CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to United States Provisional Patent Application number 63/421 ,313, filed November 1 , 2022, the entire contents of which are hereby incorporated by reference.

FIELD

[0002] The present disclosure relates generally to microgels, and methods of preparing the same

BACKGROUND

[0003] Crosslinked networks produced via free radical polymerizations are used in different applications, including sensing devices, information storage systems, coatings, scaffolds for tissue engineering, and lenses production. Polymer microgels are an example of such crosslinked networks, comprising three-dimensional (3D) network structures of hydrophilic regions which may be formed via physical or chemical crosslinking.

SUMMARY

[0004] In an embodiment of the present disclosure, there is provided:

[0005] 1. A hybrid microgel, comprising: a thermoresponsive polymer, and a polysaccharide comprising amorphous domains, the thermoresponsive polymer being grafted to the polysaccharide.

[0006] 2. The microgel of embodiment 1 , wherein the thermoresponsive polymer comprises an acrylamide backbone.

[0007] 3. The microgel of embodiment 1 or 2, wherein the thermoresponsive polymer comprises Poly(/V-isopropylacrylamide).

[0008] 4. The microgel of any one of embodiments 1 to 3, wherein the thermoresponsive polymer is present in an amount between about 75 wt% to about 95wt%, or about 80wt% to about 90 wt%.

[0009] 5. The microgel of any one of embodiments 1 to 4, wherein the polysaccharide comprises a crystallinity index (Crl) between about 20% to about 85%, or about 30% to about 80%, or about 30% to about 70%, or about 30% to about 60%, or about 30.6% to about l , or about 30.6% to about 57.1%. [0010] The microgel of any one of embodiments 1 to 5, wherein the polysaccharide comprises a beta-glucan.

[0011] 7. The microgel of any one of embodiments 1 to 6, wherein the polysaccharide comprises cellulose.

[0012] 8. The microgel of any one of embodiments 1 to 7, wherein the polysaccharide is present in an amount between about 5 wt% to about 25 wt%, or about 10 wt% to about 20 wt%.

[0013] 9. The microgel of any one of embodiments 1 to 8, wherein the microgel is a lower critical solution temperature (LCST) microgel.

[0014] 10. The microgel of any one of embodiments 1 to 9, wherein the LCST is in a range between about 30 to about 40°C; or in a range between about 32 to about 37°C.

[0015] 11. The microgel of any one of embodiments 1 to 10, wherein the microgel comprises porosity, the pores comprising an average pore size between about 5 to about 15 pm, or about 7 to about 12 pm.

[0016] 12. The microgel of any one of embodiments 1 to 11 , wherein the microgel comprises a storage modulus of about 0.01 to about 0.2 MPa, or about 0.05 to about 0.15 MPa, or about 0.06 MPa to 0.13 MPa.

[0017] 13. The microgel of any one of embodiments 1 to 12, wherein the microgel comprises an equilibrium swelling at 20 °C of about 7.5 to about 12 times its dry weight; or about 9.4 times its dry weight.

[0018] 14. The microgel of any one of embodiments 1 to 13, wherein the microgel comprises a crystallinity index (Crl) between about 30% to about 80%, or about 35% to about 75%, or about 40% to about 70%.

[0019] 15. The microgel of any one of embodiments 1 to 14, wherein the microgel comprises a volume phase transition temperature between about 30 °C to about 45°C, or about 35 °C to about 40°C.

[0020] In another embodiment of the present disclosure, there is provided:

[0021] 16. A method of synthesizing a hybrid microgel, the method comprising: providing a first mixture, the first mixture comprising an amorphized polysaccharide; providing a second mixture, the second mixture comprising a thermoresponsive monomer; mixing together the first mixture and the second mixture; reacting together the first mixture and the second mixture via free-radical polymerization; and forming the hybrid microgel. [0022] 17. The method of embodiment 16, wherein the first mixture further comprises a first initiator.

[0023] 18. The method of embodiment 16 or 17, wherein the first initiator comprises organic peroxides, azo compounds, metal alkyls, persulfates, or a combination thereof.

[0024] 19. The method of any one of embodiments 16 to 18, wherein the first initiator comprises benzoyl peroxide, peroxyacetic acid, ammonium persulfate (APS), azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), potassium persulfate (KPS), or a combination thereof.

[0025] 20. The method of any one of embodiments 16 to 19, wherein the second mixture further comprises a crosslinking agent.

[0026] 21. The method of any one of embodiments 16 to 20, wherein the crosslinking agent comprises diacrylates, bisacrylamides, dialkene-substituted aryls.

[0027] 22. The method of any one of embodiments 16 to 21 , wherein the crosslinking agent comprises an alkylene glycol diacrylate (such as ethylene glycol diacrylate), 1 ,6-Hexanediol diacrylate (HDDA), allyl methacrylate (AMA), an alkylene bisacrylamides (such as N,N-Methylenebisacrylamide), divinylbenzene, or a combination thereof.

[0028] 23. The method of any one of embodiments 16 to 22, wherein the first mixture and/or second mixture further comprises a solvent.

[0029] 24. The method of any one of embodiments 16 to 23, wherein the solvent comprises a volatile organic solvent, a bio-based solvent, or a combination thereof. [0030] 25. The method of any one of embodiments 16 to 24, wherein the solvent comprises a cyclic ether solvent, an aromatic solvent, 2-methylTHF, or a combination thereof.

[0031] 26. The method of any one of embodiments 16 to 25, wherein reacting together the first mixture and second mixture further comprises adding a second initiator.

[0032] 27. The method of embodiment 16 or 26 wherein the second initiator comprises organic peroxides, azo compounds, metal alkyls, persulfates, or a combination thereof..

[0033] 28. The method of any one of embodiments 16 to 27, wherein the second initiator comprises benzoyl peroxide, peroxyacetic acid, ammonium persulfate (APS), azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), potassium persulfate (KPS), or a combination thereof. [0034] 29. The method of any one of embodiments 16 to 28, wherein reacting together the first mixture and second mixture further comprises heating the reaction.

[0035] 30. The method of any one of embodiments 16 to 29, wherein heating the reaction comprises maintaining the reaction at a temperature between about 70 to about 80°C, or about 76 to 78°C, or about 77°C.

[0036] 31. The method of any one of embodiments 16 to 30, further comprising providing the amorphized polysaccharide, wherein providing the amorphized polysaccharide comprises: drying a polysaccharide; dissolving the polysaccharide in an ionic liquid under sufficiently anhydrous conditions and forming the amorphized polysaccharide; and isolating the amorphized polysaccharide from the ionic liquid.

[0037] 32. The method of any one of embodiments 16 to 31 , further comprising homogenizing the first mixture.

[0038] 33. The method of any one of embodiments 16 to 32, further comprising sonicating the first mixture.

[0039] 34. The method of any one of embodiments 16 to 33, wherein the ionic liquid comprises imidazolium chlorides, imidazolium acetates, or a combination thereof.

[0040] 35. The method of any one of embodiments 16 to 34, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride (B Ml MCI), 1-ethyl- methylimidazolium chloride (EMIMCI), 1-allyl-3-methylimidazolium chloride (AMIMCI), 1- butyl-3-methylimidazolium acetate (BMIMAc), 1-ethyl-3-methylimidazolium acetate (EMIMAc), or a combination thereof.

[0041] In another embodiment of the present disclosure, there is provided:

[0042] 36. A composition useful for tissue engineering, the composition comprising: the hybrid microgel of any one of embodiments 1 to 15; and at least one cell comprising a progenitor cell, stem cell, or a combination thereof.

[0043] 37. The composition of embodiment 36, wherein the composition is useful for cartilage tissue engineering, bone tissue engineering, vascular tissue engineering, pancreas tissue engineering, cardiac tissue engineering, or a combination thereof.

[0044] In another embodiment of the present disclosure, there is provided:

[0045] 38. Use ofthe hybrid microgel of any one of embodiments 1 to 15, orthe hydrogel made by the method of any one of embodiment 16 to 35 for biomedical applications, water treatment applications, waste removal applications, enhanced oil recovery applications, or a combination.

[0046] 39. The use of embodiment 38 wherein biomedical applications, water treatment applications, waste removal applications, enhanced oil recovery applications, or a combination thereof comprises tissue engineering, such as cartilage, bone, or muscle tissue engineering; 3D printed organ transplantation; controlled drug delivery vehicles, such as through encapsulation, physical adsorption or absorption, or chemical attachment; seawater desalination, and/or cargo delivery vehicle, such as for delivery of catalyst in a heavy oil reservoir.

[0047] In another embodiment of the present disclosure, there is provided:

[0048] 40. A method of synthesizing a thermoresponsive polymer, the method comprising: providing a first mixture comprising a thermoresponsive monomer and a biobased solvent; reacting the first mixture via free-radical polymerization; and forming the thermoresponsive polymer.

[0049] 41. The method of embodiment 40, wherein the first mixture further comprises a crosslinking agent, an initiator, or a combination thereof.

[0050] 42. The method of embodiment 40 or 41 wherein reacting the first mixture comprises reacting at a temperature of about 70 to about 80°C, or about 77 °C.

[0051] 43. The method of any one of embodiments 40 to 42, wherein the biobased solvent is 2-methylTHF.

[0052] In another embodiment of the present disclosure, there is provided:

[0053] 44. A method of forming an amorphized polysaccharide, the method comprising: drying a polysaccharide; dissolving the polysaccharide in an ionic liquid under sufficiently anhydrous conditions to form a mixture; heating the mixture; and forming the amorphized polysaccharide.

[0054] 45. The method of embodiment 44, further comprising isolating the amorphized polysaccharide from the ionic liquid.

[0055] 46. The method of embodiment 44 or 45, wherein the ionic liquid comprises imidazolium chlorides, imidazolium acetates, or a combination thereof.

[0056] 47. The method of any one of embodiments 44 to 46, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride (B Ml MCI), 1-ethyl- methylimidazolium chloride (EMIMCI), 1-allyl-3-methylimidazolium chloride (AMIMCI), 1- butyl-3-methylimidazolium acetate (BMIMAc), 1-ethyl-3-methylimidazolium acetate (EMIMAc), or a combination thereof.

[0057] 48. The method of any one of embodiments 44 to 47, wherein heating the mixture comprises heating at a temperature of about 100 °C.

[0058] 49. The method of any one of embodiments 44 to 48, wherein the polysaccharide has a particle size of about 15 to about 30 pm, or about 20 pm.

BRIEF DESCRIPTION OF THE FIGURES

[0059] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0060] FIG. 1 depicts schematic of possible H-abstraction mechanisms involving 2- methyltetrahydrofuran (2-MeTHF).

[0061] FIG. 2 depicts schematic of a hybrid microgel synthesis.

[0062] FIG. 3 graphically depicts storage modulus (G') and loss modulus (G") of hybrid microgel.

[0063] FIG. 4 graphically depicts a cell viability test.

[0064] FIG. 5 graphically depicts statistical analysis of relative gene expression.

DETAILED DESCRIPTION

[0065] Definitions

[0066] Unless defined otherwise, all technical and scientific terms used herein have the meaning as commonly understood in the art.

[0067] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context dictates otherwise.

[0068] “Amorphized” or “amorphous” generally refers to being partially or completely decrystallized; for example having a crystallinity index (Crl) < 100%.

[0069] “Azo compounds” include compounds comprising a -N=N- functionality (for example, a diazenyl group having the structure R-N=N-R', in which R and R' may be either substituted or unsubstituted aryl or alkyl groups).

[0070] “Metal alkyls” include compounds featuring a metal-carbon bond, such as a metal-carbon sigma bond. Examples of metal alkyls may include aluminum, lithium, boron, zinc, and magnesium alkyls or aryls, such as alkyllithium compounds (e.g., methyllithium, butyllithium, phenyllithium. Metal alkyls may also be referred to as organometallic compounds. [0071] Polymeric microgels comprising crosslinking networks are commonly produced by free radical polymerization. Such microgels comprise three-dimensional (3D) network structures of hydrophilic regions formed via physical or chemical crosslinking.

[0072] The properties of polymeric microgels, such as hydrophilicity, high- absorbency (up to 1000-fold compared to their dry weight), biodegradability, biocompatibility, softness, porosity, and viscoelastic behavior, generally resemble several properties of natural tissues. In tissue engineering, the crosslinked 3D structure of microgels may enable them to encapsulate cells in a homogeneous manner, and enhance cell-cell and cell-extracellular matrix (ECM) interactions by providing a 3D microenvironment similar to native ECM [1],

[0073] Stimuli-responsive or ‘smart’ microgels comprise a class of microgels that can undergo a change in volume, or sol-gel phase change in a reversible manner upon applying external stimuli such as temperature, pH, light, electric field, or ionic strength. For example, temperature-responsive or thermoresponsive microgels can exhibit reversible phase transition via temperature changes. This feature mimics the sensitivity of biomolecules in a biological environment.

[0074] Thermoresponsive microgels are generally classified into two categories: lower critical solution temperature (LCST) microgels that can hydrate and swell at lower temperatures; and upper critical solution temperature (UCST) microgels that can hydrate and swell at higher temperatures.

[0075] Poly(N-isopropylacrylamide) (PNIPAm) is a LCST polymer that has an LCST of approximately 32-33 °C, which is close to body temperature (37 °C) [2], PNIPAm hydrogels can form a sol state at room temperature and transform into a gel state at close to physiological temperature. This makes PNIPAm hydrogels potential candidates for use as scaffolds for tissue engineering. The structure and physicochemical properties of PNIPAm-based microgels may be controlled by using different synthesis-solvents, crosslinking methods, integrating different biomaterials, and fabrication strategies.

[0076] Cellulose, as a naturally occurring polymer, is useful for biomedical applications due to its biocompatibility, favorable mechanical strength, and ease of integration with other materials. The physical and chemical behaviour of cellulose is influenced by the arrangement of crystalline and amorphous domains. In cellulose, each anhydroglucose unit has three different hydroxyl groups. The hydroxyl groups at O(2)H, O(3)H, and O(6)H tend to be the main reactive groups amenable to chemical modification [3], During a chemical reaction, the accessibility of these hydroxyl groups on the cellulose fibril surface tends to be a factor in this reactivity. For example, crystalline cellulose (CC) tends to have highly ordered crystalline regions formed by hydrogen bonds. This compact structure of crystalline cellulose tends to limit the accessibility of reactive hydroxyl groups, while providing enhanced mechanical strength to the polymer structure. In contrast, amorphized cellulose (AC; otherwise referred to herein as modified cellulose) tends to have hydroxyl groups that are accessible and reactive, as the cellulose surface is mostly less ordered.

[0077] The mass ratio of crystallinity in cellulose can be expressed as the crystallinity index (Crl). The Crl has an influence on the stiffness and rigidity of the cellulose. Therefore, cellulose with appropriate suitable Crl (for example, between about 30.6 to about 77.7%) may enable the development of mechanically strong hybrid bioactive materials for different biomedical applications.

[0078] Ionic liquids (ILs) comprise organic salts with melting points below 100 °C and are generally regarded as green solvents. ILs tend to possess properties such as lower volatility, non-flammability, good dissolving and extracting ability, good thermal stability, easy recyclability, and lower viscosity. ILs have also been known to disrupt a native cellulose crystalline structure and break structurally important chemical linkages (e.g., |3- 1 ,4-glycosidic linkage). Furthermore, the cellulose dissolution conditions in ILs are relatively mild. 1-butyl-3-methylimidazolium chloride (B Ml MCI) is an IL that is known to exhibit some of the highest hydrogen bonding basicity among commonly used ILs. The enhanced hydrogen bond accepting ability of the Cl~ of BMIMCI can result in strong interactions with hydroxyl protons in cellulose, which may contribute to easier cellulose dissolution in said IL.

[0079] Hybrid microgels comprise crosslinked 3D polymeric networks that may be prepared by incorporating one polymer onto a second polymer chain with the assistance of a crosslinking agent, which may also acts as co-catalyst during crosslinking. Crosslinking is a key reaction in the formation of hybrid microgels. Crosslinking density can regulate swelling and mechanical properties, as well as the thermoresponsive behaviour of hybrid microgels. The control of swelling degree and mechanical stability of the polymeric 3D network are two features that are generally important for applications, such as biomedical applications (e.g., tissue engineering).

[0080] Petrochemical solvents, such as tetrahydrofuran (THF), toluene, and 1 ,4- dioxane, are often used as synthesis solvents (e.g., polymer synthesis) due to their low cost, desirable reactivity, and their ability to abstract hydrogen (H) atoms during chain transfer steps of free radical polymerization. These volatile organic solvents are considered to be toxic and are generally recognized as carcinogens, mutagens, or hazardous air pollutants [4], Moreover, these solvents may have detrimental effects during their application as it is difficult to completely remove them from the polymers. Among solvents considered suitable for pharmaceutical and biomedical applications, the solvent 2- MethylTHF is generally considered a promising candidate to replace the petrochemical solvents THF and toluene due to its properties. For example, The Hildebrand solubility parameter, a measure of the solvent power, for 2-MeTHF is 16.9 MPa ^1/2 . This value is just below that of THF (18 MPa ^1/2 ) and toluene (18.2 MPa ^1/2 ). The viscosity of 2-MeTHF (0.60 cP) is slightly higher to that of toluene (0.56 cP) and THF (0.48 cP). The polarity index (PI) of 2-MeTHF (6.97) is higher than that of THF (4.0) and toluene (2.4) [5],

[0081] Generally described herein is a crosslinked thermoresponsive hybrid microgel, and a method for making the crosslinked thermoresponsive hybrid microgel. The method for making the crosslinked thermoresponsive hybrid microgel may comprises a sustainable synthesis and modification of cellulose crystallinity. The method may comprise a sustainable, greener synthetic route for the synthesis of hybrid microgels via the use of less toxic and environment friendly conditions.

[0082] Generally described herein is a biocompatible, mechanically-stronger thermoresponsive hybrid microgel comprising amorphized cellulose and PNIPAm polymer, and a method for making the thermoresponsive hybrid microgel. The biocompatible, mechanically-stronger thermoresponsive hybrid microgel may be useful for biomedical applications. The method may comprise preparing amorphized cellulose with different crystallinity to enhance biocompatibility and viscoelastic properties of the hybrid microgel.

[0083] Generally, the present disclosure provides a crosslinked thermoresponsive hybrid microgel, and a method of preparing the crosslinked thermoresponsive hybrid microgel. The method may comprise a sustainable free radical polymerization. The method may comprise preparing amorphized cellulose (AC); preparing an aqueous solution comprising AC and NIPAm monomer in 2-MeTHF, and a crosslinking agent and a photoinitiator comprising AIBN; subjecting said solution to heat (e.g., 77 °C) and obtaining a crosslinked hybrid microgel, wherein the AC incorporate onto the PNIPAm polymer chain, and the crosslinking agent N,N’-methylenebisacrylamide (MBAA) acts as a co-catalyst of crosslinking.

[0084] Thermoresponsive monomer NIPAm may be used as a starting material. The NIPAm monomer may be used to obtain a thermoresponsive hybrid microgel that can change it phases within physiological temperatures (around 37 °C). The NIPAm monomer used may be approximately 1% w/v of the solvent in the reaction mixture. [0085] Amorphized cellulose may be used along with the NIPAm monomer. The amorphized cellulose may act as a primary network in the synthesis of the thermoresponsive hybrid microgel. NIPAm monomers polymerize and crosslink in presence of cellulose to form the crosslinked network. The amorphized cellulose may act as a primary network and NIPAm monomers may polymerize onto cellulose and crosslink with it to form a crosslinked network. The concentration of the amorphized cellulose may be chosen according to a required porosity of the final microgel, and/or to form a crosslinked hybrid microgel. The amorphized cellulose may be present in an amount of about 5 to about 25 w/w of NIPAm weight, or about 10 to about 20% w/w of NIPAm weight.

[0086] Generally described herein is a method comprising modification of cellulose crystallinity through via an ionic liquid (IL) treatment method. Ionic liquid (e.g., BMIMCI) treatment may be performed with dried cellulose (drying overnight at 50 °C). Before treatment, BMIMCI may be added in a flask, and stirred vigorously under vacuum conditions at 100 °C with nitrogen gas purging until a clear solution formed. Dried cellulose with a weight ratio of 2 % (cellulose to BMIMCI) may be added into the clear solution, and the mixture may be stirred at 100 °C under vacuum conditions and nitrogen purging for different time durations (e.g., 20 min, 1 h, 3 h, and 6 h, respectively) until the cellulose is dissolved. After dissolution of cellulose in IL, the mixture may be poured slowly into water in 2:1 (water : BMIMCI) ratio with vigorous agitation at room temperature (25°C). Vacuum filtration may be performed to collect the precipitate followed by thorough washing with water via centrifugation to remove IL. The washed, regenerated cellulose or amorphized cellulose may be dried at 60 °C for 24 h and stored at 4 °C for free radical polymerization with NIPAm monomer.

[0087] The IL treatment may be effective in reducing up to 26.5 % crystallinity of cellulose just after 20 min of treatment. The faster amorphization rate may be due to differences in the size of the untreated cellulose and the vacuum conditions during treatment. Cellulose with a lower particle size (~ 20 pm) may become more accessible to the solvent, which may increase solubility and reactivity of the cellulose. Treatment under vacuum, on the other hand, may promote creation of a moisture-free environment (e.g., sufficiently anhydrous conditions). The presence of moisture can inhibit cellulose dissolution in IL, as water causes solvation of IL anions, resulting in less sustained interaction with cellulose. A moisture-free environment, or otherwise sufficiently anhydrous conditions may be created by implementing vacuum conditions. A moisture-free environment, or otherwise sufficiently anhydrous conditions may be created by implementing vacuum conditions along with constant gas flow, such as nitrogen purging. Therefore, a relatively moisture-free environment may be created by implementing vacuum conditions along with constant nitrogen purging during treatment to transform crystalline cellulose to amorphized cellulose (AC).

[0088] Crosslinking agents as described herein may co-catalyze the crosslinking reaction, and thus, may co-catalyze the process of forming the crosslinked network. When using a crosslinking agent, such as MBAA, as a co-catalyst of crosslinking, the concentration used may be fairly low in the total reaction mixture (e.g., 0.2 g), so that the crosslinking density of the agent itself (e.g., MBAA) in the resulting hybrid microgel would be low.

[0089] Initiators as described herein may comprise organic azo-initiators, such as photoinitiator AIBN. Such initiators may generate free radicals; and may contribute to initiating free radical polymerization. For example, the labile carbon-nitrogen covalent bond of AIBN can undergo homolytic scission under thermal condition to produce free radicals that can initiate polymerization. The photoinitiator AIBN may be used in the method as described herein in an amount of about 5.7 to 6.1 % w/w of polymer mixture to initiate a free radical polymerization.

[0090] Toxicity of polymers to be used for biomedical applications is a concern. For all biomedical applications, toxicity must be low or absent in the final product. Therefore, developing a sustainable and less harmful synthesis route for the herein described thermoresponsive hybrid microgels may reduce the presence of unwanted or toxic impurities in the final hybrid microgel.

[0091] As such, generally described herein is a more sustainable, greener synthesis of thermoresponsive polymers or hybrid microgels comprising free-radical polymerization. The free-radical polymerization synthesis described herein may be more sustainable, or greener than other free-radical polymerization synthesis because it may provide a less toxic, more environmentally friendly polymer or microgel for biomedical applications. The free-radical polymerization synthesis described herein may be more sustainable, or greener relative to other free-radical polymerization synthesis through use of greener solvents. Greener solvents as used herein refer to solvents selected to minimize environmental impact resulting from use of solvents in chemical production. For example, 2-methyltetrahydrofuran (2-MeTHF) as used herein may be considered a greener solvent because can be produced from renewable resources, and/or is considered a bio-based solvent. In another example, bio-based, greener solvent ethyl lactate may not be suitable for use in the herein described synthesis of thermoresponsive polymers or hybrid microgels. In another example, volatile organic solvents such as cyclic ether solvents, or aromatic solvents may be suitable for use in the herein described synthesis of thermoresponsive polymers or hybrid microgels.

[0092] The free radical polymerization described herein may occur at a temperature of about 70 to 80°C, such as about 77°C. A temperature of about 70 to 80°C, such as about 77°C, may provide an 85% monomer conversion after 24 h. Temperatures below about 70 to 80°C, such as below about 77°C, may exhibit slower reaction rates; and temperatures above about 70 to 80°C, such as above about 77°C, may exhibit a lower monomer conversion due to decomposition of the initiator, which may halt the polymerization. Synthesis-solvent 2-MeTHF may act as a chain transfer agent during chain transfer steps in free radical polymerization. In cyclic ether solvents, the oxygen atom can stabilize polar contributions to transition states and incipient radicals. Thereby, the C-H bonds adjacent to the O atom can become reactive towards free radicals. In 2-MeTHF, radicals may be generated from the C-H bond of the 2-methyl group, and/or from the C-H bond at the C2 carbon atom by abstracting a H atom (Figure 1).

[0093] With reference to Figure 1 , H-abstraction from these two positions is denoted by transition states TS1 and TS2, respectively. H-abstraction from the 2-methyl group position involves overcoming an energy barrier of 78.2 kJ/mol through transition state TS1. In contrast for transition state TS2, H-abstraction from the C2 carbon atom involves overcoming an energy barrier of 31.2 kJ/mol [6], H-abstraction from the C2 atom position (C2) a relatively more favorable reaction pathway. Thereby, H abstraction at the C2 atom position of 2-MeTHF may lead to formation of a relatively more stable tertiary radical during a chain transfer step of free radical polymerization (Figure 1).

[0094] As such, generally described herein is a synthesis of a thermoresponsive polymer comprising free-radical polymerization. Briefly, NIPAm monomer (1.0 g) and MBAA crosslinking agent (0.3 g) may be mixed in solvent 2-MeTHF, followed by purging with nitrogen for 30 min. After nitrogen purging, AIBN initiator (0.05 g) may be added, and the reaction vessel sealed for 24 h at a constant temperature of about 77 °C. After synthesis, polymers may be precipitated by adding excess of diethyl ether and extracted from the mixture using vacuum filtration. The polymers may then be dialyzed for four days in ultrapure water (dialysis water changed every 6 h). Overnight vacuum drying may be performed to prepare dried polymer, which may then be stored at 4 °C.

[0095] Generally described herein is a synthesis of a thermoresponsive hybrid microgel. Briefly, amorphized cellulose (10-20% w/w relative to NIPAm monomer) may be added in a glass vial containing 5 mL of 2-MeTHF and 0.03 g of AIBN, and homogenized thoroughly using a homogenizer at 10,000 RPM for 25 min. 1 g of NIPAm monomer and 0.2 g of MBAA may be added in 95 mL of 2-MeTHF and stirred continuously to form a clear solution. After homogenization, the AC mixture may be placed in a water bath sonicator, and sonication may be performed for 20 minutes at 50 °C for proper fibrillation of the AC, and to make them more readily available for grafting during polymerization. After completing sonication, the mixture may be added to the solution containing NIPAm and MBAA. The final solution may be purged with nitrogen gas for 30 min followed by adding 0.05 g of the AIBN initiator and kept in a sealed reaction flask for 24 h at a constant temperature of about 77 °C.

[0096] The synthesized hybrid microgels may then be precipitated by being added dropwise into a 10-fold excess of ethyl ether and extracted from the mixture by vacuum filtration using EZFlow® membrane filter (Foxx Life Sciences, pore size: 0.22 pm). The extracted polymers may then be first dialyzed for 4 days in Milli-Q water using Slide-A- Lyzer™ G2 dialysis cassette (Thermo Scientific, MWCO: 3.5 K) and then using DiaEasy™ dialysis tubes (BioVision, MWCO: 6-8 K) for 2 days (dialysis water changed at every 6 h interval). After dialysis, purified hybrid microgels may be dried in vacuum dryer at 50 °C overnight followed by storage at 4 °C for further use. The dried microgels may be stored in sealed scintillation vial to avoid moisture absorption.

[0097] Generally, amorphized cellulose may first be homogenized and sonicated with AIBN to form free radicals on their surface. Free radical polymerization may then be initiated by adding pretreated amorphized cellulose into a reaction mixture containing NIPAm monomer, crosslinking agent MBAA, initiator AIBN, and synthesis-solvent 2- MeTHF. The 2-cyano-2-propyl radicals generated from the AIBN may initiate the hydrogen abstraction from the hydroxyl groups of cellulose to generate alkoxyl radicals. Such alkoxyl radicals, along with free end groups of the amorphous region of cellulose, may then interact with NIPAm monomers to form hybrid microgels (Figure 2).

[0098] Generally, bioactive compounds may be immobilized on, or within the hybrid microgels as described herein. Various immobilization techniques exist, including physical entrapment, electrostatic attraction, physical adsorption or absorption and chemical bonding can be used. Active biomolecules or cells may be attached to the hybrid microgel by physical adsorption. Bioactive ingredients such as growth factors, non-essential amino acids, and antibiotics may be introduced by placing prepared hybrid microgels in a solution containing these ingredients for extended periods of time. The ingredients may then diffuse into the hybrid microgel by imbibition.

[0099] The hybrid microgels as described herein may act as a scaffold and/or support for cells to grow. The hybrid microgels may act as a 2D or 3D scaffold, such as for tissue engineering. The hybrid microgels may provide a cellular-like microenvironment that facilitates cell growth and/or differentiation. The hybrid microgels may provide a cellular- like microenvironment via their mechanical properties, such as strength, stiffness, and/or porosity. The mechanical properties of the hybrid microgel may direct cell differentiation.

[00100] The hybrid microgels as described herein may act as a 2D or 3D scaffold and/or support for tissue engineering. When used for tissue engineering, stem cells or progenitor cells may be applied to a hybrid microgel. The mechanical properties of hybrid microgel, such as strength and/or porosity, may direct cell differentiation into a desired tissue (e.g., cartilage, bone, heart, etc.). Once the cells are differentiated into the desired engineered tissue, the tissue may be introduced into or onto a subject while still supported on the hybrid microgel. The engineered tissue may be isolated from the microgel, and may then be introduced into or onto a subject. The engineered tissue may be isolated from the microgel by breaking up, breaking down, or dissolving the microgel. The engineered tissue may be isolated from the microgel by subjecting the microgel to a temperature-induced phase transition. The engineered tissue may be introduced into or onto a subject through injections, e.g., for smaller areas of tissue damage; or through implantation, e.g., for larger areas of tissue damage.

[00101] Generally described herein is the use of hybrid microgels for biomedical applications. In an example of a biomedical application, ATDC5 cells may be grown in a Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium, and passaged by trypsinization at a ratio of 1 :10 due to a higher proliferation rate. ATDC5 cells may then loaded onto hybrid microgels at a concentration of 1 million cells per 100 pL of hybrid microgels in phosphate-buffered saline solution. A fixable viability stain (FVS510) may be added to the microgels at 1 pL per 1 million cells. The microgels may be incubated at about 37 °C for about 48 h with about 5 % CO ₂ and then the cells may be isolated from the microgels. Isolation of proliferated cells from the hybrid microgels may be accomplished by adding the microgels to a 70 pm strainer and washing with phosphate buffer solution (PBS) to remove unincorporated cells, followed by incubation for 5 min in 100 % ethanol (to dissolve the microgel and fix the cells). This cell/microgel mixture may then be run through a 5 pm strainerto remove the microgels and the remaining cells are then processed for further analysis.

[00102] The hybrid microgels as described herein may be useful for cartilage tissue engineering. The hybrid microgels may be useful for chondrogenic gene expression. Hybrid microgels comprising 85 wt% thermoresponsive polymers may be useful for chondrogenic gene expression, for cartilage tissue engineering. Hybrid microgels comprising 80 wt% thermoresponsive polymers may be useful for osteogenic gene expression, for bone tissue engineering. The hybrid microgels as described herein may be useful for 3D printed organ transplantation.

[00103] The hybrid microgels as described herein may be useful for controlled drug delivery. The hybrid microgels as described herein may be useful for controlled drug delivery through encapsulation, physical adsorption, physical absorption, or chemical attachment. The hybrid microgels as described herein may be useful for water treatment applications, waste removal applications, enhanced oil recovery applications or a combination thereof via encapsulation, physical adsorption or absorption, and/or chemical attachment. The hybrid microgels as described herein may be useful for water treatment applications such as seawater desalination. The hybrid microgels as described herein may be useful for enhanced oil recovery applications via cargo delivery vehicle, such as for delivery of catalyst in a heavy oil reservoir.

[00104] In an example, the present disclosure generally provides:

[00105] 1. A hybrid microgel, comprising: a thermoresponsive polymer, and a polysaccharide comprising amorphous domains, the thermoresponsive polymer being grafted to the polysaccharide.

[00106] 2. The microgel of example 1 , wherein the thermoresponsive polymer comprises an acrylamide backbone.

[00107] 3. The microgel of example 1 or 2, wherein the thermoresponsive polymer comprises Poly(/V-isopropylacrylamide).

[00108] 4. The microgel of any one of examples 1 to 3, wherein the thermoresponsive polymer is present in an amount between about 75 wt% to about 95wt%, or about 80wt% to about 90 wt%.

[00109] 5. The microgel of any one of examples 1 to 4, wherein the polysaccharide comprises a crystallinity index (Crl) between about 20% to about 85%, or about 30% to about 80%, or about 30% to about 70%, or about 30% to about 60%, or about 30.6% to about l , or about 30.6% to about 57.1%.

[00110] 6. The microgel of any one of examples 1 to 5, wherein the polysaccharide comprises a beta-glucan.

[00111] 7. The microgel of any one of examples 1 to 6, wherein the polysaccharide comprises cellulose. [00112] 8. The microgel of any one of examples 1 to 7, wherein the polysaccharide is present in an amount between about 5 wt% to about 25 wt%, or about 10 wt% to about 20 wt%.

[00113] 9. The microgel of any one of examples 1 to 8, wherein the microgel is a lower critical solution temperature (LCST) microgel.

[00114] 10. The microgel of any one of examples 1 to 9, wherein the LCST is in a range between about 30 to about 40°C; or in a range between about 32 to about 37°C.

[00115] 11. The microgel of any one of examples 1 to 10, wherein the microgel comprises porosity, the pores comprising an average pore size between about 5 to about 15 pm, or about 7 to about 12 pm.

[00116] 12. The microgel of any one of examples 1 to 11 , wherein the microgel comprises a storage modulus of about 0.01 to about 0.2 MPa, or about 0.05 to about 0.15 MPa, or about 0.06 MPa to 0.13 MPa.

[00117] 13. The microgel of any one of examples 1 to 12, wherein the microgel comprises an equilibrium swelling at 20 °C of about 7.5 to about 12 times its dry weight; or about 9.4 times its dry weight.

[00118] 14. The microgel of any one of examples 1 to 13, wherein the microgel comprises a crystallinity index (Crl) between about 30% to about 80%, or about 35% to about 75%, or about 40% to about 70%.

[00119] 15. The microgel of any one of examples 1 to 14, wherein the microgel comprises a volume phase transition temperature between about 30 °C to about 45°C, or about 35 °C to about 40°C.

[00120] In another example, the present disclosure generally provides:

[00121] 16. A method of synthesizing a hybrid microgel, the method comprising: providing a first mixture, the first mixture comprising an amorphized polysaccharide; providing a second mixture, the second mixture comprising a thermoresponsive monomer; mixing together the first mixture and the second mixture; reacting together the first mixture and the second mixture via free-radical polymerization; and forming the hybrid microgel.

[00122] 17. The method of example 16, wherein the first mixture further comprises a first initiator.

[00123] 18. The method of example 16 or 17, wherein the first initiator comprises organic peroxides, azo compounds, metal alkyls, persulfates, or a combination thereof.

[00124] 19. The method of any one of examples 16 to 18, wherein the first initiator comprises benzoyl peroxide, peroxyacetic acid, ammonium persulfate (APS), azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), potassium persulfate (KPS), or a combination thereof.

[00125] 20. The method of any one of examples 16 to 19, wherein the second mixture further comprises a crosslinking agent.

[00126] 21. The method of any one of examples 16 to 20, wherein the crosslinking agent comprises diacrylates, bisacrylamides, dialkene-substituted aryls.

[00127] 22. The method of any one of examples 16 to 21 , wherein the crosslinking agent comprises an alkylene glycol diacrylate (such as ethylene glycol diacrylate), 1 ,6-Hexanediol diacrylate (HDDA), allyl methacrylate (AMA), an alkylene bisacrylamides (such as N,N-Methylenebisacrylamide), divinylbenzene, or a combination thereof.

[00128] 23. The method of any one of examples 16 to 22, wherein the first mixture and/or second mixture further comprises a solvent.

[00129] 24. The method of any one of examples 16 to 23, wherein the solvent comprises a volatile organic solvent, a bio-based solvent, or a combination thereof.

[00130] 25. The method of any one of examples 16 to 24, wherein the solvent comprises a cyclic ether solvent, an aromatic solvent, 2-methylTHF, or a combination thereof.

[00131] 26. The method of any one of examples 16 to 25, wherein reacting together the first mixture and second mixture further comprises adding a second initiator.

[00132] 27. The method of example 16 or 26 wherein the second initiator comprises organic peroxides, azo compounds, metal alkyls, persulfates, or a combination thereof.

[00133] 28. The method of any one of examples 16 to 27, wherein the second initiator comprises benzoyl peroxide, peroxyacetic acid, ammonium persulfate (APS), azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), potassium persulfate (KPS), or a combination thereof.

[00134] 29. The method of any one of examples 16 to 28, wherein reacting together the first mixture and second mixture further comprises heating the reaction.

[00135] 30. The method of any one of examples 16 to 29, wherein heating the reaction comprises maintaining the reaction at a temperature between about 70 to about 80°C, or about 76 to 78°C, or about 77°C.

[00136] 31. The method of any one of examples 16 to 30, further comprising providing the amorphized polysaccharide, wherein providing the amorphized polysaccharide comprises: drying a polysaccharide; dissolving the polysaccharide in an ionic liquid under sufficiently anhydrous conditions and forming the amorphized polysaccharide; and isolating the amorphized polysaccharide from the ionic liquid.

[00137] 32. The method of any one of examples 16 to 31 , further comprising homogenizing the first mixture.

[00138] 33. The method of any one of examples 16 to 32, further comprising sonicating the first mixture.

[00139] 34. The method of any one of examples 16 to 33, wherein the ionic liquid comprises imidazolium chlorides, imidazolium acetates, or a combination thereof.

[00140] 35. The method of any one of examples 16 to 34, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride (BMIMCI), 1-ethyl- methylimidazolium chloride (EMIMCI), 1-allyl-3-methylimidazolium chloride (AM I MCI), 1-butyl-3- methylimidazolium acetate (BMIMAc), 1-ethyl-3-methylimidazolium acetate (EMIMAc), or a combination thereof.

[00141] In another example, the present disclosure generally provides:

[00142] 36. A composition useful for tissue engineering, the composition comprising: the hybrid microgel of any one of examples 1 to 15; and at least one cell comprising a progenitor cell, stem cell, or a combination thereof.

[00143] 37. The composition of example 36, wherein the composition is useful for cartilage tissue engineering, bone tissue engineering, vascular tissue engineering, pancreas tissue engineering, cardiac tissue engineering, or a combination thereof.

[00144] In another example, the present disclosure generally provides:

[00145] 38. Use of the hybrid microgel of any one of examples 1 to 15, or the hydrogel made by the method of any one of example 16 to 35 for biomedical applications, water treatment applications, waste removal applications, enhanced oil recovery applications, or a combination.

[00146] 39. The use of example 38 wherein biomedical applications, water treatment applications, waste removal applications, enhanced oil recovery applications, or a combination thereof comprises tissue engineering, such as cartilage, bone, or muscle tissue engineering; 3D printed organ transplantation; controlled drug delivery vehicles, such as through encapsulation, physical adsorption or absorption, or chemical attachment; seawater desalination, and/or cargo delivery vehicle, such as for delivery of catalyst in a heavy oil reservoir. [00147] In another example, the present disclosure generally provides:

[00148] 40. A method of synthesizing a thermoresponsive polymer, the method comprising: providing a first mixture comprising a thermoresponsive monomer and a biobased solvent; reacting the first mixture via free-radical polymerization; and forming the thermoresponsive polymer.

[00149] 41. The method of example 40, wherein the first mixture further comprises a crosslinking agent, an initiator, or a combination thereof.

[00150] 42. The method of example 40 or 41 wherein reacting the first mixture comprises reacting at a temperature of about 70 to about 80°C, or about 77 °C.

[00151] 43. The method of any one of examples 40 to 42, wherein the bio-based solvent is 2-methylTHF.

[00152] In another example, the present disclosure generally provides:

[00153] 44. A method of forming an amorphized polysaccharide, the method comprising: drying a polysaccharide; dissolving the polysaccharide in an ionic liquid under sufficiently anhydrous conditions to form a mixture; heating the mixture; and forming the amorphized polysaccharide.

[00154] 45. The method of example 44, further comprising isolating the amorphized polysaccharide from the ionic liquid.

[00155] 46. The method of example 44 or 45, wherein the ionic liquid comprises imidazolium chlorides, imidazolium acetates, or a combination thereof.

[00156] 47. The method of any one of examples 44 to 46, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride (BMIMCI), 1-ethyl- methylimidazolium chloride (EMIMCI), 1-allyl-3-methylimidazolium chloride (AM I MCI), 1-butyl-3- methylimidazolium acetate (BMIMAc), 1-ethyl-3-methylimidazolium acetate (EMIMAc), or a combination thereof.

[00157] 48. The method of any one of examples 44 to 47, wherein heating the mixture comprises heating at a temperature of about 100 °C.

[00158] 49. The method of any one of examples 44 to 48, wherein the polysaccharide has a particle size of about 15 to about 30 pm, or about 20 pm.

[00159] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway. [00160] EXAMPLES

[00161] Following is a description of a method of preparing crosslinked thermoresponsive hybrid microgels by free radical polymerization. The method may comprise modifying crystallinity of cellulose; preparing an aqueous solution comprising two hydrophilic polymers (modified cellulose and poly(N-isopropylacrylamide) (PNIPAm)) in a bio-based solvent (2-Methyltetrahydrofuran or 2-MeTHF), a crosslinking agent, and an initiator comprising azobisisobutyronitrile (AIBN); subjecting said solution to heat and obtaining a crosslinked microgel, wherein the modified cellulose may enhance crosslinking with PNIPAm and the crosslinking agent may act as a co-catalyst for crosslinking. The method as described herein may reduce up to 97% of carbon emissions associated with other microgel syntheses. The method may also provide a thermoresponsive hybrid microgel that is cytocompatible, has a lower toxicity; and/or is useful for biomedical applications, such as scaffold for tissue engineering.

[00162] Example 1 - Preparation of amorphized cellulose (AC)

[00163] Briefly, 1 .0 g cellulose was dried overnight in a dryer at 50 °C (P = 20 kPa).

Ionic liquid BMIMCI (49.0 g) was added and stirred vigorously in a flask under vacuum conditions at 100 °C with nitrogen gas purging until a clear solution was formed. Dried cellulose of 2 % w/w (cellulose to BMIMCI) was then added into clear solution. The mixture was stirred at 100 °C under vacuum conditions and nitrogen purging for 1 h.

[00164] After complete dissolution of cellulose, the cellulose-BMIMCI mixture was slowly poured into water in a 2:1 (water : BMIMCI) ratio with vigorous agitation at room temperature (25 °C). The precipitate was collected by vacuum filtration followed by washing thoroughly with water via repeated centrifugation (4 times) at 4000 RPM for 15 min to eliminate ionic liquid. The regenerated AC was then dried at 60 °C for 24 h in a vacuum dryer before storage for further use.

[00165] Changes in cellulose structure resulted in distinctive XRD patterns. From the XRD spectra, the peak (20) corresponding to the 002 lattice plane shifted from 22.7° to 22.4°, which reflected the 020 plane of amorphized cellulose.

[00166] From the crystallinity index (Crl) calculation, Crl of AC was reduced from 77.7% to 41.7%.

[00167] Example 2 - Hybrid microgel synthesis

[00168] Hybrid microgels composed of AC and PNIPAm were synthesized via free radical polymerization. Briefly, 0.15 g of AC (15% w/w of NIPAm weight) was added in a scintillation vial containing 5 mL of 2-MeTHF and 0.03 g of initiator AIBN. This AC mixture was homogenized thoroughly using a homogenizer at 10,000 RPM for 25 min for fibrillation of the cellulose and to make it more readily available for grafting during polymerization. After homogenization, AC mixture was placed in a water bath sonicator, and sonication was performed for 20 min at 50 °C for further fibrillation of the cellulose and to make it more readily available for grafting during polymerization.

[00169] In another flask, 1 .0 g of NIPAm monomer and 0.2 g of MBAA were added in 95 mL of 2-meTHF and stirred continuously to form a clear solution.

[00170] After sonication, the AC mixture was added to the solution containing NIPAm and MBAA. The final solution was purged with nitrogen gas for 30 min followed by adding 0.05 g of AIBN and kept in a sealed reaction flask for 24 h at a constant temperature of 77 °C. Hybrid microgels were successfully synthesized from the polymerization in 2- MeTHF after 24 h of reaction.

[00171] Synthesized AC-g-PNIPAm hybrid microgels were precipitated by being added dropwise into a 10-fold excess of ethyl ether and extracted from the mixture by vacuum filtration using EZFlow® membrane filter (Foxx Life Sciences, pore size: 0.22 pm). The extracted microgels were first dialyzed for 4 days in Milli-Q water using Slide-A-Lyzer™ G2 dialysis cassette (Thermo Scientific, MWCO: 3.5 K) and then using DiaEasy™ dialysis tubes (BioVision, MWCO: 6-8 K) for 2 days (dialysis water changed at every 6 h interval). The purified hybrid microgels were then dried at 50 °C for 24 h in a vacuum dryer before storage for further use.

[00172] Example 3 - Characterization of hybrid microgels

[00173] Equilibrium swelling was determined at a temperature between 20 °C and 45 °C to assess the thermoresponsive swelling behavior of the hybrid microgels. The swelling study was performed by swelling the purified microgels in ultra-pure water for 24 h and calculating the relative increase in uptake (at 20 °C) and release (at 45 °C) of water. Equilibrium swelling at 20 °C was 9.4 times of its dry weight, and at 45 °C, 1 .4 times of its dry weight, which indicated a thermoresponsive swelling ability of the hybrid microgels.

[00174] Internal microstructure of the microgel matrix was analyzed by preparing a cylindrical microgel disk (height: 5 mm and width: 10 mm). Microgel disk was then dipped in ultrapure water and allowed to reach equilibrium. The swollen microgel disk was then frozen quickly in liquid nitrogen for 2 min to hold the swollen structure. The frozen disk was freeze-dried for 48 h using a freeze dryer at -50 °C. The freeze-dried microgel matrix was then fractured mechanically and coated with platinum using a sputter coated for 45 sec before SEM imaging. Pore structure analysis by Imaged software (v1 ,52s) showed that the microgel matrix exhibited porosity (average pore size: 10 pm) with uniform distribution of cellulose fibers, indicating good integration between cellulose and PNIPAm molecules.

[00175] Rheological measurements were conducted in a rheometer using a measuring setup of a stainless-steel 25 mm parallel plate geometry with a gap height of 1 mm. Dried microgel of 0.5 g was added in 3 mL of deionized water to prepare a concentrated suspension for the measurements. Strain sweeps were performed within the shear strain range between 0.05 % and 100 % at 20 °C and 45 °C at a constant frequency of 1 Hz to measure the linear viscoelastic region (LVR) for oscillatory measurements. Frequency sweeps were performed within the angular frequency range of 1 .0 rad/s to 100 rad/s at 20 °C and 45 °C at a constant 1 .0 strain % (within the LVR regime).

[00176] Storage modulus (G') and loss modulus (G") of microgel at a frequency of 6.06 rad/s (~ 1 Hz) at 20 °C and 45 °C were as follows (Figure 3):

At 20 °C, G' = 24248 Pa and G" = 4605 Pa

At 45 °C, G' = 61001 Pa and G" = 33657 Pa

[00177] It was observed that at both temperatures, the microgel exhibited higher G' and lower G", which indicated elastic nature and well-crosslinked network of the microgel. Moreover, higher G' and G" values at 45 °C compared to 20 °C indicated the thermoresponsive behavior of the microgels.

[00178] Further, it was found that the hybrid microgels exhibited a storage modulus between 0.06 MPa to 0.13 MPa. This range reflected G’ (at 45 °C) of hybrid microgels synthesized using 10 wt% to 20 wt% cellulose. Upper bound: 0.13 MPa for hybrid microgels comprising 20 wt% cellulose. Lower bound: 0.06 MPa for hybrid microgels comprising 10 wt% cellulose.

[00179] Example 4 - Chondrogenesis on hybrid microgels

[00180] Chondrogenesis is generally considered the process by which cartilage is developed.

[00181] Initially, ATDC5 cells (chondroprogenitor cells derived from mouse teratocarcinoma and characterized as a chondrogenic cell line model; purchased from Millipore Sigma, Catalog No.: 99072806-1VL) were grown in DMEM/F12 medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) containing 10 % fetal bovine serum, 1 % non-essential amino acids and 1 % penicillin-streptomycin. Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 combines high concentrations of glucose, amino acids, and vitamins with F-12's wide variety of components. DMEM/F-12 contains no proteins, lipids, or growth factors. ATDC5 cells were passaged by trypsinization at a ratio of 1 :10 due to their higher proliferation rate. Trypsinization was used because it facilitated dissociation of adherent cells from the vessel (e.g., cell culture flask) in which they were being cultured initially. Otherwise, the cells could not be collected from the culture flask and transferred/loaded onto microgels.

[00182] ATDC5-loaded hybrid microgels were generated at a concentration of 1 million cells per 100 pL of biomaterial (prepared in phosphate-buffered saline or PBS solution). A fixable viability stain (FVS510) was added to the microgels at 1 pL per 1 million cells. The microgels were incubated at 37 °C for 48 h with 5 % CO ₂ , and then the cells were isolated from the microgels. This was accomplished by adding the microgels to a 70 pm strainer and washing with phosphate buffer solution (PBS) to remove unincorporated cells, followed by incubation for 5 min in 100% ethanol (to dissolve the biomaterial and fix the cells). This cell/microgel mixture was run through a 5 pm strainer to remove the microgels and separated cells for further analysis.

[00183] Example 5 - Cell viability test

[00184] Isolated cells were filtered and resuspended in 500 pL of 100 % ethanol and left for 5-10 mins at room temperature. The cells were then centrifuged, the liquid was removed, and 500 pL of 0.1 % Tween 20 was added to permeabilize the cells for 20 mins at room temperature. The cells were centrifuged again, the liquid was removed, and 50 pL of Tween buffer and 0.5 pg of antibody was added to each tube and incubated in the dark for 30-45 mins at room temperature (primary antibodies conjugated to fluorophores included: Prrxl (Thermo-Fisher # PA5-18831), Sox9 (Thermo-Fisher # 14-9765-82), Osterix (Thermo-Fisher # BS-1110R). The cells were then washed three times with FACs buffer then resuspended in FACs buffer (Flow Cytometry Staining Buffer; buffered saline solution containing fetal bovine serum (2-5%) and sodium azide (0.09%) as a preservative; purchased from ThermoFisher Scientific, Catalog No.: 00-4222-26). The cells were then analyzed using the Attune NxT system. The results were analyzed using FlowJo software. [00185] Cell viability and percentage of cell population expressing different markers were analyzed by flow cytometry. The following markers were used: Prrxl for nondifferentiated ATDC5 cells, Osterix for osteogenic gene expression in stem cells, and Sox9 for chondrogenic gene expression in stem cells.

[00186] Percentage of cells expressing Prrxl marker in microgels were around 87 %. Osterix marker was responsible for osteogenic gene expression in stem cells. Osteogenesis is generally considered the process by which bones are developed. In this demonstration, 68 % of the positive cells in microgels expressed Osterix marker at the protein level (Figure 4). Sox9 was the gene expression marker responsible for chondrogenesis. In case of Sox9 expression among cell population, around 51 % of cells obtained from the hybrid microgels were Sox9+ cells.

[00187] This test indicated the type of cells (chondrogenic, osteogenic, or nondifferentiated) that were proliferating on and/or in the hybrid microgels. This test also indicated their population when they were growing onto or into the hybrid microgels. This test indicated whether the microgel scaffold could provide an appropriate cellular microenvironment for cells to proliferate, differentiate, and upregulate the gene expression of interest for desired tissue engineering.

[00188] Example 6 - Gene and protein expression analysis

[00189] Isolated cells were lysed in TRIzol (Ambion) with an 18-gauge needle, then total RNA was extracted using EZNA® Total RNA Kit I (OMEGA). Briefly, 200 pL of chloroform per ml of TRIzol was added to the TRIzol solution. Sample was then vortexed, incubated at room temperature for 15 mins, then centrifuged for 15 min at 4 °C and 12,000 RPM. The top clear layer was transferred into a new HiBind® RNA mini column in a 2 mL collection tube. The sample was centrifuged at 10,000 g for 1 min, and the filtrate was discarded. The sample was washed with 500 pL wash buffer I, centrifuged for 30 s at 10,000 g and the filtrate was discarded. The sample was washed with 500 pL wash buffer II, centrifuged for 1 min at 10,000 g, and the filtrate was discarded. The sample was then centrifuged at 14,000 g for 2 min to remove any excess liquid. The collection tube was replaced with a new one and the sample was extracted by adding 50 pL of ultra-pure water and centrifuging at 14,000 g for 2 min. The sample was immediately stored at -80 °C until the next step could be completed.

[00190] RNA (5 pg) was converted to cDNA using a High-Capacity cDNA Reverse Transcription kit, mRNA levels were then analyzed using TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assay primers for mouse Prrxl , Sox9, Sp7 (known as Osterix) and 18S (endogenous control) (all Thermo-Fisher) on a QuantStudioTM 6 Flex Real-Time PCR System.

[00191] Sample was run in triplicate and resulting cycle threshold (Ct) values were analyzed using the AACt method (2" ^AACt ) against 18S endogenous control and undifferentiated/untreated ATDC5 cells as the reference control. The relative mRNA expression was plotted using GraphPad Prism 6.0. Statistical analysis was done on the AACt values (Figure 5).

[00192] In the analysis, baseline of the relative gene expression was fixed using control. Compared to control, relative gene expression of Prrxl in cells obtained from the microgels remained below the baseline and no significant changes in the expression was observed. Significant upregulation in the expression of Sp7 (Osterix) gene for osteogenesis was found in cells grown on microgels.

[00193] In case of Sox9 expression, it was found that the expression in cells collected from the microgels was upregulated from the baseline. This Sox9 transcription factor induced expression of cartilage-related genes like collagen type II alpha 1 and aggrecan.

[00194] From the flow cytometric analysis and qPCR analysis, the prepared hybrid microgel appeared to be a suitable scaffold candidate for chondrogenesis as indicated by the higher expression of Sox9 marker in cells collected from this microgel. The porous structure of the microgels may play a role in this, along with the stiffness in stimulating higher chondrogenic gene expression. The porosity of scaffolding material can affect the chondrogenic gene expression.

[00195] Microgels with larger pore sizes may induce chondrogenesis because of more efficient transport of nutrients, oxygen, and chondrogenic factors. The effectiveness of AC-g-PNIPAm hybrid microgel scaffold for chondrogenesis may depend on both rheological properties, as well as micro-architecture of the scaffolds. Hence, in addition to stiffness, the microgel had a large enough mean pore size and porosity which contributed to its higher Sox9 gene expression.

[00196] This gene and protein expression analysis indicated that the hybrid microgels provided sufficient cellular microenvironment for the cells to express the gene of interest (e.g., Sox9 for chondrogenesis), as well as protein required for desired biomedical applications.

[00197] Example 7 - Hybrid microgel synthesis

[00198] Hybrid microgels composed of AC and PNIPAm were synthesized via free radical polymerization. Briefly, 0.05 g of AC (5% w/w of NIPAm weight) was added in a scintillation vial containing 5 mL of 2-MeTHF and 0.03 g of initiator AIBN. This AC mixture was homogenized thoroughly using a homogenizer at 10,000 RPM for 25 min for fibrillation of the cellulose and to make it more readily available for grafting during polymerization. After homogenization, AC mixture was placed in a water bath sonicator, and sonication was performed for 20 min at 50 °C for further fibrillation of the cellulose and to make it more readily available for grafting during polymerization.

[00199] In another flask, 1 .0 g of NIPAm monomer and 0.2 g of MBAA were added in 95 mL of 2-meTHF and stirred continuously to form a clear solution. [00200] After sonication, the AC mixture was added to the solution containing NIPAm and MBAA. The final solution was purged with nitrogen gas for 30 min followed by adding 0.05 g of AIBN and kept in a sealed reaction flask for 24 h at a constant temperature of 77 °C. Hybrid microgels were successfully synthesized from the polymerization in 2- MeTHF after 24 h of reaction.

[00201] Precipitation and purification of the synthesized hybrid microgel was same as described in Example 2.

[00202] Example 8 - Hydrogel Crystallinity

[00203] To study the crystallinity of the synthesized hybrid microgels, X-ray powder diffraction (XRD) was performed using a diffractometer (Bruker®, model: D8 Advance). The dried samples (0.05 g/sample) were ground into powder and analyzed in the 20 range of 5° - 70° with 0.05° step size.

[00204] Hybrid microgels comprising amorphized cellulose resulted in distinctive XRD patterns. From the XRD spectra, peak (20) corresponding to the 110 and 020 lattice planes of amorphized cellulose (AC) were found at 15.5° and 20.5°, while peak at 22.5° reflected the presence of PNIPAm.

[00205] The crystallinity (Crl) of the microgels changed with changing the AC content in the structure. Despite the fact the PNIPAm is amorphous in nature, crystalline regions of the AC were found to play a role in determining the Crl of the hybrid microgels. It was found that the Crl of the hybrid microgels ranges between 40.3% to 68.8%.

[00206] Example 9 - Phase Transition Temperature

[00207] The lower critical solution temperature (LCST), as well as volume phase transition temperature (VPTT) of AC-g-PNIPAm microgels were measured by turbidimetric analysis in deionized water. Absorbances were collected using an UVA/is spectrophotometer (VWR® UV-3100PC) at A = 500 ± 0.5 nm wavelength. For turbidimetry, the temperature range was chosen between 20 °C and 50 °C with a 1 °C/min heating rate controlled by a constant-temperature heating system (VWR®, model: A-102).

[00208] The VPTT was considered an important parameter of PNIPAm microgels. In general, the VPTT of thermoresponsive microgels is controlled by the relative hydrophobicity of the microgel system. The VPTT of the hybrid microgels changed with changing AC content. The hybrid AC-g-PNIPAm microgels showed clear phase transition behaviour like PNIPAm microgels. It was found that, depending on the amount of ACs in the crosslinked network structure, the hybrid microgels exhibited VPTT between 35.4 °C to 39.2 °C. [00209] Example 10 - Hybrid microgel synthesis

[00210] Hybrid microgels composed of AC and PNIPAm were synthesized via free radical polymerization. Briefly, 0.2 g of AC (20% w/w of NIPAm weight) was added in a scintillation vial containing 5 mL of 2-MeTHF and 0.03 g of initiator AIBN. This AC mixture was homogenized thoroughly using a homogenizer at 10,000 RPM for 25 min for fibrillation of the cellulose and to make it more readily available for grafting during polymerization. After homogenization, AC mixture was placed in a water bath sonicator, and sonication was performed for 20 min at 50 °C for further fibrillation of the cellulose and to make it more readily available for grafting during polymerization.

[00211] In another flask, 1 .0 g of NIPAm monomer and 0.2 g of MBAA were added in 95 mL of 2-meTHF and stirred continuously to form a clear solution.

[00212] After sonication, the AC mixture was added to the solution containing NIPAm and MBAA. The final solution was purged with nitrogen gas for 30 min followed by adding 0.05 g of AIBN and kept in a sealed reaction flask for 24 h at a constant temperature of 77 °C. Hybrid microgels were successfully synthesized from the polymerization in 2- MeTHF after 24 h of reaction.

[00213] Precipitation and purification of synthesized hybrid microgel was same as described in Example 2.

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Abstraction Reactions by Hydroperoxyl Radical from 2-Methyltetrahydrofuran and 2,5- Dimethyltetrahydrofuran. The Journal of Physical Chemistry A. 2013; 117(24): 5028-5041. [00221] The embodiments described herein are intended to be examples only.

Alterations, modifications, and/or variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole. [00222] The aspects, embodiments, and/or examples of the present disclosure being thus described, it should be recognized that said aspects, embodiments, and/or examples may be varied in ways that do not depart from the spirit and scope of the present disclosure, and that said variations are intended to be included within the scope of the following claims. [00223] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.