Size-dependent aggregation of erythrocytes by low molecular weight hyaluronic acids of different sizes: bioactivity and quality control potential.

INTRODUCTION

Hyaluronic acid (HA) is a crucial biological molecule whose diverse functions are strongly influenced by its molecular weight. In particular, low molecular weight HA (LMW-HA) fragments-such as HA60 (average 60 kDa), HA35 (average 35 kDa), and HA24 (average 24 kDa)-exhibit enhanced tissue permeability and unique interactions with cell surfaces compared to high molecular weight HA (HMW-HA). This study investigates the size-dependent aggregation effects of LMW-HA on erythrocytes and examines the implications for bioactivity, quality control, and therapeutic applications.

METHODS

We investigated the effects of LMW-HA fragments on erythrocyte aggregation across molecular sizes using erythrocyte sedimentation rate (ESR) assays, CD44 receptor blocking assays, and molecular weight assessment via gel electrophoresis and GPC-MALLS. LMW-HA samples were applied at varying concentrations to measure their binding affinity to erythrocytes, while CD44 antibodies were used to assess receptor involvement. Species-specificity of aggregation was examined by comparing erythrocytes from different animals.

RESULTS

LMW-HA induced erythrocyte aggregation in a size-dependent manner, with HA60 exhibiting the strongest binding affinity, followed by HA35 and HA24. Aggregation was partially reversible and could be inhibited by CD44 antibodies, indicating a receptor-mediated interaction. Minimum effective concentrations for aggregation were inversely related to molecular weight, with lower molecular weight fragments requiring higher concentrations. Species-specific effects were also observed, highlighting variations in erythrocyte-HA interactions across different animals.

DISCUSSION

The study suggests that LMW-HA facilitates erythrocyte aggregation through CD44-mediated binding, offering insights into HA's role in erythrocyte physiology and its effects on blood rheology. The findings support the potential of LMW-HA for therapeutic applications in pain and inflammation management, given its enhanced tissue permeability and reversible interaction with erythrocytes. Additionally, the size-dependent aggregation provides a valuable parameter for quality control, enabling consistency in LMW-HA products. These results underscore the importance of molecular weight in determining HA's physiological and pharmacological activity, paving the way for further clinical research to confirm species-specific effects and optimize safe therapeutic uses of LMW-HA.