One-pot synthesis of stable antioxidant metal-ornamented polyphenol supramolecular assemblies for material engineering

Natural polyphenols (NPPs) are of widespread interest in material engineering; however, only a small fraction of NPPs have been utilized in biomedical applications because of their poor solubility and instability in aqueous conditions. Herein, we report a family of metal-ornamented polyphenol supramolecular (MOPS) assemblies that are highly stable in aqueous solutions for over 6 months. In this one-pot, green synthesis method, metal ions are reduced in water by NPPs to form NPP-capped metal nanoclusters, which then assemble with more NPP molecules to form stable hydrophilic MOPS assemblies. This strategy is generally applicable to a range of NPPs (25 polyphenols tested) regardless of their original water solubility. The resulting stable MOPS assemblies, integrating attributes of both NPPs and metal nanoclusters, possess much stronger antioxidant capabilities than the NPPs, notably a multiplied capacity in superoxide-free radical scavenging capacity. MOPS assemblies can be further engineered, based on polyphenol chemistry, into various functional materials with potential for a wide range of antioxidant applications. Natural polyphenols reduce metal ions into metal nanoclusters in water followed by subsequent self-assembly into metal-ornamented polyphenol supramolecular (MOPS) assemblies. MOPS assemblies, which integrate the merits of both the natural polyphenols and metal nanoclusters, possess excellent solubility, stability, and free radical scavenging capacity in aqueous solutions and enable further functional material engineering via polyphenol chemistry.


Introduction
Polyphenol-based supramolecular structures have attracted widespread interest due to their diverse properties, including their affinity for various materials endowed by their inherent phenolic chemistry, free radical scavenging capability imparted by the reducing nature of the phenolic hydroxyl groups and biocompatibility due to bioderived phenolic ingredients [1][2][3][4][5] . Such polyphenolic materials have been engineered with potential applications in catalysis, sensing, energy storage, biosynthesis, water purification, diagnosis, therapy, and tissue engineering [6][7][8][9][10][11][12][13][14][15] . Although natural polyphenols (NPPs) widely exist in various types of animals, plants, and microorganisms, their application in biomedical material engineering is limited by their instability in aqueous conditions, especially their sensitivity to oxidizing conditions 16 . Many NPPs with excellent chemical and biological activities have poor solubility in water [16][17][18][19] . For instance, the water solubility of curcumin (Cur), the major polyphenolic compound of the herb Curcuma longa with excellent bioactivity and health benefits, is 11 ng mL −1 , which limits its clinical applications 19 . In addition, many NPPs are sensitive to oxygen and light, especially when dispersed in water 16,18,19 . Therefore, organic solvents and additional stabilizers are generally used in the preparation of polyphenolic superstructures; however, those solvents and stabilizers can introduce additional toxicity or interfere with the desired functionality of the polyphenolic materials 17 . Polyphenolic polymers synthesized by conjugating dihydroxyphenyl (catechol) or trihydroxyphenyl (galloyl) groups on highly biocompatible polymers have been considered an appropriate replacement for NPPs in biomedical material engineering 3,20 . However, the synthesis process is typically labor intensive, involving organic solvents, multiple steps, and tedious purification 21,22 . In addition, harsh conditions are required in certain synthesis processes 23 .
To enhance the stability and bioaccessibility of NPPs for biological and biomedical applications, stable inorganic nanostructures have been utilized to hybridize with polyphenolic materials. Therefore, a wide range of polyphenol-based organic/inorganic hybrid superstructures have been developed. For instance, tea polyphenol (TP), a mixture of polyphenols extracted from tea leaves, has been utilized to synthesize polyphenol-capped Ag and Au nanoparticles (NPs) with sizes ranging from 10 nm to a few hundred nanometers 24,25 . Similar to TP, different types of NPPs were extracted from plants and used for the synthesis of polyphenol-capped Au NPs with diverse sizes and shapes [26][27][28] . However, the synthesis of metal nanoclusters (NCs), which are multinuclear aggregates with sizes less than 3 nm and typical luminescence properties, catalytic activities, stability, and magnetism 29,30 , by using NPPs in water has rarely been reported.
Herein, we report a family of metal-ornamented polyphenol supramolecular (MOPS) assemblies, which are hydrophilic nanoassemblies with NPP-capped metal NCs as ornaments and stable in aqueous conditions during long-term storage. In this one-pot and organic solventfree synthesis process, NPPs are dispersed in water to reduce metal ions (Au III , Cu II , or Ag I ) and form NPPcapped metal NCs, followed by subsequent self-assembly of NPPs and NPP-capped metal NCs in water ( Fig. 1), which produces hydrophilic MOPS assemblies with water solubility as high as 10 4 -10 6 × compared to that of the NPPs. NPPs are chosen as both the reducing agents and protective ligands to stabilize the resulting metal NCs. The rate of reduction is controlled by varying the temperature of the reaction mixture. No organic solvents, specialized equipment, or additional steps are required in this process. The phenolic hydroxyl groups from NPPs play a key role in reducing metal ions and conjugating to metal NCs. Water-soluble NPP-protected metal NCs can adsorb and interact with other NPP molecules via, for example, hydrogen bonding, π-π stacking, and hydrophobic interactions and thus form stable MOPS assemblies in water (Fig. 1). The successful application and versatility of the present MOPS synthesis strategy is demonstrated with 25 different NPPs, resulting in a large family of MOPS assemblies and indicating that this method is generally applicable to NPPs with diverse numbers of phenolic hydroxyl groups regardless of their original solubility in water. The synthesized MOPS assemblies combine the advantages of their constituentsthe metal NC ornaments (including stability and luminescence properties) and the NPP matrix (including reducibility and affinity to diverse materials)-with a multiplied free radical scavenging capacity and open up a new path for the engineering of functional metal/organic hybrid biomaterials.

Synthesis of MOPS assemblies
The general synthesis of Au NC-ornamented polyphenol supramolecular (Au@NPP) assemblies is described as follows. NPPs (3 mg) were dispersed in 9.6 mL of ultrapure water in a 20 mL glass vial and mixed vigorously by a vortex mixer for 10 s. A freshly prepared aqueous solution of HAuCl 4 (25 mM, 0.4 mL) was then added to the dispersion. The reaction mixture was vigorously mixed by a vortex mixer for 60 s immediately after the addition of HAuCl 4 . When tannic acid (TA), cyanidin (Cya), procyanidin B2 (PC-B), and procyanidin C1 (PC-C) were used as the NPP ligands, the reaction mixture turned brick red and was kept at 25°C under gentle stirring for 10 min. For the remaining 21 types of NPP ligands, which were less soluble in water, the reaction mixture was heated to 75°C to raise the reaction rate and shorten the reaction time under gentle stirring for 24 h. The reaction mixture was then centrifuged at 17,000 × g for 10 min. Large metal nanoparticles and aggregates, which were side products of the reduction reaction, could be removed from the reaction solution via high-speed centrifugation. After centrifugation, the supernatant was filtered with 220 nm diameter membranes to further remove the smaller nanoparticles and aggregates. The obtained Au@NPP solutions were then stored at 4°C for at least 6 months with negligible changes observed in their optical properties. The supramolecular solutions were lyophilized to obtain yellowish Au@NPP powders. The synthesis of Ag NC-or Cu NC-ornamented polyphenol supramolecular assemblies was performed using the same procedure as described above except that AgNO 3 (25 mM, 0.4 mL) or Cu(NO 3 ) 2 (25 mM, 0.4 mL) solution was used, respectively, as the metal ion precursor.

Stability assessment
NPP and MOPS aqueous solutions were stored under ambient conditions for 1, 3, 5, 8, and 15 days or subjected to the following treatments: (i) heating, heated to 50°C for 7 h; (ii) ultraviolet (UV) light irradiation, irradiated under 365 nm UV light for 7 h; (iii) acidification, adjusted to pH 2 and stored for 24 h; and (iv) basification, adjusted to pH 10 and stored for 14 h. Dynamic light scattering (DLS), ζpotential, total polyphenol content measurement, and free radical scavenging capacity analyses of the MOPS solutions after storage or subject to the treatments were carried out as described in the Supplementary Information.

MOPS-metal-carbohydrate hydrogel fabrication
In a typical synthesis, to obtain a carboxymethyl chitosan (CMCS)-Au@TA-Ti IV sol, 500 µL of CMCS solution (5 wt.% in water) was introduced into a 2 mL glass vial. Then, 2.2 µL of Ti IV solution (50 wt.% in water) was added to 500 µL of Au@TA solution (1.5 mM in water) in a 1.5 mL tube with vigorous vortexing for 1 min to obtain the orange-red Au@TA-Ti IV mixture. The Au@TA-Ti IV mixture (500 µL) was then added to the CMCS solution in a glass vial with vigorous vortexing for 1 min. The mixture was left to stand. An orange-red gel formed within 5 min, as confirmed by the vial inversion test. Additional gelation tests were performed using Au@Cur, Au@gallic acid (GA), or Au@caffeic acid (CA) and Zr IV .
MOPS-based metal-phenolic network (MPN) core-shell particle and capsule assembly All solutions were freshly prepared. In a typical synthesis, 300 µL of polystyrene (PS) particles (2.5 wt.% in water) was first added to 570 µL of water. Then, 30 µL of FeCl 3 solution (5 mg mL −1 in water) was added, and the dispersion was vortexed for 10 s. Immediately, 600 µL of Au@TA solution (1 mg mL −1 in water) was added followed by vigorous vortexing for 10 s. Phosphate-buffered saline (PBS) buffer (1.5 mL, 100 mM, pH 8.0) was then added, and the dispersion was vortexed for 1 min. The particles were washed with 3 mL of ultrapure water three times to remove excess Au@TA and Fe III . In the washing step, the particles were centrifuged (5000 × g, 5 min), the supernatant was discarded, and the pellets were vortexed for 10 s. To obtain capsules, the PS cores were removed by washing with tetrahydrofuran (THF) three times. In each THF washing step, the pellets were first vortexed for 10 s, 2 mL of THF was added to the pellet, and the pellet was then resuspended through gentle pipetting (at least 20×). This suspension was then kept on a gentle rotating mixer for 30 min. The particles were centrifuged (5000 × g, 10 min), the supernatant was removed, and the process was repeated three times. After the final THF washing step, the pellet was resuspended in 2 mL of ultrapure water through gentle pipetting.

Results and discussion
We first demonstrated the synthesis of gold nanoclusters (Au NCs) using Cur, a nonflavonoid polyphenol compound with poor solubility (11 ng mL −1 ) and stability in water 19 . Cur and Au III were mixed thoroughly in water. The mixture could be warmed up to 75°C to increase the reaction rate. The reaction was allowed to proceed for 24 h. Transmission electron microscopy (TEM) analysis showed that the obtained aqueous solution of Au-ornamented Cur supramolecular (Au@Cur) assemblies formed an amorphous film when added dropwise onto the copper grid, whereas ordered lattice structures were detected within the amorphous film under high-resolution TEM imaging (Fig. 2a). The lattice fringes were measured to be 2.35 Å, which was identical to the (111) lattice of face-centered cubic (FCC) Au. The presence of Au ornaments in the synthesized supramolecular assemblies was confirmed by energydispersive X-ray spectroscopy (Fig. S1). These results indicated the feasibility of engineering highly ordered materials from amorphous NPP films with the assistance of Au NC formation. The aqueous solution of the synthesized Au@Cur assemblies (1 mg mL −1 ) was transparent, which was nearly colorless under ambient light and exhibited blue fluorescence under 365 nm UV light (Fig. 2b). Fluorescence spectra showed the luminescence properties of Au@Cur assemblies in water (Fig. 2c). The Au@Cur assemblies showed maximum emission at~450 nm with excitation at 350 nm, which was different from the emission of natural Cur at~550 nm with the same excitation light (Fig. 2c and  Fig. S2). Based on the fluorescence intensity of the synthesized product, the synthesis conditions of Au@Cur assemblies could be optimized to obtain products with efficient luminescent emission (Fig. S2).
UV-visible (UV-vis) spectrophotometry and Fourier transform infrared (FTIR) spectroscopy revealed the differences in the molecular structures of the synthesized Au@Cur and natural Cur (Fig. 3a and Fig. S3). Natural Cur showed two distinct absorption bands in the UV-Vis spectrum at 250-280 and 420-450 nm, related to the n-π* and π-π* transitions, respectively. However, for the synthesized Au@Cur, the absorption band at 420-450 nm disappeared, while the absorption band attributed to the n-π* transition was less affected. In addition, there was no absorption peak in the 520-560 nm region, which is typical for Au NPs due to their surface plasma resonance properties, for the Au@Cur sample, indicating the absence of Au NPs in the Au@Cur solution. In the FTIR spectrum, the synthesized Au@Cur assemblies showed a much higher and broader absorption band at 3600-3100 cm −1 compared to natural Cur, which could be attributed to the O-H stretching of phenolic hydroxyl groups 31,32 . The broadening of this band indicated that the phenolic hydroxyl groups of Cur ligands participated in the assembly of Au@Cur assemblies via intermolecular hydrogen bonds 31 . The hydroxyl groups of Cur reduced Au III to Au 0 , while Cur ligands capped the synthesized Au NCs, forming Curcovered Au NCs. The reduction of the total polyphenol content in Au@Cur compared with the total polyphenol content in Cur confirmed the consumption of the hydroxyl groups during the synthesis of Au NCs (Fig. 3b and Fig. S4). The Cur-capped Au NCs then adsorbed unreacted Cur and subsequently assembled into supramolecular assemblies with a considerably higher free phenolic hydroxyl activity, as indicated by the FTIR spectra 31,32 . The complete disappearance of the absorption band at 420-450 nm, a typical indicative band of Cur, for the synthesized Au@Cur in the UV-Vis spectrum demonstrated that there were no free Cur molecules left in the final product. DLS analysis revealed the distribution of the Au@Cur assemblies in different aqueous solutions (Fig. 3c and Fig. S5). The diameter of Au@Cur assemblies in ultrapure water was 81 ± 4 nm and was hardly affected by the pH or salt concentration of the aqueous solutions.
Unlike hydroalcohol solutions of natural Cur that are not stable when stored in ambient environments (Fig. S6), Au@Cur solutions (1 mg mL −1 ) were highly stable when stored under ambient conditions, with neither a color change nor precipitation observed for up to 6 months. DLS analysis confirmed the physical stability of the Au@Cur aqueous solutions either stored under ambient conditions for different time intervals or subjected to various treatments (Fig. S7). Moreover, Au@Cur assemblies maintained chemical stability in aqueous solutions without changes observed in their total polyphenol content when either stored under ambient conditions for a long period or subjected to heating, UV irradiation, or acid treatment (Fig. S8). These results indicated that the incorporation of metal ornaments into the NPP supramolecular assemblies could largely increase the stability of the NPP-based nanoassemblies. However, similar to NPPs, the phenolic hydroxyl groups of the synthesized Au@Cur assemblies were vulnerable to basic conditions. The total polyphenol content of Au@Cur decreased sharply in basic solutions with a pH higher than 10. Therefore, strong alkali conditions should be avoided during the storage and processing of MOPS assemblies.
NPPs are generally recognized as efficient antioxidants that scavenge free radicals and inhibit the risks of oxidative stress 33,34 . A commonly used method to assess antioxidant ability is to measure the free radical scavenging capacity of antioxidants. In this work, the free radical scavenging capacities of natural Cur and the synthesized Au@Cur were determined by using different free radical scavenging capability determination kits (Fig. 3d-f and Fig. S9). At equal total polyphenol content levels (per gallic acid equivalent (GAE)), Au@Cur possessed a much higher free radical scavenging capacity than natural Cur (Fig. 3d). Specifically, Au@Cur exhibited doubled nitroxide free radical (NFR) scavenging capacity and almost 10-fold enhanced capability for superoxide free radical (SFR) scavenging. Moreover, the free radical scavenging capacities of Au@Cur remained constant during long-term storage under ambient conditions as well as under various treatments (Fig. 3e, f). Compared to natural Cur, the introduction of Au NCs to Au@Cur assemblies could be a key factor resulting in enhanced and more stable capacities for free radical scavenging. The detailed mechanisms are currently under investigation to determine whether there are any combined or synergistic effects between the Au NCs and Cur ligands. Stability of free radical scavenging capacities of the as-prepared Au@Cur aqueous solutions after storage at ambient temperature for various time periods (e) or after being subjected to different treatments (f). NFR nitroxide free radical; SFR superoxide free radical. n = 3, ****p < 0.0001, nonparametric one-way ANOVA.
To demonstrate the general applicability of this one-pot, organic solvent-free MOPS synthesis method to diverse NPPs, 24 other NPP compounds (Fig. S10) were examined to synthesize the corresponding MOPS assemblies. The resulting UV-Vis spectra showed the differences observed in the absorption spectra of the MOPS assemblies and NPPs, suggesting an alteration in the molecular structure of the synthesized MOPS assemblies ( Fig. 4 and Fig. S11). The luminescence properties of the series of synthesized MOPS assemblies were different due to the different ligands from which MOPS assemblies were derived (Fig. S12). In addition to Au, Ag, and Cu were examined to synthesize the corresponding Ag NC-and Cu NCornamented supramolecular assemblies. Using Cur, Ag I and Cu II were reduced in water to form Cur-capped Ag NCs and Cu NCs, which subsequently assembled into the corresponding MOPS (Ag@Cur and Cu@Cur) assemblies (Fig. 5). Because of the different types of metal NC ornaments, Ag@Cur and Cu@Cur assemblies exhibited different light absorption and luminescence properties (Fig. 5a, b, d, e). There were three absorption peaks in the range of 200-350 nm for Ag@Cur assemblies, while there were only two absorption bands in the same region for Cu@Cur assemblies. In addition, the maximum emissions of Ag@Cur and Cu@Cur assemblies were at~500 and 450 nm, respectively, with excitation at~350 nm. All these results indicated that the metal ornaments, although at a low content in the MOPS assemblies, played an important role in determining the microstructures and physiochemical properties of the supramolecular assemblies. Similar to Au@Cur, ordered lattice structures were detected within the amorphous Ag@Cur and Cu@Cur films, suggesting that metal NCs could mediate the fabrication of highly ordered structures from amorphous polyphenol assemblies (Fig. 5c, f). The lattice fringes of the Ag@Cur and Cu@Cur samples were measured to be 2.36 and 2.05 Å, respectively, which were attributed to the (111) lattice of the FCC Ag and the (111) lattice of the FCC Cu. Moreover, similar to Au@Cur assemblies, Ag@Cur and Cur@Cur assemblies showed significantly higher capacities in scavenging SFRs than natural Cur at equal total polyphenol content levels (Fig. S13). This indicated that the presence of metal NC ornaments could not only stabilize the polyphenolic ligands but also remarkably enhance the antioxidant capacities of polyphenols.
For NPPs with poor aqueous solubility, e.g., Cur, ellagic acid (EA) and quercetin (QC), continuous synthesis of MOPS was achieved through the addition of supplementary metal ions into the reaction mixture ( Fig. 6a-f). By controlling the synthesis time and cycle number, the composition of the synthesized assemblies, indicated by the UV-Vis absorption and fluorescence intensity, could be tuned (Fig. S14). For NPPs with poor solubility in water, the reaction mixture could be heated to accelerate the synthesis of metal NCs by increasing both the amount of NPP ligands in water and the oxidation-reduction rate. In addition, the synthesized NPP-capped metal NCs could "pull" more NPP molecules into water from the undissolved NPP solids to form hydrophilic MOPS assemblies, thereby increasing the solubility of the NPPs in water. Continuous synthesis could allow for a relatively complete reaction of NPPs and reduce the waste of raw materials.  Moreover, in the present study, continuous synthesis of MOPS assemblies enabled the mass production of hydrophilic polyphenolic supramolecular nanostructures. For NPPs with good solubility in water, such as TA, TP, and procyanidins, the reaction proceeded rapidly, with more polyphenol molecules dissolved and interacting with metal ions in water. Therefore, the synthesis process was completed at ambient temperature within 15 min. Mass production of MOPS by using such soluble NPPs could be achieved by increasing the amount of reactants in the reaction system proportionately. In addition, the fluorescence intensity of the synthesized MOPS could be tuned by adjusting the ratio of the reactants in the reaction system (Fig. S15).
With a relatively high content of active phenolic hydroxyl groups, MOPS assemblies possess many physicochemical properties that are similar to NPPs, including metal chelation and affinity for different materials. These polyphenolic properties, based on polyphenol chemistry, enabled the NPPs to be utilized as building blocks for the engineering of a wide range of nanostructured functional materials, including MPN-based nanoparticles, nanocapsules and hydrogels [2][3][4][5][6] . Similarly, MOPS assemblies with polyphenolic properties could be engineered into different functional materials via polyphenol chemistry, e.g., surface coatings, hollow structured capsules, hydrogels, and colloidal suspensions (Fig. 6g, h and Fig. S16). Owing to their excellent free radical scavenging capacity, these MOPS-based materials are expected to be excellent antioxidants for a wide range of biomedical applications. Free radicals, especially reactive oxygen species (ROS), are typically associated with oxidative stress in the body and are related to the development of many diseases, including cardiovascular diseases, neurological disorders, obesity, aging, and cancer [35][36][37][38][39] . Antioxidants are able to reduce free radicals, especially ROS, alleviate oxidative stress in the body and prevent or treat related disorders. Therefore, these MOPS-based materials are anticipated to have potential in various areas, including the diagnosis and treatment of various diseases.

Conclusion
In summary, the family of stable antioxidant MOPS assemblies reported herein, prepared via the simple onepot and green method, provides a generally applicable solution to increase the hydrophilicity and stability of NPPs. The MOPS assemblies integrate the advantages of both the metal NCs and NPPs, resulting in a range of metal/organic nanocomplexes with excellent stability, luminescence properties, free radicals, especially SFR scavenging capacity, and extensive affinity for biomolecules. The synthesized NPP-derived MOPS assemblies presented in this study expand the library of advanced metal/polyphenol hybrid nanomaterials that may be used in a wide range of biological and biomedical applications.