title: Hyaluronic Acid Interactions With Cell Surface Receptors - Understanding Cell Binding and Internalization of a Popular Biomaterial
Creators: name: Campolungo, Matilde
Publisher: name: The Open University
Year: 2025

description: This thesis focuses on a detailed (molecular) understanding of the biophysical and cellular interactions of hyaluronic acid (HA) with some of the proteins that can bind it. Specifically, this project has tackled the link between molecular descriptors. macromolecular properties on the one hand, and ligand-receptor interactions, cellular recognition and internalization mechanisms on the other hand.<br><br>
The best known HA-binding proteins are receptors, and most of them (e.g. CD44, LYVE-1, and TSG-6) share a domain of around 100 amino acids through which HA binding takes place. However, also several extracellular matrix proteoglycans (PGs) interact with HA, and do so through a repeated LINK (B and B’ domains) in their core protein. Together, these receptors and PGs form the so-called LINK family.<br><br>
CD44 is possibly the best known LINK receptor, and has a rich physio-pathology – in particular it is overexpressed and activated in solid tumors. HA is best known as a biomaterial, but is indeed widely studied in drug delivery for its preferential internalization in tumour cells and tumoritropic accumulation, which is often ascribed to CD44 overexpression. Therefore, here I have always used a perspective of (tumour-)targeted delivery: the possibility to design a formulation that addresses a cargo where it is needed and avoids off-target effects is necessarily based on the mechanistic understanding of the HA interaction with cell surface receptors. The aim of this thesis was therefore to: 1) quantitatively analyze (i.e., using mathematical models) the available data on HA’s physicochemical properties and thanks to them better understand HA interactions with cell surface receptors; 2) shed light on the uptake mechanism of HA, namely binding and internalization, particularly regarding the receptors involved and the influence of parameters such as molecular weight, treatment concentration in the process; 3) understand the influence of chain modification on HA physico-chemical properties and <i>in vitro</i> behaviour.<br><br>
Under biologically relevant conditions, HA is negatively charged; the strong anionic character and its pattern of intramolecular hydrogen bonds together make HA adopt a four-fold helical conformation. This secondary structure is generally referred to as a ‘twisted ribbon’: the ‘faces’ of HA saccharidic units have a markedly different polarity, and their rotation along the chain axis makes them expose an alternation of hydrophobic and hydrophilic regions. The defining point of this secondary structure is its rigidity (large persistence length), due to the combination of electrostatic repulsion, intra-chain H-bonds and scarce propensity to attractive interactions between chain segments.<br><br>
The combination of rigidity and high molecular weight make the HA’s coil dimensions excessively large and therefore HA incorporates a very large number of water molecules within its coil. This is a massive hurdle for molecular dynamics simulations, leading to the common use of short oligosaccharides instead of a full-length polymer. However, this is an oversimplification because length affects HA’s chain properties (excluded volume effects increase much its rigidity with increasing MW) and interactions (the avidity of its polymer chain, which increases nonlinearly with chain length); therefore, we have embarked on a complex study aiming to evaluate the effect of macomolecular and receptor avitidy on the interactions with predictions of the binding affinity obtaining data crucial in determining HA-receptor binding strength. During this study, and specifically in the course of our detailed analysis of LINK-family secondary structures, we have also identified a number of structural similarities between B domains and receptors, and likely functional similarities between B’ domains and some ancillary features of receptors (e.g. the ‘basic cluster’ of CD44), which leads us to hypothesize HA binding as kind of cooperative binding between a principal and a secondary binding unit.<br><br>
With cells, however, HA uptake is more complex, since a) one can identify two, possibly three separate phases, i.e. suface binding, endocytosis and possibly surface trafficking in between the two. In all this, multiple receptors may and probably are involved. Our findings show that in glioblastoma cells (the cellular model used in this study) the initial binding phase, which appears to be an equilibrium (hence reversible) process, likely involves a non-specific but yet unidentified scavenger receptor. During or after this first phase of binding, HA chains are degraded; in the successive internalization phase, which involves CD44 or is orchestrated by it, HA is already reduced to short fragments and they are the only form of exogenous HA that can be found within the cells. Interestingly, part of the material is then released by exocytosis.<br><br>
It is important to note that in drug delivery formulations, HA often is chemically modified. We have shown that chain functionalization significantly alters both HA physical chemistry and cell uptake. The derivatization typically leads to disruption of intra-chain hydrogen bonds, and as a result to a reduction in macromolecular dimensions and in static chain rigidity. We have also observed that HA binding decreased much already at very low degrees of functionalization, lower than those for which significant changes in the coil structure start being apparent. This effect may be consisten to both the involvement of surface molecules other than the postulated scavenging receptos, and a different capacity of recognition. Understanding this will be critical for the design of targeted formulations, in that a lower recognition capacity may be detrimental. However, functionalization may also have advantageous traits: for example, the endocytosis phase was more efficient for the functionalized than for the native HA, which is a positive factor for the effectiveness of a treatment.