Engineering Host-Guest Interactions for Next-Generation Heparin Biosensors.

Heparin, a polyanionic anticoagulant, is widely used clinically but requires neutralization to restore normal blood coagulation. Protamine sulfate (PS) is the standard antidote, yet limitations exist. Herein, a polycationic, cavity-containing pillar[6]arene macrocycle (P12+) demonstrates superior heparin neutralization in physiological media compared to its lower-valency analog P10+, and performance comparable to PS. MTT assays reveal low toxicity of the P12+, suggesting feasibility for in vivo use. Dynamic light scattering reveals that P12+ forms nanoscale complexes below 400 nm, smaller than PS-heparin aggregates. Remarkably, P12+ exhibits enhanced neutralization of low-molecular-weight heparin, a known limitation of PS. Additionally, heparin detection is achieved via a host-guest indicator displacement assay (IDA) using methyl orange (MO). UV-vis studies show ∼80% MO release with P12+, compared to ∼40% with P10+ in the presence of heparin. This response is detectable in undiluted human plasma for P12+, whereas P10+ shows a suitable response in 5% diluted plasma. A fluorescence-based IDA enables sensitive detection down to 0.11 U/mL, below the therapeutic range (0.80-2.00 U/mL). Molecular dynamics simulations support these findings, revealing that weaker host-guest interactions and favorable electrostatics in P12+ facilitate efficient MO release.