How Do Amphiphilic Biopolymers Gel Blood? An Investigation Using Optical Microscopy

Abstract

Amphiphilic biopolymers such as hydrophobically modified chitosan (hmC) have been shown to convert liquid blood into elastic gels. This interesting property could make hmC useful as a hemostatic agent in treating severe bleeding. The mechanism for blood gelling by hmC is believed to involve polymer–cell self-assembly, i.e., insertion of hydrophobic side chains from the polymer into the lipid bilayers of blood cells, thereby creating a network of cells bridged by hmC. Here, we probe the above mechanism by studying dilute mixtures of blood cells and hmC in situ using optical microscopy. Our results show that the presence of hydrophobic side chains on hmC induces significant clustering of blood cells. The extent of clustering is quantified from the images in terms of the area occupied by the 10 largest clusters. Clustering increases as the fraction of hydrophobic side chains increases; conversely, clustering is negligible in the case of the parent chitosan that lacks hydrophobes. Moreover, the longer the hydrophobic side chains, the greater the clustering (i.e., C12 > C10 > C8 > C6). Clustering is negligible at low hmC concentrations but becomes substantial above a certain threshold. Finally, clustering due to hmC can be reversed by adding the supramolecule α-cyclodextrin, which is known to capture hydrophobes in its binding pocket. Overall, the results from this work are broadly consistent with the earlier mechanism, albeit with a few modifications.

Foams with Enhanced Rheology for Stopping Bleeding

“…The resulting hmC will have C 16 hydrophobes with a degree of hydrophobe modification of 1.5%. That is, the reaction is expected to follow the stoichiometry, as verified in previous studies. , The hmC is soluble in water at acidic pH.…”

“…To probe the interactions between the foams and blood, we also resorted to optical microscopy. From previous studies, we know that hmC and hmA can bind and connect blood cells through hydrophobic interactions. − In the case of a foam stabilized by one or both of these polymers, evidence for such interactions should arise around the gas bubbles. This was experimentally demonstrated in our previous study with hmC foams by bright-field microscopy, and a similar procedure is adopted here.…”

“…The following procedure, adapted from our previous work, − was used. First, 1 wt % chitosan was first dissolved in 0.2 M acetic acid.…”

“…That is, the reaction is expected to follow the stoichiometry, as verified in previous studies. 17,19 The hmC is soluble in water at acidic pH.…”

“…Following expansion, the foam forms a barrier (due to its solid-like nature), allowing active ingredients (“hemostatic agents”) in the foam to interact with and coagulate blood. We have previously shown that hmC acts as a hemostatic agent due to its ability to connect blood cells into a physical network. − In addition, the amphiphilic hmC also stabilizes the bubbles in the foamthus, the foam can be created without surfactants, which are undesirable in biomedical applications because surfactants tend to denature proteins and are often cytotoxic. Surfactant-free hemostatic foams based on hmC have been used to arrest bleeding from severe, lethal injuries in pigs and rats. ,, …”