Pollen-like microstructures

Abstract

Microstructures, their use, and methods for making microstructures are provided, where the methods involve applying an external stimulus to a plurality of packed, polymeric microspheres, wherein the polymeric microspheres have a porosity of between about 20% to 99%, and wherein at least a boundary layer of the polymeric microspheres are in contact with a surface; such that the polymeric microspheres collapse into smaller and denser microspheres that join with adjacent microspheres at contact points to form a micronetwork, wherein the contact points comprise microstructures extruded from the polymeric microspheres.

Description

[0001] Pollen-Like Microstructures Statement of Government Support This invention was made with government support under N00014-21-1-2402 awarded by the Office of Naval Research. The government has certain rights in the invention. Background Hierarchical materials decorated with multifunctional nano and microornamentations are ubiquitous in nature and their replica in synthetic systems can play a pivotal role in drug delivery, energy generation and storage, sensing, and microrobotics. So far, fabrication of materials that mimic these natural systems has been based on their sacrificial replica molding or on the integration of top-down and bottom-up approaches by a combination of self- assembly and locally controlled chemical, physical and mechanical cues. However, these manufacturing strategies are unable to control material assembly at the nanoscale and simultaneous formation of microarchitectures for a broad range of materials. Three- dimensional (3D) printing of polymeric inks that self-assemble in nanostructured materials has recently been reported to seamlessly integrate nanostructuring with microfabrication; nonetheless, the sequential manufacturing approach of top-down techniques limits the amount of material that can be synthesized and negatively affect production time. Summary In one aspect, the disclosure provides methods, comprising applying an external stimulus to a plurality of packed, polymeric microspheres, wherein the polymeric microspheres have a porosity of between about 20% to 99%, and wherein at least a boundary layer of the polymeric microspheres are in contact with a surface; such that the polymeric microspheres collapse into smaller and denser microspheres that join with adjacent microspheres at contact points to form a micronetwork, wherein the contact points comprise microstructures extruded from the polymeric microspheres. In various embodiments, the polymeric microspheres comprise hydrophilic microspheres, amphiphilic microspheres, and / or thermoplastic microspheres. In some embodiments, the polymeric microspheres comprise a water-soluble polymer, including but not limited to silk fibroin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyethylene oxide (PEO), methyl cellulose (MC), carboxymethyl cellulose (CMC), chitosan, pullulan, gelatin, pectin, and combinations thereof, or the polymeric microspheres comprise silk fibroin. In some embodiments, the polymeric microspheres are freeze dried microspheres, such as freeze-dried silk fibroin microspheres. In various embodiments, the polymeric microspheres have a porosity of between about 25% to 99%; about 30% to 99%; about 35% to 99%; about 40% to 99%; about 45% to 99%; about 50% to 99%; about 55% to 99%; about 60% to 99%; about 65% to 99%; about 70% to 99%; about 75% to 99%; about 80% to 99%; or about 85% to 99%. In other embodiments, the smaller and denser microspheres comprise microspheres of less than about 70% porosity and having a diameter reduced by at least 10%, 15%, 20%, or 25%, and / or the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% porosity. In some embodiments, the surface comprises silicate or aluminum surfaces. In other embodiments, the external stimulus comprises applying a pressure of at least 0.5 MPa, or at least about 1 MPa, on the polymeric microspheres. In various embodiments, the external stimulus comprises applying a solvent vapor of about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, or between about 80% to about 99%, to the polymeric microspheres, wherein the solvent is capable of dissolving the polymer that forms the polymeric microspheres. In some embodiments, the solvent vapor is selected from the group consisting of water, ethanol (EtOH), and acetic acid (AcOH). In various other embodiments, a polymer / solvent vapor combination is selected from the group consisting of (a) silk fibroin (SF) / water or silk fibroin (SF) / AcOH; (b) methyl cellulose (MC) / water; (c) MC / AcOH, (d) CMC / water, (e) chitosan / AcOH, (f) pectin / water, (g) polyvinyl alcohol (PVA) / water (h) polyvinylpyrrolidone (PVP) / water, (i) PVP / EtOH, polyacrylic acid (PAA) / water and (j) PAA / EtOH In one embodiment, (a) the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres; and the solvent vapor comprises acetic acid (AcOH). In a further embodiment, the polymeric microspheres further comprise NaHCO3. In some embodiments, the polymeric microspheres comprise a weight % of NaHCO3of between 1 to 50%. In other embodiments, the polymeric microspheres comprise a weight % of silk fibroin of about 50% to about 99%.^ In one embodiment, the external stimulus comprises heating, including but not limited to heating the plurality of polymeric microspheres to a temperature above the glass transition temperature. In some embodiments, the polymeric microspheres are pre-loaded with a payload selected from the group consisting of soluble chemicals, insoluble nanoparticles, therapeutics, a fertilizer, and / or a plant-growth agent, optionally wherein the microspheres comprise different payloads, and wherein the different payloads interact in the microstructures. In other embodiments, the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4 MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil, and pyraclostrobin. In one embodiment, the method further comprises breaking the microstructures to produce a plurality of patterned microspheres comprising one or more fragmented microstructures. In one such embodiment, the breaking comprises applying mechanical stress, including but not limited to ultrasound, to the micronetwork. In various embodiments, the one or more fragmented microstructures comprise spines, channels, bumps, and / or helices. In one embodiment, the one or more fragmented microstructures comprise spines. In some embodiments, the spines have a diameter of between about 0.2 μm to about 15 μm, and / or a length between about 1 μm and about 25 μm. In other embodiments, the spines have a Young’s modulus of between about 4 GPa and about 10 GPa. In one embodiment, the spines comprise a pointed end. The disclosure also provides patterned polymeric microspheres produced by the method of the disclosure. In another aspect, the disclosure provides patterned polymeric microspheres, comprising a freeze-dried silk fibroin microsphere comprising one or more spines extruded from the microsphere. In some embodiments, the one or more spines have a diameter of between about 02 μm to about 15 μm and / or a length between about 1 μm and about 25 μm. In other embodiments, the one or more spines have a Young’s modulus of between about 4 GPa and about 10 GPa, and / or the one or more spines comprise a pointed end. In some embodiments, the freeze-dried silk fibroin microsphere comprises NaHCO3. In various embodiments, the freeze-dried silk fibroin microsphere comprises a weight % of NaHCO3of between 1 to 50%, and / or the polymeric microspheres comprise a weight % of silk fibroin of about 50% to about 99%. In other embodiments, the patterned polymeric microspheres comprise water present inside the freeze-dried silk fibroin microsphere and / or the one or more spine. In another embodiment silk polymers on the microsphere surface comprise water-insoluble beta-sheets. In a further embodiment, the patterned polymeric microsphere comprises a payload within the microsphere and / or the one or more spine. In some embodiments, the payload is selected from the group consisting of a therapeutic, a fertilizer, and a plant-growth agent. In other embodiments, the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4 MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil, and pyraclostrobin. In further embodiments, the payload comprises iron, including but not limited to Fe-EDTA, Fe-DTPA, Fe-EDDHA, Fe-EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3. The disclosure also provides compositions comprising a plurality of patterned polymeric microspheres of any embodiment disclosed herein. In a third aspect, the disclosure provides methods for delivering a payload to a plant by administering to the plant a patterned polymeric microsphere produced by the method of any one embodiment herein, wherein the patterned polymeric microsphere comprises a payload within the one or more spines, or by administering the patterned polymeric microsphere of any embodiment herein. In some embodiments, the payload is selected from the group consisting of a therapeutic, a fertilizer, and a plant-growth agent. In other embodiments, the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4 MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil and pyraclostrobin In further embodiments the payload comprises iron, including but not limited to Fe-EDTA, Fe-DTPA, Fe-EDDHA, Fe-EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3. Description of Figures Figure 1. Synergistic nanostructuring of microspheres in response to solvent vapors. a, Conceptual overview of vapor induced synergistic differentiation (VISDi). Polymeric microspheres exposed to solvent vapors form interconnected microspheres’ networks (IMNet) and echinate microspheres (EMSpheres) by modulating process parameters. b, Left, picture of a mm-size IMNet. Scale bar, 1 mm. Middle, relative optical microscopy (dark field) photo of IMNets. Scale bar, 20 μm. Right, optical microscope photo of EMSphere. Scale bar, 10 μm. c, Library of structures obtained by VISDi, including silk fibroin (SF), methyl cellulose (MC), polyvinyl alcohol (PVA), polyacrylic acid (PAA), and polyvinylpyrrolidone (PVP). The triggering vapor is selected from the solvents of corresponding polymers, such as water (H2O), ethanol (EtOH), and acetic acid (AcOH). Scale bar, 20 μm. Figure 2. Structural transformation and characterization of SF EMSphere. a, fixed-point high-resolution environmental SEM of SF microspheres during VISDi and in response to WV, showing the contraction induced differentiation, the formation of a pair of spines and their recoil when RH is not decreased. Time points are 0, 5, 13, and 25 min from left to right. Scale bar, 10 μm. b, Microsphere diameter reduction during VISDi. Sample size n = 12. c, SEM of SF EMSpheres with different parts marked as matrix, shoulder, and spine. Scale bar, 2 μm. d, AFM images of shoulder and matrix areas of a SF EMSpheres. Scale bar, 100 nm. e, Polarized light images of a SF EMSpheres, left, 0 deg, right, 90 deg, showing polarization at the microsphere’s shoulder. Scale bar, 10 μm. f, Distribution of beta-sheet content in different areas on SF EMSpheres. Sample size n = 5. The results show a statistical significance (* p < 0.05). g, μRaman spectra of EtOH treated SF EMSpheres obtained at the shoulder area, showing no anisotropy post disorder-to-order transition. The experimental setup and coordinate system used is detailed in Fig.12. Figure 3. Structural control and patterning from microsphere to micronetworks and echinate microstructures. a, SEM analysis of IMNets obtained from different initial SF concentrations (8.0, 5.0 and 2.5wt%). Scale bar, 20 μm. b, Morphology of fractured SF IMNets microbridges, as a function of initial SF concentration. Scale bar, 2 μm. c, Distribution of micro-bridge widths as a function of initial SF concentrations. The diameter of microbridges increases by decreasing the initial SF concentration. Sample size n = 20. The results show a statistical significance (* p < 0.05). d, Distribution of number of spines on SF EMSpheres based on different VISDi setups; No spines (EMS-0) are present when there is no stack of microspheres and a prolonged VISDi induces the recoil and resorption of the microbridges. An average number of three spines (EMS-3) is achieved when VISDi is applied to a single stack of microsphere. An average number of five spines (EMS-5) is achieved by applying VISDi on a double stack of microsphere. Sample size n = 163, 93, and 105, respectively. The results show a statistical significance (* p < 0.05). e, Morphology of a silk fibroin EMSphere and a sunflower pollen grain. Scale bar, 10 μm. Figure 4. Exemplary applications of EMSpheres and IMNets. a, Young’s modulus of the SF microsphere, EMSphere, and Et-EMS by AFM. Sample size n = 512 per test. The results show a statistical significance (* p < 0.05). b, SEM of SF EMSpheres rubbing on a flat and clean PARAFILM®. Scale bar, 20 μm. c, Scratching marks from robust microspines. Scale bar, 10 μm. d, SEM images of EMS-5 on the smooth spinach leaf. Scale bar, 10 μm. e, SEM image of EMS-0 on the spinach leaf. Scale bar, 10 μm. f-g, Fluorescence microscope images of EMS-5 and -0 retention behaviors after 1 and 2 times of gentle washing (denoted as W1 and W2). Scale bar, 500 μm. h, Retention percentage of EMS-0 and -5 after 1 and 2 times of washing. The sample size n = 7 per trial. The results show a statistical significance (* p < 0.05). i-j, Confocal microscope photos of fluorescent dye-encapsulated SF microspheres before and after responding to WV. Scale bar, 100 μm. k, Magnified area of SF IMNet. Scale bar, 20 μm. Inset, scale bar, 5 μm. Figure 5. Morphology of original natural and synthetic polymer microspheres. a, Optical image and size distribution of original silk fibroin microspheres. The average diameter is 38±6 μm. Sample size n = 509. Scale bar, 100 μm. b, SEM image of original silk fibroin microspheres. Scale bar, 20 μm. c, Cross-section SEM image of an original silk microsphere. Scale bar, 5 μm. d, SEM images of original polymeric microspheres, including MC, PVA, PAA, PVP. Scale bar, 20 μm. Figure 6. Morphologies of other polymeric EMSpheres. a, Polyvinylpyrrolidone (PVP) with an average molecular weight (MW) of 55 k. b, Polyvinyl alcohol (PVA) with an average MW of 90 k. c, PVA with an average MW of 40 k. d, Polyacrylic acid (PAA) with an average MW of 100k. Scale bar, 10 μm. Figure 7. Morphologies of silk-based echinate microcapsules by VISDi. a, Original silk fibroin EMSpheres. b, 3% fluorescein sodium salt (FSS) encapsulated silk EMSpheres. c, 33% vitamin C encapsulated silk EMSpheres. d, 1% magnetite (Fe3O4) nanoparticle encapsulated silk fibroin EMSpheres. Scale bar, 20 μm. Figure 8. Size measurement during the VISDi. The diameter of microspheres without microbridge evolution is recorded at different time points. For example, the circled microspheres are chosen for diameter measurement. Scale bar, 20 μm. Figure 9. Fractured surfaces of SF original microsphere and EMSphere. Scale bar, 10 μm. Figure 10. Beta-sheet percentage of the shoulder, matrix, and spine areas of Et- EMS. The data was collected using Raman microscopy. Sample size n = 6 per area. Figure 11. Raman spectrum of ethanol-treated silk fibroin isotropic film. Figure 12. Definition of the laboratory frame and sample orientation. Figure 13. SF IMNets with different initial SF concentrations. Scale bar, 50 μm. Figure 14. Tuning average contact points of EMSpheres. a, Optical image of the SF IMNets derived from one layer of packed original microspheres. b, SF IMNets derived from two layers of packed original microspheres. c, Optical image of SF IMNet at the edge fuse on the glass substrate after the VISDi. Scale bars of a-c, 50 μm. d, SEM image of EMSpheres with an average spine number of 0. Scale bar, 5 μm. The initial SF concentration is 8.0% for a-d. e, SEM image of the edge of a SF IMNet on Al substrate (original concentration of 2.5%). Scale bar, 20 μm. Figure 15. Histogram of Young’s modulus of the original microsphere, EMSphere, and Et-EMS. Sample size n = 512. Figure 16. Morphologies of smooth spinach leaf surface before and after sprayed with EtOH-EMSpheres. a, A trichome-free spinach leaf. b, Spinach leaf sprayed with EMS- 0. c, Spinach leaf sprayed with EMS-5. Scale bar, 20 μm. Figure 17 FSS-loaded SF EMSpheres on spinach. a, Photo of spray bottles with EMS-0 (left) and EMS-5 (right) aqueous suspensions. b, Spinach leaves after spraying on EMS-0 and -5 suspensions. The left side is sprayed with EMS-0 (white circle), and the right side with EMS-5 (circle). One half is dried and covered before spraying the other half. c, SEM images of EMS-5 on the smooth spinach leaf, where the robust spines hold onto the leaf surface. Scale bar, 10 μm. Figure 18. Composition re-distribution of Fe-loaded microcapsules during VISDi process. a, SEM and EDS mapping results of a fractured Fe-silk SpheM, showing the uniform distribution of elements including Fe, Na, C, O, and N. Scale bar, 10 μm. Inset, magnified region of fractured surfaces Scale bar 5 μm b SEM and EDS mapping results of a fractured Fe-silk spiny pollen inspired microcapsules (SPIM), showing a Fe-concentrated core. Inset, magnified region of fractured surfaces. Scale bar, 5 μm. c, Sketch of silk micelle and Fe-EDTA distribution in original spray freeze-dried microcapsule and moisture-induced spiny microcapsule. The water-soluble Fe-EDTA molecules concentrate in the center of the matrix, while amphiphilic silk polymers tend to aggregate at the surfaces. Besides, the spiny- pollen inspired microencapsulation can be extended to a variety of active cargos, including water-soluble molecules (fluorescein dyes, vitamin C) and insoluble nanoparticles (magnetic Fe3O4nanoparticles) (Figure 7). Figure 19. Fe-silk SPIMs controlled the release of iron in spinach leaves. a, Sketch of a Fe-silk SPIM on a smooth spinach leaf, releasing Fe-EDTA nutrients. b, Hydroponic setup for spinach growth, the greenish plants are grown in Hoagland’s complete nutrient solution (left), and the yellowish plants are obtained in Fe-deficient Hoagland’s solution (right). c, Release profiles of silk-Fe SpheMs, SPIM-0, and SPIM-5 in 10% PluronicTMsolutions within 7 days. Sample size n = 3 per treatment. (significance*). d, Iron deficiency visual ratings of trial spinach treated non-spiny and spiny Fe-loaded SPIM formulations in a plant growth chamber study 5 DAT. Sample size n = 3 per treatment. e, SEM and EDS mapping results of a Fe-silk SPIM on spinach leaves, showing the distribution of elements including Fe, Na, C, O, and N. Scale bar, 10 μm. Inset, magnified region of microspines releasing Fe. Scale bar, 5 μm. f, SEM and EDS mapping results of Fe-silk SPIMs releasing Fe on spinach leaves over time. Left, scale bar, 10 μm. Middle and right, magnified region of a microspine under 99% RH after 10 and 40 mins, where the highlighted sections show increased Fe concentration. Scale bar, 5 μm. Figure 20. Fabrication of biopolymer-based vascular micronetworks. a, Fabrication process of biopolymer-based, vascular micronetworks using VISDi. The concentration of biopolymers is 2.5-8.0 wt% in the feed suspensions. Feed rate is set to 3 mL min-1. b, Photo and SEM images of silk-based vascular micronetworks. c, Optical image of water filled silk-based microchannels. d, Conceptual view of tunable structural parameters of vascular micronetworks, including diameter and porosity. Figure 21. Formation and molecular transport of silk-based microvascular networks. a, SEM images of silk porous microcapsules and vascular micronetworks after VISDi, via water or AcOH vapors. The fractured surfaces show hierarchical structures. b, Sketch of the formation of a silk-based vessel. The NaHCO3-silk freeze-dried microcapsules were exposed to acetic acid vapor, generating water inside of the microcapsules. The water molecules convey into a microdroplet that solubilizes the molecules around the core region. On the other hand, the silk polymers on the surfaces transformed into water-insoluble beta- sheets under acidic conditions. Together, this vapor-responsive process leads to a vessel-like structure filled with water. c, Optical images of NaHCO3-silk microcapsules gradually transforming into an interconnected vascular channel. The opaque bridges turn into translucent due to water filling. Scale bar, 50 μm. Figure 22. Molecular dye transportation within water-filled vascular micronetworks. a, Confocal microscope images of silk-FSS microcapsules during VISDi. NaHCO3-containing microcapsules react with AcOH vapor. The water-filled microchannels enable a fast and efficient FSS transport for dye-loaded to dye-free microcapsules. Without NaHCO3, the FSS dye can only move to the bridge of the channels. Scale bar, 50 μm. b, Optical images of NaHCO3-silk microcapsules form microvascular networks with their rhodamine B-encapsulated counterparts. The dye-free and dye-loaded capsules form an interconnected network in response to acetic acid vapor. The molecular dyes are transported through the water channels and delivered to adjacent counterparts. Scale bar, 100 μm. Figure 23. Chlorophyll content of spinach leaves 5 DAT. Samples include healthy spinach leaves (control), Fe-deficient leaves (Fe-defic), and Fe-deficient leaves treated with SPIM-0 and SPIM-5. The sample size n = 9. The results show a statistical significance (* p < 0.05). Figure 24. Biodegradation profile of SF SPIMs. The enzymatic degradation was monitored for 5 days. Sample size n = 3 per treatment. One-way ANOVA test indicates a statistical significance of time effect on the SF SPIMs degradation rate (p < 0.05). Figure 25. Calibration curve of concentration-absorption of Fe-EDTA in 10% PluronicTMPE 10500 solution by UV-Vis. Detailed Description As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular. Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application. All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise. As used herein, "about" means plus or minus 5% of the particular value. In one aspect, the disclosure provides methods, comprising applying an external stimulus to a plurality of packed, polymeric microspheres, wherein the polymeric microspheres have a porosity of between about 20% to about 99%, and wherein at least a boundary layer of the polymeric microspheres are in contact with a surface; such that the polymeric microspheres collapse into smaller and denser microspheres that join with adjacent microspheres at contact points to form a micronetwork, wherein the contact points comprise microstructures extruded from the polymeric microspheres. As disclosed in the attached Appendices, the methods serve to control the formation of adhesive contact points between microspheres, and their isotropic contraction and concurrent molecular reflow to enable the formation of microstructures. Using this integrated method that works with both natural and synthetic polymers, the inventors have fabricated nanostructured cm-scale networks of compartmentalized yet interconnected microcapsules where cargo molecules can be mixed at the adhesion points. As used herein, “packed” means that microspheres in the plurality are in contact with adjacent microspheres. Any polymeric microspheres may be used as appropriate for an intended use. In one embodiment, the polymeric microspheres comprise hydrophilic microspheres. In another embodiment, the polymeric microspheres comprise amphiphilic microspheres. In a further embodiment, the polymeric microspheres comprise thermoplastic microspheres. In a specific embodiment, the polymeric microspheres comprise water-soluble polymers, which is particularly useful when using the spray freeze drying (SFD) methods disclosed herein (in which water is used as the solvent / external stimulus). In contrast, the synergistic nanostructuring microfabrication processes (SnμP) disclosed herein can apply to polymers soluble in water, ethanol, acetic acid, etc., and use of corresponding solvent. In various non-limiting embodiments, the polymer may be selected from the group consisting of silk fibroin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyethylene oxide (PEO), methyl cellulose (MC), carboxymethyl cellulose (CMC), chitosan, pullulan, gelatin, pectin, and combinations thereof. In one specific embodiment, the polymeric microspheres are freeze dried microspheres. In another specific embodiment, the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres. Prior to applying the external stimulus, the polymeric microspheres have a porosity of between about 20% to 99%. In various embodiments, the polymeric microspheres have a porosity of between about 25% to 99%; about 30% to 99%; about 35% to 99%; about 40% to 99%; about 45% to 99%; about 50% to 99%; about 55% to 99%; about 60% to 99%; about 65% to 99%; about 70% to 99%; about 75% to 99%; about 80% to 99%; or about 85% to 99%. Application of the external stimulus results in the polymeric microspheres collapse into smaller and denser microspheres (compared to the microspheres before application of the external stimulus, which may be of any suitable size, such as 2 μm to 100 μm in diameter) that join with adjacent microspheres at contact points to form a micronetwork. The external stimulus may be applied to result in collapse of the microspheres of any desired magnitude as appropriate for an intended use. In one embodiment, the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 70% porosity and having a diameter reduced by at least 10%. In various embodiments, the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% porosity. In other embodiments, the smaller and denser microspheres after application of the external stimulus comprise microspheres having a diameter reduced by at least 15%, 20%, or 25%. In one embodiment, the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 25% porosity and having a diameter reduced by at least 25%. The plurality of polymeric microspheres include at least a boundary layer of the polymeric microspheres are in contact with a surface. This boundary layer would thus be in contact with the surface as well as in contact with adjacent microspheres. Any surface may be used that is suitable for use with the polymeric microspheres being used. In non-limiting embodiments, the surface comprises silicate or aluminum surfaces. Any external stimulus may be used as appropriate for an intended micronetwork. In one embodiment, the external stimulus comprises applying a pressure of at least 0.5 MPa, or at least about 1 MPa, on the polymeric microspheres. In another embodiment, the external stimulus comprises applying a solvent vapor of between about 50% to 99%, about 55% to 99% about 60% to 99% about 65% to 99% about 70% to 99% about 75% to 99%, or between about 80% to about 99%, to the polymeric microspheres, wherein the solvent is capable of dissolving the polymer that forms the polymeric microspheres. As further shown in the attached appendices, the inventors demonstrate that this method (also referred to as “vapor-induced synergistic differentiation” or VISDi) allows the nanomanufacture of echinate pollen-like, high payload content (~50 wt%) microcapsules decorated with robust spines that mechanically disrupt the cuticle and adhere to a plant leaf. As discussed therein, the methods induce a core-shell structure in the spines, enabling the release of agrochemicals from the microparticles' body into the leaf. As proof of concept, the inventors demonstrated precise and high throughput delivery of iron fertilizer in Fe-deficient spinach plants. Spray of spiny microparticles improved leaf adhesion by mechanical interlocking, reduced wash-off by a ~12.5 fold, and enhanced chlorophyll content by ~7.3 times compared to the application of spherical particle counterparts. Together, these results show that spiny microparticles can mitigate agricultural runoff and provide a high throughput tool for precise plant drug delivery. In one embodiment, the solvent vapor is selected from the group consisting of water, ethanol (EtOH), and acetic acid (AcOH), or combinations thereof. In various other embodiments, a polymer / solvent vapor combination is selected from the group consisting of (a) silk fibroin (SF) / water or silk fibroin (SF) / AcOH; (b) methyl cellulose (MC) / water; (c) MC / AcOH, (d) CMC / water, (e) chitosan / AcOH, (f) pectin / water, (g) polyvinyl alcohol (PVA) / water (h) polyvinylpyrrolidone (PVP) / water, (i) PVP / EtOH, polyacrylic acid (PAA) / water and (j) PAA / EtOH. In one embodiment: (a) the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres; and (b) the solvent vapor comprises acetic acid (AcOH). In a further embodiment, the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres and NaHCO3. As shown in the attached appendices the inventors demonstrated that NaHCO3-silk microcapsules created a water core-insoluble wall vascular configuration in response to acetic acid vapor, where beta- sheet silk constructs the wall domains (Figure 21a-b). The inventors further demonstrated that this method serves to produce a silk-based microvascular network fabrication technology that can provide free-standing, interconnected microchannels. The in-vasculature water formation can be controlled by adjusting the contact time of microcapsules and acetic acid vapor (Figure 21c). In this embodiment, the weight % ratio of silk fibroin to NaHCO3may be any as suitable for an intended purpose, in view of the teachings herein. In one non-limiting embodiment, the polymeric microspheres comprise a weight % of NaHCO3of between about 1% to about 50%. In further embodiments, the polymeric microspheres comprise a weight % of NaHCO3of between about 10% to about 50%, about 20% to about 50%, or about 40% to about 50%. As shown in the attached appendices, the weight% of NaHCO3 determines the water content. A range of weight% of NaHCO3 between 1-20% leads to 1.0-7.5 vol / vol% of water (volume of water to volume of micronetworks); 20-40% leads to 7.5-20 vol / vol% of water; 40-50% leads to 20-30 vol / vol% of water. In these embodiments, the polymeric microspheres may comprise a weight % of silk fibroin of about 50% to about 99%.^The weight % of polymers (eg., silk fibroin) determines the diameter of the microstructures. In further embodiments, the polymeric microspheres comprise a weight % of silk fibroin of between about 50% to about 90%, about 50% to about 80%, or about 50% to about 60%. In a specific embodiment, the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres, and the external stimulus comprises applying water vapor of between about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, or between about 80% to about 99%. In a further embodiment, the external stimulus comprises heating. In one embodiment, the heating comprises heating the plurality of polymeric microspheres to a temperature above the glass transition temperature, e.g., between about 50Û to about 250Û degree C. Application of the external stimulus results in the polymeric microspheres collapsing into smaller and denser microspheres that join with adjacent microspheres at contact points to form a micronetwork, wherein the contact points comprise microstructures extruded from the polymeric microspheres. The microstructures may be of any shape and size as appropriate for an intended use, but they are integral with the microspheres (i.e., made of the same polymer as the polymeric microsphere). In some embodiments, the polymeric microspheres may be pre-loaded with a payload (i.e.: on or within the microsphere). The payload may be any moiety as appropriate for an intended use. In some embodiments, the payload may be selected from the group consisting of soluble chemicals and insoluble nanoparticles. In non-limiting embodiments, the payload may comprise a therapeutic, a fertilizer, and / or a plant-growth agent; these embodiments are particularly useful, for example, in certain methods of the disclosure for delivering a payload to a plant target. In some embodiments, different microspheres in the plurality of microspheres may comprise different payloads. In these embodiments, the different payloads may interact in the microstructures. In another embodiment, the methods further comprise breaking the microstructures to produce a plurality of patterned microspheres comprising one or more fragmented microstructures. By way of non-limiting example, the inventors demonstrated in the examples breaking the microstructures to form echinate (i.e., spined) pollen-like functional microparticles with enhanced surface adhesion and drug delivery capabilities. The breaking can be carried out using any technique suitable for an intended fragment microstructure. In one embodiment, the breaking comprises applying mechanical stress to the micronetwork. Such mechanical stress may include, but is not limited to, mechanical agitation (pressing, stirring shaking, vortexing, grinding, tapping, etc.); Air agitation (air agitator, fast air blow, etc.), or ultrasound. In one embodiment, the mechanical stress comprises ultrasound. In one embodiment, the sonication comprises sonication at a frequency of at least 40 kHz for at least 10, 15, or 20 seconds per each 0.2 mg of sample. The one or more fragmented microstructures may comprise any shape and size as appropriate for an intended purpose. In various non-limiting embodiments, the one or more microstructures comprise spines, channels, bumps, and / or helices. In various embodiments, the microstructure may have a maximum dimension of between about 0.2 μm to about 50 μm; or between about 0.2 μm to about 45 μm, about 0.2 μm to about 40 μm, about 0.2 μm to about 35 μm, about 0.2 μm to about 30 μm, about 0.2 μm to about 25 μm, about 0.2 μm to about 20 μm, about 0.2 μm to about 15 μm, about 0.2 μm to about 10 μm, about 0.2 μm to about 5 μm, about 0.2 μm to about 2.5 μm, or about 0.2 μm to about 1 μm. In a specific embodiment, the one or more fragmented microstructures comprise spines. The spines may be of any dimensions as suitable for an intended purpose. In one embodiment, the spines have a diameter of between about 0.2 μm to about 15 μm, and / or a length between about 1 μm and about 25 μm In another embodiment the spines have a diameter of between about 0.5 μm to about 10 μm. In a further embodiment, the spines have a length between about 2 μm and about 25 μm. In another embodiment, the spines have a Young’s modulus of between about 4 GPa and about 10 GPa. In a further embodiment, the spines have a Young’s modulus of between about 7 GPa and about 9.5 GPa. In one embodiment, the Young’s modulus is obtained using amplitude modulation-frequency modulation (AM-FM) viscoelastic mapping on an atomic force microscope (AFM). In another embodiment, the spines comprise a pointed end. This embodiment is particularly useful for use of the patterned microspheres for payload delivery, such as to plants. In other embodiments, the patterned polymeric microspheres comprise 2, 3, 4, 5, or more microstructures, such as spines. The disclosure also provides patterned polymeric microsphere produced by the method of any embodiment disclosed herein. In another aspect, the disclosure provides patterned polymeric microspheres, comprising freeze-dried silk fibroin microspheres comprising one or more spines extruded from the microsphere. The spines may be of any dimensions as suitable for an intended purpose. In one embodiment, the spines have a diameter of between about 0.2 μm to about 15 μm, and / or a length between about 1 μm and about 25 μm. In another embodiment, the spines have a diameter of between about 0.5 μm to about 10 μm. In a further embodiment, the spines have a length between about 2 μm and about 25 μm. In another embodiment, the spines have a Young’s modulus of between about 4 GPa and about 10 GPa. In a further embodiment, the spines have a Young’s modulus of between about 7 GPa and about 9.5 GPa. In another embodiment, the spines comprise a pointed end. This embodiment is particularly useful for use of the patterned microspheres for payload delivery, such as to plants. In other embodiments, the patterned polymeric microspheres comprise 2, 3, 4, 5, or more microstructures, such as spines. In one embodiment, the freeze-dried silk fibroin microsphere comprises NaHCO3. As shown in the attached appendices, the inventors demonstrated that NaHCO3-silk microcapsules created a water core-insoluble wall vascular configuration in response to acetic acid vapor, where beta-sheet silk constructs the wall domains (Figure 21a-b). The inventors further demonstrated that these microspheres can form free-standing, interconnected microstructures (such as spines) with adjacent microspheres. In this embodiment, the weight % ratio of silk fibroin to NaHCO3 may be any as suitable for an intended purpose, in view of the teachings herein In one non-limiting embodiment the polymeric microspheres comprise a weight % of NaHCO3 of between 1 to 50%. In one non-limiting embodiment, the polymeric microspheres comprise a weight % of NaHCO3 of between about 1% to about 50%. In further embodiments, the polymeric microspheres comprise a weight % of NaHCO3 of between about 10% to about 50%, about 20% to about 50%, or about 40% to about 50%. In these embodiments, the polymeric microspheres may comprise a weight % of silk fibroin of about 50% to about 99%.^In further embodiments, the polymeric microspheres comprise a weight % of silk fibroin of between about 50% to about 90%, about 50% to about 80%, or about 50% to about 60%. In another embodiment, the patterned polymeric microsphere comprises water present inside the freeze-dried silk fibroin microsphere and / or the one or more spine. In a further embodiment, silk polymers on the microsphere surface comprise water-insoluble beta-sheets. As shown in Appendix 2, the silk polymers on the surfaces transformed into water-insoluble beta-sheets under acidic conditions (i.e., AcOH vapor treatment), forming a vessel-like structure filled with water. In some embodiments, the patterned polymeric microspheres may comprise a payload within the microsphere and / or the one or more spine. The payload may be any moiety as appropriate for an intended use. In some embodiments, the payload may be selected from the group consisting of soluble chemicals and insoluble nanoparticles. In non- limiting embodiments, the payload may comprise a therapeutic, a fertilizer, and / or a plant- growth agent; these embodiments are particularly useful, for example, in certain methods of the disclosure for delivering a payload to a plant target. In one embodiment the payload comprises iron, including but not limited to Fe-EDTA, Fe-DTPA, Fe-EDDHA, Fe- EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3. The disclosure also comprises compositions, comprising a plurality of the patterned polymeric microsphere of any embodiment disclosed herein. In one embodiment, the composition comprises a plurality of the patterned silk- NaHCO3 microspheres of any embodiment or combination of embodiments described herein. As described above, the inventors demonstrated that NaHCO3-silk microcapsules created a water core-insoluble wall vascular configuration in response to acetic acid vapor, where beta-sheet silk constructs the wall domains (Figure 21a-b). The inventors further demonstrated that these microspheres can form free-standing, interconnected microchannels with adjacent microspheres. Thus, in a further embodiment, the plurality of the patterned polymeric microspheres comprise microspheres in fluid communication with each other via water-filled microstructures, such as spines In another aspect, the disclosure provides methods of delivering a payload to a plant by administering to the plant a patterned polymeric microsphere of any embodiment herein, wherein the patterned polymeric microsphere comprises a payload within the one or more spines. In a specific embodiment, the patterned polymeric microsphere comprises a payload within the one or more spines, and the one or more spines comprise a pointed end to facilitate puncturing a plant part (i.e, leaf, etc.). As shown in the attached examples, the inventors have shown that the spines (a) enhance microsphere adhesion on surfaces, including plant leaves which is helpful, for example, to reduce agrochemicals wash off; and (b) can be used to transfer payload to a plant. In non-limiting embodiments, the payload may be selected from the group consisting of a therapeutic, a fertilizer, and a plant-growth agent. In other embodiments, the payload may comprise one or more of the following: Fertilizers: ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4 MgSO4, KNO3, and KOH, Diammonium Phosphate (DAP), Monoammonium Phosphate (MAP). Micronutrients: Copper oxysulfate, Copper oxide, Copper chelate, Copper sulfate, zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, and zinc chloride. Fungicide: Mancozeb (contains zinc ion and manganese ethylene bisdithiocarbamate), Chlorothalonil, Fenbuconazole, Tebuconazole, Propineb (contains polymeric zinc propylenebis(dithiocarbamate)), Thiram (contains tetramethylthiuram disulfide), and Metiram (a member of the ethylene bisdithiocarbamate group of fungicides); Herbicides: Saflufenacil, Pyraclostrobin In one embodiment, the payload comprises iron, including but not limited to Fe- EDTA, Fe-DTPA, Fe-EDDHA, Fe-EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3. ^ Example 1 Here we report a synergistic nanostructuring microfabrication process (SnμP) where polymeric spherical microparticles obtained via spray freeze drying are mutualistically shaped in seconds by their adjacent neighbors in nanostructured microarchitectures, post assembly. By modulating exposure to moisture in the post-drying process and particles density, we have controlled the formation of adhesive contact points between microspheres, their isotropic contraction and concurrent moisture-induced molecular reflow to enable the formation of echinate nano-ornamentations. Using this integrated method that works with both natural and synthetic polymers, we have fabricated nanostructured cm-scale networks of compartmentalized yet interconnected microcapsules where cargo molecules are mixed at the adhesion points. By further modulating reflow, we formed individual echinate pollen-like functional microparticles with enhanced surface adhesion and drug delivery capabilities. Our new synergistic fabrication approach opens the door to manufacturing bioinspired, microarchitected materials. Seamless integration of biopolymers assembly phenomena at the nanoscale (1- 100nm) with collaborative cell-mediated remodeling at the meso- and microscales (100 nm- 100 μm) is a fundamental biofabrication process used in living systems to build macroscopic (mm to m) hierarchically structured materials with enhanced mechanical properties and augmented functionalities. We developed SnμP to enable the simultaneous fabrication and nanostructuring of mm-scale, echinate micronetworks. In SnμP, polymeric spherical microparticles obtained via spray freeze drying (SFD) are synergistically shaped in minutes by their adjacent neighbors in nanostructured microarchitectures, post assembly. Local control over particle density and solvent vapor pressure enables the synergistic formation of microbridges at the particle-to-particle contact points. Further evolution modulates the formation of networks of connected microparticles of increasing complexity. Application of mechanical stress via ultrasound can be used to break the microbridges and results in echinate microspheres with a pre-determined number of spines. Orthogonal functionalities can also be added by encapsulating actives during SFD, e.g. yielding adhesive microcapsules for drug delivery or compartmentalized networks where reactions occur at the junctions’ sites. SnμP provides a universal fabrication strategy that combines environmental cues, intermolecular adhesions, and mechanical interactions to morph materials across scales. To design such system, we manipulated low solid content (i.e. porosity > 85%) microspheres (Fig.5) obtained via SFD by modulating the formation of particle-to-substrate and particle- to-particle contact points and the concurrent exposure to solvent vapors (Fig.1a). Exposure to polymer-specific solvent vapors such as water, ethanol (EtOH), and acetic acid (AcOH) induce the microspheres in contact with the substrate to collapse on it due to a combination of solvent adsorption, release of internal stress induced by freeze-drying, and a decrease in apparent modulus, making anchor points. Concurrently, microspheres that are suspended in air and supported by particle-to-particle interactions undergo isometric contraction around their center of mass. At the particle-to-particle contact points, exposure to solvent vapors causes the formation of molecular bonds resulting in stickiness during their respective isometric contraction, thus forming elongated mesostructures by a combination of pulling, pore collapsing and molecular reflow. This vapor induced synergistic differentiation (VISDi) process was simulated using finite element analysis (FEA) assuming well-aligned axisymmetric highly porous spheres (modeled as hyperfoams) with constant contact areas. A uniform pressure increase was applied to the hyperfoam spheres to mimic their transformation under high solvent vapor content. The final pressure was set to achieve a nearly solid sphere. In the four-body model, the position of the microspheres at the edge is fixed, simulating the particle-to-substrate anchoring, while the two middle microspheres are mobile. When the four microspheres are of identical size and have the same contact area, they undergo symmetrical pulling from the adjacent ones, resulting in minimal relative movement of their center of mass and formation of even microbridges. Formation of wider microbridges is achieved for larger particle-to-particle contact areas or by changing the initial microsphere relative size. Longer and thinner microbridges are also characterized by an increased shear stress distribution at particles’ shoulders. VISDi can result in two different end structures, namely interconnected microparticles’ networks (IMNets) and echinate microspheres (EMSpheres). mm-sized IMNets are obtained when the microbridges remain intact during VISDi (Fig.1b), by lowering solvent vapor concentration upon their formation. A prolonged exposure to solvent may in fact cause microbridges to become too thin and loose structural integrity, with projections recoiling and being reabsorbed in the microspheres’ matrix during contraction. EMSpheres are achieved by breaking the microbridges in two spans with external mechanical cues such as ultrasounds, each forming one spine on the respective microsphere (Fig.1b). VISDi can be applied to a variety of natural and synthetic polymer / solvent vapor systems, including silk fibroin (SF) / H2O, methyl cellulose (MC) / H2O, MC / AcOH, polyvinyl alcohol (PVA) / H2O, polyvinylpyrrolidone (PVP) / H2O, PVP / EtOH, polyacrylic acid (PAA) / H2O and PAA / EtOH, to fabricate IMNets and EMSpheres (Fig.1c and Fig.6). Besides, microspheres obtained by SFD can be pre-loaded with a variety of cargo molecules and actives (e.g. fluorescein, vitamin C and magnetite (Fe3O4) nanoparticles) to functionalize IMNets and EMSpheres (Fig.7). Using the SF / H2O system as an example, we manipulated highly porous (f # 92%) microspheres obtained via SFD by modulating the formation of particle-to-particle contact points and the concurrent exposure to water vapors (WV), as shown in Fig.2. Real time environmental SEM of the VISDi process (Fig.2a) depicts an isometric contraction of the microspheres around their center of mass when relative humidity (RH) reaches 90%. Rehydrated microspheres possess a volume 9.1 times smaller than the original (Ddry= 38±6 μm, Dwet= 20±4μm) (Fig.2b, Fig.8) due a combination of restored mobility of the polymer chains post rehydration, increase in bulk density (rb) from rb,dry=0.23 to rb,wet =1.04 g ml-1(Fig.9), and a decrease in apparent modulus from a 1-5 GPa to a 1-5 MPa(9). Each contact point between two adjacent microspheres provides a point of nucleation for the formation of a microbridge during contraction and thus a pair of microspines (one per microparticle) after breakage in two spans (Fig.2a). The pulling forces generated by isometric contraction of adjacent bodies also induce relative particle movements. The process is stopped by lowering RH below 75%, thereby hindering molecular reflow. A prolonged exposure to WV may in fact cause the re-absorption of the projections in the microspheres’ matrix. EMSpheres are composed of spines, shoulders, and matrix regions, each with distinct features (Fig.2c). AFM investigation of EMSpheres surface topography depicts a root mean square height (Sq) of 0.54 nm for the particles’ matrix and 0.65nm for the shoulders, which represents a decrease of almost an order of magnitude when compared to Sq=3.50 nm measured for as made SFD microspheres (Fig.2d). AFM analysis also showed material anisotropy at the EMSpheres’ shoulder region, which was corroborated by the emergence of birefringent structures under light cross-polarization (Fig.2e) and can be attributed to the increase in shear stress during material reflow, as modeled by the FEA simulations. To investigate the effects of VISDi on silk polymorphism, we performed polarized microRaman microscopy on EMSpheres (Fig.2f, g and Fig.10-12) (10, 11). Silk in SFD microspheres possess a predominantly non-ordered (i.e. random coil) molecular structure, with <18% beta-sheets. Long time (t = 3600s) exposure to WV is known to induce disorderÆorder molecular reconfiguration in silk materials through a process called water annealing, also inducing water insolubility when the beta-sheet content ^t 30%. The short duration (t=600-1500s) of VISDi slightly increases beta- sheet content in EMSpheres to^a 22%, 25%, and 22% at matrix, shoulder, and spine tip, respectively, without making the end material water insoluble (Fig.2f). This provides an opportunity to broaden the modulation of materials’ properties post-fabrication, i.e. using WV to further control formation of projections or ethanol vapors to impart water insolubility by reaching a beta sheet content a 44% (Fig.10). Polarized Raman microscopy was also used to investigate material anisotropy in IMNets and EMSpheres post ethanol annealing. Interestingly, the intensity of the Amide I band in the XX – YY spectra and in XY – YX spectra pairs was found to be nearly equal, indicating isotropy also at the EMSpheres’ shoulder region. The spectra obtained are consistent with the ones of isotropic silk films (Fig. 11) and indicate a molecular reflow during the disorderÆorder transitions.(10–12) IMNets and EMSpheres’ features were further modulated by controlling parameters such as substrate used during the VISDi process, concentration of silk fibroin in the SFD formulation, and packing density of particles during VISDi, as shown in Fig.3. If a non-stick substrate such as the surface of weighting papers is used during VISDi, particles undergo an isometric contraction with no generation of microbridges or spines. Particle-to-substrate adhesion forces are reduced and as such microspheres collapse on the substrate post exposure to solvent vapor and do not provide efficient anchoring points for the whole system, thereby not transferring pulling forces to a fix substrate. The lack of anchoring points during microsphere contraction prevents the formation of projections. On the contrary, polymer- sticky substrates (e.g. such as silicate) grant good adhesion to biomaterials such as silk fibroin and methyl cellulose, thereby providing fix anchoring sites that offer mechanical support for the particle-to-particle transfer of pulling forces involved in the formation of projections during microspheres’ contraction. Initial silk concentration may be used to modulate microbridge width. SEM analysis of IMNets shows that application of VISDi on microspheres with a similar initial diameter but higher protein concentrations yields narrower microbridges (Fig.3a,b). We can speculate that for higher concentrations of the protein the particle-to-particle contact area decreases due to a combination of lower porosity and higher compression modulus, which leads to a decrease in material reflow during contraction. For example, SFD of SF solutions with concentrations of 8.0%, 5.0%, and 2.5% yield microspheres with diameter around 38 μm. Upon VISDi, the microspheres shrink to a diameter of 18±3 μm for 8.0% initial SF concentration, while an average diameter is difficult to measure for the other two SF concentrations due to a loss of spherical geometry. Microbridges’ diameters are 1.8±0.4 μm, 4.3±1.4 μm, and 6.3±2.0 μm for IMNets made from 8.0%, 5.0%, and 2.5% initial SF concentrations, respectively (Fig.3c, Fig.13), indicating that for the lowest concentration considered, the matrix of the microspheres are completely integrated into the network. As depicted in Fig.3b, microbridges also have a hollow structure with nanofibrillar walls. The number of particle-to-particle connections can be controlled by modulating their density and stacking (Fig.3d, Fig.14). Microspheres can be stacked together by applying a gentle pressure (a1MPa) on their top, e.g. weight of a coverglass. A second layer can then be deposited and compacted on top of the first, thereby forming a double stack of microspheres. Application of VISDi induces formation of microbridges and links together particles across stacks, increasing the number of particle-to-particle microbridges. One compaction step induces the formation of an average of 3 connections per particle, while further layer by layer stacking increases the average number of connections to 5. These IMNets were then treated by ultrasonication to break bridges into spans that form EMSpheres of predefined spine number, i.e. EMS-3 and EMS-5 for EMSpheress with 3 and 5 spines, respectively. The number of spines per particle can increase to more than 8 giving to EMSpheres the resemblance of the natural sunflower pollen grain. (Fig.3e) Encapsulation of actives in EMSpheres provides an opportunity for their use in drug delivery on surfaces with reduced adhesion, such as plants leaves. Off target delivery and runoff of agrochemicals like phytoprotectant, fertilizers and pesticides are among the leading causes of food production environmental impact and waste of intensive resources. At first, we investigated the effects of VISDi on the mechanical properties of EMSpheres through frequency modulation (FM) and amplitude modulation (AM) AFM and saw that the microparticles densification causes an increase in Young’s modulus from 4.5r0.1 to 8.1r0.3 GPa (Fig.4a, Fig.17). Further treatment in EtOH vapor (Et-EMS) to impart water insolubility provides a 10% increase in the Young’s modulus, which reaches 8.8GPa r0.6 GPa (Fig.4a, Fig.15). To test adhesion on plant leaves external wax (i.e. cuticle, hardness= 10-150MPa(13) in vitro, we rolled EMSpheres on the surface of a synthetic wax-based material such as PARAFILM® (hardness= 60 MPa(14). As shown in Fig.4b and c, EMSpheres plastically deformed and locally broke continuity in the PARAFILM®, leaving scratching marks and holes that enhance adhesion and indicate that spines on EMSpheres mimic the purpose of echinate in pollens. To provide a proof-of-concept for the use of EMSpheres as biodegradable microcapsules for agriculture applications, we evaluated their retention on the surface of spinach leaves (Figure 4d-h). Spinach leaves were selected as benchmark due to their trichome-free, smooth surfaces (Fig.16a) that induce low (^a 4%) retention rates. Fluorescein sodium salt (FSS)-loaded microcapsules were used to test retention as the fluorescent dye allows to image EMSpheres adherent on leaves post spray and washing cycles, hence providing a mean to measure retention (Fig.4f-h, Fig.16-17). After washing, EMSpheres decorated with 5 spines (EM-5) largely remain on spinach leaves with moderate relocation from initial adhesion sites (Fig.4f), while non-VISDi treated microsphere mostly washed off (Fig.4g). Microcapsules retention measurements post spray and two washing cycles are shown in Fig.4h. Retention of EMS-5 was 68% and 50% after first (W1) and second washing (W2) cycles, respectively. These data show a 4.0-fold increase in retention after W1 and a 12.5-fold increase in retention after W2 for EMS-5 when compared to EMS-0. One of the distinct features of VISDi is the possibility of manufacturing mm-scale materials that are composed of nanostructured micronetworks, where actives of different nature are encapsulated at the point of microsphere fabrication through SFD. Microspheres containing different cargo molecules provide compartmentalized reservoirs while microbridges established by VISDi act as microvessels where the cargos can flow in, mix, and react. As a proof-of-concept that depicts the mixing in microbridges of two cargos encapsulated in distinct microparticles, we formed IMNets with microspheres preloaded with FSS or Rhodamine dyes. Upon exposure to WV (Fig.4i-k), red and green fluorescent dyes transfer along the microbridges, where they colocalize, hence giving a bright fluorescent signal (Fig.4k). Conclusions We demonstrated a synergistic microfabrication strategy that can be applied to both natural and synthetic polymer systems using a combination of environmental stimuli and geometric control. Exposure of adjacent low solid content microspheres to solvent vapors induces the formation of nanostructured micronetworks, where the microparticles are connected by microbridges. Further mechanical treatment can break the microbridges’ spans and separate the microsphere, inducing echinate structures. Modulation of process parameters such as polymer type, polymer concentration, stacking of microspheres and post-treatments to modify the molecular structure can tune surface morphology, number of projections per particles and mechanical performance. A variety of cargo molecules can be loaded in individual microspheres at the point of material assembly. Functionally, echinate enhance microsphere adhesion on surfaces. This was tested in vitro on a wax surface and in planta on spinach leaves’ cuticle. EMSpheres effectively improved the retention percentage on leaves by a factor of 12.5 after two washing cycles, providing a proof of concept for the use of echinate microcapsules to reduce agrochemicals wash off. Furthermore, IMNets can transfer and co-localize different fluorescent dyes in the microbridges, opening the door to their use as compartmentalized systems. Together these findings provide a new fabrication tool to rapidly obtain nanostructured spiny microspheres and micronetworks that are unattainable with current fabrication techniques, at scale. We also investigated for the first time the use of solvent vapor and geometrical cues to morph natural and synthetic polymers, opening new directions to program responsive materials that are architected in space and time, and extending the capability of programming soft materials functionality for future applications in biotechnology, actuation, food, and sustainability. Materials B. Mori cocoons were purchased from a local silk farm in South Korea (Gokseong- gun, Jeollanam-do). Lithium bromide, sodium carbonate, ascorbic acid (vitamin C), fluorescein sodium salt, polyvinyl alcohol (PVA) Mwapproximately 40 and 90 k, polyvinylpyrrolidone (PVP) average Mwapproximately 55 k, polyacrylic acid (PAA) Mwapproximately 100 k, and iron (II, III) oxide nanopowder (50-100 nm in particle size) were purchased from Sigma-Aldrich. All water was deionized (18.2 MΩ, Milli-QTMpore). All reagents were used as received without further purification. Sunflower pollen grains were collected in July from Coppal House Farm, Lee, NH. Silk fibroin preparation The regenerated silk fibroin was extracted from the cocoons of B. Mori following an established protocol(1)^ In brief, silk cocoons were cut into pieces (2 × 2 cm2) and boiled in 0.02 M sodium carbonate solution for 120 min to remove the sericin coating. The degummed silk fibers were rinsed with Milli-QTMwater (18.2 MΩ cm) and dried overnight. The dried silk fibers were dissolved in 9.3 M lithium bromide for 4 h at 60 °C followed by dialysis against Milli-QTMwater in dialysis membranes (molecular weight cut-off: 3.5 kDa) for 3 days with a frequent water change. The dissolved silk fibroin suspension was then centrifuged to remove impurities (at 4800 × g, 25 min × 2). The final silk fibroin suspension was stored at 4 °C, and its concentration was determined gravimetrically. Original spray freeze-dried microsphere preparation 20 mL of silk fibroin, methyl cellulose, PVA, PVP, and PAA aqueous suspensions with different solid contents (2.5-8.0 w / v%) were well mixed and slowly filtered using paper filters (5 ^m pore size) to eliminate the bubbles. A Sono-Tek ECHOTMultrasonication generator with a 48 kHz vortex nozzle was used to spray polymeric aqueous microdroplets at 3 mL min-1. The rotational nitrogen gas for the vortex nozzle was set to 2 psi. The mechanical energy converted from ultrasound waves stimulates standing waves at the surface of the nozzle and then atomizes the feed suspension into fine microdroplets. The median diameter of the microdroplets is determined by the resonate frequency and therefore falls within a tight distribution.(2) The feed microdroplets were collected in a liquid nitrogen bath followed by lyophilization at íௗ105ௗÛC and vacuum condition using a freeze drier (FreeZoneTM, LABCONCO). The SEM and size distribution of the original silk fibroin microspheres are shown in Fig.5. All samples were stored in a container with desiccants with RH controlled below 25%. Original silk-based microcapsule preparation 20 mL of 8.0 w / v% silk – 0.25% FSS, – 0.25% Rhodamine 6G, and – 0.11% Fe3O4nanoparticles aqueous suspensions were well mixed and slowly filtered using paper filters (5 ^m pore size) to eliminate the bubbles. A Sono-Tek ECHOTMultrasonication generator with a 48 kHz vortex nozzle was used to spray polymeric aqueous microdroplets at 3 mL min-1. The rotational nitrogen gas for the vortex nozzle was set to 1-2 psi. The microdroplets were collected in a liquid nitrogen bath and followed with lyophilization. All samples were stored in a container with desiccants with RH controlled below 25%. Vapor-induced synergistic differentiation (VISDi) process and control of spine number of echinate microspheres (EMSphere) Original microspheres or loaded microcapsules were put on a cover glass, followed by a gentle press with another cover glass to the top to pack tightly. Then we put the sample into a sealed box with water at the bottom (inside with a hygro-thermometer, 99% RH, temperature 22°C) for 1-2 min. The samples were taken out and dried on the bench. The finish of the VISDi process can be read by the color change – light to dark due to densification – especially for microcapsules with colored payloads. The pictures of SF, SF- FSS, and SF- Fe3O4EMSpheres are shown in Fig.7. We break the microbridge of EMSphere by gentle pressing or grinding. The breakage can also be done by 10 sec of bath sonication when placing the EMSphere in ethanol, as described below. For an average of 0 spines, we loosely put original silk microspheres on hydrophobic weighing paper and do not apply the top pressure. The original microspheres shrink into smaller spheres without spine differentiation when placed in the 99% RH box for 1.5 min. For an average of 3 spines, we put 0.2 mg of original silk microspheres on a cover glass, followed by a gentle press with another cover glass on top. When placed in the 99% RH box for 1.5 min, the original microspheres evolved to EMSpheres with an average spine number of 3. For an average of 5 spines, we put 0.2 mg of original silk microspheres on a cover glass, followed by a gentle press with another cover glass on top; then we apply another layer of 0.2 mg of original silk microspheres with the same press to build a two times thicker packing. When placed in the 99% RH box for 2 min, the original microspheres evolved to EMSpheres with an average spine number of 5. SEM and optical images are included and Fig.14. Fabrication of ethanol-treated EMSpheres (Et-EMS) After the VISDi process of original silk microspheres or microcapsules, we soaked the EMSpheres in 90% ethanol for 5 min. Then we sonicated the suspension for 10 sec to break up the microbridge, followed by drying in the hood. Mechanical performance of Et-EMS on PARAFILM® We cut a 3 × 3 cm2piece of PARAFILM® and suspended it using a wide-mouth container. We put 0.2 mg of Et-EMS on the PARAFILM®and gently rubbed it with a gloved finger for 30 sec. The original and rubbed pieces of PARAFILM®were observed under SEM. Plant material and growth conditions The seeds of Carmel spinach (Spinacia oleracea var. Carmel) were purchased from David’s Garden. Seeds of spinach were sown in pots (2.5 × 2.5 × 3 in3) and kept in a plant growth chamber (Caron 7314-50-2) at 22 °C with long day (day / night 16 h / 8 h) conditions with a light intensity of 120 ^mol m-2s-1, and 70-90% RH. The spinach plants were watered twice per week for two weeks. We used the 3rdor 4thtrue leaves for the retention test. Microcapsule retention test on spinach leaves 0.2 mg silk-FSS EMSpheres with average spine numbers of 0 and 5 were treated with 90% ethanol for 5 min to increase hydrophobicity. We sonicated the suspensions for 10 sec to obtain free-standing Et-EMSpheres. Then the powders were fully dried in the hood and resuspended in water. For spinach leaves spraying, we used a hard paper board to cover the right half of the leaf and applied two sprays on the left half with EMS-0. After drying, we gently covered the left side and applied two sprays of EMS-5 on the right half of the same leaf. After drying, we cut the leaf into 1 × 1 cm2pieces from different sides of the leaves and marked the samples separately. We then observed the samples under a fluorescent optical microscope before and after two times of washing For washing we gently washed each sample with NanopureTMwater for 10 sec at 60° bevel and dried them on the bench. We picked seven different areas from three spinach plants for the retention test. Finite element analysis (FEA) The FEA was conducted using the commercial software ABAQUS / CAE 2021 Standard / Explicit Model via the Dynamic Implicit Procedure with Nlgeom on. An axisymmetric stress 3-node deformable element was used to discretize the shell, and a mesh convergence study was conducted to ensure appropriate mesh density. The radii of spheres are 20 and 10 μm, respectively. The connection between spheres varied and was smoothed by a radius of 1 μm fillet. Hyperfoam with modified uniaxial test data was used. An increasing uniform pressure was applied over time to simulate the transformation of microspheres under high solvent vapor content, which results from the release of internal stress due to freeze drying and surface tension due to the accumulation of solvent vapor on microspheres. The final pressure ensures the diameter of the sphere around half of its initial value, which makes the spheres close to be solid. Silk samples for the uniaxial test were prepared by freezing 120 min boiled silk fibroin solution with a concentration of 8 w / v%. A cylinder container (diameter 14.60 mm) was used to shape foam samples for compression, and a petri dish was utilized to fabricate films for tension. The lyophilized silk foam cylinder had a diameter of 13.30 ± 0.30 mm (sample size n = 3) and was cut into segments around 8 mm. Foam samples (n = 3) were compressed at a rate of 1 mm min-1using an Instron instrument with 1kN force detector. Films were cut into strips and stretched (sample size n = 3) at a rate of 1 mm min-1using a DMA 850 with under constant (~40%) and regulating RH at room temperature. The stiffness of silk decreases while ductility increases due to the presence of high-water content. Due to the technical difficulties in obtaining the actual mechanical properties of silk in a fast- changing humidity, we therefore, modified the uniaxial test data for simulation by lowering the stress in both compression and tension curves and extending the ultimate tensile strain in the tension curve. Specifically, the stress was lowered around 10 times while the ultimate tensile strain was extended by 10 times. The tension curve reaches a plateau of 0.04 MPa at a strain of 0.04. As the foam showed negligible transverse deformation within our compression strain, we simplified the nominal lateral strain to zero during compression in the simulation. The volume was assumed to be constant under tension, where Poisson's ratio was set to be 0.499. Specifically, the stress under compression was modified as Statistical analysis The beta-sheet content of silk EMSphere and Et-EMS was pre-processed by Fourier self-deconvolution analysis followed by peak fitting of the amide I region using OriginLab. The leaf spraying statistical tests were run in triplicates, and the standard errors for the analytical results were used to generate and present error bars. All sample size (n) was indicated in corresponding legends. Statistical significance was analyzed with one-way ANOVA test in Excel. Data analysis of diameter change during transformation and EMSpheres spine numbers was done in Fiji ImageJ software. Morphology and the VISDi process characterization The surface morphologies of all samples were investigated using a Zeiss Merlin High- resolution SEM operating at 0.8 kV, 80 pA for imaging. All SEM samples were used without coating. To determine the VISDi process and diameter change, we pre-recorded the coordinates of specific areas and observed them under the SEM. Then we took the samples out of the SEM chamber and quickly moved them into the 99% RH box for a certain amount of time, followed by dehydration in the vacuum chamber and observation of the recorded locations. Therefore, the hydration and dehydration in this setup extended the VISDi time compared with the continuous process of 1.5 min. SEM operations in Fig.2a required longer time points for this set of images. An example of recording diameter change is shown in Fig. 8. The in-situ VISDi videos were taken by a Thermo Scientific QuattroTMS - Environmental SEM using a gaseous secondary electron detector. To mimic the VISDi process within the ESEM range, we set the chamber temperature to 10 °C and changed the RH from 20-98%. Raman microscopy The polarized Raman spectra were obtained using a custom-built, confocal Raman microscope. The excitation source was a 785 nm distributed Bragg reflector (DBR) laser, of which the polarization was controlled by a linear polarizer (LP) and a half-wave plate (HWP). The excitation was focused on the sample through a 40X, 0.85 NA objective by adjusting an XZY piezo sample stage, while the back-scattered Raman signal was collected using the same objective. A broadband HWP was used to rotate the polarization of the Raman signal, and another LP was placed after this HWP. The polarized Raman signal was routed through a polarization-maintaining (PM) single-mode fiber into a spectrometer equipped with a 600-grooves mm-1grating and a nitrogen-cooled CCD. Notably, employing fixed LPs and PM fibers reduces the polarization-dependence of the optical setups. The primary source of this dependence comes from the grating in the spectrometer, which is minimized in this case. Polarization configurations are labeled based on the laboratory frame, defined by the coordinates ^, ^, and ^ (Fig.12) For example, in the (X, Y) configuration, the excitation laser and the collected Raman signal are polarized in ^ and y, respectively. In this work, the excitation power was approximately 20 mW on the sample. Spectra were collected at sample matrices and shoulders with 4 combinations of polarizations (XX, XY, YX, YY), with the sample lying in either x or y. The integration time was 60 seconds with 2 repetitions for the original silk microspheres and 30 seconds with 3 repetitions for all the other samples. The raw spectra in the range of 400–2000 cm-1were post-processed using cosmic ray removal, Lieber fit (order 5, 10 iterations)(3) for fluorescence background subtraction, and Savitzky-Golay filter (order 5, frame length 11) for smoothing. Nanomechanical and surface characterization Atomic force microscope (AFM) images were obtained using a Cypher S AFM (Asylum Research) with a tapping mode, gold backside-coated AFM probe (PPP-NCHAuD, NANOSENSORS). Silk original microspheres, EMSpheres, and Et-EMS were placed on 1ௗ×ௗ1ௗcm2polystyrene-coated silicon wafers for imaging. Nanomechanical tests were performed using amplitude modulation-frequency modulation (AM-FM) viscoelastic mapping. The cantilever spring constant and sensitivity were calibrated using the built-in GetReal function of the Asylum Research software. A spin-coated polystyrene film (roughness root mean squareௗ=ௗ226ௗpm over an area of 5ௗ×ௗ5ௗ^m2) on silicon was used as the reference sample (Young’s modulus of which was 6.3ௗGPa based on the nanoindentation results from previous work(4) to calibrate the tip radius before and after each sample scan. All force maps were acquired at a scan rate of 1.0ௗHz and a resolution of 256ௗ×ௗ256 pixels. Force-indentation curves were analyzed using Hertz, JKR, and Oliver-Pharr models, assuming a Poisson ratio of 0.33. Young’s modulus at each pixel was obtained by fitting the force-indentation curve with the JKR model using Gwyddion (Czech Metrology Institute). An area of 0.5ௗ×ௗ0.5ௗ^m2was performed at each location, and a minimum of three sites per sample were indented. For each modulus map, histograms of the moduli at pixel positions were plotted in Fig.15. Optical imaging Fluorescence and polarized microscopy images were obtained on an Eclipse TE2000- E Inverted Microscope equipped with a DS-Fi3 camera (Nikon, Japan). White light was coupled with DAPI and FITC filter cube sets for fluorescence imaging. Two linear polarizers were used to illuminate the samples with cross-polarized light for birefringence imaging. Confocal microscopy images were obtained on an Olympus FV1200 Laser Scanning Confocal Microscope. The VISDi process of SF microcapsules loaded with FSS and Rhodamine dyes was triggered using a household mini humidifier. Supplementary Text Fig.1b-c shows FEA results of VISDi processes with geometrical controls. We modeled a four-body system with different sizes and contact areas. The top and bottom bodies are fixed, and the two middle ones can move freely during the simulations. The microbridge differentiate evenly the four bodies are under identical size and contact area. A greater contact area results in wider microbridge (Fig.1b), and the microbridge extends longer when the contact area is smaller. The microbridge differentiate evenly when the differently sized microspheres are mixed in equal intervals. The stress distribution map indicates the shear stress induced by VISDi process mainly locates at the shoulder area. Tuning of average spine number using geometric controls is shown in Fig.14. Denser packing of original microspheres leads to a greater number of contact sites, and then results in more microbridge during the VISDi (Fig.14a-b). The whole micronetwork is stabilized by the edge microspheres, which collapse and bind tightly to the substrate (Fig.14c and e). With a hydrophobic substrate and the absence of close packing, the VISDi takes place without the microbridge evolution (Fig.14d). Example 1 References 1. Y. Wu, X. Dong, J. Kim, C. Wang, M. Sitti, Wireless soft millirobots for climbing three-dimensional surfaces in confined spaces. Sci. Adv.8, eabn3431 (2022). 2. D. Liu, T. Zhang, X. Cheng, B. Wang, Y. Guo, Z. Liu, H. Jiang, Y. Lu, Engineering pollen-derived microstructures to reveal material morpho-performance paradigm. Small 18 2200037 (2022) 3. H. Zhou, J.-P. Kim, J. H. Bahng, N. A. Kotov, J. Lee, Self-assembly mechanism of spiky magnetoplasmonic supraparticles. Adv. Funct. Mater.24, 1439–1448 (2014). 4. H.-J. Chen, T. Hang, C. Yang, D. Liu, C. Su, S. Xiao, C. Liu, D. Lin, T. Zhang, Q. Jin, J. Tao, M. X. Wu, J. Wang, X. Xie, Functionalized Spiky Particles for Intracellular Biomolecular Delivery. ACS Cent. Sci.5, 960–969 (2019). 5. T. Maric, M. Z. M. Nasir, N. F. Rosli, M. Budanoviü, R. D. Webster, N.-J. Cho, M. Pumera, Microrobots derived from variety plant pollen grains for efficient environmental clean up and as an anti-cancer drug carrier. Adv. Funct. Mater.30, 2000112 (2020). 6. J. Chen, J. Webb, K. Shariati, S. Guo, J.-K. Montclare, S. McArt, M. Ma, Pollen- inspired enzymatic microparticles to reduce organophosphate toxicity in managed pollinators. Nat. Food.2, 339–347 (2021). 7. Y.-W. Lee, J.-K. Kim, U. Bozuyuk, N. O. Dogan, M. T. A. Khan, A. Shiva, A.-M. Wild, M. Sitti, Multifunctional 3D-Printed Pollen Grain-Inspired Hydrogel Microrobots for On-Demand Anchoring and Cargo Delivery. Adv. Mater.35, 2209812 (2023). 8. W. Brandon Goodwin, I. J. Gomez, J. C. Meredith, K. H. Sandhage, Conversion of pollen particles into three-dimensional ceramic replicas tailored for multimodal adhesion. Chem. Mater.25, 4529–4536 (2013). 9. H. Sun, B. Marelli, Polypeptide templating for designer hierarchical materials. Nat. Commun.11, 351 (2020). 10. T. Lefèvre, M.-E. Rousseau, M. Pézolet, Protein secondary structure and orientation in silk as revealed by Raman spectromicroscopy. Biophys. J.92, 2885–2895 (2007). 11. T. Lefèvre, M.-E. Rousseau, M. Pézolet, Orientation-insensitive spectra for Raman microspectroscopy. Appl. Spectrosc.60, 841–846 (2006). 12. T. Lefè, F. Paquet-Mercier, J.-F. Rioux-Dubé, M. Pézolet, Review structure of silk by Raman spectromicroscopy: from the spinning glands to the fibers. Biopolymers, 322– 336 (2011). 13. I. Fiorello, F. Meder, A. Mondini, E. Sinibaldi, C. Filippeschi, O. Tricinci, B. Mazzolai, Plant-like hooked miniature machines for on-leaf sensing and delivery. Commun. Mater.2, 103 (2021). 14. J. Qu, L. Ouyang, C. C. Kuo, D. C. Martin, Stiffness, strength and adhesion characterization of electrochemically deposited conjugated polymer films. Acta Biomater 31 114–121 (2016) 15. D. N. Rockwood, R. C. Preda, T. Yücel, X. Wang, M. L. Lovett, D. L. Kaplan, Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc.6, 1612–1631 (2011). 16. M. Liu, P.-E. Millard, H. Urch, O. Zeyons, D. Findley, R. Konradi, B. Marelli, Microencapsulation of high-content actives using biodegradable silk materials. Small. 18, 2201487 (2022). 17. C. A. Lieber, A. Mahadevan-Jansen, Automated method for subtraction of fluorescence from biological raman spectra. Appl. Spectrosc.57, 1363–1367 (2003). Example 2 Abstract Decarbonizing food production and mitigating agriculture's environmental impact require new technologies for precise delivery of fertilizers and pesticides to plants. The cuticle, a waxy barrier that protects the surface of leaves, causes 60–90% runoff of fertilizers and pesticides, leading to the wastage of intensive resources, soil depletion, and water body pollution. Solutions to mitigate runoff include adding chemicals (e.g., surfactants) to decrease surface tension and enhance cuticles' permeability but have low efficacy. We use vapor- induced synergistic differentiation (VISDi) to nanomanufacture echinate pollen-like, high payload content (~50 wt%) microcapsules decorated with robust spines that mechanically disrupt the cuticle and adhere to the leaf. VISDi induces a core-shell structure in the spines, enabling the release of agrochemicals from the microparticles' body into the leaf. As proof of concept, we demonstrated precise and high throughput delivery of iron fertilizer in Fe- deficient spinach plants. Spray of spiny microparticles improved leaf adhesion by mechanical interlocking, reduced wash-off by a ~12.5 fold, and enhanced chlorophyll content by ~7.3 times compared to the application of spherical counterparts. Together, these results show that spiny microparticles can mitigate agricultural runoff and provide a high throughput tool for precise plant drug delivery. A grand challenge of the 21stcentury is increasing food production while reducing carbon footprint and mitigating environmental impact. The Agri-Food system accounts for 40% of anthropogenic greenhouse gas emissions and 70% of freshwater annual consumption, while the inefficient deployment of agrochemicals has a negative impact on soil, water streams, and ocean health. Off target delivery of fertilizers and pesticides is in fact a double threat to planetary health, given the intensity of their production through mining and energy- intensive chemical processes and the negative effects on soil and water ecosystems.[1]Precision agriculture has emerged as a set of new technologies focused on increasing crop production while efficiently delivering water and agrochemicals to plants. Successful examples of precision agriculture technologies include global positioning system (GPS) guidance and variable rate application. Despite these successes, precise delivery of fertilizers and pesticides to plants remains an unmet technological challenge. Agrochemical spray is the standard delivery method in open-field agriculture due to its ease of operation. However, 60– 90% of sprayed agrochemicals go off target and are affected by runoff, due to poor absorption through the leaves’ surface. The surface of leaves is protected by a waxy barrier, known as the cuticle. This barrier is composed of cutin, waxes, and polysaccharides, which prevent the absorption of sprayed water droplets and bounce back microparticles used to encapsulate, protect, and deliver agrochemicals in plants. Runoff of pesticides and fertilizers poses a significant threat to growers' economic investments. Small farm holders in rural areas are particularly affected, and this runoff may widen the yield gap. When economic resources allow farmers to apply only a limited amount of agrochemicals, their inefficient delivery hinders optimal crop production. Escalating extreme weather events have exacerbated agrochemical delivery inefficiencies, with more intense episodes of heavy rain enhancing the wash-off of fertilizers and pesticides, thus increasing runoff. A well-documented example concerns iron fertilizer. Iron (Fe2+) is a crucial micronutrient for chlorophyll synthesis and extensively used in agriculture. It is generally applied by soil application and foliar spraying. However, its runoff into soil systems causes acidification and limits crop yield by reducing plant growth. To cope with poor delivery efficacy, growers often apply excessive amounts of iron fertilizers, which further increases runoff and has negative effects on water bodies and aquatic life by triggering harmful algal blooms and reduce oxygen levels in water. The impact of inefficiencies in iron fertilizer delivery also affects high-tech farms in agriculture. One notable example is iron deficiency chlorosis (IDC) in soybeans, grapes, and peas in the United States. Studies indicate that IDC in soybeans alone can lead to yield losses ranging from 20 to 30%. Merely increasing the application of iron fertilizers does not significantly improve iron uptake due to the constraints imposed by soil conditions. This problem is further compounded by the expected surge in food production to meet the needs of a projected global population of 9.7 billion by 2050, which will inevitably lead to increased fertilizer application and exacerbate the issue of runoff. Developing high payload content and biodegradable capsules that anchor to and effectively regulate payload release through the cuticle remains challenging. A possible solution can be found in nature, where echinate pollen grains are produced by plants to physically adhere to pollinators and encapsulate and deliver a male gametophyte. These spiny pollen grains emerge as promising functional microcarriers due to their extraordinary physicochemical properties. Fabricating spiny pollen-inspired microcapsules with a high loading content, precise size control – less than 50 μm to retrofit existing spray technologies used in agriculture – and using biodegradable polymers remains an unmet technological challenge. These spiny geometries in echinate pollen form from the physical phase separation of a complex mixture of branched and unbranched polysaccharidesWe developed a spontaneous, vapor-induced synergistic differentiation (VISDi) process to enable the simultaneous microfabrication and nanostructuring of biopolymer-based spiny microcapsules (Figure 1). In this process, we first obtained polymeric spherical microparticles using a spray freeze drying (SFD) method. Then, the microspheres are synergistically shaped in t = 101– 102seconds by their adjacent neighbors in nanostructured microarchitectures. Local control over particle density and solvent vapor pressure enables the synergistic formation of microbridges at the particle-to-particle contact points. Further evolution modulates the formation of networks of connected microparticles of increasing complexity. Applying mechanical stress via ultrasound can break the microbridges and result in spiny microspheres with a pre-determined number of spines. To test the efficacy of spiny microspheres in delivering plant micronutrients precisely, we encapsulated ethylenediaminetetraacetic acid iron (III) sodium salt (Fe-EDTA), a common iron fertilizer, in silk fibroin (SF), a structural biopolymer that has been previously investigated as agrochemicals carrier material due to its high biodegradability, scalability, and ease of processing.[9,11,23–28]VISDi of silk materials enabled the fabrication of tens of grams of high payload content (50 wt%) Fe-encapsulated spiny microcapsules in a few hours. Adhesion and controlled release performance tests were conducted on spinach leaves under simulated rainy conditions. We chose spinach as the plant model for investigating wash-off performance after foliar application due to its quick growth cycle and the slippery nature of its leaves, which possess a large surface area and lack trichomes. Fe-silk composition re-distribution during the synergistic differentiation We pre-mixed iron fertilizer (Fe-EDTA) and silk suspensions and fabricated them into Fe-silk SpheMs using the SFD method. We achieved an optimal loading content of 50 wt% (dry sample) ensuring effective encapsulation performance while attaining a high- loading content. Exposure to high water vapors (WV) leads to the change of Fe-EDTA payload and silk fibroin distribution in SPIMs (Figure 18). As shown in Figure 18a, the fractured surfaces of a silk-Fe SpheM exhibit highly porous morphologies. SEM-EDS mapping results depict Fe-EDTA payload and silk as homogenous microcapsule distribution. After the VISDi process is completed, fractured surfaces of Fe-silk SPIMs show collapsed and dense morphologies. EDS analysis demonstrates prominent silk / payload separations, where Fe-EDTA – represented by Fe (colored red) and Na (colored green) – aggregate in the center of the SPIM (Figure 18b. and inset). This re-distribution may be induced by a phase separation process (Figure 18c). When WV enters and exits SpheMs, the silk fibroin polymer chains tend to remain at the air / particle interfaces due to their amphiphilicity. Simultaneously, the water-soluble, small molecules of Fe-EDTA become mobile during hydration. Upon drying, they subsequently move to the center of the microcapsule, where the hydration zone disappears last. Therefore, VISDi causes a change in Fe-silk SpheMs internal structure from matrix to core-shell, with the separation between SF and Fe-EDTA also resulting in denser silk shells. Controlled iron delivery of Fe-silk Spiny Pollen Inspired Microcapsules (SPIMs) in rainy conditions To investigate the release performance, we investigated the release performance of Fe-silk SPIMs on spinach leaves (scheme in Figure 19a). We prepared healthy and Fe- deficient spinach plants using hydroponic settings and refilled with Hoagland’s and Fe- deficient Hoagland’s solutions, respectively (Figure 19b). We first tested the Fe payload release performance in 10% PluronicTMsolution, characterizing the released Fe-EDTA using UV-Vis spectroscopy in a period of 7 days. As shown in Figure 19c, the original Fe-silk spherical microcapsules (SpheMs) showed a 73% of burst release of Fe-EDTA in the first 20 min, quickly reaching over 90% of total payloads in 4 hours. The EtOH-treated Fe-silk SPIMs decorated without spines (SPIM-0) exhibited an 8.5% burst release, then slowly released the Fe payload over time. The released Fe payload reached over 90% after 24 hrs and nearly 100% after a week. The EtOH-treated Fe-silk SPIMs decorated with 5 spines (SPIM-5) showed a similar release profile; however, they had a slightly increased burst release (15.6%) compared with SPIM-0. The burst release difference between SPIM-0 and -5 can be explained by the fact that the spine tips – broken by mechanical disturbance – serve as hollow microchannels, facilitating the Fe-EDTA transport in solutions (Figure 3b). To test the iron delivery efficiency we sprayed SPIM-0 and SPIM-5 aqueous solutions on Fe-deficient spinach leaves, let them dry naturally, then rinsed the sprayed leaves with NanopureTMwater. Visual evaluation and chlorophyll content of spinach leaves were made at 5 days after treatment (DAT). These methods are widely used across the agricultural industry for both greenhouse and field trials to determine the amount of chlorophyll content that is present in the desired plant after a chemical application.

[0029] In Figure 19d and 23, the SPIM-5-treated spinach leaf showed a significantly improved green color compared with the original yellowish leaf, which increased chlorophyll content by a factor of 7.29 – from 0.48 to 3.5 (u 10-2mg cm-2). However, the SPIM-0-treated spinach leaf showed slightly increased greenness and a chlorophyll content of 0.95 (u 10-2mg cm-2). The increased chlorophyll content indicates the successful iron delivery after the simulated rain wash, validating the anti-runoff properties of SPIMs with more microspines. We also characterized the time- lapsed iron delivery process on the spinach leaves. As shown in Figure 19e, after spraying and drying, the spine tip of a Fe-silk SPIM-5 presses against the cuticle layer of the spinach leaf. The elemental mapping results indicate a Fe accumulation around the tip region. The hollow microspines can act as conduits, enabling the controlled release of Fe at the points where the spines come into contact with the leaf's cuticle layers. To test Fe delivery, we placed the sample in an environment with 99% RH. Then, we monitored the Fe transport using EDS analysis over time (Figure 19f). The observed Fe concentration slowly intensified along with the expansion of the Fe distribution area in humid conditions for 40 min, indicating a targeted iron delivery process projected from the spines. Furthermore, we demonstrated the biodegradation of silk-based SPIMs by exposing them to protease XIV, a proteolytic enzyme produced by Streptomyces griseus, a Gram-positive species of bacteria commonly found in soil. Protease XIV is a mixture of at least three caseinolytic activities and one aminopeptidase activity and is well-known for its efficacy in degrading silk materials and has been used to simulate degradation in soil and in the human body.[30–32]After 5 days of treatment, the silk-based SPIMs lost 64.4r5.1 wt%, indicating that an enzymatic degradation process can effectively digest the silk microcapsules (Figure 24). Biopolymers self-assemble into free-standing microvascular networks for molecular and nanoparticle transport Engineering biopolymers with multiscale complexity is critical in sustainable design for next-generation technologies. For example, biopolymer-based microvascular networks possess great potential in advancing filtration and resource capture technologies in environmental applications, as well as regenerative medicine in biomedical applications. Nonetheless, engineering vascular networks has historically remained difficult. The state-of- the-art methods suffer from limited materials selection, fabrication resolution, and miniaturization. To address these issues, we explored a synergistic nanostructuring approach that transforms biopolymer microspheres into microvascular networks (Figure 20). The biopolymer suspensions can be spray freeze-dried into highly porous microcapsules (Figure 20a). Subsequently, through the vapor-induced synergistic differentiation approach, the microspheres can assemble into 3D vascular micronetworks with complex hierarchical structures across multiple scales, ranging from nanometers to centimeters (Figure 20b). We have modified this assembly process to enable tuning of the external and internal structures of biopolymer micronetworks, including the diameter and porosity of microchannels, as well as their water-filled microenvironments (Figure 20c). We found that NaHCO3-silk microcapsules created a water core-insoluble wall vascular configuration in response to acetic acid vapor, where beta-sheet silk constructs the wall domains (Figure 21a-b). The structures can be controlled by tuning vapor conditions and microcapsule compositions. We successfully developed a silk-based microvascular network fabrication technology that can provide free-standing, interconnected microchannels. The in- vasculature water formation can be controlled by adjusting the contact time of microcapsules and acetic acid vapor (Figure 21c). Furthermore, we conducted a vapor-triggered molecular transport from dye-loaded (e.g., Fluorescein sodium salt, Rhodamine B) to surrounding dye- free counterparts within 101-102seconds (Figure 22a-c). This technology provides a precision molecular transport and environmentally responsive device. Conclusion We demonstrated a spiny pollen-inspired microencapsulation technology for high throughput and precise iron delivery in spinach plants. This technology is enabled by a synergistic microfabrication strategy that can be applied to natural and synthetic polymer systems using a combination of environmental stimuli and geometric control. Exposure of adjacent low-solid-content microspheres to solvent vapors induces the formation of nanostructured micronetworks, where microbridges connect the microparticles. Further mechanical treatment can break the microbridges’ spans and separate the microsphere, inducing spiny structures. Modulation of process parameters such as silk polymer concentration, stacking of silk-based microspheres, and post-treatments to modify the silk protein’s molecular structure can tune surface morphology number of projections per particle, and mechanical performance. Fe-EDTA cargo molecules can be loaded in high content (up to 50 wt %) to individual silk fibroin microspheres at the point of material assembly. Functionally, spines enhance microsphere adhesion on surfaces and decrease wash off. Spines-mediated adhesion was tested in vitro on a wax surface and in planta on spinach leaves’ cuticle. The SPIM decorated with 5 spines effectively improved the retention percentage on leaves by a factor of 12.5 after two washing cycles, compared to conventional spherical counterparts. The Fe-loaded SPIMs significantly enhanced the chlorophyll content of treated spinach leaves by a factor of 7.29. Furthermore, the VISDi process induces the Fe- EDTA and silk phase separation, resulting in a matrix-to-core-shell structural transition. The SPIMs controlled the release of Fe payload in solutions over a period of 7 days. The hollow microspines act as conduits that enable the release of Fe into spinach leaves at the contact areas. Additionally, silk-based microcapsules can effectively biodegrade when exposed to enzymes commonly found in soil bacteria, making them an alternative to non-degradable products. Together, these findings provide a proof of concept for the use of spiny microcapsules to reduce agrochemical wash-off. We also investigated, for the first time, the use of solvent vapor and geometrical cues to modify natural and synthetic polymers assembly. This exploration opens new avenues for programming responsive materials that are structured in both space and time, thereby extending the functionality of soft materials. Our biodegradable microcapsules, which can be functionalized with various active payloads including water-soluble molecules and magnetic nanoparticles, offer numerous possibilities in biotechnology, actuation, food, and sustainability applications, including drug delivery, environmental remediation, biological sensing, cellular capture, and flexible electronics. Materials and Methods Original silk-based spherical microcapsules (SpheMs) preparation 20 mL of 8.0 wt% silk, 8.0 wt% silk – 8.0 wt% Fe-EDTA, or 8.0 wt% silk – 0.25 wt% FSS aqueous suspensions were well mixed separately, and slowly filtered using paper filters (5 ^m pore size) to eliminate the bubbles. A Sono-Tek ECHOTMultrasonication generator with a 48 kHz vortex nozzle was used to spray polymeric aqueous microdroplets at 3 mL min-1. The rotational nitrogen gas for the vortex nozzle was set to 1-2 psi. The microdroplets were collected in a liquid nitrogen bath and followed with lyophilization. All samples were stored in a container with desiccants with RH controlled below 25%. Release of Fe-silk SpheMs SPIM-0 and -5 in PluronicTMsolutions Around 2.0 mg of different Fe-silk microcapsules were dispersed in 20 mL of 10% PluronicTMPE 10500 aqueous suspension, wrapped in Al foil and under 20 rpm mild shaking. After certain time intervals, 0.75 mL of suspension was temporarily taken out for UV-Vis measurement to determine the concentration of released Fe-EDTA. The calibration curve is obtained in Figure 25. The characteristic UV absorption peak position of Fe-EDTA is around 253 nm. Plant material and growth conditions Hydroponic setting: The seeds of Carmel spinach were covered in wet tissue towel in a dark environment for around 3 days. The uniform seedlings were moved into the soil (using the mentioned soil setting) for 2-3 days. When the spinach grown 1stand 2ndleaves, we gently moved them out and washed the plants with NanopureTMwater. The spinach plants were then transferred to 1.2 u 1.3 u 1.8 in3clear grow sponge pots in a 3.3 u 3.3 u 3.3 in3acrylic box, filling with 250 mL quarter strength modified Hoagland’s solution (control and Fe-deficient solutions).

[0033] The plants were kept in the same plant growth chamber and conditions. The hydroponic pots were refilled with modified Hoagland’s solution three times per week for one month. We used the 5thand 6thtrue leaves for the chlorophyll content tests. Iron delivery test in spinach leaves The spinach seedlings were given the Fe-deficient Hoagland’s solution without Fe- EDTA on the 1stday following transplanting. They started showing significant iron deficiency chlorosis after around one week. Three groups of spinach leaves were sprayed 1 mL of 0.1 wt% of Fe-silk SPIM-0 and -5 water suspensions respectively. For simulated rain washing, we sprayed each sample with 5 mL of NanopureTMwater at 60° bevel and dried them on the bench. At the 5 days after treatment, we picked 3 different areas from three spinach plants for the iron delivery test. We tested the chlorophyll content using an atLEAF CHL STD chlorophyll meter. For each leaf, 3 different locations were tested and recorded. The readings were converted to total chlorophyll content (mg cm-2) using the atLEAF conversion tools.

[0029] Biodegradation of SF Et-SPIMs The enzymatic degradation of SF Et-SPIMs was done in PBS (1x, pH around 7.4) solution at 37 °C. For the control group: around 5.0 mg of SF Et-SPIMs (90% ethanol-treated for 60 min) were immersed in 1 mL of PBS solution and put in a 37 °C incubator under 200 rpm shaking. The sample was monitored for 5 days. For the experimental group: around 5.0 mg of SF Et-SPIMs (90% ethanol treated for 60 min) were immersed in 1 mL PBS solution with 3.5 U mL-1protease XIV from Streptomyces griseus and put in a 37 °C incubator under 200 rpm shaking. The old solution was centrifuged out, and the new protease XIV PBS solution was replenished daily. All control and experimental samples were washed with PBS solution (centrifuged at 200 × g, 10 sec u 4), then Milli-QTMwater (centrifuged at 20000 × g, 5 min u 4) and dried under 60 °C overnight before each weighing. The mass of empty test vial, test vial with the dried control sample, and test vial with dried experimental sample were marked as m0, mc, and mp. The weight remaining % was calculated as (mp-m0p) / (mc-m0c) u100%. B. Mori cocoons were purchased from a local silk farm in South Korea (Gokseong- gun, Jeollanam-do). Lithium bromide, sodium carbonate, sodium bicarbonate (NaHCO3), acetic acid (AcOH), fluorescein sodium salt (FSS), rhodamine B (RhB), methyl cellulose, and iron (II, III) oxide nanopowder (50-100 nm in particle size) were purchased from Sigma- Aldrich. All water was deionized (18.2 MΩ, Milli-QTMpore). All reagents were used as received without further purification. Silk fibroin preparation The regenerated silk fibroin was extracted from the cocoons of B. Mori following an established protocol.

[0038] In brief, silk cocoons were cut into pieces (2 × 2 cm2) and boiled in 0.02 M sodium carbonate solution for 120 min to remove the sericin coating. The degummed silk fibers were rinsed with Milli-QTMwater (18.2 MΩ cm) and dried overnight. The dried silk fibers were dissolved in 9.3 M lithium bromide for 4 h at 60 °C followed by dialysis against Milli-QTMwater in dialysis membranes (molecular weight cut-off: 3.5 kDa) for 3 days with a frequent water change. The dissolved silk fibroin suspension was then centrifuged to remove impurities (at 4800 × g, 25 min × 2). The final silk fibroin suspension was stored at 4 °C, and its concentration was determined gravimetrically. Original spray freeze-dried microsphere preparation 20 mL of silk fibroin or methyl cellulose aqueous suspensions with solid contents of 3.0 – 8.0 wt% were well mixed and slowly filtered using paper filters (5 ^m pore size) to eliminate the bubbles. A Sono-Tek ECHOTMultrasonication generator with a 48 kHz vortex nozzle was used to spray polymeric aqueous microdroplets at 3 mL min-1. The rotational nitrogen gas for the vortex nozzle was set to 2 psi. The mechanical energy converted from ultrasound waves stimulates standing waves at the surface of the nozzle and then atomizes the feed suspension into fine microdroplets. The median diameter of the microdroplets is determined by the resonate frequency and therefore falls within a tight distribution.

[0023] The feed microdroplets were collected in a liquid nitrogen bath followed by lyophilization at íௗ105ௗÛC and vacuum condition using a freeze drier (FreeZoneTM, LABCONCO). The SEM and size distribution of the original silk fibroin microspheres are shown in Figure 5. All samples were stored in a container with desiccants with RH controlled below 25%. Original silk-based spherical microcapsules (SpheMs) preparation 20 mL of 3.0 wt% silk – 2.0 wt% NaHCO3, 3.0 wt% silk – 2.0 wt% NaHCO3 – 0.3 wt% FSS, or 3.0 wt% silk – 2.0 wt% NaHCO3 – 0.3 wt% RhB aqueous suspensions were well mixed separately, and left in a 4 °C refrigerator overnight to eliminate the bubbles. A Sono-Tek ECHOTMultrasonication generator with a 48 kHz vortex nozzle was used to spray polymeric aqueous microdroplets at 3 mL min-1. The rotational nitrogen gas for the vortex nozzle was set to 1-2 psi. The microdroplets were collected in a liquid nitrogen bath and followed with lyophilization. All samples were stored in a container with desiccants with RH controlled below 25%. Vapor-induced synergistic differentiation (VISDi) process and control of vascular micronetworks Original microspheres or loaded microcapsules were put on a cover glass, followed by a gentle press with another cover glass to the top to pack tightly. The we put the sample into a lid-covered petri dish with a AcOH soaked sponge inside for 3-5 min. The finish of the VISDi process can be read by the color change – light to dark due to densification – especially for microcapsules with colored payloads. In this process, NaHCO3 encapsulated within the silk microcapsules react with the surrounding AcOH and generated water inside of the formed microchannels. The water generation in the microbridges can be monitored under an optical microscope. Example 2 Reference [1] D. Zaring, Harvard Environ. Law Rev.1996, 20, 515. [2] T. Xu, Y. Wang, Z. Aytac, N. Zuverza-Mena, Z. Zhao, X. Hu, K. W. Ng, J. C. White, P. Demokritou, ACS Nano 2022, 16, 6034. [3] G. Del Rossi, M. M. Hoque, Y. Ji, C. L. Kling, Annu. Rev. Resour. Econ.2023, 15, DOI 10.1146 / annurev-resource-111820-021317. [4] J. Walsh, D. Wuebbles, K. Hayhoe, J. Kossin, K. Kunkel, G. Stephens, P. Thorne, R. Vose, M. Wehner, J. Willis, D. Anderson, S. Doney, R. Feely, P. Hennon, V. Kharin, T. Knutson, F. Landerer, T. Lenton, J. Kennedy, R. Somerville, in Clim. Chang. Impacts United States Third Natl. Clim. Assess. (Eds.: J. M. Melillo, T. C. Richmond, G. W. Yohe), U.S. Global Change Research Program, Washington, DC, 2014, pp.19– 67. [5] S. Rawashdeh, Hamzeh M.; Florin, Int. J. Agric. Policy Res.2015, 3, 368. [6] M. W. Silver, S. Bargu, S. L. Coale, C. R. Benitez-Nelson, A. C. Garcia, K. J. Roberts, E. Sekula-Wood, K. W. Bruland, K. H. Coale, Proc. Natl. Acad. Sci.2010, 107, 20762. [7] N. C. Hansen, M. A. Schmitt, J. E. Anderson, J. S. Strock, Agron. J.2003, 95, 1595. [8] W. P. Falcon, R. L. Naylor, N. D. Shankar, Popul. Dev. Rev.2022, 48, 921. [9] B. Marelli, Science (80-. ).2022, 376, 146.

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Claims

We claim 1. A method, comprising applying an external stimulus to a plurality of packed, polymeric microspheres, wherein the polymeric microspheres have a porosity of between about 20% to 99%, and wherein at least a boundary layer of the polymeric microspheres are in contact with a surface; such that the polymeric microspheres collapse into smaller and denser microspheres that join with adjacent microspheres at contact points to form a micronetwork, wherein the contact points comprise microstructures extruded from the polymeric microspheres.

2. The method of claim 1, wherein the polymeric microspheres comprise hydrophilic microspheres.

3. The method of claim 1, wherein the polymeric microspheres comprise amphiphilic microspheres.

4. The method of claim 1, wherein the polymeric microspheres comprise thermoplastic microspheres.

5. The method of any one of claims 1-4, wherein the polymeric microspheres comprise a water-soluble polymer.

6. The method of any one of claims 1-5, wherein the water-soluble is selected from the group consisting of silk fibroin, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyethylene oxide (PEO), methyl cellulose (MC), carboxymethyl cellulose (CMC), chitosan, pullulan, gelatin, pectin, and combinations thereof.

7. The method of any one of claims 1-6, wherein the polymeric microspheres are freeze- dried microspheres.

8. The method of any one of claims 1-7, wherein the polymeric microspheres comprise silk fibroin.

9. The method of claim 8, wherein the polymeric microspheres are freeze-dried silk fibroin microspheres10. The method of any one of claims 1-9, wherein the polymeric microspheres have a porosity of between about 25% to 99%; about 30% to 99%; about 35% to 99%; about 40% to 99%; about 45% to 99%; about 50% to 99%; about 55% to 99%; about 60% to 99%; about 65% to 99%; about 70% to 99%; about 75% to 99%; about 80% to 99%; or about 85% to 99%.

11. The method of any one of claims 1-10, wherein the smaller and denser microspheres comprise microspheres of less than about 70% porosity and having a diameter reduced by at least 10%.

12. The method of any one of claims 1-11, wherein the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or 25% porosity.

13. The method of any one of claims 1-12, wherein the smaller and denser microspheres after application of the external stimulus comprise microspheres having a diameter reduced by at least 15%, 20%, or 25%.

14. The method of any one of claims 1-13, wherein the smaller and denser microspheres after application of the external stimulus comprise microspheres of less than about 25% porosity and having a diameter reduced by at least 25%.

15. The method of any one of claims 1-14, wherein the surface comprises silicate or aluminum surfaces.

16. The method of any one of claims 1-15, where the external stimulus comprises applying a pressure of at least 0.5 MPa, or at least about 1 MPa, on the polymeric microspheres.

17. The method of any one of claims 1-16, wherein the external stimulus comprises applying a solvent vapor of about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, or between about 80% to about 99%, tothe polymeric microspheres, wherein the solvent is capable of dissolving the polymer that forms the polymeric microspheres.

18. The method of claim 17, wherein the solvent vapor is selected from the group consisting of water, ethanol (EtOH), and acetic acid (AcOH).

19. The method of any one of claims 17-18, wherein a polymer / solvent vapor combination is selected from the group consisting of (a) silk fibroin (SF) / water or silk fibroin (SF) / AcOH; (b) methyl cellulose (MC) / water; (c) MC / AcOH, (d) CMC / water, (e) chitosan / AcOH, (f) pectin / water, (g) polyvinyl alcohol (PVA) / water (h) polyvinylpyrrolidone (PVP) / water, (i) PVP / EtOH, polyacrylic acid (PAA) / water and (j) PAA / EtOH.

20. The method of 17, wherein: (a) the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres; and (b) the solvent vapor comprises acetic acid (AcOH).

21. The method of claim 20, wherein the polymeric microspheres further comprise NaHCO3.

22. The method of claim 21, wherein the polymeric microspheres comprise a weight % of NaHCO3 of between 1 to 50%.

23. The method of claim 21 or 22, wherein the polymeric microspheres comprise a weight % of NaHCO3 of between about 10% to about 50%, about 20% to about 50%, or about 40% to about 50%.

24. The method of any one of claims 19-23, wherein the polymeric microspheres comprise a weight % of silk fibroin of about 50% to about 99%.^ 25. The method of any one of claims 19-23, wherein the polymeric microspheres comprise a weight % of silk fibroin of between about 50% to about 90%, about 50% to about 80%, or about 50% to about 60%.

26. The method of any one of claims 17-25, wherein the polymeric microspheres comprise silk fibroin, including but not limited to freeze-dried silk fibroin microspheres, and the external stimulus comprises applying water vapor of between about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, or between about 80% to about 99%.

27. The method of any one of claims 1-26, wherein the external stimulus comprises heating, optionally wherein the heating comprises heating the plurality of polymeric microspheres to a temperature above the glass transition temperature.

28. The method of any one of claims 1-27, wherein the polymeric microspheres are pre- loaded with a payload selected from the group consisting of soluble chemicals, insoluble nanoparticles, therapeutics, a fertilizer, and / or a plant-growth agent, optionally wherein the microspheres comprise different payloads, and wherein the different payloads interact in the microstructures.

29. The method of claim 28, wherein the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil, and pyraclostrobin.

30. The method of any one of claims 1-29, wherein the method further comprises breaking the microstructures to produce a plurality of patterned microspheres comprising one or more fragmented microstructures.

31. The method of claim 30, wherein the breaking comprises applying mechanical stress to the micronetwork.

32. The method of claim 31, wherein the mechanical stress comprises ultrasound.

33. The method of any one of claims 30-32, wherein the one or more fragmented microstructures comprise spines, channels, bumps, and / or helices.

34. The method of any one of claims 30-32, wherein the one or more fragmented microstructures comprise spines.

35. The method of claim 34, wherein the spines have a diameter of between about 0.2 μm to about 15 μm, and / or a length between about 1 μm and about 25 μm.

36. The method of any one of claims 34-35, wherein the spines have a diameter of between about 0.5 μm to about 10 μm.

37. The method of any one of claims 34-36, wherein the spines have a length between about 2 μm and about 25 μm.

38. The method of any one of claims 34-37, wherein the spines have a Young’s modulus of between about 4 GPa and about 10 GPa.

39. The method of any one of claims 34-37, wherein the spines have a Young’s modulus of between about 7 GPa and about 9.5 GPa.

40. The method of any one of claims 34-39, wherein the spines comprise a pointed end.

41. The method of any one of claims 34-40, wherein the patterned polymeric microspheres comprise 2, 3, 4, 5, or more spines.

42. A patterned polymeric microsphere produced by the method of any one of claims 30- 41.

43. A patterned polymeric microsphere, comprising a freeze-dried silk fibroin microsphere comprising one or more spines extruded from the microsphere.

44. The patterned polymeric microsphere of claim 43, wherein the one or more spines have a diameter of between about 0.2 μm to about 15 μm, and / or a length between about 1 μm and about 25 μm.

45. The patterned polymeric microsphere of claim 43 or 44, wherein the one or more spines have a diameter of between about 0.5 μm to about 10 μm.

46. The patterned polymeric microsphere of any one of claims 43-45, wherein the one or more spines have a length between about 2 μm and about 25 μm.

47. The patterned polymeric microsphere of any one of claims 43-46, wherein the one or more spines have a Young’s modulus of between about 4 GPa and about 10 GPa.

48. The patterned polymeric microsphere of any one of claims 43-46, the one or more spines have a Young’s modulus of between about 7 GPa and about 9.5 GPa.

49. The patterned polymeric microsphere of any one of claims 43-48, wherein the one or more spines comprise a pointed end.

50. The patterned polymeric microsphere of any one of claims 43-49, wherein the freeze- dried silk fibroin microsphere comprises NaHCO3.

51. The patterned polymeric microsphere of claim 50, wherein the freeze-dried silk fibroin microsphere comprises a weight % of NaHCO3 of between 1 to 50%.

52. The method of claim 50 or 51, wherein the polymeric microspheres comprise a weight % of NaHCO3 of between about 10% to about 50%, about 20% to about 50%, or about 40% to about 50%.

53. The method of any one of claims 43-52, wherein the polymeric microspheres comprise a weight % of silk fibroin of about 50% to about 99%^54. The method of any one of claims 43-53, wherein the polymeric microspheres comprise a weight % of silk fibroin of between about 50% to about 90%, about 50% to about 80%, or about 50% to about 60%.

55. The patterned polymeric microsphere of any one of claims 50-54 , comprising water present inside the freeze-dried silk fibroin microsphere and / or the one or more spine.

56. The patterned polymeric microsphere of any one of claims 50-55, wherein silk polymers on the microsphere surface comprise water-insoluble beta-sheets.

57. The patterned polymeric microsphere of any one of claims 43-56, wherein the patterned polymeric microsphere comprises a payload within the microsphere and / or the one or more spine.

58. The patterned polymeric microsphere of claim 57, wherein the freeze-dried silk fibroin microsphere comprises the freeze-dried silk fibroin microsphere of claim 48, or claim 47 when depending from claim 46, and wherein the payload is solubilized in the water present inside the freeze-dried silk fibroin microsphere and / or the one or more spine.

59. The patterned polymeric microsphere of claim 57 or 58, wherein the payload is selected from the group consisting of a therapeutic, a fertilizer, and a plant-growth agent.

60. The patterned polymeric microsphere of any one of claims 57-59, wherein the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4 MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil, and pyraclostrobin.

61. The patterned polymeric microsphere of any one of claims 57-60, wherein the payload comprises iron, including but not limited to Fe-EDTA, Fe-DTPA, Fe-EDDHA, Fe-EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3.

62. A composition, comprising a plurality of patterned polymeric microspheres of any one of claims 43-61.

63. The composition of claim 62, comprising a plurality of the patterned polymeric microspheres of any one of claims 50-61.

64. The composition of claim 62 or 63, wherein the plurality of the patterned polymeric microspheres comprise microspheres in fluid communication with each other via water-filled microstructures, such as spines.

65. A method of delivering a payload to a plant by administering to the plant a patterned polymeric microsphere produced by the method of any one of claims 15-41 wherein the patterned polymeric microsphere comprises a payload within the one or more spines, or by administering the patterned polymeric microsphere of any one of claims 42-61 or the composition of any one of claims claim 62-64.

66. The method of claim 65, wherein the payload is selected from the group consisting of a therapeutic, a fertilizer, and a plant-growth agent.

67. The method of claim 65 or 66, wherein the payload comprises one or more of ammonium, nitrate, ammonium nitrate and amide nitrogen fertilizers, KCl, K2SO4, K2SO4MgSO4, KNO3, KOH, diammonium phosphate (DAP), monoammonium phosphate (MAP), copper oxysulfate, copper oxide, copper chelate, copper sulfate. zinc sulphate, zinc oxide, zinc carbonate, zinc nitrate, zinc chloride, mancozeb, chlorothalonil, fenbuconazole, tebuconazole, propineb, thiram, metiram, saflufenacil, and pyraclostrobin.

68. The method of claim 65 or 66, wherein the payload comprises iron, including but not limited to Fe-EDTA, Fe-DTPA, Fe-EDDHA, Fe-EDDHMA, Fe-HEDTA, Fe-amino acids, Fe-humic / fulvic acids, Fe-lignosulfonates, vivianites, ferric citrates, FeSO4, Fe2(SO4)3, FeCl3, and Fe(NO3)3.

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