Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
  • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
  • C08J3/075—Macromolecular gels
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
  • C08J3/247—Heating methods
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/28—Treatment by wave energy or particle radiation
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2300/00—Characterised by the use of unspecified polymers
  • C08J2300/14—Water soluble or water swellable polymers, e.g. aqueous gels
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2300/00—Characterised by the use of unspecified polymers
  • C08J2300/16—Biodegradable polymers
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
  • F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
  • F25D2303/00—Details of devices using other cold materials; Details of devices using cold-storage bodies
  • F25D2303/08—Devices using cold storage material, i.e. ice or other freezable liquid
  • F25D2303/085—Compositions of cold storage materials

Definitions

• the present disclosure provides a method for making a crosslinked jelly ice cube (JIC), the method comprising: dissolving a biodegradable polymer in water to produce a homogenous solution; placing the homogenous solution in a mold; chilling the homogenous solution in the mold to form a JIC; and exposing the JIC to a crosslinking agent to produce a crosslinked JIC.
• the chilling occurs between about 20°C to about -25°C. In certain aspects, the chilling occurs between about 0°C to about -50°C.
• exposing the JIC to the crosslinking agent is by immersion, misting, a gaseous atmosphere or including in the homogenous biodegradable polymer solution.
• the surface layer of the JIC comprising the biodegradable polymer is crosslinked by immersion.
• the surface layer of the JIC comprising the biodegradable polymer is crosslinked by misting.
• the JIC comprising the biodegradable polymer is crosslinked by including in the homogenous solution.
• the crosslinking of the biodegradable polymer is spontaneous after contacting to the crosslinking agent.
• the crosslinking of the biodegradable polymer is induced by a member selected from the group consisting of heat, radio waves, infrared, visible light, UV, and X-rays.
• the biodegradable polymer is a plant-based polymer or animal- based polymer.
• the polymer is a protein.
• the polymer is a member selected from the group consisting of collagen, gelatin, soy protein, whey protein, zein protein, elastin, laminin, fibrin, silk fibroin lysozyme, bovine serum albumin, ovalbumin, alginate, starch, cellulose, modified proteins, carbohydrates, and combinations thereof.
• the biodegradable polymer is gelatin. In certain instances, using gelatin from different sources, it may be possible to obtain hydrogels with a variety of distinct and diverse physical, chemical, and biological properties. For example, gel strength and rigidity may change with gelatin from different sources.
• the biodegradable polymer is “water-swellable” or forms a “hydrogel,” which indicates that the polymer takes on and retains water within a network.
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w. In certain aspects, the biodegradable polymer content of the JIC is between about 5% to about 20% w/w.
• the crosslinking agent is a member selected from the group consisting of menadione sodium bisulfate (MSB), glutaraldehyde (GTA), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), photosensitizers, water soluble vitamin, transglutaminase, genepin, sulfonates, malemide, polyethylene glycol dendrimeric systems, dendritic polymers, hyper-branced dendritic polymers, formaldehyde, enzymatic cross-linker, glycation, glyceraldehydes, cyanamide, diimide, diisocyante, dimethyl adipimidate, carbodiimide and N,N’-carbonyldiimidazole (CDI), N,N’-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-
• a physical crosslinking agent can be low temperature such as about 4°C to about -200°C. In certain instances, low temperature physical crosslinking is by chilling the hydrogel to its phase transition temperature to generate a sol-gel. [0019] In certain aspects, crosslinking is photo-induced. [0020] In certain aspects, the photoinduced crosslinking occurs with UV light at 280-600 nm under an inert atmosphere. [0021] In certain aspects, the disclosure provides a jelly ice cube (JIC) made by the scalable methods described herein.
• JIC jelly ice cube
• the present disclosure provides, a method for making a crosslinked jelly ice cube (JIC), the method comprising: dissolving a biodegradable polymer and crosslinking agent, in water to produce a homogenous solution; placing the homogenous solution in a mold; chilling the homogenous solution in the mold to form a JIC; and exposing the JIC to a condition to induce crosslinking reaction.
• the chilling occurs between about 20°C to about -200°C.
• the JIC comprising the biodegradable polymer is crosslinked by chemical crosslinking.
• the crosslinking of the biodegradable polymer is induced by a member selected from the group consisting of heat, radio waves, infrared, visible light, UV, and X-rays.
• the biodegradable polymer is a plant-based polymer or animal- based polymer.
• the polymer is a protein.
• the polymer is a member selected from the group consisting of collagen, gelatin, soy protein, whey protein, zein protein, elastin, laminin, fibrin, silk fibroin lysozyme, bovine serum albumin, ovalbumin, alginate, starch, cellulose, modified proteins, carbohydrates, and combinations thereof.
• the biodegradable polymer is gelatin.
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w, or about 5% to about 20% w/w.
• the crosslinking agent is a member selected from the group consisting of menadione sodium bisulfate (MSB), glutaraldehyde (GTA), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), photosensitizers, water soluble vitamin, transglutaminase, genepin, sulfonates, malemide, polyethylene glycol dendrimeric systems, dendritic polymers, hyper-branced dendritic polymers, formaldehyde, enzymatic cross-linker, glycation, glyceraldehydes, cyanamide, diimide, diisocyante, dimethyl adipimidate, carbodiimide and N,N’-carbonyldiimidazole (CDI), N,N’-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-
• a physical crosslinking agent can be low temperature.
• crosslinking is photo-induced.
• the photoinduced crosslinking occurs with UV-vis light at 280- 600 nm under an inert atmosphere.
• the disclosure provides a jelly ice cube (JIC) made by the method described herein.
• the present disclosure provides a 3-D printing method for making a crosslinked jelly ice cube (JIC), the method comprising: chilling a dissolved biodegradable polymer in water to produce a sol-gel homogenous solution; extruding the sol-gel homogenous solution layer-by-layer in a mold to produce a JIC; and exposing the JIC to a crosslinking agent to produce a crosslinked JIC.
• exposing the JIC to the crosslinking agent is by immersion, misting, a gaseous atmosphere or including in the homogenous solution.
• the surface layer of the JIC comprising the biodegradable polymer is crosslinked by immersion.
• the surface layer of the JIC comprising the biodegradable polymer is crosslinked by misting.
• the JIC comprising the biodegradable polymer is crosslinked by including in the homogenous solution.
• the crosslinking of the biodegradable polymer is spontaneous after contacting to the crosslinking agent.
• the crosslinking of the biodegradable polymer is induced by a member selected from the group consisting of heat, radio waves, infrared, visible light, UV, and X-rays.
• the biodegradable polymer is a plant-based polymer or animal- based polymer.
• the polymer is a protein.
• the polymer is a member selected from the group consisting of collagen, gelatin, soy protein, whey protein, zein protein, elastin, laminin, fibrin, silk fibroin lysozyme, bovine serum albumin, ovalbumin, alginate, starch, cellulose, modified proteins, carbohydrates, and combinations thereof.
• the biodegradable polymer is gelatin.
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w. In certain aspects, the biodegradable polymer content of the JIC is between about 5% to about 20% w/w.
• the crosslinking agent is a member selected from the group consisting of menadione sodium bisulfate (MSB), glutaraldehyde (GTA), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), photosensitizers, water soluble vitamin, transglutaminase, genepin, sulfonates, malemide, polyethylene glycol dendrimeric systems, dendritic polymers, hyper-branced dendritic polymers, formaldehyde, enzymatic cross-linker, glycation, glyceraldehydes, cyanamide, diimide, diisocyante, dimethyl adipimidate, carbodiimide and N,N’-carbonyldiimidazole (CDI), N,N’-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-
• a physical crosslinking agent can be low temperature, (e.g., 0°C or at its phase transition temperature).
• the disclosure provides a jelly ice cube (JIC) made by the scalable methods described herein.
• the present disclosure provides a jelly ice cube (JIC), the JIC comprising: a biodegradable crossed-linked polymeric hydrogel network, wherein the network comprises a plurality of isolated chambers or cell-like cavities, creating a lattice.
• the lattice is substantially honeycombed.
• each chamber or cell-like cavity of the lattice entraps water.
• the shape of the JIC is a member selected from the group consisting of free form, a cube, a pyramid, a sphere, a cylinder, a prism, and cone. As it is possible to 3-D print the JIC, it can take on any free form shape. In certain instances, a customized or tailored design and/or geometry can be generated for cooling various-sized items and commodities.
• the plurality of isolated chambers or cell-like cavities comprises a dense matrix of uniformly sized, durable water-retaining cells within the biopolymer network.
• each isolated chamber or cell-like cavity of at least a portion of the plurality of isolated chambers or cell-like cavities has a pore to imbibe water.
• each pore is about 0.1 nm to about 100 ⁇ m or 0.1 nm to about 20 nm is diameter. In one aspect, smaller pores are made with a chemical cross-linking agent and a larger pore is made with a physical agent.
• the plurality of isolated chambers or cell-like cavities are nanometer-micrometer scaled. [0057] In certain aspects, the plurality of isolated chambers or cell-like cavities are substantially heterogeneously sized.
• the polymer is a member selected from the group consisting of collagen, gelatin, soy protein, whey protein, zein protein, elastin, laminin, fibrin, silk fibroin lysozyme, bovine serum albumin, ovalbumin, alginate, starch, cellulose, modified proteins, [0059]
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w or about 5% to about 20% w/w or about 1% to about 10% w/w.
• Figure 1 shows a process embodiment of the present disclosure (a) immersing solidified hydrogels in solutions containing chemical crosslinking agents that can induce crosslinking reactions within or among polymer (e.g., protein) molecules; (b) immersing solidified hydrogels in solutions containing crosslinking agents which require additional triggers to generate the crosslinks.
• polymer e.g., protein
• the additional triggers include, but are not limited to, photo-irradiation, heat, and X-rays; (c) applying sufficient amount of chemical crosslinking agents on solidified hydrogels to induce spontaneous crosslinking reactions within or among protein molecules on surface of the gels via mist or gaseous environment; and (d) applying mist or gaseous form of crosslinking agents, which requires additional conditions to induce the crosslinking reactions, to surface of solidified hydrogels, then exposing the gels to the adequate conditions, such as but not limited to photo-irradiation, heat, and X-rays, to complete the crosslinking reaction on the surfaces of the gels.
• the adequate conditions such as but not limited to photo-irradiation, heat, and X-rays
• Figure 2 shows a process embodiment of the present disclosure, (a) fabricating hydrogel precursor solutions containing both polymers and chemical crosslinking agents, which can spontaneously induce crosslinking reactions within or among polymers (protein molecules) in controlled reaction rates, and then quickly injecting the solutions to molds in any shapes to form hydrogels. Crosslinking reaction will complete after the shaped hydrogels are made; (b) fabricating hydrogel precursor solutions containing both polymers and chemical crosslinking agents, which requiring additional triggers to induce the crosslinking reactions, and then injecting the solutions to molds in any shapes to form hydrogels. Afterward, exposing the hydrogels to adequate conditions, such as photo-irradiation, heat, and X-rays, to initiate the chemical crosslinking step.
• adequate conditions such as photo-irradiation, heat, and X-rays
• Figure 3 shows the mechanical strengthening effect of immersing gelatin hydrogels (10 ⁇ 10 ⁇ 10 mm) in 5 mg/mL or 10 mg/mL EDC solutions, (a).
• (d) The compressive strength (e) and strain at break (f) of Gel-JICs at AFTC0 and after AFTC1, 5, or 10.
• Figure 4 shows the latent heat of fusion of Gel/EDC[S]-JICs (a), Gel-JICs (b), and Gel/MSB-JICs (c) at AFTC0 and after AFTC1, 5, or 10.
• the ratio of freezable water of Gel/EDC[S]-JICs g), Gel-JICs (h), and Gel/MSB-JICs (i) at AFTC0 and after AFTC1, 5, or 10.
• Figure 5 shows the compressive stress (a) and strain (b) at break of Gel/EDC[E]- JICs constructed by mixing EDC in different concentrations with gelatin in the precursor solution. Images of Gel/EDC[E]-1-JICs and Gel/EDC[E]-2-JICs before AFTC (AFTC0), (c). The compressive stress (d) and strain (e) at break of Gel/EDC[E]-1-JICs at AFTC0 and after AFTC1, 5, or 10. The compressive stress (f) and strain (g) at break of Gel/EDC[E]-2-JICs at AFTC0 and after AFTC1, 5, or 10.
• Figure 6 shows the compressive stress (a) and strain (b) at break of Gel/TA[E]-JICs constructed by mixing TA in different concentrations with 10% gelatin in the precursor solution.
• Figure 7 shows the latent heat of fusion of Gel/TA[E]-1-JICs (a) and Gel/TA[E]-2- JICs (d) at AFTC0 and after AFTC1, 5, or 10.
• Figure 8 shows the compressive stress and strain at break of Gel/AQS[E]-JICs prepared with 10% gelatin and AQS (anthraquinone sulfate) in different concentrations and photo-irradiated under UVA (365 nm) for 30 min, (a).
• the compressive stress and strain at break of Gel/AQS[E]-JICs made with 10% gelatin and 0.01% AQS and photo-irradiated under UVA (365 nm) for various durations, (b).
• Figure 11 shows schematics highlighting the distinct 3D biopolymer network structures: nanometer-scaled enclosed chambers ideal for hydrogel cooling media (a) versus open-cell structures seen in hydrogels less suited for repeated cooling applications (b). Visuals produced using OpenAI’s DALL ⁇ E model.
• Figure 12 shows structural representation of hydrogel polymeric skeletons. (a) schematic of Gel/EDC and (b) Gel/GTA. Crosslinking mechanisms of gelatin using (c) EDC and (d) GTA.
• Figure 13 shows hydrogel fabrication methods. (a) The one-step entirety crosslinking approach, which involves integrating the crosslinking agent directly into the hydrogel precursor solutions.
• FIG. 14 shows (a) CryoEM image of an 8% gelatin hydrogel (Gel [8]) taken on a glow-discharged holey carbon grid with a resolution of 3.5 ⁇ . The image underwent processing via ImageJ, incorporating a bandpass filter spanning 20 – 30 ⁇ . Within the image, black regions represent protein skeletons, whereas white regions signify water present as ice.
• FIG. 15 shows (a) SEM images s featuring the internal structures of the hydrogel sampled prior to AFTC (A0), dehydrated using CPD; yellow scale represents 50 ⁇ m. (b) Effects of electron beam etching. Rectangular-shaped dents and additional morphology features arise from electron beams, especially when observed using 5 kV or 10 kV beams in areas less than 30 ⁇ m x 20 ⁇ m. [0076] Figure 16 shows evaluating the impact of EDC concentration on crosslinking 10% gelatin hydrogels using the mixing method. (a-b) Compressive mechanical properties of Gel/EDC samples obtained through a one-step integrated entirety crosslinking approach.
• Figure 18 shows cross-sectional images of Gel [10] (a), Gel/EDC [10-0.2] (b), Gel/EDC [10-0.5] (c), Gel/MSB [10-F1] (d) and Gel/GTA [10-S] (e) hydrogels before AFTC (A0), and after 1 (A1), 5 (A5), 10 (A10) and 15 (A15) cycles of AFTCs, with white scale bars of 2 mm.
• Figure 19 shows SEM images of CPD-dehydrated samples: Gel [10], Gel/EDC [10- 0.2], Gel/EDC [10-0.5], and Gel/GTA [10-S] hydrogels before AFTC (A0), and after 1 (A1) application freezing-thawing cycle, with white scale bars of 200 ⁇ m and yellow scale bars of 50 ⁇ m.
• Figure 20 shows total water content (a – e), ratio of freezable water (f – j), and latent heat of fusion (k – o) of Gel [10] (a, f and k), Gel/EDC [10-0.2] (b, g and l), Gel/EDC [10-0.5] (c, h and m), Gel/MSB [10-F1] (d, i and n), and Gel/GTA [10-S] (e, j and o) specimens after various AFTCs. Plotted data are expressed as means ⁇ SD of three replicates.
• Figure 21 shows (a) Compressive mechanical properties of EDC-crosslinked 8% gelatin hydrogels using mixing method.
• Figure 22 shows cross-sectional images of Gel [8] (a), Gel/EDC [8-0.5] (b), and Gel/EDC [8-1.0] (c) hydrogels before AFTC (A0), and after 1 (A1), 5 (A5), 10 (A10) and 15 (A15) cycles of AFTCs.
• Figure 23 shows SEM images of CPD-dehydrated samples: Gel [8], Gel/EDC [8- 0.5], and Gel/EDC [8-1.0] hydrogels before AFTC (A0), and after 1 (A1) application freezing-thawing cycle, with white scale bars of 200 ⁇ m and yellow scale bars of 50 ⁇ m.
• Figure 24 shows total water content (a – d), ratio of freezable water (e – h), and latent heat of fusion (i – l) Gel [8] (a, e and h), Gel/EDC [8-0.5] (b, f and j), Gel/EDC [8-1.0] (c, g and k), and Gel/MSB [10-F1] (d, h and l) hydrogel specimens after various AFTCs. Plotted data are expressed as means ⁇ SD of three replicates.
• the present disclosure provides a coolant material or “jelly ice cube” (JIC) and methods of making using a biodegradable polymer such as a protein.
• JIC coolant material or “jelly ice cube”
• the JIC diminishes meltwater while still possessing high cooling efficiency of traditional ice.
• the JICs contain zero-plastic and can prevent meltwater-caused cross-contamination with high affordability, recyclability, sustainability, and biodegradability.
• the biodegradable polymer is a member selected from the group consisting of collagen, gelatin, soy protein, whey protein, zein protein, elastin, laminin, fibrin, silk fibroin lysozyme, bovine serum albumin, ovalbumin, alginate, starch, cellulose, modified proteins, carbohydrates, and combinations thereof.
• the biodegradable polymer is gelatin.
• Gelatin is a product of partial hydrolysis of collagen. During gelatin preparation, collagen is pre-treated under acidic conditions to produce type A gelatin or alkaline conditions to produce type B gelatin. More carboxylic groups are present in type A gelatin than type B gelatin.
• the sources of gelatin include bovine skin, bovine hides, cattle, and pork bones, as well as fish and poultry gelatins.
• Gelatin is a heterogeneous mixture of water-soluble proteins of high average molecular masses, present in collagen. The proteins are extracted by boiling skin, tendons, ligaments, bones, etc. in water.
• the average molecular mass (Da) of the gelatin is about 20,000 to about 100,000 such as 20,000 ⁇ 25,000 (Da), 40,000 ⁇ 50,000 (Da) or 50,000 ⁇ 100,000 (Da).
• the Bloom number is proportional to MW such as 50-125 (low Bloom) 175-225 (medium Bloom) and 225-325 (high Bloom).
• Proteins suitable for use in the present disclosure include, for example, natural proteins such as collagen, gelatin, elastin, laminin, fibrin, silk fibroin and globular proteins such as lysozyme, BSA and ovoalbumin and combinations of the foregoing.
• the biodegradable polymer such as a protein (e.g., gelatin) is at a concentration of about 1% to about 60% w/w such as about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and/or 60% w/w.
• the biodegradable polymer is made from about 1% to about 30% w/w or about 1% to about 20% w/w.
• the biodegradable polymer content is about 5% to about 16% such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16% w/w.
• the biodegradable polymer such as a protein (e.g., gelatin) is at a concentration of about 60% to about 90% w/w.
• the biodegradable polymer, biopolymer or protein is crosslinked.
• the biopolymer is a protein that forms a protein hydrogel matrix. The biopolymer matrix can be strengthened to reduce the destructive impact of temperature variations and ice crystal formations, by crosslinking the polymer matrix or polymer hydrogel matrix.
• the degree of crosslinking is well-controlled to generate stable hydrogels or cryogels.
• the present methods for forming a JIC includes crosslinking of all or a portion of the biopolymer.
• crosslinking can occur by a chemical agent, radiation crosslinking, physical crosslinking, photo induced cross-linking or combinations thereof.
• the amount of cross-linking of the biopolymer is about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and/or 100%, based on the total amount of biopolymer. II.
• Crosslinking Various chemical cross-linkers are suitable for the present invention.
• the cross- linkers can be homobifunctional having two reactive ends that are identical or heterobifunctional having two different reactive ends.
• Cross-linkers include a chemical cross- linking agent, which is a member selected from the group consisting of menadione sodium bisulfate (MSB), glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), photosensitizers, water soluble vitamin, transglutaminase, genepin, sulfonates, malemide, polyethylene glycol dendrimeric systems, dendritic polymers, hyper-branced dendritic polymers, formaldehyde, enzymatic cross-linker, glycation, glyceraldehydes, cyanamide, diimide, diisocyante, dimethyl adipimidate, carbodiimide and N
• a physical crosslinking agent can be low temperature.
• Other cross-linkers include, transglutaminase, genepin, papain, bromelain, ficin, laccase, peroxidase, sulfonates, malemide, polyethylene glycol dendrimeric systems, dendritic polymers, hyper-branced dendritic polymers, formaldehyde, enzymatic cross-linker, glycation, glyceraldehydes, cyanamide, diimide, diisocyante, dimethyl adipimidate, carbodiimide and epoxy.
• Examples of radiation cross-linking includes exposing the hydrogel to at least one of visible light radiation, infrared radiation, ultraviolet radiation, electron beam radiation, gamma radiation, or x-ray radiation.
• An example of physical crosslinking is exposing the hydrogel article to freezing and thawing.
• Crosslinking may be carried out before and/or after forming the hydrogel structure, after shaping the hydrogel into a desired shape, after in situ formation, or at any other suitable point during processing.
• Crosslinking of proteins or macromolecules in hydrogels achieves both desired mechanical properties and maximum amount of freezable water in the system.
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w. In certain aspects, the biodegradable polymer content of the JIC is between about 1% to about 20% w/w, or about 1% to about 10% w/w.
• the amount of crosslinker is at a concentration of about 0.1% to about 15% w/w such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% w/w.
• the amount of polymer to crosslinker is about 100:1, 90:1; 80:1, 70:1, 60:1, 50:1, 40:1, 30:1; 20:1; 15:1, 10:1, 5:15, 4:1, 3:1, 2:1, or 1:1.
• the longer amount of time the solidified hydrogel is in contact with the crosslinker the stronger the mechanical strength of the JIC.
• a 10 mm x 10 mm x 10 mm cube of the present disclosure has a compressive strength of about 5 kPa to about 1 MPa, such as about 5 kPa, 25 kPa, 50 kPa, 75 kPa, 100 kPa, 125 kPa, 150 kPa, 175 kPa, 200 kPa, 225 kPa, 250 kPa, 275 kPa, 300 kPa, 325 kPa, 350 kPa, 375 kPa, 400 kPa, 425 kPa, 450 kPa, 475 kPa, 500 kPa, 525 kPa, 550 kPa, 575 kPa, 600 kPa, 625 kPa, 650 kPa, 675 kPa, 700 kPa, 725 kPa, 750 kPa, 7
• the compressive strength is about 10 kPa to about 25 kPa, such as about 10 kPa to 20 kPa.
• the physically crosslinking is performed by one or more freeze- thaw cycles.
• the crosslinking is performed while the biodegradable polymer is in a frozen state or a thawed state.
• the chemical crosslinkers into the hydrogel and induce the chemical crosslinking reaction: For example, 1) establishing a physical-crosslinked hydrogel first (by chilling or freezing the solution), then achieving surface crosslinking of JICs by applying and inducing the chemical crosslinking reaction only on the surface of JICs; 2) establishing hydrogel network structures with uniform physical and chemical crosslinking in the entirety of JICs by mixing the chemical crosslinker in the precursor solutions of JICs.
• the methods disclosed herein can be used to make kilogram quantities of JICs.
• the biodegradable polymer is crosslinked in multiple steps.
• the first goal is a physically crosslinking (by chilling the hydrogel to its phase transition temperature to generate a sol-gel) and the second goal is chemically or photochemically inducing covalent crosslinking.
• the photo-induced cross-linking agent MSB on top of physical cross-linking, introduces robust mechanical properties to a JICs that act against the temperature variations and phase changes.
• III. Surface Crosslinking [0110] Various methods achieve surface crosslinking of JICs. For example, with reference to Figure 1a, a solidified or sol-gel hydrogel 101 can be immersed in a solution containing a chemical crosslinking agent 110 that can induce crosslinking reactions within or among protein molecules 112.
• Figure 1b shows solidified hydrogels 120 being immersed in solutions containing crosslinking agents 125, which require additional triggers (e.g., heat or photoinduced) to generate the crosslinks 129.
• the triggers include, but not limited to, photo- irradiation, heat, and X-rays.
• Figure 1c shows applying or exposing sufficient amount of chemical crosslinking agents 135 on solidified hydrogels 132 to induce spontaneous crosslinking reactions within or among protein molecules on surface of the gels via mist or gaseous environment and generating a crosslinked structure 138.
• Figure 1d shows providing a solidified hydrogel 152 and applying mist or gaseous form of crosslinking agents 153, which requires additional conditions to induce the crosslinking reactions 155, to surface of solidified hydrogels, then exposing the gels to the adequate conditions, such as, but not limited to, photo-irradiation, or heat, or X-rays, to complete the crosslinking reaction 159 on the surfaces of the gels.
• the surface crosslinked hydrogels are strong and have excellent performance for repeated cooling uses.
• two stock solutions, 30% gelatin and 1% EDC were prepared in water.
• the 10% gelatin solution was prepared by diluting gelatin stock solutions at 50 °C, pipetted into the 10 mm ⁇ 10 mm ⁇ 10 mm cubic molds, and chilled at 4 °C overnight for gelation (e.g. a sol-gel or solidified hydrogel). Once solidified, gelatin hydrogels were immersed in diluted EDC solutions (e.g., 5 mg/mL or 10 mg/mL) for various durations to achieve the chemically crosslinked surface layer. The prepared samples were evaluated directly (AFTC0) or after 1, 5, or 10 application freeze-thaw cycles (AFTC1, AFTC5, or AFTC10).
• FIG. 13b shows a schematic of a surface-crosslinking method, where solidified gelatin hydrogels are immersed in diluted solutions of chemical crosslinking agents.
• IV. Uniform Hydrogel Crosslinking [0116]
• hydrogels can also be chemically crosslinked uniformly as an entirety.
• the uniform crosslinking reaction can be achieved by incorporating crosslinking agents into polymer (e.g., protein) solutions.
• the solutions can be injected into any shaped containers and chilled below their gelling point to solidify.
• Figure 2a shows fabricating hydrogel precursor solutions containing polymers and chemical crosslinking agent(s) 202, which can spontaneously induce crosslinking reactions within or among protein molecules in controlled reaction rates, reducing the temperature 207 and then quickly injecting the solutions into molds 210 in any shapes to form hydrogels. Crosslinking reaction will complete 212 after the shaped hydrogels are made.
• Figure 2b shows fabricating hydrogel precursor solution containing polymer and chemical crosslinking agent(s) 220, wherein the crosslinking agents require additional triggers to induce the crosslinking reactions. The combined solution is injected into to molds 230 in any shapes to form hydrogels.
• both chemical and photo-induced chemical crosslinking reactions can occur after formation of shaped hydrogels, which determines the polymer network structures in the gel.
• the chemical crosslinking reactions occur at the sites where the reactive groups on polymer (e.g. gelatin) and the crosslinking agents contact one another.
• Figure 13a shows a schematic of a one-step entirety crosslinking approach, which involves integrating the crosslinking agent directly into the hydrogel precursor solutions.
• V. Hybrid Crosslinking Technique [0119] In certain instances, a hybrid crosslinking technique can be used.
• a hybrid crosslinking technique utilizes rapid-freezing-slow-thawing to induce physical crosslinks, and then complemented by for example, MSB-induced photo-crosslinks.
• a physical and chemical crosslinking approach was taken wherein a 1% MSB solution in the precursor solution was used.
• First the JIC was physically crosslinked by 1 fabrication freeze-thaw cycle (F1), then photo-irradiated under UVA for 10 min. Detailed steps shown in Figure 13c.
• F1 fabrication freeze-thaw cycle
• Hydrogel precursor solutions can be either extruded layer-by-layer and crosslinked by photo-induced crosslinking methods post the extrusion of each layer, or directly crosslinked by chemical crosslinking agents. Via 3D printing, JICs can be crosslinked either as entirety or generate crosslinks on the surface. [0121] During extrusion printing, the hydrogel material should have a suitable viscosity, extrudability and be easily extruded out of a nozzle. After printing, the hydrogel maintains its shape (i.e. post-printing stability) and provide adequate mechanical structural support. VII.
• the disclosure provides a jelly ice cube (JIC), the JIC comprising: a biodegradable crossed-linked polymeric hydrogel network, wherein the network comprises a plurality of isolated chambers or cell-like cavities, creating a lattice.
• the lattice is substantially honeycombed.
• Each cell-like cavity of the substantially honeycombed structure can be any shape such as a circular or a round chamber, a hexagonal, a square, or tubular.
• each chamber or cell-like cavity of the lattice entraps water.
• the shape of the JIC is a member selected from the group consisting of a free form, a cube, a pyramid, a sphere, a cylinder, a prism, and cone.
• the plurality of isolated chambers or cell-like cavities comprises a dense matrix of uniformly sized, durable water-retaining cells within the biopolymer (e.g., protein) network.
• each isolated chamber or cell-like cavity of at least a portion of the plurality of isolated chambers or cell-like cavities has a pore to imbibe water.
• each pore is about 0.1 nm to about 100 ⁇ m in diameter.
• each pore is between about 0.1 nm to about 100 nm or about 0.1 nm to about 20 nm in diameter.
• the plurality of isolated chambers or cell-like cavities are nanometer- or micrometer-scaled.
• the plurality of isolated chambers or cell-like cavities are substantially heterogeneously sized.
• the biodegradable polymer content of the JIC is between about 1% to about 60% w/w.
• the biodegradable polymer content of the JIC is between about 1% to about 10% w/w, or between about 5% to about 20% w/w.
• the idealized structure of the hydrogel-based cooling media or “Jelly Ice Cube” (JIC) as imaged is shown in FIG.11a.
• the comprehensive schematic representation of one aspect the JIC shows a reusable hydrogel-based, resilient hydrogel network replete with an extensive 3D network of nano/micro-meter-scaled isolated chambers. In certain instances, this structure, created from hydrophilic biopolymers, entraps significant volumes of freezable water within these chambers or cavities.
• the 3D structure is highly porous and when crosslinked by using crosslinking agents, can hold >80% wt of water inside with less than 20 % wt of gelatin in the closed cell network structures.
• the weight of water is between about 80% to about 99%, such as about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99%.
• the weight of biodegradable polymer e.g., gelatin
• the weight of biodegradable polymer is between about 1% to about 20%, such as about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and/or 20%.
• a closed cell chamber or cell-like cavity in the 3D structure holds water in a small volume that cannot produce large ice crystal when frozen, preventing damages to the network and prolong repeated uses (thawing-freezing).
• the hydrophilic gelatin network allows water molecules to diffuse and penetrate through cells but may not allow them to flow.
• the significance of crosslinking in sculpting the structure is underscored, emphasizing its pivotal role in enhancing the effectiveness and efficiency of the resulting hydrogel.
• the schematic in FIG.11a highlights certain fundamental structural attributes characterizing the hydrogel materials to excel as efficient, reusable cooling mediums.
• the reusable hydrogel-based coolant is a resilient hydrogel network replete with an extensive network of nanometer-scaled isolated chambers or cell-like cavities. This structure, created from hydrophilic biopolymers, entraps significant volumes of freezable water within the chambers. [0138]
• the hydrophilic polymeric lattice embodies several key characteristics.
• the hydrogel maintains an appropriate biopolymer concentration to form closed cells or cavities, which has at least one pore, or only one pore. If the concentration dips, the hydrogel exhibits open cell structures, rendering it fragile (See, figure 11b). Such a structure would struggle to retain significant water content, particularly after freeze-thaw cycles.
• Figure 11b does not contain a plurality of cell-like cavities of chambers. Conversely, an excessively high biopolymer concentration compromises the total water content in the hydrogel, which also limits the heat-absorbing ability. Therefore, in certain aspects, a balance in biopolymer concentration is desired.
• biodegradable polymer content of the JIC is between about 1% to about 60% w/w.
• resilience against repeated freeze-thaw cycles requires that the hydrogel is well crosslinked. This crosslinked network is robust enough to resist damaging effects of ice crystal formation during freezing. However, if the gel is overly crosslinked, it can diminish its hydrophilic nature, thereby reducing its ability to retain freezable water during thawing.
• EDC crosslinking agent
• GTA crosslinking gelatin hydrogels
• EDC interacts with the gelatin molecule’s carboxyl groups, particularly those in the amino acid side chains (e.g., aspartic and glutamic acid) or C-termini, resulting in the formation of an o-acylisourea intermediate, as depicted in Figure 12c.
• This intermediate is inherently unstable in aqueous solutions and easily undergoes hydrolysis.
• this intermediate meets a primary amine group (e.g., lysine, arginine) before hydrolyzing, it forms an amide bond, achieving crosslinking. However, if it hydrolyzes, it becomes non-reactive to primary amines, limiting crosslinking efficiency.
• a primary amine group e.g., lysine, arginine
• gelatin network can only be “stappled” where a carboxylic group is extremely close to an amine group.
• An advantage of EDC is its potential of forming homogeneous crosslinked structure of the gelatin network, as shown in Figure 12a. With EDC, the gelatin network can be uniformly reinforced, retaining its inherent 3D structure and maintaining abundant hydrophilic groups for freezable water retention.
• GTA reacts swiftly with gelatin’s amine groups, as highlighted in Figure 12d.
• GTA crosslinks through the formation of Schiff’s base linkages with the gelatin’s amino groups.
• GTA can introduce a slight spatial distortion in the polymer network. Coupled with the rapid crosslinking speed of GTA, during mixing, gelatin molecules can be fixed quickly upon encountering GTA, leading to potential clustering. Therefore, the resultant hydrogel might exhibit variable crosslinking density, as visualized in Figure 12b, with bundled gelatin molecules causing concentration disparities throughout the mixture.
• As-fabricated JICs can be subjected to a variety of dehydration methods, including lyophilization, critical point drying (CPD), oven drying, or room-temperature drying, to enhance their preservation and facilitate efficient shipping.
• CPD critical point drying
• the material Upon dehydration, the material achieves a lightweight and stable state under ambient conditions. Rehydration of the dehydrated material can be accomplished by immersing the dry hydrogel form in water, either at ambient conditions or chilled temperatures (0 – 20°C), until the original water content is fully restored.
• the freezing temperature of the hydrogel is not particularly limited, so long as it is suitable to freeze or solidify the biopolymer. For example, temperatures of 0° C or lower may be suitably employed.
• a temperature of 0 to -20° C is employed to freeze the hydrogel. In one aspect, a temperature of -20° C is used. [0145] Once thawed, it is better to handle JICs with a minimum exposure time of temperatures above 0 °C. After each use, freezing temperature, i.e., -20 °C, is suitable for storage to retain water content in JICs. [0146]
• the time of freezing i.e., time of storage at freezing temperature, is not particularly limited and may range from a few hours or less to 24 hours or more. This range includes all values and subranges therebetween, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hours.
• JICs can be effectively rehydrated, cleaned or sanitized with brief water or diluted bleach rinse.
• the application of JICs can reduce water consumption in the food supply chain and food waste by controlling microbial contaminations.
• the present disclosure provides a method for cooling an object using a jelly ice cube (JIC), the method comprising: providing a chilled or frozen jelly ice cube (JIC); and contacting the object with the JIC.
• the JIC is chilled before contacting the object.
• the cooling material or JIC is suitable for maintaining temperature-sensitive items, e.g., medicinal, or unstable compounds, cell or tissue samples, or other biologically or chemically active substances, at reduced temperatures.
• the JIC possesses biocidal functionality.
• a photosensitizer can be used to provide light-induced antimicrobial functions under store lighting conditions.
• Biocidal reactive oxygen species (ROS) including hydroxyl radicals, hydrogen peroxide, and singlet oxygen, produced by the JICs containing different amounts of photosensitizer (e.g., MSB) can be used to eliminate microorganisms.
• the idealized structure of the reusable hydrogel-based cooling media or “Jelly Ice Cube” can be formulated either with or without the use of a food-grade crosslinking agent, such as transglutaminase, papain, bromelain, ficin, laccase, peroxidase, genipin, yielding an edible cooling medium.
• a food-grade crosslinking agent such as transglutaminase, papain, bromelain, ficin, laccase, peroxidase, genipin
• the resulting JIC is edible.
• a gelatin solution with concentrations ranging from 5% to 20%, is combined with transglutaminase, with concentrations ranging from 0 – 100 U/g, according to the procedure depicted in Figure 13A.
• this precursor solution undergo an incubation at temperatures between 20°C and 70°C for a duration ranging from 1 minute to 24 hours. This incubation can occur either before or after the solution is dispensed into molds.
• the enzyme can be inactivated by a temperature above 70°C.
• the solution is cooled to a temperature range of 0°C to 20°C to facilitate a sol-gel transition. It is expected that the resulting hydrogel will exhibit an internal configuration akin to that illustrated in Figure 11a.
• the reusable hydrogel- based coolant is a resilient hydrogel network replete with an extensive network of nanometer- scaled isolated chambers or cell-like cavities.
• the cooling material or JIC is suitable for maintaining temperature-sensitive items, e.g., drinks, food, medicinal, or unstable compounds, cell or tissue samples, or other biologically or chemically active substances, at reduced temperatures.
• temperature-sensitive items e.g., drinks, food, medicinal, or unstable compounds, cell or tissue samples, or other biologically or chemically active substances, at reduced temperatures.
• chemically crosslinking the surface of the JICs effectively improves their mechanical stability and reusability.
• the thickness of the surface crosslinked layer of JICs is related to the amount of crosslinking agent adsorbed or absorbed, as well as the number of crosslinkers effectively reacted with the polymer (e.g., protein) chains.
• JICs show more robust performances.
• the disclosure provides at least four (4) subcategories of methods to achieve surface crosslinking of JICs.
• 1. immersing solidified JICs in solutions with chemical crosslinking agents which induces spontaneous crosslinking reactions with or among protein molecules; 2.
• 1-B first immersing solidified JICs in solutions containing crosslinking agents which requires additional conditions to generate the crosslinks, then exposing the JICs to the adequate conditions, which includes but not limited to photo-irradiation, heat, and X- rays; 3.
• Example 1 illustrates the surface modification method 1-A: Gel/EDC[S]-JICs [0156] Two stock solutions, 30% Gelatin and 1% EDC, were prepared in water.10% gelatin solution was prepared by diluting gelatin stock solutions at 50 °C, pipetted into the 10 mm ⁇ 10 mm ⁇ 10 mm cubic molds, and chilled at 4 °C overnight for gelation. Once solidified, gelatin hydrogels were immersed in diluted EDC solutions (e.g., 5 mg/mL or 10 mg/mL) for various durations to achieve the chemically crosslinked surface layer. The prepared samples were evaluated directly (AFTC0) or after 1, 5, or 10 application freeze- thaw cycles (AFTC1, AFTC5, or AFTC10).
• AFTC0 diluted EDC solutions
• Each AFTC consists of 18 hours of freezing at – 20 °C and 6 hours of thawing at 21 °C.4 °C for sample preservations.
• the mechanical strengthening effect induced by the surface crosslinked hydrogels by using EDC is characterized and shown in Figure 3 (a).
• the compressive strength of the Gel-EDC[S]-JICs increased rapidly in the initial period of immersion before reaching equilibrium, as shown in Figure 3 (a).
• the higher concentration of EDC showed a higher speed of enhancing strength due to quick chemical crosslink reactions. Before equilibrium, a longer immersion time resulted in a better mechanical strengthening effect.
• both chemical and photo-induced chemical crosslinking reactions occur after gel formation, which also will determine the polymer network structures in the gel.
• the chemical crosslinking reactions occur at the sites where the reactive groups on gelatin and the agents colloid together.
• Example 2 illustrates the Uniform Crosslinking Method 2-A-1.
• Gel/EDC[E]-JICs Two stock solutions, 30% Gelatin and 10 mg/mL EDC, were prepared in water.
• the precursor solution Gel/EDC[E]-JICs was prepared by diluting and mixing the two stock solutions at 50 °C. Different concentration of EDC was added to the protein solution while the concentration of gelatin remained at 10% for all samples.
• the precursor solutions were pipetted into the 10 mm ⁇ 10 mm ⁇ 10 mm cubic molds and chilled at 4 °C overnight for gelation, and the achieved samples were referred to as Gel/EDC[E]-JICs.
• the prepared samples were evaluated directly (AFTC0) or after 1, 5, or 10 application freeze-thaw cycles (AFTC1, AFTC5, or AFTC10).
• FIG. 5 (a-b) shows the mechanical strengthening effect of mixing EDC into the precursor solution of hydrogels. It can be observed that with the increase of EDC in the precursor solution, the resulting Gel/EDC[E]-JICs were stronger with both higher compressive strength and compressive strain at break. All Gel/EDC[E]-JICs prepared before AFTC showed the properties reached beyond the mechanical criteria of JICs.
• JICs fabricated with two representative concentrations of EDC were selected for the test of reusability of JICs, 0.2% EDC out of gelatin content (0.02 wt.% of the total JICs) and 0.5% out of gelatin content (0.05 wt.% of the total JICs), which are labeled as Gel/EDC[E]-1-JICs and Gel/EDC[E]-2-JICs, respectively.
• the appearances and stability of the mechanical performances are shown in Figure 5 (b-g).
• Example 3 illustrates Crosslinking Method Example 2-A-2.
• Gel/TA[E]-JICs [0161] Two stock solutions, 30% Gelatin and 100 mg/mL tannic acid (TA) were prepared in water, respectively. The precursor solution Gel/EDC[E]-JICs was prepared by diluting or mixing the two stock solutions at 50 °C.
• TA Different concentration of TA was added to the protein solution, and the final concentration of gelatin in the precursor solutions was diluted to either 10% or 8%.
• the pH condition of each precursor solution was adjusted to pH 7 or 8 by 1M sodium hydroxide solutions.
• the adjusted precursor solutions were pipetted into the 10 mm ⁇ 10 mm ⁇ 10 mm cubic molds and chilled at 4 °C overnight for gelation.
• the prepared samples were evaluated directly (AFTC0) or after 1, 5, or 10 application freeze- thaw cycles (AFTC1, AFTC5, or AFTC10).
• Each AFTC consists of 18 hours of freezing at – 20 °C and 6 hours of thawing at 21 °C.4 °C for sample preservations.
• Figure 6 (a-b) shows the mechanical strengthening of Gel/TA[E]-JICs prepared by the precursor solutions containing a homogeneous mixture of 10% gelatin and different concentrations of TA. It can be observed that with the increase of TA in the precursor solution, the resulting Gel/TA[E]-JICs were becoming stronger with both higher compressive strength and compressive strain at break. A similar tendency was also observed with Gel/TA[E]-JICs containing 8% gelatin, as shown in Figure 6 (c-d).
• the precursor solution Gel/AQS[E]-JICs was prepared by diluting gelatin stock solutions at 50 °C to a final concentration of 10%. The prepared precursor solutions were pipetted into the 10 mm ⁇ 10 mm ⁇ 10 mm cubic molds and chilled at 4 °C overnight for gelation. Once solidified, the hydrogels were exposed to UV irradiation under various conditions (UVA, 365 nm, in the air or N 2 ; UVB, 312 nm, in the air or N 2 ) for different durations, resulting in the entirely photo-crosslinked Gel/AQS[E]-JICs.
• FIG. 8 shows the mechanical strengthening effect of Gel/AQS[E]-JICs prepared by the precursor solutions containing 10% gelatin and different concentrations of AQS after photoirradiation under UVA (365 nm) for 10 min.
• FIG. 8 (b) illustrates that in a hydrogel containing 10% gelatin and 0.01% AQS, the AQS-induced photo-crosslinking reaction had a relatively fast speed of response and reached equilibrium within 1 min of UVA (365 nm) irradiation.
• JICs fabricated with 10% gelatin and 0.01% AQS, and photo-irradiated under UVA (365 nm) for 10 min were selected to represent the performance of Gel/AQS[E]-JICs.
• the appearances and stability of mechanical performances are shown in Figure 8 (c-e).
• the average compressive strength was 13.72 kPa and 11.41 kPa, respectively, all of which met beyond the strength criteria of JICs.
• the latent heat of fusion, total water content, and the ratio of freezable water of Gel/AQS[E]-JICs before AFTC (AFTC0) after 1, 5, and 10 AFTCs are shown in Figure 8 (f-h).
• the process of making a JIC can lead to a structural difference in the JIC.
• One process described herein is a surface crosslinking by immersing hydrogels in a solution containing a chemical crosslinking agent, and the crosslinking reaction can be triggered by temperature, time, light, or irradiation.
• a homogeneously crosslinked hydrogel is made by mixing gelatin and a crosslinking agent together in solution and then form a hydrogel solid shape at for example, 4°C.
• Crosslinking reaction can occur by controlling temperature, time, light, or irradiation.
• the entire hydrogel is crosslinked. (See, Figure 10).
• 3D printing technology is used to inject a viscous hydrogel solution containing both protein and crosslinking agent. The crosslinking reaction can be triggered by temperature and photo irradiation.
• JICs can also be fabricated via 3D printing techniques.
• JICs can be either extruded layer-by-layer and crosslinked by photo-induced crosslinking method post to the extrusion of each layer, or directly crosslinked by chemical crosslinking agents. Via 3D printing, JICs can be crosslinked either as entirety or generate crosslinks on the surface.
• Example 5 illustrates evidence of the structure of crosslinked hydrogel cooling media Materials and Methods [0171] Materials.
• Gelatin from porcine skin (gel strength 300, Type A), glutaraldehyde (grade I, 8% aqueous solution) and ethanol (anhydrous, ACS grade) were purchased from Sigma Aldrich (Milwaukee, WI).1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Spectrum Chemical MFG Corp (Gardena, CA). MatTek glass bottom dishes, P50G-1.5-30-F, were purchased from MatTek Corporation (Ashland, MA). Milli- Deionized (Milli-DI) and Milli-Q water was used in the materials fabrication and tests. 3.1.2 Fabrication of a series of examples.
• the entire gelatin was crosslinked in a single step by mixing 10% -3 Gel/EDC gelatin with 0.2% EDC (relative to the total gelatin content) in the [ 10-0.2] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the entire gelatin was crosslinked in a single step by mixing 10% -4 Gel/EDC gelatin with 0.4% EDC (relative to the total gelatin content) in the [ 10-0.4] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the entire gelatin was crosslinked in a single step by mixing 10% -5 Gel/EDC gelatin with 0.5% EDC (relative to the total gelatin content) in the [ 10-0.5] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the entire gelatin was crosslinked in a single step by mixing 10% -6 Gel/EDC gelatin with 0.6% EDC (relative to the total gelatin content) in the [ 10-0.6] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the solidified 10% gelatin hydrogel was surface crosslinked by immersing -7 Gel/EDC in EDC aqueous solutions with concentrations of either 5 [ 10-S] mg/mL or 10 mg/mL. The immersion lasted for 30 minutes for most samples, while AFM specimens were soaked for 18 hours.
• the detailed procedure is depicted in Figure 13b.
• the entire gelatin was crosslinked in a single step by mixing 8% -11 Gel/EDC gelatin with 0.1% EDC (relative to the total gelatin content) in the [ 8-0.1] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the entire gelatin was crosslinked in a single step by mixing 8% -12 Gel/EDC gelatin with 0.2% EDC (relative to the total gelatin content) in the [ 8-0.2] precursor solution at 50-60°C.
• the specific procedure is illustrated in Figure 13a.
• the entire gelatin was crosslinked in a single step by mixing 8% -13 Gel/EDC gelatin with 0.5% EDC (relative to the total gelatin content) in the [ 8-0.5] precursor solution at 50-60°C.
• the fabricated hydrogels were tested against 0 (A0, before application cycles), 1 (A1), 5 (A5), or 10 (A10) cycles of AFTCs.
• Each AFTC consisted of 18 h of freezing at ⁇ 20 °C and 6 h of thawing at 21 °C to evaluate the performance of engineered hydrogels under commonly encountered freeze ⁇ thawing conditions.
• the AFTC conditions in this study were also consistent with the testing conditions for JICs in previous studies, enabling a performance comparison between one-step crosslinked JICs and the earlier JICs.
• Material Characterizations [0173] Native Hydrogel Structures Observations by CryoEM.
• Equation 1 Compressive Mechanical Properties. As described in earlier studies, the static compression test was performed using an Instron 5566 tester (Norwood, MA) with a 5 kN or 10 kN static load cell.
• the latent heat of different frozen hydrogels was measured using a differential scanning calorimeter (DSC-60, Shimadzu Corporation, Pleasanton, CA) using the method described in Zou et al.[4]
• the heat flow (W ⁇ g -1 ) – time (s) curves were recorded in the temperature range of -20°C to 10°C, supported by liquid nitrogen with a 1°C ⁇ min -1 heating rate under 30 mL/min protective nitrogen flow.
• the latent heat of fusion was reported with the integrated heat values of the phase change peak near 0°C normalized by the mass of the specimen in J ⁇ g -1 . Ice-water phase transition heat (334.5 J ⁇ g -1 ) was used as the reference.
• the total water content of fabricated hydrogels was tested using a thermogravimetric analyzer (TGA, SDT-Q600, TA Instrument, New Castle, DE).
• TGA thermogravimetric analyzer
• the total water content (%) and the ratio of freezable water (%) were calculated according to Equation 2 and Equation 3, where ⁇ is the weight of specimen in g where the 1 st derivative of mass changes against time is below 0.01 g ⁇ s -1 , ⁇ ⁇ is the initial weight of specimen under the ambient condition in g, and H is the fusion heat of specimen in J ⁇ g -1 .
• cryoEM imaging could not be achieved for any crosslinked samples.
• the obtained cryoEM images were processed using ImageJ, applying a bandpass filter ranging from 20 – 30 ⁇ and shown in Figure 14 a, where the black areas denote protein skeletons, while the white areas correspond to water (in the form of ice). Extremely homogenous distribution of gelatin and water was observed.
• the interpenetrating gelatin molecules formed a dense water-restraining network with pore (water droplet) size of 2.93 nm ⁇ 2.77 nm.
• CPD facilitated a comprehensive analysis of all prepared samples, including Gel [10], Gel/EDC [10-0.2], Gel/EDC [10-0.5], Gel [8], Gel/EDC [8-0.5], Gel/EDC [8-1.0], and Gel/GTA [10-S], with SEM images depicted in Figure 15 a.
• all CPD-dried virgin gelatin hydrogels (Gel [10] and Gel [8]) and EDC-crosslinked specimens (Gel/EDC [10-0.2], Gel/EDC [10-0.5], Gel/EDC [8-0.5], and Gel/EDC [8-1.0]) exhibited a smooth surface morphology.
• GTA-crosslinked samples exhibited a multifaceted, uneven cross-sectional surface characterized by micrometer-scaled wave-like, interconnected ridges and valleys, aligning with our theoretical assumptions in Figure 12.
• the swift crosslinking reaction mediated by GTA led to a heterogeneous polymer network, inducing pronounced gelatin aggregation.
• Gel/GTA [10-S] possesses a dual-structured nature, comprising both nanometer and micrometer-scaled features.
• Hydrogel properties were analyzed immediately after fabrication (prior to AFTC, A 0) and post various AFTCs (A1, A5, and A10).
• the evaluations covered compressive mechanical properties, water profile, latent heat of fusion, swelling behavior, and in vitro biodegradation, illustrated in Figure 16 through Figure 20.
• Figure 16 illustrates the impact of diverse crosslinking techniques, comparing the efficacy of the one-step scalable method using EDC (examples include Gel/EDC [10-0.1] to Gel/EDC [10-0.6]) against the two-step surface crosslinking with EDC (Gel/EDC [10-S]) or GTA (Gel/GTA [10-S]), and the JICs created via a complex method that combines rapid- freezing-slow-thawing and photo-crosslinking (Gel/MSB [10-F1]). All the analysis shown in Figure 16 focused on the intrinsic properties of hydrogels prior to AFTCs (A0).
• Gel/EDC [10-0.5] and Gel/EDC [10-0.6] exhibit water profiles and thermal absorption capacities akin to Gel/MSB [10-F1]. Additionally, Gel/GTA [10-S] was examined as the most intense crosslinking scenario, achieving the overly high crosslinking degree for 10% gelatin hydrogels. Gel/GTA [10-S] serves as another benchmark, represented by yellow dashed lines in Figure 16 d-f. Consistent with our hypothesis, excessive crosslinking diminishes the material’s heat-absorbing capability. This reduction is attributed to the drastic decrease in the hydrophilicity of the polymer networks, as hydrophilic groups are replaced by non-hydrophilic covalent bonds, as illustrated in Figure 12 b.
• Figure 16 h-g further elucidates the degree of crosslinking of various samples by tracking their swelling behavior in ambient water bath. It’s evident that while the polymer network of Gel/EDC [10-0.2] and Gel/EDC [10-0.5] was reinforced, there was not markedly change in the polymer network’s interaction with water, which contrasts starkly with that of Gel/GTA [10-S]. [0188]
• EDC as a crosslinking agent offered an added advantage in preserving the material’s transparency and colorless nature, as illustrated in Figure 17 a. Additionally, despite being crosslinked, both Gel/EDC [10-0.2] and Gel/EDC [10-0.5] retained their biodegradability, as depicted in Figure 17 b.
• the in vitro enzymatic degradation test further affirms the moderate crosslinking degree achieved through the one-step entirety crosslinking method, in contrast to the extensive crosslinking seen in Gel/GTA [10-S].
• numerous findings demonstrate the effective strengthening and preservation of water profiles using the one-step entirety crosslinking method with EDC at optimal concentrations.
• the performance of the resultant specimens aligns closely with the benchmark Gel/MSB [10-F1] and significantly surpasses that of the excessively crosslinked sample, Gel/GTA [10-S].
• One-step entirety crosslinking method by incorporating EDC in the hydrogel precursor solution stands out as a solution that achieves both efficient and controllable crosslinking.
• Figure 17 (d-m) showcases the mechanical stability performances of hydrogels, both virgin (Gel [10]) and crosslinked using various methods (Gel/EDC [10-0.2], Gel/EDC [10-0.5], Gel/MSB [10-F1], Gel/GTA [10-S]).
• the evaluations were conducted before (A0) and after multiple AFTCs (A1, A5, A10). Irrespective of their crosslinking status, all samples demonstrated a decline in mechanical strength post-AFTCs. This decline was most pronounced after the first AFTC.
• Gel/MSB [10-F1] exhibited remarkable stability as a JIC, consistently maintaining a strength high above the critical threshold of 10 kPa through a minimum of 10 AFTCs with recorded values of 24.0 kPa ⁇ 4.2 kPa after A1, 26.0 kPa ⁇ 1.4 kPa after A5, and 19.8 kPa ⁇ 1.7 kPa after A10. It is desirable for Gel/EDC [10-0.2] and Gel/EDC [10-0.5] to achieve stability on par with or slightly less than that of Gel/MSB [10- F1], but they should still sustain a strength exceeding 10 kPa.
• Gel/MSB [10-F1] exhibited the least structural damage due to ice grain formation, supporting the findings in Figure 17. While Gel/GTA [10-S] demonstrated impressive mechanical strength even after A10 in Figure 17, its internal structure was detrimentally destructed as seen in Figure 18. This suggests that the robust compressive strength of Gel/GTA [10-S] was primarily due to the crosslinked polymer skeleton. However, this crosslinked network of Gel/GTA [10-S] did not offer significant resistance against ice grain formation during freezing and could not counteract the structural changes during thawing.
• Figure 19 presents SEM images of CPD-dehydrated samples, elucidating the micro-scale internal structural changes in the above-mentioned specimens due to the first freeze-thaw cycle (A1).
• the SEM images indicate the structural change of polymer networks supporting the hydrogels.
• Gel [10] both Gel/EDC [10-0.2] and Gel/EDC [10-0.5] effectively preserved the hydrogel’s inherent homogeneous structure, namely the homogenous nanometer-scaled water retaining enclosed cells.
• the internal surfaces of Gel/EDC [10-0.2] and Gel/EDC [10-0.5] exhibited fewer damages than Gel [10], even though some cracks were still evident post-A1.
• the total water content values for Gel/EDC [10- 0.2] and Gel/EDC [10-0.5] were 88.9% and 87.3% at A0, and 87.5% and 88.1% after A10, respectively; the ratio of freezable water content for Gel/EDC [10-0.2] and Gel/EDC [10-0.5] were 81.7% and 89.2% at A0, and 86.8% and 77.7% after A10, respectively.
• phase separation transpires as freezable water crystallizes into ice.
• the expansive force exerted by the forming ice grains leads to the disruption of a portion of the native water-retaining cells within the polymer networks that illustrated in Figure 11. Absent these intrinsic finely structured enclosed water-retaining cells, when the phase-separated ice melts back into water, these water molecules are readily released from the hydrogel structure, culminating in a reduction of the total water content.
• Gel/GTA [10-S] the polymer network is intensely crosslinked, particularly on the exterior due to the surface crosslinking technique employed. This tightly crosslinked outer layer preserves the overall water content, barring the separated water from escaping the hydrogel matrix.
• the one-step entirety crosslinking method was utilized to create samples such as Gel/EDC [10-0.1], Gel/EDC [10- 0.2], Gel/EDC [10-0.4], Gel/EDC [10-0.5], and Gel/EDC [10-0.6].
• the two-step surface crosslinking approach was applied in the fabrication of Gel/EDC [10-S] and Gel/GTA [10-S].
• the multi-step crosslinking method which integrates physical crosslinking (triggered by rapid-freezing-slow-thawing) and chemical crosslinking (induced by MSB- mediated photo-crosslinking reactions), was used to produce Gel/MSB [10-F1].
• the hydrogel cooling media created through the one-step entirety crosslinking method particularly Gel/EDC [10-0.5]
• performance metrics on par with those crafted using the multi- step approach such as Gel/MSB [10-F1].
• the advantage of the one-step crosslinking method lies in its enhanced efficiency, scalability, cost-effectiveness, and sustainability, requiring minimal energy, labor and time investment.
• Gel/EDC [10-0.5] exemplified the significance of engineering resilient, intricately structured polymeric networks with nanometer-scaled enclosed water-retention cells during the manufacturing phase, ensuring a consistent ability to retain freezable water across extended freeze-thaw cycles. Additionally, we presented compelling evidence highlighting the balance needed in the crosslinking reaction.
• the polymeric nano-cells should be sufficiently robust to withstand damage from ice grains during the freezing process. At the same time, the polymer skeleton should retain a high degree of hydrophilicity, allowing for the retention of a significant amount of freezable water within the cells after thawing.
• Gel [8], Gel/EDC [8-0.5], and Gel/EDC [8-1.0] were chosen as representative samples, each reflecting different degrees of crosslinking.
• the swelling behavior and in vitro enzyme degradation tests are depicted in Figure 21 b-c, respectively. Consistent with the varying degrees of crosslinking indicated by their mechanical properties, Gel/EDC [8-1.0] exhibited the highest degree of crosslinking, while Gel [8] had the lowest.
• Figure 21 d and h demonstrates that the initial 8% gelatin hydrogel (Gel [8]) was not robust enough for repeated use as a cooling medium, with a compressive strength of 48.5 kPa ⁇ 5.8 kPa, 10.9 kPa ⁇ 0.2 kPa, 6.7 kPa ⁇ 1.5 kPa and 6.6 kPa ⁇ 1.0 kPa at A0, A1, A5 and A10, respectively.
• the Gel/EDC [8-0.5] and Gel/EDC [8-1.0] hydrogels showcased a marked improvement in both absolute compressive strength and its stability over repeated cycles.
• Gel/EDC [8-0.5] displayed strengths of 141.5 kPa ⁇ 14.7 kPa at A0, 17.2 kPa ⁇ 7.0 kPa at A1, 10.0 kPa ⁇ 1.9 kPa at A5, and 9.0 kPa ⁇ 3.5 kPa at A10, while Gel/EDC [8-1.0] exhibited strengths of 470.9 kPa ⁇ 9.4 kPa, 18.1 kPa ⁇ 4.9 kPa, 16.7 kPa ⁇ 8.7 kPa, and 13.9 kPa ⁇ 2.3 kPa at the same respective stages, as shown in Figure 21 e and f, respectively.
• this nanometer-scale network was somewhat compromised, with the degree of disruption being inversely related on the extent of crosslinking.
• Gel/EDC [8-1.0] exhibited the least disruption, while Gel [8] underwent the most.
• the biopolymer matrix acquired a secondary characteristic: irregular, randomly distributed cracks that are typically above micrometers, induced by ice grains formed under the condition of AFTCs. The frequency of these larger-scale features increased with successive AFTCs.
• the latent heat of fusion values for Gel [8] were 279.0 ⁇ 7.8 J ⁇ g -1 at A0, transitioning to 222.8 ⁇ 2.5 J ⁇ g -1 after A10.
• these values spanned from 267.9 ⁇ 8.6 J ⁇ g -1 at A0 to 245.0 ⁇ 4.1 J ⁇ g -1 after A10
• Gel/EDC [8- 1.0] ranged from 286.2 ⁇ 6.6 J ⁇ g -1 at A0 to 245.0 ⁇ 4.1 J ⁇ g -1 after A10.

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• 2023
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