A synthetic biomolecular condensate for on-demand anticoagulation therapy

Abstract

A thrombin-responsive coacervate composition comprising a coacervate comprising YR-based peptides having a thrombin cleavage sequence, an anticoagulant, and a polyphenol to stabilize the coacervate structure; and methods of using the thrombin-responsive coacervate to selectively release an anticoagulant only when thrombin is present.

Description

Attorney Docket No. 009062.8550.WO00A SYNTHETIC BIOMOLECULAR CONDENSATE FOR ON-DEMAND ANTICOAGULATION THERAPYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under HL137187 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0002] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on August 12, 2025, is named 009062.8550.WO00. xml and is 27,120 bytes in size.CROSS-REFERENCE TO RELATED APPLICATION(S)

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 684,267, filed on August 16, 2024, the contents of which is incorporated by reference in its entirety.BACKGROUND

[0004] Coacervate droplets are cell-like compartments made of a condensed solution of macromolecules such as peptides (1 ), copolymers (2), RNAs (3), and a diluted phase of the remaining liquid (4). They form via liquid-liquid phase separation driven by non-covalent interactions such as electrostatic (5), hydrophobic (6), and hydrogen bonding (7). The rich concentrations of biomolecules within the coacervates often mimic the physicochemical environments of living cells. Thus, coacervates have been extensively studied to understand the early stage of cell evolution (8-10). The physical properties of coacervate droplets rely on the structural and chemical properties of their constituent building blocks (6). However, the lack of membranes often leads to a rapid coalescence or collapse of the coacervate phases (i.e., poor stability) (1 1 ,12). The absence of a physical membrane also limits their ability to mimic the selective permeability of cellular membranes (13,14). These limitations-1 -182827596.7Attorney Docket No. 009062.8550.WO00 challenge the promise of coacervate droplets for protocell models (14), biomedicine (15), drug delivery (16), and biosensing (17) applications.

[0005] To address this stability issue, researchers have largely focused on developing hybrid protocell models consisting of coacervate-based interiors surrounded by membranes such as terpolymer (2), phospholipids (18), erythrocyte (19), and polysaccharide (20) layers. These surrounding membranes either coat or encapsulate the coacervate droplet, which in turn enhances its stability. In the coating approach, a coacervate droplet is used as a template: Two opposite-charged building blocks initially form complex coacervates followed by the in situ formation of membranes (18-21 ). Alternatively, encapsulation of coacervates within liposomes is based on microfluidic techniques. Coacervates encased within liposomes have shown great potential in the development of a bio-responsive platform capable of reacting to changes in pH (22), osmotic gradient (23), and temperature (24). However, these strategies often result in limited permeability of the surrounding membrane, which can hinder the penetration and / or release of large biomolecules (22,25).

[0006] Thus, additional work is needed to establish stable coacervate droplets for therapeutic and research applications.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 illustrates a schematic of mussel foot protein (SEQ ID NO: 23). Mytilus edulis foot protein 5 found from a byssus of mussel is made of repeating units of DOPA and lysine (K) amino acids, which provide strong wet adhesive properties through hydrophobic and positive charge. (75)

[0008] FIGS. 2A-2J illustrate size-tunable coacervate driven by YR-heparin interactions. Here, Y denotes tyrosine, and R denotes arginine. FIG. 2A is a schematic illustration of coacervate design using heparin and a YR-based short peptide. FIG. 2B shows DLS data of NH2 -YRYR-Am (SEQ ID NO: 2, “C2”) -heparin coacervates, showing their nano (198 ± 3.3 nm) or micro (>1 pm) sizes. FIG. 2C is a UV-vis spectra of nano- and microcoacervates. Formation of coacervates is illustrated as a function of different peptide (FIG. 2D) and heparin (FIG. 2E) concentrations. Six different peptide sequences (details described in Table 1 ) are examined to study the impact of the charge, concentration,-2-182827596.7Attorney Docket No. 009062.8550.WG00 number, thrombin recognition site, and length of YR-based peptides for heparin coacervation. The dashed area indicates coacervate formation (i.e., phase separation). Stars and filled dots indicate nano- and micro-sized coacervate formation, while empty dots represent no coacervate formation. FIG. 2F is a photograph that shows the increased turbidity as a function of coacervate formation. FIG. 2G shows high encapsulation efficiency of nano- and micro-coacervates. Eight dots indicate the encapsulation efficiency of eight independent coacervate samples. Stability test of nano-coacervates is illustrated in different conditions (FIG. 2H) including PEG2000, citric acid, urea, triton, SDS, DMF, and DMSO, and at different pH values (FIG. 21). FIG. 2J shows M-NTA images of nano- and microcoacervates. Small dots represent monodispersed nano-coacervates, and large dots (e.g. lower panel of FIG. 2J) indicate scattered micro-coacervates. The experiment was repeated three times independently with similar results. Data in FIG. 2H and FIG. 21 represent mean ± SD (n = 3).

[0009] FIG. 3 illustrates different-sized coacervate droplets. 02 peptides at different concentrations of 0.35, 0.5, 0.625, 0.75, and 1 mM were used to induce coacervates at a fixed concentration of heparin (50 U / ml). The average hydrodynamic diameters of 0.35, 0.5, 0.625, 0.75, and 1 mM were 101 .3 ± 2.1 nm, 133.2 ± 3.8 nm, 198.5 ± 3.3 nm, 235.9 ± 4.0 nm, and 662.2 ± 1 1 .0 nm, respectively. Nano-coacervates (sizes below 300 nm) maintained their colloidal stability and size while coacervates with a size over 500 nm continuously grew and became micro-sized coacervates. Data represent mean ± SD (n = 3).

[0010] FIGS. 4A-4C illustrate UV-vis spectra of nano- and micro-coacervates. Illustrated are UV-vis spectra of nano-coacervates (FIG. 4A) and micro-coacervates (FIG. 4B) in different dilution ratios: the ratio of the sample to the solvent was 1 to 1 , 1 .25, 1 .6, 2.5, and 5, respectively. As shown in FIG. 4G, extinction (i.e., absorbance) at 500 nm of nano- and micro-coacervates is dependent on particle concentration explained by Beer’s law. Data represent mean ± SD (n = 3).

[0011] FIGS. 5A-5G illustrate mass peaks of C1 -C6 peptides. Illustrated are electrospray ionization mass spectrometry (ESI-MS) data of C1 (FIG. 5A), C2 (FIG. 5B), C3 (FIG. 5C), and C4 (FIG. 5D) peptide sequences as well as MALDI-TOF data of C5 (FIG. 5E) and C6 (FIG. 5F) peptide sequences (FIG. 5G), in addition to the chemical structure of the-3-182827596.7Attorney Docket No. 009062.8550.WQ00C1 , C2, C3, C4, C5, and C6 sequences, respectively (SEQ ID NOS: 2-6) . The aromatic rings and guanidine group indicate Y and R peptides, respectively that provide hydrophobicity and positive charges to interact with heparin.

[0012] FIG. 6 consists of photographs of C1 -, C2-, and C3-based coacervates (SEQ ID NOS: 2 and 3). C2 and C3 peptides with different concentrations ranging from 0.05 to 1 .5 mM were combined with a constant heparin concentration of 50 U / ml, thus forming coacervate droplets. In contrast, there was no formation of coacervates when C1 peptides were mixed with heparin. The photographs show that C2 and C3 peptides became turbid as a function of coacervate formation, but there was no turbidity change in 01 -heparin mixtures (i.e., they remained transparent).

[0013] FIGS. 7A-7C illustrate M-NTA data of C1 -, C2-, and C3-based coacervation. Shown are multispectral nanoparticle tracking analysis (M-NTA) images of 01 -based (FIG. 7A), 02-based (FIG. 7B), and 03-based (FIG. 70) coacervates. 02 and 03 peptides formed nano- and micro-sized coacervates, respectively, while C1 peptides failed to form coacervates. M-NTA size profiles of nano-coacervates (FIG. 7B) and micro-coacervates (FIG. 70) are shown. The average size and standard deviation are measured by M-NTA.

[0014] FIGS. 8A-8F illustrate UV-vis spectra of C1-C6-based coacervates (SEQ ID NOS: 2-6). Different peptide sequences including C1 (FIG. 8A), C2 (FIG. 8B), C3 (FIG. 8C), 04 (FIG. 8D), 05 (FIG. 8E), and 06 (FIG. 8F) were examined to form coacervates with fixed heparin concentration of 50 U / ml. The results showed that the number of YR is important to form nano- or micro-coacervates: The single YR unit failed to form coacervates due to a lack of charge and hydrophobicity. Extinction spectra of nano-coacervates increased from 900 to 300 nm while micro-coacervates showed a broad extinction spectrum likely due to increased light scattering. In addition, C4-based coacervation showed similar extinction spectra as 02- based coacervation. Likewise, 05 and 06 peptides that have the same number of YR units showed similar extinction spectra.

[0015] FIGS. 9A-9D illustrate UV-vis spectra of C1-C4-based coacervates with the fixed peptide concentrations. Shown are UV-vis spectra of 01 (FIG. 9A), 02 (FIG. 9B) (SEQ ID NO: 2), C3 (FIG. 9C) (SEQ ID NO: 3), and 04 (FIG. 9D) (SEQ ID NO: 4) coacervate formation with various heparin concentrations ranging from 0.5 to 1000 U / ml. The peptide-4-182827596.7Attorney Docket No. 009062.8550.WQ00 concentration was constant (1 mM). The C2, C3, and C4 peptides could form nano- or microsized coacervates with heparin.

[0016] FIGS. 10A and 10B illustrate calculation of heparin encapsulation efficiency. FIG. 10A shows UV-vis spectra of MB-heparin complex. MB dyes were mixed with different concentrations of heparin-inducing MB-heparin aggregates that in turn decreased the absorption peak to 666 nm and increased the peak to 580 nm. FIG. 10B shows an encapsulation efficiency formula built using the decreased extinction value at 666 nm.(76) Data represent mean ± SD (n = 3).

[0017] FIGS. 11 A and 1 1 B illustrate stability test of nano-coacervates in different environments. Nano-coacervates (average size: 200 nm) at the same concentration of 1 mM were incubated in various conditions including MQ water (as a negative control), PEG2000 (10 mg / ml), citric acid (pH 2), 10 mM of urea, NaCI, SDS (10 mg / ml), 80% of DMF, and DMSO, respectively. As shown in FIG. 11 A, UV-vis spectra illustrated a decrease in the extinction of nano-coacervates because of the disassembly of nano-coacervates, confirming internal interactions for coacervation. (61 ) FIG. 11 B is a photograph of nano-coacervates in MQ water, PEG2000, citric acid, urea, NaCI, SDS, DMF, and DMSO, respectively.

[0018] FIGS. 12A-12J illustrate thrombin-responsive nano-coacervates. FIG. 12A is a schematic illustration of heparin release through disassembly of nano-coacervates driven by thrombin proteolysis of the C5 peptide (SEQ ID NO: 5). The released heparin binds antithrombin, which induces thrombin inactivation. FIG. 12B shows decreased turbidity of nano-coacervates as a function of thrombin concentrations. FIG. 12C shows turbidity changes of C5- and 06-based nano-coacervates (SEQ ID NO: 5 and 6) with and without thrombin (5 pM). FIG. 12D shows MALDI-MOF data before and after thrombin cleavage, confirming the mass peak of the parent (Mw: 1307.91 ) and its fragment (Mw: 845.63). FIG. 12E shows 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. FIG. 12F shows time-dependent PL670 nm changes driven by thrombin cleavage. FIG. 12G shows kcat / Kw determination for Cy5.5 labeled (SEQ ID NO: 8) C5 peptide cleavage driven by thrombin proteolysis. The thrombin (20 nM) was incubated with a fluorogenic substrate ([S]o=O-3O pM, sequence shown on top in the panel box), and-5-182827596.7Attorney Docket No. 009062.8550.WO00 the product concentration at 30 min was used. Data was fit to the Michaelis-Menten equation. FIG. 12H shows a specificity test using different biological proteins including thrombin (Thr), bovine serum albumin (BSA), hemoglobin (Hemo), main protease of SARS- CoV-19 (Mpro), and a-amylase (50 ll / rnl). A sample without any proteins is referred to as a negative control. FIG. 121 shows decreased absorbance of MB dye before and after the addition of thrombin. FIG. 12J shows an aPTT test of heparin and released heparin from the disassembly of nano-coacervates. Data in FIG. 12F and FIG. 12H represent the mean value of two independent samples. Data in FIG. 12B, FIG. 12C, FIG. 12G, and FIG. 12J represent mean ± SD (n = 3).

[0019] FIGS. 13A-13C illustrate turbidity changes at different thrombin concentrations. Shown are UV-vis spectra of nano-coacervates before (FIG. 13A) and after (FIG. 13B) thrombin incubation. FIG. 13C shows time-dependent changes in extinction at 500 nm. Nano-coacervates (1 mM) were incubated with various concentrations of thrombin, ranging from 0.05 to 2.5 pM in 20 mM Tris-HCI buffer (pH 8.5) at 37 °C for 1 h. The results indicate that higher thrombin concentrations induce a rapid disassembly of the nano-coacervates. The C5 peptide was used for the formation of nano-coacervates.

[0020] FIGS. 14A and 14B illustrate thrombin proteolytic test on nano-coacervates composed of scramble sequence. Shown are UV-vis spectra of 05-based (FIG. 14A, SEQ ID NO: 5) and C6-based (FIG. 14B, SEQ ID NO: 6) nano-coacervates before and after thrombin incubation. The C5- and 06-based nano-coacervates at the same concentration of 1 mM were incubated with 500 nM of a-thrombin at 37 °C for 1 h. The results showed that thrombin was incapable of disassembling the nano-coacervates composed of a scramble sequence (i.e., C6).

[0021] FIGS. 15A-15C illustrate sulfo-Cy5.5 conjugation to 05 peptide (i.e., 07, SEQ ID NO: 7). FIG. 15A is a schematic illustration of NHS-amine coupling of sulfo-Cy5.5-NHS and C5 peptide: NH2-YRLVPRGSYR-CONH2 (SEQ ID NO: 5 and 7); shown are MALDI-TOF data before (FIG. 15B) (SEQ ID NO: 5) and after (FIG. 150) (SEQ ID NO: 7) sulfo-Cy5.5 conjugation, confirming that sulfo-Cy5.5 dyes were successfully conjugated to C5 peptide. Sulfo-Cy5.5 needs coupling with 05 peptide to be encapsulated within the nanocoacervates. Without conjugation with the 05 peptide, sulfo-Cy5.5 was not incorporated-6-182827596.7Attorney Docket No. 009062.8550.WQ00 within the nano-coacervates. A negatively charged sulfonate group on Cy5.5 is required to activate the fluorescent signal upon disassembly of the nano-coacervates. Without the sulfonate group, the positively charged Cy5.5 dyes strongly interacted with heparin, quenching its fluorescence even after disassembly. Note that sulfo-Cy5.5 without C5 peptide did not encapsulate into the nano-coacervates.

[0022] FIGS. 16A-16D illustrate Cy5.5-YRLVPRGSYRC(Cy3) preparation (i.e., 08, SEQ ID NO: 8). FIG. 16A is a schematic illustration of maleimide-thiol and NHS-amide couplings to prepare Cy5.5-YRLVPRGSYRC-Cy3 (i.e., C8, SEQ ID NO: 8) for kcat / KM measurement. Shown are MALDI-MOF data of peptide only (FIG. 16B), peptide-Cy3 (FIG. 16C), and Cy5.5-peptide-Cy3 (FIG. 16D). The peptide sequence used for dye-conjugation is NH2-YRLVPRGSYRC-CONH2 (SEQ ID NO: 9).

[0023] FIG. 17 illustrates kcat / KM measurement. Time-dependent Cy3 fluorescence activation driven by thrombin proteolysis is illustrated. Fluorogenic substrate (Cy5.5- YRLVPRGSYRC(Cy3), C8 peptide, SEQ ID NO: 8) with different concentrations from 1 to 24 pM was incubated with 20 nM of enzyme (i.e., a-thrombin). At least three replicates were performed to measure mean and standard deviations.

[0024] FIGS. 18A-18C illustrate specificity tests with different enzymes. Illustrated is PL intensity of the C7-encapsulated nano-coacervates before (FIG. 18A) and after (FIG. 18B) enzyme incubation. Different enzymes including thrombin, bovine serum albumin (BSA), hemoglobin, main protease of SARS-CoV-2, and a-amylase at the same concentration of 5 pM were incubated with nano-coacervates, respectively. The 07- encapsulated nano-coacervates activated the PL signal upon the disassembly of the nanocoacervates. FIG. 18C illustrates time-dependent signal activation induced by different enzymes, showing that thrombin can activate PL signals.

[0025] FIGS. 19A-19C illustrate heparin release confirmed by the formation of MB- heparin complex. FIG. 19A shows UV-vis spectra of the MB-heparin complex. The positively charged MB dye can form aggregates with the negatively charged heparin, thus reducing the absorption peak at 666 nm. Shown are UV-vis spectra of nano-coacervates mixed with the MB dye before (FIG. 19B) and after (FIG. 19C) the addition of thrombin. The release of heparin due to thrombin cleavage resulted in a decrease in absorbance at 666 nm, whereas-7-182827596.7Attorney Docket No. 009062.8550.WG00 intact nano-coacervates exhibited no change in absorbance. 60 pL of the supernatant was incubated with 20 pL of MB dye (100 pM) for absorbance measurement.

[0026] FIG. 20 illustrates an aPTT test of C5 peptide and tannic acid. Plasma coagulation was observed in aPTT tests using C5 peptide and tannic acid with different concentrations from 0.1 to 1 mM. Data represent mean ± SD (n = 3).

[0027] FIGS. 21 A and 21 B illustrate colloidal stability of nano-coacervates in biofluids. C7-encapsulated nano-coacervates were incubated in various biofluids, including 50% human plasma, urine, saliva, and DMEM, respectively. FIG. 21 A illustrates time-dependent PL intensity of the nano-coacervates containing C7 peptides. Disassembly of the nanocoacervates led to an increase in fluorescence. As shown in FIG. 21 B, nano-coacervates were unstable in human plasma, resulting in the disassembly of coacervate phases and subsequently activated PL intensity. However, the nano-coacervates incubated in human urine, saliva, and DMEM showed no PL activation, indicating its stability. Data represent mean ± SD (n = 3).

[0028] FIGS. 22A-22L illustrate polyphenols-encapsulated Q6 coacervates. FIG. 22A is a schematic illustration of TA encapsulation within the coacervates. Shown are DLS (FIG. 22B), UV-vis spectra (FIG. 220), and FTIR (FIG. 22D) of NCTAs. The region highlighted by an arrow in FIG. 22C and FIG. 22D indicates the peaks that appeared after TA encapsulation. Also shown is a stability test of NC-TAs at different pH values (FIG. 22E) and in different conditions (FIG. 22F), including PEG2000, citric acid, urea, triton, SDS, DMF, and DMSO. FIG. 22G shows a TEM image of NC-TA013. FIG. 22H shows bright field (BF) and HAADF images of a single NC-TA0.13 at different angles. FIGS. 22I, 22J show EDX elemental mapping of a single NC-TA0.13, showing C, N, O, and S elements, which are major components of heparin, peptide, and TA. The -dotted line indicates the region used for the EDX mapping. The scale bar in FIGS. 22H-22J represents 100 nm. FIG. 22K is a SEM image of micro-coacervates (i.e., MC-TAs). FIG. 22L is a confocal image of MC-TAs encapsulating TA-coumarin conjugates. The 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-8-182827596.7Attorney Docket No. 009062.8550.WG005 m. Data in FIG. 22E and FIG. 22F represent mean ± SD (n = 3). The experiment in FIGS. 22G-22L was repeated three times independently with similar results.

[0029] FIGS. 23A-23C illustrate photograph and surface charge of NC-TAs. FIG. 23A shows the chemical structure of a TA molecule. FIG. 23B shows the surface charge of nanocoacervates, NC-TAo.os, NC-TA0.25, and NC-TAo.s. The zeta potential (i.e., surface charge) of nano-coacervates, NC-TA0.05, NC-TA0.25, and NC-TA0.5 were -41 .1 ± 0.4 mV, -18.7 ± 0.1 mV, -15.3 ± 0.6 mV, and -12.6 ± 0.4 mV, respectively. FIG. 23C is a photograph of nanocoacervates, NC-TAo.os, NC-TA0.25, and NC-TAo.s. The color of the solvent changed from turbid white to yellowish-brown after TA encapsulation.

[0030] FIGS. 24A-24C illustrate SEM images of NC-TAs. SEM images clearly visualized the mono-dispersed NC-TA013 (FIG. 24A), NC-TA0.33 (FIG. 24B), and NC-TA1 (FIG. 240) dried on the silicon wafer.

[0031] FIGS. 25A-25D illustrate TEM images of nano-coacervates and NC-TAs. Shown are TEM images of nano-coacervates (FIG. 25A) and NC-TA0.13 (FIG. 25B) with low magnification. FIG. 25A indicates that nano-coacervates without TA were merged and deformed while the NC-TA0.13 maintained their shapes even in vacuum conditions. FIG. 25C shows a TEM image at high magnification of NC-TA013 on the dotted area in FIG. 25B. FIG. 25D shows TEM and HAADF images of NC-TA0.13 at the highest magnification. Note: the samples were stained with uranium for the TEM measurement.

[0032] FIGS. 26A-26F illustrate TEM and HAADF images at different angles. Shown are TEM (FIGS. 26A-26C) and HAADF (FIGS. 26D-26F) images of NC-TA0.13 at different angles of 0° (FIGS. 26A, 26D), 30° (FIGS. 26B, 26E), and 60° (FIGS. 26C, 26F). The monodispersed NC-TA0.13 were dried on a TEM grid and imaged at different angles clearly showing the heights of the NC-TA0.13. The scale bars indicate 500 nm.

[0033] FIGS. 27A-27E illustrate EDX mapping of NC-TAs at different angles. Shown are images of EDX mapping of NC-TA0.13 at 0° (FIG. 27A) and 60° (FIG. 27B), illustrating the highly mono-dispersed NC-TA0.13 samples and their heights. FIG. 27C shows an HAADF image of NC-TA0.13 at 0° and 60°. FIG. 27D shows a merged image of NC-TA0.13. FIG. 27E shows signal quantification of HAADF, C, N, O, and S elements represented by the pink--9-182827596.7Attorney Docket No. 009062.8550.WG00 dotted line in FIG. 27D. The results showed that NC-TAo .13 is composed of carbon, nitrogen, oxygen, and sulfur, which are major elements of the C5 peptide and heparin.

[0034] FIGS. 28A-28D illustrate optical images of micro-coacervates after polyphenol encapsulation. Shown are optical images of coacervate droplets before (FIG. 28A) and after TA encapsulation of 0.04 mM (FIG. 28B) and 0.16 mM (FIG. 28C). The images were collected after the centrifugation. After centrifugation, the average diameter of MC-TA was 2.01 ± 0.44 pm. The average and standard deviation represent 16 independent droplets. FIG. 28D shows a high concentration of micro-coacervates incubated with different amounts of TA from 0.04 to 0.2 mM overnight. The dotted area indicates increased contrast between coacervate droplets due to high TA concentrations. The addition of 0.2 mM TA resulted in the formation of solid aggregates. The images were obtained before centrifugations. The scale bar represents 20 pm.

[0035] FIGS. 29A and 29B illustrate SEM images of micro-coacervates. Shown are SEM images of low (FIG. 29A) and high (FIG. 29B) concentrations of MC-TAs with different magnifications. TA concentration with 0.16 mM was used for encapsulation. The image in the inset shows the spherical shapes of MC-TAs and size distributions. The average diameters of MC-TAs were 2.01 ± 0.44 pm when hydrated and 0.38 ± 0.9 pm when evaporated, respectively. The average diameter was calibrated using 16 individual MC-TAs from optical and SEM images, respectively. The average and standard deviation represent 16 independent droplets.

[0036] FIGS. 30A-30E illustrate TA-encapsulated micro-coacervates (MC-TAs). FIG. 30A shows UV-vis spectra of MC-TAs and TA only at the concentrations of 0.05 and 0.13 mM. MC-TAs increased extinction ranging from 400 to 900 nm due to the formation of coacervate droplets, while TA alone has minimal extinction. Decrease in the extinction of coacervate droplets is illustrated without TA (FIG. 30B) and MC-TA0.13 (FIG. 30C) before and after 3 x g centrifugation for 10 min. The coacervate droplets without TA showed a 98.7% decrease in turbidity, while MC-TA0.05 decreased by only 1 .4%. These results indicate that TA encapsulation improves the stability of not only nano-sized but also micro-sized coacervate droplets. FIG. 30D shows DLS data of MC-TA0.13. FIG. 30E shows the turbidity-10-182827596.7Attorney Docket No. 009062.8550.WG00 of MC-TAO 13 at different time points, illustrating its high stability. The turbidity was calibrated based on the extinction value.

[0037] FIGS. 31 A-31 F illustrate micro-coacervates encapsulating coumarin-TA conjugates. Shown are fluorescence (FIG. 31 A), bright field (FIG. 31 B), and merge (FIG. 31 C) images of MC-TAs that encapsulate TA-coumarin conjugates. MALDI-TOF (FIG. 31 D) and HPLC (FIG. 31 E) data confirmed polyphenol-coumarin conjugates. Hydroxyl groups in tannic acid can conjugate with coumarin boronic acid, leading to the formation of the boronate ester. After the reaction, the sample was purified using HPLC. FIG. 31 F shows normalized absorbance and fluorescence spectrum of TA-coumarin conjugates.

[0038] FIGS. 32A-32J illustrate enhanced stability of NC-TAs and their proteolytic efficiency. FIG. 32A is a schematic illustration of a trade-off between stability and proteolysisbased disassembly of NC-TAs. NC-TAs increased stability (FIG. 32B) in NaCI as a function of TA encapsulations while reducing their proteolytic efficiencies (FIG. 32C). Thrombin was unable to dissociate NC-TAi. FIG. 32D shows size profiles of NC-TA0.13 in different biological environments. FIG. 32E is a schematic illustration of monitoring either C7 peptide (FIG. 32F) or heparin-FITC (FIG. 32G) during disassembly of NC-TAs by thrombin. The left panels in FIGS. 32F, 32G show a decrease in the PL activation rate of NC-TAs compared to nanocoacervates (i.e., NC-TAo) due to improved stability in 50% human plasma. The right panels in FIGS. 32F, 32G show that the addition of thrombin rapidly activates the PL intensity of C7 peptide or heparin-FITC, indicating proteolysis-driven heparin release. FIG. 32H shows cell viability (square) and ROS intensity (circle) of HUVEC incubating with PBS, TA, heparin, C5 peptide, and NC-TAs, respectively. FIG. 32I shows prothrombin F1 + 2 peptide concentrations of NC-TA0.13 TA, C5 peptide, and NC-TA0.13 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-TA0.13 (left) and scramble NC-TA0.13 (right). FIG. 32J shows 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 FIG. 32J represent absorbance changes of nano-coacervates and NC-TA0.13 before and after 1 h incubation in 50% human serum, showing higher stability of NC-TA0.13 than pristine-11 -182827596.7Attorney Docket No. 009062.8550.WG00 nano-coacervates. Data in FIGS. 32C, 32F, 32G, and 321 represent the mean value of two independent samples. Data in FIGS. 32B, 32D, 32H, and 32J represent mean ± SD (n = 3).

[0039] FIGS. 33A-33D illustrate UV-vis spectra of NC-TA0.25 and NC-TA1. Shown are UV-vis spectra and time-dependent changes in the extinction of NC-TA0.25 (FIGS. 33A and 33B) and NC-TA1 (FIGS. 33C and 33D). The results indicate that thrombin could disassemble NC-TA0.25, leading to a decrease in extinction while NC-TA1 showed negligible changes in extinction. These results suggest that the increased stability through polyphenol- mediated supramolecular network could lead to a reduction in thrombin proteolytic efficiency. Thrombin with various concentrations ranging from 0.06 to 1 pM was used for the experiment.

[0040] FIGS. 34A and 34B illustrate colloidal stability of NC-TA0.13 in biofluids and different conditions. Illustrated is colloidal stability of NC-TA013 in biofluids (FIG. 34A) and different environments (FIG. 34B). DLS data showed that NC-TA0.13 were mono-dispersed (PDI <0.2) and maintained their structural stability in 50% human serum, saliva, urine, and DMEM and different conditions including DPBS, NaOH (pH 10), 60 °C, and NaCI of 150 mM.

[0041] FIGS. 35A and 35B illustrate stability test of nano-coacervates in the presence of BSA. Nano-coacervates (i.e., TA: 0 mM), NC-TA013, and NC-TA033 were incubated with (FIG. 35A) and without (FIG. 35B) BSA of 50 mg / ml at 37 °C for 3 h. The results showed that the coacervates were stable in the presence of the BSA.

[0042] FIGS. 36A-36C illustrate heparin-fluorescein (FITC). FIG. 36A shows the molecular structure of heparin-FITC, Mw 27k (Creative PEGWorks, NC, USA), and its absorbance (FIG. 36B) and fluorescence (FIG. 36C). Heparin-FITC was monitored during the disassembly of nano-coacervates and NC-TAs in human plasma and the addition of thrombin described in FIG. 32G.

[0043] FIGS. 37A and 37B illustrate no interference of PL intensity of C7 peptide by background medium. FIG. 37A shows PL intensity of the C7 (i.e., sulfo-Cy5.5-C5) peptide as it was evaluated in human serum, plasma, urine, saliva, and DMEM. The results revealed that the biofluids did not quench the fluorescence signal of the sulfo-Cy5.5. FIG. 37B shows PL intensity of C7-encapsulated NC-TA0.13 before and after the addition of SDS (50 mg / ml).-12-182827596.7Attorney Docket No. 009062.8550.WG00PL intensity of the C7 peptide increased as a function of the disassembly of NC-TA013 induced by the addition of SDS in 50% of human serum.

[0044] FIGS. 38A and 38B illustrate fluorescence images and cell viability of NC, NC- TA0.13, CTAB, and PBS. As shown in FIG. 38A, Hoechst and PI were used to stain the HEK 293 cells to visualize the cell nucleus and dead cells. CTAB and PBS solution were used as a control (dead versus alive cells). Nano-coacervates and NC-TA0.13 had negligible fluorescence signal of PI, indicating low cytotoxicity against HEK 293 cells. The scale bars represent 100 pm. FIG. 38B illustrates cytotoxicity test of NC-TAs, showing high biocompatibility of NC-TAs. Data represent mean ± SD (n = 3).

[0045] FIGS. 39A and 39B illustrate the standard curve and prothrombin fragment 1 +2 (F1 +2) concentration in human plasma. FIG. 39A shows a standard curve of F1 +2 concentration. Activated optical density (O.D.) at 450 nm was converted to F1 +2 concentrations using an equation from the fitting curve. FIG. 39B shows F1 +2 fragment concentrations from whole human blood at different heparin concentrations from 0.2 to 1 U / ml, showing anticoagulation activity of free heparin. Whole human blood was collected using an EDTA-treated blood collection tube. Calcium chloride was used to trigger blood coagulation. 0.4 mL of whole human blood was incubated with different heparin concentrations. Low heparin concentration (lower than 0.1 U / ml) induced the strong thrombus, increasing F1 +2 fragment concentrations due to thrombin activation. Data represent mean ± SD (n = 3).

[0046] FIGS. 40A-40C illustrate ELISA test of NC-TA0.13. FIG. 40A shows photographs of whole human blood after incubating with DPBS (-), heparin, nano-coacervates (referred to as NC), and NC-TA0.13. The results show that NC and NC-TA0.13 prevent blood clots similar to heparin. In contrast, DPBS resulted in blood clotting, thus increasing the concentration of prothrombin fragment F1 +2 peptide (expressed in ng / ml) shown in FIG. 40B. Data represent mean ± SD (n = 4). As shown in FIG. 40C, increased absorbance at 450 nm indicates the formation of an antigen-antibody complex of prothrombin fragment F1 +2 peptides. The heparin concentration used for this experiment was 40 U / ml. In this study, whole human blood was directly collected with the blood collection tubes that contain heparin, nano-coacervates, and NC-TA013, respectively. The statistical significance was-13-182827596.7Attorney Docket No. 009062.8550.WG00 calculated with the F1 +2 peptide of (-), heparin, NC, and NC-TAo -is’s t-test: **** and ns indicate p<0.0001 and non-significance, respectively. Data represent mean ± SD (n = 3).

[0047] FIGS. 41 A-41 D illustrate residual thrombin activity and stability test of NC-TA0.13 in human serum. As shown in FIG. 41 A, a standard curve indicates residual thrombin activity of alpha thrombin in different concentrations measured by thrombin chromogenic substrate. FIG. 41 B shows optical density (i.e., residual thrombin activity) of human serum samples with serial dilution. Different dilution factors (1 :2, 1 :4, 1 :8, 1 :16, and 1 :32) were examined, showing that human serum contained residual thrombin activity comparable to that of thrombin at a concentration of 42.5 nM. Shown are UV-vis spectra of pristine nanocoacervates (FIG. 41 C) and NC-TA013 (FIG. 41 D) in 50% human serum after 1 h incubation. Data in FIG. 41 A and FIG. 41 B represent mean ± SD (n = 3).

[0048] FIGS. 42A and 42B illustrate optical images of MC-TAs before and after blood incubation. TA-encapsulated micro-coacervates (MC-TAs) were coated on the glass slides. In FIG. 42A, optical images show the MC-TAs, maintaining spherical shapes on the glass slides after drying and incubating in PBS and DMEM for 1 h. These MC-TAs showed exceptional stability after incubating in whole human blood (100%) (FIG. 42B). The dotted circles each represent a single MC-TA before and after the incubation. The scale bar represents 50 pm.DETAILED DESCRIPTION

[0049] According to the embodiments disclosed herein, a stable and enzyme- responsive coacervate platform is disclosed. In certain aspects, such a platform includes compositions and materials that automatically release the anticoagulant heparin when thrombin (a clotting factor) levels rise and stop releasing heparin in its absence.

[0050] For example, in some embodiments, a nano-sized coacervate made of bioinspired peptides and the anticoagulant heparin was engineered. Anticoagulants, such as heparin play an important role in surgical and cardiovascular medicine due to its short halflife, reversible nature, and low cost (26). However, heparin is difficult to manage and requires blood draws and central labs (27); therefore, the controlled release of heparin via the enzymatic activity of clotting factors is gaining interest (28-32). Living organisms maintain-14-182827596.7Attorney Docket No. 009062.8550.WO00 hemostasis through precise molecular feedback regulations (33). For example, vascular injury triggers a coagulation cascade process where clotting factors activate prothrombin to thrombin, transforming fibrinogen into insoluble fibrin by cleavage. Together with platelet activation, this process produces stable fibrin clots to prevent excessive bleeding (33). It is envisioned by the embodiments disclosed herein that by incorporating a feedback loop system within the coacervates, they could regulate anticoagulant release based on thrombin activity. Increasing environmental thrombin levels would promptly trigger release of an anticoagulant, such as heparin, while normal physiological thrombin levels would leave the coacervates intact— thus, there would be no risk of excessive bleeding (34,35).

[0051] The embodiments disclosed herein incorporate a thrombin cleavage site within a peptide used to make nano-coacervates, resulting in the release of an anticoagulant such as heparin as a function of concentration-dependent thrombin proteolysis. Enhanced coacervate stability via polyphenol-mediated supramolecular networks was demonstrated while maintaining their thrombin proteolytic activity. This structural and colloidal enhancement is visualized by transmission electron microscopy (TEM) — they had exceptional stability in challenging conditions and various biofluids but could still specifically release the heparin cargo. The disassembly rate of nano-coacervates rapidly increased in response to thrombin proteolysis in human plasma. Overall, the approach of utilizing polyphenols to stabilize coacervates and preserve the bioactivity for enzymatic degradation is a simple yet powerful tool in the fields of biomedicine, biosensing, and enzyme-triggered drug delivery.

[0052] In some embodiments, the technology includes a coacervate comprising at a plurality of YR-based peptides and an anticoagulant, wherein each YR-based peptide comprises a thrombin cleavage sequence.

[0053] As used herein, a “YR-based peptide” is a peptide including at least two YR residues. As used herein, a “YR residue” is a tyrosine (Y) residue immediately adjoined to an arginine (R) residue. For example, a YR-based peptide may include any peptide beginning and ending with a YR residue.

[0054] In some embodiments, the YR-based peptide includes a thrombin cleavage site. Thrombin is a serine protease that specifically recognizes and cleaves peptide bonds-15-182827596.7Attorney Docket No. 009062.8550.WO00 following arginine residues within defined sequence contexts. The canonical recognition motif for thrombin is the sequence LVPRGS (SEQ ID NO: 10), where cleavage occurs at the carboxyl side of the arginine residue. However, thrombin exhibits substrate flexibility and can bind and cleave a variety of sequences containing a P1 arginine, typically preceded by hydrophobic or proline-rich residues at the P2 and P3 positions, and followed by small or neutral residues at the P1 ' position. Representative sequences include, but are not limited to, PRS, GPRG (SEQ ID NO: 13), APRG (SEQ ID NO: 14), VPRG (SEQ ID NO: 15), GGRS (SEQ ID NO: 16), and GRGN (SEQ ID NO: 17). Thrombin may also cleave sequences such as FPRG (SEQ ID NO: 18), SGRG (SEQ ID NO: 19), and GPRA (SEQ ID NO: 20), provided the P1 arginine is accessible and the surrounding residues do not sterically hinder enzyme binding. The specificity is further influenced by the presence of proline at the P2 position and glycine or serine at the P1 ' position, but variants with other amino acids at these positions may also be susceptible to thrombin cleavage. Therefore, all peptide sequences containing the motif X1-PR-X2, where X1is any amino acid and X2is a small, neutral, or hydrophilic residue (SEQ ID NO: 21 ), are considered potential substrates for thrombin binding and cleavage.

[0055] The thrombin cleavage site may be any peptide sequence that is cleavable by thrombin. For example, the thrombin cleavage site may comprise X1-PR-X2, where X1is a hydrophobic amino acid and X2is a small, neutral, or hydrophilic residue (SEQ ID NO: 21 ). In some embodiments, the cleavage site may comprise LVPRGS (SEQ ID NO: 10), however, a person of ordinary skill in the art could recognize specific variations of the LVPRGS (SEQ ID NO: 10) sequence that may be made without substantially affecting the cleavage of the peptide sequence, for example, the thrombin cleavage site may comprise any of the sequences described in Kretz et al., “High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13,” 8:2788 Nature Scientific Reports 5 (2018), the peptides described in which are incorporated herein in their entirety. The cleavage site may comprise a peptide having an amino acid sequence selected from the group consisting of LVPRGS (SEQ ID NO: 10), PRS, GPRG (SEQ ID NO: 13), APRG (SEQ ID NO: 14), VPRG (SEQ ID NO: 15), GGRS (SEQ ID NO: 16), GRGN (SEQ ID NO: 17), FPRG (SEQ ID NO: 18), SGRG (SEQ ID NO: 19), and GPRA (SEQ ID NO: 20).-16-182827596.7Attorney Docket No. 009062.8550.WQ00

[0056] The YR-based peptide including a thrombin cleavage site may include a thrombin cleavage site with a YR residue on the C- and N- termini. For example, the YR- based peptide may comprise (YR)nX1-PR-X2(YR)n where n is 1 , 2, 3, 4, or 5; X1is a hydrophobic amino acid; and X2is a small, neutral, or hydrophilic residue (SEQ ID NO: 22). The YR-based peptide may comprise (YR)nLVPRGS(YR)n, where n is 1 , 2, 3, 4, or 5 (SEQ ID NO: 11 ). The YR-based peptide may comprise YRLVPRGSYR (SEQ ID NO: 12).

[0057] Peptides described herein may be modified using a variety of techniques well known to those skilled in the art to enhance stability, bioavailability, or activity. Such modifications include, but are not limited to, N-terminal and C-terminal modifications (e.g., acetylation, amidation), incorporation of D-amino acids, non-natural amino acids, or peptidomimetics, and conjugation to polymers such as polyethylene glycol (PEGylation). Additional modifications may involve the introduction of disulfide bridges, glycosylation, lipidation, phosphorylation, methylation, or the attachment of reporter groups, such as fluorescent or affinity tags. These modifications can be employed individually or in combination.

[0058] In some embodiments, the C-terminus of the YR-based peptide is amidated (“- CONH2”). For example, the YR-based peptide may comprise NH2-(YR)nLVPRGS(YR)n- CONH2, where n is 1 , 2, 3, 4, or 5 (SEQ ID NO: 1 ). The YR-based peptide may comprise NH2-YRLVPRGSYR-CONH2 (SEQ ID NO: 5). In some aspects, the YR-based peptide comprises any one of SEQ ID NO: 1 -9.

[0059] In some embodiments a thrombin-responsive coacervate composition is provided. Such composition includes a coacervate formed from a plurality of YR-based peptides and an anticoagulant. Each YR-based peptide includes a thrombin cleavage sequence.

[0060] In some embodiments, the thrombin-responsive coacervate comprises a concentration of YR-based peptides and a concentration of an anticoagulant such that the combination forms a coacervate. The formation of a coacervate may be detected using any method known in the art, for example, by observing increased turbidity relative to a sample without a coacervate.-17-182827596.7Attorney Docket No. 009062.8550.WO00

[0061] In certain embodiments, the composition comprises one or more anticoagulant agents selected from the group consisting of heparin, low molecular weight heparins (LMWH) such as enoxaparin and dalteparin, warfarin, fondaparinux, dabigatran, rivaroxaban, apixaban, edoxaban, argatroban, bivalirudin, and coumarin derivatives. The anticoagulant may be derived from natural sources, such as porcine intestinal mucosa in the case of heparin, or may be produced synthetically or by recombinant techniques. The selected anticoagulant agents may be present individually or in combination, in an amount effective to inhibit blood coagulation or reduce the risk of thrombus formation in the composition. In some aspects, the anticoagulant is heparin.

[0062] In some embodiments, the concentration of the YR-based peptide is about 0.25 rnM to about 1 .5 mM. In some aspects, the concentration of the YR-based peptide is about 0.25 mM, about 0.5 mM, about 1 mM, or about 1 .5 mM. In some aspects, the concentration of the YR-based peptide is about 1 mM.

[0063] In some embodiments, the concentration of the heparin is about 5 U / mL to about 50 U / mL. In some aspects, the concentration of the heparin is about 2.5 U / mL, about 5 U / mL, about 12.5 U / mL, about 25 U / mL, or about 50 U / mL. In some aspects, the concentration of the heparin is about 50 U / mL.

[0064] In some embodiments, the concentration of the YR-based peptide is about 1 mM and the concentration of the heparin is about 2.5 U / mL, about 5 U / mL, about 12.5 U / mL, about 25 U / mL, or about 50 U / mL. In some embodiments, the concentration of the YR-based peptide is about 0.25 mM, about 0.5 mM, about 1 mM, or about 1.5 mM and the concentration of the heparin is about 50 U / mL. In some aspects, the concentration of the YR-based peptide is about 0.25 mM and the concentration of the heparin is about 50 U / mL. In some aspects, the concentration of the YR-based peptide is about 0.5mM and the concentration of the heparin is about 50 U / mL. In some aspects, the concentration of the YR-based peptide is about 1 mM and the concentration of the heparin is about 50 U / mL. In some aspects, the concentration of the YR-based peptide is about 1.5 mM and the concentration of the heparin is about 50 U / mL.

[0065] In some embodiments, the compositions of the present technology include nano-coacervates and / or micro-coacervates. As used herein, a “nano-coacervate” refers to-18-182827596.7Attorney Docket No. 009062.8550.WG00 a coacervate having a diameter less than about 1 pm, and a “micro-coacervate” refers to a coacervate having a diameter greater than about 1 gm.

[0066] In some embodiments, the diameter of each thrombin-responsive coacervate is about 100nm to about 300nm or about 200nm to about 300nm. In some aspects, the diameter of each thrombin-responsive coacervate is about 190 nm to about 200 nm, about 200 nm to about 210 nm, about 210 nm to about 220 nm, about 220 nm to about 230 nm, about 230 nm to about 240 nm, about 240 nm to about 250 nm, about 250 nm to about 260 nm, about 260 nm to about 270 nm, about 270 nm to about 280 nm, about 280 nm to about 290 nm, or about 290 nm to about 300 nm. In some aspects, the diameter of each thrombin- responsive coacervate is about 198 nm, about 222 nm, about 228 nm, about 253 nm, or about 276 nm.

[0067] In some embodiments, the thrombin-responsive coacervate includes a polyphenol to enhance the stability of the coacervate. In some aspects, the polyphenol is encapsulated within the coacervate to enhance stability of the coacervate. In some aspects the composition comprises one or more polyphenols selected from the group consisting of flavonoids, phenolic acids, stilbenes, lignans, and tannins. Exemplary polyphenols suitable for use in the present invention include, but are not limited to, epigallocatechin gallate (EGCG), epicatechin, catechin, quercetin, resveratrol, curcumin, rutin, hesperidin, naringenin, chlorogenic acid, caffeic acid, ferulic acid, gallic acid, ellagic acid, pterostilbene, and tannic acid. The polyphenols may be derived from natural sources such as green tea, cocoa, grapes, berries, citrus fruits, coffee, and nuts, or may be synthesized by chemical or biotechnological methods. The selected polyphenols may be present individually or in combination, in an amount effective to achieve the desired biological or therapeutic effect in the composition. In some aspects the polyphenol is a tannin. In some aspects, the polyphenol is tannic acid.

[0068] In some embodiments, the thrombin-responsive coacervate includes about 0.03 rnM to about 1 mM tannic acid. In some aspects, the thrombin-responsive coacervate includes about 0.01 mM to about 0.05mM, about 0.05 to about 0.2mM, about 0.2mM to about 0.5mM, or about 0.5 to about 1.5mM tannic acid. In some aspects, the thrombin- responsive coacervate includes about 0.03 mM, about 0.13mM, about 0.17 mM, about 0.25-19-182827596.7Attorney Docket No. 009062.8550.WO00 mM, about 0.33 mM, about 0.5mM, or about 1 mM tannic acid. In some aspects, the thrombin coacervate includes about 0.1 mM to about 0.2mM tannic acid. In some aspects, the thrombin coacervate includes about 0.13mM.

[0069] The present technology includes stabilized coacervate compositions comprising a coacervate and tannic acid. The present technology includes composition comprising the amino acid of any one of SEQ ID NOs: 1 -12.

[0070] In some embodiments, the technology includes a method of regulating heparin release from a coacervate composition is provided. Such method includes a step of mixing the thrombin-responsive coacervate composition or the polyphenol-stabilized nanocoacervate of any one of the embodiments described above with human blood, wherein the thrombin-responsive coacervate composition or the polyphenol-stabilized nano-coacervate: (a) dissociates and releases heparin when the human blood contains a supra-physiological level of thrombin, or (b) remains intact when the human blood contains a physiological level of thrombin.

[0071] The technology includes a method of making a polyphenol-stabilized thrombin- responsive coacervate comprising mixing a solution of heparin and a YR-based peptide to form thrombin-responsive coacervates; redispersing the thrombin-responsive coacervates; mixing the thrombin-responsive coacervates with tannic acid to form the polyphenol- stabilized thrombin-responsive coacervates; and centrifuging the polyphenol-stabilized thrombin-responsive coacervates to remove unreacted tannic acid molecules.Working ExamplesExample 1. Polyphenols-Stabilized Coacervates for Enzyme-Triggered Drug Delivery

[0072] Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Reported below is 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, size-tunable coacervates were developed 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.-20-182827596.7Attorney Docket No. 009062.8550.WO00Notably, 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. A pivotal polyphenol threshold was discovered 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.Methods

[0073] A human blood specimen was collected from one male subject under approval from the institutional review board (IRB) of UC San Diego and the VA San Diego (#H170005). All subjects gave written informed consent. All work was done in accordance with the Declaration of Helsinki. Sex was not investigated as a biological variable because there are currently no known sex-based differences in thrombosis.Experimental Details

[0074] Nano-Coacervates Preparation. Briefly, 4 mg of the C5 peptides was dissolved in 3 ml of deionized water. Subsequently, 300 pL of heparin solution (100 ll / rnl) was mixed with 200- or 300- pL of the C5 peptides, formulating nano- or micro-sized coacervates. The heparin concentration required to form coacervates depends on peptide concentration and the number of repetitive YR units in the peptide building block. The color of the solution became turbid once coacervation occurred. The size of coacervate droplets depends on the concentrations of the heparin and C5 peptides used for the coacervation. For example, 0.25 mM of the C5 peptide formed nano-coacervates at a constant heparin concentration of 50 U / ml while 0.5 mM of the C5 peptide formed micro-coacervates. The resulting product was purified by centrifugation at 3 x g for 10 min to remove any unreacted heparin or peptides. The pellet containing nano-coacervates was re-dispersed in MQ water for future use. However, the micro-sized coacervates were deformed, and the coacervate phase disappeared after centrifugation at 3 x g. All peptide sequences were synthesized using an AAPPTEC peptide synthesizer.-21 -182827596.7Attorney Docket No. 009062.8550.WQ00

[0075] Polyphenol Encapsulation within Coacervates. The nano-coacervates were redispersed in 200 pL of bicine buffer at pH 8.5. Subsequently, the desired amount of TA in MQ water was added to the nano-coacervates, and the mixture was gently shaken (400 rpm) at 37 °C for 6 h. During this process, the color of the solvent was changed from white to yellowish due to TA oxidization. The resulting product was once again centrifuged at 3 x g for 10 min to remove any unreacted TA molecules. The 0.03, 0.17, 0.33, and 1 mM of TA were incubated with nano-coacervates containing 1 mM of C5 peptide. Note that excess amounts of TA could lead to the formation of solid precipitates.

[0076] The micro-coacervates were first prepared by mixing 300 pL of heparin (100 U / ml) with 300 pL peptide (1.3 mg / ml) followed by the addition of TA molecules with bicine buffer overnight. The resulting product was centrifuged at 3 x g for 10 min to remove any unreacted TA molecules. The pellet was re-dispersed in MQ water for future use. Note that excessive TA molecules can trigger solid aggregates (FIGS. 28A-28D).

[0077] Sulfo-Cy5.5-Labeled C5 Peptides (i.e., C7). Sulfo-Cy5.5-NHS was coupled with the free amine from the N-terminus of the C5 peptide to encapsulate sulfo-Oy5.5 dye within the nano-coacervates. Briefly, the desired amount of the C5 peptide was dissolved in DMSO with 1 % v / v triethylamine wrapped with aluminum foil. Subsequently, sulfo-Cy5.5-NHS was added to the C5 peptides at a 1 :1 molar ratio under generous stirring for 3 h. After the amine- NHS couplings, the final product was fully dried using a vacufuge. The resulting product was re-dispersed in 50% AON for HPLC purification. MALDI-MOF MS was used to confirm the molecular weight of the final product (FIGS. 15A-15C).

[0078] For the encapsulation of C7, the nano-coacervates were dispersed in bicine buffer at pH 8.5, followed by the addition of C7 peptides under generous stirring for 3 h. The resulting product was centrifuged at 3 x g for 10min to remove any unencapsulated C7 peptides. The pellet was re-dispersed in MQ water for future use.

[0079] Thrombin Proteolysis of Nano-Coacervates. Briefly, 1 mM of the nanocoacervates was incubated with various concentrations of a-thrombin ranging from 0.03 to 4 pM in 20 mM Tris-HCI buffer solution (pH 7.4, NaC1 150 mM). The mixture was immediately transferred into a 96-well plate, and the light absorption at 500 nm was measured at 37 °C for 1 h. The fragment solution was desalted by using a C18 column (5 pm, 9.4 x 250 mm)-22-182827596.7Attorney Docket No. 009062.8550.WG00 and then applied MALDI-TOF to confirm the cleavage site. Mass peaks shown in FIG. 12D are from the acetylated 05 parent and its fragment peptides.

[0080] Likewise, the C7-encapsulated nano-coacervates and NC-TA0.13 were incubated in 50% human plasma containing the alpha thrombin (final concentration = 500 nM) at 37 °C. The mixture was immediately transferred into a 96-well plate, and the fluorescence signal at 670 nm was measured at 37 °C for 3 h.

[0081] Stability test of NC-TAs. Briefly, the nano-coacervates encapsulated with TA molecules (0.03-1 mM) were incubated with 1 M NaCI for 1 h at room temperature in a 96- well plate. The absorbance from 300 to 900 nm was measured with a step size of 2 nm before and after the incubation. The absorbances at 500 nm before and after incubation were used to calculate turbidity changes (Turbidityafter / Turbiditybefore).

[0082] For the size measurements, NC-TA0.13 was incubated for 1 h under various conditions including fibrinogen, glucose, glutamine, acetone, methanol (MeOH), citric acid (pH 2), DPBS, NaOH (pH 10), 60 °C, human albumin, NaCI of 150 mM, 50% of human serum, human urine, human saliva, and Dulbecco’s modified Eagle’s medium (DMEM), respectively. After incubation, the resulting samples were centrifuged at 3 x g to replace the medium with MQ water for DLS measurement. The average size was calibrated using three independent replicates. The human saliva, serum, and urine samples were purchased from Innovative Research.

[0083] C7 peptide / Heparin-FITC encapsulation and fluorescent monitoring in biofluids.Briefly, NC-TAs were gently mixed with either C7 peptide (20 pM) or heparin-FITC (150 pM) for the encapsulation. The desired dye-conjugates were encapsulated within NC-TAo (i.e., nano-coacervates), NC-TA0.07, and NC-TA0.13, respectively with the same number of coacervates under generous stirring overnight. Note that the coacervate concentrations used for FIGS. 22F and 22G are different. Following encapsulation, each sample was purified using centrifugation at 3 x g for 10 min. Subsequently, the samples were transferred to a 96-well plate at 37 °C for PL measurement.

[0084] For fluorescence monitoring, NC-TAs encapsulating with either C7 peptide (FIG. 22F) or heparin-FITC (FIG. 22G) were incubated in 50% of human plasma in a final-23-182827596.7Attorney Docket No. 009062.8550.WG00 volume of 100 pL. The mixture was directly transferred to a 96-well plate at 37 °C, and the PL intensity at 670 nm (for C7 peptide) and 520 nm (for heparin-FITC) was recorded for 1 h with 1 min intervals. The activated PL signal indicates the disassembly of nano-coacervates in 50% human plasma. Notably, there was no self-PL quenching of C7 peptide or heparin- FITC in biofluids (FIGS. 37A and 37B). The slope is referred to as the PL activation rate. Normal pooled plasma and normal pooled serum were purchased from Innovative Research.

[0085] Cytotoxicity test and ROS detection ofHUVEC. HUVEC cells (ATCC, PCS-100-100) were cultured in a vascular cell basal medium with an endothelial cell growth kit. Cell cultures were incubated under 5% CO2 at 37 °C. Cells were passaged when they reached 75-80% confluency using 0.25% trypsin for primary cells. DPBS and cell lysis buffer were used for negative and positive controls of healthy and dead cells. For the cytotoxicity experiments, HUVEC cells were seeded overnight in a 96-well plate at a concentration of 10,000 cells / well. Subsequently, PBS, lysis buffer, C5 peptide, TA, nano-coacervates, NCTA0.25, NC-TA0.5, and NC-TA1 were co-incubated with HUVEC at an equal concentration of 0.16 mM for 12 h, respectively. A resazurin assay was used to analyze the cytotoxicity of nano-coacervates, NC-TA0.25, NC-TA05, and NC-TA1 following a protocol. After 4 h incubation with resazurin, cell viability was calibrated by measuring the subtracted background absorbance of each well at 600 nm from resazurin absorbance at 570 nm. The absorbances of experimental wells were compared to those of the controlled wells containing healthy and dead cells.

[0086] HUVEC cells were seeded overnight in a 96-well plate for reactive oxygen species (ROS) detection test using a DCF-DA kit. Subsequently, PBS, lysis buffer, C5 peptide, TA, nano-coacervates, NC-TA0.25, NC-TA0.5, and NC-TA1 were co-incubated with HUVEC at an equal concentration of 0.16 mM for 3 h, respectively. N-acetyl Cysteine and pyocyanin were added as the negative and positive controls. The fluorescence of experimental wells was compared to those of the controlled wells containing negative and positive controls. All experiments were performed in triplicate to measure the average and standard deviations.-24-182827596.7Attorney Docket No. 009062.8550.WO00

[0087] Peptide synthesis. Peptides were synthesized using the Fmoc-SPPS (solidphase peptide synthesis) on Rink-amide-MBHA-resin (0.55 mmol / g) using an automated Eclipse™ peptide synthesizer (AAPPTec, Louisville, KY). Amino acids were linked to the resin under nitrogen conditions with 0.2 M Fmoc-amino acid, 0.2 M HBTU, 0.4 M DIPEA, and 20% (v / v) piperidine in DMF for each peptide synthesis cycle. Fmoc groups on the amino acids were removed using piperidine. HBTU and DIPEA were utilized for amide coupling. The resulting peptides were transferred to a syringe filter and washed with DCM to remove DMF. Chemical reactions such as click and acetylation on the peptide were performed on the resin before peptide cleavage. The crude peptides were then cleaved from the resin using a cocktail solution (5 mL) containing 5 mL of TFA (83%), 300 pL of H2O (5%), 300 pL of thioanisole (5%), 300 mg of phenol (5%), and 150 pL of EDDET (2%). After 3 h incubation, the resin was filtered, and the crude peptides were precipitated using a cold ethyl ether by three-time centrifugations (7,500 rpm, 5 min). The precipitated pellets were dried and resuspended using 10 mL of ACN / H2O mixtures (20-40% of ACN depending on the solubility of the pellets) for high-performance liquid chromatography (HPLC) purification. Note Over 50% ACN can interrupt peptide purification because the absorbance peaks of ACN and peptides can be overlapped.

[0088] The crude peptides were purified using a Shimadzu LC-40 HPLC system equipped with an LC-40D solvent delivery module, a photodiode detector SPD-M40, and a degassing unit DGU-403. For each cycle, 2 mL of the crude peptides dissolved in ACN / H2O mixtures was injected, and the C4 column (pore size: 5 pm, 20x200 mm, from SHMADZU) was used. Sample injection was monitored at 220 nm to collect the target peptide, and the wavelength at 680 nm was used for monitoring to collect sulfo-Cy5.5 conjugated peptides. The desired peptide was confirmed using an electrospray ionization mass spectrometry (ESI-MS) or / and matrix-assisted laser desorption / ionization (MALDI-TOF MS) from the first cycle. The purified target peptide was frozen at -80QC for lyophilization. All purified peptides achieved a minimum 90% purity, confirmed by ESI-MS or / and MALDI-TOF. The concentration of each peptide was calibrated using a NanoDrop™ UV-vis-spectrometer (Thermo Fisher Scientific, Waltham, MA). The 31 -method in Nanodrop™ was used to calibrate the peptide concentration using an absorption of coefficient of s205 (about 75 mL-mg-1-crrr1). MQ water was used as a blank. After concentration measurement, the-25-182827596.7Attorney Docket No. 009062.8550.WQ00 purified peptides were dried using a vacufuge (Eppendorf, Germany) and stored at -20QC for future use.

[0089] Sulfo-Cy5.5-labeled C5 peptide (i.e., C7). Briefly, 05 peptide (2 pmol) was mixed with sulfo-Cy5.5-NHS (1 pmol) in DMSO with 1% v / v triethylamine. The reaction was stirred for 3 h and covered with an aluminum foil to protect the dye against UV light. After a 3 h reaction, a vacufuge (Eppendorf, Germany) was used to dry DMSO at 60 °C. The dried pellet was re-dispersed using ACN / H2O solvent for HPLC purification. The conjugation yield was ~40% after HPLC purification, and MALDI-TOF was used to confirm the mass peak shown in FIGS. 15B-15C. Note Sulfo-Cy5.5 without C5 conjugation cannot be encapsulated in the nano-coacervates due to its negatively charged nature.

[0090] Fluorogenic peptide synthesis (i.e., C8). Cy5.5-peptide-Cy3. Briefly, an aqueous solution of the peptide (2 pmol, NH2-YRLVPRGSYRC-Am (SEQ ID NO: 5), in DMSO) was mixed with TCEP (0.16 pmol) under gentle stirring for 30 min. After 30 min, Cy3-maleimide (2 pmol) was added to the above mixture for 3 h sealed with an aluminum foil. Note: The addition of a few drops of DI water may homogenize the reaction. After a 3 h reaction, the solvent was dried using a vacufuge, and the pellets were re-dispersed with ACN / H2O for HPLC purification. MALDI-TOF was used to confirm the mass peak shown in FIGS. 16B- 16D.

[0091] Next, a solution of YRLVPRGSYRC-Cy3 (SEQ ID NO: 8, 0.5 pmol in 950 pL of DMSO) was mixed with triethylamine (TEA, 10 pL, 1 % v / v) and Cy5.5-NHS (1 pmol, in 50 pL of DMSO) under gentle stirring for 3 h sealed with aluminum foil for dye protections against the light. After a 3 h reaction, the sample was dried using a vacufuge (Eppendorf, Germany) at 60 °C. Then, the sample was re-dispersed using AON and purified via HPLC. MALDI-TOF MS data was used to confirm the mass peak of the final product shown in FIGS. 16B-16D.

[0092] All the above peptide-dye conjugates used the same HPLC protocol. The solvent gradient was carefully adjusted for hydrophobic dye purification. The peptide-dye conjugates were dissolved in ACN first and diluted with H2O, depending on the conjugate’s solubility. Then, 1 mL of the sample was injected into the C4 column and eluted at a flow rate of 3.0 mL / min over 60 min linear gradient from 10% to 90% ACN in water (with 0.05%-26-182827596.7Attorney Docket No. 009062.8550.WG00TFA, HPLC grade). The sample was monitored at 220 nm (peptide), 550 nm (for Cy3), and 650 nm (for Cy5.5). The dye conjugates were quantified and aliquoted using an extinction value. Their molar extinction coefficients are scy3 =1 .5 x 105M-1crrr1at Abssss nm, and scys.s =1.98 x 105M-1crrr1at Absess nm (60).

[0093] Synthesis of TA-coumarin conjugates and confocal imaging. Both TA and coumarin boronic acid were dissolved in DMSO and mixed at a 1 :1 ratio with 0.5% TEA for 18 h at room temperature. After conjugation, the sample was dried using a vacufuge (Eppendorf, USA) at 60 °C and re-dispersed in H2O / ACN for HPLC purification. After purifying the conjugates, TA-coumarin was dried again using a vacufuge for future use.

[0094] For confocal imaging, 300 pL of heparin (100 U / ml) was first mixed with 300 pL of C5 peptide (1 .3 mg / ml), forming micro-sized coacervates. After 1 h of gentle shaking, 25 pL of TA-coumarin-TA conjugates (1.1 mg / ml) was added and incubated in the shaker overnight. The sample was then centrifuged at 3 x g for purification and re-dispersed in a 96-well plate for the confocal imaging.

[0095] Encapsulation efficiency. Briefly, the encapsulation efficiency was calibrated using a supernatant collected after the C5-heparin coacervation. After mixing heparin and peptides to form complex coacervates, the sample was centrifuged at 3 x g for 15 min to collect the supernatant. The supernatant which includes unreacted heparin was incubated with 100 pM of MB dyes to measure the changes in absorbance at 680 nm. Notably, the absorbance of MB dyes decreases as a function of heparin concentration from 0 to 10 U / ml because of MB-heparin aggregation (FIGS. 10A and 10B). Encapsulation efficiency (EE%) was calculated based on the following equation (61 ):(Total heparin cone, —free heparin concj££■(%) = x 100Total heparin cone.

[0096] 20 pL of MB dye (100 pM) was mixed with 60 pL of different heparin concentrations to measure the decreased absorbance at 666 nm (FIGS. 10A and 10B). At least three replicates were performed independently to collect the mean value and standard deviations. Likewise, 60 pL of the supernatant collected from the coacervate samples was mixed with 20 pL of MB dye (100 pM). The decreased absorbance was used to calibrate the encapsulation efficiency.-27-182827596.7Attorney Docket No. 009062.8550.WO00

[0097] Limit of detection calculation. The limit of detection (LoD) was calibrated using the limit of blank (LoB).(62) The LoB indicates the highest signal generated from a sample that contains no analyte. LoB could be calculated using the mean (meanblank) and standard deviation (SDbiank) of a blank sample with the following equation (62):LoB — meanbank + 1 .645 (SDbiank)

[0098] The LoD defines the lowest analyte concentration that could differ from the LoB. LoD is calculated based on the LoB and standard deviation of the lowest concentration samples (SDiow concentration sample) with the following equation (62):LoD = LoB + 1 .645 (SDow concentration sample)

[0099] Here, YRLVPRGSYR peptides (i.e., C5, SEQ ID NO: 5) were used with unfractionated heparin to form complex coacervates. Different concentrations of thrombin were incubated with nano-coacervates to measure the LoD. The buffer condition was Tris- HCI pH 8.5 with NaC1 150 mM at 37QC. The assay was then transferred to the 96-well plate, and the absorbance at 500 nm was measured for turbidity calibration. At least three independent replicates were performed to measure the mean value and standard deviation.

[0100] Colloidal stability in different pH and conditions. Briefly, (YR)2-heparin coacervates of 1 mM were incubated at different pH values (2-14) for 1 h. The absorbance was then measured from 300 to 900 nm. 1 mM indicates the final peptide concentration used for coacervation. The turbidity was calibrated using the absorbance at 500 nm. Different pH was controlled using HCI and NaOH and measured using a pH meter.

[0101] Colloidal stability of nano-coacervates in different conditions including PEG2000, citric acid, urea, Triton-X, SDS, DMF, and DMSO. These were used to examine which interactions govern the stability of (YR)2-heparin assemblies. Nano-coacervates were incubated in 10 pM of PEG2000, urea, Triton-X, citric acid (pH 2), and 70% of DMF and DMSO, respectively. The change in absorbance at 500 nm was measured before and after the incubation. At least three replicates of each experiment were performed to measure the average and standard deviation. Disassembly value was measured with the following equation (63):-28-182827596.7Attorney Docket No. 009062.8550.WO00 100

[0102] Enzyme kinetics for fluorogenic substrate. The kinetic model of an enzyme was developed by Michaelis (64) and Menten and it was further refined by Briggs-Hadane (65) with significant improvements. In brief, the enzyme (E) and substrate (S) reversibly form a reversible enzyme-substrate complex (ES), followed by the dissociation of the intermediate complex to produce the product along with free enzyme, which can be expressed as follows:where kon, kott, kcat, and k-cat are rate constant. The relationship between the initial velocity of the complex formation (v) and the substrate concentration ([S]o) is known at the classical Michaelis-Menten (MM) equation (66):where Vmax indicates the maximum velocity and KM is the MM constant; the subscript 0 indicates the total concentration. When the substrate concentration ([S]o) is equal to KM, the initial velocity reaches 1 / 2 vmax. The kcat term corresponds to the rate-limiting step in enzymatic reactions, thus playing a critical role in the overall catalytic efficiency of the enzyme.

[0103] Dissociating the ES complex is often considered an irreversible process because of the low affinity of the enzyme to the product. As a result, k-cat is negligibly small (67).~ ^cat

[0005] or vmaxkcat[E] Q

[0104] Experimentally, the fluorogenic substrate was diluted in Tris-HCI buffer (20 mM, pH 7.4) to reach final substrate concentrations of 0, 1 , 3, 6, 12, 18, 24, 30, 36 pM in a 60 pL volume within a 96-well plate. The alpha-thrombin ([E]o = 20 nM to a 60 pL volume) was-29-182827596.7Attorney Docket No. 009062.8550.WG00 then added to each well and the total volume was brought to 60 pL. Next, the 96-well plate was incubated at 37 °C in a hybrid multi-mode microplate reader with 10 s of shaking before each cycle of readout. The PL intensity at 570 nm for Cy3 was recorded over 12 h with 1 min intervals between each cycle.

[0105] At least three replicates were performed, and the results were averaged and plotted against substrate concentrations. Error bars indicate the standard deviation of the means. The APL = PLsomin - PLomin was correlated to the product concentration using a standard curve: APLcys versus full-digested FRET probe. The data were fitted following the Michaelis-Menten equation (68).

[0106] Specificity test. Bovine serum albumin (BSA, Mw: 66463 Da), hemoglobin (Mw: 64500 Da), alpha-thrombin (Mw: 36,000 Da), a-amylase (1000 U / mL), and Mpro(Main protease of SAR-CoV-2) were used for the specificity test. Briefly, the desired amounts of BSA, hemoglobin, alpha-thrombin, a-amylase, MPro, and human saliva were spiked into Tris- HCI buffer (20 mM, pH 8.5) to reach the final concentration of 1 pM. Then, 100 pL of nanocoacervates (crinai = 1 mM) were mixed with different enzymes. The PL signal of the mixture at 670 nm was read at 37QC every 1 min for 1 h. The fluorescence spectra from 660 nm and 900 nm were measured after a 1 h incubation. The wavelength for excitation was 630 nm. At least three replicates of each experiment were performed to measure the average and standard deviation.

[0107] Heparin release confirmation. Briefly, nano-coacervates were incubated with and without 1 pM of thrombin to disassemble nano-coacervate. After centrifugation at 3 x g for 10 min, the supernatant was collected, and the released heparin of 0.2, 0.4, 0.6, 0.8, 1 , 1.2, and 1.4 U / ml was incubated with 20 pL of MB dye (100 pM) as shown in FIG. 121 and FIGS. 19A-19C. In parallel, the same concentration of heparin fresh from the vendor was incubated with 20 pL of MB dye to compare a decrease in absorbance at 666 nm. The supernatant collected from nano-coacervates without thrombin was examined as a negative control.

[0108] Stability test of NC-TA0.13 in different media. The tannic-acid encapsulated nano-coacervates (NC-TA0.13) of 1 mM were incubated in different conditions of 50% acetone, methanol (MeOH), citric acid (pH 2), DPBS, NaOH, 60 °C, NaCI of 150 mM, 50%-30-182827596.7Attorney Docket No. 009062.8550.WO00 of human urine, saliva, and DMEM, respectively. Coacervate stability was also tested using glutamine, glucose (5.6 mM), human albumin (0.6 mM), and fibrinogen (8.8 pM). The glucose (69), albumin (70), and fibrinogen (71 ) concentrations fall in physiological conditions. After 3 h incubation, the samples were centrifuged at 3 x g to replace the solvent condition with MQ water. Then, the hydrodynamic diameter of NC-TA0.13 was measured using a Malvern Instrument Zetasizer ZS 90.

[0109] ELISA test to measure F1 +2 peptide. Human blood specimens were collected under approval from the institutional review board (IRB) of UC San Diego and the UCSD VA (#H170005). All subjects gave written informed consent. All work was done in accordance with the Declaration of Helsinki. Briefly, 400 pL of human blood was incubated with heparin, NC-TA0.13, scramble NC-TA0.13, tannic acid, and C5 peptides for 15 min and centrifuged at 1 x g for 30 min to collect human plasma or serum for enzyme-linked immunoassay (ELISA) test. Heparin was titrated to measure the prevention point of blood coagulation (FIGS. 39A and 39B). The fresh blood was drawn using an EDTA-treated blood collection tube. Calcium chloride was used to activate blood coagulation by EDTA-Ca2+chelation (FIG. 32I; FIGS. 39A and 39B). The experiments were independently performed twice and obtained similar results.

[0110] In addition, blood collection tubes containing 200 pL of PBS (-), heparin, NC, and NC-coacervates were directly used to collect 2 ml of human blood (FIGS. 40A-40C). After 15 min incubation, the sample was centrifuged at 1 x g for 30 min to collect human plasma or serum. The final concentrations of heparin were 40 ll / rnl in this test. The sample was centrifuged until no precipitates appeared, and the supernatant was collected for the ELISA test.

[0111] After all the sample collection, 100 pL of each diluted sample was transferred to a test sample well. The microplate was sealed with a plate sealer and incubated at 37 °C for 1.5 h. Following the procedure described in the protocol (Abbexa, abx252419, UK), the samples were treated with reagents. Finally, TMB solvent was used to activate the enzyme. For F1 +2 peptide calculation, averaged O.D. at 450 nm readings for each reference standard and each sample was subtracted with the averaged control (zero) O.D. readings (72).-31 -182827596.7Attorney Docket No. 009062.8550.WO00(Relative O.D. at 450 nm) = (O.D. of Each Well) - (O.D. of Zero Well)

[0112] Turbidity calculation. Turbidity measurements were calibrated using a multimode microplate reader (Synergy™ H1 model, Bioteck) in a 96-well plate. The extinction (i.e., absorbance) at 500 nm of coacervate samples (peptide-heparin complex) was measured after inducing complex coacervation. Turbidity value was measured with the following equation (60):Turbidity = 100 - io^-^tinction500nm)= 100_o / oT

[0113] Cytotoxicity Test and Cell Staining of HEK 293. HEK 293 (ATCC, CRL-1573) cells were cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum. Cell cultures were incubated under 5% CO2 at 37 °C. Cells were passaged when they reached 75 to 80% confluency using 0.25% trypsin-EDTA. DPBS and CTAB were used for negative and positive controls of healthy and dead cells. For the experiments, HEK 293 cells were seeded overnight in a 96-well plate at a concentration of 10,000 cells / well. Subsequently, PBS, CTAB, nano-coacervates, NC-TA0.13, NC-TA0.33, and NC-TA1 were coincubated with HEK 293 cells at an equal concentration of 0.16 mM for 12 h. A resazurin assay was used to analyze the cytotoxicity of nano-coacervates, NC-TA0.13, NC-TA0.33, and NC-TA1 following a protocol. After 4 h incubation with resazurin, cell viability was calibrated by measuring the subtracted background absorbance of each well at 600 nm from resazurin absorbance at 570 nm. The absorbances of experimental wells were compared to those of the controlled wells containing healthy and dead cells. For cell staining, Hoechst and propidium iodide (PI) were used to stain cell nuclei and dead cells. HEK 293 cells were seeded in a 12-well plate (50,000 cells / well) overnight and then incubated with nanocoacervates, NC-TA0.13, NC-TA0.33, and NC-TA1 with the same concentration of 0.16 mM for 24 h. After incubation, a mixture solution of Hoechst and PI was added to stain the HEK 293. The fluorescence images of each sample were obtained using an EVOS FL fluorescence microscope after gently washing with PBS.

[0114] General characterizations

[0115] 1. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) images were collected using a JEOL JEM-1400 Plus operating at 80 kV in the Nano3-32-182827596.7Attorney Docket No. 009062.8550.WO00 cleanroom UCSD. Gatan 4k digital camera with installed software was used to process TEM images. Tomography TEM and EDX images with different angles were collected using an advanced tomography holder. 2 pL of each diluted sample was dropped on the carbon grids and dried. Subsequently, uranium staining was performed under a fume hood to stain the peptide samples. A single drop of diluted 0.1% uranium solution was placed on the TEM grid for 30 s. After that, the TEM grid was washed with MQ water three times independently and dried for the measurement.

[0116] 2. The hydrodynamic diameter was calibrated based on dynamic light scattering(DLS) using a Malvern Instrument Zetasizer ZS 90. Likewise, the zeta potential (i.e., surface charge) of the sample was measured using a Malvern Instrument Zetasizer ZS 90.

[0117] 3. Absorbance spectra were measured using a BioTek Synergy H1 plate reader.Each sample was measured in 96-well plates. Absorbance was collected from 300 to 900 nm with a step size of 2 nm. Photoluminescence (PL) of fluorescent dyes or peptide-dye conjugates (C7 and C8) was measured using a desired excitation and emission wavelength: 675 / 695 nm for sulfo-Cy5.5 and 555 / 570 nm for Cy3. A seal film was attached to the 96-well plate to prevent solvent evaporation when performing time-dependent measurements. Baseline correction (i.e., solvent only) was performed to subtract the background signal.

[0118] 4. Fourier transform infrared (FTIR) data was obtained using A Bruker Tensor IIFTIR spectrophotometer. The samples were dried using a lyophilizer, and the dried sample was used for the measurement.

[0119] 5. Multi-laser nanoparticle tracking analysis (M-NTA) measurement was performed using a ViewSizer 3000 (Horiba Scientific, CA, USA) to measure particle concentration, sample dispersion, and size (73). Ten videos (8-bit) were recorded with 300 frames for seconds. Particle size was measured using the recorded 10 videos through imaging analysis installed in the instrument. A quartz cuvette with a minimum sample volume of 0.8 mL was used for the measurement. Each sample was diluted in 5 mL of DI water to prevent signal saturation.

[0120] 6. The desired peptides were confirmed using electrospray ionization mass spectrometry (ESI-MS) via the Micromass Quattro Ultima Mass Spectrometer in the-33-182827596.7Attorney Docket No. 009062.8550.WG00Molecular Mass Facility (MMSF) at Chemistry and Biochemistry Department at UC San Diego. The sample for ESI-MS was prepared using a 50% MeOH / FLO mixture. 2 pL of each sample was injected into the instrument independently three times to collect ESI-MS data.

[0121] 7. The desired peptides were confirmed using a matrix-assisted laser absorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Autoflex Max) in the MMSF at UC San Diego. Each sample was mixed with an HCCA matrix (1 :3 ratio of sample to the matrix). Subsequently, 1 -2 pL of the mixture was placed on the plate and dried using a heat gun. The measurement was repeated three times to confirm the mass peaks.

[0122] 8. Scanning electron microscopy (SEM) images were taken using Zeiss Merlin.The coacervate sample was dropped on the silicon wafer and dried overnight. 10 nm of gold was coated on the coacervates through sputtering to improve image resolution.

[0123] 9. Thrombin chromogenic substrate was used to determine thrombin activity. 10 mM of stock solution was prepared using 1 mM HCI and diluted 1 :50 in PBS, producing 0.2 mM working solution. 20 pL test sample was incubated with 180 pL substrate and monitored color change at 405 nm.

[0124] 10. Confocal images were obtained using a Leica STED Sp8 with Falcon microscope (Wetzlar, Germany) with an x63 oil objective at room temperature. The sample was prepared in a Lumox 96-well cell culture plate for confocal imaging. The excitation and emission wavelengths were 405 nm and 435-600 nm, respectively.

[0125] Supplementary Notes: Thrombin (74) is a key enzyme in haemostasias and plays an important role in the body’s response to blood vessel or tissue injuries. When a blood vessel is damaged, there is an immediate surge of thrombin. This prompts the rapid formation of a plug comprised of platelets and fibrin and triggers the blood clotting process. Thrombin exists in plasma as an inactive precursor protein: prothrombin (i.e., blood clotting factor II). Tissue damage triggers the activation of factor VII, which sets off a cascade: Factor Vila activates factor X, which in turn activates prothrombin amplifying the clotting process. Factor Xa can also significantly accelerate the activation of prothrombin (by a factor of 1000- fold). The result of the thrombin generation is the clotting of fibrinogen. Thrombin cleaves two fibrinopeptides (A and B) from fibrinogen, transforming it into fibrin monomers. These-34-182827596.7Attorney Docket No. 009062.8550.WO00 monomers then spontaneously polymerize into elongated fibrin stands. Additionally, thrombin activates another enzyme factor XIII, an enzyme that crosslinks fibrin monomers to establish a stable and robust fibrin network. Consequently, this network facilitates wound closure (74). Alpha-thrombin is composed of a light chain (A chain, Mw~ 6kDa) and a heavy chain (B chain, Mw ~31 kDa). These two chains are coupled by one disulfide bond.ResultsNano-coacervates driven by a tyrosine and arginine peptide

[0126] The Mytilus edulis foot protein 5 (Mefp-5) in mussels contains repetitive DOPA and lysine (K) groups that provide positively charged residues with hydrophobic interactions (36). This enables Mefp-5 to interact with a wide array of materials through either covalent or noncovalent interactions (FIG. 1 ) (37-39). The first step of designing the system was to determine whether a short peptide composed of tyrosine (Y) and arginine (R) could form a coacervate droplet with heparin (average Mw: 15,000 Da) (FIG. 2A). Heparin is a glycosaminoglycan with repeating sulfate units that provide negative charge and a polysaccharide structure for efficient binding with antithrombin (40). Previous studies revealed that heparin could assemble with small molecular dyes via strong electrostatic and hydrophobic interactions (29,41 ) suggesting that the repetitive YR sequence might also readily trigger the formation of coacervates. To test this, a short YRYR peptide (referred to as C2) was synthesized and mixed C2 (0.05-1.5 mM) with heparin (50 U / ml). Upon interaction with heparin, the C2 peptide instantly formed coacervate droplets of varying sizes confirmed by dynamic light scattering (DLS) (FIG. 2B; FIG. 3). Micro-sized coacervates exhibited a broad extinction spectrum, likely due to increased light scattering, while the light absorption of nano-sized coacervates increased at more blue-shifted wavelengths (FIG. 2C; FIGS. 4A-4C).

[0127] To further determine how many repeating YR units are needed to induce coacervation, YR, YRYR, and YRYRYRYR peptides were synthesized (referred to as 01 , C2 (SEQ ID NO: 2), and C3 (SEQ ID NO: 3), respectively) as confirmed by matrix-assisted laser desorption / ionization (MALDI-TOF) (FIGS. 5A-5G). At a constant heparin concentration of 50 U / ml, C2 and C3 peptides formed coacervate droplets of different sizes from 70 nm to 1 pm while the 01 peptide was incapable of forming coacervates, indicating-35-182827596.7Attorney Docket No. 009062.8550.WG00 that heparin-based coacervation requires at least two YR units (FIG. 2D; FIGS. 6-7C). In addition, six glycines (G) were added between YR sequences (YRG6YR, referred to as 04) to confirm the impact of charge density and peptide length. The results showed that both 02 and 04 require comparable peptide concentrations for coacervation, suggesting that the number of YR units (i.e., valence charge) plays a key role in coacervate formations rather than the steric bulk (extra glycine units) (FIGS. 8A-8F).

[0128] In addition, various heparin concentrations from 0.25 to 50 ll / rnl were combined with a constant peptide concentration of 1 mM, confirming that the coacervation relies on the number of YR units and heparin concentration (FIG. 2E; FIGS. 9A-9D). The formation of coacervate droplets led to an increase in turbidity, thus changing the color from transparent to white (FIG. 2F). It was also observed that the strong interactions between heparin and 02 peptide resulted in high loading efficiency of nano-coacervates (99.5-100%) (FIG. 2G; FIGS. 10A and 10B). The nano-coacervates limit their growth and maintain their size and phase separation even under the centrifugation of 7 x g There was no coalescence or merging.

[0129] To confirm the interactions governing the coacervate formation of the 02- heparin complex, nano-coacervates were incubated with PEG2000, citric acid, urea, TritonX-100, SDS, DMF, and DMSO, respectively. TritonX-100 and SDS can break nonionic or ionic interactions; DMSO and DMF are organic solvents that can destroy pi-pi interaction (42); and urea can break the hydrogen bonding (43). The nano-coacervates were disassembled in urea, TritonX-100, SDS, DMF, and DMSO conditions, indicating that electrostatic, pi-pi interaction, and hydrogen bonding were involved in the formation of C2- heparin coacervates (FIG. 2H; FIGS. 1 1 A and 11 B). Nano-coacervates were stable at low pH (1-5), but they disassembled at high pH (over 9) due to the deprotonation of the guanidine group (FIG. 2I). The isoelectric point of arginine is 10.844. Finally, micro- and nano-coacervates were visually observed using multiple wavelength nanoparticle tracking analysis (M-NTA) (45) that further verified the narrow size distribution of nano-coacervates from DLS data (FIG. 2J; FIGS. 7A-7C).-36-182827596.7Attorney Docket No. 009062.8550.WG00Enzyme-responsive coacervate droplets

[0130] Thrombin is a central enzyme in hemostasis and activates the fibrin network and platelets for blood clots when damaged tissue triggers factor VII (46,47). Heparin can prevent these clotting cascades because it contains saccharide units that bind to antithrombin, inactivating a number of coagulation enzymes (48). It was envisioned that if thrombin can cleave the peptide building block and disassemble the coacervates, then the coacervates could be an enzyme-responsive platform that can release the encapsulated heparin in response to thrombin proteolysis (FIG. 12A). To achieve this, a thrombin cleavage site (LVPR J, GS) (49) was added in the middle of the C2 sequence: YRLVPRGSYR-CONH2 (referred to as C5, SEQ ID NO: 5) (Table 1 ). The thrombin proteolysis would result in fragment peptides that contain only one YR unit, which is not sufficient for phase separation as depicted in FIG. 2D. Initially, it was confirmed that the 05 peptide could form nanocoacervates with heparin, leading to an increase in turbidity (FIG. 2D; FIGS. 8A-8F).

[0131] The nano-coacervates were then incubated with various concentrations of thrombin from 0.05 to 2.5 pM. The turbidity of the nano-coacervates decreased due to thrombin proteolysis with higher concentrations of thrombin leading to rapid dissociation of the nano-coacervates (FIG. 12B; FIGS. 13A-13C). Notably, nano-coacervates composed of the scramble sequence (i.e., 06) showed negligible change in turbidity before and after thrombin incubation (FIG. 12C; FIGS. 14A and 14B). The mass peak of the fragment peptide (845.63, YRLVPR) after thrombin cleavage was confirmed via MALDI-TOF (FIG. 12D).

[0132] In addition, the photoluminescence (PL) performance of C7-encapsulated nanocoacervates was examined upon thrombin proteolysis. A sulfo-Cy5.5 dye was conjugated with C5 peptide using an amine-NHS coupling (i.e., C7) and encapsulated C7 peptides within the nano-coacervates (details described in reference to FIGS. 15A-15C). After 07 encapsulation, the PL signal of the 07 peptide was quenched, and the nano-coacervates exhibited a red-shifted absorption peak at 688 nm. This shift was likely due to increased intermolecular interactions, such as pi-pi stacking between tyrosines (50), as well as electrostatic interactions between heparin and lysine within the nano-coacervates. Thrombin cleavage released sulfo-Cy5.5, recovering an absorption peak at 676 nm and its PL intensity at 700 nm (FIG. 12E). The kinetics of PL activation increased as a function of thrombin-37-182827596.7Attorney Docket No. 009062.8550.WO00 concentration: Higher concentrations of thrombin led to a more rapid disassembly of nanocoacervates, promptly activating PL signal (FIG. 12F). Furthermore it was determined the specificity constant (kcat / KM) by thrombin using a fluorogenic substrate (Cy5.5- YRLVPRGSYRC-Cy3, referred to as C8, SEQ ID NO: 8) (FIG. 12G; FIGS. 16A-17) was 0.91 pM“1s-1, which is as fast as the thrombin-catalyzed conversion of human fibrinogen to fibrin (1 .88 pM“1s~1) (51 ). A summary of YR-peptide building blocks used to form the nanocoacervates disclosed herein is shown in Table 1 below.

[0133] Table 1 . Description of peptide building blocks for nano-coacervates-38-182827596.7Attorney Docket No. 009062.8550.WC00

[0134] The specificity of the system was further tested toward other proteins such as bovine serum albumin (BSA), hemoglobin (Hemo), SARS-CoV-2 main protease (Mpro), and a-amylase at the same enzyme concentration of 5 pM. No PL signal was activated in the presence of BSA, Hemo, and other enzymes (FIG. 12H; FIGS. 18A-18C).

[0135] Finally, a methylene blue (MB) assay was used to confirm the released heparin from the disassembly of nano-coacervates (52). Heparin (from 0.125 to 5 U / ml) had a linear decrease in the absorption peak of the MB dye at 666 nm, subsequently causing a redshift of the peak to 566 nm due to the formation of heparin-MB complex (FIGS. 19A-19C). After incubation with thrombin, the disassembled samples were centrifuged to collect the supernatant. The supernatant obtained from the disassembled nano-coacervates decreased the absorption peak of the MB dye to 666 nm. Conversely, nano-coacervates without thrombin exhibited negligible changes in absorption, thus indicating that intact nanocoacervates did not release heparin (FIG. 121). In addition, the released heparin could prevent plasma coagulation as confirmed by an activated partial thromboplastin time (aPTT) test (FIG. 12J). The C5 peptide only showed plasma coagulation due to lack of anticoagulant ability (FIG. 20). Collectively, the coacervate-based heparin delivery offers an enzyme- responsive mechanism capable of releasing heparin in response to thrombin proteolysis for controlled anticoagulant therapy.Polyphenol-stabilized nano-coacervates

[0136] A major drawback of using coacervate is their limited stability in biofluids. Human plasma, which contains diverse proteins, clotting factors, and ions, readily disrupts coacervate phases (FIGS. 21 A and 21 B). Tannic acid (TA) may enhance colloidal and structural stability because multiple catechol groups in TA could create a strong supramolecular network with tyrosine (53) and polysaccharide (54), which are major structural components in the nano-coacervates. To verify this, the nano-coacervates were encapsulated with TA molecules of 0.05, 0.25, 0.5 mM under pH 8.5 (referred to as NC- TAO.O5, NC-TAo25, and NC-TAo.s, respectively) (FIG. 22A). These NC-TAs showed narrow size distributions (polydisperse index (PDI) < 0.1 ), and similar hydrodynamic diameters (FIG. 22B). The average diameter of each NC-TAs was 253.7 ± 9.4 nm (NC-TAo), 221 .8 ± 4.8 nm (NC-TA005), 228.4 ± 7.1 nm (NC-TAo.25), and 276 ± 4.3 nm (NC-TA0.5), respectively. TA-39-182827596.7Attorney Docket No. 009062.8550.WG00 encapsulation resulted in a notable increase in the extinction value of NC-TAs in the near ultraviolet (UV) region, and the color of the sample changed from white to yellowish-brown (FIG. 22C; FIGS. 23A-23C). Fourier-transform infrared spectroscopy (FTIR) data evidenced the TA encapsulation as the appearance of C-0 vibration (1320 cm-1) and 1 , 3-disubstituted benzene rings around (1100-700 cm-1) (FIG. 22D) (55). To further understand TA encapsulation within the coacervates, NC-TAs were incubated at different pH and solvent conditions. NC-TAs remained stable until pH 10 but disassembled beyond 11 due to deprotonation (FIG. 22E). In addition, DMF, DMSO, and SDS led to the disassembly of NC- TAs, suggesting that electrostatic and pi-pi interactions were involved in TA encapsulation (FIG. 22F). NC-TAs showed higher stability in pH 9 and under urea compared to pristine nano-coacervates.

[0137] An attempt was made to image nano-coacervates before and after the TA encapsulation using TEM and scanning electron microscopy (SEM). NC-TAs maintained their size and spherical shape even in the vacuum condition confirmed by both TEM and SEM (FIG. 22G; FIGS. 24A-24C) while the nano-coacervates without TA collapsed and deformed during the drying process (FIGS. 25A-25D). Furthermore, tomography imaging at various angles ranging from -30° to 60° was used to illustrate the interface between the bottom of NC-TAo os and the underlying substrate (i.e., TEM grid). FIG. 22H clearly shows the height of a single NC-TAo.os at 60°, indicating that TA molecules formed a rigid supramolecular network and enhanced the structural integrity of the nano-coacervates (FIGS. 26A-26F). High-angle annular dark field (HAADF) and energy-dispersive X-ray spectroscopy (EDX) were also utilized to confirm the elemental components of NC-TAs (FIGS. 22I and 22J). EDX mapping revealed that C, N, O, and S signals were observed in a single NC-TAo.os which are components of TA, C5 peptide, and heparin (FIG. 22J; FIGS. 27A-27E).

[0138] It was also discovered that polyphenol encapsulation can be applied to microsized coacervates (MC-TAs). The optical image illustrates the uniformly dispersed microcoacervates after TA encapsulation (FIGS. 28A-28D). The spherical shapes and sizes of the dried MCTAoos were confirmed by the SEM technique (FIG. 22K; FIGS. 29A and 29B). Notably, significantly improved colloidal stability was observed under the centrifugation of 3-40-182827596.7Attorney Docket No. 009062.8550.WG00 x g. The coacervate droplets without TA showed a 98.7% decrease in turbidity while MC- TAO.05 decreased by only 1.4% (FIGS. 30A-30E). Lastly, coumarin boronic acid was conjugated with TA for confocal imaging to verify TA encapsulation within coacervate droplets. HPLC was utilized to remove free coumarin dyes from TA-coumarin before encapsulation (FIGS. 31 A-31 F). FIG. 22I shows a uniformly distributed fluorescent signal of TA-coumarin conjugates from inside the MC-TAs. This result indicates that TA is encapsulated within the coacervates rather than being membrane-coated (10,56).Preserving enzymatic activity of nano-coacervates with enhanced stability

[0139] The formation of polyphenol networks within the coacervates significantly enhances stability; however, highly constructed supramolecular networks could adversely affect the proteolytic efficiency (6) (FIG. 32A). To examine this, nano-coacervates encapsulated with various TA concentrations were incubated in NaCI for 1 h. A smaller decrease in turbidity (T) as a function of increased TA encapsulation while nano-coacervates without TA were dissociated within the 30 s (FIG. 32B) was observed: Turbidity (Tafter / Tbetore) of NC-TAo, NC-TAO.17, NC-TA0.33, and NC-TAi were 7%, 36%, 53%, and 92%, respectively. In contrast, high TA encapsulation led to a decrease in proteolytic activity. The turbidity changes of NC-TAs were measured after incubating different concentrations of thrombin (Mw: 37.4 kDa) ranging from 0.06 to 1 pM. NC-TAo.s showed a reduced decrease in turbidity compared to NC-TA0.25 when incubated with the same concentration of thrombin (FIG. 32C; FIGS. 33A-33D). NC-TA1 exhibited negligible changes in turbidity, indicating that the excessive TA encapsulation could prevent thrombin-driven coacervate disassembly.

[0140] After identifying a TA encapsulation point for preserving thrombin proteolytic activity, the colloidal stability of NC-TA0.13 was examined under various biological environments. The NC-TA0.13 exhibited high colloidal stability in glutamine, glucose (5.6 mM), human albumin (0.6 mM), DPBS, NaOH (pH 10), 60 °C, NaCI (150 mM), fibrinogen (8.8 pM), 50% of Dulbecco’s Modified Eagle Medium (DMEM), serum, saliva, and urine (FIG. 32D; FIGS. 34A-35B). Both pristine nano-coacervates and NC-TAs containing either C7 peptides or heparin-FITC were incubated in 50% human plasma, respectively to examine improved stability. The quenched fluorescence of the C7 peptide and heparin-FITC was activated as a function of the disassembly of nano-coacervate (FIG. 32E). NC-TA0.13-41 -182827596.7Attorney Docket No. 009062.8550.WG00 exhibited a 3.3-fold decrease in the PL activation rate of C7 peptide than NC-TAo, indicating enhanced stability in human plasma. Simultaneously, thrombin could accelerate heparin release from NC-TA0.13 (FIG. 32F). The disassembly rate of NC-TA0.13 increased by 4.2-fold upon the addition of thrombin (500 nM), which falls within the physiologic range of free thrombin concentration. Physiologic concentrations of free thrombin during coagulation reactions range over 500 nM (57). This disassembly process was monitored using heparin- FITC (FIGS. 36A-36C). NC-TAs exhibited a decrease in PL activation of heparin-FITC compared to pristine nano-coacervates in human plasma. Concurrently, the addition of thrombin rapidly increased the PL activation rate of heparin-FITC by 1 .8-fold, recovering PL intensity within 10 min (FIG. 32G). There was no fluorescence quenching of C7 peptides and heparin-FITC either by the background medium (i.e., human plasma) or by TA molecules (FIGS. 37A and 37B). The release kinetics of heparin are different than the peptide because the concentration of coacervate samples and fluorescent dye-conjugates (C7 peptide and heparin-FITC) were different; the ratio could be tuned to control kinetics. Cell viability and cellular reactive oxygen species (ROS) levels of NC-TA0.13 was then examined using human umbilical vein endothelial cells (HUVECs), respectively. NC-TAs and their structural components such as C5 peptide, heparin, and TA showed high cell viability (>83%) and minimal ROS intensities (FIG. 32H). NC-TA013 also exhibited low cytotoxicity against human embryonic kidney (HEK) 293 cells. NC-TAs led to minimal red fluorescence of propidium iodide (PI), which corresponds to cell viability of 95% like HUVECs (FIGS. 38A and 38B).

[0141] The anticoagulant performance of NC-TA0.13 was tested in whole human blood using a human prothrombin fragment 1 + 2 (F1 + 2) enzyme-linked immunosorbent assay (ELISA) kit. Fresh whole blood was collected using an EDTA-treated blood collection tube. Free heparin (0.6 U / ml), C6 peptide, TA, NC-TA013, and scramble NC-TA0.13 were incubated with whole human blood at the same concentration, respectively. Calcium chloride was then used to trigger blood clot formation. A negligible difference in F1 + 2 formation between heparin and NC-TA0.13 was observed, confirming active blood anticoagulation driven by heparin released from NC-TAo .13 (FIG. 32I; FIGS. 39A-40C). In contrast, a strong thrombus was observed in TA, C5 peptide only, and scramble NC-TA0.13. Note that scramble NC-TA0.13 was comprised of a scramble C6 peptide, serving as a non-responsive control. Lastly, the-42-182827596.7Attorney Docket No. 009062.8550.WG00 residual thrombin activity of human serum and plasma was examined using a thrombin chromogenic substrate. Human serum exhibited a 20-fold higher residual thrombin activity than plasma (FIG. 32J). The residual thrombin activity in human serum was comparable to that of 42.5 nM of the alpha-thrombin. This difference arises because human plasma is obtained by anticoagulating blood, which prevents the clotting cascade, thus inhibiting thrombin formation. In contrast, human serum is collected by allowing blood to clot, during which thrombin is generated from prothrombin, resulting in higher residual thrombin activity. Nano-coacervates without TA showed a 56% decrease in absorbance at 500 nm due to residual thrombin activity while NC-TA013 showed only an 18% decrease in 50% human serum, indicating enhanced stability through polyphenol encapsulations (FIG. 32J; FIGS.41A-41 D).Discussion

[0142] In summary, the self-assembly of YR-based peptides with heparin, form coacervates through a combination of electrostatic, hydrogen bonding, and hydrophobic interactions. This assembly can produce a range of sizes from nano- to micro-coacervates. In addition, the peptide building blocks involve a thrombin recognition site to incorporate a hemostasis feedback loop system within the coacervate for controlled heparin release. Increasing thrombin levels trigger the disassembly of the coacervates, rapidly releasing heparin, while the absence of thrombin leaves the coacervates intact.

[0143] The nano-coacervate was further stabilized via a polyphenol-mediated supramolecular network to improve its stability in human plasma. TA encapsulation improves the structural integrity of nano-coacervates as clearly visualized by TEM. NC-TAs exhibited high stability under various biological conditions. Simultaneously, the disassembly rate of NC-TAs rapidly increased upon the addition of thrombin, leading to heparin release in human plasma. NC-TAs also feature bioresponsive anticoagulant performance in the whole human blood and high biocompatibility with HLIVEC and HEK 293 cells.

[0144] Coacervates containing catechol as structural building blocks have shown significant potential in drug delivery systems, particularly for gastrointestinal diseases (58) due to their strong adhesiveness (59) capable of prolonged retention in the gastrointestinal tract. The nano-coacervates strengthened by polyphenols also showed superior coating-43-182827596.7Attorney Docket No. 009062.8550.WO00 ability on inert substrates and maintained high stability in whole human blood (FIGS. 42A and 42B). Future work will incorporate coacervates on medical devices such as a drugeluting stent for on-demand anticoagulant delivery. Studies on inflammatory aspects such as plasma viscosity, procalcitonin, and C-reactive protein levels — as well as the elimination of particles from the circulation by phagocyte update or clearance in the kidney, spleen, and liver — are needed to validate their value in translational nanomedicine. Taken together, the polyphenol-based platform to stabilize coacervates and preserve bioactivity may have a scope well beyond drug delivery, extending its applications to biomedicine, protease sensing, and hybrid protocell models.

[0145] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.-44-182827596.7Attorney Docket No. 009062.8550.WO00REFERENCESThe following references, which are cited in the specification, are hereby incorporated by reference as if fully set forth herein.1 . Sun, Y. et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat. Chem. 14, 274-283 (2022).2. Mason, A. F., Buddingh, B. C., Williams, D. S. & Van Hest, J. C. Hierarchical self-assembly of a copolymer-stabilized coacervate protocell. J. Am. Chem. Soc. 139, 17309-17312 (2017).3. Saha, R., Verbanic, S. & Chen, I. A. Lipid vesicles chaperone an encapsulated RNA aptamer. Nat. Commun. 9, 2313 (2018).4. Yewdall, N. A., Andre, A. A., Lu, T. & Spruijt, E. Coacervates as models of membraneless organelles. Curr. Opin. Colloid Interface Sci. 52, 101416 (2021 ).5. Aumiller, W. M. Jr & Keating, C. D. Phosphorylation-mediated RNA / peptide complex coacervation as a model for intracellular liquid organelles. Nat. Chem. 8, 129-137 (2016).6. Abbas, M., Law, J. O., Grellscheid, S. N., Huck, W. T. & Spruijt, E. Peptide-based coacervate-core vesicles with semipermeable membranes. Adv. Mater. 34, 2202913 (2022).7. Wei, W. et al. An underwater surface-drying peptide inspired by a mussel adhesive protein. Adv. Funct. Mater. 26, 3496-3507 (2016).8. Dyson, F. Origins of life. (Cambridge University Press, 1999).9. Novak, V. J. Present state of the coacervate-in-coacervate theory; origin and evolution of cell structure. Orig. Life 14, 513-522 (1984).10. Jin, Z. et al. Endoproteolysis of oligopeptide-based coacervates for enzymatic modeling.ACS nano 17, 16980-16992 (2023).-45-182827596.7Attorney Docket No. 009062.8550.WO001 1 . Nakashima, K. K., vanHaren, M. H., Andre, A. A., Robu, I. & Spruijt, E. Active coacervate droplets are protocells that grow and resist Ostwald ripening. Nat. Commun. 12, 3819 (2021 ).12. laneselli, A. et al. Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells. Nat. Chem. 14, 32-39 (2022).13. Abbas, M., Lipinski, W. P., Wang, J. & Spruijt, E. Peptide-based coacervates as biomimetic protocells. Chem. Soc. Rev. 50, 3690-3705 (2021 ).14. Cook, A. B., Novosedlik, S. & van Hest, J. C. Complex coacervate materials as artificial cells. Acc. Mater. Res. 4, 287-298 (2023).15. Blocher, W. C. & Perry, S. L. Complex coacervate-based materials for biomedicine.Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnology 9, e1442 (2017).16. Johnson, N. R. & Wang, Y. Coacervate delivery systems for proteins and small molecule drugs. Expert Opin. drug Deliv. 1 1 , 1829-1832 (2014).17. Sureka, H. V., Obermeyer, A. C., Flores, R. J. & Olsen, B. D. Catalytic biosensors from complex coacervate coremicelle (C3M) thin films. ACS Appl. Mater, interfaces 1 1 , 32354-32365 (2019).18. Zhang, Y. et al. Giant coacervate vesicles as an integrated approach to cytomimetic modeling. J. Am. Chem. Soc. 143, 2866-2874 (2021 ).19. Liu, S. et al. Enzyme-mediated nitric oxide production in vasoactive erythrocyte membrane-enclosed coacervate protocells. Nat. Chem. 12, 1 165-1 173 (2020).20. Ji, Y., Lin, Y. & Qiao, Y. Plant cell-inspired membranization of coacervate protocells with a structured polysaccharide layer. J. Am. Chem. Soc. 14, 12576-12585 (2023).21. Gao, N. & Mann, S. Membranized coacervate microdroplets: from versatile protocell models to cytomimetic materials. Acc. Chem. Res. 56, 297-307 (2023).22. Last, M. G., Deshpande, S. & Dekker, C. pH-controlled coacervate-membrane interactions within liposomes. ACS nano 14, 4487-4498 (2020).-46-182827596.7Attorney Docket No. 009062.8550.WO0023. Deng, N. N. et al. Macromolecularly crowded protocells from reversibly shrinking monodisperse liposomes. J. Am. Chem. Soc. 140, 7399-7402 (2018).24. Deng, N. N. & Huck, W. T. Microfluidic formation of monodisperse coacervate organelles in liposomes. Angew. Chem. 129, 9868-9872 (2017).25. Lu, T., Javed, S., Bonfio, C. & Spruijt, E. Interfacing coacervates with membranes: from artificial organelles and hybrid protocells to intracellular delivery. Small Methods 7, 2300294 (2023).26. Wang, J. et al. A nanoscale tool for photoacoustic-based measurements of clotting time and therapeutic drug monitoring of heparin. Nano Lett. 16, 6265-6271 (2016).27. Coffin, C. M. et al. Pediatric laboratory medicine: current challenges and future opportunities. Am. J. Clin. Pathol. 117, 683-690 (2002).28. Maitz, M. F. et al. Bio-responsive polymer hydrogels homeostatically regulate blood coagulation. Nat. Commun. 4, 2168 (2013).29. Yim, W. et al. Enhanced photoacoustic detection of heparin in whole blood via melanin nanocapsules carrying molecular agents. ACS nano 16, 683-693 (2021 ).30. Zhou, J. et al. A fiber optic photoacoustic sensor for real-time heparin monitoring.Biosens. Bioelectron. 196, 113692 (2022).31. Mackman, N. Triggers, targets and treatments for thrombosis. Nature 451 , 914-918(2008).32. Zhang, Y. et al. Thrombin-responsive transcutaneous patch for auto-anticoagulant regulation. Adv. Mater. 29, 1604043 (2017).33. Di Cera, E. Thrombin. Mol. Asp. Med. 29, 203-254 (2008).34. Stump, D. A., Rogers, A. T., Hammon, J. W. & Newman, S. P. Cerebral emboli and cognitive outcome after cardiac surgery. J. Cardiothorac. Vase, anesthesia 10, 1 13- 1 19 (1996).35. Whitlock, R., Crowther, M. A. & Ng, H. J. Bleeding in cardiac surgery: its prevention and treatment— an evidence-based review. Grit. Care Clin. 21 , 589-610 (2005).-47-182827596.7Attorney Docket No. 009062.8550.WQ0036. Li, Y. et al. Molecular design principles of Lysine-DOPA wet adhesion. Nat. Common.1 1 , 3895 (2020).37. Lee, H., Dellatore, S. M., Miller, W. M. & Messersmith, P. B. Mussel inspired surface chemistry for multifunctional coatings. Science 318, 426-430 (2007).38. Berger, O. et al. Mussel adhesive-inspired proteomimetic polymer. J. Am. Chem. Soc.144, 4383-4392 (2022).39. Yim, W. et al. 3D-bioprinted phantom with human skin phototypes for biomedical optics.Adv. Mater. 35, 2206385 (2023).40. Olson, S. T., Halvorson, H. & Bjork, I. Quantitative characterization of the thrombin- heparin interaction. Discrimination between specific and nonspecific binding models. J. Biol. Chem. 266, 6342-6352 (1991 ).41. Wang, J. et al. A mechanistic investigation of methylene blue and heparin interactions and their photoacoustic enhancement. Bioconjugate Chem. 29, 3768-3775 (2018).42. Jin, Z. et al. Peptide amphiphile mediated co-assembly for nanoplasmonic sensing.Angew. Chem. 135, e202214394 (2023).43. Wu, D. et al. Phenolic-enabled nanotechnology: versatile particle engineering for biomedicine. Chem. Soc. Rev. 50, 4432-4483 (2021 ).44. Pu, W., Zhao, H., Huang, C., Wu, L. & Xu, D. Visual detection of arginine based on the unique guanidino group-induced aggregation of gold nanoparticles. Analytica Chim. acta 764, 78-83 (2013).45. Moore, C., Wing, R., Pham, T. & Jokerst, J. V. Multispectral nanoparticle tracking analysis for the real-time and label-free characterization of amyloid-[3 self-assembly in vitro. Anal. Chem. 92, 11590-11599 (2020).46. Hemker, H. A century of heparin: past, present and future. J. Thrombosis Haemost. 14,2329-2338 (2016).47. Wolberg, A. S. & Campbell, R. A. Thrombin generation, fibrin clot formation and hemostasis. Transfus. Apheresis Sci. 38, 15-23 (2008).-48-182827596.7Attorney Docket No. 009062.8550.WG0048. Hirsh, J., Anand, S. S., Halperin, J. L. & Foster, V. Guide to anticoagulant therapy: heparin: a statement for healthcare professionals from the American heart association. Circulation 103, 2994-3018 (2001 ).49. Lin, K. Y., Kwong, G. A., Warren, A. D., Wood, D. K. & Bhatia, S. N. Nanoparticles that sense thrombin activity as synthetic urinary biomarkers of thrombosis. ACS nano 7, 9001-9009 (2013).50. McGaughey, G. B., Gagne, M. & Rappe, A. K. n-stacking interactions: alive and well in proteins. J. Biol. Chem. 273, 15458-15463 (1998).51. Dang, O., Vindigni, A. & Di Cera, E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proc. Natl Acad. Sci. 92, 5977-5981 (1995).52. Tan, L., Yao, S. & Xie, Q. Electrochemical determination of heparin using methylene blue probe and study on competition of Ba2+ with methylene blue for binding heparin. Taianta 71 , 827-832 (2007).53. Zhou, J. et al. Particle engineering enabled by polyphenol-mediated supramolecular networks. Nat. Commun. 11 , 4804 (2020).54. Renard, C. M., Watrelot, A. A. & Le Bourvellec, C. Interactions between polyphenols and polysaccharides: Mechanisms and consequences in food processing and digestion. Trends Food Sci. Technol. 60, 43-51 (2017).55. Wahyono, T., Astuti, D. A., Gede Wiryawan, I. K., Sugoro, I. & Jayanegara, A. in IOPConference Series: Materials Science and Engineering. 042045 (IOP Publishing).56. Jiang, L. et al. Peptide-based coacervate protocells with cytoprotective metal-phenolic network membranes. J. Am. Chem. Soc. 145, 24108-241 15 (2023).57. Allen, G. A. et al. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J. Thrombosis Haemost. 2, 402-413 (2004).58. Zhao, P. et al. Nanoparticle-assembled bioadhesive coacervate coating with prolonged gastrointestinal retention for inflammatory bowel disease therapy. Nat. Commun. 12, 7162 (2021 ).-49-182827596.7Attorney Docket No. 009062.8550.WO0059. Guo, Q. et al. Hydrogen-bonds mediate liquid-liquid phase separation of mussel derived adhesive peptides. Nat. Commun. 13, 5771 (2022).60. Jin, Z. et al. Endoproteolysis of Oligopeptide-Based Coacervates for EnzymaticModeling. ACS nano (2023).61. Yim, W. et al. Enhanced photoacoustic detection of heparin in whole blood via melanin nanocapsules carrying molecular agents. ACS nano 16, 683-693 (2021 ).62. Armbruster, D. A. & Pry, T. Limit of blank, limit of detection and limit of quantitation. The clinical biochemist reviews 29, S49 (2008).63. Jin, Z. et al. Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing.Angewandte Chemie 135, e202214394 (2023).64. Johnson, K. A. & Goody, R. S. The original Michaelis constant: translation of the 1913Michaelis-Menten paper. Biochemistry 50, 8264-8269 (201 1 ).65. Briggs, G. E. & Haldane, J. B. S. A note on the kinetics of enzyme action. Biochemical journal 19, 338 (1925).66. Shi, H. et al. Fluorescent light-up probe with aggregation-induced emission characteristics for in vivo imaging of cell apoptosis. Organic & biomolecular chemistry 1 1 , 7289-7296 (2013).67. Neumann, II., Kubota, H., Frei, K., Ganu, V. & Leppert, D. Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme. Analytical biochemistry 328, 166-173 (2004).68. Raaijmakers, J. G. Statistical analysis of the Michaelis-Menten equation. Biometrics,793-803 (1987).69. Borg Andersson, A., Birkhed, D., Berntorp, K., Lindgarde, F. & Matsson, L. Glucose concentration in. Eur J Oral Sci 106, 931 -937 (1998).70. Guthrie Jr, R. D. & Hines Jr, C. Use of Intravenous Albumin in the Critically III Patient.American Journal of Gastroenterology (Springer Nature) 86 (1991 ).-50-182827596.7Attorney Docket No. 009062.8550.WO0071 . Schlimp, C. J. et al. Rapid measurement of fibrinogen concentration in whole blood using a steel ball coagulometer. Journal of Trauma and Acute Care Surgery 78, 830-836 (2015).72. Ota, S. et al. Elevated levels of prothrombin fragment 1 + 2 indicate high risk of thrombosis. Clinical and Applied Thrombosis / Hemostasis 14, 279-285 (2008).73. McElfresh, C., Harrington, T. & Vecchio, K. S. Application of a Novel New MultispectralNanoparticle Tracking Technique. Meas. Sci. TechnoL 29, 065002 (2018).74. Di Cera, E. Thrombin. Molecular aspects of medicine 29, 203-254 (2008).75. Lee, H., Dellatore, S. M., Miller, W. M. & Messersmith, P. B. Mussel-inspired surface chemistry for multifunctional coatings, science 318, 426-430 (2007).76. Wang, J. et al. A nanoscale tool for photoacoustic-based measurements of clotting time and therapeutic drug monitoring of heparin. Nano letters 16, 6265-6271 (2016).-51 -182827596.7

Claims

1. Attorney Docket No. 009062.8550.WQ00CLAIMSWe claim:1 . A thrombin-responsive coacervate composition comprising: a coacervate comprising a plurality of YR-based peptides and heparin, wherein each YR- based peptide comprises a thrombin cleavage sequence.

2. The thrombin-responsive coacervate composition of claim 1 , wherein the thrombin cleavage sequence comprises an amino acid sequence selected from the group consisting of LVPRGS (SEQ ID NO: 10), PRS, GPRG (SEQ ID NO: 13), APRG (SEQ ID NO: 14), VPRG (SEQ ID NO: 15), GGRS (SEQ ID NO: 16), GRGN (SEQ ID NO: 17), FPRG (SEQ ID NO: 18), SGRG (SEQ ID NO: 19), and GPRA (SEQ ID NO: 20).

3. The thrombin-responsive coacervate of claim 1 or 2, wherein the YR-based peptide comprises (YR)nLVPRGS(YR)n(SEQ ID NO: 11 ), where n is 1 , 2, 3, 4, or 5.

4. The thrombin-responsive coacervate of any of claims 1 to 3, wherein the YR-based peptide comprises is YRLVPRGSYR (SEQ ID NO: 12).

5. The thrombin-responsive coacervate of any one of claims 1 to 4, wherein the concentration of the YR-based peptides is about 0.25 mM to about 1 .5 mM.

6. The thrombin-responsive coacervate of any one of claims 1 to 5, wherein the concentration of the heparin is about 5 U / rnL to about 50 U / mL.

7. The thrombin-responsive coacervate composition of any one of claims 1 to 6, further comprising a polyphenol encapsulated within the coacervate to enhance stability of the coacervate.

8. The thrombin-responsive coacervate composition of claim 7, wherein the polyphenol-52-182827596.7Attorney Docket No. 009062.8550.WO00 is tannic acid.

9. The thrombin-responsive coacervate composition of claim 8, comprising about 0.05 to 0.5 mM tannic acid.

10. The thrombin-responsive coacervate composition of any one of claims 1 to 9, wherein the coacervate is a nano-coacervate.1 1. The thrombin-responsive coacervate composition of any one of claims 1 to 10, wherein the diameter of each coacervate is about 200 to 300nm.

12. A polyphenol-stabilized nano-coacervate comprising a mixture of a peptide comprising NH2-(YR)nLVPRGS(YR)n-CONH2 (SEQ ID NO: 1 ), where n is 1 , 2, 3, 4, or 5, heparin, and tannic acid.

13. The polyphenol-stabilized nano-coacervate of claim 12, wherein the peptide comprises NH2-YRLVPRGSYR-CONH2 (SEQ ID NO: 5).

14. A method of regulating heparin release from a coacervate composition comprising mixing the thrombin-responsive coacervate composition of any one of claims 1 to 11 or the polyphenol-stabilized nano-coacervate of any one of claims 12 or 13 with human blood, wherein the thrombin-responsive coacervate composition or the polyphenol-stabilized nanocoacervate: dissociates and releases heparin when the human blood contains a supra-physiological level of thrombin, or remains intact when the human blood contains a physiological level of thrombin.

15. A polyphenol-stabilized nano-coacervate comprising a plurality of YR-based peptides and tannic acid.

16. The polyphenol-stabilized nano-coacervate of claim 15, wherein the YR-based-53-182827596.7Attorney Docket No. 009062.8550.WQ00 peptide is selected from the group consisting of SEQ ID NOs: 1 -12.

17. The polyphenol-stabilized nano-coacervate of claim 15 or 16, further comprising heparin.

18. A composition comprising a peptide selected from the group consisting of SEQ ID NOs: 1 to 12.

19. The composition of claim 18, comprising SEQ ID NO: 1 or 11 .

20. The composition of claim 18, comprising SEQ ID NO: 5 or 12.-54-182827596.7

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