Polyphenol-stabilized coacervates for enzyme-triggered drug delivery

Abstract

Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery.

Fig. 1. Size-tunable coacervate driven by YR-heparin interactions.

a Schematic illustration of coacervate design using heparin and a YR-based short peptide. b DLS data of C2-heparin coacervates, showing their nano (198 ± 3.3 nm) or micro (>1 µm) sizes. c UV-vis spectra of nano- and micro-coacervates. Formation of coacervates as a function of different peptide (d) and heparin (e) concentrations. Six different peptide sequences (details described in Table 1) are examined to study the impact of the charge, concentration, number, thrombin recognition site, and length of YR-based peptides for heparin coacervation. The blue area indicates coacervate formation (i.e., phase separation). Red and blue dots indicate nano- and micro-sized coacervate formation, while empty dots represent no coacervate formation. f The photograph shows the increased turbidity as a function of coacervate formation. g High encapsulation efficiency of nano- and micro-coacervates. Eight dots indicate the encapsulation efficiency of eight independent coacervate samples. Stability test of nano-coacervates in different conditions (h) including PEG2000, citric acid, urea, Triton X-100, SDS, DMF, and DMSO, and different pH (i). j M-NTA images of nano- and micro- coacervates. Small blue dots represent monodispersed nano-coacervates, and large white dots indicate scattered micro-coacervates. The experiment was repeated three times independently with similar results. Data in (h) and (i) represent mean ± SD (n  =  3). Figure 1/panel (a) Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 2. Thrombin-responsive nano-coacervates.

a Schematic illustration of heparin release through disassembly of nano-coacervates driven by thrombin proteolysis. The released heparin binds antithrombin which induces thrombin inactivation. b Decreased turbidity of nano-coacervates as a function of thrombin concentrations. c Turbidity changes of C5- and C6-based nano-coacervates with and without thrombin (5 µM). d MALDI-MOF data before and after thrombin cleavage, confirming the mass peak of the parent (M_w: 1307.91) and its fragment (M_w: 845.63). The N-terminus of C5 was acetylated. e UV-vis and PL spectra of C7-encapsulated nano-coacervates before and after thrombin cleavage. The quenched PL signal of sulfo-Cy5.5 dyes activated as a function of nano-coacervates’ disassembly. f Time-dependent PL_{670 nm} changes driven by thrombin cleavage. g k_cat/K_M determination for C5 peptide cleavage driven by thrombin proteolysis. The thrombin (20 nM) was incubated with a fluorogenic substrate ([S]₀ = 0–30 µM, sequence shown on top in the panel box), and the product concentration at 30 min was used. Data was fit to the Michaelis–Menten equation (see Supplementary information 2.8). h Specificity test using different biological proteins including thrombin (Thr), bovine serum albumin (BSA), hemoglobin (Hemo), main protease of SARS-CoV-19 (M^pro), and α-amylase (50 U/ml). A sample without any proteins is referred to as a negative control. i Decreased absorbance of MB dye before and after the addition of thrombin. The disassembly of nano-coacervates released heparin, leading to a decrease in the absorbance of MB dye while intact nano-coacervates showed a negligible change in absorbance (Supplementary Fig. 17). j aPTT test of heparin and released heparin from the disassembly of nano-coacervates. Data in (f) and (h) represent the mean value of two independent samples. Data in (b), (c), (g), and (j) represent mean ± SD (n  =  3).

Fig. 3. Material characterizations of polyphenols-encapsulated coacervates.

a Schematic illustration of TA encapsulation within the coacervates. DLS (b) UV-vis spectra (c) and FTIR (d) of NC-TAs. The brown region in (c) and (d) indicates the appeared peaks after TA encapsulation. Stability test of NC-TAs in different pH (e) and different conditions (f) including PEG2000, citric acid, urea, Triton X-100, SDS, DMF, and DMSO. g TEM image of NC-TA_0.13. h Bright field (BF) and HAADF images of a single NC-TA_0.13 at different angles. Supplementary Figs 22–24 show multiple NC-TA_0.13 at different angles with low magnification. i, j EDX elemental mapping of a single NC-TA_0.13, showing C, N, O, and S elements which are major components of heparin, peptide, and TA. The red-dotted line indicates the region used for the EDX mapping. The scale bar in (h–j) represents 100 nm. k SEM of micro-coacervates (i.e., MC-TAs). l Confocal image of MC-TAs encapsulating TA-coumarin conjugates. The yellow box indicates a single MC-TA with high magnification that highlights the evenly distributed fluorescent signal of TA-coumarin inside the MC-TA. This result reveals that TA is encapsulated within the coacervates. The scale bar represents 5 µm. Coumarin boronic acid was linked with hydroxyl groups in TA, forming a boronate ester, and the conjugates were purified using HPLC before encapsulation (Supplementary Fig. 28). Data in (e) and (f) represent mean ± SD (n  =  3). The experiment in (g–l) was repeated three times independently with similar results.

Fig. 4. Enhanced stability of NC-TAs and their proteolytic efficiency.

a Schematic illustration of a trade-off between stability and proteolysis-based disassembly of NC-TAs. NC-TAs increased stability (b) in NaCl as a function of TA encapsulations while reducing their proteolytic efficiencies (c). Thrombin was unable to dissociate NC-TA₁. d Size profiles of NC-TA_0.13 in different biological environments. e Schematic illustration of monitoring either C7 peptide (f) or heparin-FITC (g) during disassembly of NC-TAs by thrombin. The left panels in (f, g) show a decrease in the PL activation rate of NC-TAs compared to nano-coacervates (i.e., NC-TA₀) due to improved stability in 50% human plasma. The right panels in (f, g) illustrate the addition of thrombin rapidly activates the PL intensity of C7 peptide or heparin-FITC, indicating proteolysis-driven heparin release. h Cell viability (blue) and ROS intensity (orange) of HUVEC incubating with PBS, TA, heparin, C5 peptide, and NC-TAs, respectively. i Prothrombin F1 + 2 peptide concentrations of NC-TA_0.13 TA, C5 peptide, and NC-TA_0.13 made of scramble peptide (i.e., C6) from whole human blood incubation. The inserted photo shows a strong blood clot from blood anticoagulation from NC-TA_0.13 (left) and scramble NC-TA_0.13 (right). j Residual thrombin activity in human serum and plasma. Human serum shows higher residual thrombin activity comparable to 42.5 nM of alpha-thrombin. The graphs on the right panel in (j) represent absorbance changes of nano-coacervates and NC-TA_0.13 before and after 1 h incubation in 50% human serum, showing higher stability of NC-TA_0.13 than pristine nano-coacervates. Data in (c), (f), (g), and (i) represent the mean value of two independent samples. Data in (b), (d), (h), and (j) represent mean ± SD (n  =  3).