Hybrid nanomaterials for assisted coral recruitment and reef rehabilitation

Abstract

A hybrid nanomaterial for coral recruitment includes silica nanoparticles that encapsulate exometabolites and a hydrogel matrix comprising a photopolymerizable material. The silica nanoparticles and the hydrogel matrix are combined to form a nanoink coating for crosslinking on a substrate that can be retained in a reef framework in natural seawater. The hybrid nanomaterial mimics the biochemical attractants, i.e., the "smell," of healthy reefs, thereby enhancing coral recruitment.

Description

HYBRID NANOMATERIALS FOR ASSISTED CORAL RECRUITMENT AND REEF REHABILITATIONRELATED APPLICATIONS

[0001] This application claims the benefit of the priority of U.S. Provisional Application No. 63 / 669,650, filed July 10, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] This invention was made with government support under HR001121 SOO 12 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the inventionFIELD OF THE INVENTION

[0003] The present invention is directed to a hybrid nanomaterial that mimics biochemical attractants of healthy reefs to facilitate and encourage coral recruitment.BACKGROUND

[0004] Coral reefs are one of the most biodiverse and economically important ecosystems globally, providing a home to more species per unit area than any other marine ecosystem. The economic significance of coral reefs is vast, particularly for the 13% of the global population residing within 100 kilometers of these ecosystems. They contribute an estimated US$375 billion annually to global economies through coastal protection, aquaculture, tourism, fisheries, as well as pharmaceuticals. Over the past five decades, coral reefs have faced significant anthropogenic pressures, primarily stemming from climate change and urbanization. Estimates suggest that by 2050, between 70% to 90% of coral reefs will be severely degraded. The survival and resilience of coral reefs depend greatly upon the successful recruitment of various benthic invertebrate larvae, such as corals, sponges, and mollusks, which help build the complex structure of the reef habitat and contribute to the maintenance of reef biodiversity.

[0005] Corals exhibit a planktonic larval phase, similar to most sessile marine invertebrates, facilitating broad dispersal of the larvae, which allows them to locate an optimal habitat for settlement. While large-scale hydrodynamics influence the initial dispersal of coral larvae across open water, once flow velocity attenuates in proximity to the benthos, larvae can actively discern and select their specific microhabitats using distinct chemical cues. This selective process aids in the identification of suitable substrates forattachment, ultimately influencing post-settlement mortality rates and subsequent reef growth dynamics. On healthy coral reefs, crustose coralline algae (CCA) play a pivotal role in inducing coral larval settlement and metamorphosis. Recent advances in metabolomics and cheminformatics have identified the distinct chemical landscape of CCA. CCA metabolites exuded into the surrounding water (termed “exometabolites”) induce settlement of coral larvae even in the absence of CCA. In contrast, competitive macro- and turf algae release other chemical signals that can inhibit settlement. As coral reefs shift towards more fleshy algal-dominated systems, the chemical landscape of coral reefs changes, which contributes to recruitment failure and ultimately describes a major bottleneck for the restoration and rehabilitation of coral reefs worldwide.

[0006] The threats facing coral reefs have overwhelmed many conservation efforts. There is now a call for more active, transformative interventions, including human-assisted evolution and microbiome and probiotic therapy. Recently, it. has been suggested that adjacent engineering fields offer promising solutions via the development of functional nanomaterials or advanced 3D biofabrication approaches to accelerate ecosystem engineering processes. Coral restoration and rehabilitation can greatly benefit from integrating advancements in bioengineering and nanotechnology which have traditionally focused on biomedical applications and human tissue engineering, with substantial progress in the engineering of functional drug carrier systems, living materials, and biohybrid materials. The present invention is directed to such an approach. Our results show that SNAP-X promotes coral settlement, highlighting its potential application in accelerating reef restoration and hybrid reef engineering efforts through chemical landscape mimicry.SUMMARY

[0007] The hybrid nanomaterial (biocoating) mimics the biochemical attractants, i.e., the “smell,” of healthy reefs, thereby enhancing coral recruitment. Specifically, the inventive biocompatible nano-ink comprises silica nanoparticles that are loaded with crustose coralline algae (CCA) exometabolites and embedded in a hydrogel matrix. The resulting material is referred to as “SNAP-X”, the name coined from its ingredients of silicon nanoparticles (NPs) and exometabolites derived from CCA. The coating can be rapidly applied to reef substrates via photopolymerization, facilitating the light-assisted 3D printing of chemical microhabitats. Testing shows that SNAP-X promotes coral settlement, highlighting its potential application in accelerating reef restoration andhybrid reef engineering efforts through chemical landscape mimicry. Coral settlement was enhanced over 20-fold using SNAP-X coated substrates compared to uncoated controls. SNAP-X is designed to gradually release chemical signals slowly (> 1 month) under natural seawater conditions, and it can be rapidly applied to natural reef substrates via photopolymerization, further facilitating the light-assisted 3D printing of microengineered habitats. This hybrid nanomaterial can be widely used to enhance coral recruitment on degraded reefs and to enhance ecosystem processes on hybrid reefs.

[0008] In one aspect, a hybrid nanomaterial for coral recruitment includes: silica nanoparticles configured to encapsulate exometabolites; and a hydrogel matrix comprising a photopolymerizable material; wherein the silica nanoparticles and the hydrogel matrix combined to form a nanoink coating configured for application to a substrate. The hydrogel matrix may be configured for one or more of gradual release of the exometabolites, stability under natural seawater conditions for periods of time greater than three months; and with a porosity of greater than 30% to permit diffusion of gasses and chemical signals. The hydrogel matrix may comprise gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA). The exometabolites are configured to promote settling of coral larvae and may be extracted from crustose coralline algae (CCA). In some embodiments, the CCA may be Hydrolithon reinboldii. The hydrogel matrix may be photo-crosslinked on a top surface of the substrate. The substrate may be calcium carbonate and may be formed as a plug configured for retention in a reef structure.

[0009] In another aspect, a structure for coral recruitment includes a substrate; and a hybrid nanomaterial applied to a surface of the substrate, the hybrid nanomaterial comprising silica nanoparticles embedded in a hydrogel matrix, wherein the silica nanoparticles are configured to encapsulate exometabolites, and wherein the hydrogel matrix is configured for slow release of the exometabolites; wherein the exometabolites are configured to promote settling of coral larvae. The hydrogel matrix may be configured for one or more of gradual release of the exometabolites, stability under natural seawater conditions for periods of time greater than three months; and with a porosity of greater than 30% to permit diffusion of gasses and chemical signals. The hydrogel matrix may comprise gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA). The exometabolites are configured to promote settling of coral larvae and may be extracted from crustose coralline algae (CCA). In some embodiments, the CCA may beHydrolithon reinboldii. The hydrogel matrix may be photo-crosslinked on a top surface of the substrate. The substrate may be calcium carbonate and may be formed as a plug configured for retention in a reef structure, which may be configured to retain an array of plugs.

[0010] In still another aspect, a method for enhancing coral recruitment includes producing a structure including a substrate and a hybrid nanomaterial applied to a surface of the substrate, the hybrid nanomaterial comprising silica nanoparticles embedded in a hydrogel matrix, wherein the silica nanoparticles are configured to encapsulate exometabolites, and wherein the hydrogel matrix is configured for slow release of the exometabolites; wherein the exometabolites are configured to promote settling of coral larvae; and disposing the structure in natural seawater containing coral larvae for one or more months.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1A-1D illustrate a schematic diagram of recruitment-enhancing SNAP-X coating for coral restoration and hybrid reef engineering, where FIG. 1A shows solidphase extraction of CCA (Hydrolithon sp.)-enriched exometabolites; FIG. IB shows CCA-exometabolites encapsulated in silica nanoparticles and combined with a hydrogel mixture which was cross-linked on CaCOs-based coral restoration substrates; FIG. 1C illustrates the SNAP-X coating gradually enriching the chemical landscape responsible for coral settlement; FIG. ID illustrates an exemplary reef structure according to an embodiment.

[0012] FIGs. 2A-2B illustrate chemoinformatics of CCA-enriched exometabolites, where FIG. 2A is a sunburst plot in which inner pie wedges and outer wedges represent the relative XIC intensity of each superclass and subclass, respectively. Unclassified subclasses were not included as outer wedges; FIG. 2B is a heatmap of classified ion features of CCA-enriched exometabolite pools for two independent sampling time points (March and June). The cells of the heatmap are colored as relative chromatogram enrichment (XIC) across each sample, calculated as standard deviations from the mean (z-scored). Ion features not classified at any level were not included. Ion features and pie wedges are colored by broad chemical classification (Superclass).

[0013] FIGs. 3A-3I illustrate characterization and release kinetics of SNAP-X nanoink. Organic mass loss (% weight) determined through (FIG. 3A) TGA and (FIG. 3B) FTIR spectra for empty and exometabolite-loaded silica nanoparticles; FIG. 3C is a TEMimage of empty silica nanoparticles and exometabolite-loaded silica nanoparticles after synthesis (t=0) and after 3 weeks in seawater (t=3 weeks). The breakdown of the nanoparticles is highlighted (black dotted area) and is characterized by the loss of nanoparticle sphericity (black arrows) and the presence of agglomeration; FIG. 3D plots particle size distribution of empty silica nanoparticles vs. exometabolite-loaded silica nanoparticles assessed using dynamic light scattering; FIG. 3E plots hydrogel porosity (%) for different prepolymer formulations (n=3 hydrogels, ANOVA, Tukey post-hoc test, p <0.05, significant differences are denoted by letters); FIG. 3F shows optical coherence tomography imaging of (I) empty hydrogels (525 ± 64 pm) vs. (II) cytochrome C-loaded hydrogels (604 ± 41 pm); FIG. 3G plots release kinetics assessed via absorbance of cytochrome C. Data are means (± SEM, n=6); FIG. 3H shows fitting of reaction rates from experimental chemical cue release data; and FIG. 31 compares rheological properties of empty hydrogels and nanoparticle (NP)-loaded hydrogels.

[0014] FIGs. 4A-D show laboratory settlement assays of Montipora capitata with SNAP- X coated substrates, where FIG. 4A shows M. capitata colonies on a reef in Hawaii’i (I). Gametes (II, yellow arrow) were collected during annual spawning and fertilized in the lab and reared to competency (III); FIG. 4B plots settlement rate (% ± SE) of larvae after an overnight incubation with seawater alone uncoated control substates (CaCCh coral plug) and different loading densities of SNAP-X (n=6, Statistical significance is signified by letters, ANOVA, Tukey post-hoc, p<0.001); FIG. 4C shows an example image of swimming larvae in the seawater control with characteristic elongated shape (white arrows); FIG. 4D is an example image of attached and settled larvae (black arrows) on SNAP-X coated substrates.

[0015] FIGs. 5A-5D illustrate mesocosm experiments and 3D diffusion modeling of chemical microhabitat under natural water flow, where FIG. 5A plots settlement rates of Montipora capitata in a natural mesocosm flowthrough system (n=6, significance values are denoted by different letters, ANOVA, Tukey post-hoc testing, p<0.01); FIGs. 5B-5C show 3D chemical diffusion modeling of the distribution of a hypothetical chemical cue at an incident flow velocity of 1 cm s’1; where FIG. 5B plots distribution of chemical cues toward the surface of SNAP-X coated biomimetic substrates with varying loading concentrations and microtopographies (flat and crevices) and FIG. 5C shows spatial distribution of the halo of chemical cues for varying loading concentrations of SNAP-X coated biomimetic substrates with top and side views; FIG.5D illustrates 3D chemical modeling of exometabolite release over 30 days for different cue loading concentrations (0.1, 1, 10 mM), showing top and side view of halo formation (at 1 nM threshold).

[0016] FIG. 6 provides top view photographs of embodiments of the SNAP-X bio-coated substrates (plugs) with a crevice surface modification, without (left) and with (right) attached coral larvae.

[0017] FIG. 7A shows recruitment location (in % of total attached larvae) for SNAP-X coated substrates in 6-well plate settlement assays (Data are means ± SE, n=6); FIG. 7B plots larval disintegration (i.e., lysis, death) in percentage of total larvae added to each well plate (% ± SE, n=6 wells). Significant differences are denoted by letters (Tukey Post hoc test, p<0.05)DETAILED DESCRIPTION OF EMBODIMENTS

[0018] In nature, several species of crustose coralline algae (CCA), e.g., Hydrolithon sp., induce settlement of a wide range of coral larvae from different coral species, partially due to the production of metabolites, which promote settlement and metamorphosis of coral recruits. Referring to FIGs. 1A-1C, a bio-inspired approach was employed in the development of SNAP-X, a hybrid nanomaterial that releases settlement-inducing exometabolites from reef substrates. FIG. 1A diagrammatically illustrates solid-phase extraction of CCA (Hydrolithon ,s / z)-enriched exometabolites from a coral reef. In FIG. IB, CCA-exometabolites encapsulated in silica nanoparticles are combined with a hydrogel mixture comprising gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA). The resulting nanoink is cross-linked on the surfaces of CaCCh- based coral restoration substrates or “plugs” using photopolymerization. Exemplary plugs are essentially “T” or mushroom shaped in cross-section with a stem that mates with receptacles in the reef structures to facilitate assembly. This example is not intended to be limiting, and alternate configurations and assembly schemes will be readily apparent to those in the art. FIG. 1C shows the SNAP-X coating on multiple plugs gradually enriching the chemical landscape responsible for coral settlement and can be locally attached to larger reef restoration frameworks formed by arranging multiple reef structures over an area to be restored. Each reef structure supports a plurality of SNAP-X coated plugs. FIG. ID provides an example of a reef substrate structure for supporting the SNAP-X coated plugs within a larger reef framework. In this non-limiting example, the reef substrate structure can be on the order of 30 cm indiameter, with multiple such structures within a broader reef framework. As will be readily apparent to those in the art, the illustrated half-sphere structure is exemplary only and other geometrical shapes may be used to enhance or modify diffusion patterns and flow velocity around the substrates, which may be influenced by local currents and surrounding topography and nearby structures.

[0019] Referring to FIGs. 2A-2B, the molecular composition of the exometabolites was characterized using liquid chromatography tandem-mass spectrometry, revealing a major contribution of lipids and lipid-like molecules as well as organic acids and derivatives to the pool of molecular ion features. To capture variability between experimental months (March and June), treatments were compared respectively to their extraction month. Fifty-eight unique ion features were identified that were significantly enriched in CCA exometabolites during at least one of the timepoints (FDR corrected p-value < 0.027. Of these, 9.7% (n == 52) were significantly enriched during both time points. These enriched exometabolites were composed of 4 organic oxygen compounds, 30 organic acids, 13 lipid and lipid-like compounds. To determine whether any of these ion features were previously identified in coral larval settlement experiments which used extracted CCA exometabolites (1) - Mass Spectrometry Search Tool (MAAST) was utilized to compare the significantly enriched metabolites against all other publicly available mass spectra within the Mass Spectrometry Interactive Virtual Environment (MassIVE) Repository (University of California, San Diego, Center for Computational Mass Spectrometry, supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R24GM148372).

[0020] These features were classified into chemical classes including carboxylic acid derivatives, carbohydrates and conjugates, amino acids, steroids, fatty amides, and ethers. FIG. 2A is a sunburst plot in which inner pie wedges and outer wedges represent the relative XIC intensity of each superclass (A, B and C) and subclass, respectively. Unclassified subclasses were not included as outer wedges. The chemical classes of CCA exometabolites (Pacific sea) are consistent with the exometabolites of CCA from the Caribbean sea and both studies primarily identified ion features classified as lipids, organic acids, and organic oxygen compounds as well as CCA specific subnetworks, e. g. , gly cerophosphoethanolamine .

[0021] FIG. 2B is a heatmap of classified ion features of CCA-enriched exometabolite pools for the two independent sampling time points (March and June). The cells of theheatmap are shaded as relative chromatogram enrichment (XIC) across each sample, calculated as standard deviations from the mean (z-scored). Ion features not classified at any level were not included. Ion features and pie wedges are shaded by broad chemical classification (Superclasses A, B and C).

[0022] Based on the complex makeup of such primarily hydrophilic exometabolites, silica nanoparticles were employed as an inorganic nanocarrier. Silica nanoparticles (NPs) can be formed as non-porous, solid, or porous depending on template functionalization. For the present application, mesoporous materials were avoided because solvent diffusion into pores accelerates silica degradation. Instead, the selected silica capsules enabled a controlled, slow release of encapsulated molecules. The degradation of these NPs occurs through hydrolysis, which is influenced by saline environments. Increased ionic strength reduces electrostatic repulsion between silanol groups, promoting dissolution. In addition, cations in the medium may form a protective shielding layer around the NPs, potentially enhancing their stability while modulating the release of silicate species, with pH variations further affecting the availability of reactive silanol groups.

[0023] Comparative thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy was used to confirm the successful encapsulation of exometabolites. FIGs. 3A and 3B compare the organic mass loss (% weight) as determined through TGA) and FTIR spectra, respectively, for empty and exometabolite-loaded silica NPs. TGA (FIG. 3A) measured a mass loss of 29% at 350- 550°C for exometabolite-loaded silica nanoparticles compared to a mass loss of 12% at 110°C for empty nanoparticles. While the mass loss of empty nanoparticles is primarily attributed to the desorption of water molecules from the silica surface, the additional mass loss at higher temperatures for exometabolite-loaded silica nanoparticles originates from the thermal decomposition of organic compounds.

[0024] FIG. 3C is a TEM image of empty silica nanoparticles (upper panels) and exometabolite-loaded silica nanoparticles (lower panels) after synthesis (t=0) and after 3 weeks in seawater (t=3 weeks). The breakdown of the nanoparticles is highlighted (black dashed area) and is characterized by the loss of nanoparticle sphericity (black arrows) and the presence of agglomeration. FIG. 3D plots particle size distribution of empty silica NPs vs. exometabolite-loaded silica NPs assessed using dynamic light scattering. The mean hydrodynamic diameter of the empty and exometabolite-loaded silica nanoparticles was on average 67 nm and 42 nm, respectively. The difference insize may be attributable to the presence of complex metabolites that can modulate the supramolecular chemistry involved in the formation of nanosilica.

[0025] To enhance coral recruitment in nature, it is crucial that chemical signals are released from the biomaterial slowly over time, ideally over a timeframe of several weeks and months to coincide with major spawning events. In contrast, for most biomedical applications nanoparticles are designed to rapidly release a drug over a short time frame (i.e., burst release within minutes to hours) once they reach the target cell or location, which is facilitated by particle pore openings and / or in situ stimuli. This feature is not beneficial to coral engineering applications. Instead, a combination of a fully inorganic non-porous silica NP system combined with a hydrogel matrix was employed to provide a controlled, slow release of encapsulated exometabolites.

[0026] To achieve the desired breakdown kinetics, a GelMA-PEGDA hydrogel was optimized to ensure stability under natural seawater conditions for extended periods of time (> 3 months), while still facilitating a sufficiently high porosity (>30%) to allow for diffusion of gasses and chemical signals through the hydrogel network. FIG. 3E plots hydrogel porosity (%) for different prepolymer formulations. The GelMA component of the hybrid material is selected for its excellent biocompatibility, biodegradability, and tunable mechanical properties. GelMA is highly porous due to its enrichment in OH groups, which have a high affinity for silica, and therefore offers protection from NP hydrolysis. . PEGDA, a synthetic polymer, improves mechanical stiffness and crosslinking efficiency due to its uniform repeating units, which facilitate a high degree of polymerization and impart antifouling properties. This dual-component hydrogel approach was previously demonstrated to improve material performance by leveraging the complementary mechanical strength of PEGDA and the biological compatibility of GelMA, further supporting its application in SNAP-X. The dualcomponent hydrogel is photo-crosslinked on top of CaCCh as a thin layer (between 500- 600 pm) containing uniformly dispersed nanoparticles (FIG. 3F).

[0027] The release kinetics of dispersed nanoparticles and hybrid nanoparticle hydrogel systems can theoretically stem from a variety of factors including Ostwald ripening of silica nanoparticles, cargo diffusion within the silica framework, silica nanoparticle dissolution, hydrogel swelling, leaked cargo diffusion within the hydrogel matrix, and hydrogel framework dissolution. Release kinetics were assessed using a common model molecule (cytochrome C) and revealed up to 4-fold slower release rates for thedeveloped nanoparticle-hydrogel system (-20% mean release after 28 days) compared to the standalone nanoparticles (~ 89% mean release after 28 days).

[0028] The release profile of dispersed nanoparticles aligns with earlier studies, suggesting a complex interaction between cargo diffusion within the silica framework and nanoparticle dissolution that controls release rate. TEM images (immediately after dispersion and after 3 weeks in solution), confirmed that the dissolution of silica is very slow and that it begins from the nanoparticle core, a behavior that is also observed with comparable silica nanoparticle systems. Ostwald ripening occurs during the growth of spherical silica nanoparticles, leading to larger particles at the expense of smaller ones, eventually resulting in nearly monodisperse nanoparticles. This phenomenon is common due to reversible silica condensation and hydrolysis, leading to the release of embedded species from smaller nanoparticles without re-entrapment in larger ones. The contribution to total released species due to Ostwald ripening is generally small.

[0029] For the hybrid nanoparticle hydrogel system, silica nanoparticle dissolution, cargo diffusion within the silica framework, and Ostwald ripening are largely impeded by the restricted mobility of water and other small water-soluble species within the hydrogel network. Initially, the release profile remains nearly constant for 3-5 days, likely due to hydrogel swelling, which hinders cues from being released as liquid flows into the hydrogel. Once swelling is finished, cues then gradually leak from the hybrid material in a stepwise manner, first diffusing within the silica framework until reaching the silicahydrogel interface, and then within the hydrogel framework until reaching the hydrogelwater interface. Arguably, these processes are largely dominated by diffusion compared to the more dominant release profiles of dispersed nanoparticles, which is driven by more rapid silica dissolution.

[0030] To interpret these release kinetics under different flow environments and for extended periods in situ, spatio-temporal models were developed using COMSOL MULTIPHYSICS® (COMSOL, Inc. Burlington, MA, USA), a simulation software designed for creating and solving physics-based models through finite element methods (FEM). The spatio-temporal flow model is created using the ‘Reacting Flow, Diluted Species’ coupling feature, combining the ‘Transport of Diluted Species’ module with the ‘Laminar Flow’ model. This combination simulates the transport and mixing of chemical species within a fluid under laminar flow conditions. The models are used to predict the concentration of exometabolites after being released by the SNAP-X coatedsurfaces for up to 30 days. The input geometry consists of a plug situated in a large body of water, modeled as a box with an inflow on one side and an outflow on the opposite side. The bottom boundary, where the plug is located, and the surface of the plug are defined by a ‘no-slip’-boundary conditions. This indicated that the fluid velocity at the boundary is set to zero. The other boundaries are defined to match the pre-set flow matching in both velocity and direction, ensuring consistent flow around the boundaries of the model.

[0031] The molecular release of exometabolites is implemented as a surface reaction occurring at the top of the plug and at the bottom of the plug's crevices. This surface reaction models the release of molecules from SNAP-X. The release rate is based on the measured cytochrome C release kinetics and the data is fitted to a function that describes NP-dissolution behavior. The mathematical description of the exometabolite release within a timeframe of 5 to 30 days is based on the nanoparticle dissolution behavior (Eq 1.) and is used to model the combined processes resulting in cue release: c = cmax(l — e~at + bEq. 1

[0032] Mathematical modeling showed that the measured rates were well predicted based on silica hydrolysis in a stabilized environment, i.e., hydrogel. FIG. 3H shows fitting of reaction rates from experimental chemical cue release data based on Eq. 1, fitted to release kinetics. (Note that data fitting for the hybrid nanoparticle hydrogel system was omitted for days 1 -5, due to hydrogel swelling). By taking the derivative of this function (Eq. 2), the release rate was derived as a function of time and the initial concentration of exometabolites within the SNAP-X coating. The diffusion coefficient of the exometabolites is set to a generalized value of l - e'9m2 / s, based on the diffusion of hydrocarbons in water. This constant accounts for the wide range of different molecules found in exometabolites (1), which are collectively represented by one single term in the model.

[0033] FIG. 3F shows optical coherence tomography imaging of (I) empty hydrogels (525 ± 64 pm) vs. (II) cytochrome C-loaded hydrogels (604 ± 41 pm).

[0034] To mimic water motion, the release tests were also performed under constant shaking, which revealed comparable release rates between stationary and shakingconditions for the NP-hydrogel system . FIG. 3G plots release kinetics assessed via absorbance of cytochrome C. Data are means (± SEM, n=6; max. difference at day 22, mean stationary, 16.10% ± 2.72 SD, mean shaking, 21.06 % ± 3.56 SD, ANOVA, F(i,8)=0.17, p=0.69). These results suggest the suitability of the inventive hybrid nanoparticle-hydrogel coating for long-term (weeks to months) in situ applications.

[0035] The rheological properties of SNAP-X and control hydrogels were evaluated over time, specifically examining changes in mechanical stability under natural seawater conditions. SNAP-X exhibited a significantly higher initial storage modulus (G') of -3500 Pa (0-70 rad / s) compared with the control hydrogel (G' -250 Pa, 0-70 rad / s), highlighting the enhanced mechanical strength conferred by the incorporation of silica NPs. FIG. 31 compares the rheological properties of empty hydrogels and NP-loaded hydrogels. The NPs contributed to a denser crosslinked network, improving the stability of the hydrogel in saline environment. Over 4 weeks, the storage modulus (G') of SNAP- X gradually decreased to -500 Pa (0-70 rad / s), while that of the control hydrogel declined to -150 Pa (0-70 rad / s). This sustained mechanical performance of SNAP-X can be attributed to the reinforcing effect of the silica NPs, which helps maintain the elasticity of the hydrogel even in saline conditions. This material behavior, characterized by initial stability and gradual softening, provides an advantage for coral larvae — the firm substrate supports settlement, while the subsequent softening may promote their growth and integration into the material.

[0036] To test the efficacy of SNAP-X in settlement assays, laboratory experiments were performed with Montipora capitata, a primary reef-building coral in Hawai‘i (FIG. 4A). Wild spawned Montipora capitata gametes were collected from a natural spawning slick near reef 11 in Kaneohe Bay (Lat. 21.449248° N, Long. 157.796328° W), Oahu, Hawai’i. This species is a hermaphroditic broadcast spawner and gamete release times do not overlap with other species. Gametes were collected from several hundred square meters of patch reef dominated by M. capitata, likely encompassing hundreds of parental genotypes. Gamete bundles were returned to the Hawai’i Institute of Marine Biology and allowed to break up and fertilize in 50 mL conical tubes, rinsed and added to filtered seawater conical rearing tanks following established best practices. (See, e.g., S. A. Rahnke, et al., Optimizing sexual reproduction of Montipora capitata for restoration: effects of abiotic conditions and light acclimation on juvenile survival and growth. Mar. Ecol. Prog. Ser. 691, 41-54 (2022).) Corals were reared to competency at4 days post fertilization following the established best practices before introduction to settlement trials. Coral larvae were about -200-400 pm in size.

[0037] Compared to CaCCh-based control substrates, larvae exposed to SNAP-X exhibited up to six-fold higher settlement rates (ANOVA, F(4,42) = 40.77, p<0.001, FIG. 4B). Visual observations suggested that larvae, indicated as the light oval rings, preferred to settle in close proximity to the SNAP-X coating on top of CaCCh, and particularly near the SNAP-X coated crevices seen in FIG. 4D (see, also, FIG. 7A), suggesting the successful molecular enrichment of the substrate microhabitat. In contrast, for CaCCh- based control substrates, settlement rates were close to 10%, while seawater controls showed no settlement (FIG. 4C). These experiments demonstrate that SNAP-X coating successfully induced controlled metamorphosis and settlement to the substrate of interest, a major improvement compared to previous efforts that used cues homogeneously dissolved within the ambient water, leading largely to unattached metamorphosed larvae.

[0038] An outdoor mesocosm settlement experiment was performed using natural seawater and continuous water flow to evaluate SNAP-X under in situ-like conditions. Settlement was enhanced by up to 20-fold using SNAP-X compared with the uncoated control substrate (ANOVA, F(3,67):=:7.09, p<0.001). Under continuous water flow, a significant effect of the concentration of CCA exometabolites was detected on settlement induction, which was not observed in the laboratory experiments. Referring to FIG. 5A, substrates coated with SNAP-X 10 (i.e., 10-fold enhanced exometabolite loading concentration), showed settlement densities that were up to 170% higher compared to the medium loading concentration (SNAP-X 1). In turn, for SNAP-X 0.1 (i.e., 10-fold reduced loading concentration) there was no significant difference in settlement compared to uncoated control substrates (ANOVA, p= 0.59, FIG. 5A). 3D chemical diffusion modeling showed that the concentration of a hypothetical chemical cue (with average molecular weight of 12 kDa,) is enhanced towards the surface of the substrate (FIG. 5B) The developed SNAP-X coating can create a “halo” of settlement-inducing metabolites that extends well beyond (> 10 cm) the coating, as shown in the top and side views in FIG. 5C. Such chemical landscape enrichment is dependent on the initial loading density of exometabolites, as well as fluid flow and mass transfer properties. Thickening of the diffusive exometabolite boundary layer, via the addition of microarchitectural changes (e.g., the addition of crevices such as shown in FIGs. 4D,the lower left panel of FIG. 5B, and FIG. 6, or other surface topographical features) allows for local enhancement of the concentration of exometabolites. As SNAP-X is a photo-crosslinkable nanomaterial, it can be used with light-assisted 3D printing approaches such as those disclosed in U.S. Patent No. 12,167,720, which is incorporated herein by reference, to create on-demand micropattems that will optimize chemical signaling in different fluid flow environments.Applications for reef rehabilitation and hybrid reef engineering

[0039] Reef restoration endeavors have recently gained momentum through global pledges aimed at substantial recovery of natural ecosystem health and size (e.g. UN Decade for Ecosystem Restoration). Coral restoration typically employs either asexual fragmentation of adult corals or the production of sexually propagated juvenile corals. Importantly, the latter method offers the advantage of greater genetic diversity and is thus crucially needed to enhance long-term ecosystem resilience. Enhancing sexually propagated corals in situ so far has been hampered by low settl ement success on standard restoration substrates. The inventive scheme provides a biomaterials approach employing chemical landscape mimicry to augment recruitment microenvironments with potent settlement cues from CCA.

[0040] There has been an increasing interest in identifying specific molecules and compounds responsible for settlement and promising cue candidates include tetrabromopyrolle (produced by biofilms), cnidarian neurotransmitters, cycloprodigiosin, and CCA derived morphogens. Although the present disclosure describes a bulk approach that encapsulates a complex suite of settlement-inducing exometabolites, the inventive nanoink can be theoretically modulated for the encapsulation of different cues of interest, through, for example, the functionalization of the silica pore network or the hydrogel network to offer the ability to bind hydrophobic molecules, e.g., cycloprodigiosin and CCA derived morphogens. The described examples of chemical landscape mimicry thus provide a viable means to enhance the recruitment of specific ecosystem engineers, such as corals, but could also be more broadly employed to attract other marine life such as coral reef fish larvae.

[0041] The following examples describe methods, applications, and evaluation of embodiments and exemplary implementations of the inventive approach and materials. These examples are intended to be illustrative and non-limiting.Example 1 : Preparation of CCA exometabolites

[0042] CCA that was visually identified as Hydrolithon reinboldii, was collected from Kaneohe Bay, Hawai’i. CCA was cleaned of epiphytes and maintained in flow-through seawater outdoors at the Hawai’i Institute of Marine Biology. To extract settlementinducing exometabolites, small pieces of CCA (covering a surface area of about 200- 400 cm2) were incubated with 1 L of seawater for 12 hours during daytime. Exometabolites were extracted from metabolite enriched seawater using a solid-phase extraction. For this, seawater was filtered through 0.2 pm polyethersulfone filter cartridges (Sterivex, Millipore, UK) and acidified to pH 2 with LC-MS grade hydrochloric acid. The acidified filtrate was extracted on the solid-phase extraction (SPE) resin, Priority PolLutant (PPL, Agilent Bond Elut). PPL columns were cleaned and activated prior to extraction with LC-MS grade solvents. To identify and remove any unwanted background molecular features (e.g., due to manual handling), blanks were collected using LC-MS grade water. PPL resins were dried with nitrogen gas and stored in an ultra-low temperature freezer ( — 80 °C) for further use. SPE-bound metabolites were eluted in LC-MS grade methanol and the dried exometabolite powder was recovered by methanol evaporation using a rotary evaporator.Example 2: Liquid chromatography tandem mass spectrometry (LC-MS / MS) Testing

[0043] SPE-bound metabolites were eluted in HPLC-grade methanol and injected into a Vanquish reverse-phase ultra-high-performance liquid chromatography system using C18 core-shell column (Kinetex C18, 150 * 2 mm, 1.8 pm particle size, 100 A pore size, Phenomenex) electrospray ionization (ESI+) mass spectrometer (MS). An Orbitrap Elite Hybrid Linear Ion-Trap (Thermo Fisher Scientific) in data-dependent acquisition (DDA) of MS / MS spectra was performed to collect fragmentation spectra within a range of 150-1500 mass-to-charge ratio (m / z) in positive ion mode at a MSI resolution of 120,000 and MS2 resolution of 17,500 (79). LC-MS / MS was conducted at the Environmental and Complex Analysis Laboratory University of California, San Diego.Example 3: Metabolomic analytical pipeline

[0044] An “ion feature” (also called molecular features) refers to an ion signal at specific retention times eluted off the UHPLC for which an MS / MS spectra is assigned. After conversion of the 26 raw MS-MS files to centroid mzXML, the mass spectrometry data analysis platform MzMine 3.9.0 (mzio GmbH, Bremen, DE) was used to detect 268 unique ion features. Each ion feature was aligned to previously characterized spectrallibraries using the Global Natural Products Social Molecular Networking (GNPS) platform. Using the feature-based molecular network, GNPS additionally compared all ion features to cluster structurally similar ion features together into molecular subnetworks which were visualized using the Cytoscape™ software platform (Cytoscape Consortion). Using the in silico prediction tool CANOPUS (Lehrstuhl Bionformatik Jena, Friedrich-Schiller-Universitat Jena, DE), we predicted putative structures for each ion features. A total of 12 experimental blanks were used to define background ion features (putative contaminants), described previously as ion features that had an average logio transformed extraction ion chromatogram (XIC) intensity across all samples less than 50% max abundance from experimental blanks. This removal of background features reduced the dataset to 76 ion features. Two-sided t-tests compared the exometabolites enriched in CCA pools to those observed in the seawater controls separately for each timepoint (FDR corrected t-test p < 0.05). Fifty-eight ion features were defined as significantly enriched in CCA exometabolites.

[0045] To capture variability between experimental months (March and June), treatments were compared respectively to their extraction month. Fifty-eight unique ion features were identified that were significantly enriched in CCA exometabolites during at least one of the timepoints (FDR corrected p-value < 0.027. Of these, 81% (n = 47) were significantly enriched during both time points. These enriched exometabolites comprised 4 organic oxygen compounds, 30 organic acids, 13 lipid and lipid-like compounds. To determine whether any of these ion features were previously identified in coral larval settlement experiments which used extracted CCA exometabolites [1] - Mass Spectrometry Search Tool (MAAST) was utilized to compare the significantly enriched metabolites against all other publicly available mass spectra within the Mass Spectrometry Interactive Virtual Environment (MassIVE) Repository (University of California, San Diego, Center for Computational Mass Spectrometry, supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R24GM148372). Data were logio transformed prior to any statistical analysis to approximate gaussian distribution.

[0046] Directly assessing the stability of encapsulated exometabolites within the SNAP-X system can be technically challenging due to difficulties in fully releasing the compounds and breaking down the silica nanoparticles and hydrogel matrix. Investigation of the potential effects on exometabolites of UV exposure duringphotopolymerization was initiated because residuals from this process could interfere with ionization during mass spectrometry analysis. As a proxy for encapsulated conditions, an unencapsulated exometabolite solution was exposed to UV irradiation. Metabolomics analysis revealed a 15.1% ± 20.7% decrease in total ion intensity (TIC) in response to UV exposure. The metabolites were categorized as lipids and lipid-like compounds, organic acids, or uncharacterized. Lipid intensity decreased by 23.6% ± 3.93%, and uncharacterized metabolites showed a 21.8% ± 2.50% loss, while no organic acids were identified as UV sensitive. Notably, in the SNAP-X system, UV exposure is mitigated by the photoinitiator LAP, which efficiently absorbs UV light at 405 nm, and the encapsulation within silica nanoparticles, which further shields the exometabolite from UV light.Example 4: Nanoparticle (NP) formulation

[0047] Several protocols were evaluated for one-pot synthesis and loading of non-porous silica nanoparticles with hydrophilic species, which has previously been described for delivery of anticancer drugs. (See, e.g., S. Jiang, et al., One-pot green synthesis of doxorubicin loaded-silica nanoparticles for in vivo cancer therapy, Mater. Set. Eng. C Mater. Biol. Appl. 90, 257-263 (2018); A. Auger, et al., A comparative study of non- covalent encapsulation methods for organic dyes into silica nanoparticles, Nanoscale Res. Lett. 6, 328 (2011).). To synthesize inorganic non-porous silica nanoparticles that encapsulate exometabolites and can be incorporated into a hydrogel matrix for slow release, a water-in-oil microemulsion sol-gel method was used. This approach enables the direct entrapment of hydrophilic species within the forming silica framework. Triton X-100 (1.77 mL) and n-hexanol (1.8 mL) were dissolved in cyclohexane (7.5 mL). Separately, exometabolite dry powder extracted from one PPL cartridge was dissolved in 600 pL of MilliQ water and added to 100 pL of tetraethyl orthosilicate (TEOS) followed by vortexing for 5 min before this mixture was added to the organic solution. 50 pL of 30% aqueous ammonia solution was added and the water-oil emulsion was stirred at 250 rpm overnight at a room temperature of 20 °C. 20 mL of pure acetone was subsequently added to precipitate the exometabolite loaded silica nanoparticles. The material was recovered by centrifugation at 12000 rpm, washed twice with ethanol, and three times with water. Finally, the nanoparticles were dried in a vacuum desiccator overnight. Variations in loading amounts of exometabolites (O. lx, 0.5x, lx, 5x, lOx)were explored, where lx is equivalent to the dried metabolite powder extracted from 1 PPL cartridge, which is equivalent to 200-400 cm2of CCA surface area.Example 5: Polymer synthesis

[0048] Gelatin methacrylate (GelMA) was synthesized as described by D. Wangpraseurt, et al., Bionic 3D printed corals. Nat. Commun. 11, 1748 (2020). Briefly, porcine gelatin (Sigma Aldrich, USA) was mixed at 10% (w / v) 0.25 M carbonate-bicarbonate (CB) buffer (~pH 9.2-9.5) and stirred at 50 °C until fully dissolved. Methacrylic anhydride (MA; Sigma Aldrich, USA) was added dropwise to the gelatin solution at a ratio of approximately O.lmL of MA per gram of gelatin. The reaction continued for about 2 h at 50 °C under constant stirring. The solution was then dialyzed against distilled water using 12-14 kDa cutoff dialysis tubing (Spectrum Laboratories, CA, USA) for 7 days at 40 °C to remove any unreacted methacrylic groups from the solution. The GelMA was lyophilized at -80 °C in a freeze dryer (Freezone, Labonco) for 1 week to remove the solvent. Poly(ethylene) glycol diacrylate (PEGDA, Mn = 700 Da) was purchased from Millipore-Sigma (St. Louis, MO). The photoinitiator lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) (TCI America™) was used to obtain the photopolymerizable prepolymer mixture by dissolving it in MilliQ water at 60°C via sonication to reach a stock solution of 4%.Example 6: Free radical photopolymerization of SNAP-X coating

[0049] The lyophilized GelMA was dissolved in MilliQ water to create a 15% w / v stock solution. Exometabolite loaded silica nanoparticles were uniformly dispersed in MilliQ water by vortexing for 1 min. SNAP-X was then created by mixing the GelMA stock solution, photoinitiator LAP, and PEGDA-700 at a final concentration of 5% w / v GelMA and 0.5% w / v LAP and 10% w / v PEGDA-700 and 6.25 w / v silica nanoparticles. The nanoink was then used to coat common coral restoration substrates made from calcium carbonate (coral plugs, see, e.g., FIG. IB) via free radical photopolymerization. For each plug, 120 pL of the nanoink was used to coat the surface of the substrate, with 40 pL applied to the base of the plug and 80 pL applied to the top of the plug. Crosslinking was facilitated via rapid free radical photopolymerization induced using a 405 nm LED source (Thorlabs, New Jersey, USA) emitting 17mW cm'2s'1for a total of 45 seconds exposure time. Hydrogel porosity was measured based on weight changes following ethanol immersion.Example ?: Porosity Evaluation

[0050] The porosity of GelMA (5%), PEGDA-700 (10%) and GelMA - PEGDA-700 (5% / l 0%) hydrogels was measured by preparing circular hydrogel discs (diameter = 25 mm) through photopolymerization and immersing them in absolute ethanol for 24 h. (See, e.g., M. Akhlaq, et al., Methotrexate-Loaded Gelatin and Polyvinyl Alcohol (Gel / PVA) Hydrogel as a pH-Sensitive Matrix, Polymers 13 (2021).) The weights of the discs were obtained (~ 1.5 g) before immersion in absolute ethanol. After 24 h of immersion, each disc was removed and the excess ethanol was wiped with low-lint wipes (e.g., Kimwipes® or similar) to measure the weight.

[0051] The porosity (% ± SD) was determined using the following formula: 100 Eq. 3where M2 is the mass of the hydrogel after removal from ethanol, Ml is the mass of the hydrogel before immersion in ethanol, p represents the density of absolute ethanol and V represents the volume of the hydrogel used for photopolymerization to form the disc shape.Example 8: SNAP-X material characterization

[0052] The sizes of the nanoparticles were characterized using TEM imaging and dynamic light scattering (DLS) (FIG. 3C and 3D). For DLS measurements, 10 mg of empty silica nanoparticles and exometabolite loaded silica nanoparticles were suspended in 1 mL FSW and incubated in the dark at 25°C shaking (80 rpm) condition. The samples in seawater were centrifuged and washed three times with dH2O to avoid interference due to seawater components (such as electrolytes). The DLS measurements were performed on dispersions of silica nanoparticles in dH2O (pH 7) using a zetasizer ultra equipment with multi-angle dynamic light scattering (MADLS) technology (Malvern Panalytical, USA) and analyzed using a commercial software (ZS Xplorer, USA).

[0053] To evaluate the successful loading of silica nanoparticles with exometabolites, Fourier-transform infrared spectroscopy (FTIR) and thermo gravimetric analysis (TGA) were performed. For the FTIR analysis, silica nanoparticles were obtained as fine powder after drying and the sample amount was chosen to completely cover the small transparent window of the ATR-FTIR chamber (~1 mg). The FTIR spectra wererecorded using a high-resolution spectrometer (Shimadzu IRAffinity-1). The attenuated total reflectance spectra were collected at a spectral resolution of 1 cm1with 45 scans from 600 to 4000For TGA measurements, silica nanoparticles were heated from 25°C to 100°C at a speed of 2.6°C min'1, then heated from 100 to 800 °C at a speed ofbefore being held at this temperature for a further 30 min. Measurements were carried out under a 20 mL min1airflow using a simultaneous thermal analyzer (Netzsch model STA 449 Fl Jupiter).

[0054] Transmission Electron Microscopy (TEM) imaging was used to visually evaluate the shape and nanoarchitecture of the nanoparticles as well as their breakdown over three weeks following incubation with seawater. For TEM imaging, 10 mg of empty silica and exometabolite-loaded silica nanoparticles were suspended in 1 mL FSW and incubated under shaking conditions (at 80 rpm) at 25°C in the dark for three weeks. Aliquots (300 pL) were taken weekly during the three-week experiment. Samples were centrifuged and washed three times with dELO to avoid interference with seawater (e.g., due to the presence of salts). Diluted samples (0.5 mg nanoparticles / mL dELO, 5-10 pL) were placed on TEM copper grids for 3-5 minutes.

[0055] The grids were first placed on a filter paper to remove any excess droplets. Afterwards, they were returned to the grid holder and left overnight in a well-ventilated place to ensure complete evaporation of the residual solvent. The samples were imaged with a Talos™ L120C TEM with a LaB6 electron source operating at a voltage of 120 kV with a beam current of 5 pA.

[0056] Rheological measurements were performed by using a DHR30 from TA Instruments to assess the mechanical behavior of the hydrogel and the nanoparticle loaded hydrogels. A 25 mm parallel plate was used with a Peltier plate to hold the temperature at 25°C. An isostrain of 1% and angular frequency range of 1-100 rad / s were used with a variable gap to correct for the swelling of the samples affecting the thickness. Storage and loss modulus were plotted versus the angular frequency. The distribution of the nanoparticles within the hydrogel and the thickness of the hydrogel layer was measured through Optical Coherence Tomography (OCT) (Thorlabs GmbH, Dachau, Germany).Example 9: Cytochrome C release tests

[0057] The release of chemical cues from the SNAP-X system was investigated using cytochrome C, a commonly used model molecule. Cytochrome C was selected because its molecular weight (12 kDa) closely approximates the molecular weight ofexometabolites (50-100 kDa) as determined by LC / MS, suggesting a similar diffusion coefficient. While CCA exometabolites comprise a complex mixture of compounds, many of which are not fully characterized, cytochrome C was chosen for its hydrophilicity, making it a relevant proxy for the water-soluble fraction of exometabolites extracted from the cartridges. Although no single model molecule can fully replicate the release profile of this complex exometabolite mixture, cytochrome C provided a useful preliminary validation tool. Its strong absorption in the visible region enabled effective monitoring of release kinetics via UV / Vis absorption spectroscopy. It is important to note that the measured release kinetics reflect those of the model molecule, whereas individual exometabolites may diffuse at different rates depending on their specific properties.

[0058] To study release kinetics with cytochrome C, nanoparticles were synthesized as described above using an initial loading concentration of cytochrome C of Img / mL. To explore the benefits of stabilization through the hydrogel network, tests were conducted on both dispersed nanoparticles and the hybrid nanoparticle hydrogel system. In the case of dispersed nanoparticles, 10 mg of nanoparticles were suspended in 1 mL of FSW and incubated under static and shaking conditions at 25°C in darkness. The nanoparticle suspension was centrifuged (16000 ref) every 24 h. The supernatant was discarded and the obtained nanoparticles were re-dispersed in 1 mL FSW. A well plate reader (SpectraMax iD3) was used to quantify the peak absorbance of cytochrome C (410 nm) from the suspension. The change in absorbance intensity was used to determine the percentage of cytochrome C released. Similarly, for the hybrid nanoparticle hydrogel system, an equal amount of cytochrome C-loaded nanoparticles (10 mg per plug) were cross-linked within the hydrogel and on top of CaCCh plugs. The coated plugs were incubated in six-well plates filled with 9 mL of FSW under the same condition as the dispersed particles. Aliquots (300 pL) were taken, replaced with an equal volume of FSW, and the release of cytochrome C was measured at specific time intervals. To account for any background absorption due to the hydrogel, measurements were also performed on hydrogels alone without nanoparticles.Example 10: Coral settlement assays

[0059] Montipora capitata larvae were reared following established best practices (as described above) to three-four days post fertilization. Larval settlement was assessed in 6-well plates without water flow and in a flow-through mesocosm experiment . For 6-well plate experiments, the SNAP-X coating was tested for three different loading densities (O.lx, lx, lOx concentrations, designated as SNAP-X 0.1, SNAP-X 1, and SNAP-X 10) and evaluated against uncoated control plugs and seawater controls (n=6 plugs per treatment). Each plug was placed in a single well and filled with 7 mL of FSW. M. capitata larvae were first washed with FSW to remove debris and organic matter before 3 mL were added to each well (at a density of ~13 larvae mL’1). Settlement assays were performed overnight in a 27°C incubator. To test the efficacy of SNAP-X under in SHU-W Q conditions, we created a flow through mesocosm system that used natural seawater. Each treatment (SNAP-X 0.1, SNAP-X 1, SNAP-X 10, uncoated control) was replicated in three tanks, with six plugs per tank ( / / = l 8 per treatment). The volume of seawater in the tank was maintained at 0.5 L with an average outflow rate of ~3 mL s'1. 100 mL of washed AL capitata larvae (at a density of 10 mL’1) was added to each tank. After an incubation period of 12 hours, larval settlement was counted with the aid of a blue light excitation source (Sola Nightsea Light, USA) to improve larval / recruit visibility. Referring to FIGs. 7A-7B, larval behavior was categorized through visual observations and microscopy using a digital microscope (Dino-Lite Premier digital microscope, USA) coupled with a blue excitation source (Sola Nightsea Light, USA) and a stereo microscope (Motic SMZ-168) as settled (attached and metamorphosed), dead / disintegrated, and swimming. Larval settlement locations were also recorded as top, bottom and side of the CaCCh substrate. FIG. 7A shows recruitment location (in % of total attached larvae) for SNAP-X coated substrates in 6-well plate settlement assays. (Data are means ± SE, n=6). FIG. 7B plots larval disintegration (i.e., lysis, death) in percentage of total larvae added to each well plate (% ± SE, n=6 wells). Significant differences are denoted by letters (Tukey Post hoc test, p< 0.05).Example 11 : 3D chemical modeling

[0060] Spatio-temporal models were built to simulate the 3D distribution of exometabolites released from SNAP-X coated substrates over a 30 day period at different loading concentrations, 0.1, 1, and 10 mmol. As described above, the ‘Reacting Flow, Diluted Species’ feature of COMSOL Multiphysics software was used for this analysis. To simulate the diffusion of a pool of exometabolites, a generalized diffusion coefficient (he’9m2 / s) based on the diffusion of hydrocarbons in water was used. Exometabolites were released as a steady state flux based on measured cytochrome C release. The molecular release profile of the nanoparticle hydrogel system was modeled as a surfacereaction producing exometabolites, located at the surface of the plug covered by the hydrogel in the experimental set-up. The reaction rate was obtained by fitting the kinetic release data of the nanoparticle hydrogel system loaded with cytochrome C after the initial swelling period (2-3 days). The combined processes resulting in cue release over a timeframe of 5 to 30 days were modeled using Eq. 1 and Eq. 2 above. The 3D distribution of exometabolites released from a singular plug for each loading concentration was then estimated at an incident flow velocity of 1 cm / s, as shown in FIGs. 5B-5C. Simulations with multiple plugs attached to larger reef frameworks were also performed using higher ambient flow velocities (4 cm / s). FIG. 5D illustrates 3D chemical modeling of exometabolite release over 30 days for different cue loading concentrations (Mo = 0.1, 1, and 10 mM). Top and side views of halo formation (at 1 nM threshold) are shown for each of days 5, 15, and 30.

[0061] All statistical analysis and data plotting was performed with Origin Pro 2024 (Origin, USA). Data were verified for normal distribution using Shapiro-Wilk tests with a significance level of a = 0.05. Data were arcsine square root transformed if necessary. One-way analysis of variance (ANOVA) was used to assess statistical differences in coral settlement (p<0.05). When statistical differences were observed, Tukey’s HSD post-hoc comparisons were applied determine which of the groups and experimental treatments were significantly different with a significance level of a = 0.05.

[0062] The foregoing description, examples and results demonstrate that the inventive SNAP-X hybrid nanomaterial can enhance coral settlement over 20-fold under natural water flow, highlighting its capacity for improving coral recruitment and accelerating the construction of living hybrid reefs. SNAP-X employs a modular, flexible ecosystem augmentation approach, making it suitable for integration with existing artificial reef structures to enhance coral propagation. Strategic placement and distribution of SNAP- X coated substrates on reef infrastructures can be further used to optimize cue accumul ation and thus settlement chances in situ. The core components of the inventive hybrid nanomaterials are abundant and cost-effective to produce (e.g. silica, gelatin) and free radical photopolymerization is among the fastest existing cross-linking methods, thus suggesting a viable pathway towards scalability.

Claims

WHAT IS CLAIMED:

1. A hybrid nanomaterial for coral recruitment comprising: silica nanoparticles configured to encapsulate exometabolites; and a hydrogel matrix comprising a photopolymerizable material; wherein the silica nanoparticles and the hydrogel matrix combined to form a nanoink coating configured for application to a substrate.

2. The hybrid nanomaterial of claim 1, wherein the hydrogel matrix is configured for gradual release of the exometabolites.

3. The hybrid nanomaterial of claim 1, wherein the hydrogel matrix is configured for stability under natural seawater conditions for periods of time greater than three months.

4. The hybrid nanomaterial of claim 1, wherein the hydrogel matrix is configured with a porosity of greater than 30% to permit diffusion of gasses and chemical signals.

5. The hybrid nanomaterial of claim 1, wherein the hydrogel matrix comprises gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA).

6. The hybrid nanomaterial of claim 1, wherein the exometabolites are configured to promote settling of coral larvae.

7. The hybrid nanomaterial of claim 1, wherein the exometabolites are extracted from crustose coralline algae (CCA).

8. The hybrid nanomaterial of claim 4, wherein the CCA comprises Hydrolithon reinboldii.

9. The hybrid nanomaterial of claim 1, wherein the hydrogel matrix is photocrosslinked on a top surface of the substrate.

10. The hybrid nanomaterial of claim 1, wherein the substrate comprises calcium carbonate.

11. The hybrid nanomaterial of claim 1, wherein the substrate is formed as a plug configured for retention in a reef structure.

12. A structure for coral recruitment, comprising: a substrate; and a hybrid nanomaterial applied to a surface of the substrate, the hybrid nanomaterial comprising silica nanoparticles embedded in a hydrogel matrix, wherein the silica nanoparticles are configured to encapsulate exometabolites, and wherein the hydrogel matrix is configured for slow release of the exometabolites; wherein the exometabolites are configured to promote settling of coral larvae.

13. The structure of claim 12, wherein the hydrogel matrix is configured for gradual release of the exometabolites.

14. The structure of claim 12, wherein the hydrogel matrix is configured for stability under natural seawater conditions for periods of time greater than three months.

15. The structure of claim 12, wherein the hydrogel matrix is configured with a porosity of greater than 30% to permit diffusion of gasses and chemical signals.

16. The structure of claim 12, wherein the hydrogel matrix comprises gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA).

17. The structure of claim 12, wherein the exometabolites are configured to promote settling of coral larvae.

18. The structure of claim 12, wherein the exometabolites are extracted from crustose coralline algae (CCA).

19. The structure of claim 18, wherein the CCA comprises Hydrolithon reinboldii.

20. The structure of claim 12, wherein the hydrogel matrix is photo-crosslinked on a top surface of the substrate.

21. The structure of claim 12, wherein the substrate comprises calcium carbonate.

22. The structure of claim 12, wherein the substrate is formed as a plug configured for retention in a reef structure.

23. The structure of claim 22, wherein the reef structure is configured to retain an array of plugs.

24. A method for enhancing coral recruitment, the method comprising: producing the structure according to claim 23; and disposing the structure in natural seawater containing coral larvae for one or more months.

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