogenesis of the light chains. This study aimed to identify the fibrillogenic hotspots of the model protein 6aJL2 and in pursuing this goal, two complementary approaches were applied. One of them was based on several web-based computational tools optimized to predict fibrillogenic/aggregation-prone sequences based on different structural and biophysical properties of the polypeptide chain. Then, the predictions were confirmed with an ad-hoc synthetic peptide library. In the second approach, 6aJL2 protein was proteolyzed with trypsin, and the products incubated in aggregation-promoting conditions. Then, the aggregation-prone fragments were identified by combining standard proteomic methods, and the results validated with a set of synthetic peptides with the sequence of the tryptic fragments. Both strategies coincided to identify a fibrillogenic hotspot located at the CDR1 and β-strand C of the protein, which was confirmed by scanning proline mutagenesis analysis. However, only the proteolysis-based strategy revealed additional fibrillogenic hotspots in two other regions of the protein. It was shown that a fibrillogenic hotspot associated to the CDR1 is also encoded by several κ and λ germline variable domain gene segments. Some parts of this study have been included in the chapter “The Structural Determinants of the Immunoglobulin Light Chain Amyloid Aggregation”, published in Physical Biology of Proteins and Peptides, Springer 2015 (ISBN 978-3-319-21687-4)