Abstract
Breast cancer is one of the most common cancers in the world, diagnosed in 2.3 million women last year, 15-20 % of which have been reported to be HER2 positive. This molecular subtype is caused by an overexpression of the human epidermal growth factor (HER2) receptors on breast cancer cells that cause rapid and uncontrolled cell growth leading to a more aggressive type of cancer.In this work, selective nanoparticles for the HER2 receptor were prepared. The vectors used were dendrons, novel peptides composed of hyperbranched poly-L-lysine dendrons or linear (K16) versions with or without a HER2 targeting sequence (LSYCCK) or a scrambled version (SGen3K) were synthesised and tested. Hyperbranched peptides electrostatically bind the negative charges of nucleic acids thus, are capable of carrying either DNA or small interfering RNA (siRNA) molecules.
Different behaviours were identified based on the nature of the nucleic acid cargo when tested in MDA-MB-231 and SKBR3 cells. In the case of DNA delivery, experiments were conducted on cells overexpressing the target receptor (HER2). Both the selective peptide (TGen3K) and the branched and linear controls were found to be inadequate for efficient transfection. However, the addition of a small quantity of lipids (DOTMA:DOPE) was necessary to achieve high levels of transfection. In this case, the lipid helper (DOPE) likely facilitated endosomal-escape. Furthermore, TGen3K was shown to be selective for the receptor. However, the scramble control (SGen3K) proved to be even more effective (twice/three times more) than the reference peptide, possibly due to better DNA packaging resulting from the different arrangement of amino acids.
In the case of siRNA delivery, on the other hand, the presence of lipids was found to be unnecessary. Confocal microscopy has revealed how the structure itself has an anti-proliferative effect and how the target sequence (KCCYSL) is selective for the HER2 receptor. It has also shown that peptides, particularly RGen3K and TGen3K, are capable of internalising the siRNA.
Finally, the nanocarriers were encapsulated in alginate microparticles following various studies that optimised the gelation of alginate microparticles. As a result, the chosen preparation for the encapsulation of the nanocarriers will be based on in-chip gelation.
The encapsulation of the nanocarriers highlighted the ability of the microparticles to retain and protect the nanocarriers during the washing of the microparticles to remove the oily phase following emulsion rupture. The nanocarriers are subsequently released within 24 hours when placed in an environment simulating the extracellular environment.
| Date of Award | Feb 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Matteo Santin (Supervisor), Marco Marengo (Supervisor) & Dr Laila Kudsiova (Supervisor) |