Heparin Inclusion in Cyclodextrins: A Combined MD and ITC Study

Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides widely used as host molecules capable of forming inclusion complexes
with a variety of guest compounds. In this study, for the first time, we investigated the potential interactions between
α-, β -, and γ-CD and heparin (HP), a highly sulfated glycosaminoglycan, using a combined theoretical and experimen-
tal approach. All-atom molecular dynamics (MD) simulations showed that HP does not enter the CD cavity, instead
remaining adsorbed on the external surface at the secondary cavity of the CD. Free energy calculations using LIE
and MM-GBSA confirmed that complex formation is enthalpically unfavorable in water, with desolvation penalties
outweighing van der Waals and electrostatic contributions. The binding observed in the MD simulations could be,
therefore, driven by the entropy. Potential of mean force (PMF) analysis further demonstrated that HP translocation
through the γ-CD cavity requires overcoming a high energy barrier of ≈ 23 kcal/mol, indicating that the inclusion is
not spontaneous. Complementary isothermal titration calorimetry (ITC) measurements in aqueous buffer also showed
negligible enthalpy changes, suggesting that complex formation is not driven by detectable heat effects and is predomi-
nantly entropically controlled. Overall, our findings highlight the limitations of natural cyclodextrins in encapsulating
large, highly charged polysaccharides such as HP, emphasizing the need for their chemical modifications to enable
effective host–guest complexation in such systems potentially designed to encapsulate this cargo