Polyphenol-stabilized coacervates for enzyme-triggered drug delivery

Coacervate droplets—membraneless, phase-separated condensates of macromolecules—have attracted interest as protocell models and biomedical carriers but suffer from poor stability (coalescence/collapse) and lack of selective permeability. Prior strategies stabilize coacervates by adding external membranes (polymers, lipids, erythrocyte, polysaccharide layers) or by liposomal encapsulation; however, these limit permeability, hindering payload exchange. The authors aim to create a stable yet permeable, enzyme-responsive coacervate for controlled heparin delivery aligned with physiological hemostatic feedback. By assembling heparin with short tyrosine–arginine (YR) peptides containing a thrombin-cleavable motif, they hypothesize thrombin will trigger coacervate disassembly and heparin release proportionally to thrombin activity, minimizing bleeding risk at normal thrombin levels. Additionally, they propose integrating polyphenols (tannic acid) within the coacervate to enhance stability in biofluids without compromising proteolytic responsiveness.

The study builds on work showing coacervates formed from peptides, polymers, and RNAs as models of membraneless organelles and protocells, and prior efforts to stabilize coacervates via membranes (terpolymers, phospholipids, erythrocyte and polysaccharide layers), or liposomal encapsulation enabling stimulus responses (pH, osmotic gradients, temperature). These approaches improve stability but reduce permeability to large biomolecules. Mussel-inspired chemistries (e.g., DOPA/lysine interactions) inform the choice of YR-rich peptides to engage in electrostatic, hydrophobic, hydrogen bonding, and π–π interactions with heparin. Heparin’s clinical utility and challenges in monitoring motivate controlled, enzyme-triggered release strategies. Polyphenol-mediated supramolecular networks are known to enhance particle stability and adhesion, suggesting their potential to stabilize coacervates while preserving bioactivity.

Design and materials: Short YR-based peptides were synthesized (C1: YR; C2: YRYR; C3: YRYRYRYR; C4: YRGGGGGGYR; C5: YRLVPRGSYR with thrombin site; C6: scramble YRSLRGPVYR; C7: Sulfo-Cy5.5-labeled C5; C8: Cy5.5-YRLVPRGSYRC(Cy3) fluorogenic substrate). Heparin (avg Mw ~15 kDa) served as the polyanion cargo. Peptides were confirmed by MALDI-TOF. Coacervate formation and sizing: Peptides (0.05–1.5 mM) were mixed with heparin (0.25–50 U/ml). Phase separation was monitored by turbidity/UV–vis and size by DLS and M-NTA. Dependence on peptide valence and heparin concentration was mapped; C1 did not form coacervates, C2/C3 did (70 nm to >1 µm). C4 (with glycines) tested steric effects; coacervation required at least two YR units, indicating charge/valence importance over sterics. Interaction probes: Stability/disassembly tested in PEG2000, citric acid, urea (H-bond disruption), Triton X-100 and SDS (nonionic/ionic interactions), and organic solvents (DMF, DMSO for π–π disruption), and across pH. Findings implicated electrostatic, π–π, and H-bonding in assembly; stable at pH 1–5, disassembly >pH 9. Enzyme responsiveness: A thrombin-cleavable sequence (LVPR–GS) was inserted into C5 to yield enzyme-triggered disassembly. Coacervates of C5/heparin were incubated with thrombin (0.05–2.5 µM). Turbidity decrease kinetics were measured; specificity evaluated with C6 (scramble). MALDI-TOF confirmed cleavage fragments (parent m/z ~1307.91 acetylated; fragment m/z 845.63 for YRLVPR). Optical reporter assays: C5 was labeled (C7) to monitor disassembly by PL activation. Co-assembly quenched sulfo-Cy5.5 PL and red-shifted absorbance; thrombin cleavage restored absorbance at 676 nm and PL at 700 nm. Kinetics measured across thrombin concentrations. Enzyme kinetic parameters were determined using fluorogenic substrate C8 and Michaelis–Menten fitting to obtain kcat/KM. Specificity tests used BSA, hemoglobin, SARS-CoV-2 main protease, and α-amylase. Heparin release and function: Methylene blue assay quantified released heparin (decrease at 666 nm; redshift to 566 nm upon complex formation). Activated partial thromboplastin time (aPTT) assessed anticoagulant function of released heparin versus intact coacervates and components. Polyphenol integration (stabilization): Tannic acid (TA) was encapsulated within nano- and micro-coacervates at pH 8.5 (0.03–1 mM TA; typical NC-TA0.05, 0.25, 0.5 and optimized NC-TA0.13). Samples were purified by low-speed centrifugation (3×g). Characterization by DLS (sizes ~220–276 nm; PDI ≤ 0.1), UV–vis (near-UV increase; color shift to yellow-brown), FTIR (C–O vibration ~1320 cm−1; 1,3-disubstituted benzene ring features 1100–700 cm−1). Stability was tested versus pH (stable to pH 10; disassembly beyond 11) and solvents (DMF, DMSO, SDS). Electron microscopy: TEM/SEM imaged morphology; tomography (−30° to 60°) visualized structural integrity and interface with substrate. HAADF-EDX mapping confirmed C, N, O, S consistent with TA, peptide, heparin. Coumarin-boronic acid–TA conjugates (HPLC-purified) enabled confocal imaging, showing uniform internal TA distribution. Trade-off mapping (stability vs proteolysis): NaCl (1 M) turbidity retention quantified stability at increasing TA loadings (0 to 1 mM). Proteolytic responsiveness assessed by turbidity decrease with thrombin (0.06–1 µM) across TA levels; excessive TA reduced proteolysis. A critical TA level (e.g., NC-TA0.13) balanced stability and enzyme responsiveness. Biofluid stability and enzyme-triggered release: NC-TA0.13 was challenged in diverse conditions (glutamine, glucose 5.6 mM, human albumin 0.6 mM, DPBS, NaOH pH 10, 60 °C, NaCl 150 mM, fibrinogen 8.8 µM, 50% DMEM, human serum, saliva, urine) and 50% human plasma. Disassembly monitored by PL activation using encapsulated C7 or heparin-FITC. Thrombin (500 nM) addition probed enzyme-accelerated release. Biocompatibility: HUVEC and HEK293 cytotoxicity via resazurin; ROS via DCF-DA. Ex vivo coagulation assays: Whole human blood (EDTA; IRB-approved, one male donor). Treatments (free heparin 0.6 U/ml, C6, TA, NC-TA0.13, scramble NC-TA0.13) were added; CaCl2 initiated clotting. Prothrombin fragment F1+2 (ELISA) quantified coagulation; visual thrombus observation recorded. Residual thrombin activity in human serum vs plasma was measured with a chromogenic substrate. Coacervate stability in 50% serum compared between NC and NC-TA0.13 by absorbance at 500 nm before/after 1 h incubation.

• Coacervate formation requires at least two YR units: C2 (YRYR) and C3 form nano- to micro-coacervates (70 nm to >1 µm), whereas C1 (YR) does not. Charge/valence drives phase separation more than peptide length/sterics (C4 with glycines behaves like C2).
• Size-tunable assembly: By adjusting peptide (0.05–1.5 mM) and heparin (0.25–50 U/ml) concentrations, droplets range from ~70 nm to >1 µm. Nano-coacervates show high loading efficiency (99.5–100%) and maintain size under mild centrifugation (7×g) without coalescence.
• Interaction mechanisms: Disassembly in urea, Triton X-100, SDS, DMF, and DMSO implicates electrostatic, π–π, and hydrogen bonding; stable at pH 1–5; disassembly >pH 9 due to arginine guanidinium deprotonation.
• Enzyme-responsive disassembly: Thrombin (0.05–2.5 µM) induces concentration-dependent turbidity decrease of C5-based nano-coacervates; scramble C6 shows negligible response. MALDI-TOF confirms thrombin cleavage (parent ~1307.91; fragment m/z 845.63 YRLVPR).
• Optical reporting and kinetics: Sulfo-Cy5.5-labeled C5 (C7) PL is quenched in coacervates and reactivates upon thrombin cleavage (absorbance shift to 676 nm; PL at 700 nm). PL activation kinetics scale with thrombin concentration. kcat/KM for thrombin cleavage of C8 is ~0.91 µM⁻1, comparable to fibrinogen conversion (~1.88 µM⁻1). Specificity: No PL activation with BSA, hemoglobin, SARS-CoV-2 main protease, or α-amylase (5 µM).
• Heparin release and anticoagulant function: Methylene blue assay detects heparin release only after thrombin-induced disassembly; intact coacervates show negligible release. aPTT confirms functional anticoagulation by released heparin; C5 alone shows coagulation.
• Polyphenol stabilization: TA encapsulation yields NC-TAs with sizes ~222–276 nm (PDI ≤ 0.1) and spectral/FTIR signatures of TA (C–O at 1320 cm⁻1; benzene ring features 1100–700 cm⁻1). Stable to pH 10; disassembly in DMF, DMSO, SDS indicates electrostatic and π–π roles. TEM/SEM/tomography show preserved spherical morphology and enhanced structural integrity versus collapse without TA. EDX maps C, N, O, S in single particles. TA distributes throughout the coacervate interior (confocal with TA–coumarin).
• Stability–proteolysis trade-off: In 1 M NaCl for 1 h, turbidity retention (Tafter/Tbefore) increases with TA: NC-TA0 ~7%; NC-TA0.17 ~36%; NC-TA0.33 ~53%; NC-TA1 ~92%. However, excessive TA reduces thrombin-induced disassembly; NC-TA1 shows negligible proteolysis. An optimal TA level (e.g., NC-TA0.13) preserves proteolytic responsiveness while enhancing stability.
• Biofluid performance: In 50% human plasma, NC-TA0.13 shows a 3.3-fold slower PL activation (greater stability) than unstabilized NC; addition of thrombin (500 nM, physiologic range) accelerates disassembly 4.2-fold and rapidly restores PL (heparin-FITC PL increases 1.8-fold faster, reaching plateau within ~10 min). No background quenching by plasma or TA.
• Biocompatibility: Component materials and NC-TAs show high cell viability (>83%) and minimal ROS in HUVEC; NC-TA0.13 exhibits low cytotoxicity in HEK293.
• Ex vivo anticoagulation: In whole human blood, NC-TA0.13 reduces prothrombin fragment F1+2 to levels comparable to free heparin (0.6 U/ml), indicating effective anticoagulation via released heparin; TA alone, C5, and scramble NC-TA0.13 show strong thrombus formation.
• Serum vs plasma thrombin activity: Human serum exhibits ≥20-fold higher residual thrombin activity than plasma, comparable to ~42.5 nM α-thrombin. After 1 h in 50% serum, nano-coacervates without TA show a 56% absorbance decrease at 500 nm (instability), whereas NC-TA0.13 shows only an 18% decrease (enhanced stability).

The work addresses the core challenge of coacervate instability and lack of selective permeability by introducing internal polyphenol (tannic acid) networks that preserve droplet integrity in complex biofluids while maintaining enzymatic accessibility. Incorporating a thrombin-cleavable site into the peptide scaffold creates a feedback mechanism where elevated thrombin triggers coacervate disassembly and heparin release, directly aligning anticoagulant delivery with coagulation activity. The identification of a critical TA loading that balances stability and proteolytic responsiveness is central, enabling robust performance in plasma and serum without sealing off the interior like impermeable membranes. The system exhibits high specificity for thrombin over other proteins/enzymes, therapeutically relevant kinetics (kcat/KM ~0.91 µM⁻1), and effective anticoagulation in whole blood comparable to free heparin, supporting its translational potential. These findings advance coacervate-based platforms for responsive drug delivery, biosensing of protease activity, and construction of hybrid protocells with tunable permeability and stability.

This study introduces a peptide–heparin coacervate platform that is both enzyme-responsive and stabilized by internal polyphenol networks. Key contributions include: (1) a minimal YR valence criterion for coacervation and size-tunable droplets; (2) a thrombin-cleavable peptide enabling proportional, enzyme-triggered heparin release; (3) tannic acid–mediated stabilization that maintains permeability and proteolytic activity at an optimal loading (e.g., NC-TA0.13); and (4) validated stability and functionality across biofluids, with effective anticoagulation in whole blood and good biocompatibility. Future work will explore integration on medical devices (e.g., drug-eluting stents) for on-demand anticoagulation, assess inflammatory and hemorheological impacts (plasma viscosity, procalcitonin, C-reactive protein), and investigate in vivo pharmacokinetics and clearance (phagocyte uptake, renal/hepatic/splenic elimination), broadening applications to biomedicine, protease sensing, and hybrid protocell systems.

• The balance between stability and proteolysis is sensitive to TA loading; excessive polyphenol content inhibits enzymatic disassembly.
• Most data are in vitro or ex vivo (50% plasma/serum; whole blood from a single male donor); in vivo efficacy, biodistribution, and long-term safety were not assessed.
• Sex as a biological variable was not investigated.
• While specificity was tested against several proteins/enzymes, broader protease panels and potential off-target interactions in vivo remain to be evaluated.
• Mechanical and hemodynamic factors relevant to device coatings (shear, flow) and immunological responses were not fully characterized; future studies are needed to assess inflammatory markers and clearance pathways.