[Thesis]. Manchester, UK: The University of Manchester; 2018.
Two of the main limitations of conventional cancer drugs are their lack of ability
to discriminate between cancer and normal cells, producing the well-known side effects,
and the resistance to radio- and chemotherapy. The resistance is mainly due to the
presence of hypoxic (low oxygen) regions in tumours where the Hypoxia Inducible Factor
(HIF) transcriptional system regulates the expression of hypoxia-dependent pro-survival
and drug resistance genes. The development of a delivery system capable of specifically
targeting and penetrating tumours is a promising strategy to overcome these issues.
Currently, one of the most investigated agents for cancer targeting is hyaluronic
acid (HA), since its main receptor, CD44, is overexpressed in many cancers. However,
it is still unclear which cell-related factors influence HA binding and internalisation
(collectively called Ă˘uptakeĂ˘) into CD44 expressing cells. To address this, the
expression of CD44 (standard and variants isoforms, CD44s and CD44v respectively)
was evaluated in human dermal fibroblasts (HDF) as healthy control and in a large
panel of cancer cell lines. It was found that the expression of CD44v can negatively
influence the uptake of HA but, interestingly, the healthy control HDF that expressed
high levels of CD44s were less efficient in taking up HA when compared to high expressing
CD44s cancer cells. Starting from this knowledge, HA-coated chitosan (CS)-based nanoparticles
(NPs), engineered to contain siRNAs to knockdown HIF-1, were used to target CD44s
expressing pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). Two different NPs
were formulated using low or high molecular weight (LMW or HMW) CS evaluating differences
in their internalisation by cells and correlating that with gene silencing studies.
Cells showed a slower internalisation of HMW CS/HA NPs compared to the LMW counterpart.
However, the latter were slightly more efficient in HIF-1ĂŽÂ± and its downstream target
genes knockdown. LMW CS/HA NPs were able to penetrate deeply into multicellular spheroids,
but when injected intravenously in tumour bearing mice they resulted not completely
stable (with potential decomplexation) as 48 hours post-injection most of HA was detected
in the liver and the siRNA in the kidneys. In conclusion, these results provide some
understanding on the interplay between CD44 expression, its functionality and the
underlying mechanism(s) for HA uptake and importantly, demonstrate that factors other
than the amount of CD44 receptor can play a role in the interaction with HA. However,
other methods for preparation or in vivo administration of HA/CS NPs need to be evaluated
to assess the system stability in a route different from intravenous.